Proceedings Book

Transcription

Proceedings Book

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European Journal of Cancer
26th EORTC – NCI – AACR Symposium on
Molecular Targets and Cancer Therapeutics
Barcelona, Spain, 18 – 21 November 2014
Proceedings Book
Amsterdam • Boston • London • New York • Oxford • Paris • Philadelphia • San Diego • St Louis
Editor-in-Chief:
Editors:
Basic and Preclinical Research:
Drug Development:
Early Breast Cancer:
Advanced Breast Cancer:
Gastrointestinal Cancers:
Genitourinary Cancers:
Lung Cancer:
Gynaecological Cancers:
Head and Neck Cancer:
Sarcomas:
Melanoma:
Neuro-oncology:
Epidemiology and Prevention:
Paediatric Oncology:
Founding Editor:
Past Editors:
Editorial Office:
Alexander M.M. Eggermont
Institut Gustave Roussy
Villejuif, France
Richard Marais, Manchester, UK
Ulrich Keilholz, Berlin, Germany
Jordi Rodon, Barcelona, Spain
Kathleen I. Pritchard, Toronto, Canada
David Cameron, Edinburgh, UK
Volker Heinemann, Munich, Germany
Michel Ducreux, Villejuif, France
Karim Fizazi, Villejuif, France
Mary O’Brien, London, UK
Ignace Vergote, Leuven, Belgium
Kevin Harrington, London, UK
Jean-Yves Blay, Lyon, France
Dirk Schadendorf, Essen, Germany
Roger Stupp, Zurich, Switzerland
Jan Willem Coebergh, Rotterdam, The Netherlands
Rob Pieters, Rotterdam, The Netherlands
Henri Tagnon
Michael Peckham, London, UK; Hans-Jörg Senn, St Gallen, Switzerland; John Smyth, Edinburgh, UK
Elsevier, The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK
Tel: +44 (0) 1865 843590, Email: [email protected]
EDITORIAL BOARD
CLINICAL ONCOLOGY
J.-P. Armand (France)
A. Ayhan (Japan)
R. Blamey (UK)
M. Bolla (France)
J. Boyages (Australia)
N. Brünner (Denmark)
F. Cardoso (Portugal)
J. Cassidy (UK)
M. Castiglione (Switzerland)
L. Cataliotti (Italy)
L. Cheng (USA)
H. Cody (USA)
R. Coleman (UK)
A. Costa (Italy)
J. De Bono (UK)
M.J.A. De Jong (The Netherlands)
E. de Vries (The Netherlands)
A. Dicker (USA)
R. Dummer (Switzerland)
F. Eisinger (France)
S. Erridge (UK)
G. Ferrandina (Italy)
H. Gabra (UK)
H. Gelderblom (The Netherlands)
B. Hasan (Belgium)
J.C. Horiot (Switzerland)
C. Huber (Germany)
R. Jakesz (Austria)
J. Jassem (Poland)
D. Jodrell (UK)
V.C. Jordan (USA)
A. Katz (Brazil)
M. Kaufmann (Germany)
I. Kunkler (UK)
L. Lindner (Germany)
P.E. Lønning (Norway)
P. Lorigan (UK)
K. McDonald (Australia)
R. Mertelsmann (UK)
F. Meunier (Belgium)
T. Mok (Hong Kong)
D. Nam (Korea)
P. O’Dwyer (USA)
J. Overgaard (Denmark)
N. Pavlidis (Greece)
J. Perry (Canada)
P. Price (UK)
D. Raghavan (USA)
J. Ringash (Canada)
J. Robert (France)
A. Rody (Germany)
D. Sargent (USA)
M. Schmidinger (Austria)
S. Sleijfer (The Netherlands)
P. Sonneveld (The Netherlands)
A. Sparreboom (USA)
M. van den Bent (The Netherlands)
M. Van Glabbeke (Belgium)
G. Velikova (UK)
U. Veronesi (Italy)
A. Vincent-Salomon (France)
A. Voogd (The Netherlands)
E. Winquist (Canada)
BASIC, PRECLINICAL AND TRANSLATIONAL RESEARCH
A. Albini (Italy)
P. Allavena (Italy)
F. Balkwill (UK)
M. Barbacid (Spain)
M. Broggini (Italy)
C. Catapano (Switzerland)
J. Collard (The Netherlands)
E. Garattini (Italy)
A. Gescher (UK)
R. Giavazzi (Italy)
I. Hart (UK)
W. Keith (UK)
J. Lunec (UK)
D.R. Newell (UK)
G.J. Peters (The Netherlands)
A. Puisieux (France)
V. Rotter (Israel)
M. Schmitt (Germany)
C.G.J. Sweep (The Netherlands)
G. Taraboletti (Italy)
P. Vineis (UK)
N. Zaffaroni (Italy)
D. Forman (France)
A. Green (Australia)
K. Hemminki (Germany)
C. Johansen (Denmark)
L.A. Kiemeney (The Netherlands)
E. Lynge (Denmark)
M. Maynadié (France)
H. Møller (UK)
P. Peeters (The Netherlands)
A.G. Renehan (UK)
S. Sanjose (Spain)
M.K. Schmidt (The Netherlands)
I. Soerjomataram (France)
H. Storm (Denmark)
L.V. van de Poll-Franse (The Netherlands)
H.M. Verkooijen (The Netherlands)
E. de Vries (The Netherlands)
R. Zanetti (Italy)
G. Chantada (Argentina)
F. Doz (France)
A. Ferrari (Italy)
M.A. Grootenhuis (The Netherlands)
K. Pritchard-Jones (UK)
L. Sung (Canada)
M. van den Heuvel-Eibrink (The Netherlands)
M. van Noesel (The Netherlands)
EPIDEMIOLOGY AND PREVENTION
B. Armstrong (Australia)
P. Autier (France)
J.M. Borras (Spain)
C. Bosetti (Italy)
H. Brenner (Germany)
L.E.M. Duijm (The Netherlands)
J. Faivre (France)
S. Franceschi (France)
PAEDIATRIC ONCOLOGY
C. Bergeron (France)
A. Biondi (Italy)
E. Bouffet (Canada)
M. Cairo (USA)
H. Caron (The Netherlands)
Aims and Scope
The European Journal of Cancer (EJC) is an international multidisciplinary oncology journal, which publishes original research, reviews, and editorial
comments on basic and preclinical cancer research, translational oncology, clinical oncology – including medical oncology, paediatric oncology, radiation
oncology, and surgical oncology, and cancer epidemiology and prevention. The EJC is the official journal of the European Organisation for Research and
Treatment of Cancer (EORTC), the European CanCer Organisation (ECCO), European Association for Cancer Research (EACR) and the European Society of
Breast Cancer Specialists (EUSOMA).
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AACR-NCI-EORTC International Conference
e on
MOLECULAR TARGETS AND
CANCER THERAPEUTICS
The Premier International Meeting Featuring Novel Cancer Therapeutics
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We look forward to seeing you in Boston!
ORGANIZING COMMITTEE
SCIENTIFIC COMMITTEE CO-CHAIRPERSONS
Carlos L. Arteaga, MD
Vanderbilt-Ingram Cancer Center,
Nashville, TN (AACR)
Levi A. Garraway, MD, PhD
Dana-Farber Cancer Institute,
Boston, MA, USA (AACR)
James H. Doroshow, MD
National Cancer Institute-DCTD,
Bethesda, MD, USA (NCI)
Lee J. Helman, MD
National Cancer Institute,
Bethesda, MD, USA (NCI)
Roger Stupp, MD
University Hospital Zurich (USZ),
Zürich, Switzerland (EORTC)
Jean-Charles Soria, MD, PhD
Institut Gustave-Roussy,
Villejuif, France (EORTC)
ABOUT THIS CONFERENCE
Join us for the premier international meeting featuring novel cancer therapeutics. This meeting
brings together nearly 3,000 academics, scientists, and pharmaceutical industry representatives from
across the globe to discuss innovations in drug development, target selection, and the impact of new
discoveries in molecular biology.
WHAT PAST ATTENDEES SAID...
“Very exciting conference with excellent speakers. Great opportunity to broaden my knowledge of cancer
biology and therapeutics.”
“I felt stimulated to work in cancer and science again! Thank you!”
For more information and to bookmark this
important event, go to www.AACR.org/Targets15
v
Table of Contents
Letter of Welcome
vii
Societies’ Profiles
Conference Committees
ix
xi
Acknowledgement
General Information
xii
xiii
Travel Grants
xvii
CME Accreditation
Scientific Programme
Programme Overview
xvii
xviii
Programme Details
Abstracts
Late Breaking Abstracts
xx
1
193
Presenting Authors List
Author Index
201
205
Subject Index
229
FUTURE MEETINGS
AACR – NCI – EORTC International Conference on
Boston, MA, USA, 5 – 11 November 2015
28th EORTC – NCI – AACR Symposium on
Munich, Germany, 18 – 21 October 2016
As a significant number of cancers in Europe can be attributed to smoking a strict no-smoking policy will
be enforced within all areas used by the conference.
28TH EORTC-NCI-AACR SYMPOSIUM
EORTC
NCI
AACR
2016
‘MOLECULAR TARGETS
AND CANCER
THERAPEUTICS’
18 – 21 OCTOBER 2016
28TH EORTC-NCI-AACR SYMPOSIUM ON
‘MOLECULAR TARGETS AND
CANCER THERAPEUTICS’
The 28th EORTC-NCI-AACR Symposium on ‘Molecular Targets
and Cancer Therapeutics’ will bring together academics,
scientists and representatives from the pharmaceutical
industry to discuss innovations in drug development, target
selection and the impact of new discoveries in molecular
biology. During the last few years, numerous innovative agents
have been discovered as a result of tremendous developments
in the understanding of the molecular basis of cancer.
Further clinical progress in cancer treatment will be
accomplished mainly through the conduct of translational
research projects, efficient new drug development and the
execution of large, prospective, randomised, multicentre
cancer clinical trials. This requires a joint and global approach
and early and optimal exchange of information. Therefore, the
Symposium will ensure the maximum amount of interaction
and discussion through an exciting range of plenary sessions
and lively workshops.
MUNICH, GERMANY
The future of cancer therapy
For forthcoming announcements about the
28th EORTC-NCI-AACR Symposium on ‘Molecular
Targets and Cancer Therapeutics’ please bookmark
www.eortc.be, or contact the Secretariat directly:
c/o ECCO – the European CanCer Organisation
Avenue E. Mounier, 83
B-1200 Brussels
Belgium
Tel.: +32 (0) 2 775 02 01
Fax: + 32 (0) 2 775 02 00
E-mail: [email protected]
www.ecco-org.eu/ENA
SAVE THE DATE
vii
Letter of Welcome
Dear Colleagues,
It is our pleasure to invite you the 26th EORTC–NCI–AACR Symposium on Molecular Targets and Cancer
Therapies. This annual meeting is taking place alternatingly in the USA and in Europe (since 1978) and is
the premier international symposium on drug development, innovative approaches, targeted agents including
biomarkers and translational research. Over 2000 delegates are exchanging ideas and results to foster drug
and cancer therapeutic development, with a particular focus on early clinical application.
For this edition, we have revamped the programme, by adding more slots for oral presentations populated
by abstracts that contain brand new data that has never been presented before. We have particularly focused
our efforts in bringing more clinical data by attracting additional original phase I/II trials. The latest in
immunotherapy, oncolytic viruses, epigenetic targets as well as drug resistance will be discussed by eminent
speakers in plenary and workshop sessions. This year, to compliment the poster presentations, we will have
“posters in the spotlight” sessions, where selected poster presenters will be given a podium to present their
posters and answer any questions from delegates. There will be ample opportunities to network during the
welcome reception, evening poster viewing sessions and the DNA networking event which will be held in
the Casa Llotja de Mar, located on the sea front in the historical centre of Barcelona.
The 26th EORTC–NCI–AACR Symposium on Molecular Targets and Cancer Therapeutics is the place to
meet new collaborators and old friends; gaining new insights that will aid the development of more effective
treatments for the patients.
We welcome you to the meeting!
Jean-Charles Soria
Scientific Chair
Roger Stupp
EORTC President
ix
Societies’ Profiles
The European Organisation for Research and Treatment of Cancer (EORTC)
The EORTC, based in Brussels, Belgium, unites European cancer clinical research experts from all
disciplines for trans-national collaboration. It links a network of over 2,500 collaborators from all disciplines
involved in cancer treatment and research in more than 300 hospitals in over 30 countries and offers an
integrated approach to drug development, drug evaluation programmes and medical practices.
EORTC Headquarters in Brussels handles some 30 protocols that are permanently open to patient entry,
over 50,000 patients who are in follow-up, and a database of more than 180,000 patients.
The ultimate goal of the EORTC is to improve the standard of cancer treatment by developing new agents
and innovative approaches and to test more effective treatment strategies using commercially available drugs,
or surgery and radiotherapy.
EORTC studies have contributed to success stories in drugs development including the registration of several
drugs by the United States Food and Drug Administration and the European Medicines Agency. The EORTC
has a proven track record in establishing new standards, e.g. RECIST, QOL, etc., and in changing clinical
practice. EORTC Headquarters staff provides full clinical, scientific, operational, quality assurance, and
regulatory support for clinical and translational research projects, and augments this support with strong
expertise in biostatistics, clinical study design and methodology, endpoint definition and analysis, etc.
The EORTC fully supports the idea of Health Technology Assessment (HTA) becoming standard practice
for the approval of new drugs or new indications and, moreover, has agreed that high quality clinical trials
in the future should be HTA compliant.
The role of pragmatic data and related methodology need to be developed within multi-stakeholder platforms,
and with this goal in mind, the EORTC participates in projects such as those put forth by the Innovative
Medicines Initiative. Such activities make the EORTC one of Europe’s leading players in transforming
experimental discoveries into new treatments with a significant clinical impact.
Alongside its own scientific and clinical programme, the EORTC collaborates with a number of research
institutions including the US National Cancer Institute, the National Cancer Institute of Canada, and many
other national and international research groups worldwide.
Funders of the EORTC include the EORTC Charitable Trust (with the support of national cancer leagues),
the “Fonds Cancer”, private donations and corporate sponsorship, annual grants allocated by BELSPO (the
Belgian Federal Science Policy Office) and by the Belgian National Lottery, and, for specific research
projects, the European Commission. Since the early 1970’s, EORTC headquarters has received continuous
support from the US NCI. Support is also received from the pharmaceutical industry for specific studies.
www.eortc.org
x
Societies’ profiles
The National Cancer Institute (NCI)
The National Cancer Institute (NCI) is part of the National Institutes of Health (NIH), which is one of 11
agencies that compose the United States (U.S.) Department of Health and Human Services (HHS). The NCI,
established under the National Cancer Institute Act of 1937, is the Federal Government’s principal agency for
cancer research and training. The National Cancer Act of 1971 broadened the scope and responsibilities of
the NCI and created the National Cancer Program. Over the years, legislative amendments have maintained
the NCI authorities and responsibilities and added new information dissemination mandates as well as a
requirement to assess the incorporation of state-of-the-art cancer treatments into clinical practice.
The National Cancer Institute coordinates the National Cancer Program, which conducts and supports
research, training, health information dissemination, and other programs with respect to the cause, diagnosis,
prevention, and treatment of cancer, rehabilitation from cancer, and the continuing care of cancer patients
and the families of cancer patients. Specifically, the Institute:
• Supports and coordinates research projects conducted by universities, hospitals, research foundations, and
businesses throughout the U.S. and abroad through research grants and cooperative agreements.
• Conducts research in its own laboratories and clinics.
• Supports education and training in fundamental sciences and clinical disciplines for participation in basic
and clinical research programs and treatment programs relating to cancer through career awards, training
grants, and fellowships.
• Supports research projects in cancer control.
• Supports a national network of cancer centers.
• Collaborates with voluntary organizations and other national and foreign institutions engaged in cancer
research and training activities.
• Encourages and coordinates cancer research by industrial concerns where such concerns evidence a
particular capability for programmatic research.
• Collects and disseminates information on cancer.
• Supports construction of laboratories, clinics, and related facilities necessary for cancer research through
the award of construction grants.
The American Association for Cancer Research (AACR)
Founded in 1907, the American Association for Cancer Research (AACR) is the world’s first and largest
professional organization dedicated to advancing cancer research and its mission to prevent and cure cancer.
AACR’s membership includes 34,000 laboratory, translational and clinical researchers; population scientists;
other health care professionals; and cancer advocates residing in more than 90 countries. The AACR
marshals the full spectrum of expertise of the cancer community to accelerate progress in the prevention,
biology, diagnosis and treatment of cancer by annually convening more than 20 conferences and educational
workshops, the largest of which is the AACR Annual Meeting with more than 17,000 attendees. In addition,
the AACR publishes seven peer-reviewed scientific journals and a magazine for cancer survivors, patients and
their caregivers. The AACR funds meritorious research directly as well as in cooperation with numerous
cancer organizations. As the Scientific Partner of Stand Up To Cancer, the AACR provides expert peer
review, grants administration and scientific oversight of individual and team science grants in cancer research
that have the potential for near-term patient benefit. The AACR actively communicates with legislators and
policymakers about the value of cancer research and related biomedical science in saving lives from cancer.
For more information about the AACR, visit www.AACR.org
xi
Conference Committees
Organising Committee
EORTC
NCI
AACR
Roger Stupp
Jean-Charles Soria
James Doroshow
Lee Helman
C.L. Arteaga
Jeff Engelman
EORTC President
Scientific Chair
NCI President
Scientific Co-Chair
AACR President
Scientific Co-Chair
Executive Scientific Committee
Jean-Charles Soria (France), Scientific Chair
Lee Helman (USA), Scientific Co-Chair
Jeff Engelman (USA), Scientific Co-Chair
Josep Tabernero (Spain)
Anthony Tolcher (USA)
Kapil Dhingra (USA)
Roger Stupp (Switzerland)
Scientific Committee
Jeffrey Abrams
Carlos L. Arteaga
Udai Banerji
Philippe Bedard
Emiliano Calvo
Lewis Cantley
Anthony Chan
Jerry M. Collins
Barbara Conley
Sara A. Courtneidge
William Dahut
Kapil Dhingra
James Doroshow
Susan Galbraith
Levi A. Garraway
James Gulley
Toby Hecht
Monika Hegi
Lee J. Helman
David Hong
Axel Hoos
Maria Koehler
Scott Kopetz
Shivaani Kumar
Annette Larsen
USA
USA
UK
Canada
Spain
USA
Hong Kong
USA
USA
USA
USA
USA
USA
United Kingdom
USA
USA
USA
Switzerland
USA
USA
USA
USA
USA
USA
France
Emma M. Lees
Elaine Mardis
Paul Meltzer
Gordon B. Mills
Frits Peters
Ruth Plummer
Yves Pommier
Victoria M. Richon
Neal Rosen
Jan Schellens
Joel Schneider
Jeffrey E. Settleman
Alice T. Shaw
Kevan Shokat
David B. Solit
Patricia S. Steeg
Charles Swanton
Josep Tabernero
Beverly A. Teicher
Anthony Tolcher
Joseph E. Tomaszewski
Matthew G. Vander Heiden
Yi Long Wu
James Zwiebel
USA
USA
USA
USA
Netherlands
United Kingdom
USA
USA
USA
Netherlands
USA
USA
USA
USA
USA
USA
United Kingdom
Spain
USA
USA
USA
USA
China
USA
xii
Acknowledgement
EORTC, NCI and AACR would like to gratefully acknowledge the following companies for supporting the
26th EORTC–NCI–AACR Symposium on Molecular Targets and Cancer Therapeutics:
Major Sponsor:
Silver Partners:
Grant Support:
EORTC, NCI and AACR would like to thank the following sponsors and exhibitors for their support to this
meeting.
This list reflects the companies/organisations confirmed at the time of going to print.
Company/Organisation
Booth
Company/Organisation
Booth
Advance Cell Diagnostics
D1
Horizon Discovery
A10
Affymetrix
C9
Indivumed
B2
Almac
C6
Medical Prognosis Institute
C1
Biocrates Life Sciences
C4
Novella Clinical
A4
Cancer Research Technology
C2
Oncodesign SA
A5
Cellecta, Inc.
A15
Oncotest GmbH
B1
Champions Oncology
B9
OracleBio Limited
B3
Charles River
C10
PerkinElmer Inc.
C5
Crown BioScience
A2
Proqinase GmbH
B4
Cureline, Inc.
A6
Silicon Biosystems
A9
Dove Medical Press
A11
Studylog Systems
A1
Epistem Ltd
C3
Sysmex Inostics
C8
European Association for
Cancer Research (EACR)
C11
vivoPharm
B13
Xentech
A3
GSK
A13
xiii
General Information
Symposium Secretariat
ECCO − the European CanCer Organisation
Avenue E. Mounier, 83
B-1200 Brussels − Belgium
E-mail: [email protected]
During the Symposium, the Secretariat can be reached at +32 (0)2 880 15 21
Venue
Centre de Convencions Internacional Barcelona (CCIB)
Rambla Prim 1−17
ES-08019 Barcelona − Spain
Tel.:
+34 (0)93 230 10 00
App
All attendees may download the free ECCO App on iPhone, iPad, or Android
supported devices.
Features include EORTC–NCI–AACR 2014 related information and news.
The App contains the complete list of sessions, session types, speakers and
exhibitors. Users can save their selected sessions, notes, favourites, as well as
export sessions to their smartphone calendar. To download the App, search for
ECCO cancer in iTunes or Google Play.
Learn more at www.ecco-org.eu/app or use the QR code for direct download.
Badges
For security reasons, delegates are requested to wear their badge at all times
during the Symposium. Delegates who have lost their badge can obtain a new
one at the registration desk. A replacement fee of 75 EUR per participant will
be charged.
Catering
Coffee breaks
Coffee breaks courtesy of the organisers have been scheduled as follows:
Tuesday 18 November:
14:45–15:15
Wednesday 19 November:
09:45–10:15 and 15:20–16:00
Thursday 20 November:
10:00–10:30 and 15.35–16:00
Friday 21 November:
10:30–11:00
Coffee breaks will take place in the catering areas of the exhibition except
for Friday 21 November when the coffee break will take place in the poster
area.
Lunches
Complimentary lunches will be served in the exhibition catering areas:
12:00–13:00
12:00–13.15
12.30–13:30
During the poster viewing sessions, local tapas and drinks will be served.
Delegates can also purchase food and beverages from the cash bar near the
entrance of the Convention Centre. Free water fountains are located throughout
the Convention Centre.
xiv
General information
Certificate of Attendance
To print your certificate of attendance after the Symposium, you will need
your badge code number, so please keep your badge for this purpose. You can
print your certificate of attendance via this link:
http://www.ecco-org.eu/ENA1014 or in the Internet Zone located on the
venue ground floor (as of Wednesday 19 November). Online certificates will
be available until the end of December 2014.
Please note that the Symposium Secretariat will not post certificates to
participants after the event.
City Information
Barcelona Convention Bureau will operate a tourist and tour desk in the
registration area as of Tuesday 18 November.
Cloakroom
A cloakroom is situated in the entrance hall near the registration area.
Cloakroom Opening Hours
10:00–20:00
07:30–19:30
07:30–19:30
Friday 21 November:
08:30–13:30
The price for this service is 2 EUR per item.
Exhibition
The EORTC–NCI–AACR 2014 Exhibition is held on the ground floor of the
Convention Centre. Entrance is free for registered delegates.
Exhibition Opening Hours:
12:00–18:30
09:45–16:00
09:45–16:00
First Aid
A first aid room is located on level −1. In case of emergency, please inform
the nurse on duty via +34 697 324 433 and/or dial internal the extension
number 257 from any internal phone in the Convention Centre.
Insurance
The organisers of the EORTC–NCI–AACR 2014 Symposium do not accept
liability for individual medical, travel or personal insurance. Participants
are strongly advised to take out their own personal insurance policies.
The organisers of the EORTC–NCI–AACR 2014 Symposium accept no
responsibility for loss due to theft or negligence.
Internet WIFI access
General WIFI access is available throughout the Convention Centre. To access
WIFI, activate the WIFI network on your laptop or device, select the network
listed as ENA2014, and enter the password ENA2014.
Internet Zone
The official EORTC–NCI–AACR Internet Zone is available free of charge
during the Symposium. The terminals provide you with the following services:
Internet browsing, access to web-based mail, the Symposium searchable
programme and exhibitor information.
General information
xv
Language and translation The official language of the Conference is English. No simultaneous translation
is provided.
Lost and Found
All enquiries should be directed to the registration helpdesk in the entrance
hall. Participants are advised to mark their own Symposium bag and
programme book with their name. The organisers accept no responsibility
for loss due to theft or negligence.
Media
Press Office Opening Hours
Friday 21 November:
10:00–18:00
08:00–18:00
08:00–18:00
09:00–13:00
Entry to the Press Office is restricted to accredited media staff only.
The Press Office is located in Room M221.
Posters
Posters will be displayed in the exhibition area on the ground floor.
Poster presentations:
Poster presentations are scheduled on:
18:00–19:30
18:00–19:30
Friday 21 November:
09:00–10:30
Poster Viewing
Poster presenters will present their work and answer questions from delegates.
During this session, local tapas and drinks will be served.
Posters in the Spotlight
Session
The Scientific Committee will make a special selection of posters which will be
highlighted through short oral presentations (5 min + 5 min discussion each).
Take this opportunity to discuss some of the hottest news at the Symposium!
These spotlight sessions will take place in the exhibition area on Wednesday 19
and Thursday 20 November during the lunch break.
Registration
EORTC–NCI–AACR 2014 is open to all registered participants. Your official
delegate name badge is required for admission to the Convention Centre and
all Symposium events. For security reasons, participants are requested to wear
their badge at all times.
Registration Opening Hours:
Monday 17 November:
Friday 21 November:
Registration Package
15:00–18:00
08:00–18:00
07:00–18:00
07:00–18:00
08:00–12:00
The full Symposium registration package includes:
• Entry to all scientific sessions, and to the Welcome Reception on
Tuesday 18 November;
• Entry to the exhibition;
• Proceedings book;
• EORTC–NCI–AACR 2014 coffee breaks and complimentary lunches;
• Internet access via the internet zone and WIFI access in the Convention
Centre.
xvi
General information
The day registration package includes:
• Access to all scientific sessions on that day;
• Entry to the exhibition;
• Proceedings book (depending on availability);
• EORTC–NCI–AACR 2014 coffee breaks and lunch on that day;
• Internet access via the internet zone and WIFI access in the Convention
Centre.
Speaker Preview Room
The Speaker Preview Room is located on the ground floor. Speakers are
requested to bring their PowerPoint presentations to the speaker preview room
at least 4 hours before their session starts or one day in advance if the session
starts early in the morning. To avoid breaks between speakers, NO laptop
presentations are foreseen in session rooms.
Speaker preview room opening
Friday 21 November:
Social Events
hours:
11:00–18:30
07:00–18:30
07:00–18:30
08:00–12:00
Welcome Reception
All registered participants are invited to the Welcome Reception, organised on
Tuesday 18 November, in the exhibition hall. The Welcome Reception starts
at 17:30.
Networking Event “It’s all about DNA”
Thursday 20 November from 20:00.
Casa Llotja de Mar, Passeig d’Isabel II, 1 08003 Barcelona
Casa Llotja de Mar, located on the sea front in the historical centre of
Barcelona, is one of the most significant and splendid monuments representing
the economy and culture of the city. It was built in the second half of the
14th century, the most brilliant period of Catalan Gothic.
Price per person: 65 EUR
How to get there:
From the Convention Centre, walk to the El Maresme/Fòrum subway station
and take the L4 towards Trinitat Nova. Get off at Barceloneta station. Head
west on Pia del Palau toward Plarca de Pau Vila. Turn left onto Passeig
d’Isabel II. The Casa Llotja de Mar will be on the right.
Social media
We recommend and support Twitter as the main social platform during the
Symposium − tweet and follow the latest updates about the 26th EORTC–
NCI–AACR Symposium on Twitter using #ENA2014
Find links, tutorials and tips: www.ecco-org.eu/social
xvii
Travel Grants
Through the generosity of its sponsors, EORTC, NCI and AACR have awarded 15 travel grants to attend the 26th
EORTC–NCI–AACR Symposium in Barcelona.
Baxter, Merissa
USA
Mittal, Deepak
Australia
Bhattacharya, Bhaskar
Singapore
Pettitt, Stephen
United Kingdom
Campagne, Cécile
USA
Rose, April
Canada
Choi, Min-ah
South Korea
Sahay, Debashish
France
Eun, Young-Gyu
South Korea
Sos, Martin
USA
Goldstein, Rebecca
USA
Willetts, Lian
Canada
Kim, Jisun
Korea
Zoni, Eugenio
Netherlands
Mantaj, Julia
United Kingdom
CME Accreditation
The 26th EORTC–NCI–AACR Symposium on ‘Molecular Targets and Cancer Therapeutics’
is accredited by the European Accreditation Council for Continuing Medical Education
(EACCME) to provide the following CME activity for medical specialists. The EACCME is
an institution of the European Union of Medical Specialists (UEMS), www.uems.net
The 26th EORTC–NCI–AACR Symposium is designated for a maximum of 18 hours of European external CME
credits. Each medical specialist should claim only those hours of credit that he/she actually spent in the educational
activity. The EACCME credit system is based on 1 ECMEC per hour with a maximum of 3 ECMECs for half a day
and 6 ECMECs for a full-day event.
European Accreditation is granted by the EACCME in order to allow participants who attend the above-mentioned
activity to validate their credits in their own country.
Through an agreement between the European Union of Medical Specialists and the American Medical Association,
physicians may convert EACCME credits to an equivalent number of AMA PRA Category 1 Credits™. Information
on the process to convert EACCME credit to AMA credit can be found at
www.ama-assn.org/go/internationalcme
Live educational activities, occurring outside of Canada, recognised by the UEMS-EACCME for ECMEC credits are
deemed to be Accredited Group Learning Activities (Section 1) as defined by the Maintenance of Certification Program
of The Royal College of Physicians and Surgeons of Canada.
xviii
Scientific Programme – Overview
Tuesday18November
Wednesday19November
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Posters
AnimalModels
Cytotoxics
DrugResistanceandModifiers
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Immunotherapy(Immunecheckpoints,Vaccination,
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PreclinicalModels
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xix
Thursday 20 November
Friday 21 November
Auditorium
09.00
08.00
Plenary Session 4
10.00
Antibody-Based Therapies (ADC
and others)
Poster Viewing
(Poster Area)
10:30
Coffee Break
Coffee Break
10.30
Auditorium
Plenary session 5
12.30
Lunch 12.30-13.30
Including
Posters in the Spotlight Session
13.30
Plenary Session 6
Proffered Paper Session
15.35
Coffee Break
16.00
Plenary Session 7
Novel Mechanisms for Drug
Resistance
17.50
18.0019.30
Poster Viewing
(Poster Area)
Networking Event
Posters
Chemoprevention
Clinical Methodology
DNA Repair Modulation (including
PARP, CHK, ATR, ATM)
Drug Delivery
Drug Design
Molecular Targeted Agents I
Paediatric Oncology
Toxicology
Exhibition 09:45 - 16:00
11.00
Epigenetic Targets
Plenary session 8
Targeting RAS and Other Driver
Oncogenes
13.00
Posters
Drug Synthesis
Molecular targeted agents II
New Therapies with Pleiotropic Activity
xx
Scientific Programme – Details
Tuesday 18 November 2014
Opening Ceremony
13:00–13:15
13:15–14:00
Opening
13:00
13:05
13:10
Auditorium
Remarks
J.C. Soria (France)
L.J. Helman (USA)
J.A. Engelman (USA)
EORTC
NCI
AACR
Michel Clavel Lecture
Chair: J.C. Soria (France)
Auditorium
13:15 Rational combination therapies for cancer
R. Bernards (Netherlands)
14:00–14:45
Keynote Lecture
Chair: A.M.M. Eggermont (France)
Auditorium
14:00 Immunocheckpoints − Gateway to Immunotherapy
D. Chen (USA)
Objectives:
1. Understand how cancer immunotherapy works, the cancer-immunity cycle and what makes it different
from other forms of cancer therapy.
2. Understand what rationale approaches can be taken to monotherapy and combination therapy
involving cancer immunotherapy.
3. Understand the role for biomarkers in understanding emerging immune biology, interpreting clinical
results and identifying optimal treatment decisions.
Key Messages:
1. Our understanding of how the immune system interacts with cancer is rapidly evolving.
2. Cancer immunotherapy approaches may be able to generate durable responses in patients with
metastatic cancer.
3. Emerging biomarkers may be able to help us understand biology, define combination approaches and
choose between therapeutic options.
Plenary Session 1
15:15–17:30
Is the Genomic Landscape Changing the Outcome for Cancer Patients?
Chairs: R. Stupp (Switzerland) and J. Tabernero (Spain)
15:15 Overview of academic precision medicine trials
P. Bedard (Canada)
15:35 Lessons from SAFIR01 trial
F. Andre (France)
Main objectives:
1. Understand the pillars of personalised medicine.
2. Understand the main reasons of failures to deliver personalised medicine.
3. Understand what are the possible solutions to address these limitations.
Auditorium
Abstract number
Scientific programme – details
xxi
Key messages:
1. There is a need for randomised trials before implementing multigene tools for personalised medicine.
2. Drug availability and rules to interpret genome data are mandatory.
3. Genome analysis will not provide all the informations needed to deliver personalised medicine.
15:55 Translating gene expression signatures into clinical practice: prospects and challenges in the context
of ‘next-generation medicine’
R. Dienstmann (USA)
Key messages:
1. There are unique issues with high-dimensional data that represent obstacles to generating performant
genomic signature classifiers and translating initial research findings into robust diagnostics.
2. Better understanding of the intrinsic gene expression-based subtypes of a histopathologically defined
cancer, independent of their prognostic and predictive values, may also lead to new biological insights
and eventually to development of novel therapies directed toward homogeneous molecular subsets.
3. A rational and focused approach to the evaluation of genomic markers is needed, whereby analytically
validated assays are prospectively investigated in clinical trials with adaptive designs that take into
consideration primary–metastatic site tumour heterogeneity and clonal evolution in the decisionmaking process.
16:15 Genomic characterisation of cancer: Case studies
E.R. Mardis (USA)
Key objectives:
1. Provide detailed description of the molecular assays being used to characterise the cancer mutations
and their expression in RNA for individual patients.
2. Describe the analytical approaches that permit integrated analysis of mutations and RNA expression
toward two therapeutic ends: (1) identification of small molecule therapies that may provide relief
of tumour burden, (2) identification of immunoepitopes that may contribute toward a personalised
vaccine strategy for individual patients.
3. Illustrate the two aforementioned objectives by detailed descriptions of specific case studies from our
work in this area of applied cancer genomics.
16:35 Screening Patients for Efficient Clinical Trial Access (SPECTA)
D. Lacombe (Belgium)
Key messages:
1. Cancer drug development is undergoing profound changes and the path to registration will be
substantially altered due to challenges of big data (omics).
2. The forms and the methods of cancer clinical research are evolving with the need to integrate new
technologies to understand biology early on in development. New efficient platforms for patient access
(molecularly defined subgroups) are needed.
3. Clinical oncology will need to take into account new type of guidelines for treatment decision with
an increased role played by molecular advisory boards.
16:55 ORAL PRESENTATION: Feasibility of large-scale genomic testing to facilitate enrollment on
genomically-matched clinical trials
F. Meric-Bernstam, L. Brusco, S. Kopetz, M. Davies, M.J. Routbort, S.A. Piha-Paul, R. Alvarez,
S. Khose, J. DeGroot, V. Ravi, F. Janku, D. Hong, Y. Li, R. Luthra, K.P. Patel, R. Broaddus, K. Shaw,
J. Mendelsohn, G.B. Mills
17:10 General discussion
J. Tabernero (Spain)
17:15 Faster execution of clinical trials − has bioinformatics the solution?
G. McVie (Italy)
1
xxii
Wednesday 19 November 2014
Workshop 1
08:00–09:45
Auditorium
Management of Toxicity of Molecular Targeted Agents
Chairs: J.C. Soria (France) and P. Bedard (Canada)
08:00 Cardiac toxicities with MOA and TKI
S. Ederhy (France)
08:20 Digestive and endocrine toxicities of checkpoint inhibitors
N. Chaput (France)
Main objectives:
1. To describe digestive toxicities (clinic/endoscopic/histological) in patients treated with anti-CTLA4
and/or anti-PD1.
2. To describe immune disorders in patients developing digestive toxicities (blood and colonic biopsies).
Key messages:
1. Anti-CTLA-4 and/or anti-PD-1, monoclonal antibody treatment of cancer, are associated with several
phenotypes of enterocolitis.
2. Intestinal inflammation observed in patients who were prescribed anti-CTLA-4, anti-PD1 or both,
have distinct phenotypes: anti-CTLA-4 is associated with acute, often severe colitis, chronic
duodenitis and less often with ileal and anal lesions. Anti-PD-1 seems to be associated with mild,
chronic colitis. The combination of anti-CTLA-4 and anti-PD1 seems to lead to severe duodenal
inflammation and severe colitis.
3. Anti-CTLA-4 colitis appears as a model of T-cell induced colitis in humans as suggested by the
immune monitoring of patients.
08:40 Management of pulmonary toxicity due to targeted anticancer agents
J. Porter (United Kingdom)
09:00 Ocular toxicity of MEK inhibitors and other targeted therapies
J.P. Velazquez-Martin (Canada)
Main objectives:
1. Identify the key agents associated with ocular toxicities.
2. Identify symptoms and clinical findings associated with ocular toxicity.
3. Learn the management, follow up and prognosis of these patients.
Key messages:
1. Ocular toxicities from targeted therapies are frequent.
2. Symptoms range from none to moderate visual impairment.
3. Always consult with an ophthalmologist or retina specialist (preferable).
09:20 Discussion
Workshop 2
08:00–09:45
Room 117
Cancer metabolism
Chairs: C. Van Dang (USA) and G. Hardie (United Kingdom)
08:00 AMPK − opposing the metabolic changes in tumour cells
G. Hardie (United Kingdom)
Key messages:
1. The AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status that
is switched on by metabolic stresses causing depletion of cellular ATP, such as ischaemia (a frequent
event within solid tumours).
2. Most tumour cells (and other rapidly proliferating cells) display elevated glucose uptake and glycolysis
(the Warburg effect); this occurs in part because the TCA cycle is being used as an anabolic pathway
providing precursors for biosynthesis, and can no longer satisfy the increased demand for ATP caused
by activation of biosynthetic pathways.
xxiii
3. By inactivating most biosynthetic pathways and promoting oxidative metabolism rather than
glycolysis, AMPK opposes these metabolic changes; this explains why the pathway is often downregulated in tumour cells, but also why AMPK-activating drugs have potential in the treatment or
prevention of cancer.
08:20 IDH1/IDH2
D.P. Schenkein (USA)
Key messages:
1. Cancer metabolism, a newly validated area for oncology drug discovery.
2. Mutations in the metabolic enzyme IDH are present in a wide range of malignancies and increasingly
recognised in pre-malignant conditions: the role of 2-HG as an oncometabolite.
3. Clinical trial results from the first IDH inhibitor, AG-221: significant single agent activity in AML,
excellent safety profile, and a novel mechanism of action.
08:40 Targeting MYC-driven Glutaminolysis
C. Van Dang (USA)
09:00 Identification of novel inhibitors of GLUT1 as potent cancer cell-killing agents
A. Wise (United Kingdom)
Key objectives:
1. Demonstrate chemical tractability of the glucose transporter GLUT1 as a drug target by identifying
a lead series of potent and selective small molecule inhibitors.
2. Demonstrate in vitro validation of GLUT1 as a therapeutic target in cancer by utilising our novel
GLUT1 inhibitors as tool compounds.
3. Demonstrate in vivo validation of GLUT1 as a therapeutic target in cancer by utilising our novel
GLUT1 inhibitors as tool compounds.
09:20 Discussion
Workshop 3
10:15–12:00
Auditorium
“Liquid Biopsies” in Solid Tumours
Chairs: K. Dhingra (USA) and A. Bardelli (Italy)
10:15 Application of CTC detection technologies in oncology clinical trials
J.S. De Bono (United Kingdom)
Key objectives:
Showing that circulating blood biomarkers can have clinical utility as multi-purpose biomarkers as
1. Prognostic biomarkers.
2. Predictive biomarkers
3. Response and surrogate biomarkers.
10:35 Cancer mutation and transcriptome analysis in exosomes
J. Skog (USA)
Key objectives:
1. What is an exosome and what do they contain?
2. What is the benefit of analyzing exosome nucleic acids?
3. What do you need for successful analysis of exosomal RNA, and what types of analysis are possible?
10:55 cfDNA for detection of actionable cancer mutations
C. Caldas (United Kingdom)
11:15 Precision medicine for colorectal cancers: liquid biopsies and preclinical models
A. Bardelli (Italy)
Key objectives:
1. Understanding the molecular bases of secondary resistance to EGFR blockade in CRC is necessary
to design additional therapeutic options.
2. Molecular alterations in KRAS, NRAS, and MET are causally associated with the onset of acquired
resistance to anti-EGFR antibodies in colorectal cancers.
xxiv
3. Development of a diagnostic platform to detect ‘molecular resistance’ in the blood (liquid biopsy)
months before clinical or radiographic evidences of disease progression.
11:35 Discussion
Workshop 4
10:15–12:00
Room 117
Translational Research with Immunostimulatory Monoclonal Antibodies
Chairs: J. Tabernero (Spain) and I. Melero (Spain)
10:15 The making of new immunotherapy agents
A. Korman (USA)
10:35 Translational research of combined immunotherapies
I. Melero (Spain)
11:05 Biomarkers for new immunotherapies
M. Calahan (USA)
11:25 The interface of immunomodulation and vaccines
A. Van Elsas (NL)
Key messages:
1. Cancer vaccines enhance anti-tumour immune responses when used prophylactically, but lack robust
efficacy in therapeutic setting.
2. Several immunomodulatory receptor-ligand pathways regulate T cell responses to cancer and
vaccines.
3. Experimental combinations of cancer vaccines and immunomodulatory antibodies display efficacy in
preclinical models of established cancer.
11:45 Discussion
12:15–12:45
Moderator: R. Plummer (United Kingdom)
Exhibition Hall
The following abstracts will be discussed: 364 (poster board P144), 161 (P155), 422 (P155).
Professional Advancement Session
12:15–13:15
“Navigating Your Career with Confidence” −
A Professional Advancement Session Organized by the AACR-Women in
Cancer Research (WICR) Council
Chair: P. LoRusso (USA)
Room 117
Feel and project more confidence when seeking new career opportunities. This session will include an inspirational
Keynote Lecture from Susan M. Galbraith, MBBCh, PhD, Vice President of Oncology, AstraZeneca, as well as a panel
of professionals discussing the ways that women scientists in particular can bolster their confidence and overcome
insecurities in order to embrace new opportunities. The panel will address topics such as assessing which career
opportunities are a good fit, improving a resume or portfolio in order to secure an interview, blending personal and
career life once on the job, and more.
Although all conference attendees are invited to attend this session, it is geared toward early-career female investigators.
Welcome
P. LoRusso (USA)
Keynote
S.M. Galbraith (United Kingdom)
Panel Discussion
Susan M. Galbraith (United Kingdom), M. Foti (USA), F. Meunier (Belgium)
Audience Engagement (Q&A)
Closing Remarks and Evaluation
P. LoRusso (USA)
Plenary Session 2
13:15–15:20
Chairs: C. Arteaga (USA) and R. Plummer (United Kingdom)
xxv
Auditorium
Abstract number
13:15 Target validation as a crucial bottleneck in cancer drug discovery
P. Workman (United Kingdom)
Key objectives:
1. To illustrate the challenges of identifying and prioritising new cancer targets in the multiomics era
and to show how target validation is a critical bottleneck in cancer drug discovery.
2. To exemplify the above with case histories from our recent research discoveries concerning potential
molecular targets in the areas of oncogenic kinases and the HSP90 molecular chaperone pathway.
3. To set out criteria for robust target validation, including the use of genetic techniques and chemical
probes, so as to give the best chance of clinical impact.
13:35 ORAL PRESENTATION: Safety and early evidence of activity of a first-in-human phase I study
of the novel cancer stem cell (CSC) targeting antibody OMP-52M51 (anti-Notch1) administered
intravenously to patients with certain advanced solid tumors
A. Patnaik, P. LoRusso, P. Munster, A.W. Tolcher, S.L. Davis, J. Heymach, R. Ferraroto, L. Xu,
A.M. Kapoun, L. Faoro, J.A. Lewicki, J. Dupont, S.G. Eckhardt
2
13:50 ORAL PRESENTATION: Afuresertib (GSK2110183), an oral AKT kinase inhibitor, in combination
with carboplatin and paclitaxel in recurrent ovarian cancer
S. Blagden, A. Hamilton, L. Mileshkin, M. Hall, T. Meniawy, S. Wong, S. Anandra, M. Buck,
D. McAleer, B.A. Reedy, R.B. Noble, D.A. Smith, S.R. Morris, H. Gabra
3
14:05 ORAL PRESENTATION: Activity of galeterone in castrate-resistant prostate cancer (CRPC) with
C-terminal AR loss: Results from ARMOR2
M.E. Taplin, K.N. Chi, F. Chu, J. Cochran, W.J. Edenfield, E.S. Antonarakis, U. Emmenegger,
E.I. Heath, A. Hussain, V.C. Njar, A. Koletsky, D. Lipsitz, L. Nordquist, R. Pili, M. Rettig, O. Sartor,
N.D. Shore, D. Marrinucci, K. Mamlouk, B. Montgomery
4
14:20 ORAL PRESENTATION: Mechanism based targeted therapy for hereditary leiomyomatosis and renal
cell cancer (HLRCC) and sporadic papillary renal cell carcinoma: interim results from a phase 2
study of bevacizumab and erlotinib
R. Srinivasan, D. Su, L. Stamatakis, M.M. Siddiqui, E. Singer, B. Shuch, J. Nix, J. Friend, G. Hawks,
J. Shih, P. Choyke, W.M. Linehan
5
14:35 ORAL PRESENTATION: Imaging in cancer immunology: Phenotyping of multiple immune cell
subsets in-situ in FFPE tissue sections
J.R. Mansfield, C. Slater, C. Wang, K. Roman, C.C. Hoyt, R.J. Byers
6
14:50 LATE BREAKING ABSTRACT: Clinical safety and activity in a phase I trial of AG-120, a
first in class, selective, potent inhibitor of the IDH1-mutant protein, in patients with IDH1 mutant
positive advanced hematologic malignancies
D.A. Pollyea, S. de Botton, A.T. Fathi, E.M. Stein, M.S. Tallman, S. Agresta, C. Bowden, B. Fan,
M. Prah, H. Yang, K. Yen, R.M. Stone
1LBA
15:00 LATE BREAKING ABSTRACT: The identification of potent and selective inhibitors of oncogenes in 2LBA
medullary thyroid carcinoma and lung adenocarcinoma disease models
S. Fritzl, H. Small, B. Acton, S. Holt, G. Hopkins, S. Jones, A. Jordan, N. March, R. Newton,
I. Waddell, B. Waszkowycz, M. Watson, D. Ogilvie
15:10 Discussion Late Breaking Abstracts
C.L. Arteaga (USA)
Plenary Session 3
16:00–17:50
Oncolytic Viruses
Chairs: K. Harrington (United Kingdom) and L.J. Helman (USA)
16:00 T-VEC for the treatment of melanoma: Are we ready for prime time?
H. Kaufman (USA)
Auditorium
Abstract number
xxvi
16:20 Poliovirus oncolytic immunotherapy of glioblastoma
M. Gromeier (USA)
Key objectives:
1. Discuss the role of mechanisms of viral tumour cell killing in oncolytic immunotherapy.
2. Define principles of efficacious oncolytic immunotherapy: explain the role of the innate antiviral
response to oncolytic poliovirus.
3. Communicate the major clinical/radiographic findings from oncolytic immunotherapy of recurrent
glioblastoma with recombinant poliovirus.
16:40 Measles and vesicular stomatitis virus strains as novel oncolytic platforms
E. Galanis (USA)
17:00 Challenges in clinical development of oncolytic viruses
K. Harrington (United Kingdom)
17:20 LATE BREAKING ABSTRACT: Initial report of a first-in-human study of the first-in-class fatty acid 3LBA
synthase (FASN) inhibitor, TVB-2640
J. Infante, M. Patel, D. Von Hoff, A. Brenner, C. Rubino, W. McCulloch, V. Zhukova-Harrill, M. Parsey
17:30 LATE BREAKING ABSTRACT: A phase 1 study of first-in-class microRNA-34 mimic, MRX34, in 4LBA
patients with hepatocellular carcinoma or advanced cancer with liver metastasis
M. Beg, A. Brenner, J. Sachdev, M. Borad, J. Cortes, R. Tibes, Y. Kang, A. Bader, J. Stoudemire,
S. Smith, S. Kim, D. Hong
L.J. Helman (USA)
18:00–19:30
Animal Models
Poster board
Poster Viewing
Poster area
Abstract number
P001 Mouse clinical trial − A new preclinical study concept using patient-derived xenografts
V. Vuaroqueaux, C. Gredy, S. Gorynia, S. Baltes, H.H. Fiebig, T. Metz
P002 Imaging growth and anti-cancer activity in orthotopic patient derived tumors
M. Baugher, C. Bull, A. Cohen-Barnhouse, A. Flecha, M. Franklin, K. Guley, P. McConville,
W.R. Leopold
P003 Antineoplastic effects of auranofin in canine lymphoma
D. Thamm, B.J. Rose, J.K. Shoeneman
P004 mTOR inhibition with everolimus − a novel treatment option for head and neck cancer identified in a
translational research study using patient-derived xenografts
K. Klinghammer, J.D. Raguse, T. Plath, A.E. Albers, B. Brzezicha, A. Wulf-Goldenberg, U. Keilholz,
J. Hoffmann, I. Fichtner
P005 A panel of patient derived xenograft models of different haematological malignancies suitable for
preclinical drug screening campaigns
E. Oswald, C. Tschuch, K. Klingner, B. Hammerich, D. Lehnhard, C. Rentsch, M. Lübbert,
H.H. Fiebig, J. Schüler
P006 Next generation sequencing (NGS) guided therapy prediction for the treatment of glioblastoma
multiforme (GBM)
J. Sarkaria, D.M. Ma, S.P. Peng, S.B. Byron, D.C. Craig, J.C. Carpten, M.B. Berens, B.O. O’Neill,
N.T. Tran
P007 Whole exome sequence analysis of canine transitional cell carcinoma of the bladder
D.L. Duval, B. Hernandez, J. Brown, S.E. Lana, R. Page, K.L. Jones
P008 Mixeno mouse models for in vivo evaluation of anti-human cancer immunotherapeutics
J. Zhang, J. Qiu, M. Qiao, Q. Shi
P009 Genetic and molecular validation of uterine sarcoma patient-derived xenograft models
T. Cuppens, E. Hermans, J. Depreeuw, M. Moisse, T. Van Brussel, L. Coenegrachts, D. Lambrechts,
F. Amant
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Abstract number
P010 Allografting improves the feasibility of genetically engineered mouse models (GEMM) for anti-cancer
drug development
K. Kukuk, K. Klingner, A.L. Peille, P. Müller, A. Zipelius, J. Schüler
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P011 Studies on glycoprotein expression differences between MCF-7 and MCF-7-Z
J. Ner-Kluza, A. Drabik, M. Kubbutat, A. Lingnau, J. Silberring
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P012 Establishment and characterization of a Merkel Cell carcinoma PDX panel: Screening for potentially
useful therapies
M.J. Wick, J. Meade, M. Nehls, T. Vaught, J. Carlile, A.W. Tolcher, D.W. Rasco, A. Patnaik,
K.P. Papadopoulos
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P013 Syngeneic models for developing cancer therapeutics targeting immune system
L. Zhang, J. Zhang, Q. Shi
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P014 miR-25 is a key regulator of prostate cancer invasiveness by modulation of the cross-talk between
Notch and TGF-b signaling
E. Zoni, A.F. van de Merbel, G. van der Horst, J. Rane, T. Visakorpi, E.B. Snaar, N. Maitland,
G. van der Pluijm
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Cytotoxics
Poster board
Abstract number
P015 Pharmacogenomics of mithramycin in thoracic malignancies
W. Figg, T.M. Sissung, C.J. Peer, D. Schrump
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P016 Novel combination therapy, TAS-102 combined with the anti-EGFR antibody or the anti-VEGF
antibody showed therapeutic benefit toward colorectal cancer xenografts
K. Ishida, K. Sakamoto, N. Tanaka, K. Oguchi, K. Yamamura, A. Fujioka, F. Nakagawa, K. Matsuo,
T. Utsugi
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P017 Phase I study of lurbinectedin (PM01183) administered on days (D) 1 & 8 every 3 weeks (q3wk) in
patients (pts) with solid tumors
M.J. Ratain, L. Gore, S. Szyldergemajn, J. Diamond, D. Geary, C. Fernandez-Teruel, A. Soto-Matos,
M. Sharma, A. Jimeno
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P018 Androgen receptor (AR) expression in triple negative breast cancer (TNBC): results from a phase II
neoadjuvant trial with carboplatin and eribulin mesylate in TNBC patients
K. Siziopikou, V. Parini, V. Kaklamani
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P019 An ING1b-derived peptide that inhibits cancer cell viability and promotes apoptosis
A. Boyko, K. Riabowol
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P020 NPD926, a small molecule inducer of reactive oxygen species, kills cancer cells via glutathione
depletion
T. Kawamura, Y. Kondoh, M. Muroi, M. Kawatani, H. Osada
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P021 TAS-102 treatment results in high trifluridine incorporation into DNA with pyrimidine metabolic
pathway markedly up-regulated in cancer
K. Oguchi, K. Sakamoto, H. Kazuno, H. Ueno, K. Ishida, T. Yokogawa, K. Yamamura, R. Kitamura,
K. Matsuo, T. Utsugi
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P022 Characterization of the type of cell death induced by novel tambjamine analogs in lung cancer
A. Rodilla Martı́n, V. Soto-Cerrato, P. Manuel-Manresa, L. Korrodi-Gregório, R. Quesada, R. PérezTomàs
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P023 TAS-114 is a novel dUTPase/DPD inhibitor, its DPD inhibition reduces capecitabine dosage but does
not diminish therapeutic window in human tumor xenografts
W. Yano, H. Kazuno, T. Yokogawa, K. Sakamoto, K. Yoshisue, T. Wakasa, M. Fukuoka, K. Matsuo,
K. Noguchi, T. Utsugi
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Poster board
Abstract number
P024 The fungal-derived cyclohexadepsipeptide Destruxin E exerts multifaceted anticancer and
antiangiogenic activities
R. Dornetshuber-Fleiss, P. Heffeter, T. Mohr, P. Hazemi, K. Kryeziu, C. Seger, W. Berger,
R. Lemmens-Gruber
P025 N-Myc amplification sensitizes tumor cells to inhibition by Danusertib, an Aurora kinase inhibitor
P. Carpinelli, R. Ceruti, R. Alzani, C. Re, D. Ballinari, S. Cribioli, M. Russo, A. Degrassi, G. Texido,
M. Ciomei, E. Pesenti, A. Montagnoli, A. Galvani
P026 Replication stress is a determinant of synergy between gemcitabine and Chk1 inhibition
S.B. Koh, A. Courtin, R. Boyce, B. Boyle, F.M. Richards, D.I. Jodrell
P027 Combining the long-acting topoisomerase 1-inhibitor etirinotecan pegol with the PARP inhibitor
rucaparib to provide anti-tumor synergy without increased toxicity
U. Hoch, D. Charych
P028 Phase I, dose-escalation study of the investigational drug D07001-F4, an oral formulation of
gemcitabine HCl, in patients (pts) with advanced solid tumors or lymphoma
C. Lin, W. Su, J. Lee, C. Hsu, A. Cheng, C. Lin, H. Ho, C. Huang, S. Hsueh, J. Yang
P029 BRCA1 expression exploratory analysis in patients of the phase III trial of trabectedin vs.
doxorubicin-based chemotherapy as first-line therapy in translocation-related sarcomas
M. Aracil, P. Lardelli, A. Nieto, C.M. Galmarini
P030 Suppression of metastasis and improvement of drug distribution by eribulin mesylate
Y. Ozawa, K. Okamoto, Y. Adachi, M. Asano, K. Tabata, Y. Funahashi, J. Matsui
P031 Effect of a microtubule-targeting drug on cell–cell contacts in bladder epithelial tumour cells
L.M. Antón-Aparicio, R. Castosa, M. Haz, M. Blanco, M. Rodriguez, M. Valladares, A. Figueroa
P032 The indolyl-chalcone CDD-026 induces cancer cell death through targeting of STMN1 and mitotic
catastrophe
B. Wegiel, Y. Wang, F. Jernigan, L. Sun
P033 Phytochemical indole-3-carbinol synergizes strongly with fludarabine and induces p53-dependent and
-independent cell death in chronic lymphocytic leukemia cells irrespective of their IGHV mutation
state and treatment resistances
G. Perez-Chacon, C. Martinez-Laperche, N. Rebolleda, B. Somovilla-Crespo, C. Muñoz-Calleja,
I. Buño, J.M. Zapata
P034 Selectivity and mechanism of action studies for Polo Box-targeted, non-ATP based inhibitors of PLK1
M. Baxter, S. Craig, C. McInnes, M.D. Wyatt
P035 Clinical activity of BIND-014 (docetaxel nanoparticles for injectable suspension) as second-line
therapy in patients (pts) with Stage III/IV non-small cell lung cancer
R. Natale, M. Socinski, L. Hart, O. Lipatov, D. Spigel, B. Gershenhorn, G. Weiss, S. Kazmi,
N. Karaseva, O. Gladkov, V. Moiseyenko, J. Summa, G. Otterson
P036 Interaction of SJG-136 with cognate sequences of oncogenic transcription factors
J. Mantaj, P.J. Jackson, D.E. Thurston, K.M. Rahman
P037 CDKN1A-mediated responsiveness of MLL-AF4-positive acute lymphoblastic leukemia to Aurora
kinase-A inhibitors
L. Hung, Y. Chen, H. Lin, M. Tsai, H. Hsieh, J. Chang, N. Chen, S. Yang, T. Chen
P038 Metformin and its impact on gastric cancer patients survival after gastrectomy
C.K. Lee, M.K. Jung, I.K. Jung, S.J. Heo, J.Y. An, H.I. Kim, J.H. Chung, W.J. Hyung, S.H. Noh,
H.S. Kim, S.Y. Rha, H.C. Chung
P039 Early preclinical study of BO-2094 for treatment of human colon cancer, in combination with
5-fluorouracil
T.L. Su, T.H. Ou, M.H. Wu, Y.W. Lin, T.C. Lee
P040 Broad-spectrum preclinical combination activity of eribulin combined with various anticancer agents
in human breast cancer, lung cancer, ovarian cancer, and melanoma xenograft models
M. Asano, J. Matsui, M.J. Towle, J. Wu, S. McGonigle, T. Uenaka, K. Nomoto, B.A. Littlefield
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Poster board
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Abstract number
P041 Lurbinectedin (PM01183) specifically targets RNA Pol II for degradation via the proteasome pathway
in a transcription and TC-NER dependent fashion
G. Santamaria, J.F. Martı́nez-Leal, C. Cuevas, L.F. Garcia-Fernandez, C.M. Galmarini
P042 Trabectedin and lurbinectedin are effective against leukemic cells derived from patients affected by
chronic and juvenile myelomonocytic leukemia
M. Romano, A. Gallı̀, N. Panini, L. Paracchini, L. Beltrame, E. Bello, S.A. Licandro, C. Cattrini,
R. Tancredi, S. Marchini, V. Rosti, M. Zecca, M. Della Porta, A. Zambelli, C.M. Galmarini, E. Erba,
M. D’Incalci
P043 siRNA targeting of mitochondrial thymidine kinase 2 (TK2) sensitizes cancer cells to gemcitabine
and increases mitochondrial toxicity
C. Di Cresce, P. Ferguson, R. Figueredo, M. Rytelewski, S. Maleki Vareki, M.D. Vincent,
J. Koropatnick
P044 The effect of esomeprazole, a proton pump inhibitor, on the pharmacokinetics of the investigational
Aurora A kinase inhibitor alisertib (MLN8237) in patients with advanced solid tumors or lymphomas
X. Zhou, J. Nemunaitis, S. Pant, T. Bauer, A. Lockhart, M. Patel, B. Zhang, V. Kelly, C.D. Ullmann,
M. Bargfrede, K. Venkatakrishnan
P045 Development of rational combination therapy strategies to optimize treatment for GPNMB expressing
Her2+ and triple negative breast cancer
A. Rose, G. Maric, M.G. Annis, H. Smith, W.J. Muller, P.M. Siegel
P046 Platinum(IV) derivatives of oxaliplatin: Cellular effects and in vivo potency
S. Göschl, V. Pichler, E. Brynzak, P. Heffeter, U. Jungwirth, M.A. Jakupec, W. Berger, M. Galanski,
B.K. Keppler
P047 Adeno-associated virus (AAV) carrying diphtheria toxin a gene for pancreatic cancer therapy
M. Chen, C.Y. Ho, M. Teng, H. Chen
P048 MGMT methylation assessed by methyl-BEAMing technique is a prognostic and predictive biomarker
in glioblastoma and metastatic colorectal cancer patients
L. Barault, A. Amatu, F.E. Bleeker, C. Moutinho, A. Cassingena, F. Tosi, T. Venesio, M. Esteller,
A. Bardelli, S. Siena, A. Sartore-Bianchi, F. Di Nicolantonio
P049 Low, frequent doses of PM060184 induce remarkable in vivo antitumor activity
P. Aviles, M.J. Guillen, P.P. Lopez-Casas, F. Sarno, O. Cataluña, P. Nuñez, C. Cuevas, M. Hidalgo
P050 Radiosensitizing effect of sodium metaarsenite in a metastatic brain tumor model
W.Y. Kang, Y.M. Park, S.J. Kim
P051 Differential antitumor activity of trabectedin, lurbinectedin, Zalypsis and PM00128 against a panel of
human cells deficient in transcription and NER factors
V. Moneo, S. Avila, P. Martı́nez, B. de Castro, S. Cascajares, C. Cuevas, L.F. Garcia-Fernandez,
C.M. Galmarini
P052 Pipecolidepsin A, Stellatolide A and Irvalec: New cyclodepsipeptides of marine origin with antitumor
activity
J.M. Molina-Guijarro, V. Moneo, J.F. Martinez-Leal, C. Cuevas, L.F. Garcia-Fernandez,
C.M. Galmarini
P053 Discovery of novel inhibitor of FOXO nuclear–cytoplasmic shuttling from natural products of marine
origin
F.J. Castillo Correa, N. De Pedro, L. Rodriguez Quesada, D. Oves Costales, J.R. Tormo, J. Martin,
F. Reyes, O. Genilloud, F. Vicente, W. Link, B. Cautain
P054 Sodium metaarsenite cytotoxic activity is associated with telomere length and many types of arsenic
transporters in non-small cell lung cancer
Y.M. Park, S.J. Kim
P055 A panel of pediatric liver cancer patient-derived xenografts to improve stratification of children with
hepatoblastoma
M. Fabre, D. Nicolle, A. Gorse, O. Déas, C. Mussini, L. Brugières, M.R. Ghigna, E. Fadel,
L. Galmiche-Rolland, C. Chardot, C. Armengol, J.G. Judde, S. Branchereau, S. Cairo
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Poster board
Abstract number
P056 Antitumor and temozolomide-sensitizing effects of sodium metaarsenite in an orthotopic glioblastoma
xenograft model
W.Y. Kang, Y.M. Park, S.J. Kim
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P057 Radium-223 dichloride − Efficacy and mode-of-action in a mouse model of prostate cancer bone
metastasis
M.I. Suominen, K.M. Fagerlund, J.P. Rissanen, Y. Konkol, E. Alhoniemi, D. Mumberg, K. Ziegelbauer,
S.M. Käkönen, J.M. Halleen, R.L. Vessella, A. Scholz
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P058 Nifuroxazide halogenic derivatives induce ROS-mediated apoptosis and display antitumor activity
against metastatic melanoma
C. Fernandez de Farias, M.H. Massaoka, N. Girola, C.R. Figueiredo, R.A. Azevedo, L.C. Tavares,
L.R. Travassos
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P059 Phase II drug metabolism UGT1A enzyme affects cellular response of colon cancer cells to antitumor
triazoloacridinone C-1305 treatment
E. Augustin, E. Bartusik, A. Theus, B. Borowa-Mazgaj, Z. Mazerska
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P060 Clinical validity of new genetic biomarkers of irinotecan neutropenia: An independent replication
study
F. Innocenti, J. Ramirez, W. Qiao, A.J. de Graan, M.J. Ratain, R.H.N. van Schaik, R.H.J. Mathijssen,
G.L. Rosner, D.J. Crona
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P061 Cytotoxic response as a result of the cross-talk between UGT mediated metabolism and modulation
of UGT activity by C-1311 and C-1305 acridinone antitumor agents in selected solid tumor cell lines
Z. Mazerska, M. Pawlowska, A. Bejrowska, M. Szydlowska, B. Fedejko, E. Augustin
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P062 Can the P53 status predict the outcome of Polo-like kinase 1 inhibition in non-small cell lung cancer
cell lines?
J. Van den Bossche, A. Wouters, C. Deben, V. Deschoolmeester, P. Specenier, P. Pauwels, M. Peeters,
F. Lardon
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Drug Resistance and Modifiers
Poster board
Abstract number
P063 AKT3 activation mediates resistance to HER2-targeted therapies in HER2-amplified breast cancer
F. Carmona Sanz, F. Montemurro, V. Rossi, C. Verma, M. Berger, J. Baselga, M. Scaltriti
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P064 Antitumor activity of S116836, a novel tyrosine kinase inhibitor, against imatinib-resistant FIP1L1PDGFRa-expressing cells
J. Pan, Y. Shen, K. Ding
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P065 Novel regulation of estrogen receptor transcription by the PI3K pathway
E. Toska, M. Elkabets, A. Bosch, O. Litvin, M. Scaltriti, J. Baselga
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P066 Nrf2 as a molecular target in overwhelming chemoresistance in breast cancer therapy
N. Samadi, F. Ramezani, M. Sabzichi
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P067 Differential pathway resiliency in response to MAPK inhibition in BRAF-mutant cancer
M. Sos, R.S. Levin, J.D. Gordan, J.A. Oses-Prieto, J.T. Webber, M. Salt, B. Hann, A.L. Burlingame,
F. McCormick, S. Bandyopadhyay, K.M. Shokat
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P068 Increased EDN1 expression mediates acquired resistance to the combination therapy of PI3K and
MEK inhibitors for colorectal cancer
B. Bhattacharya, S.H.H. Low, T. Benoukraf, M.L. Chong, K.X. Koh, R. Soong
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P069 Loss of PTEN leads to acquired resistance to the PI3Ka inhibitor BYL719: a case of convergent
evolution under selective therapeutic pressure
P. Castel, D. Juric, M. Griffith, O.L. Griffith, H.H. Won, B. Ainscough, H. Ellis, S. Ebbesen,
I. Gopakumar, C. Quadt, M. Peters, D. Solit, S.W. Lowe, E.R. Mardis, M.F. Berger, M. Scaltriti,
J. Baselga
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Poster board
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Abstract number
P070 Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors
A.A. Marusiak, Z.C. Edwards, W. Hugo, E.W. Trotter, M.R. Girotti, N.L. Stephenson, X. Kong,
M.G. Gartside, S. Fawdar, A. Hudson, W. Breitwieser, N.K. Hayward, R. Marais, R.S. Lo, J. Brognard
P071 Tumor suppressive roles of miR-221 and miR-222 in lung cancer
M. Sato, R. Yamashita, T. Kakumu, T. Hase, E. Maruyama, Y. Sekido, M. Kondo, Y. Hasegawa
P072 A comprehensive in vitro screen to identify therapeutic candidates for inclusion with etoposide/platin
combinations to improve treatment of SCLC
D. Evans, R. Delosh, J. Laudeman, C. Ogle, R. Reinhart, M. Selby, T. Silvers, A. Monks, E. Polley,
G. Kaur, J. Morris, B.A. Teicher
P073 Quantitative proteomics as a tool to identify resistance mechanisms in erlotinib-resistant subclones of
the non-small cell lung cancer cell line HCC827
K. Jacobsen, R.R. Lund, H.C. Beck, H.J. Ditzel
P074 Determination of an oxidative stress gene signature in inflammatory breast cancer patient tumors
and development of a novel redox modulatory strategy in overcoming chemotherapy resistance and
mediating anti-tumor efficacy
G.R. Devi, J.L. Allensworth, M. Evans, N. Ueno, D. McDonnell, F. Bertucci, S. Van Laere
P075 Sensitization of triple-negative breast cancer to PI3K inhibition by cotargeting IGF1R
K. de Lint, J.B. Poell, J. Vidal Rodriguez, H. Soueidan, W. Nijkamp, L.F. Wessels, R.L. Beijersbergen
P076 Regorafenib resistance in colorectal carcinoma is associated with enhanced expression of type II
interleukin 1 receptor and reversed by MEK/ERK inhibitor
A.C. Mar, C.H. Chu, C.W. Shiau, T.C. Lee
P077 Acquired resistance to BET bromodomain inhibitors is associated with modulation of the apoptotic
signaling network
P. Sandy, S. Nerle, A. Conery, C. Hatton, B. Bryant, R. Sims, E. Normant
P078 Src family kinase activation is a compensatory survival mechanism for acquired resistance to EGFRTKIs in lung cancer cells
M. Ono, K. Sonoda, K. Azuma, K. Watari, M. Molina, R. Rosell, M. Kuwano
P079 Wnt secretion is required to maintain Wnt activity in colon cancer
O. Voloshanenko, G. Erdmann, T.D. Dubash, I. Augustin, M. Metzig, C.R. Ball, H. Glimm, R. Spang,
M. Boutros
P080 Influence of EGFR exon 19 mutation subtypes on survival outcomes in advanced stage Asian nonsmall cell lung cancer patients receiving TKI therapy
B. Chowbay, O. Singh, N. Sutiman, D.S.W. Tan, W.T. Lim, E.H. Tan
P081 Critical difference in development of acquired resistance to MDM2 inhibitor SAR405838 in vitro and
in vivo
G. Hoffman-Luca, C.Y. Yang, J. Lu, D. Ziazadeh, D. McEachern, L. Debussche, S. Wang
P082 Genome-wide drug sensitivity screens in haploid mouse embryonic stem cells
S.J. Pettitt, D. Krastev, H. Pemberton, Y. Fontebasso, I. Bajrami, I. Kozarewa, J. Frankum, R. Rafiq,
J. Campbell, R. Brough, A. Ashworth, C.J. Lord
P083 Tie-2 regulates the stemness of prostate cancer cells
K. Tang, M. Ling
P084 Tumor infiltrating leukocyte subpopulations as a biomarker of response and resistance to targeted
therapy in patients with BRAF mutation-positive metastatic melanoma
M.C. Kelley, D.B. Doxie, A.R. Greenplate, H. Crandall, J.A. Sosman, J.M. Irish
P085 4E-BP1 expression levels determine sensitivity of triple negative breast cancer cells to mTOR
inhibitors
K. Jastrzebski, B. Thijssen, J. Vidal Rodriguez, K. de Lint, C. Lieftink, L.F. Wessels, R.L. Beijersbergen
P086 Role of ERK nuclear translocation in cisplatin-sensitive and -resistant ovarian cancer cells
S. Dilruba, G.V. Kalayda, U. Jaehde
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Abstract number
P087 A stress induced early innate response causes multi-drug tolerance in melanoma
D.R. Menon, S. Das, C. Krepler, A. Vultur, B. Rinner, S. Schauer, K. Kashofer, K. Wagner, G. Zhang,
E. Bonyadi Rad, H.P. Soyer, B. Gabrielli, R. Somasundaram, G. Hoefler, M. Herlyn, H. Schaider
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P088 Elucidating mechanisms of resistance to FGFR inhibitors in endometrial cancer
L. Packer, S. Byron, C. Mahon, D. Loch, A. Wortmann, K. Nones, S. Grimmond, J. Pearson,
N. Waddell, P. Pollock
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Drug Screening
Poster board
Abstract number
P089 Identification of synthetic lethality compounds from natural products for cancers
K.W. Ng, K.S. Lee, V. Patel, E. Sundaramoorthy, N. Ayoub, X. Su, A. Venkitaraman, S.H. Teo
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P090 Evaluation of hormone therapies in a panel of breast PDX models: Relevance of ER status on
sensitivity to letrozole and tamoxifen
M.J. Wick, T. Vaught, L. Gamez, J. Meade, A. Diaz, K.P. Papadopoulos, D.W. Rasco, A. Patnaik,
M. Beeram, A. Lang, A.W. Tolcher
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P091 Utilization of low passage adenoid cystic carcinoma PDX models to identify novel combination
therapies
M.J. Wick, J. Meade, T. Vaught, M. Nehls, J. Flores, J. Kaufman, A.W. Tolcher, D.W. Rasco,
A. Patnaik, C.A. Moskaluk, K.P. Papadopoulos
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P092 Synergistic inhibition of HER2 positive breast cancer by triptolide and lapatinib
P. Chalugun, J.S. Shim, P. Korangath, S. Sukumar, J.O. Liu
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P093 Combination screening of investigational oncology agents
S. Holbeck, J.M. Collins, J.D. Doroshow
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P094 Small molecules selectively targeting breast cancer cells
J. Sakoff, J. Gilbert, A. McCluskey
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P095 PIK3CA mutation-targeting compounds analyses using NCI60 cell line panel
H. Bando, J. Lih, E.C. Polley, S.L. Holbeck, B. Das, D. Sims, T. Doi, A. Ohtsu, M. Williams,
N. Takebe
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P096 Comparison of platinum/taxane and anthracycline-based therapies in ovarian PDX models: Correlating
stage of biopsy collection and engrafting with in vivo drug sensitivity
J. Meade, M.J. Wick, T. Vaught, L. Gamez, M. Farley, A. Moriarty, A.W. Tolcher, A. Patnaik,
D.W. Rasco, A.J. White, K.P. Papadopoulos
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P097 The use of next generation sequencing (NGS) in the management of metastatic breast cancer (MBC):
Defining a model for genomic-driven therapies
L. Austin, M. Gooptu, T. Avery, R. Jaslow, J. Palazzo, M. Cristofanilli
103
P098 Identification of inhibitors of tryptophan metabolizing enzymes for cancer immunotherapy by highthroughput screening
G. Zaman, J.C.M. Uitdehaag, S. van Gerwen, N. Seegers, A.M. van Doornmalen, J. de Man,
R.C. Buijsman
104
P099 A platform to test multiple therapy options simultaneously in a patient’s own tumor
N. Caffo, R. Klinghoffer
105
Immunotherapy (Immunecheckpoints, Vaccination, Oncolytic viruses, Cytokines)
Poster board
Abstract number
P100 Beta-3 integrin inhibition reduces inflammatory cytokine release but not anti-cancer activity of
oncolytic adenovirus in ovarian cancer
A.K. Browne, L.A. Tookman, C.K. Ingemarsdotter, R. Bouwman, K. Pirlo, Y. Wang, K.M. HodivalaDilke, I.A. McNeish, M. Lockley
106
Poster board
xxxiii
Abstract number
P101 CIGB-247: Anti-VEGF therapeutic vaccine in patients with advanced solid tumors
F. Hernández-Bernal, J.V. Gavilondo, M. Ayala Ávila, A.V. de la Torre, J. de la Torre, K.H. SelmanHousein, Y. Morera, M. Bequet-Romero, C.M. Valenzuela, Y. Martin
P102 CD70 (TNFSF7), a receptor involved in acute immune modulation of viral infection, is frequently
overexpressed in solid and hematological malignancies
J. Jacobs, K. Zwaenepoel, P. Aftimos, C. Rolfo, S. Rottey, L. Ysebrant de Lendonck, K. Silence,
A. Awada, A. Thibault, P. Pauwels
P103 Functional activity, but not PD-1 expression level, differentiates primary CLL from healthy PD1+
T cells using SCNP
S. Liang, L. Leung, S. Putta, D. Hotson, D. Rosen, R.E. Hawtin
P104 IL-6/STAT3/Fra-1 signaling axis promotes colorectal cancer aggressiveness through epithelial–
mesenchymal transition
J. Shao, H. Liu
P105 Identification of peptides which could block PD-1 checkpoint for NSCLC immunotherapy
Y. Zhu, C. Li
P106 A novel primary human tumor explant platform provides a preclinical translational link from tissue
culture to the clinic
G. Juan, K. Paweletz, E. Trueblood, J. Rossi, M. Damore, A. Anderson, R. Loberg
P107 Preclinical activity and pharmacodynamic biomarkers of W014A, a PD-1 decoy peptide blocking both
PD-1 immune checkpoint ligands, PD-L1 and PD-L2
C. Bailly, M. Broussas, M. Ramachandra, P.G. Sasikumar, K. Shrimali, S. Adurthi, M. Ramachandra,
L.K. Satyam, A.A. Dhudashia, S. Dhodheri, K.B. Sunilkumar, N. Corvaı̈a, P. Ferre
P108 Toll-like receptor 5 agonist entolimod as a potential anticancer immunotherapeutic agent
L. Burdelya, C. Brackett, B. Kojouharov, J. Veith, A. Gudkov
P109 Anti-metastatic activity via co-blockade of PD-1 and adenosine A2A receptor
D. Mittal, A. Young, K. Stannard, M.W. Teng, B. Allard, J. Stagg, M.J. Smyth
P110 Novel targets for antibody–drug conjugate therapy
A.G. Grandi, S.C. Campagnoli, M.P. Parri, E.D.C. De Camilli, B.J. Jin, P.S. Sarmientos, G.G. Grandi,
L.T. Terracciano, P.P. Pileri, G.V. Viale, R. Grifantini
P111 “Arming” the chimeric oncolytic adenovirus enadenotucirev to deliver checkpoint inhibitors and other
therapeutics directly to tumours
B. Champion, P. Kodialbail, S. Illingworth, N. Rasiah, D. Cochrane, J. Beadle, K. Fisher,
A.C.N. Brown
P112 Major synergy between Coxsackievirus A21 (CAVATAK™) and radiotherapy or chemotherapy in
bladder cancer
G.R. Simpson, N. Annels, M. Ajaz, F. Launchbury, G. Bolton, A.A. Melcher, K.J. Harrington, G. Au,
D. Shafren, H. Pandha
P113 3-Bromopyruvate as an inducer of immunogenic cell death in colon cancer cells
K.H. Jung, J.H. Lee, J.W. Park, C. Quach, K.H. Lee
P114 Density of CD 8 +ve T cells & CD 56 +ve NK cells in follicular adenoma & papillary carcinoma of
thyroid in Pakistani population
J. Varda, N. Naseem, A.H. Nagi
P115 A modified double-deleted vaccinia virus combining viral oncolysis and potential gene therapy as a
novel therapeutic for atypical teratoid/rhabdoid tumors
Y. Ruan, A. Narendran
P116 Analysis of immune-response markers in resectable NSCLC
M. Usó, E. Jantus-Lewintre, R. Sirera, S. Calabuig-Fariñas, S. Gallach, E. Escorihuela, A. Blasco,
R. Guijarro, C. Camps
P117 Targeting tryptophan metabolism in human lung cancer
J. Deshane, C. Schafer, Y. Wang, A. Sawant, T.H. Jin, D. Zhi, S. Ponnazhagan, S. Grant
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
xxxiv
Poster board
Abstract number
P118 A WT1-derived peptide protects against metastatic melanoma in a syngeneic model by in vivo
immunomodulatory effects on dendritic cells
M.H. Massaoka, C.R. Figueiredo, N. Girola, R.A. Azevedo, L.R. Travassos
124
P119 Critical issues in the clinical development of oncolytic viruses − A regulatory perspective
M. Schuessler-Lenz
125
P120 IMCgp100: A novel bi-specific biologic for the treatment of malignant melanoma
W. Shingler, J. Harper, G. Bossi, D. Barker, J. Dukes, N. Liddy, S. Paston, T. Mahon, P. Molloy,
M. Sami, E. Baston, B. Cameron, A. Johnson, A. Vuidepot, N. Hassan, Y. McGrath, B. Jakobsen
126
P121 Correlation of clinical activity of pembrolizumab (MK-3475) with immunohistochemical staining for
programmed death-1 ligand (PD-L1) in 50% of tumor cells in a prospective non-small cell lung
cancer (NSCLC) validation population
E.G. Garon, N.A. Rizvi, N.B. Leighl, R. Hui, J.P. Eder, A. Patnaik, C. Aggarwal, L. Horn,
A.S. Balmanoukian, M.A. Gubens, E. Felip-Font, E. Carceny Costa, J.C. Soria, M.J. Ahn,
H.T. Arkenau, J.S. Lee, G. Robinet, G.M. Lubiniecki, J. Zhang, K. Emancipator, R. Rutledge,
M. Dolled-Filhart, L. Gandhi
127
P122 T cell-mediated cancer immunotherapy through OX40 agonism
M. Huseni, C. Du, J. Zhu, P. Pacheco-Sanchez, M. Moskalenko, H. Chiu, K. Dalpozzo, K. Totpal,
L. Damico-Beyer, J. Kim
128
P123 Characterization of PD-L1 expression in circulating tumor cells (CTCs) of non-small cell lung
cancer (NSCLC) patients
R. Krupa, D. Lu, M. Harvey, J. Louw, A. Jendrisak, D. Marrinucci, R. Dittamore
129
P124 Preclinical results of ProCervix, a first in class, first in indication therapeutic vaccine targeting
HPV16/18 infected women
M. Esquerre, M. Bouillette-Marussig, A. Goubier, M. Momot, H. Keller, M. Bissery
130
P125 Zfra activates novel Hyal2+ CD3− CD19− memory spleen cells to block cancer growth, stemness, and
metastasis in vivo
N. Chang
131
P126 A novel anti-PDL1 antibody-based bifunctional protein with enhanced immunological activity
Y. Wu, S. Martomo, Z. Zhong, D. Lu, Z. Polonskaya, X. Luna, Z. Zhang, H. Zhang, L. Witte,
S. Waksal, Z. Zhu
132
P127 Analysis of immune microenvironment in resectable NSCLC: Prognostic value of regulatory and
conventional T cell markers proportion
M. Usó, R. Sirera, S. Calabuig-Fariñas, A. Blasco, E. Pastor, R. Guijarro, E. Jantus-Lewintre,
J. Forteza, C. Camps
133
P128 Epigenetic immunomodulation by SGI-110 combined with immune checkpoint blockade for new
therapeutic strategies
A. Covre, C. Fazio, H.J.M.G. Nicolay, P.G. Natali, P. Taverna, M. Azab, S. Coral, M. Maio
134
P129 AD-O64.3: IFN-g–TRAIL fusion protein. Use of two independent signaling pathways for a strong
synergistic antitumor effect
B. Zerek, J.S. Pieczykolan, S.D. Pawlak, P.K. Rozga, A. Pieczykolan, M. Szymanik, A. Jaworski,
M. Galazka, K. Bukato, K. Poleszak, M. Teska-Kaminska, W. Strozek
135
P130 Tumor-infiltrating lymphocytes (TILs) following intratumoral administration of ONCOS-102 are
associated with prolonged overall survival in last line solid tumor patients
S. Pesonen, T. Joensuu, E. Jager, J. Karbach, C. Wahle, K. Kairemo, K. Partanen, R. Turkki,
A. Hemminki, C. Backman, M. von Euler, T. Hakonen, T. Ranki, L. Vassilev, A. Vuolanto,
M. Jaderberg
136
P131 Immune checkpoint blockade enhances measles virotherapy
C.E. Engeland, R. Veinalde, C. Grossardt, S. Bossow, I. Shevchenko, V. Umansky, D.M. Nettelbeck,
D. Jäger, C. von Kalle, G. Ungerechts
137
Poster board
xxxv
Abstract number
P132 Preclinical activity and safety of MGD006, a CD123xCD3 Bispecific DART® molecule for the
treatment of hematological malignancies
P. Moore, G.R. Chichili, L. Huang, H. Li, S. Burke, F. Chen, L. He, Q. Tang, L. Jin, S. Gorlatov,
V. Ciccarone, S. Koenig, M. Shannon, R. Alderson, S. Johnson, E. Bonvini
138
P133 GBR1302: a BEAT® bispecific antibody for the treatment of HER2 positive cancers
A. Croset, J. Macoin, R. Ollier, M. Pluess, C. Delon, D. Skegro, S. Blein, S. Hou, J. Back
P134 Relationship between programmed death ligand 1 (PD-L1) expression and clinical outcome in
patients (pts) with melanoma (MEL) treated with pembrolizumab (pembro; MK-3475)
A. Daud, O. Hamid, C. Robert, F.S. Hodi, J.D. Wolchok, W.J. Hwu, J.S. Weber, R. Kefford,
P. Hersey, A.M. Joshua, R. Joseph, T.C. Gangadhar, R. Dronca, A. Patnaik, H. Zarour, K. Gerigich,
J. Lunceford, K. Emancipator, M. Dolled-Filhart, X. Li, P. Kang, S. Ebbinghaus, A. Ribas
P135 Phase I study of ipilimumab with stereotactic radiosurgery for melanoma patients with brain
metastases
W. Shi, E. Wuthrick, K. Feeney, M. Werner-Wasik, D.W. Andrews, J.J. Evans, K. Judy, C. Farrell,
A.P. Dicker
P136 IL-17 as a novel molecular target for prostate cancer prevention
A. De Angulo, R. Faris, C. Jolly, L. deGraffenried
139
Preclinical Models
Poster board
140
141
142
Abstract number
P137 Regional bias of intratumoral genetic heterogeneity of nucleotide repeats in colon cancers with
microsatellite instability
C. An, Y. Choi, M. Kim, J. Lee, N. Yoo, S. Lee
P138 Association between interleukin 17/interleukin 17 receptor gene polymorphism and papillary thyroid
cancer
Y. Eun, H. Chung, Y.C. Lee
P139 ARQ 087, a novel pan FGFR-inhibitor crosses the BBB (blood–brain barrier) and distributes to the
brain of rats
R. Savage, T. Hall, B. Schwartz
P140 Establishment of patient-derived xenografts (PDX) models for triple negative breast cancer (TNBC)
as a pre-clinical platform for drug development
J. Thatte, M. Meza, J. Ricono, T. Broudy, C. Mirsaidi, P. Nair
P141 An integrated approach for identifying E-cadherin synthetic lethality networks
I. Bajrami, S.J. Pettitt, R. Brough, H. Pemberton, D. Kastrev, Y. Fontebasso, J. Frankum, J. Campbell,
A. Ashworth, C.J. Lord
P142 RAS synthetic lethal interactions from yeast to human cells
S. van Wageningen, A. Prahallad, G. Heynen, R. Rothstein, R. Bernards
P143 Quantitative mutational assessment of circulating tumor DNA using massively parallel deep
sequencing in plasma and urine from advanced colorectal cancer patients
J.C. Poole, C.R.T. Vibat, L. Benesova, B. Belsanova, S. Hancock, T.L. Lu, M.G. Erlander, M. Minarik
P144 Pirin downregulates E-cadherin gene expression and contributes to EMT
K. Komai, Y. Niwa, Y. Sasazawa, S. Simizu
P145 Impact of EGFR amplification pattern on the expression of miRNA-200c in primary glioblastoma
multiforme
L. Muñoz Hidalgo, C. López Gines, E. Serna, D. Monleon, R. Callaghan, R. Gil Benso,
H. Martinetto, A. Gregori Romero, J. Gonzalez Darder, M. Cerda Nicolas
P146 Preclinical characterization of MM-151, an oligoclonal antibody therapeutic that targets EGFR by
three distinct mechanisms of action
A. King, M. Sevecka, N. Gerami-Moayed, O. Burenkova, J. Kearns, G. Tan, C. Sloss, R. Bukhalid,
U. Nielsen, B. Wolf
143
144
145
146
147
148
149
150
151
152
xxxvi
Poster board
Abstract number
P147 Moving beyond in vitro models and addressing the challenges of pooled RNAi screens in mouse
xenografts
D. Tedesco, K. Bonneau, M. Makhanov, D. Deng, P. Sun, A. Chenchik
153
P148 Drug response database with PDX tumor models in biomarker-driven multi-drug multi-arm clinical
trial settings
J. Jiang, T.F. Yu, Y. Yan, W. Du, T.T. Tan, L. Hua, J.L. Gu, X.Q. Yang, Z.H. Liu, X.K. Ye, Z. Gu
154
P149 SNIPER(TACC3) degrades TACC3 protein via the ubiquitin–proteasome pathway and induces
apoptosis in cancer cells expressing a large amount of TACC3
N. Ohoka, K. Nagai, K. Okuhira, N. Shibata, T. Hattori, N. Cho, M. Naito
155
P150 Behaviour of platinum(IV) complexes with prodrug function in different models of hypoxia
E. Brynzak, P. Heffeter, V. Pichler, M.A. Jakupec, B.K. Keppler
156
P151 Sensitive and specific detection of 1p/19q codeletion in gliomas by next generation sequencing
E. Dubbink, P.N. Atmodimedjo, R.M. van Marion, J.M. Kros, M.J. van den Bent, W.N.M. Dinjens
157
P152 The development of a series of orthotopic solid tumour models of prostate, lung and ovarian cancer
using optical and X ray imaging
M. Batey, M. Brown, E. Bowden
158
P153 Neoadjuvant chemotherapy in breast cancer patients induces expression of miR-34a and miR-122
P. Freres, C. Josse, N. Bovy, M. Boukerroucha, I. Struman, V. Bours, G. Jerusalem
159
P154 Identification of fusion genes through kinome-centered RNA sequencing in different types of solid
tumors
L. Mittempergher, C. Sun, F.H. Groenendijk, A.J. Bosma, S.M. Willems, T. Sustic, I.J. Majewski,
W. Grernrum, N.M. Davidson, I. de Rink, H.M. Horlings, W. Theelen-Engelsman, S.F. Chin,
A. Oshlack, B.W. van Rhijn, M. van den Heuvel, M.S. van der Heijden, C. Caldas, R. Bernards
160
P155 Leveraging a novel DNA barcoding platform for integrated profiling and pharmacodynamic readouts
C. Castro, V. Peterson, A. Ullal, S. Agasti, S. Tuang, N. Miller, M. Birrer, R. Weissleder
161
P156 Drug–drug interaction predictions for MLN2480, an investigational pan-RAF inhibitor, based on
nonclinical data
S.K. Balani, A. Bulychev, L. Cohen, M. Liao, C.Q. Xia, F. Wang, P. Li, B. LeClair, T. Bohnert, L. Gan,
X. Zhou, V. Bozon, S. Prakash
162
P157 Interrogation of pharmacogenes in cancer patients using targeted DNA sequencing
F. Innocenti, N. Gillis, J. Parker, N. Hayes, D. Eberhard, K. Richards, J.T. Auman, E. Seiser
163
P158 Augmentation of NAD+ by NQO1 activation attenuates cisplatin-mediated hearing impairment
S. Yang, G.S. Oh, K.B. Kwon, R.K. Park, S.Y. Lee, S.R. Moon, H.S. So
164
P159 Effect of zoledronic acid on the post-translational modification of activated leukocyte cell adhesion
molecule (ALCAM) in cancer cells
R. Toth, G. Trombino, V. Castronovo, A. Bellahcene
165
P160 Pre-clinical and clinical activity of Anti-DLL4 (demcizumab) in combination with gemcitabine plus
nab-paclitaxel in pancreatic cancer
M. Hidalgo, A. Cubillo, R. Stagg, J. Dupont, Y. Wan-Ching, T. Hoey
166
P161 Genomic characterisation of 1003 cancer cell-lines
G. Bignall, F. Iorio, P.A. Futreal, M.R. Stratton, P. Campbell, U. McDermott
167
P162 CDCP1 as a new marker of aggressiveness in triple-negative breast cancers
M. Campiglio, F. Turdo, F. Bianchi, M. Sasso, L. De Cecco, P. Casalini, P. Gasparini, L. Forte,
R. Agresti, I. Maugeri, G. Sozzi, E. Tagliabue
168
P163 BRAF mutation testing in cell-free DNA from plasma of patients with advanced cancers using a
novel, rapid, automated molecular diagnostics prototype platform (Idylla™)
F. Janku, H.J. Huang, B. Claes, G.S. Falchook, A. Naing, S. Piha-Paul, A.M. Tsimberidou,
R.G. Zinner, D.D. Karp, S. Fu, V. Subbiah, D.S. Hong, J.J. Wheler, R.G. Luthra, S.P. Patel,
E.S. Kopetz, E. Sablon, G. Maertens, R. Kurzrock, F. Meric-Bernstam
169
Poster board
xxxvii
Abstract number
P164 Novel, ultra-deep next-generation sequencing for BRAF mutation testing using small amount of cellfree DNA from plasma of patients with advanced cancers
F. Janku, H.J. Huang, N.M. Ramzanali, X. Cai, R. Klausner, F. Meric-Bernstam, J.B. Fan
170
P165 Antagonistic interaction between gemcitabine and erlotinib is influenced by EGR1 (early growth
response 1) transcription factor expression
C.D. Hose, Y. Zhao, E.C. Polley, J. Fang, N.D. Fer, A. Rapisarda, B.A. Teicher, R.M. Simon,
J.D. Doroshow, A. Monks
171
P166 Modulation of estrogen-dependent transcription by cohesin in MCF7 human breast adenocarcinoma
cells
T. Dasgupta, J. Antony, J. Rhodes, M. McEwan, M. Eccles, J. Horsfield
172
P167 Ras-mediated activation of mitogen-activated protein kinase pathway unleashes basement membrane
damaging activity of serine protease hepsin
T. Tervonen, S. Pant, D. Belitskin, J. Englund, K. Närhi, E. Verschuren, P. Kovanen, J. Klefström
173
P168 Combination of molecular and drug response data in patient-derived xenografts to assist patient
stratification
S. Cairo, O. Déas, A. Beurdeley, V. Yvonnet, M.F. Poupon, J.G. Judde
174
P169 Functional analysis of [methyl-3 H]choline uptake in glioblastoma cells: Influence of anti-cancer and
central nervous system drugs
M. Inazu, C. Taguchi, T. Yamanaka, H. Uchino
175
P170 Resolvin D2 has mitogenic activity in estrogen receptor positive breast cancer cell lines via activation
of estrogen receptor
N. Al-Zaubai, C. Johnstone, M. Rizzacasa, A. Stewart
176
P171 Validation of 3D primary organoid cultures of colorectal carcinoma as discovery and validation
platform for personalized cancer therapy
P. Halonen, A. Kuijpers, B. Morris, B. Diosdado, S. Mainardi, R. Bernards, V. Verwaal,
R. Beijersbergen
177
P172 Establishment of patient-derived xenografts (PDX) models for small cell lung (SCL) as a pre-clinical
platform for drug development
T. Broudy, J. Ricono, C. Mullins, C. Mirsaidi, P. Nair
178
P173 Effects of human breast cancer cells secreted factors on macrophage differentiation
S. Coimbra de Sousa, R. Brion, J. Mönkkönen, H. Joensuu, D. Heymann, J. Määttä
179
P174 Identifying and monitoring somatic mutations in cell free DNA of patients with metastatic melanoma
J. Wisell, C.M. Amato, W.A. Robinson
180
P175 Aberrant Wnt signaling activation in human cancers: In vitro and in vivo models to facilitate Wnt
targeted drug development
G. Liu, C. Dong, R. Zhang, L. Zhang, S. Qian, J. Cai, J. Zhang, J. Ning
181
P176 TRAP1 represents a key mediator of stemness and glycolytic metabolism in colorectal cancer cells
G. Lettini, F. Maddalena, L. Sisinni, V. Condelli, L. Del Vecchio, M. Gemei, T. Notarangelo,
M. Landriscina
182
P177 Spatio-temporal characterization of tumor growth and invasion
A.M. Jimenez, O. Yogurtcu, M. Horn-Lee, P. Rao, S.X. Sun, D. Wirtz
183
P178 Molecular profiling of heterogeneous tumor cells
A. Chenchik, D. Deng, K. Bonneau, M. Makhanov, M. Coram, G. Dolganov, S.S. Jeffrey
184
P179 Treatment of patient-derived NSCLC xenograft preclinical models using image-guided small animal
irradiation
N. Papadopoulou, A. McKenzie, J. King, M. Page, R. Kumari
185
P180 The use of Quantitative Textural Analysis imaging biomarkers to predict response to temsirolimus
treatment in advanced HCC subjects
R. Korn, R. Osarogiagbon, R. Newbold, D. Burkett, J. Sachdev
186
xxxviii
Poster board
Abstract number
P181 Targeted genomic profiling of penile squamous cell carcinoma using the Oncomine cancer research
panel
A.S. McDaniel, D. Hovelson, A. Cani, C.J. Liu, Y. Zhang, S. Sadis, S. Bandla, P. Williams, D. Rhodes,
S.A. Tomlins
187
P182 RANBP2 knock-down is synthetic lethal with BRAF V600E in colon cancer
L. Vecchione, V. Gambino, G. d’Ario, S. Tian, A. Schlicker, S. Mainardi, B. Diosdado, I. Simon,
M. Delorenzi, C. Lieftink, R. Beijersbergen, S. Tejpar, R. Bernards
188
P183 Molecular and pharmacological characterization of primary mesothelioma tumor cell lines
orthotopically xenografted in nude mice
C. Pisano, A. Cole, A. Barbarino, E. Bianchino, M. Guglielmi, C. Melito, G. Mercadante,
A. Porciello, A. Riccio, I. La Porta, S. Orecchia, R. Libener, L. Mazzucco, S. Licandro, M. Ceccarelli,
F. D’Angelo, P. De Luca
189
P184 Differential chemosensitivity between CETCs and tumour spheroids in cancer patients with solid
tumors
D. Zimon, M. Pizon, E.L. Stein, U. Pachmann, K. Pachmann
190
P185 Homotypic and heterotypic cell signaling transduction using a dielectrophoresis microfluidic device
M. Tellez Gabriel, D. Heymann
191
P186 Extensive characterization of patient derived colon cancer xenografts for preclinical biomarker
identification
V. Vuaroqueaux, F. Kiefer, P. Bronsert, A.L. Peille, B. Zeitouni, F. Foucault, T. Kees, J. Guo, J. Schüler,
H.H. Fiebig
192
P187 The identification of new therapies for ependymoma subgroups
N. Boulos, J.D. Dapper, Y.T. Patel, M. DeCuypere, B. Bianski, K.M. Mohankumar, M.O. Jacus,
B. Freeman III, K.D. Wright, A. Gajjar, A.A. Shelat, C.F. Stewart, R.K. Guy, R.J. Gilbertson
193
P188 Pancreatic ductal organoids as a new platform for drug discovery
M. Ponz-Sarvise, V. Corbo, D. Öhlund, T. Oni, A. Handly-Santana, D. Engle, H. Tiriac, C. Chio,
M. Feigin, L. Baker, C. Ardito-Abraham, Y. Park, C. Hwang, E. Elyada, K. Yu, H. Clevers,
D.A. Tuveson
194
P189 A humanized mouse model for preclinical testing of molecules targeting immune checkpoints
G. Baia, D. Vasquez, D. Ciznadija, D. Sidransky, A. Katz, K. Paz
195
P190 Neuropilin 2 (NRP2) modifies CXCL12/CXCR4 signaling and promotes lymph node metastases in
colon cancer
H. Schneider, P. Hönscheid, S. Schölch, C. Jakob, M. Muders
196
P191 Statistical aspects of kinetic analysis of gliomas with FDG-PET
D. Hawe, F.R. Hernandez, S. Murphy, E. Wolsztynski, J. Huang, J. O’Sullivan, M. Muzi, J. Eary,
K. Krohn, F. O’Sullivan
197
P192 Genetic and pharmacological inhibition of PIM-1 reduces tumor development in a K-Ras-driven
mouse model of non-small cell lung cancer
E. Aguirre, O. Renner, M.C. Rodriguez de Miguel, M.I. Albarran, A. Cebria, D. Cebrian, F. RamosLima, J. Pastor, C. Blanco-Aparicio
198
P193 Inhibitor of differentiation 1 (Id1) expression in lung cancer cells and liver microenvironment
is required for liver metastasis (LM) development from non-small cell lung cancer (NSCLC) by
regulating EMT-related and proliferation-related proteins
E. Castanon, A. Soltermann, I. López, M. Ecay, M. Collantes, J.M. Lopez Picazo, M. Ponz, C. Rolfo,
A. Calvo, I. Gil-Bazo
199
P194 The influence of different cMET and EGFR backgrounds on the cytotoxicity of cMET and EGFR
small molecule inhibitors in vitro
N. Van Der Steen, K. Zwaenepoel, C. Rolfo, E. Giovannetti, M. Castiglia, V. Deschoolmeester,
A.P. Carreca, P. Germonpre, P. Pauwels
200
Poster board
xxxix
Abstract number
P195 Transcriptome analysis of CD133-positive stem cells and prognostic value of survivin in colorectal
cancer
S. Kim, S.H. Park, J. Lee, Y.S. Park, H.C. Kim
Radiation Interactive Agents
Poster board
201
Abstract number
P196 The novel microtubule-destabilizing drug BAL101553 (prodrug of BAL27862) sensitizes a treatment
refractory tumor model to ionizing radiation
A. Broggini-Tenzer, F. Bachmann, V. Vuong, A. Messikommer, K. Nytko-Karouzakis, T. O’Reilly,
H.A. Lane, M.N. Pruschy
202
P197 Ionizing radiation induced phosphatidylserine externalization on endothelial cell surface − a potential
target for vascular targeting
Z. Zhao, M. Johnson, B. Chen, M. Grace, J. Ukath, V. Lee, M. Stoodley
203
P198 High-throughput functional screening identifies the flavoreductase POR as a principal determinant of
sensitivity to the hypoxia-targeting prodrug SN30000
F.W. Hunter, Z. Shalev, J. Wang, J. Moffat, T. Katella, M. Koritzinsky, W.R. Wilson, B.G. Wouters
204
P199 Metformin to modulate AMP-kinase and enhance chemotherapy and radiotherapy in non-small cell
lung cancer
M.J. Troncone, S.M. Cargnelli, G. Pond, E. Tsiani, J. Wright, G. Steinberg, H. Skinner, L. Bo,
J. Bradley
205
P200 Identification of novel targets for radiosensitisation of non-small cell lung cancer by secretome
analysis
A. Sharma, S. Bender, O. Riesterer, A. Broggini-Tenzer, M. Pruschy
206
P201 The enhancement of radiotherapy efficacy with docetaxel-titanate nanotubes as a new nanohybrid for
localized high risk prostate cancer
C. Mirjolet, J. Boudon, A. Loiseau, S. Chevrier, T. Gautier, R. Boidot, J. Paris, N. Millot,
G. Crehange
207
P202 Combined treatment of a DNA-PKcs inhibitor (NU7441) and ionizing radiation causes a differential
mode of cell death in a panel of NSCLC cell lines and exhibits robust radiosensitisation
D. Saha, Y. Lan, F. Hsu, V. Tumati, Z. Zhang, Y. Lin, B. Chen
208
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Thursday 20 November 2014
Plenary Session 4
08:00–10:00
Antibody-Based Therapies (ADC and others)
Chairs: A. Tolcher (USA) and N. Gökbuget (Germany)
Auditorium
Abstract number
08:00 Antibody mediated payload delivery: Cautionary lessons from 20 years of clinical trials
A. Tolcher (USA)
Key objectives:
1. Understand the disconnect between activity with antibody drug conjugates (ADC) in preclinical
models versus clinical results.
2. Understand the multiple challenges associated with ADCs as a drug from the 3 components: antibody,
linker and payload.
3. Understand patient selection based on target and potential mitigation strategies when the target is also
expressed on normal tissues.
08:20 Challenging the dogmas: Clinical efficacy of SN38 conjugated antibodies in solid tumours
D.A. Goldenberg (USA)
Key objectives:
1. Discuss current status and challenges of antibody-drug conjugates (ADC) in solid cancer therapy.
2. Supertoxic drugs used in ADC may compromise therapeutic index.
3. A moderate cytotoxic drug in an ADC may allow higher doses and a higher therapeutic index.
08:40 Targeted alpha particle therapy for haematologic malignancies
J. Jurcic (USA)
Key objectives:
1. List the advantages and disadvantages of targeted alpha-particle therapy.
2. Summarise the results of recent clinical trials using targeted alpha-particle therapy for the treatment
for haematologic malignancies.
3. Describe the results of recent preclinical studies using alternative radioisotopes and pre-targeting
strategies.
09:00 Bispecific T-cell engaging antibodies in acute leukaemia − Recent advances and future challenges
Speaker: N. Gökbuget (Germany)
Key messages:
1. Relapsed and refractory ALL has a very poor outcome and there is an urgent medical need for new,
alternative treatment options.
2. Blinatumomab is a bispecific antibody which represents a new treatment principle based redirected
cell kill of CD19 positive target cells by T-cells.
3. Reponse rates are promising and the compound is currently studied in a randomised trial.
09:20 ORAL PRESENTATION: Pre-clinical and translational pharmacology, pharmacokinetics and
pharmacodynamics for a humanized anti-OX40 antibody MOXR0916, a T-cell agonist in the
treatment of solid tumors
S. Sukumaran, J.M. Kim, M. Huseni, J. Ruppel, H. Taylor, K. Totpal, J. Zhu, C. Zhang, H. Chiu,
E.G. Stefanich
209
09:35 ORAL PRESENTATION: A phase 1 study of KTN3379, a human anti-ErbB3 monoclonal antibody in
patients with advanced cancers
P. LoRusso, T. LaVallee, L. Kimmel, C. Lubeski, R. Gedrich, C. Sidor
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09:50 General discussion
A. Tolcher (USA)
Plenary Session 5
10:30–12:30
Epigenetic Targets
Chairs: K. Helin (Denmark) and T. Helleday (Sweden)
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Auditorium
Abstract number
10:30 Histone demethylases and methyltransferases as novel targets for cancer therapeutics
K. Helin (Denmark)
10:50 DOT1 and EZH2 targeted therapies
R. Copeland (USA)
Key messages:
1. EZH2 plays a critical role in normal B-cell maturation and is frequently dysregulated in germinal
center-derived lymphomas.
2. EPZ-6438 is a potent and selective inhibitor of EZH2 that demonstrates robust and durable antilymphoma activity in preclinical animal models both as a single agent and in combination with
standards of care.
3. EPZ-6438 is the first EZH2 inhibitor to enter human clinical studies and early observations from this
clinical trial will be discussed.
11:10 Targeting non-oncogene addiction with MTH1 inhibitors
T. Helleday (Sweden)
11:30 ORAL PRESENTATION: A novel synthetic lethal interaction between the histone mark H3K36me3
and checkpoint kinases
S.X. Pfister, E. Markkanen, Y. Jiang, S. Sarkar, V. D’Angiolella, G. Dianov, A.J. Ryan, T.C. Humphrey
211
11:45 ORAL PRESENTATION: Novel anti-tumor activity of targeted LSD1 inhibition by GSK2879552
H. Mohammad, K. Smitheman, G. Van Aller, M. Cusan, S. Kamat, Y. Liu, N. Johnson, C. Hann,
S. Armstrong, R. Kruger
212
12:00 LATE BREAKING ABSTRACT: Results of a first-in-man phase I trial assessing OTX015, an orally
available BET-bromodomain (BRD) inhibitor, in advanced hematologic malignancies
A. Stathis, B. Quesnel, S. Amorim, C. Thieblemont, E. Zucca, E. Raffoux, H. Dombret, Y. Peng,
A. Palumbo, N. Vey, X. Thomas, M. Michallet, C. Gomez-Roca, C. Recher, L. Karlin, K. Yee,
K. Rezai, C. Preudhomme, T. Facon, P. Herait
5LBA
12:10 LATE BREAKING ABSTRACT: Phase 1 first-in-human study of the enhancer of zeste-homolog 2
(EZH2) histone methyl transferase inhibitor E7438 as a single agent in patients with advanced solid
tumors or B cell lymphoma
V. Ribrag, J.C. Soria, L. Reyderman, R. Chen, P. Salazar, N. Kumar, G. Kuznetsov, H. Keilhack,
L.H. Ottesen, A. Italiano
6LBA
T. Helleday (Sweden)
12:45–13:15
Moderator: E. Calvo (Spain)
Exhibition Hall
The following abstracts will be discussed: 41 (poster board P035), 195 (P189) and one to be announced.
Plenary Session 6
13:30–15:35
Chairs: E. Calvo (Spain) and J. Doroshow (USA)
Auditorium
Abstract number
13:30 ORAL PRESENTATION: A phase I dose-finding study of BI 853520, a potent and selective inhibitor
of focal adhesion kinase (FAK), in Japanese and Taiwanese patients with advanced or metastatic solid
tumors
T. Doi, C.C. Lin, A. Ohtsu, J.C.H. Yang, K. Shitara, L.C. Pronk, A. Sarashina, A.L. Cheng
213
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13:45 ORAL PRESENTATION: Homologous recombination deficiency (HRD) score and niraparib efficacy
in high grade ovarian cancer
P. Haluska, K.M. Timms, M. AlHilli, Y. Wang, A.M. Hartman, J. Jones, A. Gutin, Z. Sangale, C. Neff,
J. Lynchbury, L. Rudolph-Owen, M.A. Becker, S. Agarwal, K.M. Wilcoxen
214
14:00 ORAL PRESENTATION: Updated clinical and preliminary correlative results of ARIEL2, a Phase 2
study to identify ovarian cancer patients likely to respond to rucaparib
E. Swisher, J. Brenton, S. Kaufmann, A. Oza, R.L. Coleman, D. O’Malley, G. Konecny, L. Ma,
M. Harrell, D. Visscher, A.W. Hendrickson, K. Lin, M. Raponi, E. Mann, H. Giordano, L. Maloney,
L. Rolfe, I. McNeish
215
14:15 ORAL PRESENTATION: Phase I study of panobinostat and fractionated stereotactic re-irradiation
therapy (FSRT) for recurrent high grade gliomas
W. Shi, Y.R. Lawrence, M. Werner-Wasik, D.W. Andrews, J.J. Evans, J. Glass, L. Kim, V. Bar Ad,
Y. Moshel, K. Judy, C. Farrell, N.L. Simon, A.P. Dicker
216
14:30 ORAL PRESENTATION: Results of a phase I, open-label, multicentre study to assess the safety,
tolerability, pharmacokinetics and preliminary anti-tumour activity of AZD9150 in patients with
advanced/metastatic hepatocellular carcinoma
Y.K. Kang, B.Y. Ryoo, T.Y. Kim, K.H. Lee, H.Y. Lim, S.J. Lee, M. Ikeda, T. Okusaka, S. Nadano,
C.C. Lin, T.P. Poon, C.J. Yen, P. McCoon, F. Neumann, K. Vishwantahan, R. DuPont, P. Lyne
217
14:45 ORAL PRESENTATION: Genomic analysis identifies novel drivers and targetable pathways in
inflammatory breast cancer patient samples
D.M. Moran, K. Rao, P. Bacon-Trusk, K. Pry, V. Weigman, V. Velculescu, M. Cristofanilli, S. Bacus
218
15:00 ORAL PRESENTATION: Phase I trial evaluating the antiviral agent Cidofovir in combination with
chemoradiation in cervical cancer patients: A novel approach to treat HPV related malignancies?
E. Deutsch, A. Levy, R. Mazeron, A. Gazzah, E.A. Angevin, V. Ribrag, R. Balheda, A. Varga,
C. Lhomme, C. Haie-Meder, J.C. Soria
219
15:15 LATE BREAKING ABSTRACT: Phase 1 dose-expansion study of AMG 900, a pan-Aurora kinase
inhibitor, in adult patients with advanced taxane-resistant solid tumors
B. Markman, D. Mahadevan, S. Hurvitz, D. Kotasek, M. Shaheen, M. Carducci, O. Goodman,
X. Jiang, V. Chow, G. Juan, G. Friberg, E. Gamelin, J. Desai
7LBA
15:25 Discussion Late Breaking Abstract
J. Doroshow (USA)
Plenary Session 7
16:00–17:50
Novel Mechanisms for Drug Resistance
Chairs: U. Banerji (United Kingdom) and S.A. Courtneidge (USA)
Auditorium
Abstract number
16:00 Genomic instability, diversity and resistance during cancer evolution
N. McGranahan (United Kingdom)
Key messages:
1. Driver mutations are often subclonal in non-small cell lung cancer (NSCLC), potentially comprising
the efficacy of targeted therapy approaches.
2. A single NSCLC tumour may follow multiple distinct evolutionary trajectories simultaneously, with
mutational processes varying over space and time.
3. NSCLCs have a long period of tumour latency prior to clinical detection.
16:20 Immune-scape to PD1/PDL1 blockade
D. Chen (USA)
Key objectives:
1. Understand what responses and durability of responses looks like for cancer immunotherapy and
PD-L1/PD-1 inhibitors to date.
2. Understand potential reasons for primary resistance.
3. Understand potential reasons for secondary resistance.
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Messages:
1. Responses to PD-L1/PD-1 inhibitors appear highly durable, but not every patient responds and not
every responding patient is “cured”.
2. Primary resistance may be driven by lack of adequate active anti-cancer T cell immune responses in
tumours.
3. It is too early to tell what might account for secondary resistance, but it could involve factors that
lead to fluctuations in immunity and a break in the cancer-immunity cycle.
16:40 EMT as mechanism of resistance to TKI
J.P. Thiery (Singapore)
Key objectives:
1. Malignant cells harbor numerous genetic alterations at the time of clinical detection. The mutational
landscape is extraordinarily complex, exhibiting considerable heterogeneity within the primary
tumour, in circulating tumour cells and in metastases.
2. These findings are in support of the original hypothesis of clonal evolution, and suggest that the
current therapeutics strategies must be revisited to delay the onset of tumour refractoriness.
3. It is imperative to develop treatment strategies that do not strictly rely on specific activating mutations.
In particular, one can leverage on the Epithelial–Mesenchymal Transition (EMT) status of a tumour,
as it has an impact on the tumour’s potential to progress and resist treatment. This can be achieved
by establishing an EMT score for each tumour along the EMT spectrum. Targeted therapeutics can
then be used to move the tumour along the EMT spectrum rather than inhibiting its growth in order
to improve its response to conventional therapeutics and to restore the body’s immune response.
17:00 The landscape of kinase fusions in cancer
C. Lengauer (USA)
Key messages:
1. We have performed a pan-cancer analysis of kinase fusions across all TCGA RNA-seq data (>7,000
samples).
2. Our analysis unveiled several new and recurrent kinase fusions. Overall, kinase fusions are a driver
event in at least 3% of solid tumours.
3. These discoveries have profound and immediate implications for the diagnosis and treatment of cancer
patients, as well as potential avenues for new drug discovery programmes.
17:20 ORAL PRESENTATION: Overcoming drug-resistance in multiple myeloma by XPO1 inhibitor
combination therapy
J. Turner, J. Dawson, S. Grant, K. Shain, C. Cubitt, Y. Dai, L. Zhoui, M. Kauffman, S. Shacham,
D. Sullivan
220
17:35 LATE BREAKING ABSTRACT: Activity of the PARP inhibitor olaparib in ATM-deficient gastric
8LBA
cancer: from preclinical models to the clinic
D. Hodgson, H. Mason, L. Oplustilova, C. Harbron, X. Yin, S.A. Im, H. Jones, L. Zhongwu,
B. Dougherty, M. McLoughlin, A. Dickinson, A. Fielding, J. Robertson, W.H. Kim, C. Womack, Y. Gu,
Y.J. Bang, A. Lau, J.C. Barrett, M.J. O’Connor
17:45 Discussion Late Breaking Abstract
U. Banerji (United Kingdom)
18:00–19:30
Poster Viewing
Chemoprevention
Poster board
Poster area
Abstract number
P001 Docosahexaenoic acid along with modulation of actin binding proteins reduces cancer cell migration
M. Ali, L.K. Rogers
221
P002 Estimating predictive values of short-term morphologic assays of cancer chemoprevention for efficacy
in animal tumor assays
B. Dunn, V.E. Steele, R.M. Fagerstrom, C.F. Topp, D. Ransohoff, C. Cunningham, R. Lubet, L.G. Ford,
B.S. Kramer
222
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Poster board
Abstract number
P003 Preclinical assessment of nintedanib for chemoprevention in hepatocellular carcinoma
V. Tovar, A. Moeini, S. Torrecilla, M. Higuera, J. Peix, I.M. Quetglas, L. Rodriguez-Carunchio,
H. Cornella, M. Sole, J.M. Llovet
223
P004 Synergic tumor growth suppression with carbohydrate-restriction diet and natural AMP-dependent
protein kinase activators
M. Choi, J. Lee
224
P005 Molecular targets of interest to the NCI PREVENT cancer preclinical drug development program
R.H. Shoemaker, B.K. Dunn, C. Suen, R.A. Lubet, D.L. Boring, B.D. Klein, M.S. Miller, V.E. Steele
225
P006 Efficacy of cancer preventing drugs administered by intermittent dosing regimens
V.E. Steele, C. Grubbs, C.V. Rao, R.A. Lubet
226
Poster board
Abstract number
P007 Multiplexed ICE COLD-PCR: A mutation detection methodology for achieving sensitivities of
<0.01% using either Sanger or NGS
G. Wu, B. Legendre, S. Cherubin, C. Cubrich, A. Dowers, S. Jensen, J. Gniffke, A. Kruempel,
P. Krzycki, E. McCutchen, E. Montagne, S. Peterson, J. Pope, K. Scott, K. Richardson
227
P008 Her2−3 heterodimer is a new and better than HER2 IHC score for clinical outcome prognosis
G. Weitsman, P.R. Barber, K. Lawler, C. Gillett, N. Woodman, B. Kholodenko, L.K. Nguyen, T. Santra,
B. Vojnovic, T. Ng
228
P009 DNA methyltransferase 1 expression in human solid tumors and lymphomas by immunohistochemistry
S.X. Yang, D. Nguyen, L. Rubinstein, S. Kummar, J.E. Tomaszewski, J.H. Doroshow
229
P010 High correlation between clinical responses to first line AML patients treated with cytarabine and
idarubicin and their pharmacological profiles in patient samples measured by ExviTech
J. Ballesteros, P. Hernandez, D. Primo, A. Robles, A.B. Espinosa, E. Arroyo, V. Garcia-Navas,
J. Sanchez-Fenoy, M. Jimenez, M. Gaspar, J.L. Rojas, J. Martinez-Lopez, J. Gorrochategui
230
P011 Clinical pharmacodynamic assay development for the first in class investigational ubiquitin activating
enzyme (UAE) inhibitor MLN7243
B. Bahamón, F. Gao, B. Stringer, Y. Yang, J. Shi, K. Burke, J. Huck, T. Traore, D. Bowman,
H. Danaee, M. Millhollen, M. Hyer, N. Bence, Y. Ishii
231
P012 Development of a targeted NGS assay system for patient enrollment to the NCI-MATCH study
M. Williams, D. Sims, J. Lih, A. Datta, S. Hamilton, A.J. Iafrate, J. Sklar, S. Sadis, N. Takabe,
J. Tricoli, J. Doroshow, B. Conley
232
P013 Analytical validation and application of the MPACT assay, a next generation sequencing based
targeted mutation detection assay for treatment selection
C. Lih, D.J. Sims, R.D. Harrington, E.C. Polley, Y. Zhao, R.M. Simon, M.G. Mehaffey, T.D. Forbes,
W.D. Walsh, V. Datta, B.A. Conley, A.P. Chen, S. Kummar, J.H. Doroshow, P.M. Williams
233
P014 Kinetic analysis of dynamic 11 C-verapamil PET study: Compartmental v adaptive mixture models
comparison
F. Hernandez, D. Hawe, S. Murphy, J. O’Sullivan, E. Wolsztynski, J. Huang, M. Muzi, J. Eary,
234
P015 Kinetic analysis of dynamic 11 C thymidine PET imaging studies: Compartmental and nonparametric
approaches
S. Murphy, D. Hawe, F. Hernandez, E. Wolsztynski, J. Huang, J. O’Sullivan, M. Muzi, J. Eary,
235
P016 Whole genomic assay on endoscopic ultrasound-guided fine needle aspiration samples of unresectable
pancreatic cancer
J.M. Ha, K.H. Lee, J.K. Lee, K.T. Lee, W.Y. Park, J.S. Bae, J.K. Jung, D.H. Park, Y.K. Seong, E. Kim
236
Poster board
xlv
Abstract number
P017 Impact of phase 1 expansion cohorts on probability of success in phase 2 and time-to-drug-approval:
analysis of 385 new drugs in oncology
D. Bugano, K. Hess, L.L. Siu, F. Meric-Bernstam, A.R.A. Razak, D.S. Hong
DNA Repair Modulation (including PARP, CHK, ATR, ATM)
Poster board
237
Abstract number
P018 Reversal of primary and acquired PARP-inhibitor resistance in BRCA-mutated triple-negative breast
cancers by inhibition of transcriptional cyclin-dependent kinases (CDKs)
S. Johnson, N. Johnson, D. Chi, B. Primack, C. Cruz, D. Stover, A.K. Greifenberg, S. Cao,
K. O’Connor, J. Baselga, J. Balmaña, V. Serra, M. Geyer, A. D’Andrea, E. Lim, G.I. Shapiro
238
P019 Niraparib, a selective PARP 1/2 inhibitor, is efficacious in pre-clinical models of small-cell lung
cancer
Y. Wang, J. Ricono, K. Admunson, S. Agarwal, R.J. Fram, T. Broudy, K.M. Wilcoxen
239
P020 The combination of Chk-1 and ATR inhibitor synergistically kills cancer cells
K. Sanjiv, A. Hagenkort, P.M. Reaper, T. Koolmeister, O. Mortusewicz, N. Schultz, M. Scobie,
U.W. Berglund, P.A. Charlton, J.R. Pollard, T. Helleday
240
P021 Epigenetic loss-of-function BRCA1 mediates tumor cure by single dose radiotherapy
C. Campagne, T.H. Thin, J.D. Fuller, K. Manova-Todorova, A. Haimovitz-Friedman, S.N. Powell,
R.N. Kolesnick, Z. Fuks
241
P022 BMN 673 as single agent and in combination with temozolomide or PI3K pathway inhibitors in small
cell lung cancer and gastric cancer models
Y. Feng, L.E. Post, R. Cardnell, L.A. Byers, B. Wang, Y. Shen
242
P023 Modulation of PI3K/mTOR pathway following PARP inhibition in small cell lung cancer
R.J. Cardnell, Y. Feng, L. Diao, Y. Fan, F. Masrorpour, S. Mukherjee, J. Shen, J. Wang, L.A. Byers
243
P024 Development of xenoimplants from germline BRCA1/2 mutant breast cancer (BC) for the
identification of predictive biomarkers, mechanisms of resistance against poly(ADP-ribose)
polymerase (PARP) inhibitors and evaluation of novel therapies
C. Cruz, Y. Ibrahim, B. Morancho, P. Anton, J. Grueso, P. Cozar, M. GuzmÁn, P.M. Avilés,
M.J. Guillén, C. Galmarini, J. Arribas, J. Baselga, J. Balmaña, V. Serra
244
P025 Chk1 is a potential novel therapeutic target that regulates cell survival and potentiates chemotherapy
in osteosarcoma (OS) models
S.J. Strauss, P. Mistry, A. Mendoza, M. Robson, H. Holme, P. Nandabhiwat, B. Kwok, M. Qadir,
R.B. Pedley, J.S. Whelan, P.H.B. Sorensen
245
P026 PARP inhibitors trap PARP1 onto damaged DNA via catalytic inhibition and not by an allosteric
mechanism
T. Hopkins, L. Solomon, Y. Shi, L. Rodriguez, C. Donawho, E. DiGiammarino, S. Panchal, A. Olson,
D. Stolarik, D. Osterling, W. Gao, E. Johnson, D. Maag
246
P027 Selective inhibitors of nuclear export (SINE) block the expression of DNA damage repair proteins
and sensitize cancer cells to DNA damage therapeutic agents
T. Kashyap, M. Crochiere, S. Friedlander, B. Klebanov, W. Senapedis, E. Baloglu, D. del Alamo,
S. Tamir, T. Rashal, D. McCauley, R. Carlson, M. Kauffman, S. Shacham, Y. Landesman
247
P028 Use of ATR inhibitor in combination with topoisomerase I inhibitor kills cancer cells by disabling
DNA replication initiation and fork elongation
R. Jossé, S.E. Martin, R. Guha, P. Ormanoglu, T. Pfister, J. Morris, J. Doroshow, Y. Pommier
248
P029 Preclinical efficacy of the PARP inhibitor rucaparib (CO-338/AG014699/PF-01367338) in pancreatic
cancer models with homologous recombination deficiencies (HRD)
L. Robillard, K. Lin, P.P. Lopez-Casas, M. Hidalgo, T.C. Harding
249
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Poster board
Abstract number
P030 Comprehensive genomic profiling of pancreatic acinar cell carcinomas identifies recurrent RAF
fusions and frequent inactivation of DNA repair genes
J. Chmielecki, K.E. Hutchinson, G.M. Frampton, Z.R. Chalmers, A. Johnson, C. Shi, J. Elvin,
S.M. Ali, J.S. Ross, O. Basturk, S. Balsubramanian, D. Lipson, R. Yelensky, W. Pao, V.A. Miller,
D.S. Klimstra, P.J. Stephens
250
P031 A combined in vitro and mathematical modelling approach for understanding the impact of an
inhibitor of ATR on DNA damage and repair after ionising radiation
J. Yates, S. Checkley, L. MacCallum, R. Odedra, J. Barnes, A. Lau
251
P032 The DNA damage response gene Schlafen 11 (SLFN11) is a transcriptional target of ETS
transcription factors in Ewing’s sarcoma and other cancers
Y. Pommier, S.W. Bilke, F. Sousa, M. Yamade, J. Murai, V. Rajapakse, L. Helman, P. Meltzer
252
P033 Phase 1 correlative study of ARQ761, a b-lapachone analogue that promotes NQ01-mediated
programmed cancer cell necrosis
D. Gerber, Y. Arriaga, M.S. Beg, J.E. Dowell, J.H. Schiller, A.E. Frankel, R. Leff, C. Meek, J. Bolluyt,
O. Fatunde, R.T. Martinez, P. Vo, F. Fattah, V. Sarode, Y. Zhou, Y. Xie, M. McLeod, B. Schwartz,
D.A. Boothman
253
P034 Molecular analysis in breast cancer: correlation with immunohistochemical classification and
pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC)
S. Baulies, M. Gonzalez-Cao, N. Karachaliou, A. Rodriguez Capitan, M.A. Molina-Vila, M.T. Cusido,
C. Teixido, S. Viteri, R. Fabregas, X. Gonzalez, R. Rosell
254
Drug Delivery
Poster board
Abstract number
P035 Pre-clinical in vivo characterization of MLN7243, an investigational ubiquitin activating enzyme
inhibitor, in solid tumor models
T. Traore, J.H. Huck, J.S. Shi, D.S. Sappal, J.D. Duffey, Y.Y. Yang, E.K. Kadakia, A.C. Chakravarty,
B.S. Stringer, Y.I. Ishii, R.G. Griffin, C.X. Xia, M.M. Milhollen, J.C. Ciavarri, P.F. Fleming,
N.B. Bence, M.H. Hyer
255
P036 Minicell packaged targeted delivery of shRNA to cancer cells
M. Jivrajani, N. Shrivastava, M. Nivsarkar
256
P037 Development of peptide-mediated drug delivery systems for colon cancer targeted imaging and
therapy
H.C. Wu, C.H. Wu, Y.H. Kuo
257
P038 Pancreatic cancer cells expressing the OATP1B3 transporter show promising sensitivity to the highly
cytotoxic microcystin-LR molecule
V. Kounnis, G. Chondrogiannis, M.D. Mantzaris, D. Fokas, N.A. Papanikolaou, I. Sainis, E. Briasoulis
258
P039 Factors that limit delivery of Cdk4/6 inhibitor palbociclib to GBM
K. Parrish, J. Pokorny, R. Mittapalli, K. Bakken, J. Sarkaria, W. Elmquist
259
P040 Hsp90 Inhibitor Drug Conjugates (HDC): Payloads and possibilities
D. Chimmanamada, W. Ying, J. Zhang, D. Proia, T. Przewloka, J. Jiang, D. Vutukuri, G. Lu,
S. Osman, S. Chen, J. Chu, P. Rao, D. Zhou, T. Inoue, L. Shin Ogawa, R. Singh, N. Tatsuta,
A. Sonderfan, C. Cortis
260
P041 Lesion characterization with ferumoxytol MRI in patients with advanced solid tumors and correlation
with treatment response to MM-398, nanoliposomal irinotecan (nal-IRI)
R.K. Ramanathan, R.L. Korn, J.C. Sachdev, G.J. Fetterly, G. Jameson, K. Marceau, V. Marsh,
N. Raghunand, J. Prey, S.G. Klinz, J. Kim, E. Bayever, J.B. Fitzgerald
261
P042 Improved cytotoxic activity of Nor-b-lapachone-loaded PLGA microcapsules in PC3M prostate cancer
cell line
C. Pessoa, A.C.S. Feitosa, M.P. Costa, F.C. Evangelista, F.A.M. Sales, I.S. Bomfim, E.N. Silva Júnior,
G.G. Dias, V.N. Freire, W.S. Caetano
262
Poster board
xlvii
Abstract number
P043 Controlled release of cisplatin using hyaluronic oligosaccharides-coated gold nanoparticles as an
efficient delivery system applied to the treatment of pancreatic tumors
H. Parkkola, L. Sobrevals Amieva, L. Vivero, R. Miñana, J. Sendra
P044 Vasculogenic mimicry in small cell lung cancer
F. Trapani, R.L. Metcalf, R. Polanski, A. Fusi, C. Hodgkinson, D. Nonaka, M.J. Hendrix, C. Morrrow,
F. Blackhall, K.L. Simpson, C. Dive
P045 Pretargeted nanoparticles to deliver both chemotherapeutics and radiation for the treatment of
lymphoma
C. Fang, J.C. Jones, S.M. Frayo, M.H. Hylarides, M. Zhang, O.W. Press
P046 Auxiliar treatment by targeting the extracellular matrix to improve drug delivery and therapeutic
response
E. Henke, F. Roehrig, H. Hoffmann, F. Escorcia, M. Wartenberg, S. Volova, S. Gaetzner,
A. Rosenwald, S. Erguen, D.A. Scheinberg, Z.V.I. Granot
P047 Novel adjuvant therapy with leptin peptide receptor antagonist-2 conjugated to nanoparticles (IONPLPrA2) to minimize chemoresistance in triple negative breast cancer
T. Harmon, A. Harbuzariu, L. Yang, R.R. Gonzalez-Perez
Drug Design
Poster board
263
264
265
266
267
Abstract number
P048 A potent and highly efficacious bivalent Smac Mimetic APG-1387 in Phase I clinical development
J. Lu, S. Rong, H. Sun, L. Liu, D. McEachern, G. Wang, J. Wen, Y. Zhai, M. Guo, D. Yang, S. Wang
P049 The NCI-60 as an effective tool for scaffold hopping: A phenotypic systems-based approach to the
design of novel chemotherapeutics
D.G. Wishka, V. Kumar, B. Teicher, G. Kaur, B. Fang, P. Risbood, M. Hollingshead, J. Zais, J. Morris
P050 Discovery, development and optimization of low molecular weight EPH−ephrin protein–protein
inhibitors
M. Tognolini, C. Giorgio, I. Hassan-Mohamed, E. Barocelli, M. Mor, D. Pala, S. Russo, M. Incerti,
A. Lodola
P051 Data integration and graph analysis for cancer genomics and drug discovery
B. Bernard, M. Miller, H. Rovira, I. Shmulevich
P052 8-(1-Anilino)ethyl)-2-morpholino-4-oxo-4H-chromene-6-carboxamides as PI3Kbeta/delta inhibitors:
structure–activity relationships and identification of AZD8186, a clinical candidate for the treatment
of PTEN deficient tumours
B. Barlaam, S. Cosulich, S. Degorce, M. Fitzek, S. Green, U. Hancox, C. Lambert-van der Brempt,
J.J. Lohmann, M. Maudet, R. Morgentin, M.J. Pasquest, A. Peru, P. Ple, T. Saleh, M. Vautier,
M. Walker, L. Ward, N. Warin
P053 Significance of serine-167 and cysteine-129 residues in the active site of the immune-suppressive
enzyme indoleamine 2,3-dioxygenase 1 (IDO1) for the binding of novel inhibitors
P. Tomek, B.D. Palmer, J.U. Flanagan, L. Ching
P054 Novel cysteine derivatives for the next generation anticancer agents acting on KSP
N. Ogo, J. Sawada, Y. Ishikawa, K. Matsuno, A. Hashimoto, A. Asai
P055 Modulation of PIP2 levels through small molecule inhibition of PIP5K
D. Andrews, S. Cosulich, N. Divecha, D. Fitzgerald, V. Flemington, C. Jones, D. Jones, O. Kern,
E. MacDonald, S. Maman, J. McKelvie, K. Pike, M. Riddick, G. Robb, K. Roberts, J. Smith,
M. Swarbrick, I. Treinies, M. Waring, R. Wood
P056 An X-ray crystal structure-based understanding of the inhibition of the MDM2−p53 protein–protein
interaction by isoindolinones
B. Anil, E. Blackburn, T. Blackburn, S. Cully, J. Liu, C.J. Drummond, J.A. Endicott, B.T. Golding,
R.J. Griffin, K. Haggerty, J. Lunec, D.R. Newell, C.H. Revill, C. Riedinger, A.F. Watson, Q. Xu,
Y. Zhao, I.R. Hardcastle, M.E.M. Noble
268
269
270
271
272
273
274
275
276
xlviii
Poster board
Abstract number
P057 The discovery and pre-clinical development of the first clinical stage EZH2-inhibitor,
EPZ-6438 (E7438)
K. Kuntz, H. Keilhack, R. Pollock, S. Knutson, N. Warholic, V. Richon, R. Chesworth, R. Copeland,
M. Porter-Scott, C. Sneeringer, T. Wigle
P058 Sentinel lymph nodes mapping of macrophage targeted mannosyl human serum albumin-indocyanine
detected by combined color and near infrared fluorescence imaging system in esophagus
Y. Quan, Y. Oh, J.I.H.O. Park, J. Park, J. Jeong, B. Kim, H. Kim
P059 Discovery of multiple kinases inhibitors, DBPR114, as the novel anti-cancer agent
H.P. Hsieh, C.C. Kuo, J.J. Chiu, T.A. Hsu, T.K. Yeh, C.T. Chen
P060 A hybrid drug design approach to overcome imatinib resistance for treating leukemia
Y.M. Wei, K.K.W. To, S.C.F. Au-Yeung
P061 Novel hybrid drug design strategy to circumvent erlotinib resistance and to optimize its
pharmacokinetic properties for treating lung cancer
D.C.S. Poon, S.C.F. Au-Yeung, K.K.W. To
P062 Membrane anchorage of Stat3 via artificial protein lipidation
M. Avadisian, S. Fletcher, B. Liu, W. Zhao, J. Turkson, C. Gradinaru, P. Gunning
P063 The development of the first selective inhibitors of the UBA5 enzyme to probe for E1 activity in
diseased cells
S.R. da Silva, S.L. Paiva, M. Bancerz, M. Geletu, A.M. Lewis, J. Chen, Y. Cai, H. Li, P.T. Gunning
P064 Poly(ADP-ribose) glycohydrolase (PARG) inhibitors increase nuclear poly(ADP-ribose) after
methylating DNA damage
A. Jordan, B. Acton, E. Fairweather, N. Hamilton, S. Holt, J. Hitchin, C. Hutton, D. James, S. Jones,
A. McGonagle, H. Small, K. Smith, A. Stowell, I. Waddell, B. Waszkowycz, D. Ogilvie
P065 A nanomolar-potency small molecule inhibitor of the STAT5 protein
A.A. Cumaraswamy, A. Lewis, M. Geletu, A. Todic, D.B. Diaz, X.R. Cheng, C.E. Brown, R. Laister,
D. Muench, K. Kerman, H.L. Grimes, M.D. Minden, P.T. Gunning
P066 Potent and selective non-sulfamate-containing small molecule inhibitors of the ubiquitin activating
enzyme
S. Paiva, S.R. da Silva, M. Bancerz, H. Quereshi, G.W. Xu, A.D. Schimmer, P.T. Gunning
P067 The discovery and optimization of small molecule antagonists of the WDR5−MLL interaction
R. Al-Awar, R.S. Al-Awar, M. Getlik, D. Smil, Y. Bolshan, G. Poda, G. Senisterra, H. Wu, A. AllaliHassani, G.A. Wasney, D. Barsyte-Lovejoy, L. Dombrovski, A. Dong, H. He, A. Seitova, I. Chau, F. Li,
J.F. Couture, E. Kuznetsova, R. Marcellus
P068 Synthetic isomalyngamide A analogs that inhibit breast cancer migration
W. Li, T. Chang, C. Hung, C. Chen, S. Jao
P069 Progress in drugging CYP1A1, 1B1 and CYP2W1 overexpressed in cancer
K. Pors, V. Le Morvan, S. Travica, S.D. Shnyder, M. Sutherland, H.M. Sheldrake, M. Searcey,
I. Johansson, S. Mkrtchian, P.M. Loadman, J. Robert, M. Ingelman-Sundberg, L.H. Patterson
P070 Exosome analysis in cancer patients: From the preclinical towards the clinical application:
Trial design
I. Mertens, M. Castiglia, A.P. Carreca, G. Baggertman, M. Peeters, P. Pauwels, C. Rolfo
Poster board
277
278
279
280
281
282
283
284
285
286
287
288
289
290
Abstract number
P071 Aflibercept has anti-tumor activity in bevacizumab-escaping tumors of colorectal cancer: Molecular
profiles and mechanisms
C. Dib, R.G. Bagley, P. Mankoo, J. Pollard, J. Watters, M. Chiron
P072 Salmonella typhimurium A1-R decoys quiescent cancer cells to cycle rendering them chemosensitive
S. Yano, Y. Zhang, M. Zhao, Y. Hiroshima, S. Miwa, F. Uehara, H. Kishimoto, H. Tazawa,
T. Fujiwara, R.M. Hoffman
291
292
Poster board
xlix
Abstract number
P073 Inhibition of the cell cycle regulated Cdc7 kinase pathway is an efficacious therapeutic approach for
hematologic malignancies and solid tumors
R. Santos, D. Shum, D. Carrillo, R. Zhang, M. Churchill, S. Mukherjee, R. Brentjens, C. Radu,
T.J. Kelly, H. Djaballah, M.G. Frattini
P074 Synthetic lethal screen identifies Aurora A as a selective target in HPV driven cervical cancer
B. Gabrielli, F. Bokhari, M. Ranall, A. Stevenson, M. Murell, M. Kelly, S. McKee, G. Leggatt,
T. Gonda, N. McMillan
P075 Combining forces: Study of the cytotoxic effect of the MDM2 inhibitor Nutlin-3 in combination with
CDDP in non-small cell lung cancer cell lines
C. Deben, C. Rolfo, V. Deschoolmeester, A. Wouters, M. Peeters, I. Gil-Bazo, F. Lardon, P. Pauwels
P076 A first-in-Asian phase I dose escalation study to evaluate the safety and pharmacokinetics of VS-6063
(defactinib), a focal adhesion kinase inhibitor in subjects with non-hematologic malignancies
T. Shimizu, H. Aida, J. Horobin, M. Keegan, M. Padval, A. Poli, C. Hashii, K. Nakagawa
P077 Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC
J. Chen, K. Amato, S. Wang, V. Youngblood, D. Brantley-Sieders, R. Cook, L. Tan, N. Gray
P078 Met degradation by SAIT301, a Met monoclonal antibody, reduces the invasion and migration of
nasopharyngeal cancer cells via inhibition of EGR-1 expression
C. Kim, B.S. Lee, K.A. Kim, Y.J. Song, K.H. Cheong
P079 Tyk2-src dependence of kidney cancer
B. Krishnan, S.C. Hanna, H.L. Wilson, S.T. Bailey, J.S. Damrauer, T. Simamura, R.L. Levine,
K.K. Wong, G.L. Johnson, W.Y. Kim
293
294
295
296
297
298
299
P080 Preclinical pharmacologic characterization of GSK2849330, a monoclonal AccretaMab® antibody
with optimized ADCC and CDC activity directed against HER3
N. Clarke, C. Hopson, A. Hahn, K. Sully, F. Germaschewski, J. Yates, C. Akinseye, B. Mangatt,
Z. Jonak, C. Matheny
P081 Using PK/PD/efficacy modeling to predict potential of AZD9291 to target brain metastases from
advanced NSCLC with EGFR sensitizing mutations (EGFRm+)
J. Yates, P. Ballard, S. Ashton, D. Cross, R. Dattani, M. Mellor, J. Wilson, P. Yang, L. Xie
P082 FAK inhibitor VS-6063 (defactinib) targets mesothelioma cancer stem cells which are enriched by
standard of care chemotherapy
J. Pachter, V.N. Kolev, P. Baas, D.T. Weaver, Q. Xu
P083 Genomic and epigenomic analysis identify potential therapeutic targets in luminal B breast cancer
molecular subtype
L. Addou Klouche, A. Belhadj, A. Bendaoud, M. Benyelles, I. Ziane, M. Chaffanet, D. Birnbaum
300
P084 PI3 -Kinase inhibition forestalls the development of drug resistance in BRAFV600E/PTENNull
melanoma
M. Deuker, V. Marsh Durban, W. Phillips, M. McMahon
P085 Selinexor, a novel selective inhibitor of nuclear export, potentiates the antitumor activity of
gemcitabine against pancreatic cancer by nuclear retention of p27
M. Amit, S. Kazim, H. Kazim, S. Shacham, M. Kauffman, M.P. Malafa
P086 CC-115 inhibits DNA damage and repair pathways in vitro
T. Tsuji, L. Sapinoso, S. Sankar, D.S. Mortensen, S. Xu
P087 Stromal delivery of long Pentraxin-3 impairs FGF/FGFR-dependent tumor growth and metastasis
A. Giacomini, E. Di Salle, D. Coltrini, S. Rezzola, M. Belleri, M. Presta, R. Ronca
P088 Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen
receptor dependent modulation of the apoptotic pathway
S. Gameiro, A. Ardiani, A.R. Kwilas, R.N. Donahue, J.W. Hodge
P089 RNA sequencing and in silico analysis identifies an unannotated antisense long non-coding RNA
involved in cancer progression
S. Inoue, K. Horie-Inoue, K. Ikeda
304
301
302
303
305
306
307
308
309
l
Poster board
Abstract number
P090 Inhibition of Trk-driven tumors by the pan-Trk inhibitor RXDX-101
D. Anderson, M. Ciomei, P. Banfi, S. Cribioli, E. Ardini, A. Galvani, G. Li
310
P091 IGF-1R inhibition induced activation of Yes/SFK acts as a by-pass resistance pathway in
rhabdomyosarcoma
X. Wan, C. Yeung, C. Heske, A. Mendoza, L.J. Hlman
311
P092 The prognostic role of mesothelin expression and its association with KRAS mutation in advanced
lung adenocarcinoma
A. Thomas, Y. Chen, S. Steinberg, J. Luo, G. Giaccone, I. Pastan, M. Miettinen, R. Hassan
312
P093 Development of a one-step isolation platform for exosomal RNA and circulating cell-free DNA from
cancer plasma samples
D. Enderle, K. Brinkmann, T. Koestler, S. Bentink, C. Berking, J. Skog, M. Noerholm
313
P094 Optimal sequencing schedules for combining BRAF inhibition with BCL-2 inhibition
A. Schalck, D.T. Frederick, M.R. Hammond, I. Ferreiro-Neira, Z.A. Cooper, J.C. Cusack,
D.P. Lawrence, K.T. Flaherty, J.A. Wargo, R.J. Sullivan
314
P095 Biological characterization of TAS3681, a new type of androgen receptor antagonist with androgen
receptor downregulating activity
K. Minamiguchi, M. Seki, H. Aoyagi, T. Mori, D. Kajiwara, N. Masuko, R. Fujita, S. Okajima,
Y. Hayashi, E. Sasaki, T. Utsugi
315
P096 Potent, partial agonists at ERalpha as selective estrogen mimics for treatment of tamoxifen-resistant
breast cancer
G. Thatcher, R. Xiong, H.K. Patel, J. Zhao, X. Liang, Y. Wang, M.E. Molloy, D. Tonetti
316
P097 IKKb inhibition suppresses sphere formation and self-renewal of lung cancer initiating cells
T. Carneiro-Lobo, A.C.P. Salviatto, A.S. Baldwin, D.S. Basseres
317
P098 Novel therapeutic targets for ARID1A mutant ovarian clear cell carcinoma (OCCC)
R.E. Miller, I. Bajrami, R. Brough, A. Konde, J. Campbell, R. Rafiq, A. Ashworth, C.J. Lord
318
P099 Genomic profiling of uterine leiomyosarcomas reveal frequent alterations in Akt/mammalian target
of rapamycin (mTOR) pathway genes and other actionable genomic abnormalities linked to targeted
therapies
J.A. Elvin, Z.R. Chalmers, J. Chiemlicki, K.A.I. Wang, N. Palma, S.M. Ali, A. Huho, C.E. Sheehan,
V.A. Miller, P.J. Stephens, J.S. Ross
319
P100 First-in-human study with ARQ 092, a novel pan AKT-inhibitor, in subjects with advanced solid
tumors or recurrent malignant lymphoma
M. Saleh, K. Papadpoulos, A. Arabnia, A. Patnaik, R.M. Stein, F. Chai, M. Lamar, R. Savage,
G. Abbadessa, A. Tolcher
320
P101 Potent and selective inhibition of EZH2 by AU-2121 leads to significant tumor growth inhibition in
mutant EZH2 dependent non-Hodgkin lymphoma
S. Ahmed, K. Narayanan, A. Gadakh, S. Dodheri, S. Surendranath, S. Nathan, S. Mukherjee,
S. Marappan, R. Sushmita, S. Dhadunia, K.M. Obilisetti, J. Anil, J. Reddy, K. Reddy, A. Basavaraju,
C. Naik, C. Pandit, M. Ramachandra
321
P102 Novel alternative splice variants as potential biomarkers and therapeutic targets in aggressive prostate
cancer in men of African descent
S. Patierno, J. Freedman, B. Wang, N. Lee, D. George
322
P103 The development of short form of mimic microRNA for lung cancer therapy
S. Ohno, K. Sudo, M. Kuroda
323
P104 Clinical pharmacokinetics (PK), translational PK/pharmacodynamics (PD), and exposure–
adverse events (AEs) relationship of MLN2480, an oral investigational pan-RAF kinase inhibitor
X. Zhou, A.J. Olszanski, M. Middleton, R. Gonzalez, M.J. Bargfrede, M. Kneissl, V. Bozón,
E. Gangolli, K. Venkatakrishnan, M. Patel, C. Zopf, D.W. Rasco
324
Poster board
li
Abstract number
P105 Identification and characterization of an irreversible inhibitor of CDK2
M. Martin, E. Anscombe, E. Meschini, D. Staunton, M. Geitmann, U.H. Danielson, L.Z. Wang,
R. Mora Vidal, T. Reuillon, B.T. Golding, D.R. Newell, S. Wedge, M.E.M. Noble, J.A. Endicott,
R.J. Griffin
325
P106 A novel non-ligand competing anti-EGFR antibody for cancer treatment
X. Wang, S. Rust, F. Comer, V. Muniz-Medina, Q. Du, A. Yuan, K. Senthil, R. Fleming, R. Minter,
S. Coats
326
P107 Combination of MDM2 antagonists with RAS pathway inhibitors in colorectal cancer
B. Higgins, C. Tovar, K. Kolinsky, Y. Zhang, S.A. Middleton, G. Nichols, K. Packman, F. Su,
L. Vassilev
327
P108 Jagged1 expression and its relevance in metastatic progression of breast cancers
N. Bednarz-Knoll, A. Efstathiou, F. Gotzhein, H. Wilkman, V. Mueller, Y. Kang, K. Pantel
328
P109 A first-in-human study evaluating the safety and pharmacology of MM-151, a novel oligoclonal antiEGFR antibody combination in patients with refractory solid tumors
M. Beeram, C. Lieu, W. Harb, L. Power, J. Kearns, R. Nering, V. Moyo, B. Wolf, A. Adjei
329
P110 Preclinical development of BIS-1602, first in class growth hormone releasing hormone antagonist
Y.Y. Maxuitenko, N.L. Block, A.V. Schally, S.J. Reich, P. Goldstein
330
P111 Biomarker discovery and validation for assessing the response to cMet inhibition and functional
inactivation
S. Jung, S. Selzer, C. Lößner, K. Kuhn, U. Fiedler, A. Klotzbücher, H.D. Zucht, S. Koncarevic,
T. Prinz, D. Hertfelder, K.A. Boehme, H. Volkmer, M.H.G. Kubbutat, P. Budde, I. Pike
331
P112 Dual PI3K delta/gamma inhibition by RP6530 accentuates bortezomib activity in multiple myeloma
cell lines
S. Viswanadha, G. Babu, S. Veeraraghavan, S. Vakkalanka
332
P113 Short antisense oligonucleotides antagonize Lin28 and enable pre-let-7 processing and suppression of
cell growth in human hepatocarcinoma cells
M. Roos, G. Civenni, M. Lucic, D. Pavlicek, U. Pradere, H. Towbin, C.V. Catapano, J. Hall
333
P114 Aplidin: first in class compound targeting EEF1A in tumor cells
A. Losada, J.F. Martinez, P. Moral, L. Carrasco, F. Gago, C. Cuevas, L.F. Garcı́a-Fernández,
C.M. Galmarini
334
P115 RICTOR amplification as a novel molecular target for the treatment of lung cancer
H. Cheng, B. Piperdi, Y. Zou, A. Verma, X. Liu, E. Schwartz, C. Zhu, C. Montagna, B. Halmos,
A. Chachoua, R. Perez-Soler
335
P116 The effect of food on the pharmacokinetics of the investigational Aurora A kinase (AAK) inhibitor,
alisertib (MLN8237), in patients (pts) with advanced solid tumors or lymphomas
X. Zhou, T.M. Bauer, S. Goel, J. Sarantopoulos, B. Zhang, V. Kelly, J. Mertz, K. Venkatakrishnan
336
P117 Sensitisation of HPV+ HNSCC to cytotoxic treatments by targeting the G2/M checkpoint with AZ1775 to improve survival
A. Osman, N. Tanaka, A. Patel, J. Wang, A. Fitzgerald, T. Xie, M. Zhao, S. Jasser, M. Gadhikar,
H. Skinner, M. Frederick, F. Johnson, J.N. Myers
337
P118 BM-1252 (APG-1252): a potent dual specific Bcl-2/Bcl-xL inhibitor that achieves complete tumor
regression with minimal platelet toxicity
L. Bai, J. Chen, L. Liu, D. McEachern, A. Aguilar, H. Zhou, C.Y. Yang, H. Wang, J. Wen, G. Wang,
Y. Zhai, M. Guo, D. Yang, S. Wang
338
P119 NAMPT inhibition as strategy to impair tumor growth
A. Cerezo, S. Jiménez, E. Lospitao, N. Bravo, R. Campos-Olivas, C. Aguilera, M. Cañamero,
R. Gilmour, S. Geeganage, G. Zhao, S. Velasco-Miguel
339
lii
Poster board
Abstract number
P120 Preclinical activity of the VEGFR, FGFR and PDGFR inhibitor lucitanib in FGFR2 aberrant
endometrial and gastric cancer models
F. Guffanti, R. Chilà, E. Bello, L. Ceriani, M. Zangarini, M. Zucchetti, C. Saba, A. Jacquet-Bescond,
M. Pierrat, G. Damia
340
P121 Dinaciclib alters cell cycle dynamics and induces cell death in Soft Tissue Sarcomas
N. Mulet Margalef, S. Rello-Varona, S. Garcia-Monclús, M. Fuentes-Guirado, O.M. Tirado, X. Garcia
del Muro
341
P122 A phase 1/2 study evaluating the safety, pharmacokinetics and efficacy of ABT-414 in subjects with
advanced solid tumors likely to over-express the epidermal growth factor receptor (EGFR)
A. Tolcher, G. Goss, M. Gordon, L. Gandhi, K.P. Papadopoulos, D. Rasco, M. Pedersen, J. Fischer,
W. Ames, H. Xiong, M. Dudley, W. Munasinghe, P. Ansell, K. Holen, E. Vokes
342
P123 Salmonella typhimurium A1-R effectively targets experimental breast cancer brain metastasis
Y. Zhang, S. Miwa, N. Zhang, R.M. Hoffman, M. Zhao
343
P124 Search for new blood biomarkers for response to antiangiogenic therapy in non-small cell lung cancer
patients
A. Rodrı́guez-Garzotto, M.T. Agulló-Ortuño, S. Ponce, C.V. Dı́az-Garcı́a, A. Agudo-López, C. Pérez,
E. Prieto-Garcı́a, H. Cortés-Funes, L. Iglesias
344
P125 Interaction of plitidepsin with eEF1A in living tumor cells
C. Garcı́a, A. Losada, J.M. Molina-Guijarro, M.A. Sacristán, J. Martinez, C.M. Galmarini, M.P. Lillo
345
P126 Inhibition of Wnt pathway by novel thiazole-based Traf2- and Nck-interacting kinase (TNIK)
inhibitor
Y. Uno, H. Moriyama, S. Kashimoto, M. Masuda, M. Sawa, T. Yamada
346
P127 Can a link between the EGFR and cMET pathway in non-small cell lung cancer explain resistance
against targeted therapies and open new therapeutic opportunities?
N. Van Der Steen, K. Van Der Steen, K. Zwaenepoel, E. Giovannetti, M. Castiglia, A.P. Carreca,
P. Pauwels, C. Rolfo
347
P128 Two NSCLC-PDXs with different EGFR exon 20-insertions respond differently to different TKIs
H. Li, M. Yang, J. Cai
348
P129 Synergistic drug combinations that target beta-catenin-driven and MYC-driven cancers
J.C.M. Uitdehaag, J.A.P. Spijkers-Hagelstein, J.A.D.M. de Roos, A.M. van Doornmalen,
M.B.W. Prinsen, J. de Man, R.C. Buijsman, G.J.R. Zaman
349
P130 Oncogenic Ras mutants differentially utilize PLC-dependent calcium flux and PKC activation for
MAPK signaling
C. Pitt, F. McCormick
350
P131 hz515H7, a humanized antibody exerts its antitumor activity via antagonism of the CXCR4/SDF-1
axis, and through effector functions
C. Klinguer-Hamour, M. Broussas, B. Akla, S. Berger, N. Boute, C. Beau-Larvor, A. Robert,
J.F. Haeuw, L. Goetsch, C. Bailly, N. Corvaia
351
P132 UNC2025, a novel small molecule MerTK and Flt3 tyrosine kinase inhibitor, has therapeutic activity
and promotes sensitivity to chemotherapy in animal models of acute leukemia
D. Graham, D. DeRyckere, X. Wang, A.A. Hill, W. Zhang, S.V. Frye, H.S. Earp
352
P133 Protein expression for receptor activator of NFkB (RANK) and its ligand (RANKL) in non-small cell
lung cancer (NSCLC)
M. D’Arcangelo, S. Ekman, W. Dougall, D. Branstetter, M. Bergqvist, P. Liv, D. Chan, J. Botling,
F. Hirsch
353
P134 PCR-based assay for BRAFV600 mutation analysis in ctDNA: clinical results from plasma and serum
samples
M. Gonzalez-Cao, C. Mayo de las Casas, M.A. Molina-Vila, L. De Mattos-Arruda, J.L. Manzano,
E. Muñoz, J. Cortes, J.P. Berros, M. Sanmamed, A. Gonzalez, C. Alvarez, N. Karachaliou, N. JordanaAriza, S. Martin Algarra, R. Rosell
354
Poster board
liii
Abstract number
P135 Altiratinib (DCC-2701): a balanced inhibitor of MET, TIE2, and VEGFR2 kinases that exhibits broad
anti-tumor and anti-angiogenic activities
D. Flynn, B.D. Smith, C.B. Leary, M.D. Kaufman, B.A. Turner, M.M. Hood, W.P. Lu, T.J. Rutkoski,
T. Samarakoon, S. Vogeti, S.C. Wise, O. Rosen
355
P136 Discovery of novel pyrido[2,3-b]pyrazine as fibroblast growth factor receptor (FGFR-1, 2, 3 & 4)
kinase inhibitors with nanomolar affinity
P. Angibaud, O. Querolle, V. Berdini, G. Saxty, A. Cleasby, H. Colombel, I. Csoka, R. Gilissen,
P. King, L. Meerpoel, C. Paulussen, I. Pilatte, V. Poncelet, D.C. Rees, B. Roux, V. Tronel, T. Verhulst,
B. Wroblowski, C.W. Murray, J. Vialard
356
P137 Multi-color flow cytometry immunophenotyping for detection of CSC in NSCLC
A. Martinez-Romero, J.M. Pardo, S. Tejedor, S. Calabuig Fariñas, R. Lucas, S. Figueroa, E. JantusLewintre, C. Camps, R. Farras
357
P138 Knockdown of beta-catenin with dicer-substrate siRNAs down-regulates Wnt/beta-catenin pathway
signaling
N. Pursell, W. Zhou, R. Diwanji, B. Holmes, N. Avitahl-Curtis, C. Dutta, H. Dudek, S. Ganesh,
M. Abrams, W. Wang, B. Ying, D. Chen, S. Shui, U. Saxena, H. Yang, A. Shah, R. Arvan, M. Koser,
C. Lai, B.D. Brown
358
P139 Correlation of tumour-associated macrophage, but not tumour-infiltrating lymphocyte, levels with
progression-free survival in patients with metastatic renal cell carcinoma treated with axitinib
J.A. Williams, J.F. Martini, B. Escudier, B.I. Rini, R.J. Motzer, J. Tarazi, S. Li, P.A. English
359
P140 Tumor suppressive microRNA-192 as a prognostic factor for recurrence of surgically resected nonsmall cell lung cancer
J.H. Hong, K.S. Roh, E.K. Jeon, J.H. Kang, S.S. Suh, S.C. Lee
360
P141 Nanoformulations of the PARP inhibitors olaparib and BMN 673 for cancer nanotherapy
S. Sridhar, S. Tangutoori, P. Baldwin
361
P142 Phenotypic plasticity in epithelial progenitors and mesenchymal carcinoma is regulated by Axl
signaling
A. Engelsen, K. Wnup-Lipinska, C. Tiron, F. Pelissier, T. Jokela, G. Haaland, G. Gausdal, T. Sandal,
R. Frink, X. Liang, S. Hinz, L. Ahmed, M. Hellesøy, D. Mickelm, J. Minna, M. LaBarge, R. Brekken,
J. Lorens
362
P143 Cytosolic p21 is a pharmacodynamic marker of CHEK1 and IKKe inhibition in ovarian cancer cells
M. Kim, D.J. Min, G. Wright, C. Annunziata
363
P144 First-in-human phase 1 study of MLN2480, an investigational oral pan-RAF kinase inhibitor, in
patients (pts) with relapsed or refractory solid tumors, including BRAF/NRAS-mutant melanoma
M. Middleton, D.W. Rasco, A.J. Olszanski, P. Corrie, P. Lorigan, R. Plummer, J. Larkin, A. Pavlick,
X. Zhou, Z. Yuan, E. Gangolli, M. Kneissl, V. Bozón, R. Gonzalez
364
P145 Pim-1 kinase: Validated as a therapeutic cancer target for MYC-driven tumours
O. Renner, Y. Cecilia, M.C. Rodriguez de Miguel, S. Peregrina, B. Garcia-Serelde, M.I. Albarran,
A. Cebria, D. Cebrian, F. Ramos-Lima, A. Carnero, J. Pastor, C. Blanco-Aparicio
365
P146 Targeting colorectal and pancreatic cancer stem cells with the LGR5 monoclonal antibody BNC101
P. Chu, F. Shojaei, K. Smith, J. Norton, C. Walsh, J. Iglesias, C. Reyes
366
P147 Induction of apoptosis and inhibition of angiogenesis by novel fusion protein − AD-O54.9 as a new
preclinical strategy in cancer treatment
P. Rozga, B. Zerek, A. Pieczykolan, M. Galazka, K. Bukato, S. Pawlak, M. Szymanik, A. Jaworski,
M. Teska-Kaminska, K. Poleszak, A. Grochot-Przeczek, W. Strozek, J. Pieczykolan
367
P148 NP137, the first humanized monoclonal antibody directed against netrin-1, exhibits antitumor activity
by inducing dependence receptors-mediated cell death
J.G. Delcros, B. Ducarouge, R. Abes, D. Goldschneider, B. Gibert, J.G. Blachier, D. Neves, P. Mehlen,
A. Bernet, S. Depil
368
liv
Poster board
Abstract number
P149 Diagnosis and molecular targeting for individualized treatment of patients with pre-neoplastic lesions
and locally advanced cervical cancer
P.M.A. Moreno-Acosta, A.R.R. Alfredo Romero-Rojas, A.H.S. Antonio Huertas Salgado, D.M. Diana
Mayorga, N.M. Nicolas Morales, J.A. Jinneth Acosta, O.G. Oscar Gamboa, N.M. Nicolas Magne,
M.M. Monica Molano
369
P150 Discovery of the genes that underpin the transition to malignant phenotype of breast tissues in a
highly consanguineous region
I. Gupta, S. Shanmuganathan, A. Ouhtit
370
P151 Selective targeting of head and neck cancer cells by perturbing reactive oxygen species homeostasis
M. Kwon, J.W. Kim, E.H. Kim, J.Y. Park, S.Y. Kim, J.L. Roh
371
P152 Specific inhibition of hTERT expression by targeting common promoter mutations which cause
quadruplex DNA instability
D. Miller, A. Sokolova, S. Thomas, F. Rezzoug, J. Chaires, W. Dean, R. Buscaglia, J. Trent
372
P153 AKT2 gene amplification is a marker for sensitivity to allosteric but not ATP-competitive AKT
inhibitors
T. Abe, K. Ichikawa, R. Fujita, M. Okada, K. Tanaka, M. Ohkubo, K. Yonekura, T. Shimomura,
T. Utsugi
373
P154 Neuregulin 1 (NRG1) expression is a predictive biomarker for response to AV-203, an ERBB3
inhibitory antibody, in human tumor models
K. Meetze, S. Vincent, S. Tyler, E. Mazsa, A. Delpero, S. Bottega, D. McIntosh, J. Gyuris, Z. Weng
374
P155 MER as a novel therapeutic target in colorectal cancer
K. Wong, A.C. Tan, T. Pitts, P. Klauck, S. Earp, S. Frye, X. Wang, D.K. Graham, S.G. Eckhardt
375
P156 Tumor-targeting Salmonella typhimurium A1-R enhances gemcitabine–bevacizumab efficacy on a
patient-derived orthotopic xenograft (PDOX) pancreatic cancer nude mouse model
Y. Hiroshima, M. Zhao, M.H.G. Katz, J.B. Fleming, S. Sato, T. Murakami, M. Yamamoto, F. Uehara,
S. Miwa, S. Yano, M. Momiyama, Y. Zhang, A. Maawy, T. Chishima, K. Tanaka, M. Bouvet, I. Endo,
R.M. Hoffman
376
P157 Plucked hair as a platform for monitoring pharmacodynamic and mechanistic consequences of
clinical exposure to the Wnt/beta-catenin inhibitor PRI-724
G. Miele, B. Reed, E. Harrison, T. Mefo, J. Read, T. Senba, T. Odagami
377
P158 A first-in-human (FIH) safety and pharmacological study of SAR405838, a novel HDM2 antagonist,
in patients with solid malignancies
V. de Weger, M.P. Lolkema, M. Dickson, A. Le Cesne, A. Wagner, M. Merqui-Roelvink, A. Varga,
W. Tap, G. Schwartz, G. Demetri, W. Zheng, G. Tuffal, S. Macé, H. Miao, J.H.M. Schellens,
M. de Jonge
378
P159 Identification and rational in silico-based targeting of a novel mediator of metastatic breast cancer
R. Clarkson, J. Soukupova, A. Wakefield, D. Turnham, W. Yang, C. Bordoni, A. Westwell, A. Brancale
379
P160 Induction of apoptosis with a novel dual cIAP1/XIAP antagonist in models of melanoma
G. Ward, G. Chessari, C.N. Johnson, J. Lewis, S. Rich, N. Thompson
380
P161 Efficacy of specific FGFR inhibitors against gatekeeper resistance mutations and shared mechanism of
cell death in FGFR2-dependent endometrial cancer cell lines
L. Packer, S. Byron, S. Stehbens, D. Loch, F. Dehkhoda, S. Stephenson, P. Pollock
381
P162 Phase Ib study of oral dual-PI3K/mTOR inhibitor GDC-0980 in combination with capecitabine and
mFOLFOX6 + bevacizumab in patients with advanced solid tumors and colorectal cancer
L. Rosen, J. Goldman, J.M. Hubbard, M. Roos, J. Capdevila, J. Maynes, W. Lin, B. O’Keeffe,
M. Lackner, J. Spoerke, J. Ware, B. Arnieri, E. Freas, S. Leong
382
P163 Identification of potent and selective tankyrase 1/2 inhibitors with activity in a subset of APC mutant
colorectal cancer
S.M. Guichard, Y. Zhang, D. Ferguson, A. Mazzola, H. Wang, L. Bao, S. Grosskurth, J. Johannes,
M. Wagoner, M. Zinda, S. Fawell, E.J.E. Pease, A. Schuller
383
Poster board
lv
Abstract number
P164 REDX04988, a novel dual B-RAF/C-RAF inhibitor and a potential therapeutic for BRAF-mutant
colorectal cancer
J. Rainard, R. Testar, R. Poonawala, H. Mason, P. Smith, H. Brooke, V. Huart, S. Frith, J. Ahmet,
J. Hall, A. Morrison, M.A. Campbell, M. Bingham, R. Armer
384
P165 Noninvasive monitoring of acquired EGFR-T790M mutation and discovery of its heterogeneity in
patients with advanced NSCLC treated with EGFR-TKI
X. Ye, D. Zheng, M.Z. Zhang, Y. Sun, J.Y. Wang, J. Ni, H.P. Zhang, L. Zhang, J. Luo, J. Zhang,
L. Tang, B. Su, G. Chen, G.S. Zhu, J.F. Xu, Y. Gu
385
P166 PI3K and MEK inhibitor combination toxicities and relative dose intensity: Vall d’Hebron experience
A. Azaro, D. Marino, A. Garrido-Castro, C. Cruz, M. Alsina, J. Perez, R. Dienstmann, G. Argiles,
C. Hierro, M. Berzosa, B. Adamo, J. Tabernero, J. Rodon
386
P167 Combination of the ERK inhibitor GDC-0994 with the MEK inhibitor cobimetinib significantly
enhances anti-tumor activity in KRAS and BRAF mutant tumor models
M. Merchant, J. Chan, C. Orr, J. Cheng, X. Wang, T. Hunsaker, M.C. Wagle, S.A. Huang,
J. Tremayne, H. Ngu, M. Solon, J. Eastham-Anderson, H. Koeppen, L. Friedman, M. Belvin, J. Moffat,
M. Junttila
387
P168 An integrated molecular analysis of lung adenocarcinomas identifies potential therapeutic targets
among TTF1-negative tumors
R.J. Cardnell, C. Behrens, L. Diao, Y. Fan, X. Tang, J.D. Minna, G.B. Mills, J.V. Heymach,
I.I. Wistuba, J. Wang, L.A. Byers
388
P169 A phase 1, dose-escalation, first-in-human study of ARQ 087, an oral pan-FGFR inhibitor, in adult
subjects with advanced solid tumors
K. Papadopoulos, A. Tolcher, M. Kittaneh, A. Patniak, D. Rasco, G. Chambers, G. Newth, R. Savage,
T. Hall, B. Schwartz, J. Kazakin, P. LoRusso
389
P170 Inhibitors of EZH2 act synergistically with type 1 interferon to induce a potent interferon-stimulated
gene response, triggering apoptosis in diffuse large B-cell lymphoma
B. Bradley, F. Zhao, C.C. Yuan, S. Balasubramanian, P. Iyer, C. Hatton, B. Bryant, E. Normant,
P. Trojer
390
P171 LOXO-101, a pan TRK inhibitor, for the treatment of TRK-driven cancers
S. Winski, B. Baer, D. Hartley, S. Rhodes, R. Wallace, S. Smith, N. Nanda, L. Kunkle, P. Lee,
K. Bouhana
391
P172 Using modelling & simulation to integrate mouse PK−PD-efficacy with preliminary human PK data
to inform the Phase II doses and schedule for the experimental c-Met inhibitor AZD6094 (Volitinib)
R. Jones, A. Cheung, T. Coleman, P. Ballard, C. D’Cruz, A. Schuller, M. Frigault, Y. Gu, Y. Sai,
S. Weiguo, Y. Ren, W. Qing, L. Lindbom, K. Petersson
392
P173 Small modification of ceritinib enhances the activity against ALK
C.H. Park, C.H. Kang, H.J. Jung, H.R. Kim, C.O. Lee, H.K. Lee, S.U. Choi
393
P174 Sensitivity of acute myeloid leukemia cells to a urokinase-activated anthrax lethal toxin (PrAgU2/lf)
is dependent on uPAR expression and phospho-MEK1/2 levels
A. Bekdash, S.H. Liu, S.H. Leppla, A.E. Frankel, R. Abi-Habib
394
P175 cMet: Proof-of-concept clinical trial with volitinib in patients with advanced papillary renal cell
cancer (PRCC)
T.K. Choueiri, B. Escudier, S. Kumar Pal, E. Jonasch, D. Heng, T. Powles, H.T. Arkenau, E. Clark,
C. D’Cruz, M. Frigault, A.F. Nash, S.R. Morgan
395
P176 Gastrointestinal stromal tumor associated with neurofibromatosis type I
T. Takahashi, T. Nishida, R. Nakatsuka, M. Kaneda, S. Hirota, Y. Miyazaki, Y. Kurokawa,
M. Yamasaki, H. Miyata, K. Nakajima, S. Takiguchi, M. Mori, Y. Doki, S. Serada, T. Naka
396
lvi
Poster board
Abstract number
P177 Characterization of molecular targets of therapy in Non-Small Cell Lung Cancer (NSCLC) utilizing a
liquid biopsy
S. Greene, D. Lu, R. Krupa, M. Harvey, J. Louw, A. Jendrisak, N. Bales, D. Marrinucci, J. Gray,
R. Dittamore
P178 TAS-119 a selective inhibitor of Aurora A kinase, potentiates taxane therapy in breast and lung
cancer models
Y. Nakatsuru, A. Hashimoto, H. Sootome, K. Ito, M. Sakuragi, A. Miura, N. Oda, H. Hirai, T. Utsugi
P179 Rho-GTPase, RAC1 and Cdc42 mediates Wnt−beta-catenin signals for metastasis associated
phenotypes in TNBC: A proof of concept study
N. Dey, P. De, B. Leyland-Jones
P180 Optimization of novel pyrido[2,3-b]pyrazine based small molecule fibroblast growth factor
receptor 1, 2, 3 & 4 (FGFR) inhibitors into a potential clinical candidate
P. Angibaud, O. Querolle, V. Berdini, G. Saxty, A. Cleasby, H. Colombel, I. Csoka, N. Esser,
R. Gilissen, L. Meerpoel, C. Paulussen, I. Pilatte, V. Poncelet, D.C. Rees, B. Roux, T. Verhulst,
V. Tronel, B. Wroblowski, C.W. Murray, J. Vialard
P181 TAS-121, a highly potent and mutant-specific EGFR inhibitor, overcomes T790M-acquired resistance
with promising antitumor activity through specific inhibition of mutant EGFR signaling
M. Kato, K. Miyadera, K. Ito, Y. Aoyagi, A. Hashimoto, K. Yonekura, Y. Iwasawa, T. Utsugi
P182 Characterization of the oncogenic properties of mutant isocitrate dehydrogenase 1 (IDH1R132H) in
human primary cells
M. Barradas, L. Diezma, C.M. Pérez-Ferreiro, A. Cerezo, E. Lospitao, S. Peregrina, S. Jiménez,
N.A. Brooks, R. Torres, S. Geeganage, R. Gilmour, S. Velasco-Miguel
P183 A novel dielectrophoretic microwell array system for detection and single cell analysis of circulating
tumor cells from breast cancer patients
T. Sawada, A. Morimoto, T. Mogami, K. Iijima, Y. Akiyama, K. Katayama, T. Futami, M. Yunokawa,
K. Tamura, M. Watanabe, Y. Koh, F. Koizumi
P184 Targeting PI3K somatic mutations reduces invasion and EMT in squamous cell carcinoma of the lung
A. Cavazzoni, M. Bonelli, F. Saccani, S. La Monica, M. Galetti, C. Caffarra, D. Cretella,
C. Fumarola, R. Alfieri, P.G. Petronini
P185 Aurora kinases A and B are required for KRAS-induced lung cell oncogenicity
E.O. Ozorio dos Santos, M.N. Aoki, E. Levantini, D.S. Bassères
P186 Aflibercept (Zaltrap) directly attenuates the migration and invasion of colorectal cancer cells
A. Bouygues, P. Mésange, M. Ayadi, V. Poindessous, M. Chiron, E. Dochy, T. André, A. de Gramont,
A.K. Larsen
P187 Structural basis for inhibition of ligand-dependent and -independent ErbB3 activation by KTN3379
D. Alvarado, S. Lee, E. Greenlee, G.F. Ligon, J.S. Lillquist, E.J. Natoli, J. Amick, Y. Hadari,
J. Schlessinger
P188 A potent and selective small molecule inhibitor of MCL-1 sensitizes DLBCL cell lines to the BCL-2
selective inhibitor ABT-199
D.C. Phillips, Y. Xiao, L. Lam, E. Litinovic, L. Roberts-Rapp, A.J. Souers, J.D. Leverson
Paediatric Oncology
Poster board
397
398
399
400
401
402
403
404
405
406
407
408
Abstract number
P189 Transcription factor activating protein 2 beta (TFAP2B) mediates neuronal differentiation in
neuroblastoma
F. Ikram, S. Ackermann, F. Roels, R. Volland, B. Hero, F. Hertwig, H. Kocak, D. Dreidax,
K.O. Henrich, F. Berthold, P. Nürnberg, F. Westermann, M. Fischer
P190 Pediatric Preclinical Testing Program (PPTP) Evaluation of the p53−MDM2 Antagonist MK-8242
M. Smith, M. Kang, P. Reynolds, R. Lock, H. Carol, R. Gorlick, A. Kolb, J. Maris, S. Keir, J. Wu,
R. Kurmasheva, P. Houghton
409
410
Poster board
lvii
Abstract number
P191 Druggability of p16 deleted pediatric leukemia: The novel cell line POETIC3 identifies potential
agents and drug combinations for mechanism based targeted therapeutics
J. Reimer, A. Kovulchuk, Y. Ruan, R. Shah, A. Jayanthan, M. Perinpanayagam, T. Truong, I. AuerGrzesiak, J. Luider, O. Kovulchuk, T. Trippett, A. Narendran
411
P192 The Pediatric Preclinical Testing Program (PPTP): Analysis of the first 10 years in vivo testing
P. Houghton, R. Lock, H. Carol, R. Gorlick, A. Kolb, J. Maris, S. Keir, J. Wu, M. Kang, P. Reynolds,
R. Kurmasheva, M. Smith
412
P193 Next-generation sequencing identifies the mechanism of tumourigenesis caused by loss of SMARCB1
in malignant rhabdoid tumours
M.A. Finetti, M. Selby, A. del Carpio Pons, J. Wood, B. Skalkoyannis, A. Smith, S. Crosier, S. Bailey,
S. Clifford, D. Williamson
413
P194 Genomic profiling using a clinical next generation sequencing (NGS) assay reveals genomic
alterations to guide targeted therapy in advanced neuroblastoma patients
S. Ali, E.M. Sanford, M.J. Hawryluk, J. Chmielecki, K. Wang, G.A. Palmer, N.A. Palma, D. Morosini,
R. Erlich, R. Yelensky, D. Lipson, J.S. Ross, Y. Mosse, P.J. Stephens, J.M. Maris, V.A. Miller
414
P195 Comprehensive next generation sequencing of solid tumors from 669 adolescents and young adults
reveals a distinct spectrum of targetable genomic alterations
D. Morosini, K. Wang, K. Wagner, B. Gershenhorn, R. Yelensky, D. Lipson, J. Chmielecki, S.M. Ali,
J.S. Ross, P.J. Stephens, V.A. Miller
415
P196 Results of phase I study of bolus 5-fluorouracil in children and young adults with recurrent
ependymoma
K.D. Wright, D.C. Turner, K.M. Haddock, M.O. Jacus, K.E. Harstead, S.L. Throm, V.M. Daryani,
G.W. Robinson, G.T. Armstrong, A. Onar-Thomas, C.F. Stewart, A. Gajjar
416
P197 Evaluating the activity of the p53−MDM2 inhibitor NDD0005 in Ewing sarcoma
J. Pecqueur, B. Vormoor, Y. Zhao, H. Newell
417
P198 Population pharmacokinetics of intravenous bolus 5-fluorouracil in a phase I trial for children and
young adults with recurrent ependymoma
D.C. Turner, K.M. Haddock, M.O. Jacus, K.E. Harstead, S.L. Throm, V.M. Daryani, C.F. Stewart,
K.D. Wright
418
P199 Targeted inhibition of casein kinase II (CK2) produces a strong therapeutic effect in pediatric
leukemia
S. Dovat, C. Song, C. Gowda, K.J. Payne
419
P200 Analysis of genomic alterations in Ewing sarcoma (German cohort) reveals cooperating mutations
and novel therapy targets
G.H.S. Richter, K. Agelopoulos, E. Schmidt, K. von Heyking, B. Moser, H.U. Klein, U. Kontny,
M. Dugas, K. Poos, E. Korsching, T. Buch, G. Köhler, C. Rössig, D. Baumhoer, H. Jürgens,
S. Burdach, W.E. Berdel, C. Müller-Tidow, U. Dirksen
420
P201 Molecular profiling for factors predicting sensitivity or resistance to therapy in relapsed child cancer
F. Saletta, C. Wadham, J. Byrne, D. Ziegler, G. McCowage, M. Haber, G. Marshall, M. Norris
421
P202 CBL0137, a novel NFkB suppressor and p53 activator, is highly effective in pre-clinical models of
neuroblastoma
M. Haber, J. Murray, L. Gamble, A. Carnegie-Clark, H. Webber, M. Ruhle, D. Carter, A. Oberthur,
M. Fischer, D. Ziegler, G.M. Marshall, K. Gurova, C. Burkhart, A. Purmal, A.V. Gudkov, M.D. Norris
422
P203 RNA helicase A is essential for 1p36 gene KIF1Bb tumor suppression in neuroblastomas
Z.X. Chen, K. Wallis, S.M. Fell, V.R. Sobrado, M.C. Hemmer, D. Ramsköld, Z. Choo, U. Hellman,
R. Sandberg, R.S. Kenchappa, T. Martinsson, J.I. Johnsen, P. Kogner, S. Schlisio
423
lviii
Poster board
Abstract number
Toxicology
Poster board
Abstract number
P204 Nonclinical safety assessment of a humanized anti-OX40 agonist antibody, MOXR0916
R. Prell, W. Halpern, J. Beyer, J. Tarrant, S. Sukumaran, M. Huseni, R. Kaiser, D. Wilkins,
S. Karanth, H. Chiu, J. Ruppel, C. Zhang, K. Lin, L. Damico-Beyer, J. Kim, H. Taylor
424
P205 Evaluation of drug reactions to anti-neoplastic agents in Phase I clinical trials
M. Bupathi, J. Hajjar, K. Hess, S. Bean, D. Karp, F. Meric-Bernstam, A. Naing
425
P206 Serum levels of CCL22 and CCL25 might predict skin rash induction the commonest adverse event
by bendamustine in the treatment of malignant lymphoma
Y. Terui, R. Kuniyoshi, Y. Mishima, K. Hatake
426
P207 Hematotoxicity potential of new drug candidates measured in hematopoietic progenitors in
bone marrow samples
J. Ballesteros, D. Primo, P. Hernandez, A. Robles, A.B. Espinosa, E. Arroyo, V. Garcia-Navas,
J. Sanchez-Fenoy, M. Jimenez, M. Gaspar, J.L. Rojas, J. Martinez-Lopez, J. Gorrochategui
427
lix
Friday 21 November 2014
09:00–10:30
Poster Viewing
Plenary Session 8
11:00–13:00
Targeting RAS and Other Driver Oncogenes
Chairs: J.A. Engelman (USA) and M. Barbacid (Spain)
Poster area
Auditorium
Abstract number
11:00 Targeting KRAS driven lung and pancreatic adenocarcinoma
M. Barbacid (Spain)
11:20 Systematic interrogation of vulnerabilities in KRAS driven cancers
W. Hahn (USA)
Key objectives:
1. Understand the concept of synthetic lethality.
2. Understand systematic genetic screens to identify targets.
3. Understand the role of innate immune regulators in KRAS induced tumorigenesis.
11:40 Targeting synthetic lethal partners of KRAS
J. Luo (USA)
Main objectives:
1. To inform the participants about therapeutic strategies in targeting KRAS mutant cancer.
2. To inform the participants about the concept of synthetic lethality and its therapeutic utilities.
3. To provide the participants examples of synthetic lethal interactions with the KRAS oncogene.
Take home messages:
1. Current difficulties in treating KRAS mutant tumours warrant exploration of new therapeutic
strategies.
2. Synthetic lethality is a powerful approach for identifying functional vulnerabilities in cancer cells.
3. Synthetic lethal partners of the KRAS oncogene could be potential targets for drug discovery.
12:00 ORAL PRESENTATION: Clinical acquired resistance to combined RAF/EGFR or RAF/MEK
inhibition in BRAF mutant colorectal cancer (CRC) patients through MAPK pathway alterations
R. Corcoran, E.M. Coffee, E. Van Allen, L.G. Ahronian, N. Wagle, E.L. Kwak, J.E. Faris, A.J. Iafrate,
L.A. Garraway, J.A. Engelman
12:15 LATE BREAKING ABSTRACT: Antitumor activity of ASP8273, an irreversible mutant
selective EGFR-TKI, in NSCLC patients with tumors harboring EGFR activating mutations and
T790M resistance mutation
H. Murakami, H. Nokihara, T. Shimizu, T. Seto, A. Keating, A. Krivoshik, K. Uegaki, S. Morita,
K. Nakagawa, M. Fukuoka
428
9LBA
12:25 LATE BREAKING ABSTRACT: Interim phase 2 results of study CO-1686-008: A phase 1/2 study 10LBA
of the irreversible, mutant selective, EGFR inhibitor rociletinib (CO-1686) in patients with advanced
non small cell lung cancer
J. Soria, L.V. Sequist, J.W. Goldman, H.A. Wakelee, S.M. Gadgeel, A. Varga, H.A. Yu, B.J. Solomon,
S.H. Ou, V. Papadimitrakopoulou, G.R. Oxnard, L. Horn, R. Dziadziuszko, B. Chao, A.I. Spira, S. Liu,
T. Mekhail, S. Matheny, J. Litten, R.D. Camidge
12:35 LATE BREAKING ABSTRACT: Phase I study of the selective BRAFV600 inhibitor
11LBA
encorafenib (LGX818) combined with cetuximab and with or without the a-specific PI3K inhibitor
alpelisib (BYL719) in patients with advanced BRAF mutant colorectal cancer
J. Tabernero, R. van Geel, J.C. Bendell, A. Spreafico, M. Schuler, T. Yoshino, J.P. Delord, Y. Yamada,
M.P. Lolkema, J.E. Faris, F.A.L.M. Eskens, S. Sharma, R. Yaeger, H.J. Lenz, Z. Wainberg, E. Avsar,
A. Chatterjee, S. Jaeger, T. Demuth, J.H.M. Schellens
J.A. Engelman (USA)
lx
Poster Sessions
Poster area
Drug Synthesis
Poster board
Abstract number
P001 Development of extracellular signal-regulated kinase 5 (ERK5) inhibitors for anti-cancer therapy
S. Myers, N. Martin, R. Bawn, T. Blackburn, L. Barrett, T. Reuillon, B. Golding, R. Griffin,
T. Hammonds, I. Hardcastle, H. Leung, D. Newell, L. Rigoreau, A. Wong, C. Cano
429
P002 Design and structure–activity relationships of highly potent and bioavailable imidazolinone
FASN KR domain inhibitors
G. Bignan, R. Alexander, J. Bischoff, P. Connolly, M. Cummings, S. De Breucker, N. Esser,
E. Fraiponts, R. Gilissen, B. Grasberger, B. Janssens, T. Lu, D. Ludovici, L. Meerpoel, C. Meyer,
M. Parker, D. Peeters, C. Rocaboy, C. Schubert, K. Smans
430
Molecular Targeted Agents II
Poster board
Abstract number
P003 Real-time pharmacokinetic (PK) results from an ongoing randomized, parallel-dose phase 1 study of
onapristone in patients (pts) with progesterone receptor (PR)-expressing cancers
F. Lokiec, J. Bonneterre, A. Italiano, A. Varga, M. Campone, T. LeSimple, A. Leary, V. Dieras,
K. Rezai, S. Giacchetti, S. Proniuk, A. Bexon, E. Gilles, J. Bisaha, A. Zukiwski, P. Cottu
431
P004 ODM-203, a novel, selective and balanced FGFR and VEGFR inhibitor with strong anti-tumor
activity in FGFR- and VEGFR-dependent cancer models
T. Holmström, A. Moilanen, T. Linnanen, G. Wohlfahrt, S. Karlsson, R. Oksala, T. Korjamo,
M. Björkman, S. Samajadar, S. Rajagopalan, S. Chelur, K. Narayan, R. Ramachandra, T. Anthony,
S. Ds, M. Ramachandra, P. Kallio
432
P005 Genomic predictors of therapeutic sensitivity to TAS-119, a selective inhibitor of Aurora-A kinase
H. Sootome, N. Fujita, A. Miura, T. Suzuki, H. Fukushima, S. Mizuarai, H. Hirai, T. Utsugi
433
P006 Bipolar androgen therapy for men with castration sensitive and castration resistant prostate cancer:
Reversing resistance and maintaining sensitivity to androgen ablative therapies
S.R. Denmeade, E.S. Antonarakis, M.A. Eisenberger, M.A. Carducci, H. Wang, C.J. Paller, J.T. Isaacs,
M.T. Schweizer
434
P007 A pan-cancer tumor-derived epithelial-to–mesenchymal transition (EMT) signature determines
patterns of drug sensitivity and enrichment in immune target expression following EMT
M.P. Mak, P. Tong, L. Diao, P.K.S. Ng, Y. Fan, R.J. Cardnell, D.L. Gibbons, W.N. William,
J.V. Heymach, K.R. Coombes, L.A. Byers, J. Wang
435
P008 Monitoring activity of RXDX-101 in Phase 1/2 patients using a pharmacodynamic assay for TrkA
activation
D. Murphy, H. Ely, R. Patel, G. Wei, A. Diliberto, R. Shoemaker, J. Christiansen
436
P009 PIM kinase inhibitor AZD1208 sensitises SCLC to BH3 mimetic AZD4320
R. Sloane, B. Bola, M. Lancashire, C. Hodgkinson, C. Morrow, K. Simpson, C. Dive
437
P010 MM-131: A bispecific antibody that inhibits c-Met signaling through avid binding to the EpCAM
tumor antigen
B.D. Harms, A. Lugovskoy, A. Abu-Yousif, A. Fulgham, M. Geddie, S.V. Su, N. Kohli, B. Johnson,
K. Masson, U.B. Nielsen, B. Schoeberl, G. MacBeath
438
P011 PI3K/mTOR inhibitor VS-5584 targets cancer stem cells and prevents tumor regrowth after
chemotherapy in preclinical models of small cell lung cancer
V. Kolev, M. Padval, Q. Wright, J. Ricono, D. Weaver, J. Pachter, Q. Xu
439
P012 Phenotypic alteration in a highly metastatic variant of the MDA-MB-231 cell line: role of
Annexin A1
Y. Tu, E. Fietz, J. Cameron, A. Stewart
440
Poster board
lxi
Abstract number
P013 The role of methylation in metastasis of oral squamous cell carcinoma: understanding the OSCC
methylome
M. Clausen, L.J. Melchers, T. De Meyer, S. Denil, W. Criekinge, G.B. Wisman, J.L.N. Roodenburg,
E. Schuuring
P014 Targeting urokinase plasminogen activator for radioimmunotherapy using an antagonistic internalizing
human antibody
A. LeBeau, H.F. VanBrocklin
P015 Notch3-targeted antibody drug conjugates have superior preclinical efficacy to Notch signaling
inhibitors
K.G. Geles, Y. Gao, L. Sridharan, A. Giannakou, T.T. Yamin, J. Golas, M. Charati, J. Lucas, K. Wang,
S. Pirie-Shepherd, M. Roy, M. Follettie, A. Maderna, X. Li, L. Tchistiakova, H.P. Gerber, P. Sapra
P016 Debio 1143 in combination with carboplatin and paclitaxel in patients with non-small cell lung cancer
(NSCLC), triple-negative breast cancer (TNBC) and platinum-refractory epithelial ovarian cancer
(EOC). Preliminary results of a Phase I dose-escalation study
C. Le Tourneau, I. Ray-Coquard, N. Isambert, C.A. Gomez-Roca, P. Cassier, M.P. Sablin, E. Ruits,
B. Gavillet, C. Zanna, P. Fumuleau, J.P. Delord
P017 MEK inhibition enhances gemcitabine efficacy by increasing MDM2-mediated ubiquitination and
degradation of RRM1
F. Vena, E. Li Causi, T. Hagemann, J.A. Hartley, S. Goodstal, D. Hochhauser
P018 The cancer stem cell inhibitors VS-6063 (defactinib) and VS-5584 exhibit synergistic anticancer
activity in preclinical models of mesothelioma
Q. Xu, W.F. Tam, C.M. Vidal, V.N. Kolev, Y. Kadariya, C.W. Menges, J.R. Testa, J.A. Pachter
P019 Novel, quantitative in vivo shRNA screening approach identifies new molecular targets to block
cancer metastasis
L. Willetts, R. Paproski
P020 Exposure to EGFR inhibitors influences release of extracellular vesicles by tumor cells
R. van der Meel, S.M. van Dommelen, P. de Corte, M. Coimbra, W.W. van Solinge, P. Vader,
R.M. Schiffelers
P021 Cytokine induces MIR-424 expression and modulates SOCS2/STAT5 signaling pathway in oral cancer
S.G. Shiah, H.Y. Peng, S.L.C. Jin, J.Y. Chang, C.C. Kuo
P022 Precise gene editing of mutant NRAS using CRISPR to determine sensitivity to trametinib
C. Hose, N.D. Fer, M. Burkett, J. Connelly, E. Harris, J. Lih, M. Williams, D. Evans, T. Silvers,
A. Monks, R. Parchment, B.A. Teicher, J.H. Doroshow, A. Rapisarda
P023 Correlative and updated clinical endpoint analysis of a multicenter phase II trial of selumetinib
(AZD6244) plus erlotinib in chemotherapy-refractory advanced pancreatic adenocarcinoma (PDAC)
A.H. Ko, A.H. Tempero, T.B. Bekaii-Saab, P. Kuhn, R. Courtin, S. Ziyeh, S. Tahiri, R.K. Kelley,
E. Dito, A. Ong, R. Linetskaya, A. Talasaz, A.P. Venook, W. Korn
P024 eIF2alpha phosphorylation determines the adaptation of tuberous sclerosis complex mutant cells to
stress and their response to anti-tumor therapies
A. Koromilas, C. Tenkerian, J. Krishnamoorthy, R. Kamindla, U. Kazimierczak, S. Wang
P025 Plasma metabolomic signature of novel signal transduction inhibitors from preclinical identification to
clinical validation
J.E. Ang, R. Pandher, Y. Asad, D.J. Skene, P. Workman, S. Eccles, J. De Bono, S. Kaye, U. Banerji,
S. Davies, F.I. Raynaud
P026 Monitoring therapy response and resistance mutations in circulating RNA and DNA of plasma from
patients with malignant melanoma
D. Enderle, K. Brinkmann, T. Koestler, S. Bentink, K.T. Flaherty, J. Skog, M. Noerholm
P027 Role of MDM2 as therapeutic target in gastroenteropancreatic neuroendocrine neoplasms
(GEP-NENs)
F. Briest, I. Grass, F. Christen, F. Lewens, H. Freitag, D. Kaemmerer, J. Saenger, M. Hummel,
B. Siegmund, P. Grabowski
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
lxii
Poster board
Abstract number
P028 SF3B1 mutations induce disease relevant aberrant mRNA splicing in cancer and confer sensitivity to
spliceosome inhibition
S. Buonamici, K. Lim, J. Feala, R. Darman, K. Myint, E. Park, D. Aird, B. Chan, P. Fekkes,
R. Furman, G. Keaney, P. Kumar, K. Kunii, X. Puyang, M. Thomas, Y. Mizui, M. Warmuth, P. Zhu,
L. Yu, P. Smith
P029 Preclinical pharmacology of AZD5312, a generation 2.5 antisense oligonucletotide targeting the
androgen receptor with differentiated activity from enzalutamide
B.R. Davies, A. Thomason, R. Ellston, H. Campbell, C. D’Cruz, A.M. Mazzola, T. Zhou, J. Schmidt,
M. Jo, Y. Kim, A.R. McLeod, D. Blakey, N. Brooks
P030 Combinational genome and proteome survey of therapeutic targets of hepatocellular carcinoma
T. Yamada
P031 Preclinical characterization of CC-115, a novel inhibitor of DNA-PK and mTOR kinase currently
under clinical investigation
D.S. Mortensen, K.E. Fultz, W. Xu, T. Tsuji, M. Hickman, M. Abbasian, G. Khambatta, B. Cathers,
P. Worland, M.F. Moghaddam, J. Apuy, S. Richardson, J. Elsner, G. Shevlin, S. Perrin-Ninkovic,
S. Canan, H.K. Raymon, R.K. Narla, S. Peng, S. Sankar
P032 Molecular biomarkers of inflammatory signature in melanoma
S. Ekmekcioglu, M. Shin-Sim, K. Tanese, V.G. Prieto, D.S. Hoon, E.A. Grimm
P033 A phase I trial of dabrafenib (BRAF inhibitor) and pazopanib in BRAF mutated advanced
malignancies
S. Haraldsdottir, F. Janku, C. Timmers, S. Geyer, L.J. Schaaf, J. Sexton, J. Thurmond, V. VelezBravo, V.M. Stepanek, E. Bertino, K. Kendra, A. Mortazavi, V. Subbiah, M. Villalona-Calero, M. Poi,
M. Phelps, M.H. Shah
P034 Clinical sequencing of cancer in real-time by digital sequencing of cell-free DNA for tailoring
targeted therapy in refractory cancer patients
A. Talasaz, S. Mortimer, B.J. Schiller, G. Mei, S. Huang, D.S.B. Hoon, H. Eltoukhy
P035 PTPN11 is a central node in intrinsic and acquired resistance to targeted cancer drugs
A. Prahallad
P036 Influence of warm and cold ischemia on molecular patterns in clinical biospecimen
F.T. Unger, N. Lange, P.C. Uhlig, H. Juhl, K.A. David
P037 Biomarker analysis in Phase 1b study of anti-cancer stem cell antibody Tarextumab (TAR) in
combination with nab-paclitaxel and gemcitabine (Nab-P+Gem) demonstrates pharmacodynamic (PD)
modulation of the Notch pathway in patients (pts) with untreated metastatic pancreatic cancer (mPC)
A. Kapoun, E. O’Reilly, A. Cohn, J.C. Bendell, L. Smith, J.H. Strickler, W. Gluck, Y.W. Liu,
B. Wallace, R. Tam, B. Cancilla, A. Brunner, D. Hill, L. Zhou, J. Dupont, C. Zhang, M. Wang
P038 IGF2 drives IGF oncogenic signaling in HCC and emerges as a potential target for therapies
I.M. Quetglas, R. Pinyol, D. Dauch, A. Portela, A. Villanueva, J. Peix, M. Higuera, A. Moeini,
J. Zucman-Rossi, M. Esteller, V. Mazzaferro, L. Zender, J.M. Llovet
P039 Two in one − Delivery of apoptotic signal into cancer cells by new class of TRAIL derived, fusion
protein
S.D. Pawlak, B.M. Zerek, P.K. Rozga, K. Bukato, M. Szymanik, M. Teska-Kaminska, A. Pieczykolan,
A. Jaworski, M. Galazka, K. Poleszak, W. Strozek, J.S. Pieczykolan
P040 Clinicopathologic implication of c-MYC gene copy number gain and overexpression in colorectal
cancer
K. Lee, Y. Kwak, G. Choe, W. Kim, D. Kim, S. Kang, H. Lee
P041 OTX015, a novel BET-bromodomain (BET-BRD) inhibitor, is a promising anticancer agent for human
glioblastoma
L. Ouafik, C. Berenguer, M. Cayol, L. Astorgues-Xerri, M. Bekradda, E. Odore, K. Rezai,
M.E. Riveiro, E. Cvitkovic
P042 Transcriptional regulation of cancer stem cells marker CD133 by p53
E.K. Park, S.Y. Bang, S.A. Yi, J.W. Han
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
Poster board
lxiii
Abstract number
P043 Screening of Champions predictive TumorGraft platform guides the clinical development of the
selective dual BRAF-EGFR inhibitor CEP-32496
B. Ruggeri, M. Wabler, E. Bruckheimer, B. Wilkinson, B. Dorsey, S. Trusko, J. Friedman
471
P044 Do we hit the target? Phospho-reactome measurements determine efficacy of targeted therapies
L. van ’t Veer, M. Moro, Z. Chen, P.R.E. Lee, B. Pan, D. Brunen, A. Prahalled, R. Bernards,
J.P. Coppe
472
P045 FDG PET/CT as imaging biomarker in the era of molecular targeting therapies: sequential FDG
PET/CT demonstrated biological response and acquisition of resistance to tyrosine kinase inhibitor
therapy for renal cell carcinoma
N. Nakaigawa, K. Namura, D. Ueno, U. Tateishi, T. Inoue, M. Yao
473
P046 Potentiation of oxaliplatin in colon cancer by JNK inhibition
I. Vasilevskaya, M. Selvakumaran, L. Cabal-Hierro, P. O’Dwyer
474
P047 The synergistic anti-proliferative effect of combining the FGFR inhibitor, ARQ 087 with the AKT
inhibitor, ARQ 092 in human cancer cell lines and PDX models
E. Marchlik, Y. Yu, E. Chiesa, D. Dransfield
475
P048 FGFR2 targeting with allosteric inhibitor RPT835
I. Tsimafeyeu, F. Daeyaert, W. Yin, J. Ludes-Meyers, M. Byakhov, S. Tjulandin
476
P049 Jab1/Csn5: a new player driving the resistance to HER2-targeted therapies for breast cancer
F. Claret, T. Vu, T.J. Shackleford, J.L. Allensworth, Q. Zhang, F.J. Esteva, E. Drakos, R. Zhang,
L. Tian, G.Z. Rassidakis
477
P050 Pharmacological disruption of the Astrocytic Elevated Gene-1 (AEG1) in anticancer intervention:
PB0412_3 (PB03) as a first-in-class AEG1 interacting agent
J. Jimeno, G. Acosta, C. Teixido, C. Olbiol, N. Karachaliou, M.A. Molina, O. Villacañas, M. SanchezRonco, J. Bertran, A. Gimenez-Capitan, J.C. Monasterio, M. Taron, R. Rosell, F. Albericio
478
P051 The discovery and development of potent and specific anti-SialylTn antibodies for the treatment of
solid tumors
K. Meetze, D. Ghaderi, M. Zhang, I. Purnajo, J. Hermann, J. Fett, J. Behrens, A. Paula Galvao da
Silva
479
P052 Identification of novel small molecules as selective PAK4 allosteric modulators (PAMs) by stable
isotope labeling of amino acids in cells (SILAC)
W. Senapedis, Y. Landesman, M. Schenone, B. Karger, S. Wu, S. Shacham, E. Baloglu
480
P053 In vitro and vivo evaluation of the pan FGFR inhibitor ARQ 087 and selective pan AKT inhibitor
ARQ 092 in endometrial cancer: potential for combination therapy
J. Meade, M.J. Wick, T. Vaught, R. Chavez, M. Rundle, K. Stanfield, B. Quattrochi, K.P. Papadopoulos,
D.T. Dransfield, Y. Yu, E. Marchlik, E. Chiesa, A.W. Tolcher
481
P054 Repurposing the antihelminthic mebendazole as a hedgehog inhibitor
A.R. Larsen, R.Y. Bai, J.H. Chung, A. Borodovsky, G.J. Riggins, F. Bunz
482
P055 Irreversible covalent pan-FGFR inhibitors are highly efficacious against FGFR-dependent cancers
V.T. Phan, E. Verner, M. Gerritsen, J.M. Bradshaw, D.M. Goldstein, R.J. Hill, D. Karr, J. LaStant,
P. Nunn, D. Tam, J. Shu, J.O. Funk, K. Brameld
483
P056 Treatment of advanced solid tumors with golvatinib (E7050) in combination with lenvatinib (E7080)
E.L. Kwak, D. Juric, J.M. Cleary, G. Cote, J.F. Hilton, K.T. Flaherty, K. Wood, C. Rance, S. Barrett,
G.I. Shapiro
484
P057 Glutathione S-transferases M1−5 reduce the aggressive behaviour in breast cancer by modulating the
PI3K/AKT pathway
A. Bergamaschi, S. Levy, A. Scott, J. Jeong, M. Kiefer, E. Beasley, J. Baker
485
P058 Development of small molecule activators of protein phosphatase 2A for the treatment of lung cancer
J. Sangodkar, S. Mazhar, D. Kastrinsky, M. Ohlmeyer, G. Narla
486
lxiv
Poster board
Abstract number
P059 EC-70124, a multi-kinase inhibitor, blocks NF-kB and STAT3 dependent signaling in prostate cancer
stem cells
G. Civenni, P. Costales, C. Garcı́a-Inclán, D. Albino, N. Longoni, L.E. Nuñez, F. Moris,
G.M. Carbone, C.V. Catapano
487
P060 Development of a publicly accessible knowledgebase to facilitate decision support for clinical cancer
genomics reporting
K. Shaw, J. Zeng, A.M. Bailey, A. Johnson, V. Holla, E.V. Bernstam, J. Butts, J. Mendelsohn,
G.B. Mills, F. Meric-Bernstam
488
P061 Development and clinical validation of a quantitative mass spectrometric assay for PD-L1 protein in
FFPE NSCLC samples
E. An, W. Liao, S. Thyparambil, J. Rodriguez, R. Salgia, I. Wistuba, J. Burrows, T. Hembrough
489
P062 Potent and selective inhibitors of the KRAS-signaling nanocluster protein, CNKSR1, block oncogenic
KRAS signaling and mut-KRAS cell growth
L. Kirkpatrick, G. Triana-Baltzer, M. Indarte, M. Scott, R. Lemos, G. Powis
490
P063 Development and characteristics of resistance to the HER family tyrosine kinase inhibitor AZD8931
V.G. Brunton, H. Creedon, M. Muir, T. Klinowska, K. McLeod, A. Byron
491
P064 Quantification of exportin-1 (XPO1) occupancy by selective inhibitors of nuclear export (SINE)
M. Crochiere, B. Klebanov, E. Baloglu, O. Kalid, T. Kashyap, W. Senapedis, D. del Alamo, S. Tamir,
D. McCauley, R. Carlson, M. Kauffman, S. Shacham, Y. Landesman
492
P065 Preclinical pharmacokinetic (PK)/pharmacodynamic (PD)/Efficacy modeling for MLN2480, an
investigational pan-RAF kinase inhibitor, in A375 and SKMEL-2 human melanoma xenografts
M. Patel, J. Chouitar, J. Mettetal, E. Gangolli, S. Balani, P. Shimoga, K. Galvin, W.C. Shyu,
A. Chakravarty, C.J. Zopf
493
P066 LPA6 promotes growth and tumorigenicity of hepatocellular carcinoma via activation of PIM-3 protooncogene kinase
C. Lopane, V. Goffredo, F. Dituri, F. De Santis, A. Filannino, R.C. Betz, Y.Y. Li, N. Mukaida, P. Winter,
C. Tortorella, G. Giannelli, C. Sabbà, A. Mazzocca
494
P067 Divergent androgen regulation of UPR pathways drives prostate cancer
Y.J. Arnoldussen, M. Storm, X. Sheng, M. Tesikova, Y. Jin, H.Z. Nenseth, S. Zhao, I.G. Mills, L. Fazli,
P. Rennie, B. Risberg, H. Wæhre, H.E. Danielsen, G.S. Hotamisligil, F. Saatcioglu
495
P068 Prevalence of MET amplification, MET expression, and MET-related genomic alterations in nonsmall cell lung cancer (NSCLC)
A.L. Ang, H. Yang, A.A. Anderson, R. Tang, M.A. Damore, R.D. Loberg
496
P069 Preclinical evaluation of dimeric IAP proteins inhibitor APG-1387, in triple negative breast cancer
both in vitro and in vivo
G. Wang, P. Min, H. Wang, Z. Du, M. Wu, H. Dong, D. McEachern, L. Liu, Y. Lin, X. Lin, J. Wen,
Y. Zhang, L. Gu, M. Guo, Y. Zhai, S. Wang, D. Yang
497
P070 CDK4/6 inhibitor (LY2835219) exhibits potent anti-tumor activity in human lung cancer cell lines
with intact retinoblastoma
J.W. Goldman, E.B. Garon, D. Conklin, D.J.L. Wong, B. Wolf, D.D. Silveira, N. Kamranpour, S. Pitts,
R.S. Finn, D.J. Slamon
498
P071 Phase 2a study of copanlisib, a novel phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with
relapsed/refractory, indolent or aggressive lymphoma
M. Dreyling, P.L. Zinzani, K. Bouabdallah, D. Bron, D. Cunningham, K. Linton, C. Thieblemont,
E. Van den Neste, U. Vitolo, J. Grunert, M. Giurescu, S. Mappa, B.H. Childs, F. Morschhauser
499
P072 Triple blockade with LEE011, everolimus, and exemestane in women with ER+/HER2−
advanced/metastatic breast cancer: results from a Phase Ib clinical trial
A. Bardia, C. Chavez-MacGregor, S. Modi, M. Campone, B. Ma, M. Kittaneh, L. Dirix, M. Motta,
V. Zhang, S. Bhansali, M.L. Fjaellskog, M. Oliveira
500
Poster board
lxv
Abstract number
P073 Immunological STAT3 knockdown associated with anti-tumor activity in pre-clinical models translates
to clinical samples, suggesting immune modulation contributes to the clinical activity of AZD9150, a
therapeutic STAT3 ASO
P. McCoon, R. Woessner, R. DuPont, K. Bell, M. Collins, L. Pablo, D. Lawson, P. Nadella, V. Jacobs,
C. Womack, C. Reimer, D. Hong, J. Nemunaitis, Y.K. Kang, T.Y. Kim, H.Y. Lim, T. Okusaka,
S. Nadano, C.C. Lin, P. Lyne
P074 MI130004, an antibody–drug conjugate including a novel payload of marine origin: Evidences of
in vivo activity
P.M. Aviles, M.J. Guillen, J.M. Dominguez, M.J. Muñoz-Alonso, L.F. Garcia-Fernandez, M. Garranzo,
V. Martinez, A. Francesch, S. Munt, C.M. Galmarini, C. Cuevas
P075 UNC2025: A small molecule inhibitor of merTK with efficacy in mouse melanoma models
S. Earp, D. Darr, A. Holtzhausen, A. Zimmermann, K. Clark, L. Hunter, N. Sharpless, X. Wang,
S. Frye, D. Graham
P076 Frequent loss-of-function mutations in MLK4 suppresses signaling in the JNK-cJUN-p21/p15
pathway to promote growth of colon cancer cells
A. Marusiak, N. Stephenson, H. Baik, E. Trotter, Y. Li, E. Testoni, K. Blyth, S. Mason, L. Puto,
C. Miller, T. Hunter, O. Sansom, J. Brognard
P077 The anti-ErbB3 antibody, EV20, counteracts vemurafenib resistance in BRAF-mutated colorectal
cancer stem cells
G. Sala, P.R. Prasetyanti, D. Barcaroli, S. Volpe, E. Capone, C. Rossi, R. Carollo, M. Todaro,
G. Stassi, J.P. Medema, S. Iacobelli, V. De Laurenzi
P078 Phase (Ph) 1/2a study of TSR-011, a potent inhibitor of ALK and TRK, in advanced solid tumors
including crizotinib-resistant ALK positive non-small cell lung cancer
J. Sachdev, H.T. Arkenau, J.R. Infante, M.M. Mita, S.P. Anthony, R.B. Natale, S. Ejadi, K. Wilcoxen,
V. Kansra, H. Laken, L. Hughes, R. Martell, G.J. Weiss
P079 Amplification of D-type cyclin genes CCND2 and CCND3 confers an oncogene addiction phenotype
in tumor cells and hypersensitivity to the CDK4/6 inhibitor LY2835219
S. Buchanan, X. Gong, M. Lallena, L. Chio, F. Merzoug, J. Dempsey, R. Beckman, C. Reinhard
P080 A phase Ib trial of AMG386 and temsirolimus in patients with advanced sold tumors
(PJC-008/NCI#9041)
J.W. Chiu, S.J. Hotte, C.K. Kollmannsberger, D.J. Renouf, D.W. Cescon, D. Hedley, S. Chow,
J. Moscow, M. Perry, I. Diaz-Padilla, D. Tan, H. Hirte, E. McWhirter, H. Chen, L.L. Siu, P.L. Bedard
P081 Detecting gene amplification in circulating tumor cells of patients with gastric cancer for clinical trial
Y. Mishima, S. Matsusaka, Y. Terui, M. Mikuniya, T. Takayama, N. Mizunuma, K. Hatake
P082 Inhibition of STAT3 enhances the radiosensitising effect of temozolomide in malignant glioma cells
in vitro and in vivo
I. Kim, E. Choi, B. Cho, T. Han, S. Song, J. Kim, S. Paek
501
P083 ComboPredictor: identification of synergy biomarkers and enrichment within tumor sample
populations
M. Tomilo, A. Bankhead III, M.E. Urick, S. Eddy, R. Rickles, J. Ledell, E.T. Bowden
511
P084 Somatically mutated ABL1 represents an actionable and essential lung cancer survival gene
E. Testoni, N.L. Stephenson, A.A. Marusiak, E.W. Trotter, A. Hudson, J. Brognard
P085 Synergistic inhibition of ovarian and endometrial cancer cell lines using combined treatment of
ARQ 092 and ARQ 087 in vitro and in vivo
Y. Yu, E. Nakuci, E. Chiesa, C.R. Chen, E. Marchlik, D.T. Dransfield
P086 Hypoxia inducible factor (HIF)-1a expression levels and p53 mutations are prognostic factors for
survival in breast cancer patients treated with neoadjuvant chemotherapy
M. Molina-Vila, S. Baulies, M. Gonzalez-Cao, N. Karachaliou, A. Rodriguez Capitan, S. Viteri,
M. Cusido, R. Fabregas, C. Teixido, R. Rosell
512
502
503
504
505
506
507
508
509
510
513
514
lxvi
Poster board
Abstract number
P087 TAS-115, a potent MET/VEGFR-targeted kinase inhibitor, is a new therapeutic approach for the
treatment of bone metastasis of lung cancer
A. Gomori, M. Sakuragi, A. Hashimoto, K. Ito, T. Haruma, T. Suzuki, H. Fujita, Y. Fujioka,
K. Yonekura, T. Utsugi
515
P088 Different genetic profiles of resistant and sensitive patients with EGFR wild type NSCLC undergoing
tyrosine kinase inhibitor (TKI) treatment
P.U. Ulivi, E.C. Chiadini, A.D. Dubini, D.C. Calistri, M.P. Puccetti, M.A.B. Burgio, A.D. Delmonte,
A.V. Verlicchi, A.G. Gamboni, M.P. Papi, W.Z. Zoli, L.C. Crinò, C.D. Dazzi
516
P089 Identification and characterization of selective MELK kinase inhibitors
P. Carpinelli, N. Amboldi, D. Ballinari, S. Re Depaolini, U. Cucchi, G. Canevari, M. Caruso,
R. Galli, D. Donati, A. Isacchi, E.R. Felder, A. Montagnoli, A. Galvani
517
P090 The HSP90 inhibitor, AT13387, overcomes resistance to crizotinib and second generation ALK
inhibitors
A. Courtin, A. Smyth, K. Hearn, V. Martins, J. Lewis, N. Thompson, J. Lyons, N. Wallis
518
P091 AD-O53.2 − novel biological anticancer therapeutic agent with a dual mechanism of action
K. Poleszak, J. Pieczykolan, S. Pawlak, B. Zerek, P. Rozga, M. Teska-Kaminska, M. Galazka,
M. Szymanik, A. Jaworski, A. Pieczykolan, K. Bukato, W. Strozek
519
P092 Validation of Nanostring for FGFR1 gene expression analysis in squamous non-small cell lung cancer
(sqNSCLC) tissue
C. Rooney, C. Geh, V. Williams, C. Cresswell, K. Al-Kadhimi, M. Dymond, T. French, P.D. Smith,
C. Barrett, E.A. Harrington, E. Kilgour
520
P093 Tisular expression of the EGFR and N-Glycolyl GM3 Ganglioside as recognized by ior egf/r3 and
14F7 Mabs in triple negative breast cancer
A. Calvo Pérez, R.I. Alvarez Goyanes, E. Rengifo Calzado, S. Franco Odio, R. Camacho Rodrı́guez
521
P094 Evaluation of the efficacy of melatonin in breast cancer metastasis mediated by ROCK-1
T. Borin, A.S. Arbab, L.C. Ferreira, G.B. Botaro, L.B. Maschio, G.M. Moschetta, N.N. Gonçalves,
G.R. Martins, D.A.P.C. Zuccari
522
P095 Antitumor activity of mTOR kinase and DNA-PK inhibitor CC-115 in a mouse model of
glioblastoma
H. Raymon, S. Peng, J. Katz, C. Zhao, C. Phan, M.F. Moghaddam, K.E. Fultz, S. Sankar,
D.S. Mortensen, R.K. Narla
523
P096 Targeting MET for a differentiation therapy of rhabdomyosarcoma
K. Skrzypek, A. Jagiello, B. Szewczyk, T. Adamus, K. Miekus, M. Majka
524
P097 Analysing Src and phosphoinositide 3-kinase pathway inhibition in a radiotherapy context − pathway
interplay and therapeutic response
E. Rowling, N. Burrows, P. Elvin, K. Williams
525
P098 A Phase I study of the dual, intravenous (IV) phosphatidylinositol 3-kinase (PI3K)/mammalian target
of rapamycin (mTOR) inhibitor PF-05212384 in combination with irinotecan in patients (pts) with
colorectal cancer (CRC) and other advanced solid tumors
Z. Wainberg, J. Tabernero, M.A. Maqueda, S. Leong, G. Del Conte, C. Britten, N. Brega, C. Davis,
B. Houk, K. Pierce, J. Vermette, L. Siu, I. Brana
526
P099 Is CRAF required for the maintenance of KRAS mutant non-small cell lung cancer?
P. Russell, J. Wickenden, K. Cadwallader, S. Maguire, J. Joel, M. Stockdale, A. Chicas, D. Banka,
R. Darman, S. Perino, P. Fekkes, P. Smith, P. Zhu, S. Buonamici, J. Moore
527
P100 A procaspase activator shows preclinical promise for glioblastoma therapy
G. Riggins, A. Joshi, P.J. Hergenrother
528
P101 The Cancer Research UK Stratified Medicine Programme: From national screening to national trial
I. Mirabile, E. Shaw, C. Lindsay, I. Walker, P.W.M. Johnson
529
Poster board
lxvii
Abstract number
P102 Anti-angiogenic therapy induces T-lymphocyte infiltration associated with poor survival in metastatic
renal cell carcinoma patients
X.D. Liu, A. Hoang, L. Zhou, S. Kalra, A. Yetil, M. Sun, Z. Ding, S. Bai, P. German, X. Zhang,
P. Tamboli, P. Rao, J. Karam, C. Wood, S. Matin, A. Zurita, N. Tannir, K. Sircar, E. Jonasch
P103 In vitro and in vivo anti-tumor activity of ARQ 092, a potent and selective pan-AKT inhibitor
Y. Yu, S. Cornell-Kennon, C.R. Chen, E. Marchlik, T. Isoyama, K. Tazaki, K. Fujiwara,
D.T. Dransfield
P104 Evaluation of a BRCAness signature as a predictive biomarker of response to veliparib/carboplatin
plus standard neoadjuvant therapy in high-risk breast cancer: results from the I-SPY 2 trial
A. Glas, J. Peeters, C. Yau, D.M. Wolf, A. Sanil, Y. Li, T. Severson, S. Linn, I-SPY 2 TRIAL
Investigators, M. Buxton, A. DeMichele, N. Hylton, F. Symmans, D. Yee, M. Paoloni, L. Esserman,
D. Berry, H. Rugo, O. Olopade, L.J. van ’t Veer
P105 Detection of recurrent novel fusion transcripts from whole transcriptome sequencing of 120 primary
breast cancer
J. Kim, S.Y. Go, S.W. Kim, S. Lee, H.S. Lee, J. Park, M.J. Lee, H.G. Moon, D.Y. Noh, S. Kim, S. Kim,
W. Han
P106 Expression of c-MET in invasive meningioma
S. Yun, J. Koh, K. Lee, A. Seo, K. Nam, G. Choe
530
531
532
533
534
P107 Tumor targeting and tissue distribution of solitomab (AMG 110; anti-EpCAM BiTE® ) in human
EpCAM-positive tumor bearing mice
F.J. Warnders, S. Waaijer, M.N. Lub-de Hooge, M. Friedrich, A.G.T. Terwisscha van Scheltinga,
P. Deegen, S.K. Stienen, P.C. Pieslor, H.K. Cheung, J.G.W. Kosterink, E.G.E. de Vries
P108 Shifts in microRNA expression pattern can facilitate the cancer cell stemness
V. Halytskiy
P109 Absolute quantitation of MET using mass spectrometry for clinical application: assay precision,
stability, and correlation with MET gene amplification in FFPE tumor tissue
F. Cecchi, W.L. Liao, S. Thyparambil, K. Bengali, J. Uzzell, M. Darflar, D. Krizman, J. Burrows,
T. Hembrough, T. Veenstra, D.P. Bottaro, T. Karrison, L. Henderson, P. Xu, B. Rambo, S.Y. Xiao,
L. Zhao, J. Hart, D. Catenacci
P110 Development of a novel anti-tumor antibody targeting CXADR
M. Kawada, H. Inoue, M. Kajikawa, M. Sugiura, S. Sakamoto, S. Urano, T. Masuda, A. Nomoto
P111 Reversing the epithelial to mesenchymal transition with N-myc downstream regulated gene-1 and
novel iron chelators in pancreatic cancer
L. Fouani, Z. Kovacevic, D. Richardson
535
P112 Vemurafenib alters glucose utilization in BRAF-driven human melanoma cells
M. Falck Miniotis, T. Delgado-Goni, S. Wantuch, P. Workman, R. Marais, M.O. Leach,
M. Beloueche-Babari
540
P113 The abnormal bone remodeling associated with prostate cancer bone metastasis is attenuated by TAS115, the dual inhibitor for HGF/VEGF signaling
H. Fujita, C. Matsumoto, K. Yonekura, K. Watanabe, M. Hirata, C. Miyaura, T. Utsugi, M. Inada
P114 Tivantinib in combination with gemcitabine shows strong antitumor activity on mesothelioma cell
lines and cytoskeletal effects via inhibition of actin
M. Simonelli, P. Zucali, M. Suter, L. Rubino, A. Santoro, C. Carlo-Stella
P115 MEK1/2 specific inhibitor, SMK-17 selectively induces apoptosis in b-catenin mutated tumors
M. Kiga, A. Nakayama, Y. Sasazawa, Y. Shikata, H. Ikeda, E. Tashiro, M. Imoto
P116 Preclinical studies of a dual Bcl-2/Bcl-xL inhibitor APG-1252 with strong anti-tumor efficacy and
significantly reduced platelet toxicity
H. Wang, G. Wang, Z. Du, M. Wu, D. McEachern, A. Aguilar, Y. Lin, X. Lin, J. Wen, L. Gu,
M.I.N.G. Guo, Y. Zhai, S. Wang, D. Yang
541
536
537
538
539
542
543
544
lxviii
Poster board
Abstract number
P117 MiRNAs related to KRAS mutational status in resectable non-small cell lung cancer
S. Gallach, S. Calabuig-Fariñas, E. Jantus-Lewintre, D. Montaner, E. Escorihuela, J. Castellano,
S. Figueroa, A. Blasco, C. Hernando, R. Guijarro, C. Camps
545
P118 The PI3Kbeta/delta inhibitor AZD8186 combines with the dual mTORC1/2 inhibitor AZD2014 to
give comprehensive PI3K pathway inhibition and drive tumour regression in vivo
B. Simon, U. Hancox, U. Polanska, L. Hanson, P. Dudley, R. Ellston, J. Maynard, M. Kraus,
J. Curwen, T. Klinowska, L. Ward, F. Cruzalegui, S. Symeonides, K. Cronin
546
P119 Preclinical activity of Debio 1347, an oral selective FGFR1, 2, 3 inhibitor, in models harboring FGFR
alterations
A. Vaslin Chessex, C. Moulon, V. Nicolas-Métral, A. Ménétrey, H. Maby-El Hajjami, S. Rigotti,
C. Zanna, G. Vuagniaux
547
P120 Development of a small molecule activator of protein phosphatase 2A for the treatment of prostate
cancer
K. McClinch, D. Callejas, M. Cooper, A. Stachnik, D. Kastrinsky, M. Ohlmeyer, M. Galsky, G. Narla
548
P121 A novel cancer marker and potential therapeutic target
M.P. Parri, S.C. Campagnoli, A.G. Grandi, A.S. Santi, E.D.C. De Camilli, G.V. Viale, P.C. Chiarugi,
L.T. Terracciano, P.P. Pileri, R. Grifantini
549
New Therapies with Pleiotropic Activity
Poster board
Abstract number
P122 Nupharidine inhibits NF-kB activity, induces apoptosis and has synergistic cytotoxic activity with
cisplatin and etoposide
J. Gopas, J. Ozer, N. Eisner, D. Benharroch, A. Golan-Goldhirsh
550
P123 Phase 1B study of CC-486 (oral azacitidine) in tumors associated with a viral etiology
D.D. Von Hoff, N. Isambert, J. Lopez-Martin, P.N. Munster, D.W. Rasco, J.C. Bendell,
J.H.M. Schellens, J. Tomaro, R. Sarmiento, K. Liu, A. Nguyen, G.L. Bray, S.R. Hatty, J.F. DiMartino,
C. Le Tourneau
551
P124 Discovery of an unexpected vulnerability of cancer stem-like cells via a functional dissection of EMT
biology
Y. Feng, E.S. Sokol, C.A. Del Vecchio, S. Sanduja, J.H. Claessen, T.A. Proia, D.X. Jin, F. Reinhardt,
H.L. Ploegh, Q. Wang, P.B. Gupta
552
P125 PRPF6, a tri-snRNP spliceosome protein, regulates the alternative splicing of a distinct oncogenic
ZAK variant and promotes colon tumor growth
R. Firestein, A. Adler, M. McCleland, M. Yaylaoglu, Z. Zhang, J. Liu, Z. Jiang
553
P126 Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast
cancer cells
R. Brown, M. Fuchter, E. Curry, I. Green, S. Kandil, F. Cherblanc, L. Payne, N. Chapman-Rothe,
E. Shamsaei, N. Srimongkolpithak, J. Snyder, M. Vedadi
554
P127 CB-5083, a first in class inhibitor of the AAA-ATPase p97/VCP, induces irresolvable ER stress that
results in antitumor activity in solid and hematological tumor models
F. Yakes, R. Le Moigne, S. Wong, D.J. Anderson, S. Djakovic, E. Valle, M.K. Menon, J. Rice, B. Yao,
F. Soriano, J. Wang, S. Kiss von Soly, M. Chesi, P.L. Bergsagel, H.J. Zhou, M. Rolfe, D. Wustrow
555
P128 Antagonizing microRNA mediated epigenetic reprogramming as therapeutic strategy for aggressive
prostate cancer
C. Dallavalle, D. Albino, G. Civenni, P. Ostano, M. Mello-Grand, R. Garcia-Escudero, G. Chiorino,
C.V. Catapano, G.M. Carbone
556
P129 A rational approach for discovery of inhibitors of YAP−TEAD interaction
L. Chene, A. Soude, C. Valaire, S. Delaporte, S. Jacquet, Y. Cambet, I. Braccini, M. Barth,
C. Montalbetti, P. Broqua, C. Fromond
557
Poster board
lxix
Abstract number
P130 RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
H. Wendel, K. Singh, A. Wolfe, Y. Zhong, P. Drewe, J. Porco, J. Pelletier, G. Rätsch
P131 Development of a unique biologic for treating cysteine-dependent malignancies
E. Stone, S. Cramer, A. Saha, S. Tiziani, J. Digiovanni, G. Georgiou
P132 ZEN3365 is a novel BET bromodomain inhibitor for the treatment of hematologic malignancies and
solid tumors
K.G. McLure, R. Jahagirdar, O.A. Kharenko, E.M. Gesner, E. Campeau, D. Gilham, J. Wu,
L. Tsujikawa, S. Attwell, C. Calosing, N. Sharma, J. Tobin, H.C. Hansen
P133 Nonclinical characterization of the first in class investigational ubiquitin activating enzyme inhibitor
MLN7243 in cellular and in vivo models of cancer in support of a phase I study
M. Milhollen, M. Hyer, J. Ciavarri, T. Traore, D. Sappal, J. Huck, J. Shi, J. Duffy, J. Gavin,
J. Brownell, Y. Yang, B. Stringer, Y. Ishii, E. Koenig, A. Lublinsky, R. Griffin, C. Xia, J. Powe,
P. Fleming, N. Bence
P134 Lysophosphatidic acid-induced breast cancer metastasis depends on LPA1/ZEB1/miR-21-activation
pathway
D. Sahay, R. Leblanc, J. Ribeiro, P. Clezardin, O. Peyruchaud
P135 Hsp90 pharmacoproteomics: Harnessing pleiotropy for therapeutic synergy
R. Goldstein, G. Chiosis, L.C. Cerchietti, A.M. Melnick
P136 OTX015, a novel BET-BRD inhibitor is active in non-small-cell lung cancer cell (NSCLC) lines
harboring different oncogenic mutations
M. Riveiro, L. Astorgues-Xerri, N. Ijaz, M. Bekradda, R. Vazquez, R. Frapolli, A. Rinaldi, I. Kwee,
E. Cvitkovic, E. Raymond
P137 Preclinical evaluation of OTX015, a novel BET-BRD inhibitor, on small cell lung cancer (SCLC) cell
lines
N. Ijaz, L. Astorgues-Xerri, E. Odore, M. Bekradda, E. Cvitkovic, K. Noel, E. Raymond, M. Riveiro
P138 GNS396 and analogues are potent new small molecules to target and kill chemotherapy-resistant
subpopulation cells in acute myeloid leukemia
F. Bassissi, R. Castellano, E. Josselin, C. Motersino, L. Pouyet, A. Goubard, A. Rostouin, G. Nicolas,
S. Brun, J. Courcambeck, C. Dubray, T. Prébet, N. Vey, A. Béret, P. Halfon, Y. Collette
P139 OTX015, a BET-bromodomain (BET-BRD) inhibitor, potentiates the in vitro effects of chemotherapy
drugs and targeted agents in human leukemic cell lines
L. Astorgues-Xerri, C. Canet-Jourdan, M. Bekradda, E. Cvitkovic, P. Herait, E. Raymond, M. Riveiro
P140 The BET bromodomain inhibitor OTX015 shows synergy with several anticancer agents in preclinical
models of mantle cell lymphoma (MCL) and multiple myeloma (MM)
E. Bernasconi, C. Tarantelli, E. Gaudio, I. Kwee, A. Stathis, E. Riveiro, P. Herait, E. Cvitkovic,
E. Zucca, F. Bertoni
P141 Biological consequences of selective inhibition of the first BET bromodomain
J.G. Seitzberg, M.H. Hansen, T.T.A. Kronborg, C.R. Underwood, V. Polyak, G. Friberg, B. Tonnesen,
M. Nørregaard-Madsen, L. Teuber, T. Franch, S. Nielsen
P142 Modulation of chromatin-related processes in DNA damage response as a potential strategy to treat
acute myeloid leukemia
K. Chomej, M. Lagergren Lindberg, D. Zong, T. Juntti, L. Kanter, L. Stenke, R. Lewensohn,
K. Viktorsson, P. Hååg
P143 Novel potent inhibitors of the Histone Demethylase KDM1A: Synthesis, pharmacological evaluation
and in vivo activity
M. Varasi, O. Botrugno, A. Cappa, R. Dal Zuffo, P. Dessanti, A. Mai, A. Mattevi, C. Mercurio,
G. Meroni, S. Minucci, F. Thaler, P. Trifiro’, S. Valente, P. Vianello, M. Villa
P144 Oral panobinostat in patients with advanced tumors and impaired renal function: Relationship
between pharmacokinetics and key safety parameters
M. Porro, S. Sharma, P.O. Witteveen, M.P. Lolkema, D. Hess, H. Gelderblom, S.A. Hussain,
E. Waldron, S. Valera, S. Mu
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
lxx
Poster board
Abstract number
P145 Suppression of c-Myc oncogene and hematological tumor growth by a novel benzazepine BET
inhibitor
K.A. Gelato, P. Lejeune, A.E. Fernández-Montalván, R. Neuhaus, N. Schmees, S. Siegel, H. Weinmann,
V. Gekeler, B. Haendler
P146 M-COPA, a Golgi disruptor, inhibits cell surface expression of MET protein and exhibits antitumor
activity against MET-addicted cancers
Y. Ohashi, M. Okamura, A. Hirosawa, A. Akatsuka, I. Shiina, K. Yoshimatsu, T. Yamori, S. Dan
P147 SF3B1 mutations are associated with alternative splicing in ER-positive breast cancer
S. Maguire, A. Leonidou, P. Wai, C. Marchio, C.K.Y. Ng, B. Weigelt, A. Sapino, A. Vincent-Salomon,
J.S. Reis-Filho, R. Natrajan
P148 Novel inhibitors of peritoneal seeding
Y. Shen, X.L. Li, X. Lu, S. Kuwada
P149 Characterization of the cellular mechanism of action of the first in class investigational inhibitor of
the Ubiquitin Activating Enzyme, MLN7243
M. Milhollen, D. Sappal, J. Duffy, K. Hoar, J. Huck, P. Sha, E. Koenig, M. Hyer, J. Ciavarri,
N. Bence
P150 Invadopodia are required for cancer cell extravasation and are a therapeutic target for metastasis
H.S. Leong, A.E. Robertson, K. Stoletov, S.J. Leith, C.A. Chin, A.E. Chien, M.N. Hague, A.L. Ablack,
K. Carmine Simmen, V.A. McPherson, C.O. Postenka, E.A. Turley, S.A. Courtneidge, A.F. Chambers,
J.D. Lewis
P151 Eph/ephrin-B interactions modulate a BAFF-R/TACI dependent survival of chronic lymphocytic
leukemia (CLL) cells mediated in vitro by bone marrow stromal cells
L.M. Alonso-Colmenar, A.G. Zapata, P. Fortea, M.A. Flores, F. Ortuño, G. Soler, M.D. Garcı́a,
J. Garcı́a-Cantalejo
P152 Evaluation of the pan-BET-bromodomain inhibitor OTX015 as a single agent and in combination with
everolimus (RAD001) in triple-negative breast cancer models
R. Vázquez, L. Astorgues-Xerri, M.E. Riveiro, M. Di Marino, L. Beltrame, M. Bekradda, E. Cvitkovic,
E. Erba, R. Frapolli, M. D’Incalci
P153 Identification of genomic and chromatin features that predict transcriptional response to BET
bromodomain inhibition
J. Mertz, H.R. Huang, N. Follmer, A. Reddy, R. Centore, B. Bryant, C. Hatton, H. Franco,
W.L. Krause, R. Sims III
P154 Rational for targeting chromatin-modifying genes in clear-cell renal cell carcinomas
G. Malouf, J. Zhang, D. Khayat, X. Su, J.P. Spano
P155 The 8p11 amplicon in luminal breast cancers harbors multiple interacting epigenome modifying
oncogenes: implications for epigenome-targeted therapy
S. Ethier, J. Irish, R. Wilson, B. Turner
P156 The correlation between EMT and cancer stemness in lung adenocarcinoma affects on its prognosis
T. Sowa, T. Menju, T. Nakanishi, K. Shikuma, N. Imamura, T. Yamada, A. Aoyama, M. Sato, T. Sato,
F. Chen, M. Sonobe, M. Omasa, H. Date, T. Sozu
P157 The investigational HSP90 inhibitor ganetespib displays robust single agent activity in gastric cancer
models both as monotherapy and in combination with standard of care therapeutics
S. He, C. Zhang, J.P. Jimenez, J. Sang, M. Sequeira, D. Smith, J. Acquaviva, M. Nagai, R. Bates,
D.A. Proia
P158 Phase I study of pan-histone deacetylase inhibitor abexinostat in combination with cisplatin in
patients with advanced solid tumors
M. Campone, N. Isambert, R. Sabatier, H. Castanie, S. Zanetta, I. Sudey, F. Cantero, J. Pauly,
E. Leroux, S. Malasse, A. Goncalves
P159 Cellular pharmacokinetics and molecular pharmacodynamics studies of the BRD-BET inhibitor
OTX015 in sensitive and resistant leukemic cell lines
E. Odore, L. Astorgues-Xerri, M. Bekradda, E. Cvitkovic, P. Herait, F. Lokiec, K. Rezai, M. Riveiro
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
Poster board
lxxi
Abstract number
P160 Neddylation as a therapeutic target in refractory pediatric malignancies: Evaluation of the activating
enzyme inhibitor MLN4924
Y. Ruan, A. Jayanthan, T. Cooper, A. Narendran
588
P161 N-myc downstream regulated gene 1 (NDRG1) as a novel anti-angiogenic and therapeutic target for
VEGF/VEGF receptor signaling by vascular endothelial cells
K. Watari, A. Shinoda, T. Shibata, A. Kawahara, T. Nakama, S. Yoshida, M. Kage, M. Kuwano,
M. Ono
589
P162 A dose dense schedule improves antitumor activity of trabectedin in myxoid liposarcoma with type III
FUS-CHOP chimera
S. Uboldi, R. Frapolli, E. Bello, S. Brich, F. Bozzi, R. Sanfilippo, P.G. Casali, A. Gronchi,
C.M. Galmarini, J.M. Fernandez Sousa-Faro, S. Pilotti, M. D’Incalci
590
P163 Mechanistic analysis of reversible FASN inhibition in preclinical tumor models identifies highly
susceptible tumor types and enriches biomarker discovery for clinical applications
T.S. Heuer, R. Ventura, J. Waszczuk, K. Mordec, J. Lai, M. Fridlib, R. Johnson, L. Hu, H. Cai,
A. Wagman, M. O’Farrell, D. Buckley, G. Kemble
591
P164 TAS-116, a highly selective inhibitor of heat shock protein 90a/b, inhibits tumor growth in biliary
tract cancer mouse models
S. Ohkubo, H. Muraoka, Y. Kodama, K. Ito, S. Ito, A. Hashimoto, C. Yoshimura, T. Utsugi
592
P165 Strategies to overcome resistance to BET bromodomain inhibitor in KRAS/LKB1 mutant NSCLC
M. Soucheray, E. Kikuchi, I. Pulido, E. Akbay, J.H. Becker, C.L. Christensen, N. Johnson, T.B. Patel,
J. Carretero, K.K. Wong, T. Shimamura
593
P166 Identification of novel EZH2 inhibitor scaffolds
X. Espanel, L. Chene, A. Soude, S. Estevez, V. Adarbes, B. Loillier, B. Boubia, P. Masson,
C. Montalbetti, P. Broqua, C. Fromond
594
P167 Improving specificity of epigenetic therapy through combined targeting of DNA and histone
methylation
T. Sato, M. Cesaroni, J. Jelinek, J.P. Issa
595
th
26 EORTC–NCI–AACR Symposium on
Plenary Session 1
15:15–17:30
PLENARY SESSION 1
Is the Genomic Landscape Changing
the Outcome for Cancer Patients?
1
ORAL PRESENTATION
Feasibility of large-scale genomic testing to facilitate enrollment on
genomically-matched clinical trials
F. Meric-Bernstam1 , L. Brusco2 , S. Kopetz3 , M. Davies4 , M.J. Routbort5 ,
S.A. Piha-Paul2 , R. Alvarez6 , S. Khose2 , J. DeGroot7 , V. Ravi8 , F. Janku2 ,
D. Hong2 , Y. Li9 , R. Luthra5 , K.P. Patel5 , R. Broaddus10 , K. Shaw11 ,
J. Mendelsohn11 , G.B. Mills12 . 1 The University of Texas MD Anderson
Cancer Center, Investigational Cancer Therapeutics, Houston Texas, USA;
2
The University of Texas MD Anderson Cancer Center, Investigational
Cancer Therapeutics, Houston Texas, USA; 3 The University of Texas
MD Anderson Cancer Center, GI Medical Oncology, Houston Texas,
USA; 4 The University of Texas MD Anderson Cancer Center, Melanoma
Medical Oncology, Houston Texas, USA; 5 The University of Texas
MD Anderson Cancer Center, Hematopathology, Houston Texas, USA;
6
The University of Texas MD Anderson Cancer Center, Breast Medical
Oncology, Houston Texas, USA; 7 The University of Texas MD Anderson
Cancer Center, Neuro-Oncology, Houston Texas, USA; 8 The University
of Texas MD Anderson Cancer Center, Sarcoma Medical Oncology,
Houston Texas, USA; 9 The University of Texas MD Anderson Cancer
Center, Biostatistics, Houston Texas, USA; 10 The University of Texas
MD Anderson Cancer Center, Pathology, Houston Texas, USA; 11 The
University of Texas MD Anderson Cancer Center, Institute for Personalized
Cancer Therapy, Houston Texas, USA; 12 The University of Texas
MD Anderson Cancer Center, Systems Biology, Houston Texas, USA
Background: We sought to determine the feasibility of performing
standardized somatic mutation analysis for patients with a variety of
advanced cancer types at a major cancer center. We report the experience
with the first 2000 patients who underwent testing on a genomic testing
protocol, including the frequency of actionable alterations across tumor
types, subsequent enrollment on clinical trials, and the challenges for trial
enrollment.
Materials and Methods: From February 2012 to July 2013, after
informed consent, 2000 patients underwent genomic testing using a
‘hot-spot’ platform, an 11-gene Sequenom assay (251 patients) or 46gene Ion Torrent (Ampliseq) assay (1749 patients). Of the 46 genes
tested, 35 were considered ‘potentially actionable’, as these genes
might be directly or indirectly targeted by approved or investigational
agents. TP53 was not considered actionable. Trials that require a
genomic alteration for enrollment were considered ‘genotype-selected’, and
biomarker-unselected trials using agents relevant to a genomic alteration
were designated ‘genotype-relevant’; either type of trial was considered
‘genomically-matched’.
Results: Of the 2000 patients, 38.4% had at least one potentially
actionable alteration. The most commonly mutated potentially actionable
genes were PIK3CA (12.8%), KRAS (11.3%), BRAF (7%), NRAS (4.2%),
EGFR (1.8%), AKT1 (1.4%) and PTEN (hot-spot mutations, 1.2%). Patients
with mutations in potentially actionable genes were more likely to be treated
on clinical trials than those without mutations (30% vs 25%, p = 0.0046). Of
the patients with mutations in potentially actionable genes, 7% were treated
on genotype-selected trials, 5% were treated on genotype-relevant trials
and 20% were treated on other therapeutic trials. Of 97 patients treated
on genotype-matched trials, 67% had PIK3CA/AKT1/PTEN or BRAF
mutations. Eighty-five patients with PI3K/AKT1/PTEN/BRAF mutations had
a discussion about genotype-matched trials documented but were not
enrolled; most common reasons were election of treatment closer-to-home
(22%), non-investigational therapy (20%), poor performance status (13%),
ineligibility for trials for other reasons (17%), stable disease (9%), lack of
relevant trials (8%) or enrollment in other trials (6%).
Discussion: Broad implementation of multiplex hot-spot testing is feasible
and identifies potentially actionable alterations. Patients with actionable
alterations are more likely to be enrolled on clinical trials; however, only
a small portion of patients with actionable alterations were enrolled on
genotype-matched trials. Novel solutions to increase awareness of test
results and therapeutic implications, increased awareness of existing trials
such as clinical trial alert systems, and increased access to targeted drugs
through basket trials and novel just-in-time clinical trial systems are needed.
3
th
Plenary Session 2
13:15–15:20
PLENARY SESSION 2
2
ORAL PRESENTATION
Safety and early evidence of activity of a first-in-human phase I study
of the novel cancer stem cell (CSC) targeting antibody OMP-52M51
(anti-Notch1) administered intravenously to patients with certain
advanced solid tumors
A. Patnaik1 , P. LoRusso2 , P. Munster3 , A.W. Tolcher1 , S.L. Davis4 ,
J. Heymach5 , R. Ferraroto5 , L. Xu6 , A.M. Kapoun6 , L. Faoro6 ,
J.A. Lewicki6 , J. Dupont6 , S.G. Eckhardt4 . 1 South Texas Accelerated
Research Therapeutics (START), San Antonio TX, USA; 2 Wayne State
University Karmanos Cancer Institute Detroit MI, Center for Translational
Therapeutics, Detroit MI, USA; 3 University of California San Francisco,
Department of Medicine, San Francisco CA, USA; 4 University of
Colorado-Denver, University of Colorado Cancer Center, Aurora CO,
USA; 5 MD Anderson Cancer Center, Houston TX, USA; 5 Oncomed
Pharmaceuticals Inc., Redwood City CA, USA
Background: The Notch pathway plays a key role in embryonic
development, the regulation of stem and progenitor cells, and is implicated
in human cancer. Notch1 (N1) signaling is activated by various mechanisms
including N1 activating mutations in certain solid tumors. OMP-52M51 is
a humanized IgG2 antibody that inhibits the signaling function of N1. As
such, OMP-52M51 is a novel anti-cancer agent that inhibits tumor growth
through direct actions on tumor cells, including CSCs, and effects on tumor
angiogenesis.
Materials and Methods: A phase I dose escalation and expansion
study was initiated in patients (pts) with certain advanced solid tumors
(cholangiocarcinoma, breast (BC), colorectal (CRC), esophageal, gastric,
pancreatic, and small cell lung cancers) that have rates of N1 activation
between 12−29%. OMP-52M51 was administered intravenously to study
safety, pharmacokinetics (PK), pharmacodynamics, preliminary efficacy,
and to determine the maximum tolerated dose. The trial has a N1 IHC
biomarker selected expansion cohort.
Results: 20 pts have been enrolled in 5 cohorts at doses of 0.25, 0.5,
1, and 2.5 mg/kg every 4 weeks (Q4W) and 2.5 mg/kg every 3 weeks
(Q3W). The most frequent adverse events (AE) were: mild to moderate
diarrhea (75%), fatigue (65%), and nausea (40%). Grade 3 or higher
AEs included diarrhea (20%), increased alkaline phosphatase (15%), and
pain (15%). Diarrhea was manageable. Two pts experienced dose-limiting
toxicity (DLTs) AEs with gr 3 fatigue (2.5 mg/kg Q4W) and gr 3 diarrhea
(2.5 mg/kg Q3W). PK exhibited dose-dependent clearance with a T 12 of
4 days at 2.5 mg/kg. One pt with an N1 activating mutation in adenoid
cystic carcinoma (ACC) had partial response (38% decrease in lesions)
after 2 doses. 1 BC and 1 CRC pt had stable disease (110 days and
280 days, respectively). Sequencing the cell-free DNA from the CRC pt
found a loss-of-function mutation in FBXW7, a negative regulator of N1. A
prototype immunohistochemistry (IHC) test showed high levels of activated
N1 in the ACC and CRC pts. Biomarker analyses revealed reductions in
circulating tumor cells with OMP-52M51 treatment. The MTD has not been
established.
Conclusions: OMP-52M51 is generally well tolerated. Diarrhea is the
primary toxicity of this antibody. Potential early efficacy consistent with the
predictive biomarker hypothesis is noted. Enrollment continues. Updated
efficacy, safety, and PK results will be presented.
Clinical trial information: NCT01778439.
7
3
ORAL PRESENTATION
Afuresertib (GSK2110183), an oral AKT kinase inhibitor, in
combination with carboplatin and paclitaxel in recurrent ovarian
cancer
S. Blagden1 , A. Hamilton2 , L. Mileshkin3 , M. Hall4 , T. Meniawy5 ,
S. Wong6 , S. Anandra2 , M. Buck5 , D. McAleer7 , B.A. Reedy7 ,
R.B. Noble7 , D.A. Smith8 , S.R. Morris9 , H. Gabra1 . 1 Imperial College
Hammersmith Hospital Campus, Department of Medical Oncology,
London, United Kingdom; 2 Royal Women’s Hospital, Department of
Medical Oncology, Parkville, Australia; 3 Peter MacCallum Cancer Centre,
Department of Cancer Medicine, East Melbourne, Australia; 4 Mount
Vernon Cancer Centre, Department of Medical Oncology, Middlesex,
United Kingdom; 5 Sir Charles Gairdner Hospital, Department of Medical
Oncology, Perth, Australia; 6 Western Hospital, Department of Oncology,
Footscray, Australia; 7 GlaxoSmithKline, GSK Alternative Discovery &
Development, Upper Merion, USA; 8 GlaxoSmithKline, GSK Clinical
Pharmacology, Modelling and Simulations; 9 GlaxoSmithKline, GSK
Alternative Discovery & Development, Research Triangle Park, USA
Background: Afuresertib is a highly selective oral pan-AKT kinase inhibitor
in development for patients with hematologic and solid malignancies.
Preclinically, AKT inhibition by afuresertib can reverse platinum resistance
in ovarian cancer cell lines isolated from patients with platinum-resistant
ovarian cancer.
Materials and Methods: Study objectives were to evaluate the maximum
tolerated dose (MTD), pharmacokinetics (PK), and clinical activity of
afuresertib in combination with carboplatin & paclitaxel in patients (pts)
with recurrent, platinum sensitive, resistant or refractory epithelial ovarian
cancer (EOC). This two part study included a dose escalation (part1) and
expansion phase (part2). Part 1 evaluated the PK and toxicity profile of
increasing doses [50 mg-150 mg daily] of afuresertib with IV carboplatin
(AUC 5) & paclitaxel (175 mg/m2 ) q3 weekly for up to 6 cycles followed by
afuresertib monotherapy until progression. Efficacy was evaluated using
GCIG CA125 and RECIST 1.1 criteria.
Results: Part 1 has completed enrolment (n = 29; mean age 59), 23
of the 29 pts had platinum-resistant or refractory EOC with a median
platinum-free interval of 4 months. Pts had a median of 3 prior therapies;
17% had 6 prior therapies. Dose-limiting-toxicities were grade 3 rash
(n = 3, at 125 mg (1) and 150 mg (2)) and grade 3 syncope (1) at 125 mg
afuresertib. MTD of afuresertib with carboplatin & paclitaxel was defined
as 125 mg daily. The most frequent adverse events (AEs) regardless of
relatedness were diarrhea (72%), nausea (72%) and fatigue (69%); all
Grade 2. The most common AEs grade 3 were neutropenia (40%),
hyperglycemia (10%), thrombocytopenia (7%), and ascites (7%). There
was no clinically relevant PK interaction between afuresertib and paclitaxel.
The ORR (overall response rate) regardless of dose was 60% (CA125) and
29% (RECIST). For those patients who received afuresertib at the MTD
dose of 125 mg, the ORR was 50% by both GCIG CA125 and RECIST
1.1 criteria; the clinical benefit rate (RECIST partial responses plus stable
disease of 6 months) was 63%.
Conclusions: Afuresertib can be safely combined with carboplatin and
paclitaxel at a MTD of 125 mg once daily. This triplet shows promising
clinical activity in a heavily pretreated patient population. Part 2 is now
open to pts with resistant or refractory EOC (in two separate cohorts) to
further evaluate the safety and clinical efficacy of afuresertib within this
treatment combination.
8
4
ORAL PRESENTATION
Activity of galeterone in castrate-resistant prostate cancer (CRPC)
with C-terminal AR loss: Results from ARMOR2
M.E. Taplin1 , K.N. Chi2 , F. Chu3 , J. Cochran4 , W.J. Edenfield5 ,
E.S. Antonarakis6 , U. Emmenegger7 , E.I. Heath8 , A. Hussain9 , V.C. Njar9 ,
A. Koletsky10 , D. Lipsitz11 , L. Nordquist12 , R. Pili13 , M. Rettig14 , O. Sartor15 ,
N.D. Shore16 , D. Marrinucci17 , K. Mamlouk18 , B. Montgomery19 . 1 Dana
Farber Cancer Institute, Department of Medicine Harvard Medical
School, Boston MA, USA; 2 BC Cancer Agency, Genitourinary Oncology,
Vancouver BC, Canada; 3 San Bernadino Urological Associates, Urology,
San Bernadino Ca, USA; 4 Urology Clinics of North Texas, Urology,
Dallas Tx, USA; 5 Cancer Centers of the Carolinas, Oncology, Greenville
SC, USA; 6 The Sidney Kimmel Comprehensive Cancer Center at John
Hopkins, Genitourinary Oncology, Baltimore MD, USA; 7 Sunnybrook
Health Sciences Centre, Oncology, Toronto ON, Canada; 8 Karmanos
Cancer Institute Wayne State University, Genitourinary Oncology, Detroit
MI, USA; 9 Univ of Maryland, Genitourinary Oncology, Baltimore MD,
USA; 10 Center for Hematology-Oncology, Oncology, Boca Raton FL,
USA; 11 Carolina Urology, Urology, Concord NC, USA; 12 Urology Cancer
Center and GU Research Network LLC, Oncology, Omaha NE, USA;
13
Roswell Park Center Institute, Genitourinary Oncology, Buffalo NY,
USA; 14 Institute of Urologic Oncology, Genitourinary Oncology, Los
Angeles CA, USA; 15 Tulane University, Genitourinary Oncology, New
Orleans LA, USA; 16 Carolina Urologic Research Center Atlantic Urology
Clinics, Urology, Myrtle Beach SC, USA; 17 EPIC Sciences, Sciences, San
Diego CA, USA; 18 Tokai Pharma, Medical Affairs, Cambridge MA, USA;
19
University of Washington, Genitourinary Oncology, Seattle WA, USA
Plenary Session 2
5
ORAL PRESENTATION
Mechanism based targeted therapy for hereditary leiomyomatosis
and renal cell cancer (HLRCC) and sporadic papillary renal cell
carcinoma: interim results from a phase 2 study of bevacizumab and
erlotinib
R. Srinivasan1 , D. Su1 , L. Stamatakis1 , M.M. Siddiqui1 , E. Singer1 ,
B. Shuch1 , J. Nix1 , J. Friend1 , G. Hawks1 , J. Shih1 , P. Choyke1 ,
W.M. Linehan1 . 1 National Cancer Institute, Urologic Oncology Branch,
Bethesda Maryland, USA
This abstract is part of the media programme and is embargoed until the
day of presentation, when it will be published online at 08:00.
6
ORAL PRESENTATION
Imaging in cancer immunology: Phenotyping of multiple immune cell
subsets in-situ in FFPE tissue sections
J.R. Mansfield1 , C. Slater2 , C. Wang1 , K. Roman1 , C.C. Hoyt1 ,
R.J. Byers2 . 1 PerkinElmer, LST, Hopkinton, USA; 2 University of
Manchester, Pathology, Manchester, United Kingdom
Background: There has been a rapid grown in the field of tumor
immunobiology in recent years as a result of recent successes in
cancer immunotherapies, and it is becoming clear that immune cells
play many sometimes conflicting roles in the tumor microenvironment.
However, obtaining phenotypic information about the various immune cells
Poster Session – Animal Models
that play these roles in and around the tumor has been a challenge.
Existing methods can either deliver phenotypic information on homogenous
samples (e.g., flow cytometry or PCR) or morphologic information on
single immunomarkers (standard IHC). We present here a methodology
for delivering quantitative per-cell marker expression and phenotyping,
analogous to that obtained from flow cytometry, but from cells imaged in
situ in FFPE tissue sections.
Materials: This methodology combines the sequential multi-marker
labeling of up to 8 antigens using antibodies all of the same species
in a single section; automated multispectral imaging (MSI) to remove
problematic FFPE tissue autofluorescence and correct cross-talk between
markers; and an automated analysis that can quantitate the per-cell marker
expression, determine the cellular phenotype, count these cells separately
in the tumor compartment and stroma, and provide high-resolution images
of their distributions. A tissue microarray comprising 35 post-transplant
lymphoproliferative disorder (PTLD) samples was immunostained for CD3,
CD8 and FOXP3. Single FFPE slides from 9 HER2+ breast cancer patients
receiving neoadjuvant chemotherapy were stained with fluorophores
targeting cytokeratin, CD8, CD4, FoxP3, CD20 and PD-L1. MSI and
analysis was used to enumerate specific phenotypes of cells, as well as to
map their spatial locations.
Results: MSI successfully captured and quantified multiple immune cell
types in all tissues. Validation of singly stained versus multiply stained
samples for PTLD showed excellent correlation (R > 0.9). Breast cancer
patients not achieving a pathologic complete response (pCR), the density
of both the CD8 (p = 0.03) and CD4 (p = 0.05) infiltrates in the stroma were
significantly greater than in the tumor. For patients achieving a pCR, there
was no significant difference in the densities of stromal and intratumoral
CD8 (p = 0.11) or CD4 (p = 0.75) infiltrates suggesting that T cell infiltration
into the tumor from the stroma is critical.
Conclusions: Multispectral imaging allows different immune cell phenotypes to be visualized and quantified simultaneously in the same tissue
section enabling further study of the relationships and distribution of these
cells within the tumor and tumor microenvironment, and their spatial
distribution and proximity to the tumor cells. This technology will enable
improved understanding of the immune infiltrate in solid tumors thereby
facilitating the rational design and use of immunotherapeutic agents in
combination with standard systemic therapies.
Poster Sessions
Animal Models
7
POSTER (Board P001)
Mouse clinical trial − A new preclinical study concept using
patient-derived xenografts
V. Vuaroqueaux1 , C. Gredy1 , S. Gorynia1 , S. Baltes1 , H.H. Fiebig1 ,
T. Metz1 . 1 Oncotest GmbH, Freiburg, Germany
Patient tumor explants passaged in immunocompromised mice (patientderived xenografts, PDXs) represent the most commonly used system for
preclinical efficacy testing of anti-cancer agents. The standard format for
efficacy tests typically uses preselected PDXs and group sizes of 8 to 12
PDX-bearing mice to test the efficacy of a treatment relative to a vehicle
control group. The strength of this approach is the high reliability of the
efficacy data obtained. However, in the face of limitations of available
resources this test format restricts the number of PDX models that can
be tested. In many studies less than 10 PDX models are used which, in
view of the genetic diversity of cancer, is often not satisfactory.
The need for in vivo efficacy tests in broader PDX panels is addressed by
the emerging mouse clinical trial (MCT) format. Ideally, this format relies
on only one mouse per PDX model and treatment arm (referred to as
xenopatient), thus enabling the investigation of efficacy in substantially
larger panels of PDX models (typically 40 or 50 models which are not
preselected) which collectively better mirror the inter-patient response
heterogeneity observed in the clinic. However, given that all PDX models
display some growth heterogeneity, results obtained for individual models
are less reliable.
To compare results obtained for individual PDXs in the standard and
in the MCT format, in the present work five SoC drugs were tested in
colorectal (cetuximab, oxaliplatin, irinotecan, 5-FU) and non-small-cell lung
cancer (cetuximab, paclitaxel) PDXs. Dosing and schedules were adapted
to clinical standards. Preliminary data for 17 PDX models suggest that
9
in approximately 80% of cases the results obtained in the MCT format
(single mouse trial) are in line with the results of the standard efficacy tests.
Furthermore, in about 10% of cases the results obtained with both formats
were similar by trend. The proportion of false positive or false negative
results obtained with tests in the MCT format was below 5%.
Our findings indicate that for the drugs tested here the risk of misjudging
the sensitivity of a given PDX model based on MCT efficacy data is
relatively low. This risk could probably be further lowered by increasing
group sizes to three mice. For the identification of biomarkers which profits
from accurate efficacy data such an intermediate format between the MCT
and the standard format may be advantageous.
8
POSTER (Board P002)
Imaging growth and anti-cancer activity in orthotopic patient derived
tumors
M. Baugher1 , C. Bull2 , A. Cohen-Barnhouse1 , A. Flecha2 , M. Franklin3 ,
K. Guley2 , P. McConville2 , W.R. Leopold4 . 1 Molecular Imaging Inc,
Pharmacology, Ann Arbor MI, USA; 2 Molecular Imaging Inc, Imaging,
Ann Arbor MI, USA; 3 Molecular Imaging Inc, Pharmaco-Imaging, Ann
Arbor MI, USA; 4 Molecular Imaging Inc, Oncology, Ann Arbor MI, USA
Background: Preclinical models that more closely mimic the clinical setting
are being sought using patient-derived tumors (PDX). Utilization of PDX
material in an orthotopic (OT) setting provides a preclinical model in a
disease-relevant location. Here we describe the growth and treatment
response data of OT pancreas, lung and breast PDX material with
conventional and non-invasive imaging techniques.
Materials and Methods: All patient-derived tumor material was obtained
in collaboration with Oncotest GmbH. SCID beige mice were used with the
appropriate material surgically implanted into the pancreas, directly injected
into the left lung or directly injected into the mammary fat pad. All dosing
is as shown.
Results: OT implantation of pancreatic PDX material resulted in robust
disease establishment with a near 100% take rate. We utilized MRI and
Fluorescence Molecular Tomography (FMT) imaging to non-invasively track
tumor progression. We found that different FMT probes had different
capabilities for detecting tumor burden. Both MRI and FMT showed that this
model was highly sensitive to treatment with docetaxel, where a significant
number of mice had complete response and increased overall survival
when compared to control mice. The NSCLC PDX material showed near
100% take rates as determined by CT evaluation. By CT we found a
minor bevacizumab response, as is consistent with this drug not being an
optimal VEGFR inhibitor in the mouse. PET imaging was performed and
demonstrated FDG avidity. However, no drug response was observed. In
the breast model, treatment with cyclophosphamide resulted in a robust
response with approximately 34 of mice having no measurable tumors
on day 77. In this setting, FDG PET and FMT imaging were utilized to
determine metabolic activity and avidity. FMT reflected the decrease in
tumor burden with treatment. At the time of imaging, no difference in FDG
SUV values was observed with treatment. In all models, disease latency
and tumor volume doubling times were consistent with expectations based
on known subcutaneous data.
Conclusions: We have shown the establishment of OT PDX pancreas,
lung and breast models. The use of multi-modality imaging non-invasively
tracked tumor burden over time and provided useful readouts of disease
progression and drug treatment response in these more disease-relevant
models. Future work continues to investigate the metastatic potential of OT
PDX material and non-invasive imaging to track this.
9
POSTER (Board P003)
Antineoplastic effects of auranofin in canine lymphoma
D. Thamm1 , B.J. Rose1 , J.K. Shoeneman1 . 1 Colorado State University,
Dept. of Clinical Sciences, Fort Collins, USA
Background: Lymphoma (LSA) is a serious condition for which there
remain unmet medical needs in humans and dogs. The gold complex
auranofin has been utilized as a human therapeutic, primarily as an
antirheumatic agent. Antiproliferative and pro-apoptotic activity has been
observed in a variety of human tumor-derived cell lines, including
carcinomas of the breast, head and neck, ovary, lung, and a variety
of hematopoietic tumors, including LSA. Putative antitumor mechanisms
include inhibition of NF-kB and STAT3 signaling, and induction of reactive
oxygen species via thioredoxin reductase (TrxR1) inhibition. The dog is a
well-established model for spontaneous LSA in humans, owing to striking
similarity in biology and gene expression. Dogs with spontaneous tumors
naturally develop therapy resistance and metastasis. In addition, tumor
burdens in spontaneous canine tumors are more similar to humans than
those in murine models, which may be important with regard to biologic
factors such as hypoxia and clonal variation. The size of canine tumors
10
also allows for serial imaging and tissue collection over time. The goal of
the current study was to investigate the antineoplastic effects of auranofin
in a panel of canine lymphoma-derived cell lines, and to identify potential
biomarkers of drug activity for future translational studies in spontaneous
canine LSA.
Materials and Methods: 4 canine LSA-derived cell lines were incubated
in varying concentrations of auranofin +/− bortezomib, elesclomol, or
the conventional antineoplastic agents doxorubicin (DOX), CCNU and
vincristine (VCR) for 24−72 hours. Relative viable cell number was
assessed using MTS. Cell cycle distribution and apoptosis induction were
evaluated cytometrically with propidium iodide staining and Annexin V/PI
staining respectively. Induction of ROS was assessed using the redoxsensitive fluorescent dye CM-H2 DCFDA.
Results: Auranofin induced dose-dependent antiproliferative effects in all
canine LSA cell lines, with 50% inhibitory concentrations between 0.1
and 1 uM. These are similar to those observed in human hematopoietic
tumor cells, and well within clinically achievable serum concentrations.
Dose- and time-dependent apoptosis induction was observed, and additive
to synergistic antiproliferative/propaptotic activity was observed with
bortezomib and elesclomol, but not DOX, CCNU or VCR. Dose- and timedependent induction of ROS was observed following auranofin treatment.
Conclusions: Auranofin-associated antitumor activity is observed in vitro
in canine LSA cells, at pharmacologically achievable concentrations. This
is similar to activity observed in human LSA and leukemia. Cooperative
activity was observed with the targeted agents bortezomib and elesclomol.
These data support translational evaluation of auranofin in canine LSA as
a model for the human disease.
10
POSTER (Board P004)
mTOR inhibition with everolimus − a novel treatment option for head
and neck cancer identified in a translational research study using
patient-derived xenografts
K. Klinghammer1 , J.D. Raguse2 , T. Plath2 , A.E. Albers3 , B. Brzezicha4 ,
A. Wulf-Goldenberg4 , U. Keilholz5 , J. Hoffmann4 , I. Fichtner4 . 1 Charité
University Medicine, Department of Hematology & Oncology, Berlin,
Germany; 2 Charité University Medicine, Department of Maxillio-Facial
Surgery, Berlin, Germany; 3 Charité University Medicine, Department of
ENT, Berlin, Germany; 4 Experimental Pharmacology & Oncology GmbH,
Berlin, Germany; 5 Charité University Medicine, Cancer Comprehensive
Center, Berlin, Germany
Background-Aims: We recently established a large panel of head and
neck squamous cell carcinoma (HNSCC) patient-derived xenografts for
exploration of novel treatment approaches and biomarker evaluation. Next
to activation of the EGFR-RAS-RAF-ERK pathway the PI3K-AKT-mTOR
axis is often amplified and activated in HNSCC. We evaluated the mTOR
inhibitor everolimus in a panel of HNSCC PDX derived from 29 different
patients. Treatment response of everolimus was compared to treatment
response of cetuximab and further correlated to gene expression of mTOR
pathway members such as mTOR, RPS6KB1, Akt1, FKBP1B and TSC1.
Since PI3K mutation has been associated with pathway activation, we
aimed to define the predictive role of PI3K mutations for everolimus
response.
Methods: Specimens from head and neck tumor surgery were transplanted
subcutaneously to immunodeficient mice. Groups of 6 mice were treated
with cetuximab or everolimus as single agent. A T/C (mean tumor volume
of treatment versus control) of less than 50% was defined as biological
meaningful activity of the treatment. Transcription of mTOR pathway
members were analyzed in detail using RT-PCR. Sequencing analysis was
accomplished on Illumina TruSeq Amplicon − Cancer Panel.
Results: Response to treatment was very individual. Response rate of
everolimus was 20/29 (68%) and for cetuximab 23/29 (79%). RPS6KB1
gene expression showed a trend to positive correlation with treatment
response to everolimus (p = 0.0784). PI3K mutations were identified in 7
models at different gene regions. All but one model harbouring mutations
within the PI3K gene showed a significant growth inhibition (6/7, 85%),
when treated with everolimus. Activity is slightly lower in the cohort with
PI3K wildtype (14/22, 63%).
Conclusion: The study demonstrated a significant therapeutic activity of
everolimus in the majority of our HNSCC models. Although not significant,
analysis of mutations in PI3K and the expression of RPS6KB1 mRNA
provide some first mechanistic evidence, that an activated PI3K/mTOR
pathway may predict sensitivity. Analysis needs to be extended to a
larger study group. In conclusion, we demonstrate a comprehensively
characterized panel of head and neck cancer PDX models, which
represent a valuable and renewable tissue resource for evaluation of novel
compounds and associated biomarkers.
11
POSTER (Board P005)
A panel of patient derived xenograft models of different
haematological malignancies suitable for preclinical drug screening
campaigns
E. Oswald1 , C. Tschuch1 , K. Klingner1 , B. Hammerich1 , D. Lehnhard1 ,
C. Rentsch2 , M. Lübbert3 , H.H. Fiebig4 , J. Schüler4 . 1 Oncotest GmbH,
In Vivo Tumorbiology, Freiburg, Germany; 2 University Hospital Basel,
Division of Urology, Basel, Switzerland; 3 University Hospital Freiburg,
Hematology and Oncology, Freiburg, Germany; 4 Oncotest GmbH,
Freiburg, Germany
In order to obtain a better understanding of hematological malignancies
and to develop improved therapeutic strategies, the generation of functional
and reproducible in vivo models is widely pursued. Here, we report
the establishment of transplantable PDX models of acute myeloid and
acute lymphoid leukemia (AML, ALL) and Non-Hodgkin Lymphoma
(NHL) growing subcutaneously as well as in a disseminated fashion in
immunocompromised mice.
Bone marrow and peripheral blood cells from 18 AML/ALL patients
were injected intratibially into NSG or NOG mice (n = 4−8/patient).
Separately, tumor tissue from 27 prostate cancer patients and one lung
cancer patient was transplanted either under the subrenal capsule or
subcutaneously into NSG or NOG mice (n = 4−10/patient), leading to
the outgrowth of nine NHLxenografts (eight from transplanted prostate
cancer and one from transplanted lung cancer tissue). Tumor growth was
monitored via determination of overall survival, flow cytometry analyses,
caliper measurement where applicable and Immunohistochemistry (IHC).
Transplantable models as well as primary in vivo cultures were further
characterized by treatment with respective standard of care drugs (SoC).
Six out of 18 AML/ALL patient-derived specimens engrafted in immunocompromised mice, following transplantation into murine bone marrow.
Human leukemic cells were detected in murine peripheral blood, bone
marrow and spleen by flow cytometry as well as by IHC. One T-ALL
and one AML could also be propagated when injected subcutaneously.
Two transplantable AMLs and one transplantable ALL were treated with
SoC drugs decitabine, cytarabine and dexamethasone. Efficacy tests using
primary AML cells were feasible although availability of tumor material
limited experiment size to maximally 25−30 mice. The eight NHL xenografts
derived from prostate cancer tissue were all Epstein-Barr-Virus-positive.
Seven were diagnosed as diffuse large B-cell lymphomas (DLBCL), one is
T-cell-derived. The NHL excised from the lung was diagnosed as a MALT
lymphoma. All lymphomas were transplantable and showed stable growth
when propagated subcutaneously. Subtyping of the DLBCL lymphomas by
IHC revealed that four of them were of the ABC-subtype.
Our results confirm that PDX models of haematological malignancies
replicate the heterogeneity and other important clinical characteristics of
the respective disease, and are valuable tools for preclinical drug testing
and investigation of tumour biology.
12
POSTER (Board P006)
Next generation sequencing (NGS) guided therapy prediction for the
treatment of glioblastoma multiforme (GBM)
J. Sarkaria1 , D.M. Ma1 , S.P. Peng2 , S.B. Byron2 , D.C. Craig3 ,
J.C. Carpten4 , M.B. Berens2 , B.O. O’Neill5 , N.T. Tran6 . 1 Mayo Clinic
Cancer Center, Radiation Oncology, Rochester Minnesota, USA;
2
Translational Genomics Research Institute (TGen), Cancer and
Cell Biology Division, Phoenix Arizona, USA; 3 Translational Genomics
Research Institute (TGen), Neurogenomics Division, Phoenix Arizona,
USA; 4 Translational Genomics Research Institute (TGen), Genomics
Division, Phoenix Arizona, USA; 5 Mayo Clinic, Neuro-oncology,
Rochester Minnesota, USA; 6 Translational Genomics Research Institute
(TGen), Cancer and Cell Biology Division, Rochester Minnesota, USA
Background: The genomic heterogeneity of glioblastoma likely underlies
the low response rates (8−24%) for targeted agents among unselected
populations. We tested whether NGS would be useful in identifying
therapeutically-actionable genetic alterations; we sought to test if this
translated into improved tumor control in a patient-derived GBM model.
Methods: Tumor specimens from each patient (n = 11) that were used
for analyte extraction contained between 70−80% viable tumor cellularity.
Genome sequence coverage was more than 30× for both tumor and
germline genomes; tumor RNA sequencing included over 100 million reads.
NGS of paired tumor and germline DNA enabled detection of single
nucleotide variations (SNVs), indels, translocations, intra-chromosomal
rearrangements, and copy number alterations. A custom drug-matching
workflow utilizing publically available databases and curated literature
on reported drug effects, pharmacokinetics, and blood–brain barrier
penetration was used to map individual gene alterations in tumors with
associated drug-response relationships. One specimen was tested in vitro
and in vivo using a matched, patient-derived xenograft model.
Results: NGS for the 11 tumor panel found ~30% of cases with EGFR
amplification, p16, and/or PTEN deletion. Novel potentially actionable
targets included a TRIM54-FGFR3 fusion, STAG2 mutation, MDM2
amplification, KIF11 and KIF15 mutation, and BRAF amplification. GBM150
is derived from a recurrent tumor and harbors the TRIM54-FGFR3 fusion,
and this line is the first to be analyzed for chemovulnerability. In a Cyquant
proliferation assay, GBM150 were relatively resistant to TMZ (IC50 300 uM)
but significantly more sensitive to pan-FGFR1−3 inhibitors as compared to
GBM108: ponatinib IC50 0.3 uM vs. 1.2 uM; AZD4547 IC50 3 uM vs. >10
uM. Similarly, in a flank tumor regrowth study, established GBM150 tumors
were randomized to therapy with placebo, temozolomide, ponatinib or
AZD4547. While TMZ-treated tumors were marginally smaller than placebo
treated tumors (mean tumor volume 1312±208 vs. 992±183 mm3 , 97 days
after therapy initiation), both ponatinib or AZD4547 resulted in sustained
tumor stasis with a mean tumor volume of 240±35 and 199±40 mm3 ,
respectively, at the same time-point.
Conclusions: These results support the concept that NGS can be used
to individualize treatment of GBM and highlight how corresponding patient
derived xenograft models can be used to validate the accuracy of potential
therapeutic predictions.
13
POSTER (Board P007)
Whole exome sequence analysis of canine transitional cell carcinoma
of the bladder
D.L. Duval1 , B. Hernandez1 , J. Brown2 , S.E. Lana1 , R. Page1 ,
K.L. Jones2 . 1 Colorado State University, Clinical Sciences, Fort Collins,
USA; 2 University of Colorado Cancer Center, Biochemistry and Molecular
Genetics, Aurora, USA
Transitional cell carcinoma (TCC) is the most common bladder cancer in
both humans and their canine companions, accounting for approximately
2% of all diagnosed malignancies in both populations. Clinical presentation
of human TCC is broken into 2 groups: superficial non-muscle-invasive
TCC (~70% of cases) and muscle invasive TCC (~30% of cases) which is
associated with a high risk of death from distant metastasis. The majority
of canine TCCs are papillary infiltrative TCCs of intermediate to high
grade at diagnosis. Similarities in risk factors, histopathology, sites of
metastasis, and other common features indicate that canine TCC may
serve as an excellent model for invasive human TCC. Previous studies of
human TCCs have identified genetic defects that may aid in the diagnosis
and therapy of human bladder cancer. To further assess the value of
spontaneous canine TCCs as a model for human TCC, we have utilized
whole exome sequencing to screen a panel of canine TCCs for cancer
gene mutations that contribute to the pathogenesis and progression of
canine bladder cancer. Genomic DNA was isolated from 11 archived canine
TCCs, 3 matched normal tissue samples, and 2 canine TCC cell lines.
Whole exome capture was conducted using the Agilent Sure-select insolution capture system designed for the canine genome, and the captured
fragments were sequenced using an Illumina HiSeq2000 next generation
sequencing platform. The sequences were mapped to the CanFam3.1
canine reference genome and single-nucleotide polymorphisms, insertions
and deletions were identified using Freebayes. Somatic mutations were
characterized and compared to the Cancer Gene Census (COSMIC).
Similar to a variety of human cancers including bladder cancers, the
mutation spectrum in these genes is dominated by C:G>T:A transitions.
Nonsense, missense, and insertion/deletion mutations were identified in
75 genes shown to be drivers or repressors in human cancer. Pathway
analysis (Pathway Studio) identified DNA damage, genomic instability, and
chromatin remodeling as the top 3 cellular pathways affected. The 10 genes
most frequently exhibiting potentially deleterious mutations were: MITF,
KDM6A, MLLT6, AKAP9, C2orf44, ROS1, NSD1, BRAF, FANCD2, and
PCM1. This mutation spectrum, including members of the RTK/Ras/Raf
pathway, histone modifying and chromatin remodeling enzymes, indicates
that similar activating pathways drive both human and canine transitional
cell carcinoma of the bladder.
14
POSTER (Board P008)
Mixeno mouse models for in vivo evaluation of anti-human cancer
immunotherapeutics
J. Zhang1 , J. Qiu2 , M. Qiao2 , Q. Shi1 . 1 Crown Biosciences, Cancer
Pharmacolgy, Santa Clara CA, USA; 2 Crown Biosciences, Cancer
Signaling, Santa Clara CA, USA
The past few years have witnessed a renaissance in the field of cancer
immunotherapy, relating largely to the clinical advances associated with
the development of immunomodulatory agents, e.g. monoclonal antibodies
targeting the immune inhibitory pathways (CTLA-4 and PD-1/PD-L1).
11
Often, the preclinical efficacy assessments are based on the evaluation
of surrogate anti-mouse target antibodies using mouse syngenic tumor
models. However, this strategy is limited due to the fact it can only be used
to test surrogate molecules, rather than directly evaluate the therapeutic
molecules that target human targets. Here we set out to validate mouse
models that harbor human immune cells by engrafting the immuno-deficient
mice with human PBMC (the MixenoTM model), and use them for efficacy
evaluation of the humanized anti-PD-1 antibody. PD-L1 high-expression
human tumor cell lines are selected using Xenobase® and FACS analysis
to develop the in vivo models. BMS-936558, a fully humanized anti-PD-1
IgG4 produced promising anti-tumor activity in the HCC827 lung cancer
MixenoTM model. Based on the preliminary result, the MixenoTM models
may be a useful tools in immunotherapeutic antibody development, and
may greatly increase the clinical translatability of animal studies.
15
POSTER (Board P009)
Genetic and molecular validation of uterine sarcoma patient-derived
xenograft models
T. Cuppens1 , E. Hermans1 , J. Depreeuw2 , M. Moisse2 , T. Van Brussel2 ,
L. Coenegrachts1 , D. Lambrechts2 , F. Amant1 . 1 Catholic University of
Leuven, Gynaecologic Oncology, Leuven, Belgium; 2 Catholic University
of Leuven-Vesalius Research Center VIB, Oncology Translational
Genetics, Leuven, Belgium
Background: Genetic and molecular heterogeneity within tumor types
directly affects a patient’s response to a given compound, warranting more
personalized therapeutic strategies based on predictive markers. However,
for rare malignant tumors in particular, randomized trials are scarce,
emphasizing the need for reliable pre-clinical tumor models. Traditional
approaches for pre-clinical evaluation of cancer therapies rely on the
use of xenograft models of commercially available cell lines. Whereas
these models are human in origin, they are unlikely to fully recapitulate
human cancers. In contrast, patient-derived tumor xenografts (PDTXs),
established directly from patient’s tumors, may better preserve the initial
tumor characteristics as they represent the heterogeneity and retain the
most important genetic features of the original tumor. Here, we aim to
establish and validate PDTX models for uterine leiomyosarcoma, a highly
aggressive tumor arising from the myometrium. We explore the genetic
and molecular stability of xenografted tumors, which will ultimately serve
as pre-clinical models for individualized treatment.
Materials and Methods: In collaboration with the PDTX platform, 8 uterine
leiomyosarcomas (obtained from surgery after patient’s informed consent)
were subcutaneously implanted into immune-deficient mice. Engrafted
tumors were re-implanted for several generations (F1, F2, F3, etc.) and then
compared to their original tumors by means of copy number analysis (SNP
arrays-Illumina), tissue-specific marker expression (immunohistochemistry)
and histology (H&E).
Results: We have established 5 uterine leiomyosarcoma models (take
rate >60%), of which one diploid and one tetraploid model have been
analyzed at present. Both tumors retained their histologic features, as well
as their expression of mesenchyme-specific (vimentin) and muscle-specific
(desmin/caldesmon) markers.
Further, copy numbers of the F4 xenograft derived from the diploid tumor
were >99% identical to the original tumor. However, the F3 xenograft of
the same model showed tetraploidy and some additional copy number
changes.
In the tetraploid PDTX model, 37% of all genes in F2 and F3 tumors had
changed in copy number when compared to the original tumor. Comparing
the F2 and F3 tumor revealed a copy number stability of only 56%. Hence,
in both models, the later xenograft generation shows greater resemblance
to the original tumor than to the prior xenograft from which it was derived.
This finding strongly suggests the presence of subclones.
Conclusions: We successfully established patient-derived tumor xenograft
models for pre-clinical individualized therapy testing. Of note, the genetic
stability and in particular the presence of subclones should be taken into
account when selecting therapies based on genetic features.
16
POSTER (Board P010)
Allografting improves the feasibility of genetically engineered mouse
models (GEMM) for anti-cancer drug development
K. Kukuk1 , K. Klingner1 , A.L. Peille2 , P. Müller3 , A. Zipelius3 , J. Schüler2 .
Oncotest GmbH, In Vivo Tumorbiology, Freiburg, Germany; 2 Oncotest
GmbH, Freiburg, Germany; 3 University Hospital Basel, Basel, Switzerland
1
Genetically engineered mouse models (GEMMs) represent an attractive
system for preclinical research since GEMM tumors develop in the
presence of a competent immune system. Unlike classical syngeneic
mouse tumor models, in GEMMs the identity of some of the oncogenic
12
alterations is known. Typical drawbacks of GEMM tumors include slow
and heterogeneous tumor development, making the use of these models
challenging for routine in vivo efficacy studies. These disadvantages can be
avoided by grafting autochthonous GEMM tumors on mice of the parental
strain. Here, we describe the establishment and characterization of such
allografts from a non-small cell lung cancer (NSCLC) and two breast cancer
models, among them an invasive lobular carcinoma (ILC) and a model
carrying a Brca-1 alteration.
Tumors were excised from their organ of origin and tumor pieces were
implanted subcutaneously (s.c.) and orthotopically (o.t.) into C57BL/6N
(NSCLC model) and FVB/N mice (breast cancer models). Arising tumors
were passaged and compared to the original in-situ tumors molecularly,
histologically, and immunohistochemically (e.g. Ki67 proliferation marker).
In parallel, cell lines were established from the in-situ tumors.
Subcutaneous allografts were passaged at least 8 times with stable and
consistent tumor growth as documented by tumor-growth-kinetics and H&E
staining of tumor sections. The allografts of the ILC model metastasized to
multiple organs when transplanted o.t.
For all three models, patho-histology of in-situ tumors and allografts was
congruent and no signs of an immunological reaction against allografts
were detected. IHC analysis revealed differences between Ki-67 and
CD31 expression among different allografts representing the same GEMM.
Efficacy tests with standard of care (SoC) drugs performed on allografted
NSCLC tumors revealed significant anti-tumor activity of the dual PI3K
and mTOR inhibitor. BEZ235 displayed good antitumoral activity both in
monotherapy and in combination with the dual EGFR and HER2 inhibitor
afatinib. The selective EGFR inhibitor erlotinib had no significant effect,
which is in line with the low mEGFR expression detected.
In conclusion, s.c. and o.t. transplantable allografts, which replicate key
aspects of the human disease, were obtained from the investigated GEMMs
and enable efficacy tests in immunocompetent mice in a suitable time
frame. Further studies will elucidate the feasibility of these models for
preclinical testing of immunomodulatory drugs.
17
POSTER (Board P011)
Studies on glycoprotein expression differences between MCF-7 and
MCF-7-Z
J. Ner-Kluza1 , A. Drabik2 , M. Kubbutat3 , A. Lingnau3 , J. Silberring2 .
1
Uniwersity of Science and Technology, Department of Biochemistry
and Neurobiology, Krakow, Poland; 2 AGH Uniwersity of Science and
Technology, Department of Biochemistry and Neurobiology, Krakow,
Poland; 3 ProQinase GmbH, Freiburg, Germany
According to the American Cancer Society, in 2014, more than 230,000
women will be positively diagnosed for breast cancer in the US,
demonstrating the urgent need for more promising anti-tumor drugs
worldwide.
The human breast cancer cell line MCF-7 has been commonly used in
subcutaneous xenograft tumor models in order to screen for novel cancer
drugs. By comparing two different variants of MCF-7 (MCF-7 and MCF7-Z), both authentic when analysed by STR profiling, we found completely
different tumor growth characteristics in mice. We excluded the possibility
that a loss in estradiol dependency was responsible for these differences,
and started analysing the proteome of the MCF-7 tumors by an approach
to discriminate between the glycosylation pattern.
Among post-translational modifications glycosylation is one of the most
important involved in tumor progression. The presented project is
based on monitoring changes in glycoprotein profiles of two cell lines
MCF-7 and MCF-7-Z. For the isolation of glycoproteins lectin affinity
chromatography (LAC) was applied, followed by one-dimensional SDSPAGE electrophoresis. After digestion, peptides extracted from the gel were
analyzed by nanoLC-MS/MS system. The identified glycoproteins were
identified by bioinformatics tools, such as Osprey and Panther databases.
Final part of the research is based on quantitative analysis using labelfree approach of the identified proteins using Bruker software called Profile
Analysis.
The research was supported by the The Polish National Science Center
2012/07/B/NZ4/01468, and EuroNanoMed ‘META’ 05/EuroNanoMed/2012.
18
POSTER (Board P012)
Establishment and characterization of a Merkel Cell carcinoma PDX
panel: Screening for potentially useful therapies
M.J. Wick1 , J. Meade1 , M. Nehls1 , T. Vaught1 , J. Carlile1 , A.W. Tolcher1 ,
D.W. Rasco1 , A. Patnaik1 , K.P. Papadopoulos1 . 1 South Texas Accelerated
Research Therapeutics, (START), San Antonio TX, USA
Background: Merkel cell carcinoma (MCC) is an uncommon, aggressive
neuroendocrine skincancer with a poor prognosis and few effective
treatment options for advanced disease. Recent studies have identified
a strong association between infection with the Merkel cell polyomavirus
(MCPyV) and MCC transformation. MCPyV antigens have been shown
to affect cell growth and proliferation through apoptosis and cell cycle
pathways. Targeted therapies have shown promise in vitro and in vivo;
however, their utility in the clinical setting is anecdotal. To better
understand which targeted therapies are effective in MCC, we established
and screened a panel of MCC patient-derived xenograft (PDX) models
evaluating approved and investigational therapies.
Methods: Methods: Merkel cell carcinoma START-PDX models were
established in immune-deficient mice from primary or metastatic patient
tissue and once established were confirmed by histologic comparative
analysis and linked with patient treatment and outcome data. Drug
sensitivity studies were performed evaluating each model to available
targeted therapies including pazopanib, everolimus and sunitinib and
investigational Akt, IAP and CDK 4/6 inhibitors. Study endpoints included
tumor volume and time from treatment initiation with tumor growth inhibition,
delay and regression reported at study completion.
Results: Five MCC models evaluated in this project demonstrated
differential responses to test agents; statistically significant (p < 0.05)
tumor growth inhibition was reported with pazopanib. Drugs targeting the
PI3K/mTOR pathway, including an Akt inhibitor and everolimus as single
agents, were not effective in these studies.
Conclusion: We have established a panel of MCC PDX models and
evaluated a panel of approved and investigational therapies. We found
differential responses to these therapies, with pazopanib the most active
as a single agent. These models may be of utility in identifying effective
single and combination regimens for MCC.
19
POSTER (Board P013)
Syngeneic models for developing cancer therapeutics targeting
immune system
L. Zhang1 , J. Zhang1 , Q. Shi1 . 1 Crown Biosciences, Cancer
Pharmacolgy, Santa Clara CA, USA
Syngeneic tumor models have long been used in cancer research, from
mechanistic study to developing cancer therapeutics, especially those that
require intact immune system, such as antibody therapeutics that have
ADCC effect. Recently, cancer immunotherapy reignited to become one
of the most promising therapies, largely because of the success of the
clinical studies of CTLA-4, PD-1/PD-L1 antibodies. Researchers are now
believe there are many novel therapeutics, both small and large molecules,
and many novel targets, to be discovered and developed in this field. To
meet this demand, Crown has established a large collection of syngeneic
models that covers most of the tumor types and mutational profiles. In
addition, we’ve also profiled the models using anti-mouse PD1/PD-L1
antibodies. The syngeneic models display very different responses towards
the immunotherapeutics, ranging from shrinking the tumor to stimulating
the tumor growth. These results emphasize the need to carefully select
models based on the development goals. A single agent development
approach would require selecting the models with the best response,
while a combination study design would require a model with suboptimal
response. Our comprehensive list of syngeneic models and profiling data
are essential in developing cancer immunotherapies that may one day
benefit the patients.
20
POSTER (Board P014)
miR-25 is a key regulator of prostate cancer invasiveness by
modulation of the cross-talk between Notch and TGF-b signaling
E. Zoni1 , A.F. van de Merbel1 , G. van der Horst1 , J. Rane2 , T. Visakorpi3 ,
E.B. Snaar4 , N. Maitland2 , G. van der Pluijm1 . 1 Leiden University Medical
Center, Department of Urology, Leiden, Netherlands; 2 YCR Cancer
research Unit, Department of Biology, York, United Kingdom; 3 Institute of
Medical Technology, University of Tampere, Tampere, Finland; 4 University
of Leiden, Department of Biology, Leiden, Netherlands
Background: Prostate cancer is the most commonly diagnosed cancer in
males and the second leading cause of death from cancer in men. Prostate
cancer stem/progenitor cells (CSCs) have been shown to play a crucial
role in carcinogenesis, metastasis and therapy resistance. Previously, we
have shown that the ALDHhigh subpopulation of human prostate cancer
cells is enriched for CSCs. ALDHhigh cells display strong clonogenicity and
migration when compared to the more differentiated ALDHlow .
Material and Methods: We performed microRNA (miR) expression
profiling of ALDHhigh vs ALDHlow subpopulation of prostate cancer cells.
We studied the functional effects of miR-25 on Notch signaling on
mRNA and proteins and used Luciferase reporters to monitor the effect
of miR-25 on TGF-b signaling and to validate the interaction between
miR-25 and Notch1. Finally we used zebrafish model to demonstrate that
Poster Session – Cytotoxics
miR-25 reduces dissemination of human prostate cancer cells in the intact
organism.
Results: In this study we have identified miRs that are differentially expressed between the metastatic subpopulation of PC-3M-Pro4
cells (ALDHhigh /a2high /CD44+ ) vs non-tumorigenic/non-metastatic ALDHlow .
miR-25 was strongly downregulated in the ‘driver’ ALDHhigh CSC
subpopulation. In both clinical prostate cancer specimens and prostate
cancer cell lines we found that miR-25 is low/absent in CSC compartment
and steadily increases during differentiation into luminal epithelial cells.
We used Targetscan to discover novel miR-25 predicted targets and
identified Notch1 as putative target of miR-25. Previously published studies
have highlighted the role of Notch and Jag1 (Notch ligand) in skeletal
metastasis. Interestingly, overexpression of miR-25 strongly decreases the
expression of Notch1 and Jag1 together with other Notch downstream
targets in two prostate cancer cell lines. Moreover, we found that miR-25
is able to decrease TGF-b signaling in prostate cancer cells and can block
the induction of Jag1 driven by TGF-b. In line with these observations,
we further demonstrate that miR-25 reduced metastasis by blocking the
extravasation of human prostate cancer cells in vivo.
Conclusion: Previously we have shown that miR-25 directly target av- and
a6-integrins and strongly reduces migration of human prostate cancer cells.
Here we show that miR-25 affects Notch pathway and can interfere with
the TGF-b signaling, reducing the TGF-b induced expression of Jag1. Our
data are supported by previous studies on the critical role of av-integrin in
activation of TGF-b signaling and its role in skeletal metastasis.
In conclusion, we suggest that miR-25 seems to be a master regulator
of invasiveness in human prostate cancer through its interaction with
Notch and TGF-b signaling pathways. The identification of miRs and
functional validation of their target genes, is crucial for the elucidation of the
mechanisms involved in tumor progression and bone metastasis formation
in human prostate cancer.
Cytotoxics
21
POSTER (Board P015)
Pharmacogenomics of mithramycin in thoracic malignancies
W. Figg1 , T.M. Sissung1 , C.J. Peer1 , D. Schrump2 . 1 National Cancer
Institute, Clinical Pharmacology Program, Bethesda, USA; 2 National
Cancer Institute, Thoracic Oncology Section, Bethesda, USA
Background: Mithramycin inhibits expression of the fusion oncogene
EWS-FLI1, and is therefore under investigation for use in thoracic
malignancies. One of the major obstacles to mithramycin therapy is
significant liver toxicity and unknown plasma concentrations.
Materials and Methods: To discover genetic variants that are associated
with these obstacles, we used the Drug Metabolizing Enzymes and
Transporters (DMET) genotyping array on germline DNA from 12 patients
with various thoracic malignancies receiving mithramycin. We also
developed a novel assay to detect mithramycin plasma concentrations in
these patients.
Results: Grade 3−4 ALT and AST rises were observed in 8 of 12 patients
that typically occurred after three daily administrations. Mithramycin plasma
concentrations remained at sub-therapeutic levels, and pharmacokinetic
parameters were not related to the LFT rises (P > 0.061). DMET analysis
revealed two polymorphisms that are related to LFT rises in genes
responsible for hepatocellular bile transport, ABCB4 (encoding PC-flopase)
and ABCB11 (encoding BSEP). Patients without LFT rises carried wild-type
alleles at both of these sites, whereas those with LFT rises carried at least
one variant allele [OR (95% CI) = 153.0 (2.6–9100); P = 0.0020]. When
DMET results were applied to pharmacokinetic data, an increasing trend
was observed in which patients carrying (TA)6 /(TA)6 at UGT1A1*2 had low
AUC that became progressively higher in those carrying (TA)6 /(TA)7 and
(TA)7 /(TA)7 respectively.
Conclusions: Taken together, the data indicate that mithramycin likely
causes liver toxicity by inhibiting bile acid flow. Mithramycin is known to
inhibit hepatic transporter expression that we suspect causes progressive
bile acid accumulation in hepatocytes and subsequent cellular damage.
Mithramycin is also most-likely glucuronidated by the liver; thus, extensive
UGT1A1 metabolizers are likely to have sub-therapeutic plasma concentrations.
13
22
POSTER (Board P016)
Novel combination therapy, TAS-102 combined with the anti-EGFR
antibody or the anti-VEGF antibody showed therapeutic benefit
toward colorectal cancer xenografts
K. Ishida1 , K. Sakamoto1 , N. Tanaka1 , K. Oguchi1 , K. Yamamura1 ,
A. Fujioka1 , F. Nakagawa2 , K. Matsuo1 , T. Utsugi1 . 1 Taiho Pharmaceutical
Co. Ltd, Tsukuba Research Center, Tsukuba, Japan; 2 Taiho
Pharmaceutical Co. Ltd, Tokushima Research Center, Tokushima, Japan
Background: TAS-102 (Lonsurf® ) consists of trifluridine (FTD) and tipiracil
hydrochloride, thymidine phosphorylase inhibitor (TPI), at a molar ratio of
1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102
and exerts its antitumor activity via FTD incorporation into DNA. FTD is
activated to F3 dTMP by thymidine kinase and further phosphorylated to
triphosphate form, F3 dTTP which is incorporated into DNA. TPI prevents
degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found
to significantly improve overall survival of patients with metastatic colorectal
cancer that is refractory/intolerable to the standard chemotherapies in a
global Phase III study. We investigated TAS-102 based combination therapy
with cetuximab (Cmab), panitumumab (Pmab), or bevacizumab (Bmab) that
are key drugs for the treatment of metastatic colorectal cancer.
Material and Method: Antitumor effects of TAS-102 combined with Cmab,
Pmab or Bmab were evaluated in human colorectal cancer xenograft
implanted SW48, HCT116 and HT-29. TAS-102 was administered orally
twice daily for 14 days, and Cmab, Pmab or Bmab were administered twice
a week intravenously or intraperitoneally. Tumor growth inhibition on day 15
and tumor growth delay was calculated from the tumor volume. Moreover,
we measured accumulation of FTD and its phosphorylated forms in tumor
with LC-MS/MS in the study of the combination with Bmab.
Results: The antitumor effects of TAS-102 were enhanced by the
combination with Cmab or Pmab toward KRAS wt tumor SW48, compared
with monotherapy with TAS-102 or anti-EGFR antibodies as measured by
both tumor growth inhibition and tumor growth delay. Similarly, tumor growth
inhibition by Bmab plus TAS-102 was stronger than monotherapy toward
both KRAS wt and mut tumors, SW48 and HCT-116 as well as in a BRAF
mut tumor, HT-29. The tumor growth delays were also extended by Bmab
combined administration in comparison with monotherapy. Interestingly, it
was observed that the level of FTD and its phosphorylated forms in the
tumor treated with TAS-102 plus Bmab were increased compared with TAS102 monotherapy.
Conclusions: TAS-102 plus anti-EGFR therapy for KRAS wt tumors
and TAS-102 plus Bmab therapy for tumors with KRAS and BRAF
mutations could be effective combinations for colorectal cancer. TAS-102
based therapy may prove to be an effective alternative to standard 5-FU
based chemotherapy in combination with antibody treatment for advanced
colorectal cancer.
23
POSTER (Board P017)
Phase I study of lurbinectedin (PM01183) administered on days (D)
1 & 8 every 3 weeks (q3wk) in patients (pts) with solid tumors
M.J. Ratain1 , L. Gore2 , S. Szyldergemajn3 , J. Diamond2 , D. Geary1 ,
C. Fernandez-Teruel3 , A. Soto-Matos3 , M. Sharma1 , A. Jimeno2 .
1
The University of Chicago, Chicago, USA; 2 The University of Colorado,
Denver CO, USA; 3 PharmaMar, Colmenar Viejo Madrid, Spain
Background: PM01183 is a new anticancer agent with broad clinical
activity against several solid tumors. The recommended dose (RD) when
administered on D1 every three weeks (q3wk) is 4.0 mg/m2 or 7.0 mg as flat
dose (FD). Reversible myelosuppression is its dose-limiting toxicity (DLT).
Here we explored a D1&8 q3wk schedule.
Material and Methods: Pts with solid tumors (other than colorectal
cancer), adequate organ function and PS 0−1 were treated at 3 dose levels
(DLs) using a 3+3 design, from 3.0 mg to 5.0 mg FD on D1&8 q3wk.
Results: 21 pts were treated and 20 were evaluable: (M/F: 5/15); median
age 63 years (39−75); BSA 1.8 m2 (1.6−2.5); albumin 3.8 g/dL (2.8−4.3);
prior lines 5 (2−13). Most pts had ovarian (8, 38%), soft tissue sarcoma
(STS) (5, 24%) or pancreatic/biliary tract (5, 24%) cancer. DL3 (5.0 mg
FD/D1&8) was defined as the RD whereas 3 of 13 (23%) evaluable pts had
DLT in Cycle 1: G4 neutropenia lasting >7 days (n = 2) and D8 omission and
C2 delay due to toxicity (n = 1). Six pts (46%) received the full RD without
delay, omissions or dose reductions; 5 (36%) pts required either a dose
reduction or G-CSF prophylaxis from Cycle 2. No dose reductions or delays
were needed in pts treated below the RD (n = 7). G4 neutropenia occurred
in 43% of pts and one pt had G4 thrombocytopenia, at the RD. Other
G3 events included nausea/vomiting and hyponatremia (1 each). Nonhematological G1/2 toxicity in 15% of pts: fatigue (64%), nausea (57%),
anorexia (36%), vomiting (28%), pyrexia (21%) and ALT/AST increase (64%
and 50%). At the RD, 8/13 pts had stable disease (4 months in 5 pts).
Two previously progressing pts with STS and ovarian cancer were on study
14
for 57+ weeks. At the RD (n = 14): mean (SD) clearance (CL) 18.1 (7.2)
L/h; AUC 318 (123) h·mg/L; Cmax 90.5 (30.7) mg/L; t1/2 30.5 (13.6) h. No
apparent association between CL and DLT or magnitude of neutropenia
was found.
Conclusions: The RD of PM01183 is 5 mg FD on D1&8 q3wk. At this RD,
myelosuppression led to frequent dosing delays or omissions, but G-CSF
support enabled the targeted dose intensity in some individuals. Two
patients had prolonged disease stabilization lasting over a year. No signs
of cumulative toxicity were observed. Interindividual variability in CL was
high, but did not appear to be associated with toxicity.
24
POSTER (Board P018)
Androgen receptor (AR) expression in triple negative breast cancer
(TNBC): results from a phase II neoadjuvant trial with carboplatin
and eribulin mesylate in TNBC patients
K. Siziopikou1 , V. Parini2 , V. Kaklamani3 . 1 Northwestern University
Feinberg School of Medicine, Pathology, Chicago IL, USA; 2 Robert H.
Lurie Comprehensive Cancer Center, Pathology Core Facility, Chicago IL,
USA; 3 Northwestern University Feinberg School of Medicine, Oncology,
Chicago IL, USA
Background: Triple negative (ER-negative/PR-negative/HER2-negative)
breast cancer remains a therapeutic challenge for the oncologist since
these carcinomas do not qualify for endocrine manipulation or HER2targeted therapy. Platinum agents were lately used in TNBC resulting
in a 16−32% pathologic complete response (pCR) in neoadjuvant trials.
Eribulin mesylate, a nontaxane microtubule dynamics inhibitor with a
novel mechanism of action also showed clinical activity in breast cancer.
In addition, different subgroups of TNBC were identified, with an AR
expressing subgroup reported to be associated with a better prognosis.
Our group recently reported that, in a phase II neoadjuvant trial, the
combination of carboplatin and eribulin mesylate in patients with TNBC was
well tolerated and showed significant efficacy. Our current study aimed to
investigate the role of AR in this patient population of TNBC cases uniquely
treated with a combination of carboplatin and eribulin mesylate.
Materials and Methods: 30 patients with stage I-III TNBC were treated in
a neoadjuvant setting with eribulin 1.4 mg/m2 over 2−5 min IV on days 1
and 8 plus carboplatin AUC6 over 30 min IV in Day 1 for four cycles. The
expression of AR (Dako, AR 441) was assessed immunohistochemically
in breast cancer specimens prior to treatment and at the time of definitive
surgery. Staining was considered negative if <1%, low positive if 1−9% and
positive if 10%.
Results: 30 patients were diagnosed with TNBC. 26 (86.7%) had a grade
III tumor and 4 (13.3%) a grade II tumor. No grade I cases were seen in
this group. All patients underwent definite surgery and were evaluated for
pathologic response. pCR was observed in 13 (43.3%); of the remaining
patients 15 (51.7%) showed a residual cancer burden (RCB) II and 1
(3.5%) patient a RCBIII as per Symmans criteria. Of the 21 patients for
whom pretreatment material was available 3 (14.2%) were positive for AR
expression. Of the 17 patients without a complete response post-treatment,
2 were positive for AR expression. Of the 3 cases that were AR-positive
pre-treatment, 2 remained positive post-treatment and 1 had a pCR.
Conclusions:
1. Combination of carboplatin and eribulin mesylate resulted in a pCR in
almost half (43.3%) of the TNBC patients.
2. Expression of AR does not appear to play a role in the promising
response rate seen with this novel neoadjuvant combination.
Additional studies are underway to further characterize the molecular
mechanisms that drive response in this challenging subtype of breast
cancer.
25
POSTER (Board P019)
An ING1b-derived peptide that inhibits cancer cell viability and
promotes apoptosis
A. Boyko1 , K. Riabowol1 . 1 University of Calgary, Department of
Biochemistry & Molecular Biology, Calgary, Canada
The ING1b protein is a type II tumor suppressor and stoichiometric
member of HDAC-containing protein complexes. ING1b contributes to
regulation of gene expression, senescence and apoptosis. Mislocalization
and decreased levels of ING1b are commonly observed in human tumors
and cancer cell lines. Multiple independent studies show that ING1b
overexpression promotes apoptosis in targeted cells. Since the inactivation
of apoptosis pathways is frequent in cancer cells, modulating ING1b
expression may serve as a viable approach for cancer therapy. We are
defining ING1b regions necessary for apoptotic function in order to design
minimal recombinant peptides with potent apoptosis-inducing properties.
Based on the predicted protein structure and published data, we produced
a number of expression constructs that encompass various portions of the
ING1b protein. Following the initial screening stage, pro-apoptotic effects
of the most successful expression constructs were further confirmed and
quantified by western blotting using the levels of PARP cleavage, and
by FACS analysis using the Annexin V assay. We have established that
ING1b-derived peptides containing its third alpha helix (A3H) and nuclear
localization sequence (NLS)/nucleolar translocation signal (NTS) domains
are able to induce apoptosis at levels comparable to those of the full length
ING1b as determined using Annexin V assays. The A3H-NLS/NTS peptide
exhibited strong nucleolar localization, characteristic of full length ING1b.
Cells overexpressing the full length ING1b and A3H-NLS/NTS peptide
showed similar changes in cell morphology characteristic of apoptosis
and exhibited increased levels of PARP cleavage. While the A3H region
was necessary but not sufficient, the NLS/NTS domain was required,
and partially sufficient, for induction of apoptosis. Adenoviral delivery of
A3H-NLS/NTS peptide led to a drastic increase in the number of cells
undergoing apoptosis and resulted in a strong dose-dependent inhibition
of cancer cells viability that was independent of p53 status. The strong
anti-tumorigenic effects were observed in a broad range of tested cancer
cell lines including those of osteosarcoma, glioblastoma and breast cancer
origin. The evaluation of the synergy between the A3H-NLS/NTS peptide
and common chemotherapeutic agents is currently ongoing. Our longterm goal is to develop ING1b-based therapeutics that can be used as
an adjuvant therapy in combination with the existing cancer treatments.
26
POSTER (Board P020)
NPD926, a small molecule inducer of reactive oxygen species, kills
cancer cells via glutathione depletion
T. Kawamura1 , Y. Kondoh1 , M. Muroi1 , M. Kawatani1 , H. Osada1 . 1 Riken,
Antibiotics Laboratory, Wako Saitama, Japan
Background: Elucidation of the mechanisms of action of bioactive
compounds may render a therapeutic regimen to be more successful.
However, this process is generally time-consuming. By a combination
of proteomic profiling and affinity purification, we recently elucidated the
mechanism of action of a newly identified cytotoxic compound, NPD926.
Here we report the mechanism of action of NPD926 and its effects on
cancer cells.
Material and Methods: Prediction of the mechanism of action of NPD926
was performed by ChemProteoBase profiling, which is based on the
proteomic perturbation in HeLa cells induced by treatment with bioactive
compounds. NPD926-binding proteins were purified using affinity matrices,
separated by SDS-PAGE, and identified by MALDI-TOF-MS.
Results: NPD926 showed cytotoxicity against 19 types of human
cancer cell lines. ChemProteoBase proteomic profiling showed that
NPD926 shared similarity with 1-chloro-2,4-dinitrobenzene, a substrate
for glutathione S-transferase (GST). By affinity purification, an isozyme
of GSTs was identified as a specific NPD926-binding protein. Based
on these findings and the subsequent biochemical assays, we elucidated the mechanism of action underlying NPD926-induced cell death:
(i) conjugation with glutathione as a substrate for GSTs, (ii) depletion of
cellular glutathione, and subsequent (iii) generation of reactive oxygen
species (ROS). NPD926 preferentially induced cytotoxicity against KRAStransformed fibroblast NIH3T3 cells, compared with their untransformed
counterparts. Furthermore, NPD926 sensitized cells to inhibitors of system
xc− , a cystine-glutamate antiporter considered as a potential therapeutic
target in cancers including cancer stem cells.
Conclusions: By the chemical biological methods, we elucidated the
mechanism of action of NPD926. Induction of excessive levels of ROS
by compounds has been considered as a potentially effective therapeutic
strategy against cancer cells. Our present study shows the effectiveness
of a newly identified ROS inducer, which targets glutathione metabolism,
in cancer cells including those harboring oncogenic KRAS.
27
POSTER (Board P021)
TAS-102 treatment results in high trifluridine incorporation into DNA
with pyrimidine metabolic pathway markedly up-regulated in cancer
K. Oguchi1 , K. Sakamoto1 , H. Kazuno1 , H. Ueno1 , K. Ishida1 ,
T. Yokogawa2 , K. Yamamura1 , R. Kitamura1 , K. Matsuo1 , T. Utsugi1 .
1
Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba,
Japan; 2 Taiho Pharmaceutical Co. Ltd., Business Development Dept.,
Chiyoda-ku, Japan
Background: TAS-102 (Lonsurf® ) consists of trifluridine (FTD) and tipiracil
hydrochloride (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog
and the antitumor component of TAS-102. TPI prevents degradation of FTD
by thymidine phosphorylase in liver. TAS-102 was found to significantly
improve overall survival of patients with metastatic colorectal cancer
that is refractory/intolerable to the standard chemotherapies in a global
Phase III study. The mechanism of FTD is considered to be based
on its incorporation into DNA, however its intracellular metabolism is
not completely defined. Here, we investigate the cellular uptake and
phosphorylation of FTD, and substrate specificity of incorporation into DNA
by DNA polymerase a.
Material and Methods: Intra-cellular uptake of FTD was evaluated
with nucleoside transporter specific inhibitors, NBMPR and dipyridamole.
Substrate specificity for nucleoside kinase and nucleotide phosphorylase
was evaluated with recombinant proteins. DNA elongation with FTD
triphosphate (F3 dTTP) by DNA polymerase a was analyzed. The levels
of FTD incorporated into DNA were analyzed with [3 H]FTD or LC-MS/MS.
Results: FTD was transported via ENT1 and ENT2 into cytoplasm,
and was phosphorylated by thymidine kinase 1 as effectively as dThd.
F3 dTTP was not recognized by dUTPase, whereas FdUrd-triphosphate was
degraded to monophosphate form. F3 dTTP was inserted at the opposite
site of adenine by DNA polymerase a. DNA extension was effectively
carried out at the site of inserted FTD, and was evident on the single strand
DNA template inserted FTD.The level of FTD incorporated into DNA was
much higher than that of FdUrd in vitro, and FTD remained in DNA after the
FTD removal from culturing medium. In vivo study also showed high FTD
accumulation in tumor implanted in nude mice. Nucleosomal abnormalities
such as swollen nuclei and decreased hetero-chromatin were observed in
the cells treated with FTD.
Conclusions: These results indicate that FTD is recognized and activated
through the thymidine salvage pathway highly up-regulated in tumor, and
incorporated into DNA during DNA replicating phase like the natural
substrate dThd. FTD might exert antitumor activity by nucleosomal
abnormalities caused by high level FTD incorporation into DNA. These
unique properties might underlie the clinical benefit of prolonged survival
of colorectal cancer patients treated with TAS-102 after failure on prior 5-FU
based chemotherapy.
28
POSTER (Board P022)
Characterization of the type of cell death induced by novel
tambjamine analogs in lung cancer
A. Rodilla Martı́n1 , V. Soto-Cerrato1 , P. Manuel-Manresa1 , L. KorrodiGregório1 , R. Quesada2 , R. Pérez-Tomàs1 . 1 University of Barcelona,
Department of Pathology and Experimental Therapeutics, Barcelona,
Spain; 2 University of Burgos, Department of Chemistry, Barcelona, Spain
Lung cancer is the leading cause of cancer death worldwide. Despite of new
advances in diagnosis and clinical care, the success of standard treatments
is still limited, especially in chemotherapy. Therefore, novel anticancer
compounds with different mechanisms of action are eagerly needed. In
this view, our research group proposes a new therapeutic strategy against
cancer that involves modulating the intracellular pH (pHi). Cancer cells have
a reversed pH gradient compared to normal cells, which allows cancer
progression by promoting proliferation and evasion of apoptosis. Hence,
anion transporter compounds, such as tambjamine analogs, have been
selected in this study for their potential as anticancer agents through the
modulation of the pHi.
First, the effect of our compounds in cell viability was evaluated in several
lung cancer cell lines by the MTT assay. A significant decrease was
observed in most of them, and two compounds (3 and 9) were chosen
for further studies. To test whether these compounds have a cytostatic
effect, cytometry assays were performed and showed that tambjamines
did not induce a significant cell cycle arrest. Then, to characterize the
type of cell death, we analyzed different molecular markers related
to apoptosis and autophagy by western blot and we observed some
evidence of caspases activation and LC3II accumulation. Furthermore,
it could be seen at the microscope, phase contrast as well as electron
microscope, that the compounds also induced massive cytoplasmic
vacuolization. We tested several markers to distinguish among different
potential organelles, which could have undergone this phenomenon. We
15
used LAMP1 and LC3II by immunofluorescence and we could exclude
lysosomes and autophagosomes, respectively. The origin of the vacuoles
could also be from the incorporation of extracellular fluid by endocytosis or
macropinocytosis, or by the swelling of organelles such as mitochondria or
endoplasmic reticulum, which we are trying to identify.
Altogether, these results show that these anion transporters have potent
cytotoxic effects in lung cancer cell lines, inducing some kind of cell death
in which traits of several cell death processes are involved.
Acknowledgements: This work was supported by a grant from the Spanish
government and the EU (FIS PI13/00089) and a grant from La Marató
de TV3 Foundation (65/C/2013). LKG holds a postdoctoral fellowship
through the Portuguese Foundation for Science and Technology-FCT
(SFRH/BPD/91766/2012).
29
POSTER (Board P023)
TAS-114 is a novel dUTPase/DPD inhibitor, its DPD inhibition reduces
capecitabine dosage but does not diminish therapeutic window in
human tumor xenografts
W. Yano1 , H. Kazuno1 , T. Yokogawa2 , K. Sakamoto1 , K. Yoshisue1 ,
T. Wakasa1 , M. Fukuoka3 , K. Matsuo1 , K. Noguchi1 , T. Utsugi1 . 1 Taiho
Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba, Japan;
2
Taiho Pharmaceutical Co. Ltd., Business Development Dept., Chiyoda-ku,
Japan; 3 Taiho Pharmaceutical Co. Ltd., CMC Center, Tokushima, Japan
Background: Deoxyuridine triphosphatase (dUTPase) is a gatekeeper enzyme for uracil misincorporation and is considered to be an important factor
for resistance to 5-fluorouracil (5-FU). Dihydropyrimidine dehydrogenase
(DPD) is a rate-limiting enzyme for 5-FU degradation. TAS-114 is a firstin class oral dUTPase/DPD inhibitor and is under clinical development in
combination with 5-FU prodrugs.
Previously, it was reported that the combination of capecitabine and a
selective DPD inhibitor could reduce the maximum tolerated dose (MTD)
of capecitabine. However, the therapeutic window of capecitabine/DPD
inhibitor was no better than capecitabine alone. In contrast, we found
that the combination of capecitabine with TAS-114, a dual inhibitor of
dUTPase/DPD, significantly enhanced the antitumor efficacy and improved
the therapeutic window. Here, we report the mode of DPD inhibition
by TAS-114, and roles of dUTPase inhibition and DPD inhibition in
capecitabine/TAS-114.
Material and Methods: Mode of DPD inhibition was determined by measuring 5-FU degradation in human liver S9 fraction. Plasma levels of 5-FU
were measured by LC/MS for pharmacokinetics. The antitumor activity
of capecitabine/TAS-114 was evaluated in xenografts of MX-1 and MC-2
(human breast cancers), and KB/TP (cancer cell stably overexpressed
thymidine phosphorylase (TP)). MX-1 is a dUTPase inhibition sensitive
tumor, and MC-2 and KB/TP are sensitive to capecitabine due to high
TP expression.
Results: TAS-114 possessed moderate DPD inhibitory activity and its
inhibition mode was reversible. In combination with capecitabine, TAS114 increased 5-FU concentration in plasma due to DPD inhibition. This
increase in 5-FU exposure led to reduction of capecitabine MTD in all
tumor models. In both MC-2 and KB/TP xenograft models, which were
highly sensitive to capecitabine, TAS-114 did not reduce maximal antitumor
activity and therapeutic window of capecitabine, despite a reduction
in the dosage. In the MX-1 xenograft model, which is inherently less
sensitive to capecitabine, TAS-114 significantly enhanced antitumor activity
of capecitabine, demonstrating the importance of dUTPase inhibition.
Conclusions: TAS-114 is a moderate and reversible inhibitor of DPD. Its
favorable profile of DPD inhibition reduces the MTD of capecitabine but
does not diminish therapeutic window. Phase 1 study of capecitabine/TAS114 is ongoing and therapeutic efficacy as a dUTPase/DPD dual inhibitor
can hopefully be demonstrated.
16
30
POSTER (Board P024)
The fungal-derived cyclohexadepsipeptide Destruxin E exerts
multifaceted anticancer and antiangiogenic activities
R. Dornetshuber-Fleiss1 , P. Heffeter2 , T. Mohr2 , P. Hazemi3 , K. Kryeziu4 ,
C. Seger5 , W. Berger2 , R. Lemmens-Gruber3 . 1 University of Vienna and
Medical University of Vienna, Institute of Pharmacology and Toxicology
and the Institute of Cancer Research Department of Medicine I and
Comprehensive Cancer Center of the Medical University, Vienna,
Austria; 2 Medical University of Vienna, Institute of Cancer Research
Department of Medicine I and Comprehensive Cancer Center of the
Medical University, Vienna, Austria; 3 University of Vienna, Department
of Pharmacology and Toxicology, Vienna, Austria; 4 Medical University
of Vienna, Institute of Cancer Research Department of Medicine I
and Comprehensive Cancer Center of the Medical University, Vienna,
Austria; 5 Leopold-Franzens University Innsbruck, Institute of Pharmacy
Department of Pharmacognosy, Vienna, Austria
Background: Destruxins − a group of secondary metabolites of the
entomopathogenic fungus Metarhizium anisopliae − recently came into
focus of interest as anticancer therapeutics for colorectal cancer. However,
the knowledge on their anticancer effects is fragmentary. Consequently,
the present study aimed to investigate the impact of the representative
derivative Destruxin E (Dtx E) on human colon cancer cell growth and
survival.
Materials
and
Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and Western blot analyses were used
to assess the cytostatic/cytotoxic activity of Dtx E on Caco-2 and HCT116
cells. Impact of cell cycle distribution was evaluated using PI-stainings and
FACS analyses. Apoptotic effects were determined with DAPI stainings
and Western blot analyses. Angiogenesis inhibition was evaluated using
scratch and tube-formation assays. To investigate the impact on the
intracellular redox potential flow cytometry with dihydroxyethidium (DHE)
and combination studies with the radical scavenger N-acetyl cysteine (NAC)
and the glutathione inhibitor BSO were performed.
Results: The experimental data showed that Dtx E exerts potent
antiproliferative activity in the nanomolar range in CaCo-2 and HCT116
cells. Moreover, Dtx E caused an imbalance of cell cycle distribution and
the cytostatic/cytotoxic effects were shown to be widely p53-independent
but reduced by p21- and bax-deletion, respectively. Cytotoxicity is based
on intrinsic apoptosis induction and associated with phosphoinositide-3kinase (PI3K)/Akt pathway inhibition. Additionally, anticancer activity of
Dtx E involves disturbance of the intracellular redox balance and finally,
Dtx E inhibits the migration and tube formation of human endothelial cells
indicating antiangiogenic potential.
Conclusion: The data of the present study indicate that Dtx E represents
a feasible, multifunctional anticancer drug candidate for preclinical
development against colorectal cancer.
31
POSTER (Board P025)
N-Myc amplification sensitizes tumor cells to inhibition by
Danusertib, an Aurora kinase inhibitor
P. Carpinelli1 , R. Ceruti2 , R. Alzani2 , C. Re1 , D. Ballinari1 , S. Cribioli2 ,
M. Russo2 , A. Degrassi2 , G. Texido2 , M. Ciomei2 , E. Pesenti2 ,
A. Montagnoli1 , A. Galvani1 . 1 Nerviano Medical Sciences Srl, Cell
Biology, Nerviano (Milano), Italy; 2 Nerviano Medical Sciences Srl,
Pharmacology, Nerviano (Milano), Italy
Amplification of N-Myc is a driving mutational event in a subset of tumor
types, particularly those of neural origin and neuroendocrine tumors such
as neuroblastoma (NB), neuroendocrine prostate cancer (NEPC), small
cell lung cancer (SCLC) and others. Thus, inhibition/reduction of N-Myc
protein levels may be of therapeutic benefit for such tumor types but
to date small molecules that specifically target this oncogene are not
clinically available. Aurora A kinase has been shown to interact directly
with N-Myc in neuroblastoma and to induce its stabilization by preventing
binding of the Fbxw7 ubiquitin ligase, which mediates its ubiquitination and
subsequent proteasomal degradation. Moreover, there is clear evidence
that pharmacological intervention with small molecule inhibitors which
target Aurora A represents a promising therapeutic approach for this tumor
subtype.
Danusertib (PHA-739358) is a small molecule ATP competitor that inhibits
Aurora A, B and C kinases. Aurora kinases inhibition by Danusertib results
in cell cycle block with concomitant inhibition of cell proliferation. The
compound is in clinical investigation in both solid tumor and hematological
malignancy settings.
Here we report the in vitro and in vivo activity of Danusertib in N-Myc
amplified tumor models. We demonstrate that in vitro, NB cell lines
bearing amplified N-Myc display greater sensitivity to Danusertib than
non-amplified cell lines. In such sensitive NB cell lines, inhibition of
histone H3 phosphorylation, a marker of Aurora kinase inhibition, is
associated with decreased levels of N-Myc and induction of apoptosis.
In NB tumor xenograft models, Danusertib induced significant in vivo
tumor growth inhibition, including tumor regression, accompanied by robust
reductions of N-Myc protein levels, again concomitant with inhibition of
phospho-histone H3 and induction of apoptosis. Additionally, Danusertib
also displayed significant in vivo activity against the TRAMP, a transgenic
mouse prostate carcinoma model, which displays several molecular and
pathological features of NEPC. Here again, treatment with Danusertib
induced decreased levels of N-Myc, associated with inhibition of histone
H3 phosphorylation.
Thus, Danusertib destabilizes N-Myc both in vitro and in vivo and therefore
merits further investigation as a potential option for the therapy of N-Myc
amplified neuroendocrine tumors, as well as other settings which are
dependent on this oncogene.
32
POSTER (Board P026)
Replication stress is a determinant of synergy between gemcitabine
and Chk1 inhibition
S.B. Koh1 , A. Courtin1 , R. Boyce2 , B. Boyle2 , F.M. Richards1 , D.I. Jodrell1 .
1
Cancer Research UK Cambridge Institute, University of Cambridge,
Cambridge, United Kingdom; 2 Sentinel Oncology Limited, Cambridge,
United Kingdom
Background: Chk1 plays multiple regulatory roles within the cell cycle and
this study interrogates the mechanism of synergy between Chk1 inhibition
and gemcitabine in cancer cells.
Methodology: We evaluated the cytotoxicity of FS105, a Chk1-specific
inhibitor (IC50 2.8nM), with gemcitabine in human and murine cancer
cells. We then employed established mathematical models to quantitatively
identify drug concentrations that yielded synergistic growth inhibition.
Subsequent mechanistic studies, achieved by quantitative high-content
imaging, immunofluorescence microscopy, flow cytometry and immunoblotting, were performed at the identified synergistic drug concentrations.
Results: Synergy with FS105 was found at sub-GI50 concentrations of
gemcitabine in all tested cell lines, e.g. Emax 91±3% growth inhibition at
3uM FS105+30nM gemcitabine in Panc-1 cells compared to Bliss predicted
33±1% growth inhibition. At these concentrations, we did not observe
substantial premature mitotic entry, contrary to the prevailing model that
inhibiting Chk1 abrogates the G2/M checkpoint. Downstream checkpoint
effector CDK1 Y15 was not inhibited by FS105 at synergistic concentrations
with gemcitabine. In agreement with this, significant S phase arrest with
hallmarks of elevated replication stress and DNA damage such as ùH2AX
was observed. For example, 63±6% MIA PaCa-2 cells were in S phase
24 hours after the combination treatment, compared to control (33±2%),
FS105 alone (35±4%) and gemcitabine alone (43±4%). The induction
of S phase arrest and ùH2AX by the combination was inhibited when
the activity of replication initiator CDC7 was suppressed by PHA-767491.
This observation is consistent with Chk1 role in regulating origin firing
and our hypothesis that Chk1 inhibition synergises with gemcitabine via
destabilisation of the replication machinery.
Conclusions: This study elucidates how Chk1 inhibition with gemcitabine
at synergistic concentrations perturbs predominantly the S-phase homeostasis in cancer cells. It also provides a molecular rationale to reassess
the current scheduling paradigm of Chk1 inhibitors with DNA-damaging
agents.
33
POSTER (Board P027)
Combining the long-acting topoisomerase 1-inhibitor etirinotecan
pegol with the PARP inhibitor rucaparib to provide anti-tumor
synergy without increased toxicity
U. Hoch1 , D. Charych2 . 1 Nektar Therapeutics, Development, San Francisco
CA, USA; 2 Nektar Therapeutics, Research, San Francisco CA, USA
Background: PARP inhibition sensitizes cells to DNA-damaging agents,
particularly topoisomerase 1-inhibitors, where the combination shows synergy in nonclinical studies. However, in the clinic, the combination of either
topotecan or irinotecan with PARP inhibitors showed severe hematologic
toxicities, requiring dose reductions beyond optimal therapeutic effects.
Etirinotecan pegol (NKTR-102) is the first long-acting Topoisomerase
1-inhibitor providing continuous exposure to active metabolite throughout
the entire chemotherapy cycle with reduced peak concentrations, resulting
in better efficacy and safety, including a low rate of Grade 3 neutropenia
(11%). Here we present in vivo combination data of etirinotecan pegol
and rucaparib in the MX-1 breast cancer model.
Methods: Mice bearing BRCA1-deficient MX-1 breast cancer tumors
(~100 mm3 ) received either vehicle, PO rucaparib, IV etirinotecan pegol,
or a combination of etirinotecan pegol and rucaparib as follows: 30 or
150 mg/kg rucaparib qd×21; 10 or 50 mg/kg etirinotecan pegol q7d×4;
or 30/10, 30/50, 150/10, or 150/50 mg/kg rucaparib/etirinotecan pegol
(n = 10/group). Anti-tumor efficacy was evaluated by tumor growth delay
(TGD) and regression responses.
Results: Control tumors grew to the 2000 mm3 endpoint in a median of
25 days. Rucaparib at 30 and 150 mg/kg resulted in TGDs of 14 and 18
days, respectively. Tumor growth was slower following rucaparib but was
continuous in all animals. Etirinotecan pegol at 10 mg/kg resulted in TGD of
32 days with one tumor-free animal. Any combination of etirinotecan pegol
and rucaparib, and single-agent etirinotecan pegol at 50 mg/kg caused
complete tumor regression in all animals that persisted for at least 10
weeks. All single-agent and combination doses were well tolerated without
clinical signs or body weight loss, except rucaparib/etirinotecan pegol
150/50 where individual animals showed BW loss of 15% (group mean:
6%).
Conclusions: Combined etirinotecan pegol and rucaparib showed clear
synergy in the BRCA1-deficient MX-1 breast cancer model with 100%
complete responses and no tumor regrowth, even 7 weeks after last dose.
The combination was well tolerated. SN38 trough and rucaparib plasma
exposure at the dose levels employed in mice are achieved clinically. Our
results suggest that this combination may diminish previously observed
toxicities of combined Topoisomerase 1- and PARP-inhibitors while
improving efficacy and support pursuing further studies with etirinotecan
pegol and PARP-inhibitors.
34
POSTER (Board P028)
Phase I, dose-escalation study of the investigational drug D07001-F4,
an oral formulation of gemcitabine HCl, in patients (pts) with
advanced solid tumors or lymphoma
C. Lin1 , W. Su2 , J. Lee1 , C. Hsu1 , A. Cheng1 , C. Lin3 , H. Ho3 , C. Huang3 ,
S. Hsueh3 , J. Yang1 . 1 National Taiwan University Hospital, Oncology,
Taipei, Taiwan; 2 National Cheng Kung University Hospital, Oncology,
Tainan, Taiwan; 3 InnoPharmax Inc., Taipei, Taiwan
Background: This phase 1 dose-escalation trial, using a 3+3 design,
was conducted to evaluate the safety, tolerability, pharmacokinetics (PK),
and preliminary efficacy of D07001-F4, an oral formulation of gemcitabine
HCl, in pts with advanced solid tumors or lymphoma. The primary
objectives were to determine the MTD, characterize DLT(s) and define the
recommended phase 2 dose of D07001-F4.
Methods: Patients aged 20 yrs with PS 0−2 were eligible. Gemcitabine
5 mg IV in cycle 0 and D07001-F4 from 2 mg PO were given on days 1, 3,
5, 8, 10, and 12 of a 21-day cycle to determine the MTD based on DLTs
in cycle 1. Plasma and peripheral blood samples were obtained for PK
analysis in cycle 1.
Results: As of May 2014, 15 pts (10 M, 5 F; median 59 years [range
42−67]) were enrolled into 5 dose escalation cohorts (2−30 mg). A median
of 3 cycles were given (range 0−13+). So far, no DLTs occurred and the
MTD has not yet been reached. Enrollment is ongoing. Two pts at cohort
4 (20 mg) experienced 3 drug-related G1 AEs (1 fever, 2 fatigue) and 2
pts at cohort 5 (30 mg) experienced 3 drug-related G1 AEs (1 vomiting, 2
nausea). PK results showed a dose proportional increase in exposure of
dFdC after repeated dosing. Plasma dFdC was fast eliminated after dosing.
The average Cmax 7474 pg/mL and AUC0−24 5603 hr·pg/ml at day 1 and
Cmax 6588 pg/mL and AUC0−24 7926 hr·pg/ml at day 12 were observed at
cohort 4 (20 mg). 10 pts had SD, 7 of whom were on study for 3 months.
One ongoing pt (5 mg) with thyroid cancer had tumor shrinkage of 28%.
Conclusions: From preliminary data, D07001-F4 appears to be well
tolerated and has shown signs of anti-tumor activity in pts with advanced
solid tumors. Accrual at the 30 mg dose level is ongoing and a
recommended phase 2 dose is expected to be reached soon. Updated
results will be presented.
Clinical trial registration: NCT01800630
35
POSTER (Board P029)
BRCA1 expression exploratory analysis in patients of the phase III
trial of trabectedin vs. doxorubicin-based chemotherapy as first-line
therapy in translocation-related sarcomas
M. Aracil1 , P. Lardelli2 , A. Nieto2 , C.M. Galmarini1 . 1 PharmaMar S.A.,
R&D, Colmenar Viejo (Madrid), Spain; 2 PharmaMar S.A., Clinical
Development, Colmenar Viejo (Madrid), Spain
Background: BRCA1 mRNA and protein expression levels were analyzed
in translocation-related soft tissue sarcoma (TRS) patients treated with
trabectedin (arm A) or doxorubicin (arm B) as first-line therapy (phase III
trial ET-C-002−07). Expression levels were analyzed in tumor tissue
samples obtained at diagnosis with the aim to determine the potential role
of BRCA1 as prognostic and/or predictive biomarker.
17
Materials and Methods: A total of 83 samples with clinical and molecular
data were subjected to biostatistical analysis in order to correlate the
expression values with the clinical outcome (ORR, PFS and OS). PFS
and objective tumor response were evaluated by an Independent External
Review Committee according to RECIST v.1.0. The IHC staining intensity
was evaluated by a pathologist and reported following a 4-level scale from
0 (no staining) to +3 (strong staining). Expression levels were categorized
using as cutoff the median value.
Results: In the trabectedin arm, lower BRCA1 protein expression (<3) was
associated with higher tumor control (PR+SD) (91.7% for low BRCA1 vs.
57.1% for high BRCA1, p = 0.0095) and longer median PFS (not reached
for low BRCA1 vs. 2.4 m for high BRCA1; p = 0.0013). In the doxorubicin
arm, lower BRCA1 protein expression was associated with longer median
PFS (18.9 m for low BRCA1 vs. 5.5 m for high BRCA1; p = 0.0156). In a
multivariate analysis including the most common clinical variables, where
treatment is not selected, BRCA1 protein expression correlated with a
worse PFS (HR = 4.539, 95% CI: 1.941–10.613; p = 0.0005).
Conclusions: The results point out the potential importance of BRCA1
in the clinical outcome of patients with TRS treated with trabectedin or
doxorubicin. In particular, high protein expression of BRCA1 seems to be
associated with a worse clinical outcome. Its potential prognostic and/or
predictive value in TRS patients needs to be further evaluated.
36
POSTER (Board P030)
Suppression of metastasis and improvement of drug distribution by
eribulin mesylate
Y. Ozawa1 , K. Okamoto1 , Y. Adachi1 , M. Asano1 , K. Tabata1 ,
Y. Funahashi2 , J. Matsui1 . 1 Eisai Co. Ltd., Oncology PCU, Tsukuba
Ibaraki, Japan; 2 Eisai Co. Ltd., BPM CFU, Andover, USA
Background: Eribulin mesylate (eribulin) has shown trends toward
greater overall survival compared to progression-free survival in late
stage metastatic breast cancer patients in the clinic. This finding
suggests that eribulin may have additional, previously unrecognized antitumor mechanisms. To investigate this possibility, eribulin’s effects on
microenvironment including tumor vessels were investigated and we
found that eribulin demonstrated improvement in tumor perfusion through
vascular remodeling in human breast cancer models.
Material and Methods: For in vitro study, cells were cultured under
1% O2 and 0.1 mg/ml glucose. EMT markers were analyzed by western blot
analysis. For in vivo model, after 15 days post-4T1 cell inoculation into the
3rd mammary fad pad of Balb/c mice, drug treatment was started, and after
28 days lung metastases were counted. In MDA-MB-231 model, treatments
with capecitabine or eribulin were started on day 1. On day 12, eribulin
treated mice were divided into no-treatment and capecitabine treatment
groups.
Results: The remodeling of tumor vessels evoked 2 possibilities, antimetastatic effect and enhancement of antitumor activity thorough the
improvement of drug influx into tumors. Our data showed that hypoxic
conditions induced EMT phenotype in breast cancer cells in vitro, and
eribulin treatment reduced hypoxic markers in xenorafts. Furthermore,
eribulin significantly reduced the lung metastasis in 4T1 OT model in which
eribulin did not have any antitumor effect against primary tumor because
of MDR over expression. We next investigated the effect of eribulin on
improvement of drug influx into tumors. We compared the antitumor effects
of capecitabine with or without pre-treatment of eribulin in MDA-MB-231
xenograft model. In eribulin pre-treated group, eribulin treatment induced
clear tumor regression, and then, capecitabine treatment was started when
tumor volumes returned to baseline prior to eribulin treatment. Interestingly,
antitumor effect of capecitabine was enhanced in eribulin-pretreated group,
compared to non-treated group. We think this enhancement of antitumor
effect is the result of improvement of capacitabine influx into tumors.
Conclusion: We have found the new effects of eribulin, anti-metastatic
effect and improvement of drug influx into tumors via the remodeling of
tumor vessels, using human and mouse breast cancer models. These
preclinical finding may provide new insights into the clinical observations.
37
POSTER (Board P031)
Effect of a microtubule-targeting drug on cell–cell contacts in
bladder epithelial tumour cells
L.M. Antón-Aparicio1 , R. Castosa2 , M. Haz2 , M. Blanco2 , M. Rodriguez2 ,
M. Valladares1 , A. Figueroa2 . 1 Complejo Hospitalario Universitario A
Coruña (INBIC-CHUAC), Medical Oncology Unit, Coruna, Spain; 2 Instituto
Investigación Biomédica A Coruña-Complejo Hospitalario Universitario A
Coruña (INBIC-CHUAC), Translational Cancer Research Group, Coruna,
Spain
Background: Bladder cancer is a common malignancy affecting the
genitourinary system that represents the fifth most common cancer in
18
the world. Transitional cell carcinoma (TCC) represents 95% of these
tumours. Vinflunine (VFL) is a microtubule-targeting drug that suppresses
microtubule dynamics, showing anti-metastatic properties both in vitro
and in living cancer cells. An increasing body of evidence underlines the
influence of the microtubules dynamics on the cadherin-dependent cell–
cell adhesions. In this report, we investigate the role of VFL on cell–cell
contacts in bladder epithelial tumour cells.
Methods: Human bladder epithelial tumour cell lines HT1376, 5637 and
UMUC3 were used to analyse cadherin-dependent cell–cell adhesions under VFL treatment. VFL effect on growth inhibition was measured by using
a MTT colorimetric cell viability assay. Western blot, immunofluorescence
and transmission electron microscopy analyses were performed to assess
the roles of VFL effect on cell–cell adhesions, epithelial-to-mesenchymal
markers and apoptosis. The role of the proteasome in controlling cell–cell
adhesion was studied using the proteasome inhibitor MG132.
Results: We show that VFL induces cell death in bladder cancer cells and
activates epithelial differentiation of the remaining living cells, leading to
an increase of E-cadherin-dependent cell–cell adhesion and a reduction
of mesenchymal markers, such as N-cadherin or vimentin. Moreover,
while E-cadherin is increased, the levels of Hakai, an E3 ubiquitin-ligase
for E-cadherin, were significantly reduced in presence of VFL. In 5637,
this reduction on Hakai expression was blocked by MG132 proteasome
inhibitor, indicating that the proteasome pathway could be one of the
molecular mechanisms involved in its degradation.
Conclusions: Our findings underscore a critical function for VFL in cell–cell
adhesions of epithelial bladder tumour cells, suggesting a novel molecular
mechanism by which VFL may impact upon EMT and metastasis.
38
POSTER (Board P032)
The indolyl-chalcone CDD-026 induces cancer cell death through
targeting of STMN1 and mitotic catastrophe
B. Wegiel1 , Y. Wang1 , F. Jernigan1 , L. Sun1 . 1 Beth Israel Deaconess
Medical Center, Surgery, Boston MA, USA
Background: Patients with metastatic prostate cancer are still in needs
of more effective treatments. Mitotic catastrophe is a tumor suppressive
process that is driven by aberrant mitosis and in many cases associated
with changes in microtubules stability. The activation of mitotic catastrophe
is a highly desirable therapeutic endpoint and the focus of this study. The
aim of this study was to investigate the anticancer activities of a series
of novel indolyl-chalcones and validate their targets in inducing mitotic
catastrophe in prostate cancer cells lines.
Materials and Methods: We have performed screen of synthetic
indolyl-chalcones in induction of apoptosis and inhibition of proliferation
of cancer cells. We have applied multiple cell biology assays to test
apoptosis (AnnexinV/PI staining, crystal violet staining), proliferation (BrDU
incorporation assay, PI cell cycle analysis), targets analysis (PCR Real time
profiler) and targets validation (real time PCR). We conducted molecular
modeling analyses to develop a QSAR model.
Results: We have identified and improved the potency of a series of
novel indoly-chalcones. Our results indicated that the lead molecule CDD026 (Formula: C20H16FNO) at 1−10 mM induced early apoptosis without
affecting incorporation of BrdU in S-phase, suggesting its critical role in
interrupting late phases of cell cycle. In contrast, the non-indolyl analog
CDD-023 inhibited cell cycle progression through G1/S phase in a dose
dependent manner (1, 10, 25 mM) but did not induce significant cell death.
We then focused our analysis on CDD-026, which at as low dose as
0.1 mM induced apoptosis in PC3 cells. In addition, CDD-026 demonstrated
potent inhibitory activity in lung and other cancer cells. To evaluate the
potential target for this drug, we have employed Cancer Pathway Finder
PCR array (Qiagen) and identified Stathmin 1 (STMN1) and adrenomedullin
genes as the most highly upregulated in response to treatment with CDD026. We have validated these targets by real time PCR in PC3 cells
treated with CDD-026. Further, we showed that in contrast to taxanes
that stabilized microtubules, CDD-026 destabilized microtubules. This effect
may be dependent on STMN1 upregulation leading to mitotic catastrophe,
which is currently being addressed in our studies.
Conclusions: CDD-026 induces mitotic catastrophe in PC3 cells via
upregulation of STMN1, which destabilizes microtubules and disrupts cell
cycle.
39
POSTER (Board P033)
Phytochemical indole-3-carbinol synergizes strongly with fludarabine
and induces p53-dependent and -independent cell death in chronic
lymphocytic leukemia cells irrespective of their IGHV mutation state
and treatment resistances
G. Perez-Chacon1 , C. Martinez-Laperche2 , N. Rebolleda1 , B. SomovillaCrespo3 , C. Muñoz-Calleja3 , I. Buño2 , J.M. Zapata1 . 1 Instituto de
Investigaciones Biomedicas “Alberto Sols” CSIC/UAM, Madrid, Spain;
2
Hospital General Universitario and Instituto de Investigacion Sanitaria
Gregorio Marañon, Servicio de Hematologia, Madrid, Spain; 3 Hospital
Universitario de la Princesa, Servicio de Inmunologia, Madrid, Spain
Background: Chronic lymphocytic leukemia (CLL) is the most common
type of leukemia found in adults in Western countries, which still is lacking
a cure. A variety of chemotherapy regimens are being used in CLL, but
unfortunately patients will eventually become refractory to treatments and
die. It is therefore a priority to develop new treatments and therapeutic
approaches that improve patient prognosis and survival. Indole-3-carbinol
(I3C) is a glucobrassicine derivative that is found in edible Cruciferae plants,
such as the broccoli and the cabbage. Phase I and II clinical trials have
demonstrated its antitumoral activity in precancerous cervix lesions and in
vulvar epidermal neoplasia, while lacking significant toxicity.
Materials and Methods: We have tested whether I3C was active against
CLL cells. We used a cohort of CLL cells from 36 patients representing
different Rai stages, IGHV mutation status, cytogenetic alterations, and
responses to treatments. The effect of increasing concentrations of I3C,
F-ara-A and combinations of both drugs (keeping a constant 50:1 ratio) on
CLL cell survival was determined. CLL samples were grouped according
to their characteristics.
Results: Our results show that I3C potently reduced the viability of CLL
cells with a LD50 of ~37 mM, while it was much less cytotoxic in PBMCs from
normal donors (LD50 ~100 mM). Moreover, I3C showed strong synergy with
F-ara-A in all cases studied (Table). Remarkably, I3C was able to synergize
with F-ara-A even in p53-deficient CLL cells (70% TP53/ATMdeletions)
and in CLL cells that showed in vitro resistance to F-ara-A. Indeed, the
concentrations of F-ara-A required to kill 90% of CLL cells in the combined
I3C/F-ara-A treatment resulted in an F-ara-A dose reduction index (DRI) of
31 for CLL cells harboring TP53/ATM deletions and of 82.5 for CLL cells
showing in vitro resistance to F-ara-A. The combination of I3C + F-ara-A
was equally active in CLL cells with mutated and unmutated IGHV and
were also active in CLL cells from patients that have developed treatment
resistances (patients that have received 2−4 treatments). Of note is that
I3C could also synergize with vincristine and chlorambucil (Table).
Conclusions: Our data indicate that I3C, alone or in combination with
F-ara-A and other chemotherapeutic drugs, is a potent inductor of apoptosis
in CLL in vitro. I3C strongly synergizes with F-ara-A in all types of CLL
tested, including CLL from refractory patients and CLL cells with deficient
p53 pathway. The striking DRI for F-ara-A in combination with I3C would
likely reduce harmful secondary effects associated to fludarabine while
having a similar or better effectiveness. Moreover, the low toxicity of I3C,
already clinically tested, and its anti-CLL activity would also support its use
as a novel neoadjuvant and adjuvant therapy in CLL, even in those patients
with relapsed or refractory disease.
40
POSTER (Board P034)
Selectivity and mechanism of action studies for Polo Box-targeted,
non-ATP based inhibitors of PLK1
M. Baxter1 , S. Craig1 , C. McInnes1 , M.D. Wyatt1 . 1 University of South
Carolina, Drug Discovery and Biomedical Sciences, Columbia, USA
Polo-like Kinase 1 (PLK1) performs critical roles in the coordination
of mitosis and is also an oncogene over-expressed in many cancer
types. ATP-binding site inhibitors of PLK1 have progressed to clinical
trials, and one was recently given FDA breakthrough status to treat
acute myeloid leukemia. However, recent data suggests that there are
major disadvantages to blocking the kinase activity of PLK1. First, PLK
family specificity is an issue with ATP-competitive compounds because
they commonly inhibit all PLK paralogs, including PLK3, a known tumor
suppressor. Second, a single point mutant in the active site of PLK1
(Cys67Val) results in dramatic resistance to structurally distinct ATPcompetitive inhibitors (Burkard et al, ACS Chem. Biol. 2012, 7, 978–
981), suggesting that the emergence of resistance in the clinic against
these agents is likely. We have validated the innovative REPLACE strategy
by discovering non-ATP competitive and PLK1-selective inhibitors as
an alternative therapeutic strategy for this important target. The Polobox domain (PBD) of each PLK is a phospho-peptide binding motif
that determines substrate recognition and subcellular localization. Our
approach targets the PBD of PLK1 to achieve desired selectivity and
improve efficacy. Through modeling and design we produced series
of fragment-ligated inhibitory peptides (FLIPs). We report three FLIPs
with an octyl-benzamide group, having the following different amino acid
C-terminus sequences: −PNGL, −AI, and −PL. In vitro binding to the
PBDs of PLK1 and PLK3 was measured by a fluorescence polarization
assay. The IC50 values for PLK1 calculated from the competition assay are
0.36 mM, 0.41 mM, and 1.2 mM, respectively, and all three are at least >1800fold more selective for PLK1. Initial testing in two cancer lines revealed
respectable anti-proliferative activity for two of the FLIPs. In addition, we
report the investigation of our PBD-inhibitors in cells expressing the mutant
C67V PLK1 that is resistant to ATP-based inhibitors. We find that our
compounds are equally active in cells expressing wild-type or C67V PLK1,
whereas cells expressing C67V PLK1 are dramatically resistant to the ATPbased inhibitor, BI 2536. Current work is characterizing the mechanism of
action for these FLIPs in cancer cells, including their ability to induce a
G2/M cell cycle arrest, and induce aberrant mitotic phenotypes associated
with PBD-inhibition. These exciting developments demonstrate the validity
of our approach to produce drug-like lead PBD-inhibitors that are PLK1
selective and are active against tumors resistant to ATP-inhibitors. PBDinhibitors could ultimately be used in combination therapy regimens with
ATP-based inhibitors for a dual attack on this important, clinically validated
oncology target.
41
POSTER (Board P035)
Clinical activity of BIND-014 (docetaxel nanoparticles for injectable
suspension) as second-line therapy in patients (pts) with Stage III/IV
non-small cell lung cancer
R. Natale1 , M. Socinski2 , L. Hart3 , O. Lipatov4 , D. Spigel5 ,
B. Gershenhorn6 , G. Weiss7 , S. Kazmi8 , N. Karaseva9 , O. Gladkov10 ,
V. Moiseyenko11 , J. Summa12 , G. Otterson13 . 1 Cedars-Sinai Medical
Center Samuel Oschin Comprehensive Cancer Center, Clinical Lung
Cancer Program, Los Angeles, USA; 2 University of Pittsburgh Cancer
Pavilion, Lung and Thoracic Malignancies Program, Pittsburgh, USA;
3
Florida Cancer Specialists, Clinical Research Director Drug Development
Program, Fort Meyers, USA; 4 Republic Clinical Oncology Dispensary,
Department of Medical Oncology, Ufa, Russian Federation; 5 Sarah
Cannon Research Institute, Lung Cancer Research Program, Nashville,
USA; 6 Cancer Treatment Centers of America Midwestern Regional
Medical Center, Department of Clinical Research and Clinical Trials,
Zion, USA; 7 Cancer Treatment Centers of America Western Regional
Goodyear, USA; 8 Cancer Treatment Centers of America Eastern Regional
Philadelphia, USA; 9 City Clinical Oncology Dispensary, Department of
Medical Oncology, Saint Petersburg, Russian Federation; 10 Chelyabinsk
Regional Clinical Oncology Dispensary, Department of Medical Oncology,
Chelyabinsk, Russian Federation; 11 N.N. Petrov Research Institute of
Oncologists, Department of Medical Oncology, Saint-Petersburg, Russian
Federation; 12 BIND Therapeutics, Department of Clinical Development,
Cambridge, USA; 13 Ohio State University Comprehensive Cancer Center,
Thoracic Oncology Program, Columbus, USA
Background: BIND-014 is a novel, polymeric nanoparticle containing
docetaxel (D) targeted to prostate-specific membrane antigen (PSMA), a
protein expressed on prostate cancer cells and on the vasculature of many
non-prostate solid tumors. BIND-014 is anticipated to improve the clinical
benefit of D by increasing its concentration and duration of exposure in
tumors. In a phase 1 study, BIND-014 was generally well-tolerated and
displayed anti-tumor activity at low doses and in tumors where solventbased D (sbD; Taxotere® ) has minimal activity.
Material and Methods: APhase 2 study was conducted of BIND-014
administered by a 60-minute intravenous infusion at 60 mg/m2 on Day 1
of a 21-day cycle as second-line therapy after a Pt-containing regimen
in pts with Stage III/IV NSCLC with characterized genomic status (EGFR
mutation, ALK rearrangement, KRAS mutation). A 2-stage design for futility
was incorporated with a maximum of 40 pts anticipated. The primary
endpoint was ORR.
Results: Forty pts were enrolled and received a median of 3 doses (range
1−12); the study is closed to enrollment with several pts continuing on
study. To date, 33 pts are evaluable for response, 5 (15%) had PR and 12
(36%) had stable disease lasting 12 weeks (SD 12 wks). Among 8
pts testing positive for a KRAS mutation, 2 (25%) had PR and 3 (38%) had
SD 12 wks. Among the 40 treated pts, 4 experienced an SAE classified
as related to study drug (Gr 2 neutropenic fever [1 pt], Gr 2 DVT [1 pt],
Gr 3 dyspnea [1 pt], Gr 3 hypoxia [1 pt]). There were 174 drug-related
AEs, 102 (59%) of which were Gr 1. Drug-related AEs Gr 2 occurring in
at least 3 pts included anemia (3 pts), dehydration (4 pts) and fatigue (5
pts). Two pts experienced Gr 3 lymphocytopenia and 2 pts experienced Gr
2 neutropenia. There was no evidence of hepatotoxicity and only 1 incident
of peripheral neuropathy (Gr 3).
19
Conclusions: BIND-014 administered at 60 mg/m2 on Day 1 of a 21-day
cycle is clinically active and well-tolerated assecond-line therapy in Stage
III/IV NSCLC pts with broad tumor mutation status. BIND-014 demonstrated
a 63% disease control rate (25% PR + 38% SD 12 wks) in pts with
KRAS mutations, a population generally unresponsive to sbD. Neutropenia,
anemia and neuropathy, commonly observed with sbD, were significantly
reduced with BIND-014. The potential safety and activity advantages of
BIND-014 compared to historical sbD data warrant further evaluation in
pts with NSCLC, including those with KRAS mutations. Final data will be
presented.
42
POSTER (Board P036)
Interaction of SJG-136 with cognate sequences of oncogenic
transcription factors
J. Mantaj1 , P.J. Jackson1 , D.E. Thurston1 , K.M. Rahman1 . 1 King’s College
London, Institute of Pharmaceutical Sciences, London, United Kingdom
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective
DNA minor-groove binding agents. They possess a ‘soft’ electrophilic
imine moiety at their N10-C11 position which can form an aminal linkage
with the C2-NH2 group of a guanine base. The PBD dimer SJG-136
is presently in Phase II clinical trials in ovarian cancer and leukaemia.
There is growing evidence that PBD monomers exert their pharmacological
effects through transcription factor inhibition, for example GWL-78, a C8linked PBD-Py-Py conjugate, has been shown to block interaction of the
transcription factor NF-Y and KMR-28−39, a GC sequence selective PBDMPB conjugate, inhibits the transcription factor NF-kB. The aim of this
study is to investigate whether PBD dimers like SJG-136 also interact with
transcription factor binding sequences and exert their antitumor activity
through this mechanism in addition to previously reported DNA strand
breakage, inhibition of endonucleases, RNA polymerases and arrest of
replication fork. We have developed an ion pair reverse phase HPLC/MS
analytical methodology and demonstrated for the first time the ability of
the PBD dimer SJG-136 to bind to specific DNA consensus sequences
of the transcription factors NF-kB, EGR-1, AP-1 and STAT3. As a dimer,
SJG-136 can form intra-, inter-strand and mono-alkylated adducts with
DNA. Interestingly, we have been able to observe that SJG-136 formed
three distinct adducts with the STAT3 consensus sequence, while only
single adducts were observed in the case of NF-kB-1, NF-kB-2, EGR-1
and AP-1. After completing the biophysical study, the effect of SJG-136
on the expression of STAT3 dependent genes was carried out by RTPCR, qPCR and Western Blot experiments using the STAT3 dependent
breast cancer cell line MDA-MB-231. Our results demonstrate a significant
dose dependent down regulation of STAT3 dependent genes cyclin D1,
survivin, NNMT, Bcl-2, STAT3 and fascin. These findings have implications
for understanding the mechanism of action of SJG-136 and can potentially
explain the differences in activity of SJG-136 against various tumour cell
lines. Future work will focus on establishing the correlation with transcription
factors NF-kB, AP-1 and EGR-1 following a similar procedure.
43
POSTER (Board P037)
CDKN1A-mediated responsiveness of MLL-AF4-positive acute
lymphoblastic leukemia to Aurora kinase-A inhibitors
L. Hung1 , Y. Chen2 , H. Lin3 , M. Tsai4 , H. Hsieh5 , J. Chang6 ,
N. Chen1 , S. Yang1 , T. Chen7 . 1 Institute of Bioinformatics and Biosignal
Transduction, Department of Life Sciences National Cheng Kung
University, Tainan, Taiwan; 2 Institute of Clinical Medicine and Division
of Hematology/Oncology, Department of Internal Medicine National
Cheng Kung University, Tainan, Taiwan; 3 Department of Pharmacology,
National Cheng Kung University, Tainan, Taiwan; 4 Eppley Institute for
Research in Cancer and Allied Diseases, University of Nebraska Medical
Center, Omaha NE, USA; 5 Institute of Biotechnology and Pharmaceutical
Research, National Health Research Institutes, Zhunan Miaoli County,
Taiwan; 6 Institute of Clinical Medicine and National Institute of Cancer
Research, National Cheng Kung University and National Health Research
Institutes, Tainan, Taiwan; 7 Institute of Clinical Medicine and Division
of Hematology/Oncology Department of Internal Medicine, National
Cheng Kung University Hospital, Tainan, Taiwan
Background: The prognosis of patients with relapsed or resistant acute
lymphoblastic leukemia (ALL) is quite poor, and resistance to chemotherapy
in ALL cells remains a challenge to successful treatment. Overexpression of
Aurora kinases is largely observed in many cancers, including hematologic
malignancies. Aurora kinases have become attractive therapeutic targets
to help overcome chemotherapy resistance.
Methods: The expression of Aurora kinases and their activators was
analyzed by Western blot analysis. Drug susceptibility was determined
by MTT assay. Expression of CDKN1A was detected by Western blot
20
and Q-PCR. The status of TP53 in ALL cells was determined by Sanger
sequencing.
Results: Nine ALL cell lines exhibited different susceptibilities to Aurora
kinase inhibitors (AKIs). Cells sensitive to AKIs underwent apoptosis at
an IC50 of approximately 10 to 30 nM and displayed a phenotype of
Aurora-A inhibition, whereas cells resistant to Aurora kinase inhibitors (with
an IC50 more than 10 mM) accumulated polyploidy, which may have resulted
from Aurora-B inhibition. Drug susceptibility was not correlated with the
expression level or activation status of Aurora kinases. RS4;11 and MV4;11
cells, which contain the MLL-AF4 gene, were both sensitive to Aurora-A
inhibitors. CDKN1A might govern the drug responsiveness of ALL cell
lines in a TP53-independent manner. Primary ALL cells with MLL-AF4 and
CDKN1A expression were sensitive to AKIs.
Conclusions: Our study suggests that Aurora-A kinase inhibitors may
have clinical utility in MLL-AF4-positive ALL. CDKN1A can be used as
a biomarker to determine drug responsiveness in MLL-AF4-positive ALL.
44
POSTER (Board P038)
Metformin and its impact on gastric cancer patients survival after
gastrectomy
C.K. Lee1 , M.K. Jung1 , I.K. Jung2 , S.J. Heo1 , J.Y. An3 , H.I. Kim3 ,
J.H. Chung3 , W.J. Hyung3 , S.H. Noh3 , H.S. Kim1 , S.Y. Rha1 , H.C. Chung1 .
1
Yonsei Cancer Center, Division of Medical Oncology, Seoul, Korea;
2
Yonsei University College of Medicine, Department of Biostatistics, Seoul,
Korea; 3 Yonsei Cancer Center, Department of Surgery, Seoul, Korea
Background: One of the most widely used oral anti-diabetes agent,
metformin has recently received attention as anti-cancer treatment with
accumulating evidences. But study showing survival benefit of metformin
for gastric cancer patients has never been reported.
Patients and Methods: We conducted a retrospective study of gastric
cancer patients who have undergone gastrectomy as curative aim from
May 2003 to December 2010 at a single institution (Yonsei Cancer Center,
Severance Hospital, Korea) based on electronic medical records. Survival
analysis was done using Kaplan–Meier plot and Cox proportional hazards
regression model.
Results: Among 1974 gastric cancer patients who underwent gastrectomy
as curative aim, 326 patients were diagnosed as diabetes and 132
patients were treated with metformin. During a median follow up duration
of 5.5 years (IQR, 3.8−7.4 years), 381 (19.3%) patients died, and 302
(15.3%) patients died as a result of gastric cancer. Gastric cancer
patients diagnosed with diabetes who were treated with metformin showed
significant survival benefit compared to diabetic gastric cancer patients who
were not treated with metformin (Overall survival: P = 0.021, HR = 0.584,
95% CI = 0.369–0.926; cancer-specific survival: P = 0.038, HR = 0.57,
95% CI = 0.334–0.975). Non-diabetes group (n = 1648) showed significant
better recur-free survival, overall survival and cancer-specific survival
compared to diabetes group (n = 326), and metformin usage prolonged
diabetes patients’ survival comparable to that of non-diabetes patients.
In multivariable analysis using Cox proportional hazard model with timedependent covariate, each cumulative 6 months of metformin use was
significantly associated to decreased risk of recur, cancer-specific mortality
and all-cause mortality (HR = 0.847, 95% CI = 0.769–0.934; HR = 0.850,
95% CI = 0.770–0.939; HR = 0.861, 95% CI = 0.794–0.933).
Conclusions: Our results show that increased cumulative duration
of metformin use decrease recur rate, all-cause and cancer-specific
mortality among gastric cancer patients with diabetes who had undergone
gastrectomy. Further prospective study of evaluating metformin for adjuvant
therapy in gastric cancer will be needed.
45
POSTER (Board P039)
Early preclinical study of BO-2094 for treatment of human colon
cancer, in combination with 5-fluorouracil
T.L. Su1 , T.H. Ou1 , M.H. Wu1 , Y.W. Lin1 , T.C. Lee1 . 1 Institute of
Biomedical Sciences, Academia Sinica, Taipei City, Taiwan
Among cancers, colon cancer is the second leading cause of cancer
deaths in the United States. Every year, about 140,000 Americans are
diagnosed with colon. Surgery, radiotherapy, and chemotherapy are the
main strategies for treating CRC patients. However, the mortality risk
associated with CRC is metastasis leading to the diminishing of systemic
treatments. Therefore, there is an urgent need of finding a better agent
to treat CRC. Recently, we have designed a series of novel water soluble
and chemically stable phenyl N-mustard-benzenealkylamide conjugates.
Of these derivatives, we found that compound BO-2094 exhibits a broad
spectrum of antitumor activity against a panel of human leukemia and
solid tumor cell lines in vitro and potent therapeutic efficacy in various
tumor xenograft models. We found that BO-2094 exhibited potent antitumor
activity in xenograft models. It also demonstrated that that the combination
of BO-2094 and 5-FU at ratios of 1:3 persuaded synergistic cytotoxicity
to HCT-116 cells in vitro. Notable, more than 98% of tumor suppression
was achieved in HCT-116-bearing nude mice when co-treated compound
BO-2094 (30 mg/kg, QD×6, iv. inj.) with 5-FU [75 mg/kg, every 7 days
for 2 doses (Q7D×2), intraperitoneal injection (ip. inj.)]; all mice (n = 5).
The early preclinical studies showed that BO-2094 has LD50 value of
115.4 mg/kg 14-day acute toxicity of in mice via a single administration
by iv injection. This agent is likely to have no cardiac arrhythmic side effect
based on hERG assay. BO-2094 showed high degree protein binding in rat
plasma, indicating that can be as drug reserve and allows drug to be slowly
released. Additionally, BO-2094 has an acceptable PK profile in rats. The
present studies indicate that the combination of compound BO-2094 + 5-FU
has great potential benefit for the treatment of advanced colon cancer.
46
POSTER (Board P040)
Broad-spectrum preclinical combination activity of eribulin combined
with various anticancer agents in human breast cancer, lung cancer,
ovarian cancer, and melanoma xenograft models
M. Asano1 , J. Matsui1 , M.J. Towle2 , J. Wu2 , S. McGonigle2 , T. Uenaka3 ,
K. Nomoto2 , B.A. Littlefield2 . 1 Eisai Co. Ltd., Oncology PCU, Tsukuba,
Japan; 2 Eisai Inc., Oncology PCU, Andover MA, USA; 3 Morphotek Inc.,
Oncology, Exton PA, USA
Eribulin is a pharmaceutically optimized, fully synthetic analog of the
marine sponge natural product halichondrin B. As its mesilate salt
(E7389, Halaven® ), eribulin is approved in 54 countries worldwide for
treatment of certain patients with advanced breast cancer. Eribulin is
a microtubule dynamics inhibitor that binds to high affinity sites on
microtubule plus ends, a distinct binding profile compared to most other
tubulin-targeted agents. Previous preclinical studies have shown that
under monotherapy conditions, eribulin has broad spectrum anticancer
activity against a wide variety of human cancer cell types in vitro and
human tumor xenograft types in vivo. The present preclinical studies
were undertaken to evaluate eribulin’s in vivo antitumor activity when
combined with a variety of cancer drugs in various human tumor xenograft
types. In xenograft models of triple negative (MDA-MB-231, MDA-MB-436,
MX-1) and HER2+ (UISO-BCA-1) breast cancer, eribulin variously showed
combination activity (defined as greater activity than either agent alone
with acceptable tolerability) with bevacizumab, capecitabine, everolimus,
and the experimental PARP inhibitor E7449, although it failed to show
combination activity with doxorubicin in the triple negative MDA-MB-435
model. In xenograft models of non-small cell lung cancer (NCI-H1993,
NCI-H322M, NCI-H522, PC-9), eribulin variously showed combination
activity with erlotinib, gemcitabine, and two experimental drugs, the PI3K
inhibitor BKM-120 and the multi-targeted tyrosine kinase inhibitor lenvatinib.
Eribulin also showed combination activity with bevacizumab in the SKOV-3 ovarian cancer model, and with lenvatinib in the A375 melanoma
model. Taken together, these preclinical results suggest that eribulin can
exert combination activity in a variety of tumor types with acceptable safety
profiles when administered together with a wide range of combination
agents having different mechanisms of action.
47
POSTER (Board P041)
Lurbinectedin (PM01183) specifically targets RNA Pol II for
degradation via the proteasome pathway in a transcription and
TC-NER dependent fashion
G. Santamaria1 , J.F. Martı́nez-Leal1 , C. Cuevas1 , L.F. Garcia-Fernandez1 ,
C.M. Galmarini1 . 1 PharmaMar S.A.U., Cell Biology Department, Colmenar
Viejo (Madrid), Spain
Lurbinectedin (PM01183) is a synthetic tetrahydroisoquinoline alkaloid
currently evaluated as single agent and in combination in phase I and
II clinical trials for solid tumors and hematological malignancies. In living
cells, PM01183-DNA adducts stall replication and transcription giving rise
to double strand breaks, inducing accumulation of cells in the S-phase
of the cell cycle and triggering apoptosis. In this work, we examined the
effects of PM01183 on the activity and stability of the RNA Pol II as well
as on other factors of the transcriptional machinery, including TBP (TFIID),
p62 (TFIIH) and XPD. Our results showed that PM01183 induced a rapid,
time- and concentration-dependent degradation of RNA Pol II in a panel of
different human tumor cell lines, including HCT-116 (colon), A549 (NSCLC),
HeLa (cervix) and A763 (sarcoma). This degradative process was efficiently
abrogated in the presence of transcriptional (DRB), ubiquitination (PYR41), and proteasome inhibitors (MG132), demonstrating that PM01183
specifically targets the transcriptionally active RNA Pol II for degradation
via the proteasome pathway. In addition, it was also shown that the effect
of PM01183 on the RNA Pol II was dependent on the presence of a
functional TC NER repair machinery. PM01183 induced degradation of
RNA Pol II in global NER (XPC) deficient cells, but failed to do it in TC
NER (CSB, XPD and XPG) deficient cells. Importantly, these effects were
confirmed to be specific for the Rpb1 subunit of RNA Pol II, since other
subunits were not affected (Rpb2 and Rpb4) as well as other factors of the
transcriptional machinery, such as TBP (TFIID), p62 (TFIIH), XPD or the
RPA194 subunit of the RNA Pol I. Finally, it was also demonstrated that,
contrary to what occurs after DNA damage with UV light, the transcription
of p53 target genes important for DNA repair, including p21 or mdm2, was
irreversibly inhibited after PM01183 treatment. Together, these results show
the mechanism by which PM01183 inhibits trans-activated transcription
process on tumor cells.
48
POSTER (Board P042)
Trabectedin and lurbinectedin are effective against leukemic
cells derived from patients affected by chronic and juvenile
myelomonocytic leukemia
M. Romano1 , A. Gallı̀2 , N. Panini1 , L. Paracchini1 , L. Beltrame1 ,
E. Bello1 , S.A. Licandro1 , C. Cattrini3 , R. Tancredi3 , S. Marchini1 ,
V. Rosti4 , M. Zecca5 , M. Della Porta2 , A. Zambelli3 , C.M. Galmarini6 ,
E. Erba1 , M. D’Incalci1 . 1 IRCCS − Istituto di Ricerche Farmacologiche
Mario Negri, Oncology, Milano, Italy; 2 Fondazione IRCCS Policlinico San
Matteo, Onco-Hematology, Pavia, Italy; 3 IRCCS Fondazione Salvatore
Maugeri, Oncology, Pavia, Italy; 4 Fondazione IRCCS Policlinico San
Matteo, Center for the Study of Myelofibrosis Research Laboratory of
Biotechnology, Pavia, Italy; 5 Fondazione IRCCS Policlinico San Matteo,
Pediatric Onco-Hematology, Pavia, Italy; 6 PharmaMar S.A., Research
and Development, Colmenar Viejo, Italy
Background: Chronic (CMML) and juvenile myelomonocytic leukemia
(JMML) are mixed myelodysplastic/myeloproliferative (MDS/MPN) haematological diseases of elderly and childhood patients, respectively. Any kind
of chemotherapy is unable to improve the outcome of these patients, so
allogeneic stem cell transplantation remains the only curative option for
MDS/MPN neoplasms.
Trabectedin and its analogue lurbinectedin are DNA minor groove
binders acting as modulators of transcription and interfering with DNA
repair mechanisms. They cause selective depletion of tumor-associated
macrophages and circulating monocytes, activating caspase-8-dependent
apoptosis and TRAIL receptors (TRAIL-Rs) expression.
These findings offer strong proof-of-concept evidence for monocyte
targeting in humans and provide the rational for testing these two drugs
in myelomonocytic leukemias.
Material and Methods: Patient samples (20 CMML; 12 JMML) were
collected from peripheral blood. The drugs cytotoxicity was evaluated
by growth inhibition; apoptosis (Annexin-V/P.I), TRAIL-Rs and cleaved
caspase-8 expression were investigated by flow cytometric assay. In vivo
experiments were performed using athymic nude mice transplanted with
MV-4−11 cells.
Results: Both trabectedin and lurbinectedin showed a strong cytotoxic
effect on malignant CD14+ monocytes in vitro and were able to induce
marked apoptosis, at nanomolar concentrations, much more effective than
azacitidine (5-AZA), used at micromolar concentrations as positive control.
In addition, trabectedin and lurbinectedin were found to efficiently inhibit
the in vitro growth of bone marrow hematopoietic precursors (CFU-GM) of
CMML and JMML patients.
In a CMML cellular model, the MV-4−11 cell line, we found an
increased TRAIL-Rs and cleaved caspase-8 expression after trabectedin or
lurbinectedin exposure suggesting that apoptosis induced by these drugs
followed the extrinsic pathway. Preliminary in vivo experiments, in MV4−11 model, showed a marked antitumor activity of both trabectedin and
lurbinectedin that was much higher than that of 5-AZA.
Conclusions: Our data suggest that trabectedin and lurbinectedin exert a
more specific cytotoxic effect on malignant CMML and JMML monocytes
than other drugs commonly used in MDS/MPN diseases and this makes
these two drugs good candidates for clinical studies in CMML and JMML.
Studies are in progress to elucidate the molecular mechanisms behind
these therapeutically antitumor effects of these drugs.
49
POSTER (Board P043)
siRNA targeting of mitochondrial thymidine kinase 2 (TK2) sensitizes
cancer cells to gemcitabine and increases mitochondrial toxicity
C. Di Cresce1 , P. Ferguson2 , R. Figueredo2 , M. Rytelewski1 , S. Maleki
Vareki1 , M.D. Vincent3 , J. Koropatnick1 . 1 Western University, Microbiology
and Immunology, London, Canada; 2 London Regional Cancer Centre,
Cancer Research Laboratories, London, Canada; 3 Western University,
Oncology, London, Canada
Background: Mitochondrial thymidine kinase 2 (TK2) preferentially
phosphorylates thymidine to generate thymidine monophosphate (dTMP).
21
TK2 also phosphorylates deoxycytidine to generate dCMP, a precursor
for dCTP. dCTP negatively regulates deoxycytidine kinase (dCK), which
primarily phosphorylates deoxycytidine as well as the anticancer drug
gemcitabine. Because phosphorylation by dCK is required to activate
the drug, there is a therapeutic advantage to high dCK in tumour cells
treated with gemcitabine. Antisense knockdown of TK2 could reduce
TK2-produced dCMP, thus decreasing dCTP levels and its inhibition of
dCK, and lead to increased dCK activity, gemcitabine activation, and
anticancer effectiveness. Importantly, gemcitabine is a poor substrate
for phosphorylation by TK2. Given the ability of TK2 to phosphorylate
deoxycytidine and thus down-regulate dCK activity, we hypothesized that:
(1) TK2 can mediate human tumour cell resistance to gemcitabine, and
(2) antisense (siRNA) downregulation of TK2 can inhibit this mechanism
of resistance. We also hypothesized that anti-TK2 siRNA-induced drug
sensitization is mediated by mitochondrial damage.
Materials and Methods: siRNA downregulation of TK2 followed by
gemcitabine treatment of MCF7, HeLa and A549 human tumour cell lines
(which express high, medium, and low levels of TK2, respectively). Cell
lines were analyzed for proliferation (alamarBlue viability staining), levels
of various mRNAs (qPCR) and proteins (immunoblotting), mitochondrial to
nuclear DNA ratio (mtDNA:nDNA) (qPCR), and mitochondrial membrane
potential (MitoTracker CMXROS and flow cytometry).
Results: Downregulation of TK2 using siRNA sensitized MCF7 and HeLa
cells (high and medium TK2 expressers, respectively) to gemcitabine, but
did not sensitize A549 cells (low TK2 expresser). siRNA knockdown of
TK1 and/or thymidylate synthase (TS) in combination with TK2-siRNA
and gemcitabine did not cause further sensitization; this phenomenon
is specific to targeting of TK2. The combined treatment with TK2
siRNA and gemcitabine increased dCK enzyme levels in TK2 high- and
medium-expressing cell lines. The addition of TK2 siRNA treatment
to gemcitabine treatment of TK2-expressing tumour cells specifically
decreased mitochondrial DNA (mtDNA:nDNA) and mitochondrial activity.
Conclusions: This is the first demonstration of a direct role for
TK2 in resistance to gemcitabine or any other anticancer drug, and
distinguishes TK2 from other dTMP-producing enzymes (cytosolic TK1 and
TS). Decreased mitochondrial function is related to TK2 siRNA-induced
sensitization to gemcitabine.
50
POSTER (Board P044)
The effect of esomeprazole, a proton pump inhibitor, on the
pharmacokinetics of the investigational Aurora A kinase inhibitor
alisertib (MLN8237) in patients with advanced solid tumors or
lymphomas
X. Zhou1 , J. Nemunaitis2 , S. Pant3 , T. Bauer4 , A. Lockhart5 , M. Patel6 ,
B. Zhang7 , V. Kelly8 , C.D. Ullmann8 , M. Bargfrede9 , K. Venkatakrishnan9 .
1
Millennium: The Takeda Oncology Company, Clinical Pharmacology,
Cambridge, USA; 2 Mary Crowley Medical Research Center, Oncology,
Dallas, USA; 3 Oklahoma University Medical Center, Hematology/Oncology,
Oklahoma City, USA; 4 Sarah Cannon Research Insitute, Tennessee
Oncology, Nashville, USA; 5 Washington University, Dept of Med. Oncology
Div. Med Onc Section, St. Louis, USA; 6 Florida Cancer Specialists,
Sarasota Cattlemen, Sarasota, USA; 7 Takeda Pharmaceuticals
International Co, Statistics, Cambridge, USA; 8 Takeda Pharmaceuticals
International Co, Oncology Clinical Research, Cambridge, USA; 9 Takeda
Pharmaceuticals International Co, Clinical Pharmacology, Cambridge, USA
Background: Alisertib is an investigational, orally available, selective
Aurora A kinase inhibitor currently in clinical development for multiple
oncology indications. Alisertib enteric-coated tablets are designed to
bypass the acidic gastric pH and delay dissolution until entry into the
upper small intestine. Alisertib exhibits pH-dependent solubility. This study
characterized the effects of esomeprazole, a proton pump inhibitor (PPI)
on the pharmacokinetics of single-dose alisertib in advanced cancer
patients (pts).
Materials and Methods: Eligible pts were 18 years of age with ECOG PS
0−1. In this fixed-sequence, 2-cycle, open-label study, a single oral dose of
alisertib 50 mg was administered on cycle 1, day 1 (C1D1) and cycle 2, day
8 (C2D8). Cycle 1 was 24 days, and cycle 2 was 31 days. Esomeprazole
40 mg (delayed release) was given once daily in cycle 2 from days 1−10.
Plasma pharmacokinetic samples were collected predose and up to 72
hours post alisertib on C1D1 and C2D8. Ratios of geometric mean Cmax ,
AUC0−last , and AUC0−inf in the presence of esomeprazole (C2D8) versus
in its absence (C1D1) were calculated and 90% confidence intervals (CI)
were estimated. Secondary endpoints were safety and tolerability.
Results: Of 25 pts enrolled (male, 44%; white, 88%; median age, 61 years;
and mean weight, 82 kg), 18 pts were evaluable. Following a single, oral
dose of alisertib, median Tmax was 3 and 4 hours in the presence and
absence of esomeprazole, respectively. The geometric mean AUC0−inf of
alisertib in the presence of esomeprazole was 129% of that in its absence
(90% CI, 107–156; N = 17). The geometric mean Cmax in the presence of
22
esomeprazole was 114% of that in its absence (90% CI, 96–136; N = 18).
At data cutoff, treatment-related adverse events (AE) were reported in 24
pts (96%); the most common AEs were diarrhea (52%), alopecia (52%),
neutropenia (36%), and fatigue (36%). Treatment-related grade 3/4 AEs
were reported in 15 pts (60%); the most common (>10%) were neutropenia
(36%) and leukopenia (12%). The observed AEs and their incidence were
generally consistent with the alisertib safety profile observed to date.
Conclusions: Esomeprazole produced an approximately 30% increase in
alisertib systemic exposure. These results support the recommendation
that gastric acid-reducing agents (e.g., PPIs, H2 receptor antagonists)
be avoided in pts receiving alisertib.
51
POSTER (Board P045)
Development of rational combination therapy strategies to optimize
treatment for GPNMB expressing Her2+ and triple negative breast
cancer
A. Rose1 , G. Maric2 , M.G. Annis2 , H. Smith2 , W.J. Muller2 , P.M. Siegel2 .
1
McGill University Department of Medicine, Goodman Cancer Research
Centre, Montréal, Canada; 2 McGill University, Goodman Cancer
Research Centre, Montréal, Canada
Background: Glycoprotein NMB (GPNMB) is a cell surface protein that
promotes tumor growth and metastasis in murine breast cancer (BC)
models. CDX011 is a GPNMB-targeted antibody drug conjugate that has
shown robust clinical activity and is being investigated in late stage clinical
trials for BC. Data from early clinical trials with CDX011 reveals that
response rate is directly linked to tumoral GPNMB expression levels. The
goal of this project is to define the prognostic and functional relevance
of GPNMB in basal/triple-negative (TN)/Her2-positive BC, and to devise
combination therapy strategies, which could improve CDX011 efficacy for
the treatment of BC.
Methods: GPNMB was ectopically expressed in breast cancer cells. Cell
invasion was assessed by boyden chamber assays and tumor growth
and spontaneous metastasis were assessed with in vivo mouse models.
The Cancer Genome Atlas (TCGA) human breast cancer gene expression
data (n = 1160) was interrogated for associations between GPNMB levels
and intrinsic subtype and overall survival. Lapatinib-resistant cells were
generated from several Her2+ cell lines. GPNMB mRNA levels were
determined by qPCR and protein by immunoblot and FACS.
Results: Ectopic GPNMB enhanced invasion by 2−4 fold, and in vivo tumor
growth was enhanced ~2-fold in both Hs578T and NIC-Her2 models. NIC
cells expressing GPNMB displayed a 4-fold increase in spontaneous lung
metastases. In the TCGA data set GPNMB expression was significantly
higher in basal and Her2 than it was in the luminal A and B subtypes.
Tumors with high GPNMB expression were associated with significantly
shorter overall survival (HR = 2.0295, 95% CI = 1.3476 to 3.0567. Treatment
with kinase inhibitors (KI) EGFR/Her2-family inhibitors (gefitinib, lapatinib)
led to significantly up-regulated GPNMB in basal-like BC cells: MDA-468,
MDA-436, MDA-157, and Her2+ cells: SkBR3, HCC1954, BT474, MDA361. Moreover, Mek inhibition with trametinib led to increased GPNMB
expression in both basal and Her2+ BC. This induction was transcriptionally
mediated, and led to increased GPNMB protein at the cell surface.
Moreover, 34 Lapatinib-resistant cells expressed higher levels of GPNMB
than corresponding parental cells. Finally, CDX011 as a single agent
caused a significant reduction in MDA-468 tumor growth.
Conclusions: GPNMB is highly expressed in Her2+ and Basal breast
cancer − it promotes tumor growth, invasion and metastasis and correlates
with poor survival. As such it represents a promising therapeutic target.
GPNMB is up-regulated by many KI that are currently used to treat BC.
Our data suggest that combination of KI with CDX011 will lead to improved
tumor regression; our efforts to test these combinations in vivo will be
discussed.
52
POSTER (Board P046)
Platinum(IV) derivatives of oxaliplatin: Cellular effects and in vivo
potency
S. Göschl1 , V. Pichler1 , E. Brynzak1 , P. Heffeter2 , U. Jungwirth2 ,
M.A. Jakupec1 , W. Berger2 , M. Galanski1 , B.K. Keppler1 . 1 University
of Vienna, Institute of Inorganic Chemistry, Vienna, Austria; 2 Medical
University of Vienna, Institute of Cancer Research, Vienna, Austria
Platinum(IV) complexes are an attractive way to overcome some disadvantages of platinum(II)-based anticancer drugs such as severe side
effects or resistances, because they are kinetically more inert to ligand
substitution and their higher lipophilicity may improve the cellular uptake.
Four platinum(IV) complexes derived from oxaliplatin reported herein have
shown activity in the colorimetric MTT assay in vitro in HCT15, HCT116 and
HCT116oxR cells (all colon carcinoma). The extent of induced apoptosis
(AnnexinV-FITC/PI staining) and the influence of the complexes on the
cell cycle were measured by flow cytometry. Two of the complexes (1 and
2) were tested in vivo in murine L1210 leukemia, and 2 was tested in a
colon tumor (CT26) mouse model. The platinum levels in the organs were
determined by ICP-MS measurements. These platinum(IV) compounds,
mostly yielding IC50 values in the micromolar range, are up to 25 times more
active in HCT116 cells than in the oxaliplatin resistant subline, whereas
oxaliplatin is 66 times less potent in HCT116oxR cells. After 24 h incubation
with 2 a strong increase of the G2 /M phase fraction and a decrease of
the S phase fraction was found in HCT15 cells, whereas the effects were
somewhat less pronounced in HCT116 cells. After 24 h incubation with 1 an
up to 5-fold increase of apoptosis was found in HCT116 but not in HCT15
and HCT116oxR cells. 48 h incubation of 1 or 2 resulted in a total increase
of up to 50% and 15% apoptosis in HCT15 and 50% and 25% in HCT116
cells, respectively. In L1210 murine leukemia an increase in life span up
to 60% was observed. 2 was tested in immuno-competent and immunodeficient mice to determine whether it is active in a solid tumor model and
whether the immune system has an influence on the efficiency as found for
oxaliplatin. The immune system seems to be relevant for activity because
2 was found to be active in Balb/c but not in SCID mice, although the tumor
platinum levels in both mouse models are the same. Overall, these results
demonstrate the high potency of modified platinum(IV) compounds. For a
deeper insight into their suitability as prodrugs activated by reduction, their
reduction properties and their activity under hypoxic conditions in 2D and
3D cell culture will be studied.
53
POSTER (Board P047)
Adeno-associated virus (AAV) carrying diphtheria toxin a gene for
pancreatic cancer therapy
M. Chen1 , C.Y. Ho1 , M. Teng1 , H. Chen1 . 1 Virovek Incorporation, R&D,
Hayward, USA
Pancreatic cancer is an aggressive malignancy with morbidity rates almost
equal to mortality rates because of the current lack of effective treatment
options. Here, we describe a DNA-based therapy for pancreatic cancer
using the AAV vector that expresses the diphtheria toxin A chain (DT-A)
under the control of the survivin gene regulatory sequences. We produced
the AAV vectors carrying DT-A coding sequence that was disrupted by the
human growth hormone intron so that its expression was abolished during
AAV vector production in insect cells and used these vectors for the study.
In vitro experiments showed that the AAV vector was effective in inhibiting
protein synthesis in pancreatic carcinoma cell lines but not in normal
cell line. In vivo experiment results demonstrated tumor growth arrest
in a xenograft mouse model for pancreatic cancer in a dose-dependent
manner. Differences in tumor size between the control and high dose group
were significant (P = 0.015). These AAV vectors provide a new option for
cancer gene therapy. The protocol and any amendment(s) or procedures
involving the care and use of animals in this study were reviewed and
approved by the Institutional Animal Care and Use Committee (IACUC) of
CrownBio prior to conduct. During the study, the care and use of animals
were conducted in accordance with the regulations of the Association for
Assessment and Accreditation of Laboratory Animal Care (AAALAC).
Table 1. Antitumor activity of test article AAV6-DTA in the treatment of
subcutaneous PANC-1 human pancreatic cancer xenograft model
T/C
(%)
T-C
(days) at
786 mm3
P valueb
Group 1. AAV6-SURV-GFP 1.5×1012 vg/mouse 1720±258
Group 2. AAV6-SURV-DTA 1.5×1012 vg/mouse 786±234
Group 3. AAV6-SURV-DTA 1.5×1011 vg/mouse 808±218
−
−
−
45.72
12.5
0.015
46.98
10.5
0.017
Group 4. AAV6-SURV-DTA 1.5×1010 vg/mouse
60.84
6
0.072
Treatment
a Mean±SEM; b vs. vehicle control.
Tumor size (mm3 )a
on day 48
1046±301
54
POSTER (Board P048)
MGMT methylation assessed by methyl-BEAMing technique is a
prognostic and predictive biomarker in glioblastoma and metastatic
colorectal cancer patients
L. Barault1 , A. Amatu2 , F.E. Bleeker3 , C. Moutinho4 , A. Cassingena2 ,
F. Tosi2 , T. Venesio5 , M. Esteller4 , A. Bardelli6 , S. Siena2 ,
A. Sartore-Bianchi2 , F. Di Nicolantonio6 . 1 Candiolo Cancer Institute −
FPO IRCCS, Experimental Clinical Molecular Oncology, Candiolo,
Italy; 2 Ospedale Niguarda Ca’ Granda, Department of Hematology
and Oncology, Milan, Italy; 3 Academic Medical Center University of
Amsterdam, Department of Clinical Genetics, Amsterdam, Netherlands;
4
Bellvitge Biomedical Research Institute (IDIBELL), Cancer Epigenetics
and Biology Program (PEBC), Barcelona, Spain; 5 Candiolo Cancer
Institute − FPO IRCCS, Investigational Clinical Oncology, Candiolo,
Italy; 6 University of Torino, Department of Oncology, Candiolo, Italy
Background: O6-methylguanine DNA methyltransferase (MGMT ) silencing by promoter methylation is a common alteration found in different cancer
types. This has been shown to be both a prognostic and a predictive
marker of sensitivity to alkylating agent-based therapy like dacarbazine
and temozolomide in glioblastoma. However in other malignancies its
value remains controversial. This might be due to sampling issues, tumor
heterogeneity or to the use of inadequate detection methods. In this study,
we present a new assay to reliably measure MGMT methylation both in
tumor and plasma samples.
Material and Methods: Methylation of MGMT has been assessed
by an ultra-sensitive digital PCR technique, in which a two-step PCR
is followed by detection via fluorocytometer (Methyl-BEAMing). Results
were compared to two other commonly used techniques (Methylation
Specific PCR, MSP and pyrosequencing). Two samples datasets have
been evaluated: tumors from a cohort of 98 newly diagnosed glioblastoma
patients from the pre-temozolomide era, and specimens from a cohort of 68
metastatic colorectal cancer patients treated with dacarbazine in a phase II
clinical trial (DETECT-01 trial, EUDRACT number 2011–002080−21). The
prognostic and/or predictive value of MGMT methylation has also been
evaluated. As a proof of concept, the three methods were assessed in a
subset of colorectal cancer patients’ plasma derived DNA to evaluate their
performance as a liquid biopsy test.
Results: Methyl-BEAMing showed high reproducibility across independent
experiments, as well as high sensitivity (up to 0.09% methylation detected)
and specificity. In the glioblastoma cohort, Methyl-BEAMing methylated
status (>50%) was associated with a decreased hazard ratio for death
(HR = 0.35; p < 0.0001) compared to MSP (HR = 0.54; p = 0.006) or
pyrosequencing (HR = 0.61; p = 0.059). In mCRC, tissue where tumor
heterogeneity is possibly higher, both Methyl-Beaming and pyrosequecing
assays provided better prediction of objective response to dacarbazine
than MSP. Progression free survival was also improved in metastatic
colorectal cancer with methylated status when samples were assessed with
Methyl-BEAMing (p = 0.0012) or pyrosequencing (p = 0.0005). Quantitative
evaluation of MGMT methylation in circulating tumor DNA was effective
with Methyl-BEAMing.
Conclusions: MGMT methylation testing based on BEAMing technology
outperforms commonly used methods and might allow the non-invasive
follow-up of patients, upon alkylating agent treatments using blood
circulating DNA.
55
POSTER (Board P049)
Low, frequent doses of PM060184 induce remarkable in vivo
antitumor activity
P. Aviles1 , M.J. Guillen1 , P.P. Lopez-Casas2 , F. Sarno3 , O. Cataluña4 ,
P. Nuñez4 , C. Cuevas4 , M. Hidalgo3 . 1 PharmaMar S.A., Preclinical,
Colmenar Viejo (Madrid), Spain; 2 Centro Nacional de Investigaciones
Oncológicas and Hospital de Madrid, Colmenar Viejo (Madrid), Spain;
3
Centro Nacional de Investigaciones Oncológicas and Hospital de
Madrid, Madrid, Spain; 4 Pharmamar S.A., Preclı́nica, Colmenar Viejo
(Madrid), Spain
Background: PM060184 is a synthetic marine-derived compound originally isolated from the marine sponge Lithoplocamia lithistoides. PM060184
induces disorganization and disruption of the microtubule network as
well as aberrant mitotic spindle multipolarization and chromosome
missegregation. These effects give rise to prometaphase arrest and
formation of multinucleated cells. Then, cells enter to caspase-driven
apoptosis or are arrested in a pseudo-senescent state. PM060184 is
currently under evaluation in Phase I clinical studies in patients with
advanced cancer diseases. The objective of the present work was to
explore the in vivo anticancer efficacy of PM060184 administered at low
daily doses (0.5 to 2 mg/kg).
23
Material and Methods: Athymic female nu/nu mice were subcutaneously
implanted with different tumors: MDA-MB-231 (breast), H460 (NSCLC)
and several pancreas patient-derived (AVATAR) tumors, namely JH010, JH-015, Panc-291, and Panc-039. Tumor (ca. 300 mm3 ) bearing
animals (N = 6−10/group) were randomly allocated to receive PM060184
or placebo. Treatments (0.5 to 2 mg/kg, iv) were administered daily for
20 consecutive days. Antitumor effect was calculated using DT/DC (%),
defined as a percentage of the change in tumor size for treated (T) and
placebo (C) groups during the placebo-treated survival time (D). Complete
tumor regression (CR) was defined when tumor volume <63 mm3 for 2 or
more consecutive measurements.
Results: The treatment with PM060184 produced lowest DT/DC values as
summarized in the table.
Tumor
Breast
MDA-MB-231
NSCLC
H460
Pancreas
JH-024
JH-010
JH-015
Panc-291
Panc-039
Daily dose
(mg/kg)
Minimal
DT/DC
(%)
On Day
2.0
1.0
0.5
2.0
1.0
0.5
2.0
2.0
2.0
2.0
2.0
7.3
14.1
24.0
5.5
7.8
17.7
19.7
14.8
38.9
12.0
6.1
21
21
21
11
11
11
21
21
28
24
28
Conclusion: The treatment with PM060184 at low, frequently given doses
demonstrated significant in vivo antitumor activity in breast, NSCLC and
pancreas xenografted tumors.
56
POSTER (Board P050)
Radiosensitizing effect of sodium metaarsenite in a metastatic brain
tumor model
W.Y. Kang1 , Y.M. Park2 , S.J. Kim2 . 1 Institute for refractory cancer
research Samsung medical center, Samsung Biomedical Research
Institute, Seoul, South Korea; 2 Komipharm International Inc. Co.,
Pharmaceutical Division, Seoul, South Korea
Background: Brain metastases are found in about 10% of lung cancer
patients at the time of diagnosis, and about 40% of all lung cancer
patients develop brain metastases during their disease progression. The
chemotherapy is limited because of little or no effectiveness due to the
blood–brain barrier. The radiation therapy is the most frequently used, and
sensitizing agents, which synergize with radiation, can improve the efficacy
of the therapy.
Material and Methods: Sodium metaarsenite (KML001® ) is an orally
bioavailable arsenic compound that has entered phase I/II clinical trials in
solid tumors and hematopoietic malignancies. In this study, we elucidated
the radio-sensitizing effect of sodium metaarsenite (KML001® ) in an animal
model of metastasis of lung cancer to the brain.
Results: The clonogenic assay showed that treatment with sodium
metaarsenite (KML001® ) inhibited clone formation in radio-sensitive
(H23) and radio-resistant lung cancer cells (A549 and PC14PE6) in a
concentration-dependent manner. The combined irradiation and sodium
metaarsenite (KML001® ) treatment significantly reduced colony formation
in H23 (p < 0.01), A549 (p < 0.05) and PC14PE6 lung cancer cells
(p < 0.05), compared with the radiation alone group.
In the metastatic brain cancer model with H23 cells, sodium metaarsenite
(KML001® ) treatment (5 mg/kg/day) and radiation therapy (5 Gy) showed
54.5% and 67.6% reduction in tumor volume, respectively, compared with
control group (p < 0.001 vs. control). The combined irradiation and sodium
metaarsenite (KML001® ) treatment induced 88.1% decrease in tumor
volume (p < 0.001 vs. control).
In the metastatic brain cancer model with PC14PE6 cells, the single
irradiation (15 Gy) and the combined irradiation (15 Gy) and sodium
metaarsenite (KML001® ) treatment (7 mg/kg/day) significantly increased
median survival day of the mice to 22 and 26 days, respectively, compared
to control group (median survival day = 19) (p < 0.001). The combination
improved survival significantly with regard to the radiation only group
(p < 0.001).
Conclusions: This study demonstrated that sodium metaarsenite
(KML001® ) may have potential as an alternative therapeutic agent,
24
especially in combination with radiation therapy in lung cancer patients
with brain metastases and provide a rationale for testing the combination
of sodium metaarsenite (KML001® ) and radiotherapy in the clinic.
59
POSTER (Board P053)
Discovery of novel inhibitor of FOXO nuclear–cytoplasmic shuttling
from natural products of marine origin
57
POSTER (Board P051)
Differential antitumor activity of trabectedin, lurbinectedin, Zalypsis
and PM00128 against a panel of human cells deficient in transcription
and NER factors
F.J. Castillo Correa1 , N. De Pedro1 , L. Rodriguez Quesada1 , D. Oves
Costales1 , J.R. Tormo1 , J. Martin1 , F. Reyes1 , O. Genilloud1 , F. Vicente1 ,
W. Link2 , B. Cautain1 . 1 Fundacion Medina, Screening and Target
Validation, Granada, Spain; 2 Universidad do Algarave, Ciencias
Biomedicas e Medicina, Faro, Portugal
V. Moneo1 , S. Avila1 , P. Martı́nez1 , B. de Castro1 , S. Cascajares1 ,
C. Cuevas1 , L.F. Garcia-Fernandez1 , C.M. Galmarini1 .
1
PharmaMar S.A.U., Cell Biology Department, Colmenar Viejo (Madrid),
Spain
Trabectedin, lurbinectedin, Zalypsis and PM00128 are synthetic marinederived anticancer drugs that covalently bind to the minor groove of
DNA through via a common pentacyclic skeleton. The compounds differ
in an additional chemical moiety attached to the pentacyclic skeleton
that protrudes out from the double helix. Trabectedin, lurbinectedin and
Zalypsis present a tetrahydroisoquinole, a tetrahydro b-carboline and a
trifluorocinnamic group, respectively, while PM00128 lacks such additional
moiety. To gain insight into how the presence of those different chemical
groups contribute to the selectivity of the four compounds, we tested their
cytotoxicity profiles against a panel of 43 fibroblast cell lines derived from
patients with genetic diseases caused by mutations in transcription/NER
factors, including CSA, CSB, XPC, XPA, XPE, XPB, XPD, XPG. In
general, trabectedin and lurbinectedin presented a similar, although not
identical, response pattern against the cell panel. However, these two
compounds have a different behavior when compared to Zalypsis and
PM00128. Four out of seven fibroblast cells lines carrying truncations
in the XPG endonuclease gene were more resistant to trabectedin than
normal fibroblasts, with RI values ranging from 3.4 to 6.7. Also interesting,
a cell line (GM15754) carrying a missense mutation (R112H) in the
XPD helicase gene, showed a significant resistance to both trabectedin
and lurbinectedin, showing a RI of 41.0. Thus, the proper functionality
of XPG and XPD factors in transcription and/or DNA repair processes
may be important for the antiproliferative action of these compounds
Remarkably, the sensitivity profiles obtained with standard DNA binders,
such as cisplatin and mitomycin C, were completely divergent to those of
trabectedin and lurbinectedin. On the other hand, no significant differences
in the sensitivity of these cell lines were observed against Zalypsis and
PM00128, suggesting that the repair of the DNA damage generated by
these compounds is not exclusively dependent on the NER pathway. In
summary, the differences in the chemical moieties attached to the common
DNA binding scaffold in these compounds, seemed to correlate with the
differential biological activity found against different tumoral cells.
58
POSTER (Board P052)
Pipecolidepsin A, Stellatolide A and Irvalec: New cyclodepsipeptides
of marine origin with antitumor activity
J.M. Molina-Guijarro1 , V. Moneo1 , J.F. Martinez-Leal1 , C. Cuevas1 ,
L.F. Garcia-Fernandez1 , C.M. Galmarini1 . 1 PharmaMar S.A.U., Cell
Biology Department, Colmenar Viejo (Madrid), Spain
Pipecolidepsin A and Stellatolide A, along with Irvalec, are three synthetic
marine-derived cyclic depsipeptides that show antitumor activity in vitro
against a wide variety of human tumor cell lines. In dose–response curves
against a panel of 24 human cancer cell lines, representative of 11 different
tissues, the compounds showed GI50 values in the low micromolar range.
Using the mechanism of action of Irvalec as a model, the cellular effects
of Pipecolidepsin A and Stellatolide A were evaluated. Preliminary studies
with these compounds were conducted in A549 (NSCLC) and HCT-116
(colon adenocarcinoma) cell lines as tumor models. The results pointed
out that all of them share a common set of cellular effects. Phase contrast
and fluorescence microscopy as well as plate fluorimetry analyses, showed
that the compounds caused very rapid cell membrane destabilization,
causing rapid and dramatic morphological changes, including cell blebbing,
severe swelling, and plasma membrane permeabilization (as detected
by propidium iodide) that irrevocably lead to cell lysis and necrotic cell
death. Interestingly, membrane damage was observed only after reaching
a threshold concentration, variable depending on the compound, in the
culture medium. Strikingly, all the compounds caused a notable inhibition of
cell viability after 30 min exposure, indicating that their cellular effects were
produced very rapidly upon treatment. The efficacy of Pipecolidepsin A and
Stellatolide A is currently being evaluated in in vivo models. In summary,
these results indicate that the compounds Pipecolidepsin A, Stellatolide A
as well as Irvalec, exert their potent antitumor activity by inducing rapid and
severe membrane damage in tumor cells.
The nucleo-cytoplasmic transport of certain tumor suppressor and
oncoproteins is disrupted in cancer cells resulting in their aberrant
subcellular localization and their respective inactivation or over-activation.
This subcellular localization occurs actively via the nuclear pore complex
that spans the nuclear envelope and is mediated by transport receptors.
Regulation of the FoxO (forkhead box O) factors is receiving increasing
attention as their activation in the nucleus has been linked to cell-cycle
arrest and apoptosis, acting as tumor suppressors.
Microbial natural metabolites have played a major role as one of the
most important sources for the discovery of novel drugs. In this work,
we applied a previously established high-throughput and high-content
cellular-imaging assay that monitors the nuclear–cytoplasmic translocation
of a GFP–FOXO3a fusion protein in U2OS cells to screen a library of
extract from marine actinobacteria, one of the most efficient groups of
secondary metabolite producers. A total of 3,300 extract, fractions and
pure compounds were screened. The nuclear accumulation of fluorescence
triggered by the pan-PI3K inhibitor, LY294002 (at 250 mM), was defined
as 100% activity and used as a reference to define primary hits in
U2foxRELOC assay. A final hit rate of 0.04% (13/3,300) was achieved;
defining hits as non-toxic, specific FoxO Relocators. Two news FOXO
relocators were isolated as pure compounds. MDN-XX is an alkaloid that
has been previously described as a compound with antifungal activity.
The other one is a new structure actually under characterization. As a
general result, we validate the strategy used to identify activators of FoxO
based on the screening of a collection of natural products, such as marine
actinomycetes using the U2foxRELOC assay. Furthermore, the discovery
of MDN-XX, with an IC50 of 10 nM, proves that marine actinomycetes can
be used as an attractive and unexplored source of FoxO activators.
60
POSTER (Board P054)
Sodium metaarsenite cytotoxic activity is associated with telomere
length and many types of arsenic transporters in non-small cell lung
cancer
Y.M. Park1 , S.J. Kim1 . 1 Komipharm International Co. Ltd., Pharmaceutical
Division, Shiheung, South Korea
Background: Sodium metaarsenite (KML001® ) is an orally bioavailable
arsenic compound that has entered phase I/II clinical trials in solid tumors
and hematopoietic malignancies. The underlying mechanisms of anticancer
effects of KML001® have been shown to be telomere poisoning. Because
arsenic resistance could limit the efficacy of KML001® in patients with nonsmall cell lung cancers (NSCLC), expression of membrane pore proteins
involved in arsenic influx into cells, energy-dependent transporters that
detect and eject arsenic from cells, and arsenic-detoxifying machinery
proteins need to be evaluated to predict the sensitivity and resistance of
human non-small cell lung cancers to KML001® .
Material and Methods: In this study, we characterized six NSCLC
cells with regards to the cytotoxicity by KML001® and expression of
arsenic influx/efflux machinery proteins. We profiled expression of human
aquaglyceroporin (AQP) 1−9, glucose transporter(GLUT) 1−4, LRP and
MRP1−3 in six NSCLC cell lines. In addition, the telomere restriction
fragment (TRF) lengths were measured and compared among six NSCLC
cell lines. Finally we examined in vivo antitumor activity of KML001® in two
human NSCLC xenografts transplanted with H292 cells with a low IC50 and
PC14PE6 cells with a high IC50 for KML001® .
Results: MTT assay revealed that H292, H23, and H2009 were sensitive
to KML001® (IC50 = 2.2, 2.4, and 9.8, respectively), whereas A549,
PC14PE6, and H1792 were resistant to KML001® (IC50 = 29.6, 31, and 35,
respectively). Arsenic influx activity for arsenic-sensitive cell lines was
higher than that for arsenic-resistant cell lines. However, compared to
arsenic-sensitive cell, arsenic-resistant cells showed a rapid reduction in
arsenic accumulation over 24 h coupled with a increase in the rate of
arsenic efflux. TRF length analysis demonstrated that the resistant A549,
H1792, and PC14PE6 cells displayed longer telomeres compared to the
sensitive H2009, H292 and H23 cells. H23 and H292 with higher expression
levels of AQP9, AQP2, AQP3, GLUT3 and GLUT4 showed significantly
enhanced arsenic uptake in compared to A549 and PC14PE6 cell lines with
lower expression levels of these membrane pore proteins. Arsenic contents
were significantly low in PC14PE6 and A549 cell lines with increased
MRPs and LRP expression. In H292 and PC14PE6 NSCLC xenograft
models, administration of 3.5 and 7 mg/kg KML001® significantly reduced
tumor volume and tumor weight without additional toxicity to the mice. The
combination with cisplatin and KML001® showed synergic inhibitory effects
on the growth of H292 and PC14PE6 xenografts.
Conclusions: These results suggest that telomere length, membrane pore
proteins and drug transporters responsible for arsenic influx/efflux, such
as AQPs, glucose transporters, LRP and MRPs may be used to predict
the sensitivity and/or resistance of human NSCLC cells to KML001® and
sodium metaarsenite (KML001® ) may have potential as an alternative
therapeutic agent, especially in combination with cisplatin in NSCLC
patients.
61
POSTER (Board P055)
A panel of pediatric liver cancer patient-derived xenografts to
improve stratification of children with hepatoblastoma
M. Fabre1 , D. Nicolle2 , A. Gorse2 , O. Déas2 , C. Mussini3 , L. Brugières4 ,
M.R. Ghigna5 , E. Fadel6 , L. Galmiche-Rolland7 , C. Chardot8 ,
C. Armengol9 , J.G. Judde2 , S. Branchereau10 , S. Cairo2 . 1 Institut Gustave
Roussy, Biology and Medical Pathology Department, Villejuif, France;
2
Xentech, R&D, Evry, France; 3 Bicêtre Hospital, Pathology Department,
Le Kremlin Bicêtre, France; 4 Institut Gustave Roussy, Pediatric Oncology
Department, Villejuif, France; 5 Marie Lannelongue Hospital, Pathology
Department, Le Plessis Robinson, France; 6 Marie Lannelongue Hospital,
Surgery Department, Le Plessis Robinson, France; 7 Necker Hospital,
Pathology Department, Paris, France; 8 Necker Hospital, Pediatric Surgery
Department, Paris, France; 9 Health Sciences Research Institute Germans
Trias i Pujol (IGTP), Translational Liver Oncology, Badalona, Spain;
10
Bicêtre Hospital, Pediatric Surgery Department, Le Kremlin Bicêtre,
France
Despite being the predominant type of pediatric liver malignancies,
hepatoblastoma (HB), with a world-wide incidence of 1 case per million
persons per year, is a rare tumor. The high rate (>60%) of b-catenin
activating mutations places HB as one of the human tumors most tightly
associated with activation of the Wnt/b-catenin pathway. Evidence for
(epi)genetic origin of HB is provided by its association with congenital
anomalies, Beckwith–Wiedemann syndrome, and familial adenomatous
polyposis, a disorder caused by germline mutation of APC, involved in
b-catenin degradation. Like other rare diseases, rare cancers are particular
challenging due to their low incidence, particularly for the identification of
novel therapies. The rarity and the heterogeneity of childhood liver cancers
hamper the development of reliable research tools that recapitulate each
disease.
To tackle this issue, we have launched a program aimed at the constitution
of liver cancer patient-derived xenografts (PDXs). At present, 13 HBs and
2 HB/hepatocellular carcinoma (HCC) transitional liver cell tumors (TLCTs)
have been successfully grown in immunocompromised mice out of 48
tumors grafted.
HB and TLCT PDXs maintain the histological features of primary human
tumors, and the heterogeneity of AFP levels in mouse blood correlate with
that observed in patients. Comparative analysis of the clinical parameters
associated to tumors from which PDX could or could not be established has
been performed. This analysis clearly shows that tumor take is associated
with high AFP level post-chemotherapy, with low % of treatment-induced
necrotic/fibrotic area in the resected tumor, and, most important, with
poor prognosis. Among HB PDXs models that have been established
several are, in accordance with the clinical history, resistant to cis platinum
and doxorubicin-based standard of care. In vivo anti-cancer screening in
histologically different HB/TLCT PDX subtypes show unique profiles of
response to the drugs tested, and identified the combination irinotecan/
temozolomide as a promising second line combination for a subset of HB
PDXs. The identification of molecular biomarkers predictive of HB response
to irinotecan/temozolomide combination will be discussed.
Development of a panel of childhood liver tumor PDXs will endow the
scientific community with an innovative and versatile research tool that
will decisively contribute to improve our understandings on pediatric
liver malignancies. These models constitute an unperishable reservoir of
biological samples that strongly recapitulate the human tumor biology,
and they can be used in several research domains such as functional
genomics, cancer stem cell biology and pharmacogenomics, notably for
the identification of Wnt/b-catenin inhibitors. In the long run, improved
knowledge in all these research fields will be translated in improved cures
for kids.
25
62
POSTER (Board P056)
Antitumor and temozolomide-sensitizing effects of sodium
metaarsenite in an orthotopic glioblastoma xenograft model
W.Y. Kang1 , Y.M. Park2 , S.J. Kim2 . 1 Institute For Refractory Cancer
Research Samsung Medical Center, Samsung Biomedical Research
Institute, Seoul, South Korea; 2 Komipharm International Inc. Co.,
Pharmaceutical Division, Shiheung, South Korea
Background: Glioblastoma is one of the most malignant types of primary
brain tumors and the treatment of glioblastomas remains difficult in that no
contemporary treatments are curative. Temozolomide (TMZ) was approved
and has been used for the treatment of glioblastoma as adjuvant or
concomitant therapy with radiotherapy.
Material and Methods: Sodium metaarsenite (NaAsO2 , KML001® ) is
an orally available arsenic compound that has entered phase I/II clinical
trials in solid tumors and hematologic malignancies. In this study, we
investigated the antitumor activity and TMZ-sensitizing effects of KML001®
in glioblastoma cell lines and a glioblastoma orthotopic xenograft mice
model.
Results: KML001® inhibited colony formation in TMZ-sensitive (U87MG,
U373MG) and TMZ-resistant (U138MG and U251MG) glioblastoma cells
in a concentration-dependent manner. The combined KML001® and TMZ
treatment significantly decreased clonogenecity of glioblastoma cells,
compared with TMZ treatment only (p < 0.05 or less). The combinational
treatment of KML001® and TMZ increased the amount of cleaved PARP
and caspase-3 that occur at the onset of apoptosis, in U87MG- and
U251MG-glioblastoma cells, when compared with TMZ treatment only.
Immunocytochemistry (ICC) assay showed that the combinational treatment increased g-H2AX levels which is a DNA damage response factor
in U87MG and U251MG-glioblastoma cells, compared with KML001®
and TMZ treatment only. The administration of 5 mg/kg of KML001®
inhibited tumor growth (46%) and induced apoptosis (p < 0.05 vs. Control)
in the U87MG-glioblastoma orthotopic xenograft mice model. And the
combination therapy of KML001® and TXM significantly reduced tumor
mass volume in the U87MG-glioblastoma orthotopic xenograft model,
compared with KML001® monotherapy (p < 0.05). In the U251MGglioblastoma orthotopic xenograft mice model, the combination significantly
decreased tumor volume, compared with KML001® treatment only group
(p < 0.05) and TMZ treatment only group (p < 0.05).
Conclusions: In conclusion, KML001® showed similar or more potent
antitumor effect than TMZ. The combinational treatment of KML001® and
TMZ was synergistic or additive in a glioblastoma orthotopic xenograft
mice model. These results provide a rationale for clinical testing of the
combination therapy of KML001® and TMZ.
63
POSTER (Board P057)
Radium-223 dichloride − Efficacy and mode-of-action in a mouse
model of prostate cancer bone metastasis
M.I. Suominen1 , K.M. Fagerlund1 , J.P. Rissanen1 , Y. Konkol1 ,
E. Alhoniemi1 , D. Mumberg2 , K. Ziegelbauer2 , S.M. Käkönen3 ,
J.M. Halleen1 , R.L. Vessella4 , A. Scholz2 . 1 Pharmatest Services Ltd,
Turku, Finland; 2 Bayer Healthcare, Global Drug Discovery TRG-Onc/GT,
Berlin, Germany; 3 University of Turku, Department of Cell Biology and
Anatomy, Turku, Finland; 4 University of Washington, Departments of
Urology and Microbiology, Seattle, USA
Background: Radium-223 dichloride (Xofigo® ) is an alpha-emitting
calcium mimetic that improves overall survival in prostate cancer patients
with bone metastases (ALSYMPCA trial, NCT00699751, Parker et al.
NEJM 18:213−33, 2014). Here, we clarified radium-223 dichloride modeof-action in an osteoblastic prostate cancer mouse model mimicking the
devastating bone metastasis seen in prostate cancer patients.
Material and Methods: The therapeutic effects of radium-223 dichloride
were investigated in a clinically relevant patient-derived prostate cancer
xenograft model exhibiting PSA expression, osteoblastic growth and
systemic metastasis when inoculated into the bone marrow cavity. Human
LuCaP 58 prostate cancer cells were inoculated into a tibia of 6−8-week-old
male SCID mice (n = 17−18/group) and radium-223 dichloride (300 kBq/kg,
iv injection) or vehicle control were administered once serum PSA levels
reached 1 ng/ml and again 4 weeks later. X-rays and serum samples were
obtained biweekly and at sacrifice 6 weeks after the first dose. Micro-CT
measurements were performed on the tibiae (n = 8−11 mice/group) prior
to preparation of decalcified paraffin sections for histomorphometry and
immunohistochemistry. Soft tissues were collected for histology to observe
visceral tumors, followed by macroscopical evaluation.
Results: The inhibitory effect of radium-223 dichloride on tumor-induced
osteoblastic bone growth was clearly visible in x-rays and in tibial weight
26
measurement. Radium-223-treated mice exhibited reduced bone volume
and surface in addition to decreased tumor volume compared to untreated
animals. Metastatic spread to visceral organs was evident in 53% and 33%
of vehicle- or radium-223 dichloride-treated mice, respectively. Importantly,
both serum PSA and a bone formation marker PINP were reduced in
radium-223 dichloride-treated mice. Moreover, the significantly lower PSA
values were apparent as early as two weeks following the first dose,
indicating that in addition to reducing osteoblastic bone growth, radium223 dichloride constrains tumor growth in metastatic prostate cancer.
Conclusions: In conclusion, radium-223 dichloride (Xofigo® ) therapy
exhibits a dual mode-of-action that impacts on tumor cells and on tumorinduced bone reaction, both important players in the destructive vicious
cycle of osteoblastic bone metastasis in prostate cancer.
64
POSTER (Board P058)
Nifuroxazide halogenic derivatives induce ROS-mediated apoptosis
and display antitumor activity against metastatic melanoma
C. Fernandez de Farias1 , M.H. Massaoka1 , N. Girola1 , C.R. Figueiredo1 ,
R.A. Azevedo2 , L.C. Tavares3 , L.R. Travassos1 . 1 Federal University
of São Paulo, Department of Microbiology Immunology and Parasitology,
São Paulo, Brazil; 2 Butantan Institute, Department of Biochemistry and
Biophysics, São Paulo, Brazil; 3 University of São Paulo, Department
of Biochemical and Pharmaceutical Technology, São Paulo, Brazil
Background: Metastatic melanoma is a highly aggressive and therapyresistant malignancy. At the metastatic stage the disease is highly resistant
to conventional therapy, and the 5-year survival is rather low. Therefore,
novel therapeutic interventions are stimulated. Nifuroxazide is a widely used
antibiotic, and its mechanism of action depends on ROS production. In this
work we report on the antimelanoma activity of two halogenic derivatives
of nifuroxazide.
Material and Methods: Cell viability was examined by MTT assay on
B16F10-Nex2 murine melanoma cells after treatment with N-Br and N-I.
Superoxide anion production was evaluated by dihydroethidium (DHE)
assay in B16F10-Nex2 cells co-incubated or not with N-acetylcysteine
(Nac). Chromatin condensation was analyzed by Hoescht 33342 staining.
DNA degradation was evaluated in B16F10-Nex2 cells lysed in TELT
buffer and subjected to electrophoresis in 1% Agarose gel. Apoptosis, in
B16F10-Nex2 cells, was evaluated by annexin V-positive cells, measured
by FACS. Mitochondrial membrane permeability of melanoma cells, coincubated with Nac and treated with N-Br and N-I, was assessed using
the fluorescent probe tetramethylrhodamine ethyl ester (TMRE), and then
analyzed by FACS. Therapeutic efficacy was examined in a syngeneic
model of metastatic melanoma using C57BL/6 mice endovenously injected
with B16F10-Nex2 cells and treated i.p. with N-Br and N-I. After 14
days, animals were sacrificed and the lungs were examined for metastatic
colonization.
Results: Both derivatives induced cell death in vitro in a dose-dependent
manner. The IC50 values were 16mM and 12mM for N-Br and N-I, respectively, against murine melanoma cells. In the presence of Nac cytotoxicity
of both compounds decreased. Furthermore, treatment with N-Br and N-I
increased superoxide anion production by 87.5% and 55.65%, respectively.
Co-incubation with Nac completely inhibited ROS production upon treatment with both compounds. Apoptosis cell death was suggested by a ladder
pattern of DNA fragmentation and a drug concentration-dependent phosphatidyl serine surface translocation. Moreover, TMRE staining showed
that N-B and N-I were able to induce the loss of mitochondrial membrane
potential in a time-dependent manner, which was inhibited by Nac within
6 h of treatment. Hoescht staining confirmed that cells treated with both
compounds exhibited chromatin condensation as in apoptotic nuclei.
Remarkably, i.p. doses of N-Br and N-I were protective against metastatic
melanoma, raised by endovenous injection of B16F10-Nex2 tumor cells.
Conclusion: Nifuroxazide halogenic derivatives induce apoptosis in
melanoma cells in a ROS-dependent manner at micromolar doses. Both
N-Br and N-I were protective against metastatic B16F10-Nex2 melanoma
in a syngeneic model.
Supported by Fapesp no. 2014/05107-8.
65
POSTER (Board P059)
Phase II drug metabolism UGT1A enzyme affects cellular response of
colon cancer cells to antitumor triazoloacridinone C-1305 treatment
E. Augustin1 , E. Bartusik1 , A. Theus1 , B. Borowa-Mazgaj1 , Z. Mazerska1 .
1
Gdansk University of Technology, Pharmaceutical Technology and
Biochemistry, Gdansk, Poland
Background: The essential limitation of a drug’s effectiveness in a
living organism is the individual’s level of enzymes, which catalyze drug
metabolism. Furthermore, the expression level of metabolic enzymes in
tumor tissues (in vivo) or tumor cells (in vitro) may affect the final effect of
the drug treatment. C-1305 is a promising antitumor agent with high activity
against many experimental cellular and tumor models. Studies on C-1305
metabolism indicated that the compound is conjugated by several uridine
diphosphate-glucuronyltransferase (UGT) isoforms, the most active being
extrahepatic UGT1A10. Here, we investigated whether and how UGT1A
isoenzyme affects the cellular response of colon cancer cells following
C-1305 treatment.
Materials and Methods: C-1305 activity was evaluated in two human
colon carcinoma cell lines, HT29 (UGT1A positive) and HCT116 (UGT1A
negative). To determine whether UGT1A expression affects the final cellular
response upon C-1305 treatment, HT29 and HCT116 cells were exposed
to EC50 concentration for 24 h to 120 h. DAPI staining was used to analyze
cellular morphology. Flow cytometry for annexin V/PI assay and subG1 DNA fraction was applied to identify apoptosis. Analysis of cellular
morphology and expression of SA-b-galactosidase were performed to
identify cells with senescence-like phenotype. Acridine orange staining was
performed to detect acidic vesicular organelles (AVOs) as a marker of
autophagy.
Results: Treatment of colon cancer cells with C-1305 for 72 h suppressed
cell proliferation with EC50 concentration equal to 0.5 mM for HT29 and
0.8 mM for HCT116 cells, respectively. C-1305 induced apoptosis, necrosis
and mitotic catastrophe in both cell lines as evidence by chromatin
condensation, the presence of multinucleated cells, phosphatydilserine
externalization and presence of sub-G1 fraction. In all tests, the number
of apoptotic cells was low and increased in a time-dependent manner and
reached ~23% after 120 h of drug exposure in both cell lines. Starting
from 72 h, HCT116 cells which did not die by apoptosis or necrosis,
underwent cellular senescence, preceded by autophagy. Interestingly,
pharmacological inhibition of autophagy by chloroquine (10 mM) sensitized
HCT116 cells to C-1305 and profoundly intensified the senescence
phenotype. HT29 cells did not undergo cellular senescence. Moreover,
rapid autophagy observed in those cells after 24 h decreased following
prolonged drug treatment.
Conclusions: Our results indicate that in colon cancer cells treated with
C-1305, the lack of UGT1A expression promotes autophagy followed by
cellular senescence. UGT1A status had no effect on apoptosis, mitotic
catastrophe or necrosis induced by C-1305.
66
POSTER (Board P060)
Clinical validity of new genetic biomarkers of irinotecan neutropenia:
An independent replication study
F. Innocenti1 , J. Ramirez2 , W. Qiao3 , A.J. de Graan4 , M.J. Ratain2 ,
R.H.N. van Schaik4 , R.H.J. Mathijssen4 , G.L. Rosner5 , D.J. Crona6 .
1
University of North Carolina at Chapel Hill, Linberger Comprehensive
Cancer Center & Center for Pharmacogenomics and Individualized
Therapy Eshelman School of Pharmacy, Chapel Hill NC, USA; 2 University
of Chicago, Department of Medicine, Chicago IL, USA; 3 MD Anderson
Cancer Center University of Texas, Houston TX, USA; 4 Erasmus University
Medical Center, Erasmus MC Cancer Institute, Rotterdam, Netherlands;
5
Johns Hopkins University, The Sidney Kimmel Comprehensive Cancer
Center at Johns Hopkins, Baltimore MD, USA; 6 University of North
Carolina at Chapel Hill, Center for Pharmacogenomics and Individualized
Therapy Eshelman School of Pharmacy, Chapel Hill NC, USA
Purpose: This study aimed to provide evidence for the clinical validity of
germline genetic variants previously associated with irinotecan neutropenia
and pharmacokinetics.
Methods: Ten germline variants from six genes, whichassociated with
absolute neutrophil count (ANC) nadir and/or irinotecan pharmacokinetics
in a discovery cohort of 78 cancer patients, were genotyped in an
independent replication cohort of 108 cancer patients. Patients from both
cohorts received single-agent irinotecan every three weeks. Pharmacokinetic data and neutrophil counts were collected during cycle 1 of treatment.
Associations between the germline variants and ANC nadir or irinotecan
pharmacokinetics were analyzed using linear regression. Univariate and
multivariate models adjusted for sex, age, irinotecan dose, and ANC nadir
models also adjusted for baseline ANC.
Poster Session – Drug Resistance and Modifiers
Results: For ANC nadir, we replicated UGT1A1*93, UGT1A1*28, and
SLCO1B1*1b. In both univariate and multivariate models, SLCO1B1*1b
had a protective effect against neutropenia, independent of the effect of
either UGT1A1*28 or UGT1A1*93. For irinotecan AUC0−24 , we replicated
ABCC2 −24C>T and HNF1a 79A>C; however ABCC2 −24C>T predicted
only a small fraction of the interindividual variability in irinotecan exposure,
and HNF1a 79A>C had a 75% lower estimate of effect compared to
the discovery cohort. For SN-38 AUC0−24 and the glucuronidation ratio,
we replicated UGT1A1*28, UGT1A1*93, and ABCC1 1684T>C; however
ABCC1 1684T>C had an 85% lower estimate of effect compared to the
discovery cohort.
Conclusion: In addition to UGT1A1*28, this study independently validated UGT1A1*93 and SLCO1B1*1b as new predictors of irinotecan
neutropenia. Further demonstration of the clinical utility of SLCO1B1*1b
and UGT1A1*93 will help further optimize irinotecan therapy for cancer
patients.
67
POSTER (Board P061)
Cytotoxic response as a result of the cross-talk between UGT
mediated metabolism and modulation of UGT activity by C-1311 and
C-1305 acridinone antitumor agents in selected solid tumor cell lines
Z. Mazerska1 , M. Pawlowska1 , A. Bejrowska1 , M. Szydlowska1 ,
B. Fedejko1 , E. Augustin1 . 1 Gdansk University of Technology, Department
of Pharmaceutical Technology and Biochemistry, Gdansk, Poland
Background: UDP-glucuronyltransferases (UGTs, EC 2.4.1.17) transform
nearly 35% drugs to metabolites, which are usually of lower toxicity or
limited therapeutic effects. Anticancer agents are of special interest in
this respect, because the glucuronidation may represent a mechanism
of intrinsic drug resistance and there were reported the differences in
UGT expression between normal and tumour tissues. Moreover, the UGT
level and activity in normal and tumour cells can be modulated by other
chemotherapeutics, what is crucial in antitumor multidrug therapy.
Considering above we aimed here to know whether antitumor acridinone
derivatives C-1305 and C-1311 are glucuronidated in selected solid tumour
cell lines and what is the impact of UGT1A10 overexpression on cytotoxicity
of these compounds. We also answered whether C-1305 and C-1311 are
able to influence UGT enzymatic activity.
Materials and Methods: The studied compounds, 5-diethylamino-8hydroxyimidazoacridinone, C-1311, and 5-dimethylamino-8-hydroxytriazoloacridinone, C-1305, were developed in our laboratory, and C-1311 was
selected for II phase of clinical studies. Cell lines of human hepatoma,
HepG2, colon tumours, HCT116 and HT29 and breast cancer, MCF-7, were
studied and they (except HT29) were tranfected with UGT1A10 vector by
electroporation. UGT1A10 was selected as the most effective UGT isoenzyme towards studied compounds in vitro. Metabolic glucuronidation in
tumour cells was followed by HPLC analysis. The concentration of standard
UGT substrates 7-hydroxy-4-(trifluoromethyl)coumarin (7-TFK) and 7-ethyl10-hydroxycamptothecin (SN-38) were the markers of UGT activity.
Results indicated that both compounds were metabolized in HT-29 wild
type cells (natural UGT expression) and in UGT1A10 overexpressed
tumour cells to O-glucuronides on 8-hydroxyl group. Cytotoxicity of C-1311
in MCF-7 cells overexpressed with UGT was lower than in MCF-7 wild type,
whereas it was not changed in HepG2 and HCT116. At the same time the
activity of C-1305 was slightly, but significantly, higher in the presence of
higher level of UGT in HepG2 and MCF-7 cells. Compounds C-1305 and
C-1311 were similar in the profile of UGT modulation. They activated UGT
only at low concentration and after short time of incubation, whereas higher
concentration and long time resulted in the inhibition of UGT mediated
metabolism.
Conclusions: The cross-talk between the glucuronidation and UGT induction/inhibition demonstrated here seems to be responsible for the fact that
the final influence of UGT on cytotoxicresponse depended on the type of tumor cells and the type of compound. Nevertheless, the results showed that
glucuronidation of C-1305 antitumor agent in MCF-7 cells did not result in
lower cytotoxicity, as is usually observed for majority of drugs, but we even
observed the activation effect of C-1305 glucuronide toward tumour cells.
68
POSTER (Board P062)
Can the P53 status predict the outcome of Polo-like kinase 1
inhibition in non-small cell lung cancer cell lines?
J. Van den Bossche1 , A. Wouters1 , C. Deben2 , V. Deschoolmeester2 ,
P. Specenier3 , P. Pauwels4 , M. Peeters3 , F. Lardon1 . 1 Antwerp University,
Oncology, Antwerp, Belgium; 2 Antwerp University, Pathology, Antwerp,
Belgium; 3 Antwerp University Hospital, Oncology, Antwerp, Belgium;
4
Antwerp University Hospital, Pathology, Antwerp, Belgium
Introduction: Polo-like kinase 1 (Plk1), a key regulator of multiple steps
during mitosis, is considered as one of the most promising therapeutic
27
targets for mitotic intervention due to its overexpression in a number of
human malignancies, including non-small cell lung cancer (NSCLC). TP53
is the most frequently mutated gene in human cancer and plays a role
in many cellular processes including cell growth and response to DNA
damage. Previous studies suggest a negative interplay between Plk1 and
P53, leading to the hypothesis that P53 status might be a predictive
biomarker for response to Plk1 inhibition. As such, we investigated the
cytotoxic effect of the small molecule inhibitor BI6727 (volasertib) in a series
of NSCLC cell lines differing in P53 status.
Material and Methods: Three isogenic NSCLC cell lines (A549 (P53 wt),
A549-NTC (empty vector control, p53 wt) and A549–920 (P53 deficient))
were generated using a P53 GIPZ shRNA lentiviral vector. The CRL5908 NSCLC cell line, harboring a P53 mutation (R273H), was included
too. All cell lines were treated with BI6727 (0−85nM) for 24 or 72 hours.
Treated cells were incubated under both normal and reduced oxygen
conditions (hypoxia, <0.1% O2 ). Cell survival was assessed using the
sulphorhodamine B (SRB) assay and IC50 values were calculated using
WinNonlin software. The effect of Plk1 inhibition (0−10nM) on cell cycle
distribution of A549 cells was determined flow cytometrically using the
Vindelov method.
Results: Treatment with BI6726 established a dose-dependent growth
inhibition under both normoxic and hypoxic conditions. As shown in Table 1,
a significantly reduced sensitivity to volasertib was observed in P53
deficient cells compared to p53 wt cell lines (p < 0.001). For example, in
A549–920 cells, 24 hours of Plk1 inhibition (normoxia) resulted in an IC50
value of 27.59±5.77 nM in contrast to 17.87±0.40 nM for A549-NTC cells.
Table 1 demonstrates also a decreased effect of BI6727 under hypoxic
conditions (p < 0.001). However, this could not be observed in the P53
mutant cell line. Considering the cell cycle distribution under normoxic
conditions after treatment with 10nM volasertib, a significant G2 /M phase
block was induced in the A549 cell line (p < 0.022). Furthermore, a sub-G1
peak could be observed when treated with high concentrations of BI6727,
suggesting induction of apoptosis. Similar experiments will be performed in
the cell lines lacking functional P53 in order to detect possible differences
in cell fate after Plk1 inhibition.
Conclusion: Our results show that A549 NSCLC cells with functional P53
seem to be more sensitive to Plk1 inhibition compared to isogenic P53
deficient cells. Further experiments are warranted to further clarify this
observation. In addition, combination studies of volasertib with conventional
chemotherapeutics and radiotherapy will be initiated.
Table 1. IC50 values ± standard deviations after 24 and 72 hours of
treatment with volasertib under both normoxic and hypoxic conditions
Cell line
A549
NTC
920
5908
Normoxia
24 h
72 h
Hypoxia
24 h
72h
15.44±2.49
17.87±0.40
27.59±5.77
61.90±3.94
18.44±1.83
17.33±1.32
28.00±6.32
72.38±9.87
79.91±15.41
66.39±14.31
115.46±27.60
76.43±10.83
54.46±5.58
63.89±22.30
112.52±34.19
68.66±9.83
Drug Resistance and Modifiers
69
POSTER (Board P063)
AKT3 activation mediates resistance to HER2-targeted therapies in
HER2-amplified breast cancer
F. Carmona Sanz1 , F. Montemurro2 , V. Rossi2 , C. Verma3 , M. Berger1 ,
J. Baselga1 , M. Scaltriti1 . 1 Memorial Sloan-Kettering Cancer Center, New
York City, USA; 2 Institute for Cancer Research and Treatment, Candiolo,
Italy; 3 A STAR’s Bioinformatics Institute (BII), Singapore, Singapore
Background: HER2 targeted therapies have improved the clinical benefit of
HER2-amplified breast cancer when diagnosed before metastatic spread.
Nevertheless, a substantial percentage of patients with advanced HER2
positive disease are intrinsically refractory to these agents, and many
initially responding patients acquire resistance over time. Activation of
PI3K/AKT pathway via mutation of PIK3CA or loss of PTEN has been
associated with resistance to anti-HER2 therapy, and combination regimes
with PI3K inhibitors are now under investigation in the clinical setting. We
hypothesize that also aberrations in AKT3 concur in limiting the sensitivity
to anti-HER2 agents.
Material and Methods: We used targeted exome-sequencing to analyze
clinical specimens obtained from HER2-amplified breast cancer patients
28
treated with trastuzumab and/or lapatinib monotherapy. Further protein
modeling studies and functional in vitro and in vivo assays were used to
validate our findings. Genetically engineered AKT 1/2 null cells were also
used to assess the contribution of AKT3 activation in the context of drug
resistance.
Results: We have identified gain-of-function genomic alterations in
AKT3 associated with therapy refractoriness in HER2-amplified breast
cancer patients treated with anti-HER2 agents in a chemotherapy-free
setting. Specifically, copy-gain and genomic mutation were associated with
intrinsic resistance to trastuzumab or lapatinib. Protein modeling predicts
an activating role for the mutation identified, providing a mechanism
for pathway activation beyond those previously described. Treatment
combination with specific AKT inhibitors might improve treatment efficacy
and overcome resistance to HER2-directed therapy.
Conclusions: Genomic analyses of selected patient biopsies provide
relevant biomarkers that can be used for clinical decision making. The
involvement of AKT3 in the development of resistance to anti-HER2
agents provides an actionable target for efficient therapeutic intervention.
Moreover, the confirmation of AKT3 as a reliable prognostic biomarker
would improve patient stratification beyond the assessment of routinely
assayed pathway alterations.
70
POSTER (Board P064)
Antitumor activity of S116836, a novel tyrosine kinase inhibitor,
against imatinib-resistant FIP1L1-PDGFRa-expressing cells
J. Pan1 , Y. Shen2 , K. Ding3 . 1 Sun Yat-sen University Zhongshan
Ophthalmic Center, State Key Laboratory of Ophthalmology, Guangzhou,
China; 2 Sun Yat-sen University Zhongshan School of Medicine,
Department of Pathophysiology, Guangzhou, China; 3 Guangzhou
Institute of Biomedicine and Health Chinese Academy of Sciences, Key
Laboratory of Regenerative Biology and Institute of Chemical Biology,
Guangzhou, China
The FIP1-like-1-platelet-derived growth factor receptor alpha (FIP1L1PDGFRa) fusion oncogene is the driver factor in a subset of patients with
hypereosinophilic syndrome (HES)/chronic eosinophilic leukemia (CEL).
Most FIP1L1-PDGFRa-positive patients respond well to the tyrosine kinase
inhibitor (TKI) imatinib. Resistance to imatinib in HES/CEL has been
described mainly due to the T674I mutation in FIP1L1-PDGFRa, which
is homologous to the imatinib-resistant T315I mutation in BCR−ABL.
Development of novel TKIs is imperative to overcome resistance to imatinib.
We synthesized S116836, a novel TKI. In this study, we evaluated
the antitumor activity of S116836 in FIP1L1-PDGFRa-expressing cells.
The results showed that S116836 potently inhibited PDGFRa and its
downstream signaling molecules such as STAT3, AKT, and ERK1/2.
S116836 effectively inhibited the growth of the WT and T674I FIP1L1PDGFRa-expressing neoplastic cells in vitro and in nude mouse xenografts.
Moreover, S116836 induced intrinsic pathway of apoptosis as well as the
death receptor pathway, coincided with up-regulation of the proapoptotic
BH3-only protein Bim-EL through the Erk1/2 pathway.
In conclusion, S116836 is active against WT and T674I FIP1L1-PDGFRaexpressing cells, and may be a prospective agent for the treatment of
HES/CEL.
71
POSTER (Board P065)
Novel regulation of estrogen receptor transcription by the PI3K
pathway
1
1
1
1
1
1
E. Toska , M. Elkabets , A. Bosch , O. Litvin , M. Scaltriti , J. Baselga .
1
MSKCC, New York, USA
Background: Alterations in the PI3K/Akt/mTOR pathway occur in up to
50% of estrogen receptor (ER) positive breast tumors. Clinical data showed
that dual inhibition of the ER and mTOR pathways results in greater disease
control. Although previous work has pointed to the existence of crosstalk
between ER and certain nodes of the PI3K pathway, the mechanism
by which both pathways regulate each other remains to be elucidated.
Thus, we aim to investigate the mechanisms by which ER-dependent
transcriptional program is regulated by the PI3K pathway.
Material and Methods: ER-positive cell lines with different genomic
alterations in the PI3K pathway were treated in vitro with the p110a-specific
PI3K inhibitor BYL719. RNA expression was evaluated by gene expression
microarray analysis and RT-PCR. Promoter occupancy of ER to its target
genes was assessed by chromatin immunoprecipitation (ChIP) assay.
A transposon activation mutagenesis screen was developed to identify
modulators of resistance to PI3K inhibitors in ER-positive breast cancer
cells. This approach employs the transfection of transposon containing
CMV promoters that are randomly integrated in the genome and drive
activation of nearby gene expression.
Results: We observed that inhibition of the PI3K pathway by BYL719
triggers ER gene expression, which in turn results in increased ERdependent transcriptional activity accompanied by an ER-driven re-wiring
of the cell transcriptome. The transposon activation mutagenesis screen
followed by high throughput sequencing successfully identified genes that
regulate ER expression/activity and putatively cause resistance to PI3K
inhibitors in ER+ breast cancer cell lines. Interestingly, among the genes
identified we found a number of key transcription factors known to modulate
ER expression. Importantly, these results were confirmed in breast cancer
patients treated with BYL719.
Conclusions: Our findings suggest that PI3K inhibition activates the ER
pathway by modulating ER expression levels. Moreover, we have developed
a transposon activation mutagenesis screen that identified a number of ER
co-factors as possible responsible for resistance to PI3K inhibition. The
molecular mechanisms by which these factors enter into play upon PI3K
inhibition are currently being investigated.
72
POSTER (Board P066)
Nrf2 as a molecular target in overwhelming chemoresistance in
breast cancer therapy
N. Samadi1 , F. Ramezani1 , M. Sabzichi1 . 1 RCPN, Clinical Biochimestry,
Tabriz, Iran
Background: Nuclear factor erythroid 2-related factor 2 (Nrf2) has
been recognized as a transcription factor that control mechanisms of
cellular defense response by regulation of three classes of genes,
including, endogenous antioxidants, phase II detoxifying enzymes and
transporters. Previous studies have revealed the role of Nrf2 in resistance
to chemotherapeutic agents and high level expression of Nrf2 have
found in many types of cancer. At physiological concentrations, luteolin
as a flavonoid compound can inhibit Nrf2 and sensitized cancer cells
to chemotherapeutic agents. We reported luteolin loaded in phytosome
as an advanced nanoparticles carrier sensitized MDA-MB 231 cells to
doxorubicin.
Material and Methods: Luteolin-loaded phytosome was prepared by
thin film hydration technique. Cell culture/cytotoxicity studies of luteolin,
doxorubicin, and phytosome were performed by means of MDA-MB231
human cancer cells and MTT assay. Determination of the gene expression
levels of Nrf2, HO1, NQO1 and MDR1, used by Real-Time polymerase
chain reaction (RT-PCR).
Results: In this study, we prepared nano phytosoms of luteolin to enhance
the bioavailability of luteolin and improve passive targeting in breast cancer
cells. Our results showed that co-treatment of the cells with nano particles
containing luteolin and doxorubicin has the highest percentage of cell death
in MDA-MB 231cells (p < 0.05).
Furthermore, luteolin-loaded nanoparticles reduced Nrf2 gene expression
at mRNA level in the cells higher than luteolin alone (p < 0.05). Similarly,
expression of downstream genes for Nrf2 including Ho1 and MDR1 were
reduced significantly (p < 0.05). Inhibition of Nrf-2 expression caused a
marked increase in cancer cell death (p < 0.05).
Conclusions: Taken together, these results suggest that phytosome
technology can improve the efficacy of chemotherapy by overcoming
resistance and enhances permeability cancer cells to chemical treatment
and may thus be considered as a potential delivery system to increase the
therapeutic protocols in cancer patients.
73
POSTER (Board P067)
Differential pathway resiliency in response to MAPK inhibition in
BRAF-mutant cancer
M. Sos1 , R.S. Levin1 , J.D. Gordan1 , J.A. Oses-Prieto2 ,
J.T. Webber3 , M. Salt4 , B. Hann4 , A.L. Burlingame2 , F. McCormick4 ,
S. Bandyopadhyay3 , K.M. Shokat1 . 1 UCSF, Howard Hughes Medical
Institute and Department of Cellular and Molecular Pharmacology
University of California San Francisco, San Francisco CA, USA; 2 UCSF,
Department of Pharmaceutical Chemistry University of California San
Francisco California USA, San Francisco CA, USA; 3 UCSF, Department
of Bioengineering and Therapeutic Sciences University of California
San Francisco, San Francisco CA, USA; 4 UCSF, Helen Diller Family
Comprehensive Cancer Center Department of Medicine University of
California San Francisco San Francisco, San Francisco CA, USA
Background: Despite the development of potent RAS-RAF pathway
inhibitors, only a fraction of BRAF-mutant patients responds to the
treatment with these drugs.
Materials and Methods: Here, we applied a combined chemo-genomics
and chemo-proteomics approach to gain insights into the dynamics of
primary resistance signaling and to define novel cancer vulnerabilities in
these tumors.
Results: These analyses uncovered differential pathway resiliency in
response to MAPK inhibition in BRAF-mutant cancer cells and identified
how high-dose MEK inhibition as a viable strategy to overcome this source
of resistance signaling. Mapping of global phosphorylation dynamics using
orthogonal mass-spectrometry based methods revealed multi-layered
MAPK pathway feedback-loop release and autocrine cytokine secretion
as complementary drivers of adaptive signaling in these tumors.
Conclusions: Overall, our data provide a kinome- and phosphoproteomewide view of the adaptive process of primary resistance to MAPK inhibitors
in BRAF-mutant tumors that may be of importance for future clinical trials
involving these targeted drugs.
74
POSTER (Board P068)
Increased EDN1 expression mediates acquired resistance to the
combination therapy of PI3K and MEK inhibitors for colorectal
cancer
B. Bhattacharya1 , S.H.H. Low1 , T. Benoukraf1 , M.L. Chong1 , K.X. Koh1 ,
R. Soong1 . 1 National University of Singapore, Cancer Science Institute,
Singapore, Singapore
Background: Previously we reported that acquired resistance (AQR) to
the combination of BKM120 (PI3K inhibitor) and AZD6244 (MEK inhibitor)
can be generated in PIK3CA and KRAS mutant HCT116 colorectal cancer
(CRC) cells, in addition to AQR to the individual agents. Using gene
expression analysis we demonstrated that the differentially expressed
genes in the combination AQR cells are distinctly different to those
expressed in single agent AQR and parental cells, with EDN1 (endothelin-1)
gene being the most highly expressed (~33-fold). Currently, we generated
AQR to the same combination in LoVo CRC cell line and explored
determinants of resistance.
Methods: LoVo CRC cells with KRAS mutation were exposed continuously
with combined (1 IC50 dose of each agent) or single agent (2 doses of
IC25 ) concentrations of MEK (AZD6244) and PI3K (BKM120) inhibitors, or
DMSO (parental). Combination Indices (CI) was calculated by the method
of Chou and Talalay. Differentially expressed genes were determined using
Affymetrix Gene 1.0ST analysis.
Results: LoVo cells with AQR to combination treatment were obtained after
12 months ([email protected] of ‘LoVoCR’ cells 2.5±0.3 vs parental 0.19±006,
p = 0.03). LoVo cells with AQR to single agent treatment with AZD6244
(IC50 of ‘LoVoAR’ 67.1±0.02mM vs parental 5.1±0.01mM; p = 0.02)
and BKM120 (IC50 of ‘LoVoBR’ 5.2±0.11mM vs parental 0.9±0.21mM;
p = 0.002) were also generated. Consistent with our previous findings
LoVoCR cells were resistant to apoptosis, wound healing response and
cell cycle arrest compared with parental cells. Furthermore, these cells
also displayed resistance to the combination of an alternative PI3K
inhibitor (BYL719) and MEK inhibitor (GDC0973). No cross-resistance
was observed with 5-FU or carboplatin in LoVoCR cells, arguing against
a multi-drug resistance phenotype. Consistent with our gene expression
data in HCT116 cells AQR to the combination (HCT116CR) cells a
7-fold overexpression of EDN1 was also observed in LoVoCR cells. Both
HCT116CR and LoVoCR cells displayed significantly elevated phosphorylation levels of AKT and ERK compared to parental cells, suggesting some
feedback mechanism. Silencing EDN1 by siRNA significantly reduced the
proliferation rates of HCT116CR and LoVoCR cells compared to control
siRNA, which was accompanied by a decrease in phosphorylation of AKT
and ERK. Both the combination AQR cells were significantly sensitive
to bosentan (endothelin receptor antagonist) induced growth inhibition
(HCT116 IC50 = 67.4±0.9mM vs HCT116CR IC50 = 39.2±1.2mM; p = 0.01:
LoVo IC50 = 83.2±2.5mM vs LoVoCR IC50 = 41.1±3.4mM; p = 0.03). Furthermore, in HCT116CR and LoVoCR cells the antagonism was converted
to synergy in the presence of a non-growth inhibitory concentration (1/5th
IC50 ) of bosentan. Finally, pre-stimulation with exogenous endothelin-1
conferred resistance to the combination in both parental HCT116 and
LoVo cells, along with an increase in AKT and ERK phosphorylation.
Current work aims to uncover the mechanism of transcriptional regulation
of endothelin in AQR.
Conclusions: EDN1 is a specific mediator of AQR to the use of the PI3K
and MEK inhibitor combination in CRC, irrespective of the genetic lesions
involved. Endothelin receptor antagonists can potentially circumvent such
resistance.
29
75
POSTER (Board P069)
Loss of PTEN leads to acquired resistance to the PI3Ka inhibitor
BYL719: a case of convergent evolution under selective therapeutic
pressure
P. Castel1 , D. Juric2 , M. Griffith3 , O.L. Griffith4 , H.H. Won1 , B. Ainscough5 ,
H. Ellis1 , S. Ebbesen6 , I. Gopakumar1 , C. Quadt7 , M. Peters7 , D. Solit1 ,
S.W. Lowe6 , E.R. Mardis5 , M.F. Berger1 , M. Scaltriti1 , J. Baselga1 .
1
Memorial Sloan Kettering Cancer Center, HOPP, New York, USA;
2
Massachusetts General Hospital, Cancer Center, Boston, USA;
3
Washington University School of Medicine, Department of Genetics,
St Louis, USA; 4 Washington University School of Medicine, Department
of Medicine, St Louis, USA; 5 Washington University School of Medicine,
The Genome Institute, St Louis, USA; 6 Memorial Sloan Kettering Cancer
Center, Cancer Biology and Genetics, New York, USA; 7 Novartis Pharma
AG, Basel, Switzerland
Background: Activating mutations of PIK3CA, the gene encoding the
p110a subunit of PI3K, are frequent in breast cancer and selective inhibitors
of this enzyme have shown promising clinical activity in breast tumors
harboring these mutations.
Material and Methods: We studied the case of a patient with metastatic
breast cancer harboring a PIK3CA mutation that was treated in a clinical
trial with BYL719, a highly selective PI3Ka inhibitor. The treatment
resulted in partial tumor regression that lasted 9.5 months followed by
progression and rapid death of the patient. A rapid autopsy was performed
with collection of tissue samples from 16 different metastatic sites. We
compared by whole genome and exome sequencing the original primary
tumor, a rapidly progressing lung metastasis and a periaortic lesion that
was still responding to BYL719 at time of death. Besides several common
alterations, PTEN loss and a missense mutation were detected only in the
lung metastasis. Using targeted exome sequencing we analyzed all the
other available samples.
Results: Strikingly, we observed a consistent loss in PTEN (via different
mechanisms such as deletion, splice site mutation and frameshift
mutations) in all the lesions refractory to BYL719 but not in the responding
ones. We were able to build a dendrogram showing the phylogenetic
evolution of the lesions and the evolutionary convergence of the PTEN
alterations.
To validate PTEN loss as a possible mechanism of acquired resistance
to selective PI3Ka inhibition, we generated inducible PTEN shRNA clones
starting from three different BYL719-sensitive cell lines. In all the studied
models, induction of PTEN shRNA resulted in resistance to BYL719.
Since PTEN deficient genetic models have been shown to rely on the
b subunit of the PI3K holoenzyme, we tested whether the concomitant
inhibition of both p110a and p110b was sufficient to revert the resistant
phenotype. BKM120 (a pan-PI3K inhibitor) or the addition of AZD6482
(p110b inhibitor) to BYL719 re-sensitized the cells to BYL719.
To expand our findings in vivo, we used a patient-derived xenograft
(PDX) model generated from a lung PTEN-null non-responding lesion.
Consistently, this PDX model was refractory to the antitumor activity
of BYL719 but conserved sensitivity to BKM120 or the combination of
AZD6482 and BYL719. In both cases, IHC analysis revealed a decrease
in PI3K/AKT downstream effectors pAKT (473) and pS6 (240/4) staining
with BKM120 or AZD6482+BYL719, but not with BYL719 alone.
Preliminary analyses of other specimens collected from patients treated
with BYL719 showed homozygous PTEN loss in another sample upon
therapy progression.
Conclusion: Taken together, the different mechanisms that inactivate
PTEN in the tumor treated with BYL719 can be explained by convergent
phenotypic evolution in a heterogeneous tumor and highlight the importance of PTEN and PI3Kb in acquired resistance to PI3Ka inhibitors.
76
POSTER (Board P070)
Mixed lineage kinases activate MEK independently of RAF to mediate
resistance to RAF inhibitors
A.A. Marusiak1 , Z.C. Edwards1 , W. Hugo2 , E.W. Trotter1 , M.R. Girotti3 ,
N.L. Stephenson1 , X. Kong2 , M.G. Gartside4 , S. Fawdar1 , A. Hudson1 ,
W. Breitwieser5 , N.K. Hayward4 , R. Marais3 , R.S. Lo2 , J. Brognard1 .
1
Cancer Research UK Manchester Institute, Signalling Networks in
Cancer, Manchester, United Kingdom; 2 Jonsson Comprehensive Cancer
Center, Division of Dermatology, Los Angeles, USA; 3 Cancer Research
UK Manchester Institute, Molecular Oncology, Manchester, United
Kingdom; 4 QIMR Berghofer Medical Research Institute, Oncogenomics
Research, Brisbane, Australia; 5 Cancer Research UK Manchester
Institute, Cell Regulation, Manchester, United Kingdom
Melanoma is a form of skin cancer that displays a particularly aggressive
and malignant phenotype. Over 50% of melanoma cases involve mutation
30
of the BRAF protein, and of these more than 90% carry the amino
acid substitution V600E. This is an activating mutation that constitutively
activates the ERK pathway, promoting survival and proliferation. BRAF
inhibitors such as vemurafenib initially give good results, with a high rate
of objective response and improved survival rates; however, resistance
develops after around six months. We have identified a novel mechanism
of resistance to BRAF inhibition, mediated by the mixed lineage kinase
(MLK) family of proteins. MLKs are MAP3Ks, which are known to activate
the JNK pathway. We observed that MLK1−4 can directly phosphorylate
MEK in vitro and activate the ERK pathway in cells. Expression of
MLK1−4 in melanoma cell lines harbouring BRAFV600E reactivates the
ERK pathway and promotes cell survival despite treatment with BRAF
inhibitors. Furthermore, MLKs are found to be upregulated in 9 of 21
melanoma patients with acquired drug resistance. Consistent with this
observation, MLKs promote resistance to RAF inhibitors in mouse models,
and contribute to acquired resistance in a cell line model. Lastly, we observe
that a majority of MLK1 mutations identified in patients are gain-of-function
mutations. In conclusion these results suggest a role for MLKs as direct
activators of the MEK/ERK pathway with implications for melanomagenesis
and resistance to RAF inhibitors.
77
POSTER (Board P071)
Tumor suppressive roles of miR-221 and miR-222 in lung cancer
M. Sato1 , R. Yamashita1 , T. Kakumu1 , T. Hase1 , E. Maruyama1 ,
Y. Sekido2 , M. Kondo1 , Y. Hasegawa1 . 1 Nagoya University Graduate
School of Medicine, Department of Respiratory Medicine, Nagoya, Japan;
2
Aichi Cancer Center Research Institute, Division of Molecular Oncology,
Nagoya, Japan
Background: Bimodal roles of miR-221 and miR-222 microRNAs are
reported in several types of human cancers. A previous study suggested
their oncogenic role in invasiveness in lung cancer, albeit only one cell line
(H460) was used.
Material and Methods: To further evaluate involvement of miR-221 and
miR-222 in lung cancer, we investigated the effects of miR-221 and miR222 overexpression on six lung cancer cell lines, including H460, as well
as one immortalized normal human bronchial epithelial cell line, HBEC4.
Results: miR-221 and miR-222 overexpression induced epithelial-tomesenchymal transition (EMT)-like changes in morphology as well as
expression levels of EMT-associated genes in HBEC4 but did not confer
EMT-associated oncogenic phenotypes, anchorage-independent growth
and invasiveness. Consistent with the prior report, miR-221 and miR222 promoted growth in H460; however, miR-221 suppressed growth in
four other cell lines with no effects in one, and miR-222 suppressed
growth in three cell lines but promoted growth in two. Cell cycle and
apoptosis analyses revealed that growth suppression by miR-221 and
miR-222 occurred through intra-S-phase arrest and/or apoptosis resulting
from DNA double strand breaks. Microarray analysis suggested that the
JAK/STAT signaling pathway is involved in the apoptosis-induced by miR221 and miR-222. Finally, lung cancer cell lines transfected with miR-221 or
miR-222 became more sensitive to the S-phase targeting drugs, possibly
due to an increased S-phase population.
Conclusions: Our data are the first to show tumor-suppressive effects
of miR-221 and miR-222 on lung cancer, suggesting their potential as
therapeutics for the disease.
78
POSTER (Board P072)
A comprehensive in vitro screen to identify therapeutic candidates
for inclusion with etoposide/platin combinations to improve
treatment of SCLC
D. Evans1 , R. Delosh1 , J. Laudeman1 , C. Ogle1 , R. Reinhart1 , M. Selby1 ,
T. Silvers1 , A. Monks1 , E. Polley2 , G. Kaur2 , J. Morris2 , B.A. Teicher2 .
1
Frederick National Laboratory for Cancer Research, MPB/DCTD,
Frederick MD, USA; 2 NCI, DCTD, Rockville MD, USA
Background: SCLC initially shows a good response to first line
chemotherapeutic regimens but recurrent disease is highly resistant to
treatment. Consequently, the SCLC 5-year survival rate is only 5−10%. In
an attempt to improve therapeutic efficacy for SCLC, various combination
treatments have been tested; however, the two-drug combination of
etoposide and a platinum drug (carboplatin or cisplatin), with equivalent
clinical activity, has remained the standard of care for 30 years.
Improving the activity of the platin/etoposide backbone in SCLC, by
adding a third, non-cross-resistant agent, has had little success. Empirical
approaches in the clinic have not been able to evaluate the many
investigational and approved agents that could serve as the third drug to
make an efficacious triplet regimen.
Materials and Methods: We established a panel of 67 human SCLC cell
lines for use in single agent and combination screens. In phase 1, the
SCLC lines were screened with ~100 approved anticancer drugs and 433
investigational agents. The single agent data were used to select agents for
the combination screen seeking a third drug to add to the platin/etoposide
backbone. The screen used fixed concentrations of carboplatin (3.7uM) and
etoposide (0.3uM) and exposure time (96 h). The effect of simultaneously
adding a third agent (from the approved and investigational small molecule
collection), at varying concentrations, on the viability of each of the SCLC
lines was examined.
Results: Based on single agent activity in the primary screen of 67 SCLC
lines, ~180 compounds from the Approved and Investigational Oncology
Agent libraries were examined in the combination screen. The third agents
were tested from a maximum concentration corresponding to the clinical
CMax concentration with 3-fold serial dilutions over 8 dilutions. The in vitro
screening data were analyzed using two methods: the Combination Index
Method and the Bliss Independence model.
Conclusions: The goal was to identify compounds producing greater-thanadditive cytotoxicity in the majority of SCLC lines. The results suggest that
compounds targeting BCL2 and the JAK pathway improve the efficacy of
the triplet across a number of cell lines. Combined with the gene expression
and miRNA expression data for the lines, the data obtained will help
identify novel therapeutic combinations, may help in matching patients with
treatments and may identify novel molecular targets in SCLC.
Funded by NCI Contract No. HHSN261200800001E. This research was
supported in part by the Developmental Therapeutics Program in the
Division of Cancer Treatment and Diagnosis of the National Cancer
Institute.
79
POSTER (Board P073)
Quantitative proteomics as a tool to identify resistance mechanisms
in erlotinib-resistant subclones of the non-small cell lung cancer cell
line HCC827
K. Jacobsen1 , R.R. Lund1 , H.C. Beck2 , H.J. Ditzel1 . 1 Molecular
medicine, Cancer & Inflammation, Odense, Denmark; 2 Center for Clinical
Proteomics, Clinical Biochemistry and Pharmacology, Odense, Denmark
Background: Erlotinib (Tarceva® , Roche) has significantly changed the
treatment of non-small cell lung cancer (NSCLC) as approximately 70%
of patients show significant tumor regression when treated. However, all
patients relapse due to development of acquired resistance, which in
43−50% of cases are caused by a secondary mutation (T790M) in EGFR.
Importantly, a majority of resistance cases are still unexplained. Our aim
is to identify novel resistance mechanisms − and potentially new drug
targets − in erlotinib-resistant subclones of the NSCLC cell line HCC827.
Materials and Methods: We established 3 erlotinib-resistant subclones
(resistant to 10, 20, 30 mM erlotinib, respectively), and performed
comparative quantitative proteomic analysis of these and the parental
HCC827 cell line. The resistant subclones were examined both in absence
and presence of erlotinib, and in biological triplicates on a Q-Exactive mass
spectrometer. Only proteins identified with minimum 2 unique peptides and
in minimum 2 of 3 replicates were used for further analysis.
Results: Importantly, the resistant clones did not acquire the T790M
mutation or other EGFR or KRAS mutations, potentiating the identification
of novel resistance mechanisms in these subclones. We identified 2875
cytoplasmic proteins present in all 4 cell lines. Of these 87, 56 and 23
were upregulated >1.5 fold; and 117, 72 and 32 were downregulated
>1.5 fold, respectively, in the 3 resistant clones compared to the parental
cell line. By network analysis, we found cell survival, proliferation and
migration to be induced, and apoptosis and adhesion to be repressed
across the 3 resistant clones vs the parental cell line. Furthermore,
networks involved in DNA repair and adherens junction signaling were
inconsistently repressed in the 3 cell lines. More specifically, EGFR was
consistently downregulated, indicating a bypass signaling mechanism to
achieve resistance, and mitogen-activated protein kinase 1 (MAPK1) and
synovial apoptosis inhibitor 1 (SYNV1) were upregulated.
Conclusions: In conclusion, cancer-related networks such as proliferation
and apoptosis were found to be regulated, supporting the validity of
the model. EGFR was consistently downregulated and MAPK1 activated,
indicating a bypass resistance mechanism, likely leading to activation of
downstream proteins obviating EGFR. Generally, the overlap of regulated
proteins between the 3 subclones was low, indicating the subclones have
become resistant by different mechanisms.
80
POSTER (Board P074)
Determination of an oxidative stress gene signature in inflammatory
breast cancer patient tumors and development of a novel redox
modulatory strategy in overcoming chemotherapy resistance and
mediating anti-tumor efficacy
G.R. Devi1 , J.L. Allensworth2 , M. Evans2 , N. Ueno3 , D. McDonnell4 ,
F. Bertucci5 , S. Van Laere6 . 1 Duke University, Surgery and Duke Cancer
Institute, Durham NC, USA; 2 Duke University, Surgery, Durham NC, USA;
3
University of Texas MD Anderson, Oncology, Houston, USA; 4 Duke
University, Pharmacology, Durham NC, USA; 5 Institut Paoli-Calmettes,
Oncology, Marsellie, France; 6 General Hospital Sint-Augustinus,
Oncology, Antwerp, Belgium
Background: Cancer cells often have increased levels of reactive oxygen
species (ROS); however, acquisition of redox adaptive mechanisms allows
for evasion of ROS-mediated cell death. Chemotherapies including the
anthracyclines, taxanes, alkylating agents, platinum compounds as well as
radiation therapy all rely heavily on the induction of oxidative stress-induced
apoptosis for their anti-tumor activities; thus, redox adaptation can confer
resistance to many breast cancer therapies. Inflammatory breast cancer
(IBC) is an advanced and distinct breast cancer subtype characterized by
high rates of residual disease and regional recurrence despite advances
in multimodality treatment. We hypothesize that the poor response of IBC
tumors to common treatment strategies, many of which rely heavily on ROS
induction, is the result of redox adaptation.
Experimental Design: Metagene analysis was conducted to identify an
oxidative stress response (OSR) gene signature in IBC cells treated with an
acute dose of a strong ROS inducer and then allowed to recover. This OSR
signature was applied to normal breast samples, pre-treatment IBC and
nonIBC patient tumor samples to assess their oxidative stress response
(OSR). A dithiocarbamate, disulfiram (DSF), currently approved as an antialcoholism drug was applied to therapeutically resistant IBC cells alone or
in combination with copper (Cu); effects on viability, antioxidants, survival
factors, and tumor progression were assessed.
Results: Analysis of patient samples using the unique OSR metagene
generated revealed significantly higher OSR scores in IBC tumor samples
compared to normal or non-IBC tissue. In addition, we have identified a
novel mechanism of DSF to act as a Cu ionophore in a Cu-transporter
Ctr1-independent manner, target the Cu signaling redox axis in inducing
oxidative stress-mediated apoptosis. This resulted in a potent inhibition
of NF-úB signaling, aldehyde dehydrogenase activity, 3D mammosphere
formation, in vivo orthotopic mammary tumor growth in murine IBC models
and in overcoming chemotherapy resistance.
Conclusions: These results indicate that IBC tumors are highly redox
adapted, which may render them resistant to ROS-inducing therapies. DSF,
through redox modulation, may be a useful approach to enhance chemoand/or radio-sensitivity for advanced BC subtypes where therapeutic
resistance is a major challenge to durable responses to current standard
of care.
81
POSTER (Board P075)
Sensitization of triple-negative breast cancer to PI3K inhibition by
cotargeting IGF1R
K. de Lint1 , J.B. Poell1 , J. Vidal Rodriguez1 , H. Soueidan1 , W. Nijkamp1 ,
L.F. Wessels1 , R.L. Beijersbergen1 . 1 Netherlands Cancer Institute − Antoni
van Leeuwenhoek, Molecular Carcinogenesis, Amsterdam, Netherlands
Background: Targeted therapies have proven invaluable in the treatment of
breast cancer, as exemplified by tamoxifen treatment for hormone receptorpositive tumors and trastuzumab treatment for HER2-positive tumors. In
contrast, a subset of breast cancer negative for these markers (triplenegative breast cancer) has met very limited success with pathwaytargeted therapies. A large fraction of triple negative breast cancers depend
on the PI3K pathway for proliferation and survival, however, inhibition of
PI3K alone generally has limited clinical benefit. It is of interest to identify
potential combination therapies that can enhance the response and thereby
improve clinical outcome for triple-negative breast cancer.
Materials and Methods: We have analyzed the response of a panel of
triple-negative breast cancer cell lines to the PI3K inhibitor GDC-0941.
We have generated a breast cancer cell line resistant to PI3K inhibition
due to an adaptive response. We have applied large-scale pooled shRNA
screening for the identification of genes required for cell proliferation under
PI3K inhibition in the adapted triple-negative cell line.
Results: We have identified IGF1R as crucial component of the adaptive
response to PI3K inhibition. Knock-down of IGF1R potently restores the
sensitivity of these cells to the PI3K inhibitor GDC-0941. Pharmacological
inhibition of IGF1R prevents colony outgrowth of adapted cells. In
treatment-naive cells IGF1R inhibition sensitizes cells to PI3K inhibition and
induces apoptosis. In a subset of triple-negative breast cancer cell lines,
31
IGF1R inhibition shifts the dose–response curve of GDC-041 up to 5-fold.
In particular we find that breast cancer cell lines with high expression of
IGF2-processing and signaling genes are responsive to the combination of
PI3K and IGF1R inhibitors, whereas high expression of ERBB3 or mutated
RAS are associated with a limited benefit of IGF1R inhibition.
Conclusions: Our results suggest that combination treatment with PI3K
and IGF1R inhibitors may prove beneficial over monotherapy in a subset
of triple-negative breast cancers.
82
POSTER (Board P076)
Regorafenib resistance in colorectal carcinoma is associated with
enhanced expression of type II interleukin 1 receptor and reversed
by MEK/ERK inhibitor
A.C. Mar1 , C.H. Chu2 , C.W. Shiau3 , T.C. Lee1 . 1 Institute of
Biomedical Sciences, Academia Sinica, Taipei, Taiwan; 2 Institute of
Pharmacology, National Yang-Ming University, Taipei, Taiwan; 3 Institute of
Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan
Targeted therapy initially shows promising tumor regression but the
development of drug resistance quickly results in failure of treatment.
Among a batch of colorectal cancer (CRC) cell lines, we have preliminarily
found that the expression levels of type II interleukin 1 receptor (IL1R2), an
IL-1 decoy receptor, were closely associated with Regorafenib resistance.
In addition, IL1R2 expression was associated with poor prognosis of
patients with CRC cancer. Regorafenib was the newly approved multikinase inhibitor used for treatment of late-stage metastatic CRC by
US FDA. In this study, we conducted experiments to elucidate the
mechanism underlying which IL1R2 is involved in Regorafenib resistance.
We first demonstrated that silencing of IL1R2 in HT29 cells overcame
its resistance to Regorafenib, whereas ectopic expression of IL1R2 in
HCT116 cells reduced its sensitivity to Regorafenib in both in vitro and
in vivo systems. In addition, enhanced expression of IL1R2 was observed
in Regorafenib-resistant DLD-1 colon cancer cells (DLD-1-R), which were
established by growing DLD-1 cells in the presence of Regorafenib.
In IL1R2 overexpressing cells, such as HT29, IL1R2-overexpressing
HCT116, and DLD-1-R cells, Regorafenib treatment significantly resulted
in activation of MEK/ERK signaling, which is crucial for survival. We
also revealed that increased p-ERK levels in Regorafenib treated IL1R2overexpressing cells were likely due to decreased expression of MKP-3,
one of the phosphatases of ERK. Furthermore, pretreatment of HT29,
IL1R2-overexpressing HCT116, and DLD-1-R cells with MEK/ERK inhibitor
U0126 significantly reversed their Regorafenib resistance in in vitro
and in vivo systems. Taken together, our present study suggested that
enhanced IL1R2 plays certain roles on Regorafenib resistance and the
combination of Regorafenib and MEK/ERK inhibitor is a rationale regime
to overcome Regorafenib resistance in CRC patients.
83
POSTER (Board P077)
Acquired resistance to BET bromodomain inhibitors is associated
with modulation of the apoptotic signaling network
P. Sandy1 , S. Nerle1 , A. Conery2 , C. Hatton3 , B. Bryant3 , R. Sims2 ,
E. Normant1 . 1 Constellation Pharmaceuticals, Pharmacology, Cambridge,
USA; 2 Constellation Pharmaceuticals, Biology, Cambridge, USA;
3
Constellation Pharmaceuticals, Bioinformatics, Cambridge, USA
Background: Bromodomain and extraterminal (BET) family proteins
function as chromatin readers that recognize and bind acetylated lysine
residues on histones and play an essential role in the regulation of
transcription of key oncogenes, including c-Myc and Bcl-2. Potent and
selective small molecule inhibitors of BET bromodomains (BETi) induce
growth arrest and apoptosis in a wide range of tumor types, in both in vitro
and in vivo models, and are currently being evaluated in Phase I clinical
trials. In this study we sought to model acquired resistance to long term
BETi treatment and elucidate the underlying mechanisms in different tumor
types.
Materials and Methods: We used two orthogonal approaches to
generate cell lines with reduced phenotypic sensitivity to BET bromodomain
inhibition. In one, A375 melanoma cells were treated in culture with a
BETi at a concentration where the majority of cells were eliminated by
apoptosis. After several months, colonies able to proliferate in the presence
of the inhibitor were obtained. In parallel experiments, mice inoculated
with MV4−11 acute myeloid leukemia cells were dosed with a BETi
at its maximum tolerated dose and the surviving tumors were serially
transplanted into similarly treated recipient animals. Two BETi-resistant
clones maintained decreased sensitivity to BETi in vitro when compared to
parental cells in a 72-hour viability assay.
Results: Detailed phenotypic analysis revealed an attenuated apoptotic
response to high-dose BETi in the resistant clones as compared to parental
cells. Transcriptional profiling and proteomic analysis revealed altered
32
expression of Bcl-2 family members in both models, suggesting that two
vastly different tumor types have converged upon the apoptotic signaling
network as a mechanism of modulating the phenotypic response to BET
bromodomain inhibition. Functional experiments demonstrated that these
transcriptional changes are relevant in dictating the response to BETi.
Conclusions: As BETi advance toward clinical use, it becomes increasingly important to understand potential mechanisms of acquired resistance
and use this information to identify predictive biomarkers for maximal
therapeutic benefit in patients. We will discuss strategies for stratifying
patients based on the activity of the apoptotic signaling network, and
present data on the use of pharmacological inhibition of the apoptotic
response as a way to modify the phenotypic response to BETi.
84
POSTER (Board P078)
Src family kinase activation is a compensatory survival mechanism
for acquired resistance to EGFR-TKIs in lung cancer cells
M. Ono1 , K. Sonoda1 , K. Azuma2 , K. Watari1 , M. Molina3 , R. Rosell4 ,
M. Kuwano5 . 1 Kyushu University, Dept. of Pharmaceutical Oncology
Graduate Sch. of Pharmaceutical Sci., Fukuoka, Japan; 2 Kurume
University School of Medicine, Dept. of Internal Medicine Div. of
Respirology Neurology and Rheumatology, Kurume, Japan; 3 Pangaea
Biotech, Barcelona, Spain; 4 Catalan Institute of Oncology, Barcelona,
Spain; 5 Kyushu University, Lab. of Molecular Cancer Biology, Fukuoka,
Japan
Background: Most NSCLC patients harboring activating EGFR mutations
benefit from treatment with EGFR-TKIs, but the clinical efficacy of EGFRTKIs is limited by the appearance of tumor drug resistance. Multiple kinase
inhibitors of EGFR family proteins have been developed to overcome such
drug resistance. To develop further personalized therapeutics and drug
resistance modifiers, we should understand how drug resistance to EGFRTKIs including multiple kinase inhibitors is acquired at molecular basis.
In our present study, we present a novel finding that Src family kinase
activation is alternatively responsible for acquired drug resistance to afatinib
in lung cancer cells harboring activating EGFR mutation. Our afatinibresistant clones does not harbor T790M, Ras mutation, PTEN loss, mutant
EGFR loss, IGFIR activation, BIM and others.
Materials and Methods: We have established afatinib-resistant subclones
from human lung cancer cell lines, HCC827, harboring activating EGFR
mutations after stepwise exposure to afatinib. We have characterized
biochemical properties of drug-resistant subclones as compared with
their drug-sensitive counterparts by Western blot, RT-PCR and microarray
analysis.
Results: We characterized two independent afatinib-resistant subclones
isolated from HCC827. (1) Afatinib-resistant subclones, BR1−8 and BR2−3,
showed markedly decreased expression of EGFR, HER2, HER3, c-Met
and PDGFRb as compared with HCC827, and Akt phosphorylation was
highly resistant to afatinib; (2) the activation of Src family kinase (SFK) was
augmented in resistant subclones, and combination of afatinib with c-Src
siRNA or with dasatinib suppressed cell growth and Akt phosphorylation;
(3) among SFK, expression of Fyn was also upregulated in resistant
subclones, and treatment with Fyn siRNA with afatinib partially suppressed
Akt phosphorylation. In addition to above results, our recent relevant
study showed that activation of Src along with enhanced expression of
integrin b1, a2 and a5 was observed in erlotinib-resistant subclones derived
from PC9. And also Src silencing or SFK inhibitor (dasatinib) restored
erlotinib sensitivity through reduced activation of Akt.
Conclusions: Together, our present study strongly support the novel
finding that SFK activation including Src and/or Fyn could be one key
mechanism responsible for acquired resistance to afatinib, and also that
combination with SFK-targeted drug could be useful to overcome drug
resistance to afatinib.
85
POSTER (Board P079)
Wnt secretion is required to maintain Wnt activity in colon cancer
O. Voloshanenko1 , G. Erdmann1 , T.D. Dubash2 , I. Augustin1 ,
M. Metzig1 , C.R. Ball2 , H. Glimm2 , R. Spang3 , M. Boutros1 .
1
Deutsches Krebsforschungszentrum, Division Signaling and Functional
Genomics/B110, Heidelberg, Germany; 2 National Center for Tumor
Diseases (NCT) and German Cancer Research Center (DKFZ),
Department of Translational Oncology, Heidelberg, Germany; 3 University
of Regensburg, Computational Diagnostics Group, Regensburg, Germany
Aberrant activation of the canonical Wnt/b-catenin pathway occurs in almost
all colorectal cancers (CRCs) and contributes to their growth, invasion and
survival. It is widely believed that mutations in APC or b-catenin aberrantly
activate Wnt signalling, independent of upstream ligands. Yet, several
recent experimental findings have challenged this notion, for example
secreted inhibitory proteins of the SFRP family are still able to attenuate
Wnt signalling in colon cancer cells. Still the direct role of canonical Wnts
in CRCs has never been studied.
Surprisingly, we found that the Wnt cargo receptor Evi/Wls is highly
expressed in CRCs as well as Wnt3 protein. We show that independent of
the mutations in APC or b-catenin Wnt pathway activation is still responsive
to stimulation by Wnt secretion and receptor-proximal signalling. Silencing
of Evi leads to inhibition of clonal capacity of colon cancer cells and their
proliferation.
To identify pathways, which are synthetically lethal upon silenced Evi
we performed the genome-targeted siRNA screen upon three different
conditions − Wnt signalling high (shAPC), Wnt signalling normal for CRCs
(shCtrl) and downregulated Wnt signalling (shEvi). We could not identify
any pathways, which are synthetically lethal upon downregulation of APC
but we found several pathways, which are synthetically lethal upon silencing
of Evi. These results were confirmed by downregulation of Evi or b-catenin
by siRNA and treatment with the drugs, which inhibits identified pathways.
To conclude we showed that even in the presence of APC or b-catenin
mutations CRCs are still dependent on upstream canonical Wnt signalling
regulation. These findings potentially open new avenues for therapeutic
interventions by targeting Wnt secretion via Evi/Wls. We identified several
pathways, which are targeted by the drugs and are synthetically lethal upon
inhibition of Wnt secretion in CRCs.
86
POSTER (Board P080)
Influence of EGFR exon 19 mutation subtypes on survival outcomes
in advanced stage Asian non-small cell lung cancer patients
receiving TKI therapy
B. Chowbay1 , O. Singh1 , N. Sutiman2 , D.S.W. Tan3 , W.T. Lim3 , E.H. Tan3 .
1
National Cancer Centre, Laboratory of Clinical Pharmacology Division
of Medical Sciences, Singapore, Singapore; 2 The Academia SingHealth,
Clinical Pharmacology Core, Singapore, Singapore; 3 National Cancer
Centre, Department of Medical Oncology, Singapore, Singapore
Background: Mutations in epidermal growth factor receptor (EGFR) gene
have been shown to affect the treatment outcomes in patients. In this study
we aimed to explore the influence of EGFR exon 19 mutation subtypes
on survival outcomes in advanced stage Asian non-small cell lung cancer
(NSCLC) patients receiving tyrosine-kinase inhibitor (TKI) treatment as 1st
line therapy.
Material and Methods: Of 937 Asian NSCLC patients, 148 patients with
stage IIIB or IV, harboring EGFR exon 19 mutations and receiving TKI
as 1st line treatment, were selected. The majority of the patients were of
Chinese descent (83.1%), followed by Malays (10.8%), Indians (3.4%) and
others (2.7%). The median age of patients was 60 years (range: 29−86
years). Kaplan–Meier analysis, log-rank test and Cox regression methods
were implemented to evaluate the survival outcomes between groups
Results: The EGFR exon 19 mutation subtype 15-nucleotide deletion (c.2236_2250del, ELREA) was observed most frequently (62.8%)
followed by mixed insertion/substitution/deletion (23%), 18-nucleotide
deletion (c.2240_2257del, 7.4%) and 15-nucleotide deletion (non-ELREA,
6.8%). Patients harboring exon 19 ELREA deletion and mixed insertion/substitution/deletion had significantly longer median overall survival
(37.1 months), compared to those with c.2240_2257del [18 months;
hazard ratio, 2.5; 95% CI (1.1−5.6); p = 0.027] and non-ELREA deletion
[13 months; hazard ratio, 4.08; 95% CI (1.9−8.8); p < 0.001]. Similar trends
were observed for progression free survival (PFS) outcomes. Patients harboring exon 19 ELREA deletion and mixed insertion/substitution/deletion
had the longest median PFS (14.9 months) compared to those with
c.2240_2257del [9.13 months; hazard ratio, 2.01; 95% CI (0.91−4.4);
p = 0.08] and non-ELREA deletion [6.87 months; hazard ratio, 3.12; 95% CI
(1.39–6.98); p = 0.006].
Conclusions: EGFR exon 19 mutation subtypes showed significantly
different survival outcomes after 1st line TKI treatment in advanced stage
NSCLC patients. Further studies are recommended in larger cohort of
advanced stage NSCLC patients to improve the personalized therapy
based on patients’ EGFR mutation status.
87
POSTER (Board P081)
Critical difference in development of acquired resistance to MDM2
inhibitor SAR405838 in vitro and in vivo
G. Hoffman-Luca1 , C.Y. Yang1 , J. Lu1 , D. Ziazadeh1 , D. McEachern1 ,
L. Debussche2 , S. Wang1 . 1 University of Michigan, Pharmacology, Ann
Arbor MI, USA; 2 Sanofi, Oncology, Vitry/Seine, France
SAR405838 is a potent and specific MDM2 inhibitor currently being
evaluated in Phase I clinical trials for the treatment of human cancer as
a single agent and in combination. In the present study, we investigated
the acquired resistant mechanisms for SAR405838 using the SJSA-1
osteosarcoma cell line in vitro and in vivo. The SJSA-1 cell line possesses
an amplified MDM2 gene and wild-type p53 and is sensitive to SAR405838
and other MDM2 inhibitors in vitro and in vivo. In vitro treatment of the
SJSA-1 cells with SAR405838 using two different treatment protocols
resulted in acquired resistance to the drug. Analysis of these resistant
cells showed that p53 is mutated in the DNA binding domain and
cannot be activated by SAR405838. Treatment of mice bearing the
parental SJSA-1 xenograft tumors with SAR405838 led to rapid tumor
regression, but tumors eventually returned after treatment cessation. A
number of cell lines were established by culturing the regrown tumors
and, surprisingly, these sublines showed minimal loss of sensitivity to
SAR405838 as compared to the parental SJSA-1 cell line. Consistently,
analysis of thesesublines showed that p53 retains its wild-type status, with
the exception of one subline, that harbored a single heterozygous mutation
C176F. Computational modeling suggested that the p53 C176F mutant
may still be capable of binding to target DNA to elicit gene transcription,
which was confirmed by in vitro treatment with SAR405838. Xenografts
of representative sublines lacking p53 C176F mutation obtained from
regrown tumors treated with one-round SAR405838 were still responsive
to SAR405838 treatment. Interestingly, after a second round of in vivo
treatment with SAR405838, all sublines established from harvested tumors
uniformly harbored a heterozygous C176F p53 mutation, which were
still responsive to SAR405838, albeit with reduced sensitivity. Our study
showed that the SJSA-1 tumor cells acquired profound resistance to MDM2
inhibitor SAR405838 in vitro and developed inactivating p53 mutations in
its DNA binding domain. But in vivo, SJSA-1 tumor cells only acquired
minimal or modest resistance when treated with either one- or two-rounds
of SAR405838. Our study suggests that tumor cells respond differently to
drug treatment under in vitro and in vivo environments and consequently
develop different types of resistance.
88
POSTER (Board P082)
Genome-wide drug sensitivity screens in haploid mouse embryonic
stem cells
S.J. Pettitt1 , D. Krastev1 , H. Pemberton1 , Y. Fontebasso1 , I. Bajrami1 ,
I. Kozarewa1 , J. Frankum1 , R. Rafiq1 , J. Campbell1 , R. Brough1 ,
A. Ashworth1 , C.J. Lord1 . 1 Institute of Cancer Research, Division of
Breast Cancer, London, United Kingdom
Background: We have screened a genome wide collection of mutants
induced by transposon insertion in haploid cells with a variety of targeted
agents and chemotherapies in order to find new genetic determinants of
sensitivity.
Materials and Methods: Haploid mouse embryonic stem cells were
mutagenised with a piggyBac gene trap transposon. To enable accurate
counting of mutants, a random 25-nt barcode was inserted into the
transposon donor plasmid. This can be amplified in a minimal PCR step
and the products analysed by next-generation sequencing, in a manner
similar to pooled shRNA screens. Barcodes were associated with genomic
insertion sites by inverse PCR. We generated a library of approximately
100,000 mutants and treated with DMSO (solvent control) or drug at SF50
dose. The mappings for barcodes showing significant change in abundance
between treated and control arms were analysed and hits called based on
the criteria of multiple insertions in a gene showing a phenotype.
Results: In a screen for poly(ADP-ribose) polymerase (PARP) inhibitor
sensitivity using the clinical agents olaparib and BMN 673, we detected
known determinants of sensitivity including Brca2 and Cdk12. We identified
loss of Ewsr1 as a novel determinant of PARP inhibitor sensitivity and
showed that knockdown of EWSR1 in MCF7 breast cancer cells also
sensitises to BMN 673. Since Ewing’s sarcoma cells with EWSR1-ETS
fusions are reported to be PARP inhibitor sensitive, we are investigating a
possible contribution of loss of wild type EWS function to this effect.
We have also screened this library against 25 other drugs and will report
our latest results.
Conclusions: Pooled haploid mutant libraries can be used to identify
genetic determinants of sensitivity at a genome-wide scale without prior
assumptions of the mechanism.
89
POSTER (Board P083)
Tie-2 regulates the stemness of prostate cancer cells
K. Tang1 , M. Ling1 . 1 Institute of Health and Biomedical Innovation,
Brisbane Qld, Australia
Background: Prostate cancer (PCa) is the most commonly diagnosed
male cancer in Western countries. Patients with advanced PCa are
normally treated with hormone ablation therapy. This therapy is effective
initially as PCa cells require androgen to grow and survive; however,
the cancer cells eventually become androgen independent and develop
metastatic, castration-resistant tumors. At this stage, chemotherapy and
33
radiotherapy exhibit only small benefits. Ample evidence supports the idea
that a rare population of cancer cells known as cancer stem cells (CSCs) is
responsible for the development of treatment resistant and disease relapse.
Unfortunately, little is known about the identity of these cells, making it
difficult to target them. Here we reported that a rare population of PCa
cells expresses the Tie-2 protein, a tyrosine kinase receptor that regulates
the stemness and bone homing of hematopoietic stem cells.
Material and Methods: Here, using fluorescence-activated cell sorting
(FACS), we have isolated the Tie-2+ population from the PCa cell line
PC-3. cDNA microarray analysis was then performed to characterize the
gene expression profile of these cells. Meanwhile, cell adhesion assay
was performed to examine the ability of the Tie-2+ cells to adhere to
osteoblasts and endothelial cells. Furthermore, quiescent staining and drug
sensitivity assay was carried out to examine whether Tie-2+ cells are more
quiescent, and thus become resistant to chemotherapeutic drug. Finally,
we have injected both Tie-2+ and Tie-2− PC-3 cells intracardiacly into the
NOD-SCID mice to determine if Tie-2 expression promote prostate tumor
metastasis under in vivo condition.
Results: Characterization of Tie-2+ PCa cells revealed that these cells
express higher level of prostate CSC markers when compared to the Tie-2−
population. Meanwhile, Tie-2+ cells are highly adhesive to osteoblasts
and endothelial cells, a characteristic necessary for the development of
tumor metastasis. We also found that Tie-2+ cells are more quiescent and
resistant to the chemotherapeutic drug cabazitaxel, further support that
these cells possess CSC-like characteristics. More importantly, we found
that Tie-2+ cells, but not the Tie-2− cells population, developed metastatic
tumor in vivo.
Conclusions: Our data suggested that Tie-2+ population represent
the PCa stem cells population which play an importantly role in the
development of drug resistance and prostate tumor metastasis. Thus, Tie-2
might be a novel therapeutic target for treatment of advanced PCa patients.
90
POSTER (Board P084)
Tumor infiltrating leukocyte subpopulations as a biomarker of
response and resistance to targeted therapy in patients with BRAF
mutation-positive metastatic melanoma
M.C. Kelley1 , D.B. Doxie2 , A.R. Greenplate2 , H. Crandall1 , J.A. Sosman3 ,
J.M. Irish2 . 1 Vanderbilt University, Surgical Oncology, Nashville TN, USA;
2
Vanderbilt University, Cancer Biology, Nashville TN, USA; 3 Vanderbilt
University, Hematology-Oncology, Nashville TN, USA
BRAF mutation-positive metastatic melanoma responds to BRAF and MEK
targeted therapy, then progresses within 6−9 months. Many resistance
mechanisms have been identified in tumor cells, but little is known
about changes ocurring in the tumor microenvironment during resistance.
Dysfunctional immune responses are linked to tumor progression, so we
evaluated the tumor infiltrating leukocyte profile in melanoma metastases
responding and progressing on targeted therapy.
Samples were obtained during surgical excision of soft tissue metastases
in a patient with BRAF V600E mutation-positive melanoma receiving
therapy with a BRAF inhibitor (dabrafenib) and MEK inhibitor (trametinib).
Responding lesions were defined by: stable reduction in tumor volume
during therapy and the physical characteristics of the lesion. Novel high
dimensional mass cytometry (CyToF) panels were used to characterize
tumor and immune cell subsets. Machine learning tools (visNE) classified
individual cells according to a multidimensional signature of 30+ proteins
with roles in melanoma biology, immune regulation, and metastasis. Key
biomarkers of melanoma and immune cell subsets included melanoma
cell adhesion molecule (MCAM), NGFR, KIT, nestin, CD3, CD4, CD8,
CD14, CD16, CD33, CD34, CD44, CD45, CD49F, MHC class II (HLA-DR),
and chemokine receptors (CCRs & CXCRs) known to play a role in
metastasis. Intracellular signaling (pERK, pAKT, pSTAT-1, 3, 5 and 6,
pCREB), proliferation (Ki67) and apoptosis (cleaved caspease3) were also
evaluated. Live cells were identified by length and DNA content and gated
into melanoma cell (CD45−, MCAM+) and leukocyte (CD45+, MCAM−)
populations. The abundance and phenotype of subpopulations of immune
cells were compared in responding and progressing lesions.
Responding lesions contained nearly 3 fold more T-cells than progressing
lesions, and 63% of these cells were CD8+ effector T cells compared
to 14% in progressing lesions. In contrast, progressing lesions contained
almost 3 fold more MMHC-II expressing cells, including a nearly 2 fold
increase in CD14−, CD33+, HLA-DR+ myeloid-derived suppressor cells
(Table 1). In depth analysis of leukocyte subpopulations in additional
melanoma metastases responding and progressing on therapy is underway.
These preliminary findings confirm that maladaptive immune responses,
including reduced CD8+ T-cell and increased myeloid subpopulations, are
present in melanoma metastases progressing on BRAF targeted therapy.
It is unclear if these changes occur in response to resistance developing
within the tumor or are directly responsible for tumor progression. Further
investigation of combinations of targeted therapy and immunotherapy
34
are supported by these findings. Analysis of tumor infiltrating leukocyte
subpopulations may be a useful biomarker for monitoring response to
targeted therapy.
Table 1.
% CD45+ cells
% MHC+ cells
% CD3+ cells
CD3+/MHC-II− CD3−/MHC-II+ CD14− CD14−/CD33+ CD8+
Responding 74*
Progressing 27*
26*
73*
41
49
3.8
6.2
63*
14*
*p < 0.05.
91
POSTER (Board P085)
4E-BP1 expression levels determine sensitivity of triple negative
breast cancer cells to mTOR inhibitors
K. Jastrzebski1 , B. Thijssen1 , J. Vidal Rodriguez1 , K. de Lint1 , C. Lieftink1 ,
L.F. Wessels1 , R.L. Beijersbergen1 . 1 NKI-AvL, Molecular Carcinogenesis,
Amsterdam, Netherlands
Background: In contrast to other breast cancer subtypes, triple negative
breast cancers are characterized by an absence of recurrent genetic
alterations. However, alterations in the PI3K pathway are frequently
observed, either through mutation or amplification of PIK3CA or the loss
of the negative regulator PTEN. As a result, inhibitors of this pathway,
including inhibitors of mTOR, the downstream target of the PI3K pathway,
have been explored in the clinic. It has been found that mutational activation
of PI3K represents an important determinant of sensitivity to mTOR
inhibitors. Nevertheless, a number of PI3K wild-type cell lines also show
sensitivity to these inhibitors, although the regulators of this response are
still unknown. We set out to find such modulators of response to mTOR
inhibitors in a panel of triple negative breast cancer cell lines initially using
high throughput approaches.
Material and Methods: We determined the sensitivity of a panel of
triple negative breast cancer cell lines to AZD8055 (mTOR inhibitor) and
BEZ235 (mTOR/PI3K dual inhibitor). This data was then used together
with additional datasets generated for this panel, including RNA expression
(by RNA-seq), (phospho-)protein levels (by Reverse Phase Protein Arrays)
and mutational status (by post-capture DNA-seq), to carry out regression
analysis to identify features correlated with response.
Results: We found that the protein expression levels of 4E-BP1, a
downstream target of mTOR involved in regulating cap-dependent protein
synthesis, correlated with response to both inhibitors. In poorly responding
cell lines expressing low levels of 4E-BP1, overexpression of the protein
increased sensitivity. Conversely, in sensitive cell lines expressing high
levels of 4E-BP1, knock-down of 4E-BP1 led to reduced sensitivity to mTOR
inhibition.
Conclusions: These results indicate that effective inhibition of protein
synthesis downstream of PI3K/mTOR is an important determinant of mTOR
inhibitor activity. Given that approximately 20% of breast cancer tumors
annotated in The Cancer Genome Atlas show an amplification of the
4E-BP1 locus, such patients would likely benefit from mTOR inhibitor
treatment. Furthermore, we hypothesize that mTOR inhibitor response can
be improved even in patients expressing low 4E-BP1 by co-treating with
agents that induce its expression. To this end, we are currently screening
for genes and drugs that modulate 4E-BP1 levels.
92
POSTER (Board P086)
Role of ERK nuclear translocation in cisplatin-sensitive and
-resistant ovarian cancer cells
S. Dilruba1 , G.V. Kalayda1 , U. Jaehde1 . 1 Institute of Pharmacy, Clinical
Pharmacy, Bonn, Germany
Background: The clinical use of the anti-cancer drug cisplatin is limited
due to the rapid development of resistance against this drug. However, the
mechanisms of resistance are not completely understood yet. Extracellular
signal regulated kinase1/2 (ERK1/2) is activated in response to cisplatin
treatment in ovarian cancer cells. Activated ERK1/2 induces the expression
of survival genes upon translocation to nucleus, which may confer
resistance to this drug.
Objectives: This projects aims at investigating the importance of ERK1/2
nuclear translocation for cisplatin resistance in ovarian cancer cells.
Methods: EFO27 and A2780 ovarian carcinoma cells and their corresponding cisplatin-resistant variants (EFO27/CDDP and A2780cis)
were investigated. The cytotoxicity of cisplatin was estimated using
the MTT assay. Phosphorylated ERK1/2 translocation to the nucleus
was confirmed by immunofluorescence staining. Selected proteins were
overexpressed by lipofectamine-mediated transfection. Phosphorylation
of ERK1 (T202/Y204) and ERK2 (T185/Y187) and transfected protein
expression were detected by Western blot.
Results: Cytotoxicity of cisplatin was assessed with and without inhibition
of ERK1/2 by U0126. Whereas U0126 increased cisplatin sensitivity of
EFO27 and EFO27/CDDP cells, the opposite effect was found in A2780
and A2780cis cells. Translocation of ERK1/2 to the nucleus in response to
cisplatin was detected in both EFO27 cells, but not in A2780 cells. Further,
PEA-15, a small protein which inhibits ERK1/2 translocation to the nucleus
was used to investigate the relevance of ERK1/2 cytoplasmic sequestration
for cisplatin resistance. PEA-15 was overexpressed in A2780cis cells
and the cytotoxicity of cisplatin in transfected cells was measured.
The pEC50 values obtained were 5.15±0.21, 5.04±0.15, 5.00±0.09 in
A2780cis, A2780cis-HA (empty vector), and A2780cis-PEA-15wt (n = 4−6)
respectively.
Conclusion: ERK1/2 nuclear translocation differs between the ovarian
cancer cell lines. Further studies will reveal the most suitable cell line pair to
study the association between ERK1/2 nuclear translocation and cisplatin
resistance.
93
POSTER (Board P087)
A stress induced early innate response causes multi-drug tolerance
in melanoma
D.R. Menon1 , S. Das2 , C. Krepler3 , A. Vultur3 , B. Rinner4 , S. Schauer2 ,
K. Kashofer2 , K. Wagner4 , G. Zhang3 , E. Bonyadi Rad5 , H.P. Soyer1 ,
B. Gabrielli6 , R. Somasundaram3 , G. Hoefler2 , M. Herlyn3 , H. Schaider1 .
1
Translational Research Institute, Dermatology Research Centre,
Woolloongabba, Australia; 2 Medical University of Graz, Institute of
Pathology, Graz, Austria; 3 The Wistar Institute, Molecular Oncogenesis,
Philadelphia, USA; 4 Medical University of Graz, Center for Medical
Research, Graz, Austria; 5 Medical University of Graz, Dermatology,
Graz, Austria; 6 Translational Research Institute, Diamantina Institute,
Woolloongabba, Australia
Background: Acquired drug resistance constitutes a major challenge
for effective cancer therapies with melanoma being no exception. The
dynamics of early drug resistance leading to permanent resistance are
poorly understood.
Material and Methods: Melanoma cell lines were exposed to molecular
targeted inhibitors like BRAF or MEK inhibitors or chemotherapy at
sublethal drug concentrations for over 90 days. Alternatively melanoma
cells were exposed to hypoxic conditions or low glucose media. Cells
surviving drug exposure, hypoxia or nutrient starvation were monitored for
the expression of CD271, ALDH activity, differentiation markers, ABCB5,
chromatin remodeling, histone demethylases and markers for angiogenesis
to characterize cells exposed for a minimum of 12 days. Further gene
expression analyses, RPPA analyses and in vivo tumorigenicity were
performed in these cells.
Results: Drug exposure, hypoxia or nutrient starvation leads to an
early innate cell response in melanoma cells resulting in multi-drug
resistance, termed induced drug tolerant cells (IDTC). Transition into the
IDTC state seems to be an inherent stress reaction for survival towards
unfavorable environmental conditions or drug exposure independent of any
subpopulation or cancer stem cell. The response comprises chromatin
remodeling, activation of signaling cascades, and markers proposed to
be stem cell markers with higher angiogenic potential and tumorigenicity.
These changes are characterized by a common increase in CD271
expression concomitantly with loss of differentiation markers such as
melan-A and tyrosinase, enhanced ALDH activity and upregulation of
histone demethylases. Accordingly, IDTCs show a loss of H3K4me3,
H3K27me3 and gain of H3K9me3 suggesting activation and repression of
differential genes. Drug holidays at the IDTC state allow for reversion into
parental cells re-sensitizing them to the drug they were primarily exposed
to. However, upon continuous drug exposure IDTCs eventually transform
into permanent and irreversible drug resistant cells. Knockdown of CD271
or KDM5B decreases transition into the IDTC state substantially but does
not prevent it.
Conclusions: Our results suggest a phenotypic shift of parental cells to the
induced drug tolerant cell (IDTC) state irrespective of a given subpopulation
thus not representing cancer stem cells. Targeting IDTCs would be crucial
for sustainable disease management and prevention of acquired drug
resistance.
Poster Session – Drug Screening
94
POSTER (Board P088)
Elucidating mechanisms of resistance to FGFR inhibitors in
endometrial cancer
L. Packer1 , S. Byron2 , C. Mahon1 , D. Loch3 , A. Wortmann1 , K. Nones4 ,
S. Grimmond4 , J. Pearson5 , N. Waddell4 , P. Pollock1 . 1 Translational
Research Institute, Queensland University of Technology, Brisbane Qld,
Australia; 2 Translational Genomics Research Institute, Phoenix, USA;
3
Queensland University of Technology, Brisbane Qld, Australia; 4 Institute
of Molecular Biology, University of Queensland, Brisbane Qld, Australia;
5
QIMR Berghofer Medical Research Institute, Brisbane Qld, Australia
Background: Fibroblast Growth Factor Receptor 2 (FGFR2) is mutated
in 10−20% of endometrial cancer patients and is associated with a
higher risk of recurrence. Preclinical studies by our lab and others have
demonstrated FGFR2 inhibition is a viable therapeutic strategy in FGFR2mutant endometrial cancer. A significant clinical issue that we aim to
address is that of acquired resistance to anti-FGFR therapies in the context
of endometrial cancer.
Materials and Methods: The endometrial cancer cell lines AN3CA and
JHUEM-2 express mutant FGFR2 and are sensitive to FGFR inhibitors.
We have generated subclones of AN3CA and JHUEM-2 cells resistant to
the FGFR inhibitors PD173074 and NVP-BGJ398, respectively. Phosphoprotein arrays, gene expression profiling, exome sequencing and SNP
analysis were performed to identify genes and signalling pathways that
mediate resistance to FGFR inhibitors.
Results: Loss of FGFR2 protein expression was identified in one
resistant subclone, but the remaining subclones retain FGFR2 expression.
Upregulation of downstream survival pathways such as pERK and pAKT
was observed in some of the JHUEM-2resistant and AN3CAresistant subclones.
To further characterise these subclones we are currently analysing data
from exome sequencing, expression arrays and copy number analysis,
to determine the molecular mechanisms of resistance in these cell lines.
Like most endometrial cancer cells, AN3CA and JHUEM-2 cells have
constitutive activation of the PI3K pathway, due to mutations in PIK3R1,
PTEN or PIK3CA. Our results show that combined inhibition of the PI3K
pathway and the MEK/ERK pathway by trametinib or an FGFR inhibitor
causes cell death in these cells.
Conclusions: This research will provide insight into possible combination
therapies that may combat drug resistance in FGFR-mutant endometrial
cancer.
Drug Screening
95
POSTER (Board P089)
Identification of synthetic lethality compounds from natural products
for cancers
K.W. Ng1 , K.S. Lee1 , V. Patel2 , E. Sundaramoorthy3 , N. Ayoub3 , X. Su3 ,
A. Venkitaraman3 , S.H. Teo4 . 1 Cancer Research Initiatives Foundation,
Drug Discovery Research Group, Selangor, Malaysia; 2 Cancer Research
Initiatives Foundation, Nasopharyngeal Cancer Research Group,
Selangor, Malaysia; 3 University of Cambridge, The Medical Research
Council Cancer Cell Unit, Cambridge, United Kingdom; 4 Cancer Research
Initiatives Foundation, Breast Cancer Research Group, Selangor, Malaysia
Background: Many cancers, such as basal breast and serous ovarian
cancers, are characterized by genomic instability, through the loss of
function of TP53 and BRCA2. Until recently, there have been limited
successes in developing efficient targeted therapies for these forms of
solid cancers. In 2005, two research groups independently demonstrated
that inhibition of Poly-(ADP-ribose)-polymerase (PARP) can selectively
kill BRCA2-deficient cancer cells through synthetic lethality. However, no
study has hitherto explored natural products that are synthetic lethal to
the BRCA2-deficient cancers. Here, we describe the development of a
cell-based assay to search for natural products with synthetic lethality
properties.
Methods: We have developed a cell-based assay using an isogenic pair of
BRCA2-proficient and deficient cell lines. The sensitivity of the assay was
evaluated using Olaparib, Mitomycin C and Irinotecan, either in isolation
or in combination as ‘spike-in’ controls with natural product extracts, to
investigate their inhibitory activities in the presence of potential interfering
compounds. The assay was then used to screen for our in-house natural
product library of plant, microbial and fungus extracts.
Results: We report that this assay format is able to detect the selective
cytotoxic effects of Mitomycin C, Olaparib and Irinotecan with 22, 17 and
16 fold difference of EC50 in BRCA proficient cells compared to that of
BRCA deficient cells. When added as spike-in controls with natural product
35
extracts, the detection limits were 1% (w/w) for Olaparib and Irinotecan and
0.1% (w/w) for Mitomycin C. Screening of our in-house library of 454 plant,
305 microbial and 62 fungus extracts, identified 2 plants, 1microbial, 6
fungus extracts with EC50 less than 5 mg/mL on the BRCA2 deficient cells
and fold difference of >6.
Conclusion: Our data represents the first systematic identification of
potential synthetic lethal natural products using a cell based assay. We
anticipate from our on-going work to unravel and identify the key chemical
components of the 9 extracts conferring synthetic lethality properties.
96
POSTER (Board P090)
Evaluation of hormone therapies in a panel of breast PDX models:
Relevance of ER status on sensitivity to letrozole and tamoxifen
M.J. Wick1 , T. Vaught1 , L. Gamez1 , J. Meade1 , A. Diaz1 ,
K.P. Papadopoulos1 , D.W. Rasco1 , A. Patnaik1 , M. Beeram2 , A. Lang2 ,
A.W. Tolcher1 . 1 South Texas Accelerated Research Therapeutics,
(START), San Antonio TX, USA; 2 START Center for Cancer Care, San
Antonio TX, USA
Background: Breast cancer classification and treatment strategy is
currently based on immunohistochemical staining and pathological scoring
for the estrogen (ER), progesterone (PR) and ERBB2 (HER2) receptors.
Based on threshold ER scores, breast cancers are designated hormonereceptor positive and treated with endocrine therapy including ER antagonists and aromatase inhibitors (AI). However, whether breast cancers
with ER scores below threshold would benefit from endocrine therapy is
unclear. To better understand the role of endocrine therapy in breast cancer,
we stratified a panel of breast PDX models using ER percentage (ER%):
ER<10% and ER10%. Drug sensitivity studies were performed evaluating
models response to letrozole and tamoxifen and efficacy compared to the
ER% for each model.
Methods: Breast PDX models were developed in immune-deficient mice
from primary or metastatic patient tissue and established models confirmed
by histologic comparative analysis and linked with patient treatment and
outcome data. Each model was assigned an ER% from the clinical biopsy
score at the time of sample collection and engraftment. Drug sensitivity
studies were performed evaluating models response to chronic dosing of
letrozole or tamoxifen; study endpoints included tumor volume and time
from treatment initiation as study endpoints. T/C values, growth delay and
regressions were reported for each model.
Results: Letrozole treatment resulted in statistically significant (p < 0.05)
tumor growth inhibition in several ER<10% models including ST518
and ST1077 (ER = 0%) while several ER 10% models including ST565
(ER = 100%), developed from a chemo naı̈ve patient, was insensitive to
therapy. Letrozole tested in HER2 (3+) models was inactive in ST1339
(ER = 0%) model but efficacious in ST340 (ER = 20%). Activity of tamoxifen
was similar to letrozole in evaluated models.
Conclusion: We evaluated a panel of breast patient-derived xenografts
and compared model sensitivity to letrozole and tamoxifen with ER% in
each model. Overall sensitivity of either agent did not correlate with higher
ER% values, suggesting patients with ER% below threshold may benefit
from these therapies in combination with novel targeted agents or approved
standards of care.
97
POSTER (Board P091)
Utilization of low passage adenoid cystic carcinoma PDX models to
identify novel combination therapies
M.J. Wick1 , J. Meade1 , T. Vaught1 , M. Nehls1 , J. Flores1 , J. Kaufman2 ,
A.W. Tolcher1 , D.W. Rasco1 , A. Patnaik1 , C.A. Moskaluk3 ,
K.P. Papadopoulos1 . 1 South Texas Accelerated Research Therapeutics,
(START), San Antonio TX, USA; 2 Adenoid Cystic Carcinoma Research
Foundation, (ACCRF), Needham MA, USA; 3 University of Virginia,
Charlottesville VA, USA
Background: Adenoid Cystic Carcinoma (ACC) is an uncommon cancer
of the head and neck which typically originates in the salivary glands
with limited treatment options and once metastatic no approved standard
of care. To identify potentially useful therapies we utilize a panel of low
passage ACC xenograft models for in vivo screening of FDA-approved
and investigational therapies with potential benefit towards ACC. Based
on these results, combination treatments are evaluated looking for additive
or synergistic effects. Previously we reported activity of the multi-tyrosine
kinase inhibitor dovitinib in models of ACC. However, whether combination
with other targeted therapies would result in additive or synergistic activity
was unclear. To test this we evaluated dovitinib in combination with two
classes of agents with anecdotal clinical activity, an HDAC inhibitor and
IGF-1R inhibitor; for these studies we utilized vorinostat and linsitinib. RNA
and DNA-based sequence analysis was also performed on the ACC panel
models to correlate sensitivity or resistance with molecular markers.
36
Methods: Low passage ACC models were established in immune-deficient
mice from primary or metastatic patient tissue and once established were
confirmed by histologic comparative analysis. Drug sensitivity studies were
performed evaluating dovitinib, vorinostat and linsitinib. Study endpoints
included tumor volume and time from treatment initiation with tumor growth
inhibition, delay and regression reported at study completion.
Results: To date over eighty FDA-approved and investigational therapies
have been evaluated. In the current study additive effects were reported
in dovitinib/vorinostat and dovitinib/linsitinib combination groups compared
with single agents. Statistically significant (p < 0.05) tumor growth inhibition
was reported in two models comparing the combination to single agents
using a submaximal dovitinib concentration. Sequence based analysis is
currently underway to correlate sensitivity and resistance to these and other
therapies.
Conclusion: Low passage ACC models have been used to identify agents
potentially useful in the treatment of ACC. Currently we have identified
preclinical combination benefit with dovitinib/vorinostat and dovitinib/
linsitinib combinations and RNA and DNA sequence based analysis is
currently underway to correlate sensitivity and resistance to these and other
therapies.
mechanisms of action produced supra-additive activity with the same
compounds in the ‘Combo Set’. Examples of these interactions included:
(1) ‘Test Agents’ inhibiting DNA damage repair checkpoints (p53, ATR, or
GSK3-b) that all produced similar patterns of combination activity when
combined with ‘Combo Set’ alkylating agents, and to a lesser extent with
Topoisomerase poisons; (2) Wee 1 and Chk1 inhibitors that demonstrated
optimal therapeutic benefit when combined with gemcitabine; this result
was striking because gemcitabine, a member of the ‘Combo Set’, was an
infrequent combination hit with other ‘Test Agents’; (3) EGFR inhibitors,
when combined with dacarbazine, were all supra-additive in a single cell
line: PC-3.
We also found multiple antagonistic interactions, including: (1) Src inhibitors
were antagonistic with ATRA and oxaliplatin in all three tested cell lines;
(2) HDAC inhibitors were antagonistic with many antimetabolites in the
‘Combo Set‘.
These results may aid in selection of combinations for further preclinical
investigation, leading to clinical studies.
98
POSTER (Board P092)
Synergistic inhibition of HER2 positive breast cancer by triptolide
and lapatinib
J. Sakoff1 , J. Gilbert2 , A. McCluskey3 . 1 Calvary Mater Newcastle,
Medical Oncology, Newcastle NSW, Australia; 2 Calvary Mater Newcastle
Hospital, Medical Oncology, Newcastle NSW, Australia; 3 The University
of Newcastle, Chemistry, Newcastle NSW, Australia
P. Chalugun1 , J.S. Shim1 , P. Korangath1 , S. Sukumar1 , J.O. Liu1 . 1 Johns
Hopkins University, Pharmacology, Baltimore MD, USA
HER2 positive breast cancers are poorly differentiated and have a higher
potential for metastasis. Although trastuzumab and lapatinib have been
used for the treatment of HER2 positive breast cancer, resistance of both
drugs has dampened the long-term benefit of those drugs. As cancer has
multiple genetic aberrations and can readily develop resistance to individual
drugs with specific targets, we employed synergistic drug combination as
a strategy to overcome drug resistance and to enhance to efficacy of
existing drugs. From a screen of the Johns Hopkins Drug Library, we
identified triptolide as a strong synergistic hit with lapatnib in HCC1954,
a drug-resistant HER2-positive breast cancer cell line. The combination
of lapatinib and non-toxic doses of triptolide synergistically inhibited the
HER2 signaling pathway. We found that lapatinib increased both mRNA
expression and the half-life of HER2 protein, whereas triptolide countered
the effects of lapatinib by downregulating the expression of HER2, leading
to the synergistic inhibition of HER2 signaling pathway. The synergy in
cell proliferation was observed in all HER2-positive breast cancer cell lines
regardless of drug resistance status, but was absent in HER2-negative
ones. In mouse xenograft models, the combination of lapatinib and triptolide
at their minimum effective doses almost completely inhibited the growth
of the drug-resistant HER2-positive breast cancer cells. Together, these
findings suggest that the combination of triptolide and lapatinib could be a
promising new therapeutic regimen for drug-resistant HER2-positive breast
cencer.
99
POSTER (Board P093)
Combination screening of investigational oncology agents
S. Holbeck1 , J.M. Collins2 , J.D. Doroshow3 . 1 National Cancer Institute,
Rockville, USA; 2 National Cancer Institute, DCTD, Rockville, USA;
3
National Cancer Institute, DCTD, Bethesda, USA
Treatment with combinations of drugs forms the backbone of most
cancer treatment regimens. With the advent of newer therapies targeting
specific drivers or molecular defects present in tumor cells come new
opportunities and challenges in developing drug combinations. Many
molecularly targeted drugs elicit profound responses in patients as single
agents, but it is common for resistance to arise in a matter of months.
There is thus an urgent need for strategies to develop combinations
that incorporate these new agents. We have previously reported on our
systematic approach to the screening of all pair-wise combinations of
approved small molecule oncology drugs (~5000 drug pairs) in a panel of
60 human tumor cell lines, the NCI-60. Our combination screening has now
been expanded to include ~9000 drug pairs, including many investigational
agents, initially screened against a small panel of cell lines (3−5 lines). With
some overlap between the 2 screens, a total of 12,660 unique drug pairs
has been tested.
In the current screen, we established a ‘Combo Set’ panel of ~70 approved
and investigational drugs. Agents of interest (‘Test Agents’) were then
evaluated in combination with each of the drugs in the ‘Combo Set’ in
each of 3−5 cell lines. Additional testing in the NCI-60, and/or xenografts,
was performed for 55 combinations with greater than additive activity in the
smaller cell line panel.
By examining multiple representatives of a mechanistic class as ‘Test
Agents’, we identified interactions wherein ‘Test Agents’ with similar
100
POSTER (Board P094)
Small molecules selectively targeting breast cancer cells
Breast cancer is one of the most common cancers among women. Despite
advances in therapy, drug resistance is often induced, tumour selectivity is
poor and drug off-targets culminate in limited efficacy. Indeed metastatic
advanced breast cancer is deemed incurable. Better, more effective
treatments for this disease are clearly needed. We have discovered a
structurally distinct class of small molecules that target breast cancer cell
lines while having little to no effect on normal breast cancer cells or on cell
lines derived from other tumour types including colon, ovarian, lung, skin,
prostate and pancreatic carcinomas, neuroblastoma and glioblastoma.
Indeed these molecules show more than 500-fold selectivity towards breast
cancer cells compared with other tumour types, while maintaining nM
potency, as determined by the MTT growth inhibition (GI50 ) assay with
GI50 values of 0.1−0.7uM (72 h exposure). Moreover, the sensitive breast
cancer cell lines represent tumour types from the four main breast cancer
classifications including ER+ luminal A (MCF-7, T47-D and ZR-75−1 cells),
ER+ luminal B (BT-474), HER2+ (SKBR-3), and most importantly the Basal
(triple negative MDA-MB-468 cells) classification which traditionally carries
a very poor prognosis. Our novel class of molecules also retain activity in
MCF-7/VP16 cells (GI50 0.2uM) which overexpresses the drug resistance
ABCC1 gene. Only one breast cancer cell line has shown insensitivity,
MDA-MB-231 (Basal triple negative), which unlike all of the other breast
cell types has amplifying mutations in KRas and BRaf activity, a genotype
found in less than 5% of breast cancer tumours. Furthermore, our novel
compounds induce minimal effects on the growth of normal MCF10A breast
cells. Cell cycle analysis in the most sensitive breast cancer cell line, MDAMB-468, shows an increase in the proportion of cells in the S-phase of
the cell cycle within 24 h of exposure, concomitant with a decrease in the
proportion of cells in the G1 -phase, and preceding an increase in the subG1 cell death population. The ability to specifically target breast tumours,
while having little or no effect on normal breast cells, or other tumour types
is a unique finding. Elucidating the mechanism controlling this phenomenon
is now the focus of our research efforts.
101
POSTER (Board P095)
PIK3CA mutation-targeting compounds analyses using NCI60 cell
line panel
H. Bando1 , J. Lih2 , E.C. Polley3 , S.L. Holbeck4 , B. Das2 , D. Sims2 ,
T. Doi5 , A. Ohtsu5 , M. Williams2 , N. Takebe1 . 1 National Cancer Institute
National Institute of Health, Cancer Therapy Evaluation Program
Division of Cancer Treatment and Diagnosis, Rockville, USA; 2 National
Cancer Institute National Institute of Health, The Frederick National
Laboratory for Cancer Research, Frederick, USA; 3 National Cancer
Institute National Institute of Health, Biometric Research Branch
Division of Cancer Treatment and Diagnosis, Rockville, USA; 4 National
Cancer Institute National Institute of Health, Developmental Therapeutics
Program Division of Cancer Treatment and Diagnosis, Rockville, USA;
5
National Cancer Center Hospital East, Department of Gastroenterology
and Gastrointestinal Oncology, Kashiwa, Japan
Background: The US National Cancer Institute 60 (NCI-60) human tumor
cell line panel was developed as an in vitro drug-discovery tool, and it
is a unique publicly accessible platform of various datasets. Through the
CellMiner website, we could access genomic datasets of NCI-60. We could
also obtain the activities of various test compounds to the NCI-60 cell
line panel from NCI Developmental Therapeutics Program (DTP) website.
Using this capability, we screened for therapeutic agents against the cell
lines with PIK3CA mutation, the second most frequently mutated actionable
oncogene among various types of tumors.
Material and Methods: Utilizing NCI-60 cell line GI50 (50% growthinhibitory levels) data of PI3K/AKT/mTOR pathway compounds, we
calculated the differences in mean log GI50 between the PIK3CA mutated
cell lines and non-mutated cell lines (delta log GI50) to investigate
promising compounds. The volcano plot (the x-axis depicts delta log GI50
and the y-axis depicts statistical significance level) was used to search for
the promising test compounds.
Results: Seven cell lines had PIK3CA mutation (3 p.E545K, 3 p.H1047R,
and 1 p.R38C mutation). We could collect GI50 data on 35
PI3K/AKT/mTOR targeting compounds (7 pan-class PI3K inhibitors,
7 isoform-selective PI3K inhibitors, 5 rapalogs, 3 mTOR kinase inhibitors,
6 panPI3K/mTOR inhibitors, and 7 AKT inhibitors). In our analysis, 1 panclass PI3K inhibitor, 2 isoform-selective PI3K inhibitors, 1 rapalog and 1
AKT inhibitor showed promising trends by both delta log GI50 and statistical
significance.
Conclusions: NCI-60 cell line panel may be a valuable tool to identify
various actionable mutation–drug relationships and to discover promising
drug candidates.
102
POSTER (Board P096)
Comparison of platinum/taxane and anthracycline-based therapies
in ovarian PDX models: Correlating stage of biopsy collection and
engrafting with in vivo drug sensitivity
J. Meade1 , M.J. Wick1 , T. Vaught1 , L. Gamez1 , M. Farley1 , A. Moriarty1 ,
A.W. Tolcher1 , A. Patnaik1 , D.W. Rasco1 , A.J. White2 , K.P. Papadopoulos1 .
1
South Texas Accelerated Research Therapeutics, (START), San Antonio
TX, USA; 2 START Center for Cancer Care, San Antonio TX, USA
Background: Standard first-line treatment for ovarian cancer is a platinumbased therapy often including a taxane, with various salvage therapies
for recurrent disease. We and others have established and validated
low passage ovary patient-derived xenograft (PDX) models from biopsies
obtained at diagnosis and following clinical treatment progression; however,
whether drug sensitivity to each model correlates to the clinical context
was unclear. To test this we stratified thirty-nine ovary PDX models using
clinical outcome data at the time of biopsy collection into four groups:
chemo naı̈ve, first-line platinum responding, first-line platinum refractory
and anthracycline pretreated. Drug sensitivity studies were performed,
evaluating model response to platinum, platinum plus taxane and liposomal
doxorubicin and results compared between each group.
Methods: Ovary PDX models were developed inimmune-deficient mice
from primary or metastatic patient tissue and established models confirmed
by histologic comparative analysis and linked with patient treatment and
outcome data. Drug sensitivity studies were performed evaluating models
response to three regimens with tumor volume and time from treatment
initiation as study endpoints. T/C values, growth delay and regressions
were reported for each model.
Results: For the thirty-nine models evaluated: 18/39 were from chemonaı̈ve patients, 6/39 from first-line platinum responding, 10/39 from firstline platinum refractory and 5/39 from anthracycline pretreated patients. In
these studies chemo-naı̈ve and first-line platinum responding models were
sensitive to the platinum based regimens with few exceptions including
ST036 which harbors known AKT and KRAS mutations. Most first-line
platinum refractory models including ST419 and ST884 were resistant to
platinum alone and platinum/taxane combination. Interestingly sensitivity to
liposomal doxorubicin did not correlate with clinical pretreatment as several
models from chemo-naı̈ve patients including ST024 and ST511 were
insensitive while some models established from anthracycline pretreated
patients including ST255B and ST467 were sensitive to the therapy.
Conclusion: We have established a panel of ovary patient-derived
xenografts and correlated model sensitivity to platinum and anthracyclinebased treatments to the clinical context at the time of biopsy collection and
engraftment. Overall platinum sensitivity in models from chemo-naı̈ve and
first-line platinum responding patients, with few exceptions, correlated with
clinical response, irrespective of activating mutations. Preclinical response
to anthracyclines was less predictive and not predicated on the clinical
context.
37
103
POSTER (Board P097)
The use of next generation sequencing (NGS) in the management of
metastatic breast cancer (MBC): Defining a model for genomic-driven
therapies
L. Austin1 , M. Gooptu1 , T. Avery1 , R. Jaslow1 , J. Palazzo2 ,
M. Cristofanilli1 . 1 Thomas Jefferson University Hospital, Medical
Oncology, Philadelphia PA, USA; 2 Thomas Jefferson University Hospital,
Pathology, Philadelphia PA, USA
Background: Metastatic breast cancer is an incurable disease treated
with palliative intent. The limited benefit from standard therapies could
be related to the fact that the disease is biologically heterogeneous and
not properly managed by current treatments. Increased understanding of
the molecular complexity using next generation sequencing (NGS) allows
for individual tumor mutation analysis which provides better selection of
treatments based on genomic abnormalities driving the disease.
Methods: We retrospectively analyzed 33 patients with advanced or
metastatic breast cancer treated at Thomas Jefferson University, diagnosed
between 2003–2013, on whom next generation sequencing (NGS) by
Foundation One™ was sent for genomic analysis. Immediately actionable
mutations for this analysis were considered those for which FDA-approved
agents for breast cancer were identified. We also reported potentially
actionable mutations which were defined as mutations with investigationaltherapeutic agents.
Results: The majority of patients (88%) had inflammatory breast cancer
(IBC). According to disease subtype, 36% were ER+/HER− (Luminal A),
18% ER+/HER+ (Luminal B), 15% ER−/HER+ (HER2+), and 30% were
ER−/HER− (triple negative). NGS genomic analysis revealed a total of
78 unique mutations with a total of 178 mutational events. The five
most commonly mutated genes in order of frequency were TP53 (55%
of patients), MYC amplification (39%), PIK3CA mutations (36%), ERBB2
amplification (30%) and PTEN loss or mutation (15%). Eleven of 78
mutations (14%) were immediately actionable (based on our definition)
and comprised of PIK3CA mutations/amplifications, ERBB2 amplifications,
AKT mutations and PTEN loss/mutations. In total, 21 of 33 patients
(64%) had at least one immediately actionable mutation and 97% (32)
of patients had either an immediately actionable or potentially actionable
mutation. The most common potentially actionable mutations were TP53,
MYC amplification, FGFR1 amplification and MDM2 amplification. All
but one of the HER2+ patients (IHC/FISH) had ERBB2 amplification
on NGS. Interestingly, of the 10 patients that had ERBB2 amplification,
70% had a concomitant PIK3CA mutation and all had IBC. Two patients
with ERBB2 amplification and PIK3CA mutations who had progressed
on trastuzumab ((Herceptin® ) combinations were started on everolimus
(Afinitor® ), Trastuzumab and vinorelbine (Navelbine).
Conclusion: In conclusion, in this cohort of 33 patients, the Foundation
One™ panel detected unique mutations of which 14% were immediately
actionable and 97% of patients had at least one mutation that was
immediately or potentially actionable. Actionable mutations were detected
across all receptor groups and there was a high incidence on concomitant
ERBB2 amplification and PIK3CA mutation. The Foundation One™ panel
is a useful tool which can be utilized for tailoring targeted therapy for breast
cancer patients. This warrants further investigation with a prospective trial
to evaluate the clinical impact of this added information and its effect on
decision making and clinical outcomes.
104
POSTER (Board P098)
Identification of inhibitors of tryptophan metabolizing enzymes for
cancer immunotherapy by high-throughput screening
G. Zaman1 , J.C.M. Uitdehaag1 , S. van Gerwen1 , N. Seegers1 ,
A.M. van Doornmalen1 , J. de Man1 , R.C. Buijsman1 . 1 Netherlands
Translational Research Center B.V., Oss, Netherlands
Background: The amino acid tryptophan is an important regulator of the
immune system by regulating the activation of T cells. Tumor cells express
enzymes that oxidize tryptophan, thereby dampening the local T cell
immune response against cancer cells. These enzymes are indoleamine
2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO). Despite
the substantial evidence of the importance of IDO1 and TDO as small
molecule drug targets, only a handful of different chemical scaffolds have
been reported. The lack of chemical matter is explained by the lack of robust
assays for high-throughput screening. We have developed new assays for
IDO1 and TDO, which we have used to screen a compound library, and to
characterize the selectivity of reference inhibitors.
Materials and Methods: NFK Green™ is a chemical probe that specifically
reacts with N-formyl kynurine, the reaction product of the enzymatic
conversion of tryptophan by IDO1 and TDO. The reaction is quantified by
measuring fluorescence on a 384-well plate multimode reader. Biochemical
assays were developed for IDO1 and TDO using recombinantly expressed
38
proteins, and a library of 87,000 diverse lead-like molecules was screened.
NFK Green™ was also used to determine the tryptophan metabolizing
activity in a collection of human cancer cell lines and was related to the
expression levels of IDO1 and TDO by western blot analysis.
Results: Biochemical and cell-based screening assays were developed
for IDO1 and TDO using a new fluorescent read-out. High-throughput
screening of libraries of small chemical compound libraries yielded novel
selective inhibitors of either IDO1 or TDO. Side-by-side comparison of
published reference compounds revealed significant, previously unnoted
cross-reactivity of a widely used hydroxyamidine-based inhibitor of IDO1
(Compound 5l) with TDO. The selectivity of other reference IDO1 or TDO
inhibitors was confirmed, leading to definition of a new tool compound set.
Biochemical selectivity of compounds correlated with inhibition of cellular
tryptophan metabolizing activity and expression of IDO1 or TDO.
Conclusions: We have developed new biochemical and cell-based assays
for IDO1 and TDO, to enable the identification of novel small molecule
inhibitors and to support lead optimization. Side-by-side comparison of
published inhibitors revealed novel, unanticipated cross-reactivity of IDO1
inhibitor scaffolds with TDO.
105
POSTER (Board P099)
A platform to test multiple therapy options simultaneously in a
patient’s own tumor
N. Caffo1 , R. Klinghoffer1 . 1 Presage Biosciences Inc., Seattle, USA
While investigational cancer drugs must ultimately be validated in clinical
trials, most early drug discovery is performed under in vitro conditions in
cell-based models that poorly represent the disease they are intended to
represent. To enable in vivo analysis of anti-cancer agent efficacy at earlier
stages of drug development, and to potentially enable toxicity-sparing
assessment of novel agents in the oncology clinic, we have developed
a technology platform called CIVOTM . CIVOTM allows for simultaneous
assessment of up to eight drugs or drug combinations in a single solid
tumor. Controlled microinjection-based delivery of doxorubicin, docetaxel,
mafosfamide, and gemcitabine both as single agents and combinations was
tested in the canine sarcoma clinic. Drugs were co-injected in a columnar
array with UV fluorescent beads resulting in easy-to-identify bands of drug,
each at a distinct position of the patient’s tumor. Tumors were resected 72 h
following microinjection and were examined for responses using standard
histology methods. The CIVO introduced drug microdoses induced spatially
defined, graded, and mechanism-specific cellular changes around sites
of drug exposure in a patient-specific manner. Consistent with the use
of doxorubicin as first line therapy in the soft tissue sarcoma clinic, the
frequency and extent of response of localized responses to doxorubicin
exceeded those of all other agents tested. This preclinical data, along with
early responses observed in the human clinic, set the stage for clinical
application of this technology to identify which novel agents are likely to
succeed or fail in subsequent clinical trials.
Immunotherapy (Immunecheckpoints,
Vaccination, Oncolytic viruses,
Cytokines)
106
POSTER (Board P100)
Beta-3 integrin inhibition reduces inflammatory cytokine release but
not anti-cancer activity of oncolytic adenovirus in ovarian cancer
A.K. Browne1 , L.A. Tookman1 , C.K. Ingemarsdotter1 , R. Bouwman1 ,
K. Pirlo1 , Y. Wang1 , K.M. Hodivala-Dilke2 , I.A. McNeish3 , M. Lockley1 .
1
Barts Cancer Institute, Centre For Molecular Oncology, London, United
Kingdom; 2 Barts Cancer Institute, Centre For Tumour Biology, London,
United Kingdom; 3 University of Glasgow, Institute of Cancer Services,
Glasgow, United Kingdom
Background/Introduction: The potential of oncolytic adenoviruses as
anti-cancer therapy has repeatedly been demonstrated. A consistent and
worrying feature of adenoviral gene therapy is the rapid cytokine release
that occurs after viral administration. These cytokines give rise to dose
limiting inflammatory toxicities, which can be severe and have hindered further investigation and clinical development of these promising anti-cancer
therapies. Using primary ovarian cancer cells and genetically modified
mice, we show that the E1A-CR2 deleted replicating oncolytic adenovirus,
dl922–947, induces cytokines via beta-3 integrin in macrophage-rich
tissues. We present new evidence that co-administration of a Cilengitidelike integrin inhibitor controls the inflammatory cytokines and hepatic toxicity
Poster Session – Immunotherapy
induced by dl922–947 in tumour bearing mice. Importantly, although alphav/beta-3integrin functions as a secondary adenoviral receptor, we found no
evidence that beta-3 inhibition compromised viral infectivity and oncolysis
in vitro or anti-cancer efficacy in vivo.
Material and Methods: We quantified production of cytokine mRNA (qRTPCR) and protein (Mesoscale Discovery System) following dl922–947 in
ovarian cancer cells lines, tumour cells harvested from the ascites of
women with ovarian cancer and in murine tissues, peritoneal cells and
serum. Murine models included nude mice bearing intraperitoneal ovarian
cancer xenografts and non-tumour bearing, immunocompetent beta-3 null
mice. Pharmacological inhibition of beta-3 integrin was achieved using
H2574, a cyclic RGD mimetic inhibitor of alpha-v/beta-3andalpha-v/beta-5
integrins.
Results: Primary and established ovarian cancer cell lines in vitro
did not release inflammatory cytokines in response to adenovirus. In
contrast, intraperitoneal delivery of dl922–947 caused rapid, systemic
cytokine induction in ovarian cancer xenografts. Cytokines originated
predominantly in macrophage-rich murine tissues (liver, spleen and
peritoneal macrophages), rather than the injected malignant cells, and
was independent of viral replication. Adenoviruses are known to induce
cytokine release via beta-3 integrin-expressing macrophages. We found
that co-administration of the integrin inhibitor, H2574, controlled production
of inflammatory cytokines in the circulation of tumour-bearing mice. Cotreatment also reduced pathological features of viral hepatic toxicity such
as eosinophilic degeneration and liver enzyme elevation. Importantly,
combining dl922–947 with H2574 did not compromise anti-cancer activity
in vitro or in vivo.
Conclusions: Combining oncolytic adenoviruses with pharmacological
inhibition of beta-3 integrin enables safe systemic delivery of replicating
adenoviruses, without compromising anti-cancer activity. This novel
approach could have a major impact on the future development of these
effective anti-cancer agents.
107
POSTER (Board P101)
CIGB-247: Anti-VEGF therapeutic vaccine in patients with advanced
solid tumors
F. Hernández-Bernal1 , J.V. Gavilondo2 , M. Ayala Ávila2 , A.V. de la Torre3 ,
J. de la Torre4 , K.H. Selman-Housein4 , Y. Morera2 , M. Bequet-Romero2 ,
C.M. Valenzuela1 , Y. Martin5 . 1 Center for Genetic Engineering and
Biotechnology (CIGB), Clinical Trials, Havana City, Cuba; 2 Center for
Genetic Engineering and Biotechnology (CIGB), Pharmaceuticals, Havana
City, Cuba; 3 “Celestino Hernández” Hospital, Oncology, Santa Clara,
Cuba; 4 Medical and Surgical Research (CIMEQ), Oncology, Havana City,
Cuba; 5 “Celestino Hernández” Hospital, Oncology, Havana City, Cuba
Background: The CIGB-247 is a vaccine preparation for the therapy
of cancer that combines the recombinant antigen p64K-hVEGFKDR− ,
produced in Escherichia coli and the VSSP adjuvant. This antigen is a
representative molecule of the human Vascular Endothelium Growth Factor
(VEGF-A), in its isoform 121. Vaccination with CIGB-247 in experimental
animal models has been safe, with anti-tumor and anti-metastatic effects.
Material and Methods: A non-controlled, dose up scaling phase I clinical
trial was performed in patients with advanced solid tumors, which previously
received the best onco-specific treatment available without response. The
general objective of the trial was to study the safety profile of the vaccine
at three antigen dose levels. A maximum of 10 patients per group were
planned. Individuals were subcutaneously immunized for 8 consecutive
weeks with 50, 100 or 400 mg of antigen (all in the same amount of
VSSP adjuvant), and re-immunized on week 12. On week 16, evaluations
of safety, tolerance, clinical status, and immunogenicity (seroconversion
for anti-VEGF IgG, serum VEGF/KDR-Fc blocking ability, and gamma-IFN
ELISPOT with blood cells stimulated in vitro with mutated VEGF) were
done.
Results: Vaccination was shown to be safe at the three dose levels, with
only grade 1−2 adverse events. CIGB-247 was immunogenic and higher
numbers of individuals positive to the three immune response tests were
seen with increasing antigen dose.
Conclusions: This is the first clinical testing report of a cancer therapeutic
vaccine based on a human VEGF related molecule as antigen. The CIGB247 vaccine is safe, immunogenic, and merits further clinical development.
108
POSTER (Board P102)
CD70 (TNFSF7), a receptor involved in acute immune modulation of
viral infection, is frequently overexpressed in solid and hematological
malignancies
J. Jacobs1 , K. Zwaenepoel1 , P. Aftimos2 , C. Rolfo3 , S. Rottey4 ,
L. Ysebrant de Lendonck2 , K. Silence5 , A. Awada2 , A. Thibault5 ,
P. Pauwels1 . 1 Universitair Ziekenhuis Antwerpen, Pathology, Antwerp,
Belgium; 2 Institut Jules Bordet, Oncology, Brussels, Belgium; 3 Universitair
Ziekenhuis Antwerpen, Oncology, Antwerp, Belgium; 4 Ghent University
Hospital, Oncology, Ghent, Belgium; 5 arGEN-X BVBA, Research,
Zwijnaarde, Belgium
Tissue expression of CD70 (TNFSF7), a receptor of the TNF superfamily
normally involved in transient, rapid-onset immune response, is highly
restricted to small subsets of T-, B-, and dendritic cells. It is often chronically
over-expressed in patients with hematological and solid malignancies in
whom it mediates tumor cell growth and immune escape. To date, the
incidence of CD70 positivity has not been systematically characterized in
patients with advanced malignancies.
We developed an immunohistochemistry (IHC) method to detect CD70 on
paraffin-embedded tumor biopsies in support of a Phase 1 trial of ARGX110, a monoclonal IgG1 SIMPLE Antibody™ endowed with enhanced
ADCC properties (POTELLIGENT® ). A total of 182 samples (120 solid
tumors and 62 hematological malignancies) were selected at random from
a university hospital tissue bank and tested for CD70 expression.
Samples were considered positive for CD70 (cut-off: 10% cells staining)
in 33% and 58% of cases, respectively. As of March 2014, CD70
positivity has also been documented in 147 of 296 patients (50%) who
underwent IHC screening as an eligibility requirement for study participation
(ClinicalTrials.gov Identifier: NCT01813539). EBV-induced malignancies
(e.g.: Hodgkin’s and Burkitt’s lymphoma, nasopharyngeal carcinoma)
consistently overexpressed CD70. A high incidence of CD70 positivity
was observed in T- and B-cell lymphomas (64%), as well as renal cell
carcinoma (90%) and esophageal cancer (64%). Consistent with the
intracellular trafficking of CD70, staining patterns in malignant cells included
membranous, cytoplasmic and perinuclear distribution.
In conclusion, the expression of CD70 is commonly increased in a wide
variety of solid and hematological malignancies.
109
POSTER (Board P103)
Functional activity, but not PD-1 expression level, differentiates
primary CLL from healthy PD1+ T cells using SCNP
S. Liang1 , L. Leung1 , S. Putta1 , D. Hotson1 , D. Rosen1 , R.E. Hawtin1 .
Nodality Inc., Research, So San Francisco CA, USA
1
Background: Drugs inhibiting PD-1 signaling have demonstrated clinical
efficacy in the treatment of cancer. To increase understanding of PD-1+
T cell biology, PD-1 expression was profiled across CD4/CD8 T cell subsets
in peripheral blood mononuclear cells (PBMC) of CLL and healthy donors
(HD), and the functional responsiveness of these PD-1+/− subsets to
cytokine modulation or T cell receptor (TCR) modulation +/− PD-1 blockade
was interrogated.
Materials and Methods: Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry based technology that enables simultaneous
analysis of signaling networks in multiple immune cell subsets. CLL (n = 9)
and HD (n = 8) PBMC were profiled by SCNP to interrogate CD4/CD8 +/−
T cell subsets for PD-1 expression, cell subset specific signaling following
modulation with IL-2, IL-7, IL-15, IL-21 or TCR (a-CD3/a-CD28), and
proliferation following TCR modulation +/− PD-1 blockade. CLL and HD
data were compared to identify dysfunctional signaling associated with
disease and PD-1 expression.
Results: CLL and HD did not differ significantly in median PD-1 expression,
although CLL samples were more heterogeneous. Significant differences
were detected in signaling capacity in CLL vs HD, particularly in PD-1+ CD8
T cells. Unmodulated p-STAT5 was elevated in CLL in PD-1+ and PD-1−
CD8 T cells and in PD-1+ CD4−/CD8− T cells. IL-2, IL-7 and IL-15 induced
lower p-STAT5 levels in PD-1+ CD8 T cells and PD-1+ CD4−/CD8− T cells
of CLL donors, indicating dysfunctional signaling through these common
g-chain cytokines in these PD-1+ subsets. In contrast to reduced cytokine
responsiveness, increased TCR modulation of p-ERK was observed in
PD-1+ CD8 T cells in CLL vs HD. Conversely, CLL T cells demonstrated
decreased proliferation in response to TCR modulation, which was partially
reversed with PD-1 blockade.
Conclusions: The dysregulated cytokine signaling, elevated TCR responsiveness and reduced proliferation observed in CD8, but not CD4, PD-1+
T cell subsets in CLL is consistent with the reported ‘pseudo-exhausted
state’.
These data establish the application of SCNP to interrogate PD-1+
T cell subset signaling and the functional consequences of tumor–
39
microenvironment interactions. Building upon and verifying these data,
SCNP will be applied to both developing predictive and prognostic markers
of response in hematologic and solid tumor indications, and interrogating
the efficacy of therapeutics to restore T cell function.
110
POSTER (Board P104)
IL-6/STAT3/Fra-1 signaling axis promotes colorectal cancer
aggressiveness through epithelial–mesenchymal transition
J. Shao1 , H. Liu1 . 1 Zhejiang University School of Medicine, Pathology
and Pathophysiology, Hangzhou City, China
Fos-related antigen-1 (Fra-1), a member of the Fos family, is aberrantly
expressed in several types of human cancer, but its functional roles in
cancer development and the regulatory mechanisms are still not well understood. In this study, we investigated the significance of Fra-1 expression
in human colorectal cancer (CRC). Fra-1 levels were positively correlated
with the local invasion depth as well as lymph node and liver metastasis in a
total of 229 CRC patients. Intense immunohistochemical staining of Fra-1
was observed at the tumor invasive front adjacent to inflammatory cells
and in parallel with secretion of the pro-inflammatory cytokine interleukin-6
(IL-6) in the CRC tissues. Exogenous IL-6 administration to CRC cell
lines significantly upregulated Fra-1 expression in a manner dependent
on signal transducer and activator of transcription 3 (STAT3), during
which both phosphorylated and acetylated post-translational modifications
were required for STAT3 activation to promote the transcription of Fra-1
gene by directly binding to its promoter. Importantly, Fra-1 upregulated by
IL-6/STAT3 signaling endowed the CRC cells with properties of epithelial–
mesenchymal transition (EMT), an essential step for tumor progression
and metastasis. RNA interference-based attenuation of STAT3 or Fra-1
prevented IL-6-induced EMT, cell migration and invasion, whereas ectopic
expression of Fra-1 markedly reversed the inhibitory effect of STAT3knockdown on the EMT process. Collectively, this study uncovered the
existence of an aberrant IL-6/STAT3/Fra-1 signaling axis leading to EMT
and aggressiveness in colorectal cancer and suggested novel therapeutic
opportunities for the treatment of malignant disease.
111
POSTER (Board P105)
Identification of peptides which could block PD-1 checkpoint for
NSCLC immunotherapy
Y. Zhu1 , C. Li1 . 1 Suzhou Institute of Nano-tech and Nano-bionics CAS,
Nanobiomedicine, Suzhou, China
Background: Non-small cell lung cancer (NSCLC) has been reported
responding to programmed death-1 (PD-1) immune checkpoint blockade,
thus inhibitory peptides blocking PD-1/programmed death ligand-1 (PDL-1)
pathway may contribute the effective therapy to NSCLC patients.
Materials and Methods: X15 bacteria peptide library was used to
screening and selecting binding peptides to PDL-1 and PD-1. Focused
library was designed and constructed according to the peptide screening
results from X15 random bacteria peptide library. Affinity and specificity
of binding peptides for PDL-1 and PD-1 were examined with methods
of SPR and competition experiments. In vitro assessment of peptides for
blocking the PD-1/PDL-1 pathway was performed with MDA-MB-231 cell
lines. Metastatic mice model of A549 cells were constructed with nude
mice after 2 months since A549 cells were injected into the lung position
of mice. The evaluation of peptides for their anti-tumor effect was done
via measuring and comparing tumor sizes and life spans of mice injected
either with binding peptides or scramble peptides. Immunohistology were
performed with anti-CD31 antibody and blood of mice was collected for
circulating tumor cells detection.
Results:
1. Mice model with metastatic lung cancer was established;
2. Peptides binding with PDL-1 were obtained from random X15 library;
3. Conserved sequences for peptides binding with PDL-1 were identified
and their physiochemical properties were examined roughly;
4. Focused library was established according to the conserved binding
peptide sequences;
5. Peptides interacted with PDL-1 could block proliferation of MDA-MB-231;
6. Life span of tumor mice got elongated and tumor size was decreased
after mice injected with binding peptides;
7. The amount of CTC was alleviated and CD-31 positive signals got
decreased in binding peptides-treated mice group.
Conclusion: Peptides binding to PDL-1 could behave as potential drugs
for metastatic lung cancer therapy since they can block signal pathway
initiated by PD-1/PDL-1 interaction, inhibit angiogenesis and metastasis of
tumor cells.
40
112
POSTER (Board P106)
A novel primary human tumor explant platform provides a preclinical
translational link from tissue culture to the clinic
G. Juan1 , K. Paweletz1 , E. Trueblood2 , J. Rossi1 , M. Damore1 ,
A. Anderson1 , R. Loberg1 . 1 Amgen, Medical Sciences, Thousand Oaks
CA, USA; 2 Amgen, Pathology, Seattle WA, USA
Background: To explore a candidate immunotherapeutic (BiTE® ), a
translational explant platform was developed consisting of cultures of thinly
sectioned tissues from freshly resected solid tumors. The tumor explants
are being used to study treatment responses in a model system that
preserves the tumor architecture and microenvironment, including immune
effector populations.
Material and Methods: 18 freshly resected epithelial tumors were sectioned and treated in culture for 48 or 72 h with increasing concentrations
(0, 5, 50 and 500 pM) of an EGFR-BiTE® or a MEC14-BiTE® (negative
control). The culture supernatant was then collected to quantitate cytokines
released as a measure of T-cell activation using Myriad’s CytokineMAP®
A v1.0 panel. CD25 and CD3 enumeration to demonstrate in situ T-cell
activation was performed by IHC in FFPE tissues derived from the
treated explants. In addition, the parental tumors were profiled by IHC to
characterize the EGFR target levels together with the initial tumor T-cell
infiltrate. Finally, gene expression profiles of the parental tumors were
generated to explore potential resistance mechanisms.
Results: Tumor resident T-cells are activated ex vivo by BiTE® . A dose
dependent increase of several cytokines including IFN-g, IL-2 and TNF-a
was observed in conditioned media harvested from EGFR-BiTE® treated
but not in MEC14-BiTE® treated explants. In addition, a dose dependent
increase of granzyme B into media as measured by ELISA was observed.
Importantly, a dose dependent increase of total CD25 counts and the
%CD3CD25 is observed in the EGFR-BiTE® explants vs. those treated
with the control BiTE® .
EGFR-BiTE®
(N = 18)
0 pM
IFN-g (pg/mL)
IL-2 (pg/mL)
TNF-a (pg/mL)
%CD3CD25
5.6±0.9
6.8±0.7
24.4±4.2
28.5±15
5 pM
28.9±9.8
55.9±23
43.6±7.3
41.2±17.7
50 pM
101±37
114.4±43.5
88±23.6
57.9±26.2
mode of action, its activity in selected pre-clinical models, and to identify
pharmacodynamic biomarkers for response monitoring. In vitro functional
tests using lymphocytes from various species were conducted. A series
of in vivo syngenic mouse tumor models were selected based on
immune checkpoint ligand expression. Immune-related biomarkers and
drug concentrations were assessed from circulating blood and tumor
samples from those models.
Results: A first-in-class therapeutic peptide, designated W014A (formerly
AUR-012), was characterized. W014A is a 29-amino acid branched peptide
designed as a PD-1 decoy using selected portions of the human PD-1
receptor. W014A displayed equipotent antagonism towards PD-L1 and PDL2-mediated T cell exhaustion. Robust activity in rescue of proliferation
and effector functions (IFN-g secretion) from mouse, non-human primate
and human lymphocytes was demonstrated. W014A also prevented the
interaction of PD-L1 with B7.1. A complete rescue of CD4+ and CD8+
T cells and suppression of regulatory T cells were observed using proliferation assays of immune cells stimulated with anti-CD3/anti-CD-28. Different
in vivo models, including melanoma, colon, breast and kidney cancers
(B16, CT26, 4T1 and Renca) demonstrated the potent effects of W014A
on both primary tumor growth and metastasis. Active drug concentration
levels in tumour (Renca model), as well as intratumoral recruitment (CT26
model) of CD4+ and CD8+ T cells, and a reduction in PD-1+ T cells (both
CD4+ & CD8+ ) were observed concomitantly with antitumoral activity.
Conclusions: Altogether, the unique mechanism of action and preclinical
activity of W014A support the set-up of clinical trials in the near future
and suggest potential pharmacodynamic biomarkers for use in the clinic.
The synergy with other treatments that enhance endogenous antitumor
immunity will also be investigated.
114
POSTER (Board P108)
Toll-like receptor 5 agonist entolimod as a potential anticancer
immunotherapeutic agent
L. Burdelya1 , C. Brackett1 , B. Kojouharov1 , J. Veith1 , A. Gudkov1 .
1
Roswell Park Cancer Institute, Cell Stress Biology, Buffalo NY, USA
500 pM
455.3±118.8
605.8±207.7
272.9±61.8
94.9±51.3
Conclusions: Infiltrating T cells can be activated by BiTE® molecules, as
demonstrated by cytokine secretion and T-cell activation in the explant
culture system. Using a novel preclinical ex vivo model of human primary
tumors, the explants provide mechanistic insights into the modulation of
the tumor immunoresponse upon administration of a BiTE® .
113
POSTER (Board P107)
Preclinical activity and pharmacodynamic biomarkers of W014A, a
PD-1 decoy peptide blocking both PD-1 immune checkpoint ligands,
PD-L1 and PD-L2
C. Bailly1 , M. Broussas2 , M. Ramachandra3 , P.G. Sasikumar3 ,
K. Shrimali3 , S. Adurthi3 , M. Ramachandra3 , L.K. Satyam3 ,
A.A. Dhudashia3 , S. Dhodheri3 , K.B. Sunilkumar3 , N. Corvaı̈a4 , P. Ferre5 .
1
Institut de Recherche Pierre Fabre, Head of Research IRPF, Toulouse
Cedex 1, France; 2 Institut de Recherche Pierre Fabre, Experimental
Oncology Department, Saint-Julien en Genevios, France; 3 Aurigene
Discovery Technologies Ltd, Bangalore, India; 4 Institut de Recherche
Pierre Fabre, Saint-Julien en Genevois, France; 5 Institut de Recherche
Pierre Fabre, Toulouse, France
Background: Cancer immunotherapy is starting to change the way cancer
is treated. The goal is to re-activate patient’s immune system to specifically
destroy tumor cells. Recent clinical trials in various settings have confirmed
that several monoclonal antibodies blocking either the programmed cell
death-1 (PD-1) receptor on infiltrating T cells, or its ligand PD-L1 re-activate
tumor-specific T cell cytotoxicity, and induce impressive and durable
clinical responses. However, the immune evasion of tumor cells cannot
be restricted to the sole interaction between PD-1 and PD-L1. PD-1 also
interacts with a higher binding affinity with PD-L2, and PD-L1 is capable of
binding to B7.1 (CD80) on the T cell surface. Similarly as PD-1/PD-L1, both
PD-1/PD-L2 and PD-L1/B7.1 interactions result in deactivation of T cells.
Antagonizing those 3 interactions might induce a more robust anti-tumor
activity.
Materials and Methods: A novel drug candidate was designed to
antagonize both PD-L1 and PD-L2. Our goal was to characterize its
Mobilization of innate immunity by Toll-like receptor (TLR) agonists and
the subsequent development of adaptive immunity have been considered
as an attractive approach to cancer immunotherapy. Unfortunately, the
majority of TLR agonists induce prohibitive acute inflammatory responses
limiting their clinical use. In this regard, TLR5 stands alone since the
TLR5 agonist, bacterial flagellin, induces much less severe systemic
inflammation than agonists of other TLRs. Entolimod is a flagellin derivative
and a clinical stage experimental drug initially developed as a candidate
radiation antidote capable of protection from and mitigation of radiation
injury in mice and non-human primates. We found that entolimod, in
addition to its radioprotective properties, had anticancer effects in a variety
of TLR5-positive tumors in mice. We hypothesized that entolimod can
induce antitumor defense if tumors are located in a TLR5-responsive
microenvironment such as the liver independently of the TLR5 status of
tumor cells. This hypothesis was tested in three syngeneic spontaneous
and experimental liver metastasis models: CT26 and MC38 colorectal
and 4T1 breast adenocarcinomas. The results demonstrate that entolimod
treatment was efficient in suppressing metastatic disease when injected
systemically either before or after surgical removal of primary tumors in all
tested mouse models of metastatic colon, breast, and melanoma cancers.
This effect was mediated by mobilization of immune effectors to the liver, the
major primary target organ for TLR5 agonists, and did not depend on the
TLR5 status of the tumors. Rapid and massive recruitment of neutrophils,
NK, CD4/CD8 T cells and other types of immune cells to the liver
was followed by eradication of dormant tumor cells and micrometastases
and development of protective T cell memory. Importantly, entolimod has
passed through two Phase I clinical trials in 150 healthy volunteers and
is currently undergoing a Phase I trial in cancer patients with advanced
solid tumor liver metastases with the expectation to be used in adjuvant or
neoadjuvant settings for prevention of metastatic disease.
115
POSTER (Board P109)
Anti-metastatic activity via co-blockade of PD-1 and adenosine A2A
receptor
D. Mittal1 , A. Young1 , K. Stannard1 , M.W. Teng1 , B. Allard2 , J. Stagg2 ,
M.J. Smyth3 . 1 QIMR Berghofer Medical Research Institute, Immunology
in Cancer and Infection, Brisabane, Australia; 2 Faculté de Pharmacie
et Institut du Cancer de Montréal Montréal Québec Canada, Centre de
Recherche du Centre Hospitalier de l’Université de Montréal, Montreal,
Canada; 3 QIMR Berghofer Medical Research Institute, Immunology
in Cancer and Infection, Brisbane, Australia
Adenosine targeting is an attractive new approach to cancer treatment, but
no clinical study has yet examined adenosine inhibition in oncology despite
the safe clinical profile of adenosine A2A receptor inhibitors (A2ARi) in
Parkinson’s disease. Monoclonal antibodies (mAb) that block programmed
death (PD)-1 or cytotoxic T lymphocyte antigen (CTLA-4) receptors have
been associated with durable clinical responses against a variety of cancer
types and hold great potential as novel cancer therapeutics. Metastasis
is the main cause of cancer related deaths worldwide, and therefore we
have studied experimental and spontaneous mouse models of melanoma
and breast cancer metastasis to demonstrate the efficacy and mechanism
of a combination of A2A receptor inhibitor in combination with various
immune check point inhibitors. The combination of anti-PD-1 and A2A
receptor inhibitor significantly reduces metastatic burden and prolongs the
life of mice compared with either monotherapy alone. Importantly, the
combination was only effective when the tumor expressed high levels of
CD73, suggesting a tumor biomarker that at a minimum could be used to
stratify patients that might receive this combination. The mechanism of the
combination therapy was critically dependent on NK cells and interferon
gamma, and to a lesser extent, CD8+ T cells and the effector molecule,
perforin. Consistent with the anti-metastatic role of NK cells, we observed
significantly high number of NK cells in the lungs of tumor bearing mice after
combination immunotherapy. Overall, our preclinical data provide a strong
rationale to use A2ARi with anti-PD-1 mAb for the treatment of minimal
residual and metastatic disease.
116
POSTER (Board P110)
Novel targets for antibody–drug conjugate therapy
A.G. Grandi1 , S.C. Campagnoli1 , M.P. Parri1 , E.D.C. De Camilli2 , B.J. Jin3 ,
P.S. Sarmientos1 , G.G. Grandi4 , L.T. Terracciano5 , P.P. Pileri1 , G.V. Viale2 ,
R. Grifantini6 . 1 Externautics, R&D, Siena, Italy; 2 European Institute of
Oncology, Pathology, Milan, Italy; 3 Fourth Military University, Immunology,
XI’an, China; 4 Novartis Vaccines, R&D, Siena, Italy; 5 Basel Medical
University, Pathology, Basel, Switzerland; 6 Externautics, Siena, Italy
The study focuses on two novel potential therapeutic targets identified
by a systematic immune-histochemistry (IHC) screening with a large
collection of polyclonal antibodies (approximately 1600) raised against
marginally characterized human proteins. Here we describe the molecular
characterization of two surface-associated proteins (EXN36 and EXN91)
associated to different cancer types. EXN36 is mainly over-expressed
in ovary and breast cancers (frequency of approximately 30−40%).
Interestingly, it is also over-expressed in triple negative breast cancer.
The protein is involved in cell proliferation, migration and invasiveness.
Concerning EXN91, it is an adhesion molecule and it acts as a signaling
receptor, likely to be important in developmental processes and cell
communication. The protein is mainly detected in colon cancer with high
frequency (more than 80%), both in early and advanced stages, in high
and low grade cancers. Interestingly, EXN91 is over-expressed in KRAS
and BRAF mutant colon cancers with significant frequency (approximately
50%). Finally, it is also detected in esophagous SCC and ccRCC (10−20%).
Murine monoclonal antibodies able to recognize EXN36 and EXN91 on the
surface of cancer cells have been selected and characterized to assess
their potential for specific therapeutic indications. In particular, five antiEXN36 mAbs are able to recognize the target protein in breast (Her2+ ,Er+
and triple negative cells) and ovary cell lines. A murine monoclonal
antibody is able to recognize EXN91 on the surface of colon cancer cells.
This antibody also shows the ability to inhibit growth of colon cancer in
xenograft mouse models. Finally, it specifically binds cancer tissues by
IHC, suggesting that it could be also developed as companion diagnostic
tool for EXN91-based therapies.
EXN36 and EXN91 monoclonal antibodies show a high number of
binding sites on the cancer cell surface, ranging from 10,000 to 100,000
sites per cells. The antibody specificity has been confirmed in different
immunoassays (Western blot, FACS, IHC) by gene silencing experiments
and/or competition with peptides containing the antibody epitopes. Thes
eantibodies have high affinity for their target epitopes (KD : 10−9 ,10−10 nM).
Moreover, they show limited IHC reactivity in normal tissues (FDA tissue
panel).
Some antibodies are efficiently internalized by cancer cells, suggesting
that they can be exploited for the development of Antibody–Drug Conjugate
(ADC). The analysis of their potential for ADC is ongoing. Initial results from
in vitro studies show that these antibodies, indirectly linked to commercially
available drugs (e.g. auristatin-based, DM1, and duocarmycin), show
significant anti-tumor activity with specific drugs and linker chemistry.
Overall, the results indicate that EXN36, EXN91 and their specific
monoclonal antibodies could be developed for the targeted therapy of
cancer indications at high medical need, either alone or in combinatorial
strategies.
41
117
POSTER (Board P111)
“Arming” the chimeric oncolytic adenovirus enadenotucirev to
deliver checkpoint inhibitors and other therapeutics directly to
tumours
B. Champion1 , P. Kodialbail1 , S. Illingworth1 , N. Rasiah1 , D. Cochrane1 ,
J. Beadle1 , K. Fisher1 , A.C.N. Brown1 . 1 PsiOxus Therapeutics Ltd,
Abingdon Oxford, United Kingdom
Enadenotucirev (EnAd; formerly called ColoAd1) is a potent, chimeric
Ad11p/Ad3 adenovirus active against a range of epithelial cancer cells,
with a shorter time-to-lysis than either wild type Ad11p, Ad3 or Ad5.
In normal cells, EnAd is attenuated and shows little or no activity by
either cytotoxicity or by qPCR. In vivo, EnAd shows efficacy in a range
of xenograft human tumour models following intra-tumoural, intravenous
and intra-peritoneal injection, and is currently being evaluated clinically
for treatment of several different epithelial cancers. Data from an ongoing
clinical mechanism of action study have shown that i.v. dosed EnAd infects
and replicates in tumour cells, producing significant amounts of viral protein
(hexon), indicating that transgene encoded proteins will also be made in
significant amounts by tumours following i.v. delivery of an armed EnAd
virus.
To develop ‘armed’ variants for delivery of therapeutic agents that enhance
EnAd’s anti-tumour activity, we have developed a system for rapid
generation of modified viruses that can be dosed systemically to deliver
immunomodulatory antibodies into tumours. We chose to first encode antiVEGF antibodies since, unlike immunomodulators, they could be readily
evaluated in vivo in immunodeficient mouse human tumour xenograft
models. We have successfully produced EnAd variants encoding fulllength (NG-135) and ScFv (NG-76) forms of anti-human VEGF antibodies
which have similar virus activity profiles to EnAd in cancer cell lines
in vitro (virus replication, gene expression and oncolytic action), but
also express and release the respective anti-VEGF antibody forms into
the culture supernatant. Using either HCT-116 or DLD human colon
carcinoma xenograft models we have shown that the virus infection profile
following intra-tumoural injection is similar to the parental EnAd virus (virus
replication and Hexon gene expression). Anti-VEGF antibody expression by
these tumours could be detected in the tissue as both mRNA and functional
antibody. Antibodies were detectable early (within 3 days of infection) and
expression was sustained over several weeks. Furthermore, low levels
of anti-VEGF antibody were detectable in the blood. Production and
evaluation of viruses similarly expressing checkpoint inhibitor antibodies
is now in progress, together with evaluation of anti-VEGF armed oncolytic
viruses for their impact on the growth and microenvironment of tumour
xenografts.
118
POSTER (Board P112)
Major synergy between Coxsackievirus A21 (CAVATAK™) and
radiotherapy or chemotherapy in bladder cancer
G.R. Simpson1 , N. Annels1 , M. Ajaz1 , F. Launchbury1 , G. Bolton1 ,
A.A. Melcher2 , K.J. Harrington3 , G. Au4 , D. Shafren4 , H. Pandha1 . 1 The
University of Surrey, Faculty of Health and Medical Sciences, Guildford,
United Kingdom; 2 St James’s University Hospital Leeds, Institute
of Molecular Medicine, Leeds, United Kingdom; 3 Institute of Cancer
Research, Targeted Therapy Team, London, United Kingdom; 4 Viralytics
Ltd & The University of Newcastle, Viralytics Ltd & The University of
Newcastle, Newcastle, Australia
Introduction: There are still no treatments for superficial bladder cancer
(SBC) which alter its natural progression, where 20% of patients develop
metastatic disease. SBC is often multifocal, has high recurrences after
surgical resection and recurs after intravesical live BCG.As this is a clinical
setting in which local live biological therapy is already well established,
it presents intriguing opportunities for oncolytic virotherapy. Coxsackievirus
A21 (CVA21) has recently been shown to be an efficient oncolytic agent that
specifically targets and rapidly lyses human malignant melanoma, multiple
myeloma, prostate and breast tumours, which express high levels of the
CVA21 cellular uptake receptors both in vitro and in vivo. In addition, a
Phase I clinical trial in late stage melanoma patients has recently been
completed, and has demonstrated that intratumorally administered CVA21
is well tolerated in humans, and that 55.55% of patients experienced
stabilization or reduction in injected tumour volumes.
Materials and Methods: Infection Radio- & Chemotherapy Synergy
assays, Combination index analyses, QPCR and IHC for ICAM-1/DAF,
Culture of SBC.
Results: Characterization of CVA21 cytotoxicity in a panel of cell lines
yielded a range of sensitivities. CVA21 cytotoxicity seems to correlate
expression of viral receptors ICAM-1 & DAF. The addition of radiotherapy
or chemotherapy resulted in significantly increased cytotoxicity over CVA21
alone. When 5637 or T24 cells were irradiated (4−10 Gy) then 24 hours
42
later exposed to CVA21 (multiplicities of infection 0.961–12.6), clear
synergy was seen. Dose matrix analysis showed that combination indices
reached minima of approximately 0.4. Similar analyses showed synergy
between Mitomycin C (1.4–0.021ug/ml) and CVA21 at multiplicities of
infection between 0.78 and 50 on 5637 cells, which was confirmed on
T24 cells. Crucially exposure to Mitomycin C or radiotherapy up-regulates
the expression of viral receptors ICAM-1 & DAF in bladder cancer cell
lines at both the RNA and protein level. Ru19−19 cells showed low ICAM-1
expression and almost no cytotoxicity when infected with CVA21. Exposure
to Mitomycin C increased Ru19−19 ICAM-1/DAF expression, resulting in
enhanced cytotoxicity killing, higher than each agent alone. Changing the
sequence of treatment with CVA21 and Mitomycin C on bladder cancer
cell lines does not appear to change the cytotoxicity killing. Patient derived
primary bladder cancer cell lines appear to be highly susceptible to CVA21
infection.
Conclusions: Combining CVA21 with either radiotherapy or chemotherapy
synergistically enhances cytotoxicity in bladder cancer cell lines. Radiation
and chemotherapy enhanced CVA21 oncolysis, likely by increased viral
receptors ICAM-1 and DAF expression. These results offer strong support
for translational clinical trials of CVA21 plus chemotherapy or radiotherapy
that have been initiated in the clinic
119
POSTER (Board P113)
3-Bromopyruvate as an inducer of immunogenic cell death in colon
cancer cells
K.H. Jung1 , J.H. Lee1 , J.W. Park1 , C. Quach1 , K.H. Lee1 . 1 Samsung
Medical Center Sungkyunkwan University School of Medicine, Nuclear
Medicine, Seoul, South Korea
Background: As cancer cells tend to resist cell death and evade
immune surveillance, there is a need to develop therapeutic strategies
that induce apoptosis and immune-mediated clearance of cancer cells.
3-Bromopyruvate (3-BrPA) is a halogenated alkylating analog of pyruvic
acid that has been shown to inhibit tumor proliferation. The anti-cancer
effect is presumed to occur by compromised ATP synthesis through
inhibition of glycolysis and mitochondrial complex II activity. In this study,
we evaluated the ability of 3-BrPA to induce immunogenic cell death (ICD)
in colon cancer cells.
Materials and Methods: CT26 murine colon cancer cells were treated
with 10 to 20 mM of 3-BrPA for 24 h. Well-known ICD inducers including
doxorubicin (DX; 20 mM) and combined mitomycinC/tautomycin (MitoC;
30 mM/TTM; 150 nM) were used as positive controls. Cell surface exposed
calreticulin (CRT) was detected by confocal microscopy and fluorescenceactivated cell sorting (FACS) analysis using alexa488 anti-CRT antibody.
The magnitude of CRT exposure was measured by binding of 125 I labeled
anti-CRT antibody prepared by the iodogen-method. Apoptotic and necrotic
cells were identified by FACS using FITC-annexinV and PI staining.
Results: Confocal microscopy showed increased binding of alexa488 antiCRT antibodies to the surface of CT26 cells treated with 3-BrPA, in
magnitudes comparable to that by DX and MitoC/TTM. FACS analysis
confirmed a significant right-shift of fluorescent signals in cells treated
with 3-BrPA, in a fashion similar to that by DX and MitoC/TTM. Hence,
3-BrPA treatment increased fluorescent signal-positive cells from 2.7±0.8%
to 16.9±0.9%. The amount of cell bound 125 I-labeled anti-CRT antibody
increased to 246.1±109.4 % of control level by 3-BrPA treatment.
Furthermore, FITC-annexinV and PI staining of 3-BrPA-treated cells
showed that CRT was exposed on early apoptotic (AnnexinV+ PI− ) but
not secondary necrotic (AnnexinV+ PI+ ) cells.
Conclusion: This study provides evidence that, in addition to its glycolysis
inhibiting property, 3-BrPA induces early apoptotic CRT exposure on the
surface of cancer cells, indicating a potential role of ICD in its anti-cancer
effects.
120
POSTER (Board P114)
Density of CD 8 +ve T cells & CD 56 +ve NK cells in follicular
adenoma & papillary carcinoma of thyroid in Pakistani population
J. Varda1 , N. Naseem1 , A.H. Nagi1 . 1 University of Health Sciences
Lahore, Morbid Anatomy & Histopathology, Lahore, Pakistan
Background: In Pakistan, papillary thyroid carcinoma (PTC) is the
commonest (69−71%) of all thyroid malignancies, while follicular adenoma
is the most common variety of benign lesions accounting for 60% of
all thyroid nodules. In tumour microenvironment, CD 8 +ve cytotoxic
T lymphocytes (CTLs) & CD 56 +ve natural killer (NK) cells play a key
role in progression from benign to malignant neoplasm as well as in the
metastatic potential of tumour.
Objectives: To determine the density of CD 8 +ve CTLs & CD 56 +ve NK
cells in biopsies of patients with follicular adenoma and papillary thyroid
carcinoma
Material and Methods: A total of 50 patients presenting with various
histological subtypes of follicular adenoma and different grades of PTC
were recruited through non-probability convenient sampling during study
period of January–December 2013 from different clinical centers of Lahore,
Pakistan. Relevant clinical data was recorded and morphological findings
including subtypes of follicular adenoma and histological grades of PTC
was ascertained microscopically. Density of CD 8 +ve CTLs & CD 56 +ve
NK cells was determined immunohistochemically and related with other
histological parameters.
Results: The mean age of the patients with thyroid lesions was
36.72±14.04 years (Age range: 14−84 years) with 74% females and 26%
males and a female to male ratio of 2.8: 1. Among n = 25 cases each of
follicular adenoma and PTC, 76.0% and 72.0% were females and 24.0%
and 28.0% were males with a female to male ratio being 3.1: 1 and 2.1:1
respectively (p = 1.0).
The ages of patients were divided into three age groups; Group 1 (10−34
years), Group 2 (35−59 years) & Group 3 (60−84 years).
Among n = 25 cases each of follicular adenoma and PTC 64.0% and 56.0%
cases were present in Group 1, 32.0% and 16.0% cases were present in
Group 2 while 4.0% and 28.0% cases were seen in Group 3 respectively
(p = 0.046).
The density of CD 8 +ve CTLs was scored from 1−5 where increased
frequency of score 3 (56%) and score 5 (40%) were observed in cases
of follicular adenoma and PTC respectively (p = 0.01).
Whereas when the density of CD 56 +ve NK cells was related to the
total number of follicular adenoma and PTC, increased frequency of score
3 (96%) was observed in cases of follicular adenoma as compared to
PTC (52%) (p = 0.000).
Score 3 for CD 8 +ve CTLs and CD 56 +ve NK cells was most frequent
among the microfollicular variety of follicular adenoma while this density
increased with the increasing histological grades of PTC.
Conclusion: The density of CD 8 +ve CTLs and CD 56 +ve NK
cells was related strongly with the microfollicular subtype of follicular
adenoma and grade II of PTC. Hence, the lymphocytic infiltrate in tumour
microenvironment might be an independent prognostic factor if studied on
a comparatively larger sample size with follow up of the patients.
121
POSTER (Board P115)
A modified double-deleted vaccinia virus combining viral oncolysis
and potential gene therapy as a novel therapeutic for atypical
teratoid/rhabdoid tumors
Y. Ruan1 , A. Narendran2 . 1 Alberta Children’s Hospital, Calgary Alberta,
Canada; 2 Alberta Children’s Hospital, Pediatrics and Oncology, Calgary
Alberta, Canada
Background: AT/RT is a highly malignant brain tumour of infancy that has
a poor outcome. The tumor is characterized by the loss of tumor suppressor
gene SNF5, a core subunit of the SWI/SNF chromatin remodeling
complex, which consequently leads to substantial epigenetic changes and
tumorigenesis. Due to the unacceptable toxicity of current chemotherapy
on children, safe and effective novel therapies are urgently needed.
Oncolytic virotherapy has emerged as a promising approach that uses
replication-competent virus to provide anti-neoplastic activity, through the
action of direct killing via cell lysis and the additional effect via the
activation of the host anti-tumour immune response. We have previously
demonstrated the efficacy of double-deleted vaccinia virus (vvDD), a virus
with improved safety and tumor specificity, in the ability of homing to and
killing AT/RT xenografts. Vaccinia virus, in particular, has the additional
advantage of having non-essential genes on its genome that can be
replaced with foreign DNA to enhance cytotoxicity. We therefore generated
a modified vvDD that expresses wild-type SNF5 gene and tested whether
this virus possesses better efficacy against AT/RT cells.
Methods: Modified virus, vvDD-hSNF5, was generated by cloning wild type
human SNF5 gene into shuttle vector pSC65-mCherry. This construct was
transfected and co-infected with vvDD-GFP to HEK293 cells to generate
recombined viral particles. Successfully recombined viruses, in which the
mCherry and hSNF5 genes replaced GFP in the TK locus, were selected by
FACS (sorting of mCherry+ GFP− cells). Viral titre was determined by plaque
assay and cytotoxicity against AT/RT cells was determined by crystal violet
staining. The ability of vvDD-hSNF5 to express SNF5 gene in AT/RT cells
was determined by western blotting.
Results: We successfully generated the modified vvDD-hSNF5 that
expresses high levels of SNF5 protein in AT/RT cells. Arming of vvDDhSNF5 does not affect the replication of the virus. SNF5 protein is primarily
localized to the cell nucleus but is also present in the cytoplasm. vvDDhSNF5 is capable of decrease cell cycle progression in AT/RT cells, in
contrast to control virus (vvDD). The ability to cause effective cytotoxicity
remains unchanged in vvDD-hSNF5 virus.
Conclusions: We generated a modified oncolytic virus that can potentially
combine oncolytic virotherapy with gene therapy. As a proof-of-concept,
we demonstrated that vvDD-hSNF5 possesses similar replication potency
and in vitro cytotoxicity. The expression of SNF5 can induce cycle arrest in
AT/RT cells. These preliminary observations grant further investigation of
vvDD-hSNF5 in in vivo testing.
122
POSTER (Board P116)
Analysis of immune-response markers in resectable NSCLC
M. Usó1 , E. Jantus-Lewintre1 , R. Sirera2 , S. Calabuig-Fariñas1 ,
S. Gallach1 , E. Escorihuela1 , A. Blasco3 , R. Guijarro4 , C. Camps5 .
1
Fundación de Investigación Hospital General de Valencia, Molecular
Oncology, Valencia, Spain; 2 Universitat Politècnica de València,
Biotechnology, Valencia, Spain; 3 Hospital General de Valencia, Oncology,
Valencia, Spain; 4 Hospital General de Valencia, Thoracic Surgery,
Valencia, Spain; 5 Universitat de València, Medicine, Valencia, Spain
Background: Several markers have been indentified to be involved in
pro- and anti-tumor immune responses, and some of them may have a
prognostic impact. In this study we have analyzed gene expression of a
set of markers related to different immune responses in samples from
resectable NSCLC patients.
Material and Methods: RNA was isolated from fresh-frozen lung
specimens (tumor and normal lung) from resectable NSCLC patients
(n = 178). RTqPCR was performed to analyze the expression of CCL2,
CCL22, CD1C, CD209, CTLA4, IL10, IL23A, LGALS1, LGALS2, and
TGFB1 by the use of hydrolysis probes. Relative gene expression was
assessed by Pfaffl formula and normalized by the use of CDKN1B,
GUS and ACTB as endogenous genes (selected by GeNorm algorithm).
Statistical analyses were considered significant at p < 0.05.
Results: Patient’s median age was of 65 [26−85], 86.5% were male,
47.2% with squamous (SCC) histology and 11.2% presented a mutation in
KRAS gene. Mann-Whitney test revealed significant differences between
histology and TGFB1 expression (p = 0.020), being higher in patients with
SCC. Moreover, higher levels of LGALS2 (p = 0.023) were found to be
associated with absence of lymph node involvement and higher IL23A
expression was associated with KRAS WT status (p = 0.026). Survival
analyses revealed that higher levels of IL23A and LGALS2 were associated
with longer overall survival (OS) (p < 0.001 and 0.007, respectively) and
progression free survival (PFS) (p = 0.003 and p = 0.002, respectively)
(Table 1). Furthermore, survival analyses restricted to adenocarcinoma
(ADC) patients revealed that higher levels of IL10 and CTLA4 were both
correlated with longer OS (p = 0.017 and 0.028, respectively) and PFS
(p = 0.015 and p = 0.006), as well as IL23A and LGALS2 (Table 1).
Table 1.
Marker
NSCLC (n = 178)
IL23A
Low
High
LGALS2
Low
High
ADC (n = 74)
IL10
Low
High
CLTA4
Low
High
IL23A
Low
High
LGALS2
Low
High
OS
Median (months)
p
PFS
Median (months)
0.001*
42.6
NR
0.003*
23.4
81.23
0.007*
46.6
NR
0.002*
26.2
NR
0.017*
37
81.2
0.015*
17.8
49.3
0.028*
37.5
NR
0.006*
18.8
81.2
0.025*
42.9
NR
0.015*
19.2
81.2
0.001*
37.4
NR
p
0.005*
19.2
NR
Gene expression levels were dichotomized as high (median or higher) and
low (less than the median).
Conclusions: Our data indicated that the expression of certain immuneresponse markers could be associated with better outcome in resectable
NSCLC patients.
Supported by grants PS09/01149, RD06/0020/1024 and RD12/0036/0025
from RTICC, and Instituto de Salud Carlos III.
43
123
POSTER (Board P117)
Targeting tryptophan metabolism in human lung cancer
J. Deshane1 , C. Schafer1 , Y. Wang1 , A. Sawant2 , T.H. Jin1 , D. Zhi3 ,
S. Ponnazhagan2 , S. Grant4 . 1 UAB (University of Alabama Birmingham)
Cancer Center, Medicine/Pulmonary, Birmingham AL, USA; 2 UAB
(University of Alabama Birmingham) Cancer Center, Pathology,
Birmingham AL, USA; 3 UAB (University of Alabama Birmingham)
Cancer Center, Biostatistics, Birmingham AL, USA; 4 UAB (University
of Alabama Birmingham) Cancer Center, Medicine-Hematology Oncology,
Birmingham AL, USA
Background: The purpose of the study is to determine whether combination chemotherapy-mediated targeting of tryptophan (Trp) metabolism
by inhibition of indoleamine 2,3-dioxygenase (IDO) enzyme activity in
immunosuppressive myeloid-derived suppressor cells (MDSCs) predicts
improved clinical outcome in lung cancer patients.
Methods: Percentages of circulating MDSCs were determined using
CD33+ myeloid cells enriched from peripheral blood samples collected
from Stage III−IV Non-Small Cell Lung Cancer (NSCLC) patients and their
normal healthy relatives. Quantitation of L-Kynurenine (L-kyn), a read out
for IDO enzymatic activity, was performed in serum and purified circulating
MDSCs.
Results: Serum IDO activity (levels of L-kyn) was found to be significantly
correlated (P = 3.0×10−5 ) with disease status of patients (n = 11) and their
normal relatives (n = 8) using a linear mixed model with family ID as
random effect. The percentages of circulating granulocytic (G-MDSCs)
and monocytic-MDSCs (Mo-MDSCs) immunosuppressive subsets are
significantly associated with disease status. Using linear mixed model,
we found G-MDSCs (P = 0.004) and Mo-MDSCs (P = 3.7×10−5 ) are both
higher in cancer patients compared to normal healthy subjects. Following
the second cycle of treatment with combination chemotherapies, serum
L-Kyn was reduced significantly (n = 5, p = 0.026, paired t test). Mean
effect of chemo is −55.0 nmol kynurenine/hour, suggesting response
to combination chemotherapy strategies. Percentages of subsets of
MDSCs are also significantly reduced after chemotherapy treatments
(n = 5, p = 0.030 for G-MDSCs, p = 0.026 for Mo-MDSCs, paired t test).
Mean effects of chemotherapy treatments are −2.9% for G-MDSCs and
−12.4% for Mo-MDSCs. Ongoing studies will determine correlations of
change in IDO activity in MDSCs with reduction in tumor burden and overall
survival of lung cancer patients.
Conclusions: These preliminary studies suggest that the IDO enzymatic
pathway and tryptophan metabolism may serve as important therapeutic
targets and diagnostic markers in predicting clinical outcomes of combination chemotherapies in the treatment of NSCLC in humans.
Supported by ACS-IRG-60–001−53.
124
POSTER (Board P118)
A WT1-derived peptide protects against metastatic melanoma in a
syngeneic model by in vivo immunomodulatory effects on dendritic
cells
M.H. Massaoka1 , C.R. Figueiredo1 , N. Girola1 , R.A. Azevedo2 ,
L.R. Travassos1 . 1 Experimental Oncology Unit, Department of Microbiology
Immunology and Parasitology, São Paulo SP, Brazil; 2 Butantan Institute,
Department of Biochemistry and Biophysics, São Paulo SP, Brazil
Background: The cell-penetrating peptide, WT1-pTj, derived from a zinc
finger domain of the Wilms Tumor Protein 1 (WT1), displays antimelanoma
activity in vitro by suppression of cell growth and induction of cellular
senescence. We now report that WT1-pTj-mediated antitumor effects
in vivo rather depend on the immune system primarily involving dendritic
cells (DCs).
Materials and Methods: In vivo protection experiments with WT1-pTj
were made in mice challenged endovenously with B16F10-Nex2 melanoma
cells. Syngeneic C57Bl/6 or immune deficient animals were used. The
peptide significantly reduced lung metastatic nodules in syngeneic animals
when injected i.p. In the present work: (a) The same protocol was used
in immune deficient NOD/Scid/IL-2rgnull mice; (b) To investigate the role
of DCs, the syngeneic melanoma model was used. Mice were injected
e.v. with B16F10-Nex2 melanoma cells to induce lung metastasis, and
seven days after tumor inoculation animals received a single dose of
syngeneic bone-marrow DCs primed with melanoma lysate and treated
ex vivo with WT1-pTj for two days. Lung nodules were counted and
compared with the untreated control; c) CD11c+ DCs from the cervical
lymph nodes of WT1-pTj-treated metastatic melanoma-bearing mice were
examined for the expression of co-stimulatory molecules by FACS and
production of cytokines by ELISA. Additionally, the levels of CD8+ and
CD4+ T lymphocytes and NK1.1+ cells in the lung and spleen of treated
and untreated mice with metastatic disease were determined.
44
Results: Systemic WT1-pTj treatment protected against metastatic
melanoma in immune competent but not in immune deficient animals.
Remarkably, peptide-activated syngeneic DCs ex vivo protected against
metastatic melanoma, even in animals with established pulmonary nodules.
By culturing CD11c+ cells isolated from cervical lymph nodes of WT1pTj-treated animals developing metastatic melanoma, we found that the
peptide induced phenotypic maturation of DCs (e.g. upregulation of CD40,
MHC-II and CD86) and enhanced production of type-1 cytokines, such as
IL-6 and IL-12. Simultaneously, a marked decrease in TGF-b1 production
was observed in DCs retrieved from WT1-pTj-treated mice. Moreover,
the WT1-pTj-mediated antitumor activity was associated with significantly
augmented frequency of tumor-infiltrating lymphocytes (CD8+ and CD4+)
and NK1.1+ cells, and down-regulation of splenic CD4+ FoxP3+ regulatory
T cells.
Conclusions: The present results show the immunomodulatory role of
WT1-pTj and indicate that the WT1-derived peptide may act as a potent
adjuvant in cancer immunotherapy.
Supported by FAPESP no. 2010/51423-0 and the Brazilian National
Research Council (CNPq).
125
POSTER (Board P119)
Critical issues in the clinical development of oncolytic viruses −
A regulatory perspective
M. Schuessler-Lenz1 . 1 Paul-Ehrlich-Institute, Langen, Germany
Oncolytic viruses are claimed to replicate selectively and preferentially
in tumor tissue, without causing excessive damage to normal tissues. In
Europe there has been a steady increase in the number of clinical trials
performed with oncolytic viruses, and some development candidates have
reached the pivotal stage of clinical development. As oncolytic viruses
are replication competent there is a need to balance the therapeutic
promises linked to this new treatment concept against the risks that may
be associated with replication competent viruses.
Most of the oncolytic viruses are classified as gene therapy medicinal
products, based on genetic modifications to change the viral tissue tropism
and to increase tumor specificity. From a European regulatory perspective,
gene therapy medicinal products are in the scope of the Advanced Therapy
Medicinal Product (ATMP) regulation. This implies that for the development
of oncolytic viruses towards marketing authorisation, both national and European regulatory and legal requirements apply, based on the different key
players for clinical trial authorisation and central marketing authorisation. At
the European Medicines Agency the Committee for Advanced Therapies
formulates the draft opinion on quality, safety and efficacy of oncolytic
viruses for final approval by the Committee for Human Medicinal Products.
In Germany clinical trial authorisations with oncolytic viruses are approved
by the Paul-Ehrlich Institute.
A review of clinical trials shows that oncolytic viruses are at different
stages of clinical development. They constitute one of the main innovative
treatment concepts in cancer therapy, as can be seen from the number
of clinical trials submitted to the Paul-Ehrlich Institute. We review the
main issues in early clinical development, based on our experience with
clinical trial assessment and national advice. We also review the interaction
between the national competent authority and the Committee for Advanced
Therapies.
126
POSTER (Board P120)
IMCgp100: A novel bi-specific biologic for the treatment of malignant
melanoma
W. Shingler1 , J. Harper2 , G. Bossi2 , D. Barker2 , J. Dukes2 , N. Liddy3 ,
S. Paston2 , T. Mahon3 , P. Molloy3 , M. Sami1 , E. Baston3 , B. Cameron3 ,
A. Johnson1 , A. Vuidepot3 , N. Hassan1 , Y. McGrath1 , B. Jakobsen1 .
1
Immunocore Ltd, Development, Abingdon Oxford, United Kingdom;
2
Immunocore Ltd, Cell Biology, Abingdon Oxford, United Kingdom;
3
Immunocore Ltd, Protein Science, Abingdon Oxford, United Kingdom
Background: Despite significant advances in the treatment of metastatic
melanoma, long-term remission for the majority of patients remains
elusive. Kinase inhibitors provide potent but short-term responses for
a significant proportion of patients and immunotherapy elicits long-term
responses with the prospect of cure, but only in a minority. IMCgp100
comprises an affinity-enhanced T cell receptor (TCR) specific for the HLAA2 restricted melanoma gp100 peptide (YLEPGPVTA) fused to an anti-CD3
antibody fragment. Binding of IMCgp100 to melanoma cells redirects T cell
cytotoxicity allowing them to kill even HLA down-regulated melanoma cells
otherwise invisible to cancer specific T cells.
Materials and Methods: Preclinical efficacy and safety testing of
IMCgp100 has been performed using a range of in vitro assays of
immunological activity against human tissue, including ELISpot, cytotoxicity
assay and Incucyte. A Phase I clinical study is currently underway to
determine the safety and tolerability of IMCgp100 in patients with metastatic
melanoma, with extensive laboratory testing accompanying this first in
human study to try to identify pharmacodynamic markers.
Results: Here, we present data which provides the foundation for the
clinical observations. In vitro, IMCgp100 is demonstrated to potently
redirect T cells from late stage cancer patients to target melanoma tumours
exhibiting HLA down-regulation, even in the presence of high numbers of
regulatory T cells. Target cell killing is observed within hours and is specific
for gp100. In addition killing is associated with the release of various proinflammatory cytokines and chemokines as well as cross-presentation of
gp100 and other melanoma-associated antigens by dendritic cells. Thus,
IMCgp100 demonstrates the potential to elicit potent short-term responses
and trigger longer-term anti melanoma activity in vivo. Maximum tolerated
dose has been established for weekly dosing with the drug being well
tolerated and showing evidence of tumour shrinkage. Analyses of trial
patient samples provide evidence for drug mediated T cell mobilisation
and transient cytokine and chemokine release, including those reported to
play a key role in anti-melanoma responses.
Conclusion: These data support the potential of IMCgp100 as a new
immunotherapy against advanced melanoma. In-vitro data demonstrate the
drug is specific for melanoma cells, and preliminary early phase clinical
data suggest acceptable toxicity and promising durable efficacy.
127
POSTER (Board P121)
Correlation of clinical activity of pembrolizumab (MK-3475) with
immunohistochemical staining for programmed death-1 ligand
(PD-L1) in 50% of tumor cells in a prospective non-small cell lung
cancer (NSCLC) validation population
E.G. Garon1 , N.A. Rizvi2 , N.B. Leighl3 , R. Hui4 , J.P. Eder5 , A. Patnaik6 ,
C. Aggarwal7 , L. Horn8 , A.S. Balmanoukian9 , M.A. Gubens10 ,
E. Felip-Font11 , E. Carceny Costa12 , J.C. Soria13 , M.J. Ahn14 ,
H.T. Arkenau15 , J.S. Lee16 , G. Robinet17 , G.M. Lubiniecki18 , J. Zhang19 ,
K. Emancipator21 , R. Rutledge22 , M. Dolled-Filhart23 , L. Gandhi20 . 1 David
Geffen School of Medicine at UCLA, Dicision of Hematology/Oncology,
Los Angeles, USA; 2 Memorial Sloan Kettering Cancer Cente, Department
of medicine, New York, USA; 3 Princess Margaret Cancer Centre,
Department of Medicine, Toronto, Canada; 4 Westmead Hospital
University of Sydney, Department of Medical oncology, Sydney, Australia;
5
Yale University, Department of medical oncology, New Haven, USA;
6
South Texas Accelerated Research Therapeutics, Clinical research,
San Antonio, USA; 7 Abramson Cancer Center of the University of
Pennsylvania, Department of medicine, Division of Hematology/Oncology,
Philadelphia, USA; 8 Vanderbilt Ingram Cancer Center, Department of
Medicine, Nashville, USA; 9 The Angeles Clinic and Research Institute,
Department Lung and Thoracic cancer, Los Angeles, USA; 10 University of
California San Francisco, Thoracic Tumours Group, San Francisco, USA;
11
Hospital General Universitari Vall d’Hebron, Department of medical
oncology, Barcelona, Spain; 12 Catalan Institut of Oncology − Badalona,
Badalona, Spain; 13 Gustave Roussy, Drug Development Department
(DITEP), Villejuif, France; 14 Samsung Medical Center, Department of
Internal Medicine, Samsung, South Korea; 15 Sarah Cannon Research UK,
Oncology, London, United Kingdom; 16 Seoul National University Bundang
Hospital, Department of Hematology/Oncology, Seongnam-si, South
Korea; 17 Hopital Morvan, Institute de Cancerologie, Brest, France; 18 Merck
& Co. Inc., Clinical Research, North Wales, USA; 19 Merck & Co. Inc.,
BARDS, North Wales, USA; 20 Dana-Farber Cancer Institute, Medical
Oncology, Boston, USA; 21 Merck & Co., Inc., Molecular Biomarkers and
Diagnostics, Whitehouse Station, NJ, USA; 22 Merck & Co., Inc., Oncology
Clinical Researchs, Whitehouse Station, NJ, USA; 23 Merck & Co., Inc.,
Molecular Biomarkers and Diagnostics, Whitehouse Station, NJ, USA
Background: The phase I KEYNOTE-001 study evaluated the safety,
tolerability, and clinical activity of pembrolizumab, a selective anti-PD-1
antibody that blocks the interaction between programmed death-1 (PD-1)
on T cells and its ligands PD-L1 and PD-L2 on tumor cells. Of those patients
(pts) with tumor tissue evaluable for PD-L1 status by immunohistochemistry
(IHC) in the initial 38-pt NSCLC cohort, all pts with clinical response by
immune-related response criteria (irRC) were in the group with the highest
PD-L1 expression. Tissue from these pts and an additional 119 pts were
evaluated to generate a formalized training set. Among the 129 pts with
tumor evaluable for PD-L1 expression and measurable disease at baseline
per RECIST v1.1 as assessed by independent central review, a highly
significant correlation between strong PD-L1 expression (i.e., staining in
50% of tumor cells) and the overall response rate (ORR) and progressionfree survival (PFS) was observed.
Materials and Methods: 92 treatment-naı̈ve and 216 previously treated
pts with measurable NSCLC whose tumors expressed detectable PD-L1
(1% cutoff) using a prototype IHC assay using the 22C3 antibody were
randomized to receive pembrolizumab at 10 mg/kg every 2 or 3 weeks;
2 pts were never treated. A new tumor biopsy 60 days prior to or,
with Amendment 8, any time after the most recent therapy before the
first pembrolizumab dose was required. Tumor response was assessed
every 9 weeks by RECIST v1.1 by independent central review. All pts will
have a minimum of 6 mo of follow-up. PD-L1 expression was subsequently
evaluated in the study eligibility tumor samples by a clinical trial IHC assay
using the 22C3 antibody for assessment of the relationship with outcomes
with a goal of validating the utility of IHC staining in 50% of tumor cells
as a biomarker to predict the efficacy of pembrolizumab.
Results: After selection of the training set population, over 500 additional
pts signed consent between 20 May 2013 and 12 May 2014 and
provided tissue for PD-L1 assessment. Based on PD-L1 staining using the
prototype IHC assay and other study eligibility criteria, 306 pts received
pembrolizumab.
Conclusions: At the meeting, we will present data evaluating whether the
cutoff of PD-L1 staining by IHC in 50% of tumor cells that was selected
based on our training set correctly predicts ORR and PFS in the validation
set of 306 pts with advanced NSCLC treated with pembrolizumab.
128
POSTER (Board P122)
T cell-mediated cancer immunotherapy through OX40 agonism
M. Huseni1 , C. Du2 , J. Zhu2 , P. Pacheco-Sanchez3 , M. Moskalenko3 ,
H. Chiu4 , K. Dalpozzo1 , K. Totpal3 , L. Damico-Beyer5 , J. Kim2 .
1
Genentech, Oncology Biomarkers, South San Francisco CA, USA;
2
Genentech, Cancer Immunology, South San Francisco CA, USA;
3
Genentech, Translational Oncology, South San Francisco CA, USA;
4
Genentech, Biochemical and Cellular Pharmacology, South San
Francisco CA, USA; 5 Genentech, Portfolio Management and Operations,
South San Francisco CA, USA
Background: Productive immune responses to tumors are hypothesized
to require T cell costimulation. OX40 is a costimulatory molecule
that is expressed on activated effector (Teff) and regulatory T cells
(Treg). Agonistic antibodies targeting OX40 are predicted to counteract
the immunosuppressive tumor microenvironment and promote T cell
dependent anti-tumor immunity via two distinct mechanisms − activation
and expansion of antigen experienced T cells and inhibition of T cell
suppression. An anti-human OX40 antibody MOXR0916, and a surrogate
anti-mouse OX40 antibody were tested for their ability to costimulate
effector T cells and inhibit Treg function in vitro and in vivo, respectively.
Materials and Methods: MOXR0916 is a humanized agonist OX40
antibody, which binds to human OX40 with subnanomolar affinity. Due
to sequence divergence between human and mouse OX40, a murine
agonist OX40 antibody was employed to assess anti-tumor efficacy, Teff
costimulation and Treg dynamics in murine syngeneic tumor models.
The role of Fc effector function was determined utilizing Fc receptor
knockout mice and isotype variants of anti-mouse OX40. In vitro analysis
of MOXR0916 and isotype variants of MOXR0916 in Teff costimulation
and Treg suppression assays were conducted with sorted peripheral blood
T cell populations from healthy donors.
Results: In syngeneic mouse tumor models, anti-mouse OX40 treatment
induced durable tumor regression which were associated with intratumoral
Treg depletion and CD8+ T cell infiltration, and enhancement of interferon
gamma (IFN-g) production. Tumor regression and pharmacodynamic
modulation of Teff and Tregs required antibody crosslinking via Fc
receptors. MOXR0916 costimulated CD4+ memory T cell proliferation and
IFN-g production following T cell receptor engagement in a dose dependent
manner. Additionally, MOXR0916 impaired the suppressive function of
Tregs in naı̈ve T cell coculture assays. Memory CD4+ T cell costimulation
was dependent on MOXR0916 effector function. In in vitro T cell activation
assays, MOXR0916 costimulated Teff production of IFN-g at comparable
levels as anti-mouse OX40.
Conclusions: Anti-mouse OX40 antibody induced durable anti-tumor
immunity in multiple syngeneic tumor models. Potent single agent activity
correlated with Teff costimulation and modulation of Treg dynamics.
MOXR0916 and anti-mouse OX40 induced comparable levels of IFN-g
production, supporting the clinical investigation of MOXR0916 as a T cell
directed therapy for advanced malignancies.
129
POSTER (Board P123)
Characterization of PD-L1 expression in circulating tumor
cells (CTCs) of non-small cell lung cancer (NSCLC) patients
R. Krupa1 , D. Lu1 , M. Harvey1 , J. Louw1 , A. Jendrisak1 , D. Marrinucci1 ,
R. Dittamore1 . 1 Epic Sciences Inc., San Diego CA, USA
Background: Novel PD-1 and PD-L1 targeting immunotherapies are
demonstrating efficacy in multiple tumor types. Recent data have suggested
45
that increased progression free survival (PFS) is observed in patients
whith higher expression of the PD-L1 protein in tumor tissue. However,
many NSCLC patients have insufficient tumor sample or have high
co-morbidities preventing access to tissue IHC to determine PD-L1
expression and potential benefit to novel PD-1/PD-L1 immunotherapies. We
developed a protein assay for PD-L1 protein and examined CTCs and CTC
subpopulation incidence and molecular characterization in blood samples
from newly diagnosed NSCLC patients.
Material and Methods: 16 samples from newly diagnosed NSCLC patients
prior to therapy were recruited and blood specimens were collected and
shipped to Epic Sciences. All nucleated cells were plated onto glass slides
and subjected to immunofluorescent (IF) staining and CTC identification
by fluorescent scanners and algorithmic analysis. CTCs, defined as
traditional (CK+, CD45− with intact and morphologically distinct DAPI+
nuclei), apoptotic (CK+, CD45−, non-intact nuclei) and CK− (CK−, CD45−,
intact and distinct nuclei). CTC subtypes were characterized with PD-L1 IF
to assess expression.
Results: Assays for the PD-L1 protein were developed and specificity
confirmed utilizing Colo205, SU-DHL-1 and A549 cells spiked into donor
blood and run through the Epic Assay. Additionally, expression analysis of
Colo205, SU-DHL-1 and A549 cell lines show increased differential PD-L1
expression when cells were exposed to interferon gamma. PD-L1 positive
CTCs were detected in 8/16 (50%) of NSCLC patients. PD-L1 positivity was
seen in traditional and CK− CTCs, as well as apoptotic and CTC clusters.
Conclusion: PD-L1 protein assessment of CTCs and CTC subpopulations
from NSCLC patients at diagnosis is feasible on the Epic CTC platform.
This test demonstrates sensitivity and specificity and may aid in the
identification of patients suitable for clinical trial studies with novel PD-1 or
PD-L1 therapies. The identification of PD-L1 positive CTC subpopulations
identifies unique tumor cell morphology and suggests evidence of epithelial
plasticity in some patients. Further analysis of pharmacodynamics of CTCs
and PD-L1 expression on CTCs in the setting of PD-1/PD-L1 therapies is
warranted.
130
POSTER (Board P124)
Preclinical results of ProCervix, a first in class, first in indication
therapeutic vaccine targeting HPV16/18 infected women
M. Esquerre1 , M. Bouillette-Marussig1 , A. Goubier1 , M. Momot1 ,
H. Keller1 , M. Bissery1 . 1 Genticel, Labege, France
With the availability of HPV diagnostic tests, it is now possible to identify
HPV infected women and to develop treatment in order to clear the
infection and prevent the onset of neoplasia and cancer. ProCervix is a
bivalent therapeutic vaccine consisting of two adenylate cyclase (CyaA)
recombinant proteins originating from Bordetella pertussis into which
HPV16 E7 and HPV18 E7 have been inserted. The CyaA targets CD11b
expressed by Antigen Presenting Cells (APC). It delivers the vaccine
antigens to both MHC-I and MHC-II pathways in the APC for presentation
to CD4+ and CD8+ T cells. We report here on the preclinical properties
of ProCervix therapeutic vaccine designed to treat women infected with
HPV16 and/or 18 before they develop high grade cervical lesions.
ProCervix binding to CD11b was performed using a cell line expressing
human CD11b in a competition assay using biotinylated-CyaA HPV16 E7.
The immunogenicity was evaluated in C57BL/6 mice vaccinated i.d.
with ProCervix adjuvanted with Imiquimod 5% cream. E7-specific T cell
responses were measured using IFN-g ELISpot and TH 1/TH 2 cytokines,
using Cytometric Bead Array and FACS analysis. Cytotoxic T lymphocytes
(CTL) were evaluated by in vivo killing assays. Therapeutic efficacy
was evaluated in mice bearing 106 HPV16 E7-expressing tumour cells
implanted s.c. and vaccinated on day 11 and 39. Tumour growth was
measured with a caliper twice a week.
Results obtained with an in vitro functional cell-based assay demonstrated
that ProCervix binds to human CD11b-expressing cells. In vivo, using
C57BL/6 mice, intradermal administration of ProCervix in combination with
a TLR7 agonist, resulted in the development of both HPV16 E7-specific
and HPV18 E7-specific T cell responses with a clear TH 1 cytokine profile.
Using an in vivo killing assay, we demonstrated that ProCervix-induced
CD8+ T cells are functional CTLs. Using syngeneic TC-1 tumour cells, our
results demonstrated that these CTLs were able to eradicate HPV16-E7expressing cells. Finally, using this tumour-rejection model, we showed that
even a low dose of E7 antigens lead to strong therapy when it delivered
via ProCervix.
These preclinical data prompted a phase I clinical study in HPV16/18
infected women with normal cytology which indicated that ProCervix was
well tolerated, and exhibited a higher frequency of peripheral HPV-specific
interferon-gamma associated T-cells and a higher viral clearance in the
treated group compared to the placebo group.
46
131
POSTER (Board P125)
Zfra activates novel Hyal2+ CD3− CD19− memory spleen cells to
block cancer growth, stemness, and metastasis in vivo
N. Chang1 . 1 National Cheng Kung University, Tainan City, Taiwan
Zfra is a 31-amino-acid zinc finger-like protein, which participates in tumor
necrosis factor signaling.
Here, we determined that synthetic full-length Zfra1−31 peptide selfpolymerized in degassed buffers without catalytic enzymes. When nude
mice and BALB/c mice were pre-injected with micromolar levels of Zfra1−31
or truncated Zfra4−10 via tail veins, these mice became resistant to the
growth, metastasis and stemness of prostate, breast, and lung cancer
cells, melanoma cells, and many malignant cancer cells. Alteration of
the Ser8 phosphorylation site to Gly8 abolished Zfra polymerization and
cancer suppression in vivo. Injected Zfra was deposited mainly in the
spleen. Transfer of Zfra-stimulated spleen cells to naı̈ve mice conferred
resistance to cancer growth. Mechanistically, Zfra bound membrane
hyaluronidase Hyal-2 and suppressed the TGF-b/Hyal-2/WWOX/Smad4
signaling, via down-regulation of Hyal-2 and activated WWOX (with Y33
phosphorylation), in the spleen for generating novel non-T/non-B memory
cells, designated Hyal2+ CD3− CD19− cells. Transfer of these cells to
naı̈ve mice also induced anticancer response. Similarly, agonist anti-Hyal-2
antibody mimicked the effect of Zfra in causing cancer suppression.
In conclusion, Zfra self-polymerizes in the spleen to activate Hyal2+ CD3−
CD19− cells for blocking cancer growth, stemness and metastasis in vivo.
Supported in part by NSC and NHRI, Taiwan, and DoD, USA
132
POSTER (Board P126)
A novel anti-PDL1 antibody-based bifunctional protein with enhanced
immunological activity
Y. Wu1 , S. Martomo1 , Z. Zhong1 , D. Lu1 , Z. Polonskaya1 , X. Luna1 ,
Z. Zhang1 , H. Zhang1 , L. Witte1 , S. Waksal1 , Z. Zhu1 . 1 Kadmon
Corporation LLC, NYC, USA
Preclinical and clinical evidence suggest that immunotherapies based on
immunomodulating agents that inhibit immunosuppressive pathways (e.g.,
PD-1/PD-L1), or that stimulate the immunity of T-cells and NK cells (e.g., by
cytokines such as IL-12, IL-15, IL-21), hold great potential for the effective
treatment of cancer. The anti-PD1/PDL1 approach, via attenuation of
inhibitory checkpoints, has shown success in certain clinical settings across
multiple cancer types. Its full potential may be limited however by impaired
overall immunity within the tumor micro-environment. The broad clinical
application of immune-stimulating cytokines has been greatly hindered by
their limited bioavailability, short half-life and systemic toxicities due to their
lack of target specificity. It is therefore desirable to develop biotherapeutic
agents that are capable of both inhibiting immunosuppressive pathways
and simultaneously stimulating and expanding immune effector cells
thereby increasing both innate and adaptive immune responses within the
tumor environment for greater antitumor activity. To this end, we engineered
a panel of recombinant bifunctional proteins that combine an anti-PD1
antibody with an immune-stimulating cytokine. These bifunctional proteins
were genetically constructed and produced by mammalian cell cultures.
One of the bifunctional proteins, KD-033, was efficiently expressed in
mammalian cells and was fully stable when incubated in serum samples.
The bifunctional protein retained binding and functional activity of both
its parental components as tested by ELISA and cell-based assays.
Further, KD-033 showed strong activity in relieving PD1/PDL1-meditated
suppressive effect on CD4 T cell proliferation and cytokine (IL-2, IFN)
production, and simultaneously showed significant enhancement of the
killing activity of CD8 T cells and NK cells.
133
POSTER (Board P127)
Analysis of immune microenvironment in resectable NSCLC:
Prognostic value of regulatory and conventional T cell markers
proportion
M. Usó1 , R. Sirera2 , S. Calabuig-Fariñas1 , A. Blasco3 , E. Pastor4 ,
R. Guijarro4 , E. Jantus-Lewintre1 , J. Forteza5 , C. Camps6 . 1 Fundación
de Investigación Hospital General de Valencia, Molecular Oncology,
Valencia, Spain; 2 Universitat Politècnica de València, Biotechnology,
Valencia, Spain; 3 Hospital General de Valencia, Oncology, Valencia,
Spain; 4 Hospital General de Valencia, Thoracic Surgery, Valencia, Spain;
5
Instituto Valenciano de Patologı́a, Valencia, Spain; 6 Universitat de
València, Medicine, Valencia, Spain
Background: Immune cells present in tumor microenvironment have been
demonstrated to play an important role in tumor progression, and therefore,
in patient’s outcome.
Methods: FFPE samples from 117 early-stage NSCLC patients of primary
tumor tissue were used. We assessed the mRNA expression levels of
11 genes (CD127, CD25, FOXP3, CTLA-4, IL-10, TGFB-1, LAG-3, GITR
and TNFA as well as CD4 and CD8) in microdisected tumor and tumor
stroma areas by RTqPCR. The presence of CD4+, CD8+ and FOXP3+
(+ = positive) lymphocytes was also assessed in 84 of these FFPE samples
by immunohistochemistry (IHC). All statistical analysis were considered
significant at p < 0.05.
Results: Survival analyses revealed that patients with lower expression
levels of CD8 in tumor areas have worse overall survival (OS) and
progression free survival (PFS). Lower expression levels were also
associated with worse OS for CD4 and LAG-3 in tumor. We also found
that those patients with higher levels of FOXP3 stroma/FOXP3 tumor ratio
had worse OS and the same was observed for FOXP3 stroma/CD4 tumor
ratio and for FOXP3 stroma/CD8 tumor ratio. Furthermore, the presence
of FOXP3+ and CD8+ lymphocytes assessed by IHC was associated with
prognosis (Table 1).
Gene expression levels were dichotomized according to the median in high
and low.
Infiltration of FOXP3+ lymphocytes was defined as high (10% of total
lymphocytes) and low (<10%).
Infiltration of CD8+ lymphocytes was dichotomized in high and low according to the median of the absolute number of CD8+ lymphocytes (X400).
Conclusion: The presence of immune biomarkers in tumor microenvironment have a prognostic impact in resectable NSCLC patients.
Supported by grants PS09–01149 and RD12/0036/0025 from ISCIII.
Table 1 (abstract 133). Survival analysis (log-rank test).
Analyzed by RTqPCR
CD8 in tumor, low vs high
CD4 in tumor, low vs high
LAG3 in tumor, low vs high
FOXP3 stroma/FOXP3 tumor ratio, low vs high
FOXP3 stroma/CD4 tumor ratio, low vs high
FOXP3 stroma/CD8 tumor ratio, low vs high
Analyzed by IHC
CD8+ lymphocytes in tumor, low vs high
High FOXP3+ lymphocytes stroma/Low FOXP3+ lymphocytes tumor, vs others
High FOXP3+ lymphocytes stroma/Low CD8+ lymphocytes tumor, vs others
OS
Median (months)
p
PFS
Median (months)
p
37.2 vs 81.23
42.9 vs 81.23
36.2 vs 69
NR vs 42.9
81.23 vs 46.66
74.33 vs 46.43
<0.001*
0.026*
0.017*
0.002*
0.025*
0.017*
19.43 vs 81.23
23 vs 37.8
22.1 vs 30.1
NR vs 19.43
37.8 vs 22.13
37.8 vs 22.13
0.002*
0.058
0.085
<0.001*
0.027*
0.022*
40.4 vs 73.98
36.2 vs 69
17.43 vs 68.8
0.021*
0.020*
0.011*
23 vs 58.83
17.49 vs 35
15.3 vs 35.9
0.026*
0.16
0.035*
134
POSTER (Board P128)
Epigenetic immunomodulation by SGI-110 combined with immune
checkpoint blockade for new therapeutic strategies
A. Covre1 , C. Fazio1 , H.J.M.G. Nicolay2 , P.G. Natali3 , P. Taverna4 ,
M. Azab4 , S. Coral2 , M. Maio1 . 1 University Hospital of Siena, Medical
Oncology and Immunotherapy, Siena, Italy; 2 Epigen Therapeutics s.r.l.,
Pordenone, Italy; 3 University of Chieti, Lab CINBO, Chieti, Italy; 4 ASTEX
Pharmaceuticals Inc., Dublin, USA
Background: SGI-110 is a dinucleotide of decitabine (DAC) and
deoxyguanosine formulated as a low volume SQ injection that extends DAC
exposure compared to DAC IV. Our in vitro and in vivo evidences identified
a strong immunomodulatory activity of SGI-110 on human cancer cells
of different histotype and on human melanoma xenografts, and showed
a remarkable anti-tumor effect once combined with anti-CTLA-4 mAb in
a syngeneic mouse model. In this study we evaluated the contribution of
anti-tumor immune responses in the reduction of tumor growth achieved
by this therapeutic combination.
Materials and Methods: The mammary carcinoma cells TS/A (2×105 )
were implanted SQ in Balb/c mice. Animals bearing palpable tumors were
treated with 3 mg/kg of SGI-110 (days 1−5), alone or combined with 100 mg
of anti-murine CTLA-4 mAb (days 8, 11 and 14). The immunomodulatory
effects of treatment were studied on tumor and normal tissues by RTPCR and by quantitative RT-PCR analysis of murine CTA expression.
Immunohistochemical evaluation of tumor infiltrating immune cells was
also performed. P1A-promoter methylation was tested by quantitative
Methylation-Specific PCR (qMSP) on genomic DNA from tumor tissues.
Results: The expression of P1A and Mage-a family members was induced
in tumor tissues from animals treated with SGI-110, either alone or in
combination with anti-CTLA-4 mAb, but not from mice treated with antiCTLA-4 mAb alone. Levels of P1A-specific mRNA were similar in tumors
from mice treated with SGI-110 alone (3.18×10−04 P1A/b-actin molecules)
or combined with anti-CTLA-4 mAb (1.18×10−04 P1A/b-actin molecules).
The DNA hypomethylating effect of SGI-110 was sustained by the reduction
of P1A promoter methylation in cancer tissues from SGI-110- (16%) and
combination- (7%) treated mice vs control. Epigenetic remodelling was
restricted to tumor tissue leaving almost unaltered normal ones. The
contribution of immune cells in the therapeutic effectiveness of treatment
was supported by the increased frequency of tumor infiltrating CD3+ cells
in the combination arm (11±1.9) vs control (3.7±1.4) or single agent, antiCTLA-4 mAb (3±1.1) and SGI-110 (4.1±1.7), treated mice.
Conclusion: These data highlight the involvement of the immune system
in the anti-tumor effect of SGI-110 combined with CTLA-4 blockade. Based
on these experimental evidences, an exploratory phase I trial to evaluate
safety and immunobiologic activities of the combination is being activated
in advanced melanoma patients.
135
POSTER (Board P129)
AD-O64.3: IFN-g–TRAIL fusion protein. Use of two independent
signaling pathways for a strong synergistic antitumor effect
B. Zerek1 , J.S. Pieczykolan1 , S.D. Pawlak1 , P.K. Rozga1 , A. Pieczykolan1 ,
M. Szymanik1 , A. Jaworski1 , M. Galazka1 , K. Bukato1 , K. Poleszak1 ,
M. Teska-Kaminska1 , W. Strozek1 . 1 Adamed Sp. z.o.o., Drug Discovery,
Warsaw, Poland
Background: Interferon gamma (IFN-g) is a dimeric soluble cytokine, an
representative of Type II interferons group. Secreted by NK, NKT, Th1, Tc
and dendritic cells IFN-g ligand binds to two types of receptors and acts by
activation the JAK-STAT pathway.
Generally IFN-g antitumor action is mediated through the immunomodulatory function which results from the expression and action of many
different classes of IFN-stimulated genes (ISGs) responsible for antigen
processing, leukocyte trafficking and tumor cytotoxicity. IFN-g stimulates
antibody-dependent toxicity and potentiates the process of connecting the
cells with tumor cells. Additionally, it is able to induce an anti-proliferative
effects and activates caspases, thereby inducing apoptosis in many cancer
cell lines. IFN-g may cause Bak upregulation, cytochrome C secrection
and Bax translocation. In many tumor lines showing resistance to TRAILstimulated apoptosis, interferon gamma acted synergistically, contributing
to their sensitivity to TRAIL. Additionally, one of IFN-g effects is an intense
stimulation of human monocytes to produce TRAIL protein.
We developed the novel fusion protein AD-O64.3 consists of artificial dimer
of IFN-g, recombinant variant of human TRAIL fragment and sequences
recognized by tumor-specific proteases (MMP’s, uPa) in between.
Materials and Methods: AD-O64.3 protein was expressed in E. coli and
purified by IEC. Obtained protein was characterized biochemically and
biophysically using CD spectroscopy, SEC-HPLC, protease cleavage and
MTT cell cytotoxicity assays. Interaction with the respective receptors was
47
confirmed with SPR method. The proapoptotic effect was tested using
active caspase 3 staining. For in vivo potential we examined the efficacy
on mice xenograft models of human renal cell carcinoma (ACHN), human
colon adenocarcinoma (SW620) and human hapatoma (PLC/PRF/5) cell
lines.
Results: Obtained protein has well-defined secondary and quarternary
structure and partially verified mechanism of action. The molecule showed
in vitro specific cytotoxic effect on various cancer cell lines (IC50 below
0.1 ng/ml). New protein showed very low activity on normal cells. In vivo
scINFTR3.0 showed promising effect displaying significant tumor volume
inhibition.
Conclusions: We developed very promising molecule with high antitumor
activity showing synergistic effect with TRAIL.
136
POSTER (Board P130)
Tumor-infiltrating lymphocytes (TILs) following intratumoral
administration of ONCOS-102 are associated with prolonged overall
survival in last line solid tumor patients
S. Pesonen1 , T. Joensuu2 , E. Jager3 , J. Karbach3 , C. Wahle4 ,
K. Kairemo2 , K. Partanen2 , R. Turkki5 , A. Hemminki6 , C. Backman1 ,
M. von Euler1 , T. Hakonen1 , T. Ranki1 , L. Vassilev1 , A. Vuolanto1 ,
M. Jaderberg1 . 1 Oncos Therapeutics Ltd., Helsinki, Finland;
2
Docrates Cancer Center, Helsinki, Finland; 3 Krankenhaus Nordwest,
Hämatologie-Onkologie, Frankfurt, Germany; 4 Krankenhaus Nordwest,
Hämatologie-Onkologie, Helsinki, Finland; 5 Institute for Molecular
Medicine Finland, Helsinki, Finland; 6 University of Helsinki, Cancer Gene
Therapy Group Haartman Institute, Helsinki, Finland
Intratumoral administration of adenovirus activates Toll-like receptor signalling leading to production of pro-inflammatory cytokines and activation
of the innate immune system. Adenovirus causes immunogenic cancer
cell death and the subsequent release of tumor antigens in the presence of
danger signal results in the priming of a potent adaptive anti-tumor immune
response. This effect may be further enhanced by immune-stimulating
transgenes expressed by the virus. We present immunomonitoring results
from a phase I study with ONCOS-102, a chimeric oncolytic adenovirus
coding for GMCSF, in 12 patients with refractory injectable solid tumors.
A total of 9 intratumoral injections were given at three different dose levels.
Peripheral blood mononuclear cells (PBMCs) were collected at baseline
and during the study to assess the antigen specificity of CD8+ T cells by
IFN-gamma ELISPOT (analysis ongoing). The presence of tumor-infiltrating
lymphocytes (TILs) were analyzed at baseline and 1 and 2 months after
treatment initiation by immunohistochemistry (IHC). A computer assisted
quantification of IHC samples was performed by calculating the fraction of
a positively stained cellular region in the whole biopsy. In an exploratory
analysis, a correlation between the post-treatment increase in different subpopulations of TILs and overall survival (OS) was assessed by Spearman’s
rank correlation analysis.
Intratumoral ONCOS-102 triggered an innate immune response in every
patient as measured by a transient increase in systemic pro-inflammatory
cytokines and induction of fever within 6 to 10 hours post administration.
Infiltration of innate immune cells into tumors post-treatment was detected
in 11 out of 12 patients. Concomitant infiltration of T cells was detected in 11
out of 12 patients with the most prominent increase seen in CD8+ T cells.
Two patients showing the most robust increase in tumor-infiltrating CD8+
T cells post-treatment showed a prominent induction of tumor specific
CD8+ T cells in peripheral blood. Biopsies from a non-injected distant tumor
were obtained from one patient and a 2.5-fold increase in CD8+ T cells
following ONCOS-102 treatment was detected. The correlation between
OS and increase in TILs was analyzed. Among all sub-populations, CD8+
T cells (correlation coefficient (r) 0.74, p = 0.008) and CD68+ cells (r = 0.84,
p = 0.001) showed the most significant correlation with OS. A correlation
between post-treatment changes in CD8+ and CD68+ cells in tumors was
also detected (r = 0.59, p = 0.046).
Concomitant trafficking of innate and adaptive immune cells to the tumors
and the induction of tumor specific CD8+ T cells suggest that ONCOS102 is able to induce de novo anti-tumor immune responses in advanced
cancer patients. Correlation between TILs following treatment and OS, as
well as CD8+ T cell infiltration into a non-injected distant lesion, suggest
an involvement of a systemic immune activation.
48
137
POSTER (Board P131)
Immune checkpoint blockade enhances measles virotherapy
C.E. Engeland1 , R. Veinalde1 , C. Grossardt1 , S. Bossow1 ,
I. Shevchenko2 , V. Umansky2 , D.M. Nettelbeck3 , D. Jäger4 , C. von Kalle1 ,
G. Ungerechts5 . 1 National Center for Tumor Diseases (NCT), Translational
Oncology, Heidelberg, Germany; 2 German Cancer Research Center
(DKFZ), Clincal Cooperation Unit Dermato-Oncology, Heidelberg,
Germany; 3 German Cancer Research Center (DKFZ), Heidelberg,
Germany; 4 National Center for Tumor Diseases (NCT), Medical Oncology,
Heidelberg, Germany; 5 National Center for Tumor Diseases (NCT),
Translational Oncology and Medical Oncology, Heidelberg, Germany
139
POSTER (Board P133)
GBR1302: a BEAT® bispecific antibody for the treatment of HER2
positive cancers
A. Croset1 , J. Macoin1 , R. Ollier1 , M. Pluess1 , C. Delon1 , D. Skegro1 ,
S. Blein1 , S. Hou1 , J. Back1 . 1 Glenmark Pharmaceuticals S.A., R&D,
La Chaux de Fonds, Switzerland
Background: We hypothesized that combining oncolytic Measles virus
(MV) with immune checkpoint blockade using antibodies against CTLA-4
and PD-L1 can provide synergistic anti-tumor effects.
Material and Methods: Targeted immunomodulatory MV vectors encoding
anti-CTLA-4 and anti-PD-L1 antibodies were cloned and characterized
in vitro. Oncolytic efficacy was addressed in human melanoma xenografts.
Immunotherapeutic efficacy was evaluated in a fully immunocompetent
murine model of subcutaneous syngeneic malignant melanoma (B16CD20) and compared to parental MV with or without systemic administration of anti-CTLA-4 and anti-PD-L1.
Results: Viral replication and oncolysis of MV anti-CTLA-4 and MV antiPD-L1 were not impaired. In the B16-CD20 model, treatment with MV antiCTLA-4 and MV anti-PD-L1 led to an increase in tumor-infiltrating cytotoxic
T cells as well as a decrease in regulatory T cells. Therapeutic benefits in
terms of delayed tumor growth and prolonged median overall survival were
observed for animals treated with MV anti-CTLA-4 and MV anti-PD-L1,
respectively. Further investigations in preparation of a Phase I trial are
currently ongoing.
Conclusions: Combining oncolytic MV with immune checkpoint modulation is a promising approach with direct implications for future clinical
application.
Background: Human epidermal growth factor receptor 2 (HER2) is highly
expressed in a significant proportion of breast, ovarian and gastric cancers.
Since the discovery of its role in tumorigenesis and metastasis, HER2 has
received great attention in cancer research over the past two decades.
HER targeting therapies (such as Herceptin) have demonstrated efficacy
but are limited by several resistance mechanisms. Targeting, recruiting,
redirecting or bridging the cytotoxic T cells to HER2 tumor cells has
the potency to trigger a more efficient elimination of tumor cells. Using
the Glenmark BEAT® platform, we have produced a bispecific antibody,
GBR1302, targeting human CD3e and HER2 which is designed to recruit
cytotoxic T lymphocytes against HER2 positive cancer cells.
Material and Methods: Re-directed lysis (RDL) in vitro killing assays
using non-activated human peripheral blood mononuclear cells (PBMC)
as effector cells and multiple carcinoma cell lines over-expressing HER2
at different levels were performed to determine the potency of GBR1302.
In vivo, the potency of GBR1302 was evaluated using tumor models, in
which a mixture of human HER2 expressing tumor cells and human PBMCs
were xenografted subcutaneously in immuno-deficient mice (NOD-SCID).
Results: GBR1302 was able to bridge cytotoxic T lymphocytes to HER2
tumor cells and trigger killing of the tumor cells at a very low concentration.
A clear correlation between GBR1302 killing and HER2 expression levels
on target cells was demonstrated. In in vivo xenograft experiments,
GBR1302 induced a strong antitumor effect which was also dependent
on HER2 expression levels.
Conclusion: These data highlight a very good potency of GBR1302 for
HER2 overexpressing cancers and warrant further clinical studies with this
antibody.
138
POSTER (Board P132)
Preclinical activity and safety of MGD006, a CD123xCD3 Bispecific
DART® molecule for the treatment of hematological malignancies
140
POSTER (Board P134)
Relationship between programmed death ligand 1 (PD-L1) expression
and clinical outcome in patients (pts) with melanoma (MEL) treated
with pembrolizumab (pembro; MK-3475)
P. Moore1 , G.R. Chichili1 , L. Huang1 , H. Li1 , S. Burke1 , F. Chen2 , L. He1 ,
Q. Tang1 , L. Jin1 , S. Gorlatov1 , V. Ciccarone1 , S. Koenig1 , M. Shannon1 ,
R. Alderson1 , S. Johnson1 , E. Bonvini1 . 1 MacroGenics, Rockville MD,
USA; 2 MacroGenics, South San Francisco CA, USA
T-cell directed killing of tumor cells using bispecific molecules may hold
promise for cancer treatment. MGD006 (also encoded as S80880 and
known as CD3xCD123-DART) is a bispecific diabody-based molecule
consisting of two polypeptides, each composed of the VH of one antibody
in tandem with the VL of the second antibody and covalently linked
at the COOH termini via a disulfide bridge. MGD006 simultaneously
binds T lymphocytes and cells expressing CD123(IL3RA), an antigen
up-regulated in several hematological malignancies and differentially
expressed in AML blasts compared to normal hematopoietic stem and
progenitor cells. MGD006 mediates dose-dependent T-cell redirected killing
of CD123-positive cell lines as well as primary AML blasts. Furthermore,
MGD006 demonstrates potent activity in tumor-bearing mice engrafted
with human peripheral blood mononuclear cells or when tumors were coinoculated with human T lymphocytes. MGD006 binds to both human and
cynomolgus monkey antigens and can redirect T cells from either species
to kill CD123-expressing cells. Preclinical safety and pharmacology was
therefore assessed in monkeys that were infused with escalating doses of
MGD006 on continuous or intermittent schedules over a period of 4 weeks.
Monkeys infused with MGD006 showed depletion of circulating CD123positive cells, an activity biomarker, as early as 72 h after treatment initiation
and persisting throughout the 4-week treatment period. Furthermore,
T cells from treated monkeys exhibited efficient ex vivo redirected target cell
lysis, indicating no functional exhaustion. Transient release of cytokines,
particularly IL-6, was observed following the first MGD006 infusion, but
not after subsequent administrations even when the dose was escalated.
A reversible, decrease in red cell mass with concomitant reduction in
CD123-positive progenitors in the bone marrow was also observed.
No significant changes in circulating platelets or neutrophil levels were
observed.
The preclinical safety and efficacy profile of MGD006 supports advancement to clinical evaluation. A phase 1 safety study of MGD006 in relapse/refractory AML is currently recruiting patients (http://clinicaltrials.gov/
ct2/show/NCT02152956?term=mgd006&rank=1).
Note: Studies involving animal subjects were performed following institutional IACUC approval.
A. Daud1 , O. Hamid2 , C. Robert3 , F.S. Hodi4 , J.D. Wolchok5 , W.J. Hwu6 ,
J.S. Weber7 , R. Kefford8 , P. Hersey9 , A.M. Joshua10 , R. Joseph11 ,
T.C. Gangadhar12 , R. Dronca13 , A. Patnaik14 , H. Zarour15 , K. Gerigich16 ,
J. Lunceford16 , K. Emancipator16 , M. Dolled-Filhart16 , X. Li16 , P. Kang16 ,
S. Ebbinghaus16 , A. Ribas17 . 1 University of California San Francisco,
Medicine Division of Hematology and Medical Oncology, San Francisco
CA, USA; 2 The Angeles Clinic and Research Institute, Melanoma Center,
Los Angeles CA, USA; 3 Gustave Roussy and INSERM U 981, Medical
Oncology, Villejuif Paris-Sud, France; 4 Dana-Farber Cancer Institute,
Medicine, Boston MA, USA; 5 Memorial Sloan-Kettering Cancer Center,
Medicine, New York NY, USA; 6 The University of Texas MD Anderson
Cancer Center, Melanoma Medical Oncology, Houston TX, USA; 7 H. Lee
Moffitt Cancer Center, Cutaneous Oncology, Tampa FL, USA; 8 Crown
Princess Mary Cancer Centre Westmead Hospital and Melanoma Institute
Australia University of Sydney, Medicine, Sydney, Australia; 9 University of
Sydney, Medicine, Sydney, Australia; 10 Princess Margaret Cancer Centre,
Medical Oncology, Toronto, Canada; 11 Mayo Clinic, Medical Oncology,
Jacksonville FL, USA; 12 Abramson Cancer Center of the University of
Pennsylvania, Medicine, Philadelphia PA, USA; 13 Mayo Clinic, Oncology,
Rochester MN, USA; 14 South Texas Accelerated Research Therapeutics,
Phase I Research, San Antonio TX, USA; 15 University of Pittsburgh,
Medicine, Pittsburgh PA, USA; 16 Merck & Co. Inc., Molecular Biomarkers
and Diagnostics, Whitehouse Station NJ, USA; 17 University of California
Los Angeles, Hematology/Oncology, Los Angeles CA, USA
Background: In an initial analysis of the relationship between PD-L1
expression and efficacy of the anti-PD-1 monoclonal antibody pembro
(training set; n = 125), the preliminary cutpoint for PD-L1 positivity was
an Allred proportion score (APS) 2 (ie, staining in 1% of tumor cells).
Using this cutpoint, the PD-L1+ pts (71%) had a significantly higher ORR
(P = 0.0007) and longer PFS (P = 0.0051) than PD-L1− pts. We report the
results of an independent validation set to confirm the cutpoint and provide
pooled results of the training and validation sets.
Methods: The validation set included 150 PD-L1-evaluable pts with
ipilimumab naı̈ve and treated MEL treated with pembro 10 mg/kg Q3W
or 2 mg/kg Q3W in KEYNOTE-001. Response was assessed every 12
wk by RECIST 1.1 by independent central review. PD-L1 expression
was assessed in pretreatment tumor biopsies by IHC using the 22C3
Poster Session – Preclinical Models
49
Table (abstract 140): Relationship between tumor PD-L1 expression and clinical activity
Measurable disease and interpretable PD-L1 IHC
N
Validation Set
PD-L1+ (APS 2)
PD-L1− (APS <2)
HR (95% CI), PD-L1+ vs PD-L1−
2-sided P c
Pooled Set
PD-L1+ (APS 2)
PD-L1− (APS <2)
HR (95% CI), PD-L1+ vs PD-L1−
2-sided P
a
b
c
ORRa , n (%)
Interpretable PD-L1 IHC
N
PFSa
24-wk,
%
Median (range),
wk
OSb
12-mo,
%
Median (range),
mo
111
24
−
−
40 (36)
1 (4)
−
0.002
123
27
−
−
45
19
−
−
24
12
0.43 (0.27–0.69)
0.0002
67
43
−
−
NR
8
0.43 (0.24–0.78)
0.0042
194
54
−
−
81 (42)
5 (9)
−
<0.0001
212
63
−
−
51
29
−
−
24
12
0.50 (0.36–0.69)
<0.0001
72
53
−
−
NR
13
0.49 (0.33–0.74)
0.0005
Analysis cutoff: Oct 18, 2013.
Analysis cutoff: May 6, 2014.
PD-L1 association test.
antibody. APS included staining in tumor cells. Response rate, PFS, and
OS were compared between PD-L1+ and PD-L1− pts using Miettinen and
Nurminen’s method (ORR) and Cox regression (PFS, OS).
Results: 82% of validation set pts were PD-L1+ (APS2). There was a
significantly higher response rate and significantly longer PFS and OS for
PD-L1+ pts in the validation set (Table). The positive (PPV) and negative
(NPV) predictive values for ORR in the validation set were 36% and 96%.
77% of pooled set pts were PD-L1+ (Table). PPV and NPV for the pooled
set were 42% and 91%.
Conclusions: Tumor PD-L1 expression was associated with higher ORR
and prolonged PFS and OS in advanced MEL pts treated with pembro.
Although NPV is >90%, the high prevalence of PD-L1 positivity, durable
responses in PD-L1− pts, and unknown prognostic value and dynamic
nature of tumor PD-L1 expression in MEL suggest PD-L1 expression may
not be useful for selecting MEL pts for pembro treatment.
141
POSTER (Board P135)
Phase I study of ipilimumab with stereotactic radiosurgery for
melanoma patients with brain metastases
W. Shi1 , E. Wuthrick2 , K. Feeney3 , M. Werner-Wasik4 , D.W. Andrews5 ,
J.J. Evans5 , K. Judy5 , C. Farrell5 , A.P. Dicker4 . 1 Thomas Jefferson
University Hospital, Radiation Oncology, Philadelphia PA, USA; 2 Ohio
State University, Radiation Oncology, Philadelphia PA, USA; 3 Thomas
Jefferson University, Medical Oncology, Philadelphia PA, USA; 4 Thomas
Jefferson University, Radiation Oncology, Philadelphia PA, USA; 5 Thomas
Jefferson University, Neurological surgery, Philadelphia PA, USA
Background: Brain metastases (BM) occur in more than half of patients
with advanced melanoma. Stereotactic radiosurgery (SRS) is an effective
treatment for symptom palliation and provides a benefit in local control
for patients with limited BM. Ipilimumab (Bristol-Myers Squibb) is a
human monoclonal antibody targeted to cytotoxic T-lymphocyte Antigen 4
antibody (CTLA-4) receptor with survival benefit in patients with metastatic
melanoma. We hypothesize that combining SRS with Ipilimumab will yield
a synergistic effect. However, the toxicities of this combined therapy are
unknown. This is a phase I study is to assess the safety profile of combining
escalating doses of Ipilimumab with standard dose SRS.
Methods: Eligible patients had 4 BM with the largest lesion 4 cm,
or resection cavity 4 cm. Ipilimumab starting dose was 3 mg/kg once on
weeks 1, 4, 7, and 10. SRS was 15−24 Gy, based on the size, delivered on
day 1 of week 1. The primary endpoint is maximum tolerated dose (MTD)
assessed 30 days following SRS. Secondary endpoints include the rate of
new BM, extra-cranial disease response, overall survival (OS), progression
free survival (PFS), and adverse events.
Results: Nine patients with a median 1 BM (range, 1−3) were enrolled
from November 2011 to March 2014. Eight (89%) patients had extra-cranial
metastases. Five received ipilimumab 3 mg/kg and four patients received
10 mg/kg. The median follow up was 4 months (range, 2−19) and no doselimiting toxicities have been observed. All patients have local control of
treated index lesins, 80% of assessable patients have developed new BM,
and 80% of assessable patients have stable extra-cranial disease. Median
OS was not reached. Intracranial PFS at 4 months was 38.9%. PFS of any
site was 22% at 4 months. Adverse events were mild, grade 1−2 and grade
3 in six (66%) and one (11%) patient, respectively.
Conclusions: Concurrent ipilimumab and SRS is well tolerated in patients
with BM from melanoma. This phase I study will continue to assess the
MTD of ipilimumab for future phase II trials.
142
POSTER (Board P136)
IL-17 as a novel molecular target for prostate cancer prevention
A. De Angulo1 , R. Faris2 , C. Jolly1 , L. deGraffenried1 . 1 University of
Texas at Austin, Nutritional Sciences, Austin, USA; 2 Texas A&M University
Health Science, Microbial Pathogenesis and Immunology, Bryan, USA
Background: Prostate cancer represents 14% of all new cancer cases in
the US and is the fifth leading cause of cancer death. Incidence is closely
associated with aging, but the mechanisms by which aging promotes
prostate cancer development are unclear, limiting the development of
effective preventive interventions. Aging is accompanied by immune
dysfunction and a progressive increase in pro-inflammatory cytokines,
including interleukin 17 (IL-17). Aging-associated increases in circulating
IL-17 promotes pro-inflammatory signaling in prostate epithelial cells,
possibly contributing to tumorigenesis.
Methods: Serum and splenic CD4+ T-lymphocytes from young T cell
aging-mimic mice as well as young and aged wild-type mice were collected.
Surface markers and intracellular levels of IL-17, IFN-g and IL-4 in isolated
CD4+ T-cells were measured using flow-cytometry. shRNA was used to
knock down the IL-17 receptor in LNCaP prostate cancer cells and RWPE-1
non-transformed prostate epithelial cells, which were then exposed to
mouse sera or conditioned media from stimulated T-lymphocytes. NFkB and STAT3 activation, NF-kB and STAT3 target gene expression, and
epithelial cell transformation were all measured in prostate epithelial cells.
Results: T cells from aging and aging-mimic mice secrete elevated levels
of IL-17, possibly due to an imbalance in the TH17/TH1 and TH17/TH2 cell
ratios. T-lymphocyte-secreted IL-17 from aging-mimic mice induced NF-kB
and STAT3 activity and target gene expression in LNCaP and RWPE-1
cells. Importantly, IL-17 also promoted pro-tumorigenic changes in RWPE-1
cells, leading to a pre-transformed phenotype. Inhibition of IL-17 signaling
blocked age-induced changes in both the cancer and non-transformed
prostate epithelial cells.
Conclusion: Aging of the immune system is associated with increased
levels of IL-17, which stimulates the pro-inflammatory NF-kB and STAT3
pathways in prostate epithelial cells and activates programming associated
with cellular transformation. Inhibition of IL-17 signaling blocks the protumorigenic activity induced by aging T cells. These findings provide
evidence that the dysregulation of IL-17 production in aged T-cells may
directly contribute to the increased risk for prostate cancer. Future novel
immunotherapies for prostate cancer could target the increase production
of IL-17 associated with aging.
Preclinical Models
143
POSTER (Board P137)
Regional bias of intratumoral genetic heterogeneity of nucleotide
repeats in colon cancers with microsatellite instability
C. An1 , Y. Choi2 , M. Kim2 , J. Lee1 , N. Yoo2 , S. Lee2 . 1 Uijongbu St. Mary’s
Hospital, Surgery, Uijongbu City, Korea; 2 Catholic University of Korea,
Pathology, Seoul, Korea
Background: Intratumoral heterogeneity (ITH) may produce regional
biases in genotype and phenotype evaluation in a single tumor and may
impede proper cancer diagnosis.
50
Material and Methods: To evaluate the extent of ITH in colorectal
cancer (CRC) with microsatellite instability (MSI), we obtained 4−7 biopsies
from 39 CRCs followed by MSI analysis either using the Bethesda MSI
evaluation system or Promega system with 5 mononucleotide markers.
Results: We found decreased prevalence of MSI (+) by the Promega
system compared to the Bethesda system. The overall discordance
between the two systems was 54%. In contrast to the previous studies that
had shown discordance only in low MSI (MSI-L), our results showed the
discordance not only in MSI-L, but also in high MSI (MSI-H) cases. Among
the MSI (+) CRCs, ITH of MSI status was identified in 41.7% of CRC by
the Bethesda system and 22.2% by the Promega system. In terms of MSI
markers, the ITH originated from dinucleotide markers in most cases (69%),
but it originated from mononucleotide markers (31%) as well. Pooling of
DNA from a regional biopsy with MSI (+) with additional biopsies from
stable MSI (MSS) showed that this approach was beneficial to increase
the sensitivity of MSI detection.
Conclusions: Our results indicate that ITH of MSI phenotype by
the Bethesda system is more overestimated than previously identified.
However, because there was considerable ITH of MSI subtypes and
markers even by the Promega system, our data suggest that analysis of
MSI status in multiple regional biopsies is needed for a better evaluation
of MSI status in CRC.
144
POSTER (Board P138)
Association between interleukin 17/interleukin 17 receptor gene
polymorphism and papillary thyroid cancer
Y. Eun1 , H. Chung1 , Y.C. Lee1 . 1 Kyung Hee University, Otolaryngology,
Seoul, South Korea
Background: Interleukin 17 (IL17) is an important cytokine for inflammatory and autoimmune disease. Recently, although numerous studies have
been suggested the role of IL17 in tumor development, the mechanism
remains to be unknown. The aim of this study is to determine whether
single nucleotide polymorphisms (SNPs) in IL17 and IL17R contribute the
development of papillary thyroid cancer (PTC) and assess the relationship
between IL17 and IL17R SNPs and the clinicopathologic characteristics of
PTC.
Material and Methods: Eight SNPs located within the three genes of
IL17A, IL17RA and IL17RB were genotyped using direct sequencing in 94
patients with PTC and 213 patients without PTC (controls). Genetic data
were analyzed using commercially available software. And, the statistical
analyses were performed according to clinicopathologic characteristics of
PTC.
Results: Genotyping analysis demonstrated that the SNP rs4819554
of IL17RA (codominant model 1, OR = 0.39, P = 0.001; and dominant
model, OR = 0.45, 95% CI, P = 0.002) and the SNP rs1025689 of IL17RB
(dominant model, OR = 0.59, P = 0.043) were significantly associated with
the development of PTC. The SNP rs2275913 of IL17A (codominant
model 2, OR = 0.19, P = 0.034; dominant model, OR = 0.34, P = 0.033)
was significantly associated with multifocality. Furthermore, IL17RA SNP
rs4819554 (dominant model, OR = 0.25, P = 0.010) was significantly
associated with bilaterality of cancers.
Conclusion: In our case–control study of SNPs in the IL17 and IL17R
gene in patients with PTC, we demonstrated that IL17RA polymorphism
have the possibilities of developing PTC and can influence the bilaterality
of PTC.
145
POSTER (Board P139)
ARQ 087, a novel pan FGFR-inhibitor crosses the BBB (blood–brain
barrier) and distributes to the brain of rats
R. Savage1 , T. Hall1 , B. Schwartz1 . 1 ArQule Inc., Woburn, USA
Background: ARQ 087 is a potent multi-kinase inhibitor with pan-FGFR
activity against FGFR1, FGFR2, FGFR3 and FGFR4 kinases. Preclinical
data from human cell lines and xenograft models support the exploration
of its anti-tumor activity across a broad range of human solid and
hematological malignancies.
Material and Methods: QWBA (Quantitative Whole Body Autoradiography)
and metabolite profiling studies were conducted with 14 C-ARQ 087 in rats.
Free concentrations of 14 C-ARQ 087 related radioactivity were determined
in rat brain via microdialysis. The brain regions sampled by microdialysis
were the right striatum and left ventricle. The concentrations sampled in
the brain were compared to those obtained in peripheral blood sampled
from the right jugular vein.
Results: From QWBA studies, 14 C-ARQ 087 derived radioactivity was
found to distribute to the cerebellum, cerebrum, choroid plexus, corpus
callosum, lateral ventricle, meninges and spinal cord (as well as broadly
distributing to the rest of the body) after 5-Day repeat dosing in SpragueDawley rats. At 24 hours post dose on Day 5, 14 C-ARQ087 derived
radioactivity ranged from 0.540 mg equiv./g (cerebellum) to 4.12 mg equiv./g
(choroid plexus) versus 0.756 mg equiv./g in plasma. In brain tissues
AUC(0−24 h) ranged from 12.4 (cerebellum) to 408 (choroid plexus)
mg equiv.·hr/g versus 34.2 mg equiv.·hr/g in plasma. Metabolite profiling
data in rat brain showed that ARQ 087 is present primarily as parent drug.
Preliminary microdialysis data in rat brain showed that free unbound ARQ
087 (based on AUC) is present in the striatum (17.45%) and in the left
ventricle (16.30%) compared to that in systemic circulation.
Conclusions: ARQ 087 distributes to the brain of rats as well as broadly
to the rest of the body. Metabolite profiling and preliminary microdialysis
studies confirmed that in brain primarily parent ARQ 087 is present and
available in part as free ARQ 087.
146
POSTER (Board P140)
Establishment of patient-derived xenografts (PDX) models for triple
negative breast cancer (TNBC) as a pre-clinical platform for drug
development
J. Thatte1 , M. Meza1 , J. Ricono1 , T. Broudy1 , C. Mirsaidi1 , P. Nair1 .
1
Molecular Response, San Diego California, USA
TNBC comprises 15−20% of breast cancers in the United States. TNBC
lacks expression of estrogen receptor, progesterone receptor and receptor
tyrosine protein kinase ERBB2 or Her-2/neu oncogene amplification. As
a result, this type of breast cancer is difficult to treat as most of the
chemotherapies target these 3 receptors. TNBC is an aggressive form
of cancer associated with high morbidity, mortality and shorter diseasefree survival. Prognosis and management of TNBC is complicated due to
its heterogeneous clinical presentation, histology and response to therapy.
PDX models in immune compromised mice have become a valuable tool for
preclinical drug development in recent years. Here, we report development
and characterization of the TNBC PDX model. We used patient derived
TNBC tissues to generate 5 new TNBC models in NOD-SCID mice. Patient
tumors were pre-screened for their ER, PR and Her-2/Neu expression
by IHC, prior to inoculation in mice. We report efficacy of two standard
of care drugs, cisplatin and vinorelbine, in the TNBC PDX model which
demonstrates the potential utility of the TNBC model in drug discovery
effort in oncology for treatment of TNBC.
147
POSTER (Board P141)
An integrated approach for identifying E-cadherin synthetic lethality
networks
I. Bajrami1 , S.J. Pettitt1 , R. Brough1 , H. Pemberton1 , D. Kastrev1 ,
Y. Fontebasso1 , J. Frankum1 , J. Campbell1 , A. Ashworth1 , C.J. Lord1 .
1
Institute of Cancer Research, Division of Breast Cancer, London, United
Kingdom
The E-cadherin (CDH1) tumour suppressor gene encodes a calciumdependent cell–cell adhesion glycoprotein, which has roles in maintaining cell polarity, differentiation, cell migration and survival. E-cadherin
dysfunction is a feature common to many epithelial tumours, with the
highest incidence occurring in diffuse gastric cancer (50%) and lobular
breast cancer (56%) and can occur via CDH1 mutation, deletion or
epigenetic silencing. Although E-cadherin dysfunction is relatively common,
approaches to target this pathogenic alteration do not as yet exist.
We have taken an integrated functional genomics approach to identifying
E-cadherin synthetic lethality effects that exploits siRNA. Using a
combination of Achilles’ Heel siRNA and small molecule inhibitor screens
in histologically and genetically diverse tumour cell line panels, we have
identified a compendium of genes whose disruption selectively targets
E-cadherin deficient cells. Alongside the Achilles’ Heel screens in tumour
cell models, complementary screens in isogenic systems with shRNA
and CRISPR-engineered E-Cadherin defects will also be presented. As
an orthogonal approach, we have also exploited piggyBac transposonbased mutagenesis in haploid ES cells to generate a diverse mutant library
consisting of 100,000 mutants which are now being used in E-cadherin
synthetic lethal screens.
Together, these systems provide a framework for identifying candidate
synthetic lethal effects suitable for mechanistic dissection and subsequent
validation. The results from these complementary approaches and
subsequent validation of selected effects will be presented.
148
POSTER (Board P142)
RAS synthetic lethal interactions from yeast to human cells
S. van Wageningen1 , A. Prahallad1 , G. Heynen1 , R. Rothstein2 ,
R. Bernards1 . 1 Netherlands Cancer Institute Antoni van Leeuwenhoek
hospital, Molecular Carcinogenesis, Amsterdam, Netherlands; 2 Columbia
University, Genetics Department, New York, USA
Background: Synthetic lethal (SL) interactions are used to develop
targeted cancer therapy. However, novel SL interactions discovered in
mammalian cell cultures are often cell type specific and are therefore only
relevant to a small, or difficult to define, subset of patients. We developed a
strategy in which we prioritize potential SL drug targets using the genetically
tractable model system Saccharomyces cerevisiae.
Material and Methods: Weperformed a SL screen by expressing a
constitutively active RAS allele, RAS2(V19), in ~4800 S. cerevisiae strains
in which each individual gene is deleted. Next we tested if SL interactions
were conserved in human cancer cell lines.
Results: The yeast screen yielded a hit list highly enriched for mutants
with a defect in ‘endoplasmic reticulum (ER)-to-Golgi-to-vacuole’ transport.
Moreover, we found that this list had a significant overlap with strains
sensitive to b-mercaptoethanol, DTT and tunicamycin. We hypothesized
that ER homeostasis was disturbed in these cells. The two gene deletion
mutants most sensitive to ER stress are IRE1 and HAC1. These genes
make up the unfolded protein response (UPR) in yeast; the signaling
pathway that restores ER homeostasis. Both UPR genes were SL with
RAS2(V19). Next we asked if we could detect a SL interaction between
oncogenic RAS and the UPR in human cells. We find that a SL interaction
between oncogenic RAS and the UPR is dependent on specific RAS
effector pathways in human cell cultures.
Conclusions: The UPR is conserved in evolution. However, signaling
pathways downstream of RAS have diverged over time. We will present
how the interaction between oncogenic RAS and the UPR has evolved
in human cells and how this interaction can be exploited for therapeutic
intervention.
149
POSTER (Board P143)
Quantitative mutational assessment of circulating tumor DNA using
massively parallel deep sequencing in plasma and urine from
advanced colorectal cancer patients
J.C. Poole1 , C.R.T. Vibat1 , L. Benesova2 , B. Belsanova2 , S. Hancock1 ,
T.L. Lu1 , M.G. Erlander1 , M. Minarik2 . 1 Trovagene Inc., R&D, San Diego,
USA; 2 Genomac Research Institute, Center for Applied Genomics of
Solid Tumors, Prague, Czech Republic
Background: Technologies enabling the assessment of circulating tumor
DNA (ctDNA) in biofluids expand the clinical utility to detect and monitor
cancer patient oncogenic mutations by minimally invasive and non-invasive
liquid biopsy methods. Mutational tumor load quantification with high
clinical sensitivity is vital for robust individualized assessment of systemic
therapeutic responsiveness and resistance. A quantitative ctDNA assay
using a massively parallel deep sequencing approach was developed to
determine patient ctDNA mutational status.
Material and Methods: Initial assay development was for the simultaneous
detection of 13 known (7 reported) oncogenic mutations in KRAS
codons 12/13. An ultrashort 31bp region encompassing KRAS codons
12/13 was PCR amplified; G12A/C/D/R/S/V, and G13D mutations were
enriched by suppressing wild-type (WT) sequence amplification with a
WT blocking oligo. Barcoded adaptor primers were added for compatibility
with massively parallel deep sequencing. Limits of detection (LOD) were
independently determined for each of the 7 KRAS mutations by spiking
5–500 copies of each mutant into 60 ng of a WT genomic background.
Limits of quantitation (LOQ) were confirmed with 7 copies of each mutation
in an increasing WT genomic DNA background of 60–360 ng. Archived,
matched plasma and urine samples (stored between 3−5 years prior to
ctDNA extraction) from 20 treatment naı̈ve, advanced cancer patients with
known tumor tissue KRAS mutations determined by an accredited clinical
laboratory, were used in a retrospective setting for a blinded pilot study.
These samples were used to compare KRAS status in urine and plasma
to tumor tissue, and assess clinical sensitivity of the ctDNA assay.
Results: LOD data for 5–500 KRAS G12A/C/D/R/S/V, and G13D mutant
copies in 60 ng WT DNA showed a highly correlative response with an
average R2 of 0.90 for the 7 mutations evaluated. LOQ assessed for each
mutation in an increasing WT DNA background, revealed a robust signal for
each mutation versus WT alone; an estimated analytical LOD of 7 copies
per ~100,000 genome equivalents (0.007%) was observed. Of 20 blinded
retrospective plasma ctDNA samples evaluated, 19 (95%) displayed the
KRAS mutation concordant with tumor tissue. Of 20 matched urine samples
tested, 16 were deemed evaluable; 15 (94%) had a significant sequence
call consistent to tumor and to plasma.
51
Conclusion: The developed method for quantitative massively parallel
deep sequencing of ctDNA for KRAS mutational assessment has reported
high clinical sensitivity in plasma and urine. This technical approach is
scalable and has the potential for detecting and quantifying a multitude
of genomic alterations indicative of therapeutic responsiveness and
resistance. Expansion of retrospective colorectal patient cohort described
herein will be reported.
Partially supported by the Czech Ministry of Health grant NT 13660.
150
POSTER (Board P144)
Pirin downregulates E-cadherin gene expression and contributes to
EMT
K. Komai1 , Y. Niwa1 , Y. Sasazawa1 , S. Simizu1 . 1 Keio University, Faculty
of Science and Technology, Yokohama, Japan
Background: Downregulation of E-cadherin, a cell–cell adhesion protein,
induces epithelial–mesenchymal transition (EMT), which plays crucial roles
in metastatic progression. A nuclear protein pirin enhances NF-úB related
transcription by binding to Bcl3-p50 complex, which is important for
the SLUG expression and melanoma migration. Additionally, proteomics
analysis indicated that pirin expression was decreased in metastatic
adenoid cystic carcinoma cells. Although these reports suggest that pirin
may be involved in tumor metastasis, there are no reports which directly
demonstrate the contribution of pirin to metastasis. Here, we investigated
the effects of pirin on EMT which associates with metastasis, and its
mechanisms.
Material and Methods: Pirin was overexpressed or silenced in HeLa cells,
and then EMT-related genes and protein expressions were detected. The
morphological changes of pirin stably-expressing HeLa cells were measured by employing parameter of ‘circularity’, [4p(area)/(perimeter)2 ]×100,
which decreases by morphological changes from cobblestone-like epithelial
cells to spindle-like mesenchymal cells. The effect of pirin on cell migration
and anticancer drug resistance was measured by wound healing assay and
MTT assay, respectively. The binding of wild-type pirin or its mutant to Bcl3
was confirmed by GST pull-down assay using recombinant GST-Bcl3.
Results: Knockdown of pirin increased E-cadherin gene expression
whereas overexpression of pirin decreased its level. Pirin stably-expressing
HeLa cells exhibited spindle-like morphology and loss of cell–cell adhesion,
which are reminiscent of EMT. From the result of MTT assay, we
demonstrated that pirin contributed to acquire anticancer drug resistance.
Furthermore, RNAi experiment revealed that pirin positively regulated A549
cell migration. Next, we examined whether Bcl3, a binding partner of pirin, is
involved in EMT induction by pirin overexpression. GST pull-down analysis
indicated that Pirin/E103A mutant was decreased its binding ability to Bcl3;
however, as with wild-type pirin, this mutant also downregulated E-cadherin
gene expression, suggesting that pirin decreases E-cadherin expression in
Bcl3-SLUG-axis-independent manner.
Conclusions: Pirin downregulates E-cadherin gene expression in Bcl3
independent manner, and contributes to EMT and cancer malignancy.
These data provide evidence that pirin may be a potent target toward
cancer therapy.
151
POSTER (Board P145)
Impact of EGFR amplification pattern on the expression of
miRNA-200c in primary glioblastoma multiforme
L. Muñoz Hidalgo1 , C. López Gines2 , E. Serna3 , D. Monleon1 ,
R. Callaghan2 , R. Gil Benso2 , H. Martinetto4 , A. Gregori Romero2 ,
J. Gonzalez Darder5 , M. Cerda Nicolas2 . 1 Fundation HCU-INCLIVA,
Pathology, Valencia, Spain; 2 University of Valencia, Pathology, Valencia,
Spain; 3 University of Valencia, UCIM, Valencia, Spain; 4 Institute FLENI,
Neurological, Buenos Aires, Argentina; 5 Clinical Hospital, Neurosurgery,
Valencia, Spain
Glioblastoma Multiforme (GBM) is the most common tumor in the primary
tumors of the central nervous system, accounting for 60% of neoplasms
in this location. It is a highly aggressive tumor with a median survival of
twelve months. Heterogeneity in the biological behavior of this neoplasm of
astrocytic glial origin is expressed in the infiltrative nature, this is a critical
feature in glioblastoma.
Several miRNAs have been related with different types of cancer, some of
them related with ability of modulation behavior neoplastic cells expressed
epithelial/mesenchymal changes. In this regard, miRNA expression is
deregulated in most, if not all, types of cancer. Based on the literature
the most common dysregulation of miRNAs in GBM is over-expression;
more than two hundred miRNAs have been found to be significantly overexpressed.
We have evaluated whole-genome miRNA expression profiling associated
with different EGFR amplification patterns in primary glioblastoma multiforme, whose clinicopathological and immunohistochemical features have
52
also been analyzed. All samples used for histopathological examination
were fixed in neutral-buffered formalin, embedded in paraffin, sectioned
and stained with hematoxylin-eosin. The immunohistochemical study
was performed on paraffin-embedded sections using the avidin–biotin
peroxidase method. To evaluate EGFR gene status, dual-color FISH
analysis was performed on paraffin tissue arrays from 30 samples.
Epigenetic analysis study of miRNAs and mRNA in 30 samples was
performed using miRNA and mRNA Genechip Array (Affymetrix, Santa
Clara, CA, USA). Expression levels of the selected miRNA (mir-200c)
and mRNAs (CDH1, EGFR and ZEB1) were quantified using real-time
reverse transcription-PCR (RT-PCR) analysis. MirRNA-200c showed a very
significant difference between tumors having or not EGFR amplification.
With respect to EGFR status our cases were categorized into three groups:
high level EGFR amplification, low level EGFR amplification, and no EGFR
amplification. Our results showed that microRNA-200c and E-cadherin
expression are down-regulated, while ZEB1 is up-regulated, when tumors
showed a high level of EGFR amplification. Conversely, ZEB1 mRNA
expression levels were significantly lower in the group of tumors without
EGFR amplification. Tumors with a low level of EGFR amplification showed
ZEB1 expression levels comparable to those detected in the group with
a high level of amplification. In this study we provide what is to our
knowledge the first report of association between mirRNA-200c and EGFR
amplification in glioblastomas.
The mirRNA-200c plays an important role in epithelial–mesenchymal
transition, but its implication in the behavior of glioblastoma is largely
unknown, and we suggest that microRNA-200c may act as a potential
regulator of glioblastoma migration and invasion by targeting ZEB1 mRNA.
152
POSTER (Board P146)
Preclinical characterization of MM-151, an oligoclonal antibody
therapeutic that targets EGFR by three distinct mechanisms of action
A. King1 , M. Sevecka1 , N. Gerami-Moayed1 , O. Burenkova1 , J. Kearns1 ,
G. Tan1 , C. Sloss1 , R. Bukhalid1 , U. Nielsen1 , B. Wolf1 . 1 Merrimack
Pharmaceuticals, Cambridge, USA
Background: EGFR is a well-documented driver of solid tumor growth
and a validated therapeutic target. However, currently approved EGFRtargeting monoclonal antibodies have limited clinical benefit, indicating the
need for improved antibody therapeutics. We hypothesized that sub-optimal
inhibition of the EGFR network and engagement of the immune system
may limit the activity of current monoclonal antibody therapeutics, such
as cetuximab and panitumumab. MM-151, an oligoclonal combination of
three fully human IgG1s with subnanomolar affinities and non-overlapping
epitopes, was developed using a systems biology approach to address
these unmet needs.
Methods: Preclinical characterization studies were performed, including
signaling, proliferation, and immune effector assays to elucidate the
mechanisms of action of MM-151 and to differentiate it from current
monoclonal antibody therapeutics.
Results: Our characterization assays demonstrate that MM-151 has
three distinct mechanisms of action: ligand antagonism, receptor downregulation, and the pathway extrinsic activities of complement-dependent
cytotoxicity and antibody-dependent cellular cytotoxicity. MM-151 displayed
an advantage over cetuximab for all three mechanisms in the preclinical
assays.
Seven redundant ligands with a wide range of affinities can activate
EGFR. We therefore examined the ability of MM-151 to antagonize
ligand-driven signaling and proliferation in detail. Initially, we compared
MM-151 with cetuximab and observed similar inhibition of signaling and
proliferation driven by each of the three low-affinity ligands, but MM-151
showed increased inhibition against each of the four high-affinity ligands.
A bioinformatics analysis using primary tumor data from The Cancer
Genome Atlas revealed expression of distinct ligand mixtures comprised
of both low- and high-affinity ligands across indications. MM-151 provided
superior inhibition of proliferation with indication-specific ligand mixtures.
MM-151 was also active against a cetuximab-resistant cancer cell line with
upregulated expression of high-affinity ligands.
Conclusions: MM-151 is a EGFR-targeting antibody combination designed using systems biology to overcome ligand redundancy and
signal amplification within the EGFR network. MM-151’s novel oligoclonal
composition enables enhanced antagonism of EGFR ligands, EGFR downregulation and immune effector function. These preclinical studies provide
a rationale for clinical study of MM-151.
153
POSTER (Board P147)
Moving beyond in vitro models and addressing the challenges of
pooled RNAi screens in mouse xenografts
D. Tedesco1 , K. Bonneau1 , M. Makhanov1 , D. Deng1 , P. Sun2 ,
A. Chenchik1 . 1 Cellecta Inc., Mountain View CA, USA; 2 The Scripps
Research Institute, La Jolla CA, USA
RNAi loss-of-function screening with pooled shRNA expression libraries
has proven to be a useful tool to identify essential cancer genes and
drug resistance mechanisms in numerous cell lines in vitro. The effects
of knocking down large numbers of genes on cancer cell growth and
viability can be measured simultaneously using this approach. Now there
is considerable interest to adapt RNAi viability screens to ex vivo xenograft
mouse models. However, small take rates, growth rate variance of cells,
and differing cell microenvironments in these more complex tumor models
impose significant challenges to this sort of assay. We have developed a
novel approach based on the use of pooled shRNA libraries with clonal
barcodes that enables proliferation measurements of clonal populations
produced by the individual shRNA-expressing founder cells that produce
the tumor. This RNAi clonal analysis provides a basis to separate, across
large cell populations, the external parameters that strongly influence cell
growth rates from shRNA-induced growth inhibition in these systems. We
will present in vivo RNAi screening validation data for several cancer
models.
154
POSTER (Board P148)
Drug response database with PDX tumor models in biomarker-driven
multi-drug multi-arm clinical trial settings
J. Jiang1 , T.F. Yu1 , Y. Yan1 , W. Du1 , T.T. Tan1 , L. Hua1 , J.L. Gu1 , X.Q. Yang1 ,
Z.H. Liu1 , X.K. Ye1 , Z. Gu1 . 1 GenenDesign, Oncology, Shanghai, China
Recent advances in affordable genome sequencing and molecular
profiling provide opportunities to better understand the complexity and
heterogeneity of cancer, and perform multi-biomarker diagnosis in a
single comprehensive test for personalized clinical treatment. A new
biomarker-driven multi-drug multi-arm clinical trial in lung squamous cell
carcinoma (Lung-MAP) uses cutting-edge genomic profiling to match
patients with investigational treatments targeting their unique tumor
mutations. However, there are still difficulties in enrolling enough patients
with specific parameters, comparing different regimes on same clinical
tumor and validating predictive biomarkers, especially those for drugs in
early development stage.
Patient derived xenograft (PDX) tumor models have been proved to
recapitulate the complexity and heterogeneity of their corresponding human
tumors by phenotypic and genomic characterization, and thus become
to be widely used in recent years in preclinical setting to facilitate drug
discovery, translational studies and clinical trials support. To further meet
the increasing needs and complement the current clinical practice for
precision medicine, better characterized PDX models with genomic profiles
and drug response information to multiple chemo/targeted therapies are in
demand.
GenenDesign has established over 700 PDX tumor models and derived
around 100 resistance models to drugs of interest. Through our in-house
efforts, PDX models of different tumor types were tested with related SOCs
and clinical candidates in biomarker-driven multi-drug multi-arm clinical
trial settings. So far, more than 800 data sets have been generated,
including responses to chemotherapy drugs, targeted inhibitors against
HER2, EGFR, FGFRs, c-Met, MEK and Ras/Raf pathway, as well as
PI3K/Akt pathway etc. Moreover, genomic profiling data of many PDX
model have also been acquired at hot-spot mutation, gene expression,
gene copy number and RNA/Exome sequence levels. The combination
of functional and molecular information will help to design and support
clinical trials, discover predictive biomarkers and unravel underlying drug
resistance mechanisms.
155
POSTER (Board P149)
SNIPER(TACC3) degrades TACC3 protein via the ubiquitin–
proteasome pathway and induces apoptosis in cancer cells
expressing a large amount of TACC3
N. Ohoka1 , K. Nagai2 , K. Okuhira1 , N. Shibata1 , T. Hattori1 , N. Cho2 ,
M. Naito1 . 1 National Institute of Health Sciences, Division of Biochemistry
and Molecular Biology, Tokyo, Japan; 2 Takeda Pharmaceutical Co. Ltd.,
Medicinal Chemistry Research Laboratories Pharmaceutical Research
Division, Fujisawa, Japan
Microtubule inhibitors are widely used as anti-cancer drugs. They arrest
cancer cells and often induce mitotic catastrophe and cell death. However,
they also affect microtubule function in non-dividing cells, which limit their
utility. Recently, inhibitors of spindle-regulatory proteins have attracted
considerable attention, and transforming acidic coiled-coil-3 (TACC3) is a
spindle-regulatory protein overexpressed in many human cancers.
We have developed a protein knockdown system to induce degradation of
target proteins via the ubiquitin–proteasome system in cells with hybrid
molecules named SNIPER (Specific and Non-genetic IAP-dependent
Protein ERaser). In this study, we designed and synthesized novel
SNIPER(TACC3)s that target TACC3 for degradation, and evaluated their
activity in vitro.
SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation
of TACC3, and reduce the TACC3 protein level in cells. Mechanistic
analysis indicated that the ubiquitin ligase APC/CCDH1 mediates the
SNIPER(TACC3)-induced degradation of TACC3. Cancer cells express
larger amount of TACC3 than do normal fibroblasts, and SNIPER(TACC3)
selectively induced cell death in cancer cells.
These results suggest protein knockdown of TACC3 by SNIPER(TACC3)
is a potential strategy to treat cancers overexpressing the TACC3 protein.
156
POSTER (Board P150)
Behaviour of platinum(IV) complexes with prodrug function in
different models of hypoxia
E. Brynzak1 , P. Heffeter2 , V. Pichler1 , M.A. Jakupec1 , B.K. Keppler1 .
1
University of Vienna, Institute of Inorganic Chemistry, Wien, Austria;
2
Medical University of Vienna, Institute of Cancer Research, Wien, Austria
Background: Solid tumours frequently contain regions of low oxygen
concentration, which have been identified as important component for
drug modification, therapy resistance and metastasis formation. Selective
targeting of this tumour hypoxia might be a proper strategy to overcome
current therapeutic limits. Therefore platinum(IV) compounds with prodrug
function, which require the reductive environment of tumor hypoxia for their
biological activation, can be highly selective therapeutics.
Material and Methods: To investigate the cytotoxicity of platinum(IV)
compounds we used the AlamarBlue assay in hypoxic spheroid models
of CH1 (ovarian carcinoma), HT1080 (fibrosarcoma) and HCT116 (colon
carcinoma). Evidence of hypoxia has been provided by antibody staining
of HIF-1alpha, and propidium iodide staining revealed necrotic regions in
spheroid centres. Distribution of the drug within the spheroids has being
determined by LA-ICP-MS. Further investigations of compound activity in
an in vivo mouse model are ongoing.
Results: Two test compounds were found to be at least 2−6 times more
potent in hypoxic CH1 and HT1080 spheroid models than in monolayer
culture, while satraplatin, another platinum(IV) compound, was 2 times less
active. Furthermore platinum(II) complexes such as cis- and oxaliplatin
were found to be 2−11 times less active in hypoxic models. Distribution
studies showed adequate spheroid penetration for all tested compounds
and give evidence of platinum(IV) accumulation within hypoxic regions.
Conclusions: In this study we showed an advantage of platinum(IV)
prodrugs over platinum(II) therapeutics currently used in the clinic and
pointed out relevance of spheroid based in vitro models for drug screenings.
157
POSTER (Board P151)
Sensitive and specific detection of 1p/19q codeletion in gliomas by
next generation sequencing
E. Dubbink1 , P.N. Atmodimedjo1 , R.M. van Marion1 , J.M. Kros1 ,
M.J. van den Bent2 , W.N.M. Dinjens1 . 1 Erasmus MC, Pathology,
Rotterdam, Netherlands; 2 Erasmus MC Cancer Insitute, Neuro-oncology,
Rotterdam, Netherlands
Molecular subtyping of malignancies from the central nervous system
becomes increasingly important to establish histological diagnosis and
to predict differential treatment outcome of histologically similar tumors.
One of these molecular markers is deletion of chromosomal arms 1p
and 19q. Current methods to detect 1p/19q codeletion involve fluorescent
in situ hybridisation (FISH) and loss of heterozygosity (LOH) analysis
using polymorphic microsatellite markers. We will present a novel method
to detect these aberrations based on single nucleotide polymorphism
(SNP) analysis using next generation sequencing (NGS) on an Ion Torrent
platform. We show that targeted NGS analysis of multiple heterozygous
SNPs spread over the entire chromosomal arms 1p and 19q allows
sensitive (down to 40% of tumor cells) and reliable detection of 1p and/or
19q deletion in gliomas. The strength of this method is that it accurately
detects allelic imbalance in small amounts of suboptimal quality DNA
derived from routine formalin-fixed, paraffin-embedded (FFPE) tissue in a
fast turnaround time and that it can be easily adapted to perform additional
mutational analysis of genes of interest. The test is therefore perfectly
suited to become standard practice for routine glioma diagnostics.
53
158
POSTER (Board P152)
The development of a series of orthotopic solid tumour models of
prostate, lung and ovarian cancer using optical and X ray imaging
M. Batey1 , M. Brown1 , E. Bowden1 . 1 Epistem Ltd, Manchester, United
Kingdom
Background: One of the most important aspects in anti cancer drug
development is the availability of robust preclinical models which allow
the efficacy of novel therapies to be examined. Several orthotopic models
have been described where cancer cells are grown at clinically relevant
sites. The objective is that these models, compared to more conventional
subcutaneous models, will provide a more realistic environment, and
will be superior models for therapeutic evaluation. A major difficulty with
such techniques however is assessment of disease burden, with deep
lying or metastatic tumours not being amenable to the usual methods
of measurement. The introduction of non invasive small animal imaging
has opened up the possibility of using such models in preclinical drug
development programs, and in time the prospect of lower drug attrition
rates in human trials. Here we discuss the development of three preclinical
imaging models of solid human cancers, using the prostate line LnCAP, the
ovarian line A2780, and the lung line A549.
Materials and Methods: Following stable lentiviral transduction of these
cell lines with a vector expressing firefly luciferase and GFP, LnCAP cells
were implanted directly into the testis of male SCID mice, A2780 cells
were implanted into the intraperitoneal space of female CD-1 nude mice,
and A549 cells were implanted into the left lung of female CD-1 nude
mice. Using the Bruker In Vivo Xtreme System, animals were monitored
following initial transplantation, and disease burden quantified longitudinally
over several weeks using co-registered 2D and 3D bioluminescent and
X-ray detection.
Results: Establishment of disease can be measured in each individual
animal prior to commencement of therapy, and can be tracked in each
animal throughout the duration of the study, with sites of metastasis readily
visualised. In untreated animals the A2780 model runs over approximately
8 weeks, with multiple sites of solid tumour formation and frequent
associated tumour ascites. Control mice in the A549 model are viable for
approximately 6 weeks, with metastatic tumour development common in
the model and a third of mice developing a pleural effusion. The LnCAP
model runs over approximately 10 weeks, with the local development of
dense, highly vascularised tumours, which show some invasion into the
surrounding tissues. In all models visualisation of engrafted disease by
imaging is possible at an early stage, and all show consistently high take
rates.
Conclusion: The ability to quickly identify engrafted animals, and the
predictable development of disease demonstrated allows for improved
randomisation based on disease signal, early therapeutic intervention,
and extension of the treatment window. Efficacy of treatment can be
easily followed by optical imaging, and as such, these models provide an
attractive, clinically relevant environment for the preclinical assessment of
novel therapeutics and combination treatment regimens.
159
POSTER (Board P153)
Neoadjuvant chemotherapy in breast cancer patients induces
expression of miR-34a and miR-122
P. Freres1 , C. Josse1 , N. Bovy2 , M. Boukerroucha3 , I. Struman2 ,
V. Bours3 , G. Jerusalem1 . 1 C.H.U. Liege, Laboratory of Medical
Oncology, Liège, Belgium; 2 GIGA-Research, Unit of Molecular Biology
and Genetic Engineering, Liège, Belgium; 3 GIGA-Research, Human
Genetics, Liège, Belgium
Background: Circulating microRNAs (miRNAs) are extensively studied in
cancer as biomarkers but little is known about the influence of anti-cancer
drugs on their expression. In this article, we describe the modifications
of circulating miRNAs profile under neoadjuvant chemotherapy (NAC) for
breast cancer.
Methods: The expression of 188 circulating miRNAs was assessed by
RT-qPCR in plasma of 25 patients before and after NAC. 2 miRNAs
significantly increased under NAC, miR-34a and miR-122, were measured
in the tumor tissue before and after the chemotherapy for 7 patients
with pathological partial response (pPR) to NAC. These 2 chemotherapyinduced miRNAs were further studied in the plasma of 22 patients with
adjuvant chemotherapy (AC) as well as in 12 patients who did not receive
any chemotherapy and 20 healthy women.
Results: 25 plasma miRNAs are found significantly modified by NAC.
Tumor suppressor miR-34a and miR-122 are highly upregulated at the
end of the NAC, notably in pPR patients with aggressive breast cancers.
Furthermore, miR-34a level is elevated in remaining tumor tissue after
NAC treatment. Studying the kinetic of circulating miR-34a and miR-122
expression during NAC reveals that their levels are especially increased
54
after anthracycline-based chemotherapy. AC induces lower levels of plasma
miR-34a and doesn’t modify miR-122. The tumorectomy alone doesn’t
deregulate miR-34a and miR-122. Circulating miR-34a and miR-122 are
downregulated in NAC treated breast cancer patients compare to controls
and normalized after treatments.
Conclusion: This study demonstrates for the first time that NAC specifically
induces expression of tumor suppressor miRNAs in plasma and tumor
tissue that might be involved in the anti-tumor effect of the chemotherapy.
160
POSTER (Board P154)
Identification of fusion genes through kinome-centered RNA
sequencing in different types of solid tumors
L. Mittempergher1 , C. Sun1 , F.H. Groenendijk1 , A.J. Bosma1 ,
S.M. Willems1 , T. Sustic1 , I.J. Majewski1 , W. Grernrum1 , N.M. Davidson2 ,
I. de Rink3 , H.M. Horlings1 , W. Theelen-Engelsman4 , S.F. Chin5 ,
A. Oshlack6 , B.W. van Rhijn7 , M. van den Heuvel4 , M.S. van der Heijden1 ,
C. Caldas5 , R. Bernards1 . 1 Antoni van Leeuwenhoek − Netherlands Cancer
Institute, Molecular Carcinogenesis, Amsterdam, Netherlands; 2 Murdoch
Childrens Research Institute Royal Children’s Hospital, Bioinformatics
Division, Victoria, Australia; 3 Antoni van Leeuwenhoek − Netherlands
Cancer Institute, Genomics Core Facility, Amsterdam, Netherlands;
4
Antoni van Leeuwenhoek − Netherlands Cancer Institute, Thoracic
Oncology, Amsterdam, Netherlands; 5 University of Cambridge, Oncology,
Cambridge, United Kingdom; 6 The Walter and Eliza Hall Institute, Cancer
and Haematology, Victoria, Australia; 7 Antoni van Leeuwenhoek −
Netherlands Cancer Institute, Urology, Amsterdam, Netherlands
Background: Oncogenic fusion genes involving kinases are effective
therapeutic targets in different tumor types. However, diagnostic screening
for kinase fusion genes in solid tumors is particularly challenging, as many
occur with a low frequency. To overcome this, we developed a capture
enrichment strategy to enable high throughput transcript sequencing of
the human kinome. Using this approach, we recently identified novel
translocation events involving the FGFR3 and ALK genes in lung squamous
cell and adenocarcinoma (Majewski IJ et al, 2013).
Methods: To date we screened with this approach 192 non-small cell lung
carcinomas, 480 breast cancers of different subtypes from the METABRIC
cohort (Curtis C et al, 2012), 80 head and neck carcinomas and 80
muscle-invasive urothelial carcinomas. Patient material was available from
frozen or formalin-fixed paraffin-embedded tissues. Sequencing libraries
were constructed with a TruSeq mRNA library preparation kit using
poly-A enriched RNA (Illumina). Capture enrichment was performed with
the human kinome DNA capture baits (Agilent). Captured libraries were
sequenced on an Illumina HiSeq2000 platform with a paired-end 51 base
protocol. Sequences were aligned to the human genome (Hg19) with
TopHat (Trapnell C et al, 2009). Two pipelines were used to identify and
rank candidate fusion genes: TopHat-fusion (Kim D et al, 2012) and de
novo transcript assembly with Trinity (Grabherr MG et al, 2011). In order to
validate the fusion events, PCR primers were designed to amplify across
the fusion breakpoints and fusion breakpoints were confirmed by PCR
amplification from cDNA and subsequently by capillary sequencing.
Results: About 90% of the fusion events predicted with TopHat and de novo
assembly algorithm were validated by capillary sequencing. Intriguingly, a
gene was found recurrently fused at the exact same breakpoint in the breast
cancer set. Fusion genes identified in the discovery set will be screened
in independent sets of samples in order to determine their frequency. In
addition, association of genomic rearrangements with clinico-pathological
parameters has been assessed. Detailed results and functional validations
will be presented at the meeting.
Conclusion: Our kinome capture enrichment strategy for systematically
profiling kinase fusion genes across different cancer types can be reliably
used to identify novel translocation events in any expressed kinase.
161
POSTER (Board P155)
Leveraging a novel DNA barcoding platform for integrated profiling
and pharmacodynamic readouts
C. Castro1 , V. Peterson2 , A. Ullal2 , S. Agasti2 , S. Tuang2 , N. Miller2 ,
M. Birrer3 , R. Weissleder4 . 1 Massachusetts General Hospital
Cancer Center, Medicine/Center for Systems Biology, Boston, USA;
2
Massachusetts General Hospital, Center for Systems Biology, Boston,
USA; 3 Massachusetts General Hospital Cancer Center, Medicine,
Boston, USA; 4 Massachusetts General Hospital, Center for Systems
Biology/Radiology, Boston, USA
Background: Studies cite modest overlap between DNA, RNA, and
protein levels within tumor cells. Integrated testing could generate valuable
and complementary mechanistic insight. We recently developed a DNA
barcoding platform to quantify ~100 protein markers using scant clinical
trial samples down to a single cell. Here, we expand this tool to predict
drug testing by enabling simultaneous testing of tumor DNA, RNA, and
protein.
Materials and Methods: We highlight two ovarian cancer cases with
contrasting clinical characteristics (low and high grade). Besides profiling,
biopsies were also treated ex-vivo with cytotoxic (carboplatin, paclitaxel)
or targeted drugs (PKI-587 [PI3K/mTOR], selumetinib [MEK]). Antibodies
conjugated to unique DNA barcodes were used for multiplexed proteomic
measurements using one fine needle aspirate (FNA) pass. After labeling
with Ab-DNA conjugates, cells were lysed and unique barcodes cleaved
from antibodies. Lysates contained cellular DNA and mRNA along with
cleaved DNA barcodes measurable by automated detection platforms with
attendant high femtomolar sensitivity.
Results: Integrative analyses and expanded drug testing were feasible on
clinical specimens.
Profiling: Subject 1 (low grade) had increased KRAS (~3 fold) and
MYC (~5 fold) copy number suggesting MAPK pathway dysregulation.
In contrast, Subject 2 (high grade) had increased copy number of
PI3KCA (~3 fold) and AKT2 (~11 fold) genes suggesting PI3K pathway
dysregulation. Concordant with DNA results, Subject 1 had multiple
unregulated proteins in the MAPK pathway (p38 MAPK, MET) along with
MAPK1 and MET mRNA levels. Subject 2 had high AKT expression, pS6,
and MTOR at both protein and mRNA levels.
Drug testing: Both subjects downregulated EpCAM, S6RP, and p44/42
MAPK protein levels following carboplatin, selumetinib, or paclitaxel.
Subject 1 was very sensitive to selumetinib (IC50: ~20 nM), supporting our
profile testing. While Subject 1 showed limited in vitro sensitivity to PKI-587,
as predicted, we noted acquired sensitivity with HM30181 (MDR1 inhibitor)
use; results were validated with cell line testing.
Conclusion: Our novel platform’s potential for expanded ex-vivo target
modulation testing using scant specimens and various drug dosages or
combinations could render it a feasible preclinical drug screening tactic.
Our data support recent ovarian cancer MEK inhibitor trial evidence
and uncover potential synergism with MDR1, poised for further clinical
exploration.
162
POSTER (Board P156)
Drug–drug interaction predictions for MLN2480, an investigational
pan-RAF inhibitor, based on nonclinical data
S.K. Balani1 , A. Bulychev1 , L. Cohen1 , M. Liao1 , C.Q. Xia1 , F. Wang2 ,
P. Li3 , B. LeClair3 , T. Bohnert3 , L. Gan3 , X. Zhou4 , V. Bozon5 ,
S. Prakash6 . 1 Takeda Pharmaceuticals International Company,
DMPK, Cambridge, USA; 2 Takeda Pharmaceuticals International
Company, Cancer Pharmacology, Cambridge, USA; 3 Biogen Idec Inc,
DMPK, Cambridge, USA; 4 Takeda Pharmaceuticals International Co.,
Clinical Pharmacology, Cambridge, USA; 5 Takeda Pharmaceuticals
International Co., Oncology Clinical Research, Cambridge, USA; 6 Takeda
Pharmaceuticals International Co., DMPK, Cambridge, USA
Background: MLN2480 is an investigational small molecule pan-RAF
inhibitor in clinical trials in patients with metastatic melanoma and other
solid tumors. Non-clinical assessments were made to predict the potential
for drug–drug interactions (DDIs), and pH and formulation effects in the
clinic.
Material and Methods: In vitro assessments of enzymes involved in
metabolism, their quantitative phenotyping, and CYP inhibitions were
carried out using human liver microsomes. Freshly isolated human
hepatocytes were used to assess CYP induction and mRNA expression.
Caco-2 cells were used to assess membrane permeability and transporters’
inhibition potential. Pharmacokinetic (PK) studies used mice to assess the
effect of pH and cremophor on PK of MLN2480.
Results: Studies with human liver microsomes indicated that MLN2480
is metabolized by multiple enzymes, with aldehyde oxidase and CYP2C8
playing predominant roles. Due to multiple metabolic enzymes involved in
MLN2480 clearance, CYP2C8 inhibitor and inducer drugs are expected
to have a low potential to alter exposure to MLN2480. Additionally, the
compound is a significant inhibitor of CYP2C8, hence a potential to affect
PK of CYP2C8 drugs. MLN2480 did not exhibit time-dependent inhibition
of CYP3A, or induction of CYPs.
The compound exhibits high membrane permeability in Caco-2 model and
does not appear to be an efflux pump substrate, reducing any concerns of
DDIs with efflux pump inhibitor drugs. Additionally, it is not an inhibitor of
Pgp, but of BCRP. Thus, a DDI potential exists with BCRP substrate drugs.
Physicochemical-based interactions also have been assessed in mice.
Consistent with the fact that the solubility of MLN2480 is not pH dependent,
there was no effect on exposure to MLN2480 in an alkaline formulation in
rodents. Additionally, since paclitaxel would be tested as a combination
agent, and Cremophor® EL (BASF SE) in Taxol is known to affect PK
of some drugs, the effect of cremophor on MLN2480 PK was assessed
in mice. The results showed that with Cremophor concentration similar to
that at the planned Taxol dose of 80 mg/m2 in the clinic, the increase in
MLN2480 exposure (AUC) in mice was only 14%.
Conclusions: Overall, based on nonclinical data, there is a DDI potential
for MLN2480 with CYP2C8 substrates/inhibitors/inducers and BCRP
substrate drugs in the clinic. The possible alteration of MLN2480 PK by
Cremophor in Taxol (80 mg/m2 ) is likely to be minimal.
163
POSTER (Board P157)
Interrogation of pharmacogenes in cancer patients using targeted
DNA sequencing
F. Innocenti1 , N. Gillis1 , J. Parker1 , N. Hayes1 , D. Eberhard1 ,
K. Richards1 , J.T. Auman1 , E. Seiser1 . 1 University of North Carolina
at Chapel Hill, Chapel Hill NC, USA
Background: Germline sequence variation and somatic alterations in
genes associated with drug processing can generate individualized
pharmacogenetic data for cancer patients that may aid in understanding
therapeutic response.
Material and Methods: Targeted sequencing of over 200 genes, including
known druggable targets and genes relevant to cancer pathogenesis, was
performed for matched germline and tumor DNA from over 200 patients
representing many common cancer types. Aligned sequence data was
used for genotype variant calling in both germline and tumor DNA and
for mutation and copy number identification within tumors. Genotype data
was used to determine genetic ancestry and to provide summary statistics
for known pharmacogenetic variants in germline DNA. Additionally, somatic
alterations within genes relevant to the pharmacology of anticancer agents
were examined in the represented cancer types: brain/central nervous
system, breast, gastrointestinal, genitourinary, gynecologic, head and neck,
and hematologic.
Results: The use of genetic markers to determine ancestry identified
patients that were mainly Caucasian (78%) and African American (16%),
and provided a means to correct for errors and ambiguity in self
reporting for nine individuals. The most common cancer types included
gastrointestinal (19%), genitourinary (18%), and breast (13%). A total of 38
pharmacogenetic variants in 18 genes, including cytochrome P450 genes,
demonstrated similar minor allele frequencies when compared to 1000
Genomes data and were predominantly in Hardy-Weinberg equilibrium.
Observed genotype concordance of these variants in matched germline
and tumor DNA was greater than 95% for the majority of loci. Analysis
of 16 genes associated with anticancer agents identified mutations 15
genes (22% of patients exhibited at least one mutation in one or more
genes), including predicted non-synonymous coding changes and gains of
stop codons in ABCB1 and DPYD. Aberrant tumor copy numbers were
sporadically observed in most of these genes, although recurrent deletions
of DPYD and SULT1A1 were present across tumor types.
Conclusions: Next generation sequencing of pharmacogenes in both nonmalignant and tumor tissue from an individual provides a comprehensive
catalog of germline variants and both known and novel somatic alterations.
The integration of genetic data from germline and tumor may aid in the
elucidating the pharmacology underlying therapeutic effect. Analysis of the
effect of these variants and molecular alterations on drug response for a
subset of individuals is ongoing and will be presented at the meeting.
164
POSTER (Board P158)
Augmentation of NAD+ by NQO1 activation attenuates cisplatinmediated hearing impairment
S. Yang1 , G.S. Oh2 , K.B. Kwon2 , R.K. Park2 , S.Y. Lee3 , S.R. Moon4 ,
H.S. So2 . 1 Wonkwang University School of Medicine, Department
of Internal Medicine, Iksan-city Jeonbuk, South Korea; 2 Wonkwang
University School of Medicine, Center for Metabolic Function Regulation,
Iksan-city Jeonbuk, South Korea; 3 Wonkwang University School of
Medicine, Department of Thoracic Surgery, Iksan-city Jeonbuk, South
Korea; 4 Wonkwang University School of Medicine, Department of
Radiation Oncology, Iksan-city Jeonbuk, South Korea
Introduction: Cisplatin [cis-diaminedichloroplatinum-II] is an extensively
used chemotherapeutic agent, and one of its most adverse effects is
ototoxicity. A number of studies have demonstrated that these effects
are related to oxidative stress and DNA damage. However, the precise
mechanism underlying cisplatin-associated ototoxicity is still unclear. The
cofactor nicotinamide adenine dinucleotide (NAD+ ) has emerged as a key
regulator of cellular energy metabolism and homeostasis. Although a link
between NAD+ -dependent molecular events and cellular metabolism is
evident, it remains unclear whether modulation of NAD+ levels has an
impact on cisplatin-induced hearing impairment.
55
Material and Methods: To investigate whether augmentation of NAD+
by NQO1 activation using b-Lapachone (b-Lap) attenuates cisplatinmediated hearing impairment, male C57BL/6mice and NQO1 knockout
mice on a C57BL/6 background were used. For analysis of the
auditory threshold, auditory brainstem response (ABR) was recorded.
For biochemical analysis, we measured the enzymatic activity of SIRT1,
PARP1, ROS production, NAD+/NADH ratio, mRNA levels of miR-34a
and pro-inflammatory cytokines. Immunohistochemistry and western blot
analysis were also performed.
Results and Discussion: We have demonstrated for the first time that both
the protein expression level and the activity of SIRT1 were suppressed
by the reduction of intracellular NAD+ levels in cisplatin-treated cochlear
tissue. We also found that the decrease in SIRT1 protein expression
and its activity after cisplatin exposure were mediated by the increase in
transcriptional activity of p53 for miR-34a expression and PARP-1 activation
causing NAD+ -depletion, respectively. However, the increase in cellular
NAD+ levels by NQO1 activation using b-Lap prevented mice from cisplatininduced cochlear damage and hearing impairment through the modulation
of PARP-1, SIRT1, p53, and NF-kB.
Conclusion: Considering that b-Lap itself did not attenuate the tumoricidal
effect of cisplatin (Kidney International, 2014, in press), these results
suggest that the direct modulation of the cellular NAD+ level by
pharmacological agents could be a promising therapeutic strategy for
enhancing the efficacy of cisplatin chemotherapy without its adverse
effects.
165
POSTER (Board P159)
Effect of zoledronic acid on the post-translational modification of
activated leukocyte cell adhesion molecule (ALCAM) in cancer cells
R. Toth1 , G. Trombino1 , V. Castronovo1 , A. Bellahcene1 . 1 C.H.U. Liège,
Metastasis Research Laboratory, Liège, Belgium
Background: Activated leukocyte adhesion molecule (ALCAM/CD166) is
a 105kDa protein that is a member of the immunoglobulin superfamily.
This family consists of proteins that are involved in recognition binding and
adhesion processes of the cells. Zoledronic acid (ZA) is a bisphosphonate
that has been extensively studied as an osteoclast inhibitor. ZA decreases
bone turnover by inhibiting the mevalonate pathway in cells. This pathway
has been notably implicated in post-translational protein modifications such
as prenylation and N-linked glycosylation.
Methods: MDA-MDA-231 cells have been treated with different concentrations of ZA for 48 h. Enzymatic digestion was performed with PNGaseF on
protein extracts from treated and non-treated cells. Western blot analysis
and cell aggregation assays have been performed upon ZA treatment.
Results: We observed the appearance of a 95kDa form of ALCAM after
48 h of treatment with ZA in a dose dependent manner. MDA-MB-231
cytosolic/membrane protein fractionation showed that the 95kDa form of
ALCAM was detectable in the membrane fraction. PNGaseF treatment of
MDA-MB-231 cell extracts resulted in a single 68kDa band in both treated
and non-treated conditions, indicating that the 95kDa form potentially
represents an intermediate glycosylation form of ALCAM. We showed that
ALCAM is crucial for MDA-MB-231 cell aggregation. Next, we observed
that ZA treated-cells presented with a decreased aggregation capacity
potentially linked to their expression of the 95kDa ALCAM form.
Conclusion: Our results suggest for the first time that ZA affects the
glycosylation of ALCAM in MDA-MB-231 cells in vitro, which will be further
investigated in breast cancer tumor models in vivo. Reduced N-linked
glycosylation of ALCAM was associated with a decreased aggregation
potential in ZA-treated cancer cells. Cell aggregation is associated with
anchorage independent growth and proliferation in MDA-MB-231 cells.
Ongoing experiments will help to explore the potential implication of the
95kDa ALCAM form in these processes.
166
POSTER (Board P160)
Pre-clinical and clinical activity of Anti-DLL4 (demcizumab) in
combination with gemcitabine plus nab-paclitaxel in pancreatic
cancer
M. Hidalgo1 , A. Cubillo2 , R. Stagg3 , J. Dupont3 , Y. Wan-Ching3 ,
T. Hoey3 . 1 CNIO, Madrid, Spain; 2 CIOCC, Madrid, Spain; 3 Oncomed
Pharmaceutical, California, USA
DLL4-Notch signaling plays a key role in cancer stem cell (CSC) biology in
numerous tumor types including pancreatic cancer. We have developed an
anti-DLL4 antibody (demcizumab) that blocks Notch signaling and inhibits
tumor growth though multiple mechanisms including a reduction of CSC
frequency and the inhibition of productive angiogenesis. Previous studies
have shown broad spectrum activity of anti-DLL4 & gemcitabine in a panel
of patient derived pancreatic cancer xenografts. Data from a Phase 1b
clinical study of demicizumb & gemcitabine in PDAC resulted in a 25%
56
partial response rate in 16 evaluable patients. Based on the emergence
of nab-paclitaxel & gemcitabine as a standard of care regimen for the
treatment of pancreatic cancer, we have tested anti-DLL4, gemcitabine
& nab-paclitaxel in PDAC PDX models, and observed enhanced antitumor activity in all seven pancreatic cancer xenografts tested. Based
on serial transplantation experiments, the triple combination of anti-DLL4,
gemcitabine, & nab-paclitaxel reduced CSC frequency whereas treatment
with the chemothearpy agents alone did not. Interestingly, the combination
activity of anti-DLL4 appeared to be greater with gemcitabine & nabpaclitaxel than with gemcitabine alone. In a co-clinical study, a PDX model
derived from a Phase 1b patient who progressed rapidly on treatment with
demcizumab & gemcitabine was, as expected, insensitive to gemcitabine
or the combination of anti-DLL4 & gemcitabine, correlating well with the
lack of clinical response. Notably, this tumor was more sensitive to nabpaclitaxel, gemcitabine & anti-DLL4 treatment. As a result of these data
and the emergence of nab-paclitaxel & gemcitabine as a new standard of
care for pancreatic cancer, the Phase 1b clinical study mentioned above
was amended to test the triple combination of demcizumab, gemcitabine
& nab-paclitaxel in patients with 1st line pancreatic cancer. Early data
from the 1st 14 patients treated with this combination revealed a partial
response in 6 (43%) patients and stable disease in another 6 (43% pts).
Related AEs observed in 20% in the 38 patients in the Phase 1b study
were nausea (37%), fatigue (34%), vomiting (32%), decreased appetite
(24%) & hypertension (21%). Additional cohorts of patients are being
treated with demcizumab, gemcitabine & nab-paclitaxel to further assess
the preliminary safety, efficacy, pharmacokinetics and impact on biomarkers
of this combination.
167
POSTER (Board P161)
Genomic characterisation of 1003 cancer cell-lines
G. Bignall1 , F. Iorio2 , P.A. Futreal3 , M.R. Stratton1 , P. Campbell1 ,
U. McDermott1 . 1 Wellcome Trust Sanger Institute, Cambridge, United
Kingdom; 2 EMBL-EBI, Cambridge, United Kingdom; 3 MD Anderson
Cancer Centre, Houston, USA
Over the last twenty years there has been a paradigm shift in cancer
treatment, moving away from the cytotoxic effects of chemicals targeting
all cells going through cell division to a more targeted approach aimed at
inactivating specific cellular components upon which the cancers rely to
drive cell growth; a process termed oncogene addiction. The ‘poster child’
for such therapies is ‘imatinib’ a small molecule inhibitor of ABL kinase
activity targeting the BCR−ABL fusion protein in CML. Here we present the
genomic characterisation of 1003 cancer cell-lines currently being used as
reagents in a high-throughput screen of anti-cancer agents. This screen
is aimed at identifying novel biomarkers with which to stratify patients,
identifying those most likely to respond to specific cancer therapies.
To characterise the cell-lines we performed exome sequencing, copy
number, expression and methylation analysis. Since most cell-lines are
unmatched we screened out germline variants by comparison to ~8000
normal exomes. To further reduce the ‘genetic space’ for the subsequent
downstream correlation to drug response data we included only ‘clinically
relevant’ variants. The list of clinically relevant variants was identified by
looking for recurrence across the ‘systematic screen data’ in COSMIC and
by reducing the gene set to those genes identified as ‘frequently mutated’
across a series of >7000 clinical exomes/genomes from 29 cancer types
using IntOGen, MutSigCV and dN/dS analysis. Tissue specific and pan
cancer gene sets were identified for downstream analysis.
In total we identified ~500,000 putative somatic non-synonymous variants
across the set of 1003 cancer cell-lines, of which ~6000 were classed as
clinically relevant. Analysis is currently on-going correlating this ‘clinically
relevant’ variant data to the drug response.
Comparison of the mutation frequencies between the clinical samples and
the filtered variant data from the cancer cell-lines indicate that the cell-lines
are likely to represent good model systems for downstream correlation to
drug response. The variant data for the cancer cell-lines is available via
the COSMIC web portal.
168
POSTER (Board P162)
CDCP1 as a new marker of aggressiveness in triple-negative breast
cancers
M. Campiglio1 , F. Turdo1 , F. Bianchi1 , M. Sasso1 , L. De Cecco1 ,
P. Casalini1 , P. Gasparini1 , L. Forte1 , R. Agresti1 , I. Maugeri1 , G. Sozzi1 ,
E. Tagliabue1 . 1 Fondazione IRCCS Istituto Nazionale Tumori, Dept.
Experimental Oncology and Mol. Med., Milano, Italy
Background: Triple-negative breast cancer (TNBC) is an aggressive breast
cancer subtype showing high recurrences and mortality rate, for which
no therapies besides chemotherapy are available to date. Lacking specific
markers for an effective targeted therapy, TNBCs still represent the most
important challenge for clinical oncologists. In an effort to identify specific
markers responsible for aggressiveness of TNBCs that may serve as
target(s) of therapy, we used post-surgery wound-healing fluids (WHFs)
from breast cancer patients, known to be extremely enriched in growth
factors and cytokines, as a tool mimicking the pro-tumorigenic post-surgery
host microenvironment to stimulate TNBC cells lines.
Material and Methods: Cell lines knock-down for CDCP1 by specific
siRNA were analyzed for migration, invasion and proliferation in vitro.
Immunohistochemistry on FFPE TNBC specimens were performed using
a polyclonal antibody. Cases were classified as positive when plasmamembranes were stained in at least 10% of tumor cells. FISH analysis was
performed using a pool of 3 BAC (bacterial artificial chromosome) clones
that cover the CDCP1 gene at chromosome 3p21.31 and a commercial
chromosome 3 enumeration probe. Kaplan–Meier methods were used to
calculate the disease free survival (DFS) and distant DFS (DDFS) and
differences in survival curves were evaluated with the log-rank test.
Results: Gene expression profiling (GEP) on the Illumina platform using
TNBC cell lines derived after stimulation with WHFs identified among
the surface receptors the non-catalytic receptor CDCP1 (CUB domaincontaining protein 1) as the most significantly up-modulated gene. CDCP1
protein was found basally highly expressed in 6 of 8 TNBC cell lines. Its
silencing in the most highly expressing TNBC lines (MDA-MB-231 and BT549) strongly impaired both their migration (~70%) and invasion (~50%)
ability but did not affect their in vitro proliferation, suggesting a role for
CDCP1 in TNBC dissemination. IHC analysis of CDCP1 in 126 human
primary TNBC FFPE specimens revealed intense membrane staining in
60% of cases. CDCP1 expression was found to be a risk factor that
significantly reduces both DFS (log-rank p = 0.0115) and DDFS (log-rank
p = 0.0063) of TNBC patients. To evaluate whether the high expression
level of CDCP1 in human TNBC specimens can depend on a genetic gain,
FISH analysis of FFPE sections from 30 human TNBC cases (20 CDCP1positve and 10 CDCP1-negative in IHC) was performed, revealing that in
more than 50% of CDCP1-positive cases CDCP1 gene was polysomic.
Additionally, polysomy strongly correlated with CDCP1 protein expression
levels.
Conclusions: Our data identify CDCP1 as a marker of extremely
aggressive TNBCs, suggesting its candidacy as a target of novel
therapeutic strategies against this disease.
Supported by AIRC
169
POSTER (Board P163)
BRAF mutation testing in cell-free DNA from plasma of patients
with advanced cancers using a novel, rapid, automated molecular
diagnostics prototype platform (Idylla™)
F. Janku1 , H.J. Huang1 , B. Claes2 , G.S. Falchook1 , A. Naing1 ,
S. Piha-Paul1 , A.M. Tsimberidou1 , R.G. Zinner1 , D.D. Karp1 , S. Fu1 ,
V. Subbiah1 , D.S. Hong1 , J.J. Wheler1 , R.G. Luthra3 , S.P. Patel4 ,
E.S. Kopetz5 , E. Sablon2 , G. Maertens2 , R. Kurzrock6 , F. Meric-Bernstam1 .
1
The University of Texas MD Anderson Cancer Center, Investigational
Cancer Therapeutics (Phase I Clinical Trials Program), Houston, USA;
2
Biocartis, Mechelen, Belgium; 3 The University of Texas MD Anderson
Cancer Center, Molecular Diagnostic Laboratory, Houston, USA; 4 The
University of Texas MD Anderson Cancer Center, Melanoma Medical
Oncology, Houston, USA; 5 The University of Texas MD Anderson
Cancer Center, Gastrointestinal Medical Oncology, Houston, USA; 6 The
University of California San Diego, Moores Cancer Center, La Jolla, USA
Background: Cell-free (cf) DNA from the plasma of cancer patients
offers an easily obtainable, low-risk, inexpensive and repeatedly applicable
source of biologic material for mutation analysis of druggable targets and
monitoring molecular changes in tumor(s) during and after therapeutic
interventions. Novel, fast, and accurate diagnostic systems are needed for
further development of plasma cfDNA testing in personalized therapy.
Methods: cfDNA from plasma samples of patients with advanced
cancers who progressed on systemic therapy was purified and 50–
100 ng DNA was used for testing for V600 BRAF mutations using the
prototype molecular diagnostics (IdyllaTM ) fully integrated real-time PCRbased platform (Biocartis, Mechelen, Belgium) and BRAF V600 mutation
prototype cartridges with a quick turnaround time (<60 minutes for cfDNA).
The IdyllaTM platform and the BRAF V600 mutation prototype assay were
used for research purposes only. Results were compared to mutation
analysis of archival primary or metastatic tumor tissue obtained at different
points of clinical care from a CLIA-certified laboratory if available.
Results: cfDNA was extracted from plasma samples of 127 patients with
advanced cancers (colorectal, n = 54; melanoma, n = 28; non-small cell
lung, n = 12; breast, n = 6, Erdheim-Chester disease, n = 6; thyroid, n = 7;
appendiceal, n = 3; ovarian, n = 3; endometrial, n = 3; other cancers, n = 5).
BRAF mutations were detected in 29% (36/127) of plasma samples and in
43% (50/117) of available archival tumor samples, resulting in concordance
for 102 (87%) of 117 patients who had both plasma and tissue tested
cases (kappa = 0.73, 95% confidence interval 0.61–0.85) with sensitivity
72%, specificity 99%, positive and negative predictive value 97% and
83%, respectively. In all 15 discrepant cases identical plasma cfDNA
samples were tested using an alternative cfDNA BRAF mutation PCRbased method (BEAMing, Sysmex Inostics, Baltimore, MD), which yielded
100% agreement with the IdyllaTM platform. Longitudinally collected plasma
samples were available in 13 patients (appendiceal, n = 2; colorectal,
n = 2; melanoma, n = 4; papillary thyroid cancer, n = 2; other, n = 3) with
plasma BRAF V600 mutations treated with predominantly BRAF targeting
combinations and changes in the amount of BRAF-mutant cfDNA tracked
changes in tumor markers and disease burden visualized via imaging.
Conclusions: Detecting V600 BRAF mutations in cfDNA from plasma
using the IdyllaTM platform and BRAF V600 mutation prototype cartridges
is a fast and noninvasive alternative to mutation testing of tumor tissue
with an acceptable level of concordance and sensitivity, and should be
investigated further for testing and monitoring of BRAF mutation status in
patients with cancer.
57
170
POSTER (Board P164)
Novel, ultra-deep next-generation sequencing for BRAF mutation
testing using small amount of cell-free DNA from plasma of patients
with advanced cancers
associated with sensitivity or resistance, and other cell line characteristics.
EGR1 was identified as one of the top genes for which drug-induced
changes in expression were associated with sensitivity to gemcitabine
(gem; 32 cell lines up regulated >2-fold) and to erlotinib (17 cell lines
down regulated >2-fold) in at least two treatment conditions. In the clinic,
combinations of gem and erlotinib have been tested with mixed results.
We hypothesized that divergent response of EGR1 to these two drugs
might form the basis for antagonism. Treatment with the gem/erlotinib
combination in 6 cell lines generated a range of antagonism in vitro,
which depended on sensitivity to either the gem or erlotinib. Cell lines
that were more sensitive to erlotinib, with greater EGR1 down regulation,
demonstrated greater antagonism in response to the drug combination. In
support of a role for EGR1, we found that transient down regulation of
EGR1 in some gem-responsive cell lines led to decreased sensitivity to
gem, while overexpression of the gene led to increased gem sensitivity.
The measured antagonism was cell line dependent, but independent of
drug scheduling. Sensitivity to gem appears mediated by EGR1 and can
be antagonized by other pharmacologic effects on EGR1 as evidenced by
the antagonism of growth inhibition between gem and erlotinib. These data
may provide a rationale for the poor clinical response to this combination
in some tumor types.
Funded by NCI Contract No. HHSN261200800001E and supported in part
by the Developmental Therapeutics Program in the Division of Cancer
Treatment and Diagnosis, NCI.
F. Janku1 , H.J. Huang1 , N.M. Ramzanali1 , X. Cai2 , R. Klausner2 ,
F. Meric-Bernstam1 , J.B. Fan2 . 1 MD Anderson Cancer Center,
Investigational Cancer Therapeutics (Phase I Clinical Trials Program),
Houston, USA; 2 Illumina, San Diego, USA
172
POSTER (Board P166)
Modulation of estrogen-dependent transcription by cohesin in MCF7
human breast adenocarcinoma cells
Background: Plasma cell-free (cf) DNA of cancer patients offers an
easily obtainable and repeatedly applicable source of DNA for mutation
analysis, which provides attractive alternative to tumor tissue testing. Novel
ultrasensitive technologies using small amounts of DNA are needed for
further development of plasma cfDNA testing in personalized therapy.
Methods: We have developed a next-generation sequencing method for
somatic BRAF mutation detection in cfDNA with high sensitivity and
specificity. The protocol was designed specifically to utilize small DNA
fragments, using a very low DNA input of 5 ng. Each cfDNA fragment
was uniquely barcoded and amplified prior to Illumina target enrichment
workflow, followed by ultra-deep sequencing (>10,000×). Proprietary data
processing and analysis tools were developed to enable sensitive detection
of rare mutant molecules over high wild-type background (i.e. detection
of 1 in 1000 molecules). Results were compared to mutation analysis of
archival primary or metastatic tumor tissue obtained at different points of
clinical care from a CLIA-certified laboratory
Results: cfDNA was extracted from plasma samples of 24 patients with
advanced cancers (melanoma, n = 9; colorectal, n = 5; non-small cell lung,
n = 2; papillary thyroid, n = 2; other cancers, n = 6) and 5 ng were used for
BRAF mutation analysis. BRAF mutations were detected in 71% (17/24)
of plasma samples and in 88% (21/24) of archival tumor samples, resulting
in concordance in 87% (20/24) of cases.
Conclusions: Detecting V600 BRAF mutations using ultra-deep sequencing of small amounts of cfDNA (5 ng) from plasma is feasible with an
acceptable level of concordance with BRAF testing of tumor tissue obtained
at different time points and should be investigated further for testing and
monitoring of BRAF mutation status in patients with cancer.
171
POSTER (Board P165)
Antagonistic interaction between gemcitabine and erlotinib is
influenced by EGR1 (early growth response 1) transcription factor
expression
C.D. Hose1 , Y. Zhao2 , E.C. Polley2 , J. Fang2 , N.D. Fer1 , A. Rapisarda1 ,
B.A. Teicher2 , R.M. Simon2 , J.D. Doroshow2 , A. Monks1 . 1 Frederick
National Laboratory for Cancer Research, Leidos Biomedical
Research Inc., Frederick MD, USA; 2 National Cancer Institute, DCTD,
Frederick MD, USA
Drug-induced transcriptional changes may help to ascertain mechanisms
of action and possible bystander effects, and can suggest potentially
favorable or adverse drug combinations. We developed a database of
gene expression changes that were observed following treatment with
15 commonly used anticancer agents in 60 human tumor cell lines,
across 2 drug concentrations and 3 incubation times. Gene expression
profiles were measured using Affymetrix HTA array plates 133A and 133B,
and drug-induced fold-changes were calculated and compared to timematched controls. These dynamic transcriptional response data will be
provided on a public website that can be facilely accessed and explored
using the Transcriptional Pharmacology Workbench, a powerful web-based
tool-set designed by the NCI for finding drug-modulated genes that are
T. Dasgupta1 , J. Antony1 , J. Rhodes1 , M. McEwan1 , M. Eccles1 ,
J. Horsfield1 . 1 University of Otago, Pathology, Dunedin, New Zealand
Background: RAD21, a component of the evolutionary conserved chromosome cohesion protein cohesin, is amplified in breast cancer. Amplification
of RAD21 is correlated with poor prognosis and endocrine resistance in
luminal breast cancers. However, the reasons for this correlation are not
understood. Our lab discovered that depletion of RAD21 reduces estrogen
induction of the proto-oncogene c-MYC, and impairs binding of estrogen
receptor a (ERa) to its enhancers. Chromosome binding of RAD21
coincides with ERa in estrogen-induced breast cancer cells suggesting
cohesin could modulate estrogen-dependent gene transcription. This study
aimed to identify estrogen-sensitive genes that are dysregulated upon
loss of cohesin, and determine the mechanisms of regulation mediated
by cohesin.
Material and Methods: To map global changes in gene expression, a
microarray analysis was carried out in RAD21-depleted MCF7 cells in
the presence/absence of estrogen. Chromatin immunoprecipitation (ChIP)
and quantitative PCR (qPCR) was used to determine if RAD21 depletion
influenced binding of ERa at selected altered genes (IL20 and SOX4).
Results: Microarray analysis revealed that cohesin depletion affected
transcription of not all, but a subset of estrogen-sensitive genes. Loss
of cohesin resulted in bidirectional (up or down) regulation of ERa target
genes. The most significantly regulated genes were co-bound by both ERa
and cohesin and were over-represented in oncogenic pathways such as
ErbB and PI3K/mTOR. Preliminary ChIP analysis suggests that depletion
of RAD21 alters binding of ERa at sites previously identified3 for genes,
SOX4 and IL20.
Conclusion: Cohesin can promote or inhibit expression of estrogenresponsive genes in a context-specific manner. Ongoing experiments are
aimed at confirming the ChIP findings, to further elucidate cohesin’s role
in regulation of the ER transcription program.
173
POSTER (Board P167)
Ras-mediated activation of mitogen-activated protein kinase pathway
unleashes basement membrane damaging activity of serine protease
hepsin
T. Tervonen1 , S. Pant1 , D. Belitskin1 , J. Englund1 , K. Närhi2 ,
E. Verschuren2 , P. Kovanen3 , J. Klefström1 . 1 University of Helsinki,
Translational Cancer Biology Research Program, Helsinki, Finland;
2
University of Helsinki, Institute for Molecular Medicine Finland, Helsinki,
Finland; 3 University of Helsinki, Haartman Institute and Department
of Immunology and Pathology, Helsinki, Finland
Membrane-anchored serine proteases have emerged as novel players
in invasion and metastasis of epithelial cancers. Among these, type II
transmembrane serine protease hepsin is prominently upregulated in
prostate and breast cancer both in mRNA and protein level. In agreement
what was previously observed in ovarian cancer, normal human mammary
gland expresses low levels of pericellularly and basally localized hepsin
58
contrasting to diffuse, cytosolic expression observed in more than 80%
(n = 49) of breast cancer specimens representing all major subtypes.
Mutations in hepsin gene don’t explain widespread deregulation of hepsin
protein in breast cancer, suggesting a critical role for upstream factors as
culprits for hepsin deregulation.
Hepsin expression in breast cancer was studied in paraffin sections and
fresh tissue lysates from breast cancer patients. We also generated 2D
and 3D human and mouse non-malignant mammary epithelial and cancer
cell lines stably transduced with recombinant retro- or lentivirus vectors
endowing cells with constitutive or inducible hepsin expression. Hepsin
activity was probed with cell-based peptide substrate cleavage assay. We
used lox-stop-lox (LSL)-KrasD12; p53−/− mice where sporadic lung tumors
are induced with inhaled adeno-cre virus. Furthermore, mammary epithelial
cells from LSL-KrasD12 mice were isolated and Kras activated ex vivo with
adeno-cre virus to investigate pattern of 3D structure formation in Matrigel.
To elucidate factors upstream of hepsin, we explored the effects of
oncogenic Ras proteins, c-Myc, E2F-1, dominant negative p53 and Lkb1
silencing on hepsin protein expression levels in non-malignant mammary
epithelial cells. We demonstrate that HrasV12, KrasV12, ectopic wildtype Kras, endogenous mutant LSL-KrasD12 and E2F-1 deregulate
and enhance the proteolytic activity of hepsin. Oncogenic Ras proteins
also reduced expression levels of HAI-1, the negative regulator of
hepsin. Concomitantly, oncogenic Ras expression led to disappearance
of desmoplakin and desmoglein from desmosomal junctions, which
correlated with mislocalization of hepsin from its predominant localization
in desmosomes to cytosol. shRNA-mediated silencing or function blocking
Ab25 antibody for hepsin partially rescued HrasV12 induced transformation
in 2D and 3D cultures of MCF10A cells and preserved the basal lamina
integrity. Furthermore, MEK and Erk1/2 inhibitors prevented HrasV12dependent hepsin alterations as well as desmosomal and basement
membrane defects.
Our results couple Ras-MAPK pathway to deregulation of hepsin, revealing
a possible role for hepsin in mediating Ras transformation of epithelial
structures.
174
POSTER (Board P168)
Combination of molecular and drug response data in patient-derived
xenografts to assist patient stratification
S. Cairo1 , O. Déas1 , A. Beurdeley1 , V. Yvonnet1 , M.F. Poupon1 ,
J.G. Judde1 . 1 Xentech, R&D, Evry, France
Patient-derived xenografts (PDXs) are the preclinical models that most
closely resemble tumors in patients. PDX and the tumor of origin show
strikingly similar histological and molecular features, and when tested they
possess very similar drug-response profile. Thanks to these characteristics,
PDXs are potent surrogates of human tumor to investigate the impact of the
genetic background of transformed cells on tumor response to treatment.
Tumor heterogeneity is likely the reason why anticancer treatments fail to
display the same efficacy in different patients. This diversity is probably
due to the variability observed in tumor genetics, and in particular to
the combination of genetic alterations in each tumor. In patients, the
relationship between the degree of response to treatment and tumor
genetic features is often loose. One of the difficulties in identifying genetic
markers associated with anticancer treatment efficacy is that as each
patient is subjected to only one therapeutic strategy, chosen as the best
adapted based on the clinical parameters, it is impossible to know how this
same patient would have responded to a different treatment.
XenTech holds a large PDX collection, with more than 100 validated PDXs
models characterized so far. These models represent tumors occurring
in different tissues such as breast, lung, colon, prostate, liver and brain
among others. Most of these modes have been subjected to standardof-care (SOC) treatments to characterize their response profile. As the
studies are performed on the same tumor model, the genetic background
heterogeneity that can bias molecular comparison of patient cohorts
subjected to different therapeutic options is eliminated. The use of PDXs for
this type of studies enables to investigate the response of the same tumor to
different treatments by generating as many preclinical arms of xenografted
mice as desired. These studies, besides providing a personalized profiling
of tumor sensitivity to treatment, allow the investigation of the associated
molecular features.
In parallel, extensive molecular characterization has been performed,
including gene expression array-CGH microarrays, and mutational profile of
73 genes most frequently mutated in the tumor types available in our PDX
collection by microarray-based exon trapping coupled to high-throughput
sequencing. For each model, mutational, gene expression and SNP/aCGH
data of each PDX have been associated with treatment response profile in
order to check for biomarkers associated with tumor sensitivity or resistance
to treatment. The results obtained have been crossed with genomic data
from patient cohorts with annotated response to SOC, and the results of
these analyses will be discussed.
The use of each PDX as representative of a patient subset carrying the
same overall genomic alteration could be the good compromise between
the panel-driven and personalized use of PDXs to stratify patients.
175
POSTER (Board P169)
Functional analysis of [methyl-3 H]choline uptake in glioblastoma
cells: Influence of anti-cancer and central nervous system drugs
M. Inazu1 , C. Taguchi2 , T. Yamanaka3 , H. Uchino2 . 1 Tokyo Medical
University, Institute of Medical Science, Tokyo, Japan; 2 Tokyo Medical
University, Department of Anesthesiology, Tokyo, Japan; 3 Tokyo Medical
University, Department of Molecular Preventive Medicine, Tokyo, Japan
Positron emission tomography (PET) and PET/computed tomography (PETCT) studies with 11 C- or 18 F-labeled choline derivatives are used to
differentiate between malignant and benign lesions in various regions of the
body, including the brain, head, bone, and soft tissue. Concerning glioma,
choline PET may make it possible to differentiate between low-grade
and high-grade gliomas. Moreover, untreated and recurrent high-grade
gliomas can be visualized with sharp delineation, without any influence by
reactive inflammatory changes caused by choline PET. Further, a recent
in vitro study suggested that choline PET may be a useful indicator of the
response to chemotherapy agents that act by inhibiting signal transduction.
However, the nature of the choline transport system in glioblastoma is
poorly understood. In this study, we performed a functional characterization
of [methyl-3 H]choline uptake and sought to identify the transporters that
mediate choline uptake in the human glioblastoma cell lines A-172 and
U-251MG. In addition, we examined the influence of anti-cancer drugs
and central nervous system drugs on the transport of [methyl-3 H]choline.
High- and low-affinity choline transport systems were present in A-172
cells, U-251MG cells and astrocytes, and these were Na+ -independent
and pH-dependent. Cell viability in A-172 cells was not affected by
choline deficiency. However, cell viability in U-251MG cells was significantly
inhibited by choline deficiency. Both A-172 and U-251MG cells have two
different choline transporters, choline transporter-like protein 1 (CTL1)
and CTL2. In A-172 cells, CTL1 is predominantly expressed, whereas
in U-251MG cells, CTL2 is predominantly expressed. Treatment with
anti-cancer drugs such as cisplatin, etoposide and vincristine influenced
[methyl-3 H]choline uptake in U-251MG cells, but not A-172 cells. Central
nervous system drugs such as imipramine, fluvoxamine, paroxetine,
reboxetine, citalopram and donepezil did not affect cell viability or [methyl3
H]choline uptake. The data presented here suggest that CTL1 and CTL2
are functionally expressed in A-172 and U-251MG cells and are responsible
for [methyl-3 H]choline uptake that relies on a directed H+ gradient as
a driving force. Furthermore, while anti-cancer drugs altered [methyl3
H]choline uptake, central nervous system drugs did not affect [methyl3
H]choline uptake.
176
POSTER (Board P170)
Resolvin D2 has mitogenic activity in estrogen receptor positive
breast cancer cell lines via activation of estrogen receptor
N. Al-Zaubai1 , C. Johnstone2 , M. Rizzacasa3 , A. Stewart1 . 1 University
of Melbourne, Pharmacology and Therapeutics, Melbourne Victoria,
Australia; 2 Peter Maccallum Cancer Centre, Research Division,
Melbourne Victoria, Australia; 3 University of Melbourne, School of
Chemistry, Melbourne Victoria, Australia
Background: Inflammation has been implicated in tumour initiation,
angiogenesis and metastasis and linked to the development of more
aggressive, therapy-resistant estrogen receptor positive breast cancer.
Resolvin D2 (RvD2) is a potent anti-inflammatory lipid mediator. As
RvD2 is present in plasma at bioactive concentrations and may be
synthesized within breast tumours by both tumour and stromal cells, we
have characterized the impact of RvD2 on cell processes underlying breast
tumour growth and spread.
Materials and Methods: Viable cells were enumerated by Trypan-blue
exclusion. Transactivation of estrogen response element was assessed by
transient transfection with estrogen response element (ERE) reporter and
pGL3 vector constructs. RT-qPCR was used to examine gene expression.
Binding to the estrogen receptor was investigated by competitive
radioligand binding assays. Western blotting and immunofluorescence were
the techniques used to ascertain estrogen receptor a nuclear localization.
Results: Unexpectedly, whilst RvD2 (10–1000 nM) supported the proliferation of the ER-positive breast tumour, MCF-7, cells, it did not affect the
ER-negative, MDA-MB-231 cell number. The proliferative effect of RvD2
in MCF-7 cells was attenuated by the estrogen receptor antagonist ICI
182,780 (fulvestrant). Furthermore, RvD2 increased ERE transcriptional
activity in a number of ER positive breast and ovarian tumour cell lines.
This activation was also inhibited by ICI 182,780. RvD2 altered the
expression of a subset of estrogen-responsive genes. Prior exposure of
MCF-7 cells to RvD2 resulted in a significant reduction in the apparent
cytosolic ER density. However, binding experiments showed that RvD2 did
not directly compete with 3 [H]-17b-estradiol (E2) for ER binding. Confocal
immunocytochemistry and western blotting studies showed that RvD2
promoted nuclear localization of ERa with a corresponding decrease in
cytosol ER density.
Conclusions: These observations indicate that RvD2 displays significant
but indirect estrogenic activities and that it has the potential to play a role
in estrogen-dependent breast cancer progression.
177
POSTER (Board P171)
Validation of 3D primary organoid cultures of colorectal carcinoma
as discovery and validation platform for personalized cancer therapy
P. Halonen1 , A. Kuijpers2 , B. Morris1 , B. Diosdado1 , S. Mainardi1 ,
R. Bernards1 , V. Verwaal2 , R. Beijersbergen1 . 1 Netherlands Cancer
Institute − Antoni van Leeuwenhoek Hospital, Molecular Carcinogenesis,
Amsterdam, Netherlands; 2 Netherlands Cancer Institute − Antoni van
Leeuwenhoek Hospital, Colorectal Surgery, Amsterdam, Netherlands
Background: Primary organoid cultures derived from patients’ tumors
hold promise for the improvement of cancer therapy. For this, organoid
cultures should accurately represent primary tumors with respect to their
morphological and genomic characteristics. We have developed a 3D
organoid culturing platform for primary and metastatic colorectal carcinoma
derived from human tissues to conduct comparative studies of the primary
tumors and the organoid culture counterparts.
Material and Methods: Surgical specimens of primary and metastatic
colorectal carcinoma were used to derive the primary tissue for culturing.
3D cultures were established based on the protocol by Sato et al. and
adjusted for human colorectal cancer cells. Phenotypic comparison of
primary tissue and organoids was done by immunohistochemistry. Genomic
analysis consisted of copy number variation analysis by low-resolution full
genome sequencing and exome sequencing by kinome capture; RNAseq
was used to analyze gene expression. Signaling pathway status and
drug responses were quantified using western blot analysis and Luminex
technology combined with viability read-outs and fluorescence microscopy.
Results: A high degree of similarity between the 3D cultures and original
patient tissues was observed by immunohistochemistry using general and
differentially expressed colorectal cancer markers. Genomic alterations
identified in the primary tumor were also present in the organoid cultures
derived thereof. Although some mutations were enriched in the organoids,
we did not observe the appearance of specific mutations within the organoid
panel. Comparison of short and long term organoid cultures indicated the
preservation of the genomic alterations with some additional mutations
indicative of the genomic instability of tumors. Gene expression analysis
by RNAseq revealed clustering of organoids with the primary tissue and a
concordance with subtype classification.
Conclusions: This study shows that organoid cultures are highly
representative of primary tumors and maintain these characteristics upon
prolonged culturing. This establishes patient derived organoids as a
discovery and validation platform for the prediction of drug response and
a platform for the discovery of treatment options for personalized therapy.
178
POSTER (Board P172)
Establishment of patient-derived xenografts (PDX) models for small
cell lung (SCL) as a pre-clinical platform for drug development
T. Broudy1 , J. Ricono1 , C. Mullins1 , C. Mirsaidi1 , P. Nair1 . 1 Molecular
Response, San Diego, USA
With over 150,000 deaths in 2012, lung cancer is the deadliest carcinoma
in the United Sates. Small cell lung cancer, encompassing about 15−20%
of all lung cancer cases, is more invasive and has a higher rate of
proliferation with respect to non-small cell lung cancer, leading to a higher
mortality rate. Most cases are responsive to chemotherapy, however there
is a high rate of recurrence with treated patients and those in advanced
stage of the disease often have a refractory response to treatment.
Due to the fast growing nature of this cancer, the long term survival
rate is low whilst advanced stage cancers typically will lead to expiry
within 1 year of diagnosis. Preclinical PDX models have become valuable
tools for novel drug development in recent years. Here, we used patient
derived tissue to generate 8 new SCLC models in NOD-SCID mice.
Patient tumors were pre-screened for their myc amplification status, before
inoculating into immunocompromised animals. Once established as PDX
models, cells were also grown in 3D cultures to compare morphology
and to perform drug treatments to assess anti-proliferative potential of
various compounds. Further characterization of the models were done
by H&E, immunohistochemistry, and mutation analysis by next-generation
59
sequencing. Efficacy of standard of care drugs irinotecan, and cisplatin–
etoposide combination was demonstrated in these models. Current studies
are underway to derive correlations between in vivo drug response and
mutational status of these models. Our data strongly suggests the potential
utility of these unique PDX models in drug development efforts in oncology.
179
POSTER (Board P173)
Effects of human breast cancer cells secreted factors on macrophage
differentiation
S. Coimbra de Sousa1 , R. Brion2 , J. Mönkkönen1 , H. Joensuu3 ,
D. Heymann2 , J. Määttä1 . 1 University of Eastern Finland, School of
Pharmacy, Kuopio, Finland; 2 INSERM, UMR957, Nantes, France; 3 Helsinki
University Central Hospital, Department of Oncology, Helsinki, Finland
Background: Tumour associated macrophages (TAM) are a macrophage
(Mf) population recruited and educated by tumour cells. Therefore, TAM
exposed to IL-10, TGF-b, M-CSF and other immunosuppressive stimuli are
more closely related to the M2 than M1 type macrophages. TAMs perform
preferentially trophic tasks promoting epithelial outgrowth and invasion,
common to the Mf role in development, rather than immune roles. The
current study focuses on the in vitro differentiation of human Mfs in the
presence of breast cancer conditioned medium (CM) and the differential
expression of M1 and M2c markers by TAMs in tissue samples from breast
cancer patients.
Materials and Methods: CD14+ freshly isolated human monocytes were
in vitro differentiated to M1 (IFN-g), M2a (IL-4) and M2c (IL-10) Mfs in the
presence or absence of human breast cancer cell line CM (MCF-7, T47D
and MDA-MB231). Differentiated cells were analysed by flow cytometry for
the expression of CD14, CD16, CD64, CD86, CD200R and CD163. CD68,
HLA-DRIIa and CD163 were analysed by immunohistochemistry in tissue
microarray (TMA) samples from a large cohort of human breast cancer
patients.
Results: All the breast cancer CM enhanced M2a differentiation, by
increasing the percentage of cells in the population of CD200Rhigh CD86med
that normally results from IL-4 stimulus. Further, MDA-MB231 CM was
sufficient to increase CD163 expression levels to the same extent as
IL-10, the M2c inducing cytokine. In conjugation IL-10 and MDA-MB231
CM further increased CD163 expression. From the ongoing TMA analysis
we expect to find out the effect of breast tumour Mf infiltration (assessed
by CD68), M1 (HLA-DRIIa) and M2c differentiation (CD163) on recurrencefree survival of breast cancer patients treated in a randomised clinical trial
(the FinXX trial).
Conclusions: Breast cancer cell lines affect alternative Mf differentiation,
inducing more matrix remodelling and immune-suppressive status, respectively M2a and M2c. Clinically this effect is relevant as human breast cancer
TAM differentiation status may influence metastases formation, therapy
efficacy and ultimately patient outcome.
180
POSTER (Board P174)
Identifying and monitoring somatic mutations in cell free DNA of
patients with metastatic melanoma
J. Wisell1 , C.M. Amato2 , W.A. Robinson2 . 1 University of Colorado,
Pathology, Aurora CO, USA; 2 University of Colorado, Medical Oncology,
Aurora CO, USA
Background: Within the last decade, significant advances have been made
in understanding the molecular pathogenesis of melanoma, and several
mutations such as those found in KIT, a tyrosine kinase receptor, and
BRAF proto-oncogene, play a significant role in the disease. Patients with
melanoma benefit from somatic mutation screening, particularly when it
comes to predicting for therapeutic response. Such mutational analysis is
performed on tumor samples, and with recent advancements in molecular
biology, it is now possible to detect genetic changes, i.e. somatic mutations,
in patient blood. In this study, our laboratory explored possible candidates
for monitoring disease progression in patient blood samples.
Material and Methods: The University of Colorado Skin Cancer
Biorepository hosts an extensive collection of clinically annotated blood and
tumor samples. We screened 175 metastatic melanoma tissue samples for
37 separate mutations in 10 different genes to identify somatic mutations
suitable for monitoring in matched patient blood samples. Cell free DNA
(cfDNA) was isolated from patient plasma samples, and somatic mutations
were measured using digital PCR (dPCR).
Results: In our tissue screen, approximately 65% of the samples contained
at least one mutation, BRAF being the most commonly mutated oncogene,
while RAS (KRAS and NRAS) and KIT mutations were second and third. In
our cfDNA analysis, BRAFV600E levels in patient plasma correlated with
disease burden.
Conclusion: We demonstrate that somatic mutations are detectable
in cfDNA isolated from patient blood samples. Our results show that
60
BRAFV600E mutation levels correlate with tumor burden and suggest a
possible role as a prognostic indicator. Studies are currently underway
to examine RAS mutations in patient blood as a possible marker for
acquired resistance to BRAF inhibitors, and to monitor disease progression
in patients with BRAF wildtype tumors.
181
POSTER (Board P175)
Aberrant Wnt signaling activation in human cancers: In vitro and
in vivo models to facilitate Wnt targeted drug development
1
1
1
1
1
1
1
G. Liu , C. Dong , R. Zhang , L. Zhang , S. Qian , J. Cai , J. Zhang ,
J. Ning1 . 1 Crown Bioscience Inc., Molecular and Cellular Biology,
Taicang, China
Activation of Wnt signaling has emerged as one of the major oncogenic
aberrations in human cancers, which has been demonstrated to play critical
roles in the maintenance of the undifferentiated cancer stem/progenitor
cell phenotype, as well as to directly stimulate the malignant growth of
tumors. Mutations in the downstream signaling components, including APC,
AXIN, and b-catenin, have been well described in several cancer types,
and recent studies have further extended the Wnt activation mechanisms
beyond these downstream mutations to upstream signaling molecules in
this pathway. By screening primary cancer cells established from the patient
derived xenograft models, we identified multiple cancer types that secrete
Wnt stimulating ligands. We also successfully established primary cancer
cell line harboring recurrent R-spondin fusion mutations. Inhibition of the
activated Wnt signaling in these cancer cells results in reduced cancer cell
growth, indicating the critical dependence of cancer cells on the autocrine
Wnt signaling. Thus, these in vitro and in vivo models provide a valuable
resource for the high throughput screening of Wnt antagonists, efficacy
assessment of candidate Wnt inhibitors, biomarker analysis, as well as the
preclinical development of Wnt targeted therapeutics.
182
POSTER (Board P176)
TRAP1 represents a key mediator of stemness and glycolytic
metabolism in colorectal cancer cells
G. Lettini1 , F. Maddalena1 , L. Sisinni1 , V. Condelli1 , L. Del Vecchio2 ,
M. Gemei2 , T. Notarangelo1 , M. Landriscina3 . 1 CROB − IRCCS,
Laboratory of Pre-Clinical and Translational Research, Rionero in Vulture
(PZ), Italy; 2 CEINGE, Biotecnologie Avanzate, Napoli, Italy; 3 University
of Foggia, Clinical Oncology Unit Department of Medical and Surgical
Sciences, Foggia, Italy
Background: Tumor cells undergo a metabolic shift from mitochondrial
oxidative phosphorylation to a preferential glycolytic metabolism, known
as Warburg effect. Furthermore, cancer stem cells (CSCs), known for
being responsible for tumor initiation and growth, are located in a hypoxic
microenvironment, heavily relying on anaerobic glycolysis. Recently,
tumor necrosis factor receptor-associated protein 1 (TRAP1), a HSP90
mitochondrial molecular chaperone up-regulated in colorectal, prostate,
breast and lung carcinomas, has been identified as a key regulator of
tumor cell metabolism, being responsible for suppression of mitochondrial
oxidative phosphorylation and post-translational stabilization of HIF1a. This
study was designed to address the hypothesis that TRAP1 is relevant in
determining specific features of the CSC phenotype and, more specifically,
the reprogramming of their metabolism.
Methods: TRAP1 levels were investigated in CD166-positive CSC fractions
of HCT116 and HT29 colorectal carcinoma cells (CRCs) and primary stem
cells derived from dental pulp by flow cytometry, cell sorting and upon
colony formation assay. Interfering strategies were used to evaluate TRAP1
role in regulating glucose uptake and glycolytic metabolism.
Results: TRAP1-interfered CRC HCT116 cells showed lower clonogenic
potential than scramble cells in soft agar assay. Furthermore, CD166positive CSCs derived from CRC cell lines exhibited higher TRAP1
levels compared to non-CSC subpopulations and, consistently, TRAP1
stable interference resulted in the down-regulation of CD166 expression
in HCT116 cells. In parallel experiment, TRAP1 expression was downregulated upon differentiation of primary stem cells derived from dental
pulp. Finally, TRAP1 interference resulted in increased O2 consumption
and in reduced glucose uptake, and the parallel inhibition of the expression
of glucose transporters 1 and 2 and lactate production in CRC cells.
Conclusion: These preliminary data suggest a potential role of TRAP1
in the maintenance of the CSC phenotype, likely due to its capacity
to enhance glucose uptake and glycolytic metabolism, thus favoring the
adaptation to a hypoxic microenvironment.
183
POSTER (Board P177)
Spatio-temporal characterization of tumor growth and invasion
A.M. Jimenez1 , O. Yogurtcu2 , M. Horn-Lee1 , P. Rao1 , S.X. Sun2 ,
D. Wirtz1 . 1 Johns Hopkins University, Chemical and Biomolecular Eng,
Baltimore MD, USA; 2 Johns Hopkins University, Mechanical Engineering,
Baltimore MD, USA
Background: Tumors are a complex arrangement of tissues made up
of several components, including dense masses of cancer cells and
extracellular matrix (ECM). Recent studies have revealed the crucial role
extracellular matrix components have on single cancer cell behavior, but
how the interaction of ECM components affect the growth dynamics of an
entire tumor is not well understood.
Materials and Methods: We designed a novel three-dimensional (3D)
in vitro system and used human derived fibrosarcoma cell (HT1080)
aggregates embedded in 3D collagen matrices in combination with live cell
imaging, cryo-stat sectioning, and immunostaining to characterize tumor
growth and invasion.
Results: Multicellular aggregates grow linearly with a rate that decreases
with increasing collagen concentration. The spatio-temporal cell density
distribution of the aggregates differs with differing collagen concentrations.
Both cell migration and proliferation are highly dependent on collagen
concentration and local cell density.
Conclusion: We identified cell migration as a key contributor to multicellular
aggregate size and demonstrate that different cell phenotypes correlate
with different invasion patterns of the cell aggregates at various collagen
concentrations.
184
POSTER (Board P178)
Molecular profiling of heterogeneous tumor cells
A. Chenchik1 , D. Deng1 , K. Bonneau1 , M. Makhanov1 , M. Coram2 ,
G. Dolganov2 , S.S. Jeffrey2 . 1 Cellecta Inc., Mountain View CA, USA;
2
Stanford University, Palo Alto CA, USA
Molecular profiling of heterogeneous circulating tumor cells (CTCs) and
tumor biopsy samples at the single-cell level is critical for identifying
different cancer cell subpopulations and understanding their value in
predicting metastatic cancer progression and responses to treatment. Unfortunately, the isolation and comprehensive characterization of hundreds
to thousands of individual cancer cells present formidable analytical and
technical challenges.
To this end, we developed a high-throughput (HT) HiCellex technology to
obtain the genome-wide expression and mutation profiles of hundreds to
thousands of epithelial cancer cells at the single cell level. Importantly,
HiCellex allows selective molecular profiling of a complete set of viable cells
at the single-cell level in the presence of contaminating leukocytes without
physical separation of individual cells. The genetic profiling technology
uses unique cell-specific molecular barcodes to label mRNAs in individual
viable cancer cells by a spliceosome-mediated trans-splicing mechanism.
Barcoded trans-splicing constructs, necessary for the labeling of the entire
population of pre-mRNAs, are delivered and expressed in a pool of target
cancer cells with lentiviral vectors. Digital expression data are generated
by HT sequencing of barcoded amplified cDNAs and easily cluster to each
cell in silico using cell-specific barcodes.
This presentation will provide results of development and validation of
genome-wide and multiplex RT-PCR profiling assay of the 500 most
informative subtyping and metastatic signature genes (MGS) in model
cancer cell lines, CTC and tumor samples. The developed HiCellex
500 MGS profiling assay is a cost-effective approach to effectively
characterize, subtype and identify prognostic and diagnostic biomarkers
in heterogeneous cancer cells at the single-cell level.
185
POSTER (Board P179)
Treatment of patient-derived NSCLC xenograft preclinical models
using image-guided small animal irradiation
N. Papadopoulou1 , A. McKenzie1 , J. King1 , M. Page1 , R. Kumari1 . 1 Precos
Ltd, Crown Bioscience, Loughborough Leicestershire, United Kingdom
Background: Radiotherapy is a primary, adjuvant or neoadjuvant treatment
for a number of different cancers such as glioblastoma, breast, lung and
prostate. Image-guided micro-irradiation (IGMI) is widely used to treat
cancer patients providing more accurate treatment plans and reduced
side effects. However in the preclinical setting the use of IGMI is less
common with traditional irradiation studies utilising whole body irradiation
with lead shielding to focus the radiation to a specific area on the animal
or simple single beam techniques. The development of the image-guided
small animal radiation research platform (SARRP) allows the treatment
of animal models of cancer more accurately and with planned protocols
similar to those utilised in the clinic. We have established a panel of
Caucasian NSCLC patient-derived xenograft (PDX), which are sensitive
or have acquired resistance to standard of care treatments.
Methods: NSCLC samples obtained from untreated patients undergoing
surgery were collected with ethical consent, disaggregated and implanted
subcutaneously in MF-1 nude mice (Harlan, UK) admixed with a human
stromal cell component to generate patient-derived xenograft (PDX)
models. Resistant models were generated in vivo through repeated cycling
of treatment for up to 10 weeks with standard of care or targeted agents.
Patient-derived xenografts were maintained in vivo and monitored by
calliper measurements three times weekly and mice recruited to the study
when mean tumour volume was approximately 100–200 mm3 . Irradiation
protocols and dose calculation were designed to deliver appropriate
irradiation dose (2−3 Gy/day/mouse) to the tumours either alone or in
combination with a targeted agent/chemotherapeutic, whilst sparing the
surrounding normal tissue. Response to treatment was evaluated by tumour
growth inhibition and clinical condition of mice monitored daily.
Results: Mice treated with IGMI using the SARRP tolerated irradiation
doses either alone or in combinations. Response and resistance to
irradiation across a panel of Caucasian NSCLC PDX and resistant models
will be reported along with any outcome of the combination treatments to
re-sensitize resistant cancer to radiotherapy.
Conclusions: The SARRP platform allows the evaluation of irradiation
alone or in combination with anti-cancer agents in small animals with
reduced side effects and improved safety outcome, allowing these novel
preclinical models to be used effectively for drug discovery programmes
and to derive irradiation schedules and regimens suitable for testing
subsequently in clinical trials.
186
POSTER (Board P180)
The use of Quantitative Textural Analysis imaging biomarkers
to predict response to temsirolimus treatment in advanced HCC
subjects
R. Korn1 , R. Osarogiagbon2 , R. Newbold3 , D. Burkett4 , J. Sachdev5 .
1
Scottsdale Healthcare Research Institute TGEN, Imaging Endpoints,
Scottsdale, USA; 2 Baptist Cancer Center, Oncology, Memphis, USA;
3
Scottsdale Medical Imaging, Radiology, Scottsdale, USA; 4 Imaging
Endpoints Core Lab, Radiology, Scottsdale, USA; 5 Scottsdale Healthcare
Research Institute-TGEN, Virginia G Piper Cancer Center, Scottsdale, USA
Background: The treatment of advanced HCC can be challenging. Being
able to predict who will respond to therapy would represent a major step
forward in disease control. We have developed an exploratory CT imaging
biomarker that may predict treatment response to temsirolimus (T) after
sorafenib (S) failure using quantitative textural analysis (QTA) of hepatic
tissue and tumors on CT scans.
Material and Methods: Pre and Post venous phase contrast CT scans
through the abdomen were obtained in 10 subjects enrolled in a IIT from
a single institution prior to treatment with T after S failure. QTA was
performed on the axial slice of the liver containing both uninvolved hepatic
tissue and tumor in the same slice. QTA parameters were generated at
a fine filter level (SSF 3) and displayed as histogram derived features of
mean, SD, mean positive pixel (MPP), entropy, skewness and kurtosis.
Statistical Correlation between QTA parameters and subsequent CT tumor
responses by mRECIST criteria, Barcelona score, tumor markers, OS, PFS
were performed using spearman correlations, regression analysis and ROC
analysis. P values <0.05 were considered significant.
Results: A single arm Simon two-stage phase II trial was conducted to
test the activity of T in previously S treated HCC patients. Of the 26
subjects enrolled 10 subjects qualified for QTA analysis as having both pre
and post contrast venous phase CT scans available for evaluation. Of the
10 subjects, 7 had PR and 3 had SD as best response by mRECIST. The
mean change in tumor size was −26.7% (median −34.4%, range −52.5% to
11.3%) for the 10 subjects. Using QTA there was a significant correlation
between baseline tissue-tumor texture and best mRECIST response as
measured by MPP (rho = 0.7551, p = 0.0011). ROC analysis showed that a
MPP cutoff value of 7.95 had the best separation of predicting PRs with a
85% sensitivity and 100% specificity. No significant correlation was noted
between QTA and PFS, OS, serum tumor markers, Barcelona scores.
Conclusion: This limited analysis of responding HCC patients indicates
that textural analysis from CT scans may provide an imaging biomarker for
predicting response to T therapy following S failure. Although preliminary,
these findings merit further investigation.
61
187
POSTER (Board P181)
Targeted genomic profiling of penile squamous cell carcinoma using
the Oncomine cancer research panel
A.S. McDaniel1 , D. Hovelson1 , A. Cani1 , C.J. Liu1 , Y. Zhang1 , S. Sadis2 ,
S. Bandla2 , P. Williams2 , D. Rhodes2 , S.A. Tomlins1 . 1 University
of Michigan, Pathology, Ann Arbor MI, USA; 2 ThermoFisher Scientific,
Ann Arbor MI, USA
Background: Penile squamous cell carcinoma (PeSCC) is a rare cancer
notable for significant morbidity and mortality as well as an incomplete
understanding of the underlying molecular alterations and lack of effective
non-surgical therapeutic approaches.
Material and Methods: A retrospective cohort of 60 formalin fixed,
paraffin embedded (FFPE) tumor samples from 44 PeSCC cases (including
14 matched primary/metastasis pairs) was subjected to targeted next
generation sequencing (NGS) using the Oncomine Cancer Research Panel
encompassing actionable recurrent somatic alterations in ~125 oncogenes
and tumor suppressors identified by analysis across multiple cancer types.
Sequencing of multiplexed templates was performed using the IonTorrent
Proton system. Sequence analysis was performed in Torrent Suite 4.0, with
sequence alignment by TMAP, and variant calling using the Torrent Variant
Caller plugin. HPV infection status for each sample was assessed using
additional genomic DNA for PCR with the GP5/6 and My09/11 consensus
primer sets for viral detection and typing.
Results: Highly multiplexed targeted NGS yielded an average of 1,136,032
mapped reads per sample with high coverage (mean >450×) over targeted
bases using 20 ng of input genomic DNA. High risk HPV was detected in
five cases (Four with HPV 16, one with HPV 33). All classes of genomic alterations were evaluated, including single nucleotide variants (SNVs), insertions, deletions, stopgains, and copy number variants (CNVs). Frequently
altered genes included CDKN2A (20 of 44), TP53 (19 of 44), NOTCH1
(9 of 44), FBXW7 (8 of 44), PIK3CA (7 of 44), NFE2L2 (6 of 44), and HRAS
(6 of 44). Notably, tumor specimens from four cases harbored amplifications
of EGFR and one case demonstrated CDK4 amplification; genes for which
approved and investigational targeted therapies are available. Importantly,
cases with multiple tumor samples profiled showed significant differences
in 4 of 14 paired samples, indicating heterogeneity for actionable mutations
such as EGFR amplification between primary tumors and metastases.
Conclusions: We evaluated a cohort of PeSCC FFPE specimens using
an NGS panel of recurrently altered cancer-associated somatic variants,
providing detailed molecular analysis of this disease for the first time. The
scope of mutations identified was similar to squamous cell carcinomas from
other locations such as the lung and head and neck region. We identified a
subset of cases harboring mutations with immediate therapeutic potential,
including EGFR amplifications. This research suggests that NGS profiling
of PeSCC may have utility as part of a precision medicine approach to aid
clinical decision making.
188
POSTER (Board P182)
RANBP2 knock-down is synthetic lethal with BRAF V600E in colon
cancer
L. Vecchione1 , V. Gambino1 , G. d’Ario2 , S. Tian3 , A. Schlicker1 ,
S. Mainardi1 , B. Diosdado1 , I. Simon3 , M. Delorenzi2 , C. Lieftink1 ,
R. Beijersbergen1 , S. Tejpar4 , R. Bernards1 . 1 NKI/AVL, Division of
Molecular Carcinogenesis, Amsterdam, Netherlands; 2 Swiss Institute
for Bioinformatics, Bioinformatics Core Facility, Lausanne, Switzerland;
3
Agendia BV, Agendia BV, Amsterdam, Netherlands; 4 KULeuven,
Laboratory of Digestive Oncology, Leuven, Belgium
Background: Approximately 8−15% of colon (CC) patients carry an
activating mutation in BRAF. This CC subtype is associated with poor
outcome and with resistance to chemotherapeutic treatments.
We recently showed that BRAF (V600E) colon cancers have a characteristic gene expression signature, which is found also in subsets of KRAS
mutant and KRAS-BRAF wild type (WT2) tumors. Tumors having this
gene signature, referred to as ‘BRAF-like’, have a similar poor prognosis
irrespective of the presence of the BRAF (V600E) mutation.
By using a shRNA-based genetic screen in BRAF mutant CC cell lines we
aimed to identify genes necessary for survival and growth of BRAF mutant
CC in order to reveal novel targets for therapy.
Method: We selected 363 genes that are specifically overexpressed in 89
BRAF mutant tumors as compared to 608 WT2 type tumors, based on
gene expression profiles generated in two independent datasets
The 363 genes list was used to generate a shRNA library consisting of
1815 hairpins targeting these genes (BRAF library) selected from the TRC
human genome-wide shRNA collection (TRC-Hs1.0). Vaco432 and WIDR
(BRAFV600E) CC cell lines and Lim1215 (WT2) CC cell line were infected
with the BRAF library and screened for shRNAs that are selectively synthetic lethal with BRAFV600E mutation. Cells stably expressing the shRNA
62
library were cultured for 13 days, after which shRNAs were recovered by
PCR. Deep sequencing was applied to determine the relative abundance
of each shRNA in BRAF (V600E) cells as compared to WT2 CC cells.
Results and Conclusions: Based on the results of the pooled shRNA
screen, we were able to identify six candidate synthetic lethal genes in
BRAF mutant CC cell lines. In particular, further validation showed RANBP2
gene knock-down to be synthetic lethal with BRAFV600E and BRAFlikeness in CC. Experiments addressing the identification and the biological
characterization of RANBP2 will be presented.
189
POSTER (Board P183)
Molecular and pharmacological characterization of primary
mesothelioma tumor cell lines orthotopically xenografted in nude
mice
C. Pisano1 , A. Cole1 , A. Barbarino1 , E. Bianchino1 , M. Guglielmi1 ,
C. Melito1 , G. Mercadante1 , A. Porciello1 , A. Riccio1 , I. La Porta1 ,
S. Orecchia2 , R. Libener2 , L. Mazzucco2 , S. Licandro3 , M. Ceccarelli4 ,
F. D’Angelo4 , P. De Luca1 . 1 Biogem, Preclinical Research&Development
Service, Ariano Irpino (AV), Italy; 2 Azienda Ospedaliera, Hematology,
Alessandria (AL), Italy; 3 IRCCS-Istituto di Ricerche Farmacologiche
Mario Negri, Oncology, Milano (MI), Italy; 4 Biogem, Bioinformatic, Ariano
Irpino (AV), Italy
Background: Mesothelioma is estimated at less than 1% of all cancers,
however its incidence is increasing, with an expected peak in the next
10−20 years.
In order to setup in vivo models of human mesothelioma, putatively relevant
to studies of the human pathology in terms of histology, antigen expression,
and pharmacological response to chemotherapy, we have established
in vivo models of mesothelioma, using primary tumors, having different
histotypes.
Material and Methods: primary cells derived from nine mesothelioma
patients were xenografted orthotopically in immunodeficient nude mice.
Cells derived from three of nine xenografted mice (MM432: sarcomatoid,
MM473: epithelioid and MM487: biphasic) were stably transfected with
luciferase expression vector. These cells and the nine original cell lines
were used for further molecular and biochemical characterization.
Results: The selected MM473 and MM487 high luc-expressing clones
were intrapleurally reinoculated in immunodeficient nude mice. They
successfully invaded and proliferated within the murine host. In particular,
the epithelioid histotype presented tumour growth in 100% of mice, after
a short latency period. The biphasic histotype showed tumour growth
in 30% of the cases, although it was far more aggressive than the
epithelial histotype in engrafted mice. Immunohystochemistry evaluation
of resultant tumour masses confirmed the histotype from the parental
patient tumours (i.e. for CEA, EMA, Mesothelin, Podoplanin, calretinin).
Interestingly, biochemical characterization (i.e. EGFR, VEGF, Top1, TKs
pathways) of the three histotypes, revealed histotype-related differences.
The exposure of the nine cell lines to a panel of pharmaceutical drugs (i.e.
Doxorubicin, Topotecan, Cyclophosphamide, Dacarbazine, Gemcitabine,
Temozolomide, Bortezomib, 5-Azacitidine, Paclitaxel, Etoposide, 5-FU,
Cisplatin, and AZD-2281) revealed a different not histotypes-related
sensitivity.
Western blot and RT-PCR analysis revealed that mesothelioma cell lines
express elevated levels of EGFR (RNA and protein) not correlate with
downstream signaling pathways. Thus, the sensitivity to gefitinib of these
cells was not correlated with the EGFR expression. Finally, gene expression
analysis between the three histotypes (MM432, MM473 and MM487)
revealed that more than 1000 genes are differentially expressed.
Conclusions: Taken together, these results have led to expand our
knowledge on the molecular and biochemical features of various type of
mesotheliomas. In addition, our in vivo models, being established from
primary cells from patients, enlarged the available models of mesothelioma
that, with the limitations intrinsic to the experimental models, can be
predictive about the efficacy of new drugs or new therapeutic approaches
for this incurable disease.
190
POSTER (Board P184)
Differential chemosensitivity between CETCs and tumour spheroids
in cancer patients with solid tumors
D. Zimon1 , M. Pizon2 , E.L. Stein2 , U. Pachmann2 , K. Pachmann2 . 1 Simfo
GmbH, Bayreuth, Germany; 2 Simfo GmbH, Research, Bayreuth, Germany
Background: In vitro chemosensitivity testing of circulating epithelial
tumor cells (CETCs) provides real-time information about the sensitivity
of the tumor cells present in the patient and correlates with treatment
success. Nevertheless, a fraction of CETCs can survive after conventional
chemotherapy and grow into distant metastasis. A subpopulation of CETCs
with proliferation activity has the ability to form spheroids in suspension
culture. Spheroids exhibit stem cell-like properties and may be responsible
for chemo therapeutic resistance. Therefore, the aim of our study was
the comparison of the efficacy of chemotherapeutics on CETCs and on
spheroids originated from the same individuals.
Methods: The enumeration of CETCs collected from patients with solid
tumors in clinical stage 1−4 were performed using the maintrac® method.
Subsequently, viable CETCs were cultured in suspension culture system
allowing for spheroid formation. To evaluate the cytotoxic effect CETCs and
spheroids we exposed to anticancer drugs in short time culture in different
concentrations and for different periods of time.
Results: The response to chemotherapeutics was different between
CETCs and spheroids. In contrast to CETCs, spheroids from the same
patients were significantly more chemoresistant. Whereas active drugs
led to membrane permeability in single CETCs with subsequent staining
of the nuclei with propidium iodide, the same drugs led to disintegration
of tumorspheres with destruction of part of the cells but often part
of the cells in the spheres were able to survive. Epirubicin and,
interestingly, and especially salinomycin, a polyether ionophore antibiotic
isolated from Streptomyces albus, showed the best effects. Docetaxel,
cyclophosphamide and 5-fluoruracil showed almost no cytotoxic effects
onto the cells in the spheres.
Conclusion: Our results show, for the first time, that stem cells circulating
in peripheral blood, capable of forming spheroids are way more resistant to
anticancer drugs than the remnant circulating tumor cells. We, furthermore,
demonstrate that salinomycin efficiently destroy spheroids cultured from
CETCs, strengthening its role as promising anti-cancer therapeutic.
191
POSTER (Board P185)
Homotypic and heterotypic cell signaling transduction using a
dielectrophoresis microfluidic device
M. Tellez Gabriel1 , D. Heymann1 . 1 Université de Nantes, UMR 957,
Nantes, France
Studying cell signaling transduction in homotypic and heterotypic cell
interactions can have a great impact in clinics as could be useful
to determine which cells are able to establish communication (e.g.
tumoral niche). In this study, taking the advantage of a dielectrophoresis
based microfluidics device (the DEPArray), we show the possibility
of observing homotypic (Human osteosarcoma cells) and heterotypic
(Human osteosarcoma cells vs. Mesenchymal Stem Cells) cell signaling
transduction either single cells or clusters.
We prepared two different cell populations − acceptors and donors −
stained with two different fluorescent markers, one with DiI − a red
non transferable molecule − and the other one with Calcein-AM − a green
fluorochrom, able to be transferred through GAP junctions- and mixed them.
We predetermined spatial coordinates by software-guided routing for establishing cell interactions − at different ratios of populations − within the chip,
and we programmed a time-lapse for taking images of the interacting cells.
The analysis of the images showed a decreasing in time of the green
fluorescence intensity in donor cells, concomitant with an increasing of
green fluorescence in acceptor cells, indicating the existence of cell
signaling transduction between cells in both homotypic and heterotypic
interactions. These findings are promising to study which specific cell–cell
communications are established between different populations present in
a microenvironment.
192
POSTER (Board P186)
Extensive characterization of patient derived colon cancer xenografts
for preclinical biomarker identification
V. Vuaroqueaux1 , F. Kiefer1 , P. Bronsert2 , A.L. Peille1 , B. Zeitouni1 ,
F. Foucault1 , T. Kees1 , J. Guo3 , J. Schüler3 , H.H. Fiebig4 . 1 Oncotest
GmbH, Biomarker Development and Bioinformatics, Freiburg, Germany;
2
Institute of Pathology University Hospital, Freiburg, Germany; 3 Oncotest
GmbH, In vivo, Freiburg, Germany; 4 Oncotest GmbH, Freiburg, Germany
The use of large collections of preclinical models with extensive molecular
characterization is essential for improving preclinical drug sensitivity testing
and biomarker identification. We established a collection of 70 patient
derived xenografts (PDX) of colorectal cancer and analyzed them for
morphological features, genomic alterations (Whole Exome Sequencing
& Affymetrix SNP6), transcriptomic profiles (affymetrix U133 2 plus) and
sensitivity to various anticancer agents. Furthermore, we set up an ‘R’
based bioinformatics process for integrative analyses and automated
biomarker identification. Here, we used an integrative OMICS approach
to characterize our colon cancer PDX collection and we evaluated their
relevance for biomarker identification.
At the histological level, colon PDX tumor tissues closely resembled the
patient tumors and retained similar differentiation grades. Furthermore,
we observed heterogeneity in between the different colon PDX regarding
63
stroma content and vascularization. At the transcriptomic level, we identified
several PDX clusters similar to Goblet-like, Enterocyte, Transit-Amplifying,
Inflammatory and Stem-like subtypes recently reported in CRC patients. In
addition, well and moderately differentiated PDX showed a different gene
expression profile than poorly and undifferentiated models. At the genomic
level, the colon PDX models exhibited alteration profiles comparable
to those reported for patient tumors regarding signatures of mutational
processes (patient-age related or associated to mismatch repair gene
deficiencies), chromosomal rearrangement and mutated genes (e.g. APC,
TP53, KRAS and TP53). Moreover, we identified 6 PDX models with
microsatellite instability (MSI), 5 of them being hyper-mutant with a loss of
MLH1 expression. Interestingly, colon PDX displaying the MSI phenotype
showed gene expression patterns of the Globlet-like or Inflammatory
subtypes. To demonstrate the potential of our approach, we searched for
molecular determinants of cetuximab sensitivity in colon PDX by combining
in vivo drug sensitivity and molecular data. The study identified the
Transit Amplifying subtype, AREG/EREG expression and KRAS mutation
among the best predictors of response. Similarly, predictive biomarkers for
oxaliplatin and irinotecan were found, and their associations with particular
PDX subtypes are under investigation. The accuracy in predicting patient
outcome following treatment with FOLFOX and FOLFIRI will be evaluated
by assessing publicly available datasets.
The comprehensive characterization of our colon PDX models confirmed
their similarities with patient tumors and their inter-tumoral diversity. The
combined use of PDX molecular and drug sensitivity data represents
a promising approach for biomarker identification and tumor response
prediction at a preclinical stage.
with advanced PDA have improved their overall survival, but the disease
still inexorably progresses. The most commonly used system for drug
discovery/screening and development are PDA monolayer cell lines
coupled to preclinical studies in mice. However, this approach has not been
successful and new ones are needed.
Material and Methods: In the last year our lab has developed an organotypic three-dimensional culture system to study PDA. Murine organoids
derived from normal ductal, preneoplastic or neoplastic pancreatic cells
are cultured in semisolid media. In the case of human, normal and tumor
are also grown in this semisolid media. Following orthotopic transplantation,
these cultures form the normal, premalignant or malignant ductal structures
from which they were derived and they are termed ‘pancreatic ductal
organoids’. Using different methods that assess cell viability (ATP based
luminescence and fluorescence), we have measured the effect of single
drugs and combinatorial strategies.
Results: This platform allows us to compare the IC50 of different drugs
and search for those with a higher therapeutic index by comparing
normal vs tumor. We have evaluated several chemotherapeutic drugs
(Gemcitabine, 5-Flurouracil, paclitaxel, irinotecan and oxaliplatin) as well as
some targeted agents (eg. Selumetenib, Neratinib, and MK-2206). We have
also included tumor microenvironment cell types in our cultures to evaluate
their role in drug responsiveness. Furthermore, organoid cultures have
been transplanted orthotopically into immune-competent mice allowing for
the study of therapeutic responses in vivo.
Conclusions: We present this versatile ex vivo system as a promising
experimental platform for achieving further insight into biologically and
clinically important questions regarding cancer therapeutics in PDA.
193
POSTER (Board P187)
The identification of new therapies for ependymoma subgroups
195
POSTER (Board P189)
A humanized mouse model for preclinical testing of molecules
targeting immune checkpoints
N. Boulos1 , J.D. Dapper1 , Y.T. Patel2 , M. DeCuypere1 , B. Bianski3 ,
K.M. Mohankumar1 , M.O. Jacus2 , B. Freeman III4 , K.D. Wright5 ,
A. Gajjar5 , A.A. Shelat6 , C.F. Stewart2 , R.K. Guy6 , R.J. Gilbertson1 .
1
St Jude Children’s Research Hospital, Developmental Neurobiology,
Memphis TN, USA; 2 St Jude Children’s Research Hospital,
Pharmaceutical Sciences, Memphis TN, USA; 3 St Jude Children’s
Research Hospital, Radiologic Sciences, Memphis TN, USA; 4 St Jude
Children’s Research Hospital, Preclinical Pharmacokinetics Shared
Resources, Memphis TN, USA; 5 St Jude Children’s Research Hospital,
Oncology, Memphis TN, USA; 6 St Jude Children’s Research Hospital,
Chemical Biology and Therapeutics, Memphis TN, USA
Current treatment for ependymoma is limited to surgery and radiation;
existing chemotherapies are generally ineffective. We aimed to identify new
therapies for ependymoma subgroups using accurate mouse models driven
by ependymoma oncogenes, including the highly recurrent C11orf95RELA fusion (RELA+ ). First, we performed unbiased, high throughput
drug screening against tumor cells generated from a RTBDN-driven
ependymoma (mEPRTBDN ) mouse model. Initial screens performed at a
single concentration and dose response screen of 6500 compounds,
comprising FDA-approved drugs and developmental candidates, identified
377 compounds with 50% activity. Of these, 30 potent compounds
(with EC50 1mM) were selected for rigorous preclinical pharmacokinetics
(PK) and efficacy studies that included mouse tumor neurosurgery and
cranial irradiation. Our lead compound gemcitabine proved highly active
in vitro against RELA fusion-negative (RELA− ) ependymoma (mEPRTBDN
and mEPEPHB2 ) and RELA+ ependymoma. Applying stringent preclinical
PK with consideration of pediatric clinical dosing and toxicity, we identified
the optimal route of administration, dose and schedule for gemcitabine
(fixed dose rate infusion of 0.805 mg/kg/min over 3 hrs). Treatment of mice
harboring murine RELA− and human RELA+ ependymoma with this PK
guided regimen significantly prolonged survival. Moreover, combination of
gemcitabine infusions with surgery further improved survival that we are
now testing in a triple treatment regimen in mice that includes surgery,
radiation and gemcitabine. We currently are developing gemcitabine as a
new treatment for children with both RELA+ and RELA− ependymoma.
194
POSTER (Board P188)
Pancreatic ductal organoids as a new platform for drug discovery
M. Ponz-Sarvise1 , V. Corbo1 , D. Öhlund1 , T. Oni1 , A. Handly-Santana1 ,
D. Engle1 , H. Tiriac1 , C. Chio1 , M. Feigin1 , L. Baker1 , C. Ardito-Abraham1 ,
Y. Park1 , C. Hwang1 , E. Elyada1 , K. Yu1 , H. Clevers2 , D.A. Tuveson1 .
1
Cold Spring Harbor Laboratory, Cancer Center, Cold Spring Harbor,
USA; 2 Hubrecht Institute for Developmental Biology and Stem Cell
Research, Utrecht, Netherlands
Background: Pancreatic ductal adenocarcinoma (PDA) is a lethal disease
with a 5-year survival rate of 6%. Newer cytotoxic regimens for patients
G. Baia1 , D. Vasquez1 , D. Ciznadija1 , D. Sidransky2 , A. Katz1 , K. Paz1 .
1
Champions Oncology, Baltimore MD, USA; 2 Johns Hopkins University,
Baltimore MD, USA
Background: The blockade of immune checkpoints is a promising
therapeutic avenue for cancer therapy, with durable objective responses
observed in patients with a variety of solid tumors. Despite these
successes, current animal models do not reliably identify targets with
the greatest clinical potential due in part to differences between human
and murine immune systems. Hence, robust preclinical tools to test these
drugs directly against human cancers in the context of a human immune
system are required. To circumvent this limitation, Champions Oncology
has developed the ImmunoGraft, whereby two innovative technologies, the
Champions TumorGraft (patient-derived xenograft) and humanized mice
(immunodeficient mice reconstituted with a human immune system), are
combined in a single platform.
Materials and Methods: Thirty-nine melanoma, colorectal, breast, and
lung TumorGrafts were characterized with respect to HLA expression,
mutation status, and expression of PD-L1, a pathway targeted by
several therapeutics currently in clinical trial. Immune-compromised NOG
(Prkdcscid Il2rgtm1Sug ) mice were reconstituted with human CD34+ cells and
the animals monitored for cell engraftment and expansion. Humanized and
non-humanized NOG animals were subcutaneously implanted with tumor
fragments and growth rates were compared between the two groups.
Results: As early as 6 weeks after humanization was initiated, mature
human CD45+cells could be detected in the circulation of humanized
animals. We found at least 70% of reconstituted animals had >15%
hCD45+ cells in the peripheral blood 10 weeks post-hCD34+ reconstitution.
Of the hCD45+ cells present in the peripheral circulation of humanized
animals, 19% were hCD3+ (T cells), of which 67% were hCD4+ helper
T cells, and 25% were hCD8+. Although still early in the growth phase,
tumor volumes in humanized animals are comparable to those in nonhumanized animals. Assessing ImmunoGraft responses to checkpoint
inhibitors is underway.
Conclusion: Our study has demonstrated the potential of combining
humanized mice with Champions TumorGrafts to generate a novel and
unique preclinical model. The ImmunoGraft will allow direct assessment
of immuno-modulatory agents on tumor growth and progression in a
platform more reflective of the complete human microenvironment. It will
also facilitate research examining the critical interplay between tumors and
the immune system, potentially leading to identification of the most active
drugs, and perhaps additional drug targets.
64
196
POSTER (Board P190)
Neuropilin 2 (NRP2) modifies CXCL12/CXCR4 signaling and promotes
lymph node metastases in colon cancer
H. Schneider1 , P. Hönscheid1 , S. Schölch2 , C. Jakob1 , M. Muders1 .
1
Universitätsklinikum Carl Gustav Carus, Institute of Pathology, Dresden,
Germany; 2 Universitätsklinikum Carl Gustav Carus, Department of
Surgery, Dresden, Germany
Aims: Dissemination of cancer cells to regional lymph nodes is one of the
most important prognostic factors for colorectal cancer. To be able to form
lymph node metastases tumor cells need a supportive microenvironment.
One important part of this niche are lymph vessels that are located inside
the lymph node. This lymphovascular niche has been shown to be an
independent prognostic factor in rectal cancer patients after neoadjuvant
radiochemotherapy (Jakob et al., Plos One 2011). These intranodal lymph
vessels produce chemoattractant factors like CXCL12, that can bind to
CXCR4-positive tumor cells and mediate cell migration. We investigated
if the receptor Neuropilin-2 (NRP2) mediates lymph node metastasis by
modulating the CXCR4/CXCL12 signaling pathway.
Methods: In a cohort of 113 node negative colon cancer patients the
lymph vessel density in regional lymph nodes was quantified by standard
immunohistochemistry staining against D2−40, which labels lymphatic
endothelium. In vitro experiments were performed with standard cell lines.
Protein and mRNA levels were assessed by Western Blot and quantitative
real time PCR. After stimulation with human recombinant CXCL12, RNAi
mediated depletion of NRP2 and CXCR4 as well as after addition of
recombinant Semaphorin 3F (Sema3F) Boyden Chamber Migration Assays
were performed. To confirm our in vitro results a cohort of 78 patients
with locally advanced node positive colon cancer was analyzed for NRP2expression in primary tumors and lymph node metastases by standard
immunohistochemistry. Parallely, a nude mouse model with orthotopically
implanted colorectal cancer cells was assessed.
Results: In node negative colon cancer patients, intranodal lymphangiogenesis scorrelated with disease-free survival (P < 0.05). siRNA mediated
depletion of NRP2 decreased CXCR4-mRNA and protein expression, while
CXCR4 had no influence on NRP2 expression. Blocking NRP2 abrogated
CXCL12-induced AKT phosphorylation. Increase of CXCR4 mRNA after
stimulation with CXCL12 (‘feed forward loop’) was inhibited after NRP2
depletion. Tumor cell migration towards a CXCL12-gradient was reduced
when NRP2 function was blocked by siRNA or Sema3F (P < 0.001). Our
in vitro results were confirmed by an up-regulation of NRP2 in lymph
node metastases of advanced colon cancer patients’ tissue (P < 0.001)
as well as an increased expression of NRP2 in metastasis after orthotopic
implantation of colorectal cancer cells in nude mice.
Conclusion: NRP2 modulates the CXCL4/CXCL12 signaling axis in
colorectal cancer cells and influences cell migration. Therefore, NRP2
might have an impact on the metastatic potential of colorectal cancer cells.
Additionally, the detection of intranodal lymh vessels in node negative colon
cancer patients by standard immunohistochemistry against D2−40 might
offer a new tool for oncologists to decide whether an adjuvant treatment is
recommended.
197
POSTER (Board P191)
Statistical aspects of kinetic analysis of gliomas with FDG-PET
D. Hawe1 , F.R. Hernandez1 , S. Murphy1 , E. Wolsztynski1 , J. Huang1 ,
J. O’Sullivan1 , M. Muzi2 , J. Eary3 , K. Krohn2 , F. O’Sullivan1 . 1 University
College Cork, Statistics, Cork, Ireland; 2 University of Washington,
Radiology, Seattle, USA; 3 University of Alabama, Radiology, Birmingham,
USA
Background: Positron emission tomography (PET) is used to examine
the distribution of radio-labelled molecules in vivo, which allows the status
of healthy and pathological tissue to be examined by studying how it
processes substances of interest.
Material and Methods: The interpretation of dynamic PET time-course
data is complicated because the measured signal is a combination of
vascular delivery and tissue retention effects. The tissue time-course
can be expressed as a convolution between the arterial time-course
(directly measurable) and the tissue residue. In light of this, estimating
the residue is essentially a survival analysis problem, which has been
examined in great detail in the literature. Kinetic analysis of PET data is
concerned with estimation of the residue function and parameters such
as flow, flux and volume of distribution. This is generally done using
compartmental models, however as these models assume that the area
being studied is homogenous, and that there is instant mixing within
compartments, its validity has been questioned. Accordingly, we examine
both a nonparametric model, and a mixture model approach, which do not
require the same assumptions of the compartmental model and compare
these alternatives with the classic compartment model approach to the
estimation of the residue function.
Results: We evaluate the improvement in the mixture and nonparametric
models relative to compartment models using simulations and crossvalidation. Estimates of key functionals including flow, flux and volume of
distribution for the three models are compared. This is illustrated with data
from ongoing 18 F-fluorodeoxyglucose-PET studies in a series of 46 brain
tumour subjects.
Conclusions: Significant statistical evidence against the compartment
model in favour of both the nonparametric and mixture models has been
found.
Supported by Science Foundation Ireland under SFI-PI 11/27 and by the
National Institute of Health (NCI) under PO1-CA-42045.
198
POSTER (Board P192)
Genetic and pharmacological inhibition of PIM-1 reduces tumor
development in a K-Ras-driven mouse model of non-small cell lung
cancer
E. Aguirre1 , O. Renner1 , M.C. Rodriguez de Miguel1 , M.I. Albarran1 ,
A. Cebria1 , D. Cebrian1 , F. Ramos-Lima1 , J. Pastor1 , C. Blanco-Aparicio1 .
1
Spanish National Cancer Research Centre (Cnio), Experimental
Therapeutics Program, Madrid, Spain
Lung cancer is the leading cause of cancer deaths worldwide, and 40%
of these cases are diagnosed as adenocarcinomas, a subtype of nonsmall cell lung cancer (NSCLC). Activating mutations in the K-Ras protooncogene have been found in 30% of human NSCLC, and these tumors are
still an unmet medical need as in most of the cases the current treatments
failed. K-Ras G12V is one of the commonest mutations observed in
NSCLC. Several mouse models have been created to understand the
role of K-Ras in tumor initiation and progression. In this study, we have
evaluated the effect of inhibiting PIM proteins in tumor progression in a
mouse model in which K-Ras (G12V) mutation is conditionally activated
resembling human NSCLC.
The PIM proteins are a family of highly homologous serine/threonine
kinases that are implicated in the regulation of apoptosis, metabolism,
the cell cycle, and homing and migration, which makes these proteins
interesting targets for anti-cancer drug discovery. To evaluate Pim1
inhibition as a therapeutic target for NSCLC, a conditional knock-in
mouse model had been used in which Pim1 kinase activity was ablated,
controlling its expression using a Cre/loxP recombination system. This
kinase-dead (KD) mutant was crossed with K-Ras (G12V) mice, and 7
months after intra-tracheal administration of Cre-recombinase adenovirus,
tumor development was assessed by computer tomography. The images
showed that K-Ras (G12V) Pim1-KD mice developed later and less lung
tumors than K-Ras (G12V) mice, significantly increasing their survival rate.
Besides, histological examination of these tumors revealed that only 14% of
K-Ras (G12V) Pim1-KD mice developed adenocarcinomas, whereas 73%
of K-Ras (G12V) mice presented this malignant type of lung cancer.
Having genetically validated PIM1 kinase as a molecular target for NSCLC
treatment, we reinforced these results by pharmacological inhibition of the
PIM proteins activity. For this purpose, a selective and potent proprietary
panPIM inhibitor was generated in our Programme. In the PK/PD assay,
this compound revealed that 24 hours after oral single dose administration
(100 mg/kg), a 60% specific inhibition of phospho-4E-BP1 (Thr37/46) was
detected in K-Ras (G12V) lung tumors samples. After that, the compound
efficacy was evaluated by orally administrating a daily dose of 50 mg/kg
for three weeks to K-Ras (G12V) mice. Pre- and post-treatment computer
tomography images revealed that tumor growth inhibition rate was 61%
in treated mice when compared to control mice. Besides, when the
administered dose was of 80 mg/kg, tumor growth inhibition reached the
value of 70%.
In summary, in this study we have confirmed that genetic or pharmacological inhibition of Pim-1 kinase is an effective strategy to be considered in
the treatment of NSCLC using a transgenic mouse model. In the future,
further studies will be needed to establish the role of Pim inhibitors in the
treatment of human NSCLC.
Poster Session – Radiation Interactive Agents
199
POSTER (Board P193)
Inhibitor of differentiation 1 (Id1) expression in lung cancer cells
and liver microenvironment is required for liver metastasis (LM)
development from non-small cell lung cancer (NSCLC) by regulating
EMT-related and proliferation-related proteins
E. Castanon1 , A. Soltermann2 , I. López3 , M. Ecay4 , M. Collantes4 ,
J.M. Lopez Picazo1 , M. Ponz5 , C. Rolfo6 , A. Calvo7 , I. Gil-Bazo1 .
1
Clinica Universidad de Navarra, Department of Oncology, Pamplona,
Spain; 2 University Hospital Zurich, Institute of Surgical Pathology, Zurich,
Switzerland; 3 Centro de Investigación Médica Aplicada, Laboratorio
de Nuevas Dianas Terapéuticas, Pamplona, Spain; 4 Clinica Universidad
de Navarra, Nuclear Medicine, Pamplona, Spain; 5 Cold Spring Harbour,
Cold Spring Harbour Laboratory, New York, USA; 6 University of Amberes,
Phase I Clinical Trials Unit, Antwerp, Belgium; 7 Centro de Investigación
Médica Aplicada, Department of Oncology, Pamplona, Spain
Introduction: Id1 is involved in carcinogenesis. We previously showed Id1
as an independent prognostic factor in NSCLC. Id1 and Id3 are required for
breast cancer metastasizing to the lungs. We studied the potential role of
Id1 in LM in an in vivo model of NSCLC and its correlation with EMT-related
proteins.
Methods: We selected 40 Id1 wild-type mice (Id1+/+) and 40 Id1 knockout
mice (Id1−/−). We intrasplenically injected 5×105 Id1-silenced (Id1s),
Id1/Id3-silenced (Id1/Id3s) or wild-type (Id1Id3wt) Lewis Lung Carcinoma
(LLC) cells. Mice were followed by weekly microPET for 4 weeks or
until death. Microscopic LMs were studied in all necropsies. A gene
expression study (Affymetrix) was performed to assess differences in LM
from Id1+/+ and Id1−/− mice. Three tissue microarrays (TMA) containing
538 NSCLC human samples were stained with against Sox10, Betacatenin, E-cadherin, Vimentin, Slug, Podoplanin, S6, ERCC1, Id1 and Id3.
An H-score was calculated. Multivariable analysis investigated potential
predictive factors for LM colonization in mice. Spearman’s Rho test for
measuring the possible correlation between Id1, Id3 and the proteins
studied in the TMA was used.
Results: Among Id1+/+ mice the LM rate was: 57.1% when Id1/Id3wt
cells were injected, 33.3% for animals injected with Id1s cells and 0%
in those in which Id1/Id3s cells were used (p < 0.001). Nonetheless, no
significant differences in LM were found regarding the cell type injected
among Id1−/− mice (p > 0.05). In the logistic regression model, Id1 silencing
in tumor cells reduced the risk of LM in an 84.9% independently of
the mice strain used (p = 0.03). We also found an 84.5% reduction in
the probability of developing LM among Id1−/− mice compared to Id1+/+
animals regardless the expression of Id1 and Id3 in the tumor cells injected
(p = 0.006). In the LM gene expression profile, the loss of Id1 expression in
mice produced a significant downregulation of different genes involved in
proliferation such as FGFGR1 (p < 0.001), Myc (p < 0.001), Akt (p = 0.03)
and Hoxd10 (p = 0.02). EMT-related genes such as Versican (p = 0.003),
Vimentin (p = 0.007), Snail (p = 0.04), Podoplanin (p = 0.02) and IntegrinBeta 1 (p = 0.04) were also downregulated by Id1 loss in the tumor
microenvironment compared to Id1+/+ mice (p < 0.001). Coincidentally in
the NSCLC samples studied, Vimentin, Podoplanin and Akt expression was
positively correlated with Id1 levels. In human NSCLC samples a positive
and significant correlation between Beta-catenin, E-cadherin, Slug, ERCC1
and S6 with Id1 expression was found too.
Conclusions: Id1 expression in lung cancer cells and liver microenvironment may be required for LM. Id1 expression would favor the migration
and colonization capacity of lung cancer cells by regulating EMT-related
and proliferation-related proteins in mice and humans. If proven in further
mechanistic experiments, Id1 and Id3 targeting might help prevent and treat
liver metastasis from lung cancer.
200
POSTER (Board P194)
The influence of different cMET and EGFR backgrounds on the
cytotoxicity of cMET and EGFR small molecule inhibitors in vitro
N. Van Der Steen1 , K. Zwaenepoel2 , C. Rolfo3 , E. Giovannetti4 ,
M. Castiglia2 , V. Deschoolmeester1 , A.P. Carreca2 , P. Germonpre5 ,
P. Pauwels2 . 1 University Antwerp, Center for Oncological Research,
Antwerp, Belgium; 2 Antwerp University Hospital, Molecular Pathology
Unit Department of Pathology, Antwerp, Belgium; 3 Antwerp University
Hospital, Phase I − Early Clinical Trials Unit Oncology Department,
Antwerp, Belgium; 4 VU Amsterdam, Pharmacology Lab Cancer
Center, Amsterdam, Netherlands; 5 AZ Maria Middelares, Department
of Respiratory Medicine, Ghent, Belgium
Background: Despite recent advances lung cancer shows poor prognosis
and high incidence of recurrence. Today, targeted therapies against the
epidermal growth factor receptor (EGFR) are frequently used in the clinic.
Besides EGFR, the cMET receptor and its ligand hepatocyte growth factor
65
(HGF) play a role in cancer growth, as cMET amplification is a known
resistance mechanism against these EGFR-targeted therapies. In vitro,
the combination of anti c-Met synergistically enhanced gefitinib-induced
growth inhibition in all NSCLC cMET-expressing cell lines. The current study
determines the influence of cMET amplification, expression and autocrine
stimulation, and EGFR mutational status and expression on the cytotoxicity
of small molecule inhibitors.
Materials and Methods: Therefore 4 non small cell lung cancer
(NSCLC) and 1 myeloma cell line were selected: HCC827 (EGFR
exon 19 deletion), H1975 (EGFR L858R and T790M substitutions), EBC-1
(cMET amplification), LUDLU-1 (EGFR and cMET wild-type) and the
JJN3 myeloma cell line (HGF secreting). Furthermore, 4 tyrosine kinase
inhibitors (TKI) were selected: erlotinib (EGFR-TKI), PHA665752 (cMETTKI), crizotinib (dual ALK and cMET-TKI) and 17-AAG [Heat shock
protein 90 (Hsp90) inhibitor]. To determine the cytotoxic effect of these
inhibitors, a sulforhodamine B assay was performed. Each cell line was
treated using monotherapy for 72 h under normoxic conditions.
Results: The cMET amplified EBC-1 cell line seemed to be very sensitive
to the Hsp90 inhibitor (IC50 = 90 nM), possibly because Hsp90 folds the
cMET-protein. The JJN3 cell line showed moderate sensitivity to cMETTKIs (IC50 = 3000–4000 nM) and very low sensitivity to Hsp90 inhibition
(IC50 = 941 nM) compared to the other cell lines. The EGFR mutant cell
lines (sensitizing and resistance mutations) are relatively sensitive to
the Hsp90 inhibitor (IC50 = 200–250 nM) in comparison with the wild-type
LUDLU cell line (IC50 = 780 nM), which showed low sensitivity to the whole
panel of inhibitors.
Conclusions: These observations can possibly open the door for
combination therapy that targets both EGFR and cMET. Further research
will be needed to investigate the sensibility of this cMET amplified cell line
to Hsp90 inhibition.
201
POSTER (Board P195)
Transcriptome analysis of CD133-positive stem cells and prognostic
value of survivin in colorectal cancer
S. Kim1 , S.H. Park2 , J. Lee2 , Y.S. Park2 , H.C. Kim3 . 1 Korea University
Hospital, Seoul, Korea; 2 Samsung Medical Center, Medical Oncology,
Seoul, Korea; 3 Samsung Medical Center, Surgery, Seoul, Korea
CD133 is an important, but not exclusive, biomarker of colorectal cancer
(CRC) stem cells. In order to identify other CRC stem cell-specific
genes, we performed a comparative expression profiling of CD133+
and CD133− cell populations in primary and metastatic tumors from
four patients with CRC. CD133+ and CD133− CRC cells were isolated
using MagSweeper and were used for whole transcriptome analysis
with RNA-Seq. We found thatin CD133+ cells, 17 genes (RNASE2,
PRB2, IL4, MGC27382, CLEC4C, SALL3, GIMAP1, ISG15, LOC728875,
ZIK1, ICAM2, CCDC7, CDYL2, LRRC2, ZEB1, OSTF1, and CCDC144B)
were significantly upregulated compared to CD133− CRC cells. Among
them, IL4 has been known as an inducer of survivin implicated in the
survival and proliferation of cancer cells. However, the prognostic value
of survivin in CRC is controversial. We evaluated survivin expression in
formalin-fixed paraffin-embedded tumor samples of 188 patients with CRC
by immunohistochemistry. Survivin overexpression was detected in 85
patients (45.2%) and was significantly associated with primary tumor sites
(P = 0.028), lymph node metastasis (P = 0.029), and advanced III/IV CRC
stages (AJCC 7; P = 0.001). Furthermore, survivin upregulation correlated
with reduced disease-free survival (DFS; P = 0.021) and overall survival
(OS; P < 0.000) and was proved to be an independent prognostic factor for
both DFS and OS in multivariate analysis. Collectively, our data suggest
that CD133+ CRC stem cells have a distinct expression pattern and
that survivin, upregulated by differentially expressed IL-4, is a candidate
biomarker for the prediction of recurrence and survival in CRC.
Radiation Interactive Agents
202
POSTER (Board P196)
The novel microtubule-destabilizing drug BAL101553 (prodrug of
BAL27862) sensitizes a treatment refractory tumor model to ionizing
radiation
A. Broggini-Tenzer1 , F. Bachmann2 , V. Vuong1 , A. Messikommer1 ,
K. Nytko-Karouzakis1 , T. O’Reilly2 , H.A. Lane2 , M.N. Pruschy1 .
1
University Hospital Zurich (USZ), Radiation Oncology, Zürich,
Switzerland; 2 Basilea Pharmaceutica International Ltd, Basel, Switzerland
Background: Microtubule-targeting agents (MTAs) are widely used for
cancer treatment, both alone and in combination. BAL27862 is a novel
66
microtubule-destabilizing drug with a dual action against human tumors;
targeting tumor cells refractory to standard MTAs as well as tumor blood
supply. The water soluble prodrug BAL101553 has completed Phase I
clinical evaluation administered i.v. but is also orally bioavailable. We
have investigated the effect of ionizing radiation (IR) in combination with
BAL27862 in vitro in human cancer lines resistant to standard MTAs, and
with BAL101553 in a genetically defined paclitaxel-, epothilone- and IRrefractory xenograft model. Different routes of BAL101553 administration
were compared.
Methods: Alamar blue/crystal violet proliferation, clonogenic survival and
apoptotic death assays were performed in human lung carcinoma A459,
b-tubulin-mutated A549EpoB40 and Pgp-overexpressing colon carcinoma
SW480 cells. Antitumor response was determined in SW480- and
A549EpoB40-derived tumor xenografts with BAL101553 administered
i.v. (21.3 mg/kg/wk) or p.o. (15 mg/kg QD×5 or 38 mg/kg/wk) and IR
fractionated over a week (3×5 Gy).
Results: BAL27862 reduced the proliferation/survival of wild type A549
cells, paclitaxel/epothilone-resistant A549EpoB40 cells and paclitaxelresistant SW480 cells with similar potency (anti-proliferative IC50s: 28nM,
22nM & 9nM, resp). BAL27862 combined with IR resulted in additive
cytotoxicity with no apoptosis induction. In SW480 xenografts, BAL101553
exhibited antitumor activity when administered i.v. or p.o., with daily
and weekly oral administration eliciting equivalent antitumor responses
(final D%T/C: 36 & 41, resp). Equipotent single i.v. bolus and daily
p.o. BAL101553 regimens were identified which partially inhibited tumor
growth (%T/C: 66 & 52, resp) in microtubule stabilizing agent-resistant
A549EpoB40 tumor xenografts, as did IR (%T/C: 35). Interestingly, both
BAL101553 regimens induced a profound antitumor effect when combined
with IR, associated with almost complete tumor stabilization (%T/C: 4 &
7, resp) over 5 weeks. Based on the in vitro data, involvement of tumor
microenvironment in antitumor response to this combination is likely.
Conclusions: BAL101553 single bolus (i.v.) or daily (p.o.) treatment
regimens positively interact with IR in a human cancer model refractory
to clinically relevant MTAs and IR, demonstrating the potential of this
combination therapy for the treatment of cancer patients.
203
POSTER (Board P197)
Ionizing radiation induced phosphatidylserine externalization on
endothelial cell surface − a potential target for vascular targeting
Z. Zhao1 , M. Johnson2 , B. Chen1 , M. Grace3 , J. Ukath3 , V. Lee1 ,
M. Stoodley1 . 1 Macquarie University, Australian School of Advanced
Medicine, Sydney NSW, Australia; 2 University of Technology Sydney,
Faculty of Science, Sydney NSW, Australia; 3 Genesis Cancer Care,
Macquarie University Hospital, Sydney NSW, Australia
Background: Vascular targeting agents can deliver anti-cancer drugs
specifically to tumor sites by binding to unique markers/targets on endothelial surface of tumors. The discriminating power of the markers/targets
relative to normal tissues determines the specificity of the technique.
Stereotactic RadioSurgery (SRS) can precisely deliver focused ionizing
radiation to a target tumor site. Radiation-induced molecular changes
should be restricted within the tumor tissue, and be good targets for
vascular targeting. This study investigated radiation-induced externalization
of phosphatidylserine (PS) in endothelial cells, a potential target for
vascular targeting.
Materials and Methods: An immortalized cell line generated from mouse
brain endothelium, bEnd3 cells, were cultured and irradiated at different
radiation doses using a linear accelerator (LINAC) Elekta Synergy. Then
PS externalization in the cells was visualized using pSIVA-IANBD, a polarity
sensitive probe for PS. Live cell imaging was used to monitor the PS
externalization in real time.
Results: Ionizing radiation has remarkable effects on the cells and
the effects are found to be dose dependent. The cell proliferation rate
decreased after exposure to 5 Gy radiation whereas higher radiation doses
(15 Gy and 25 Gy) totally inhibited proliferation. In comparison with shamradiation treated cells, the irradiated cells showed distinct pseudopodial
elongation with little or no spreading of the cell body. The percentages
of pSIVA positive cells were significantly higher in the cells that received
25 Gy and 15 Gy radiation 24 hours after treatment. This effect sustained
until the end of the experiment (3 days). Radiation at 5 Gy did not induce
significant PS externalization compared with the sham-radiation control at
any time points.
Conclusions: Ionizing radiation can cause remarkable cellular changes
in the endothelial cells. Significant PS externalization can be induced by
the radiation at dose levels 15 Gy and above. Given the precise focusing
of the radiation beams, the radiation-induced markers/targets may have
high discriminating power to be harnessed in vascular targeting for cancer
treatment.
204
POSTER (Board P198)
High-throughput functional screening identifies the flavoreductase
POR as a principal determinant of sensitivity to the hypoxia-targeting
prodrug SN30000
F.W. Hunter1 , Z. Shalev2 , J. Wang1 , J. Moffat3 , T. Katella4 ,
M. Koritzinsky3 , W.R. Wilson1 , B.G. Wouters3 . 1 University of Auckland,
Auckland Cancer Society Research Centre, Auckland, New Zealand;
2
University Health Network, Princess Margaret Cancer Centre, Toronto,
Canada; 3 University of Toronto, Department of Radiation Oncology,
Toronto, Canada; 4 University of Toronto, Donnelly Centre and Banting
and Best Department of Medical Research, Toronto, Canada
Background: Hypoxia constitutes an attractive therapeutic target owing
to its prevalence in tumours and its contribution to disease progression
and treatment resistance. Several hypoxia-selective prodrugs have been
developed to address this target, including the preclinical agent SN30000
which is an optimised analogue of the well-studied prodrug tirapazamine.
These prodrugs are enzymatically activated under hypoxic conditions and
thus selectively toxic to viable hypoxic cells. Biomarkers that are predictive
of prodrug activation and tumour sensitivity are urgently needed to support
their clinical development.
Figure: SN30000.
Materials and Methods: We deployed whole-genome lentiviral shRNA
screens interrogating 82,017 unique hairpins targeted to 16,019 human
genes, and a high-representation focused library enriched for oxidoreductases (1,821 hairpins targeting 359 genes), in tandem with massively
parallel sequencing, to identify determinants of sensitivity to SN30000
in HCT116, HT-29 and PANC-1 cells under hypoxic conditions. Potential
determinants were evaluated by RNAi-mediated knockdown and zinc finger
nuclease-mediated genetic knockout in cell lines and xenografts.
Results: Our functional screens identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of
sensitivity to SN30000 in all cell lines evaluated, except a POR knockout
clone derived from HCT116, with no other genes consistently selected.
Specific hairpins against POR were validated by qPCR and immunoblotting,
where POR suppression directly correlated with the degree of enrichment
in the high-throughput screens. Knockdown or knockout of POR inhibited
metabolic activation of SN30000 and enhanced clonogenic survival of cells
exposed under hypoxic conditions. We are currently assessing effects
of POR knockout on the activity of SN30000 against hypoxic cells in
xenografts.
Gene
shRNA clone
Z-score
HT-29
PANC-1
HCT116
POR
POR
POR
TRCN0000046524
TRCN0000046526
TRCN0000046527
3.3
3.2
2.6
3.5
3.1
2.2
5.0
3.3
2.3
Conclusions: POR acts as a critical determinant of tumour cell sensitivity
to SN30000 by catalysing reductive activation of the prodrug. As a single
gene apparently responsible for a major component of SN30000 activity,
tumour expression of POR may be a clinically tractable predictive marker
of response to SN30000. The methodology established in this study may
be repurposed to study determinants of sensitivity to additional hypoxiaactivated prodrugs such as the clinical agents TH-302 and PR-104.
205
POSTER (Board P199)
Metformin to modulate AMP-kinase and enhance chemotherapy and
radiotherapy in non-small cell lung cancer
M.J. Troncone1 , S.M. Cargnelli1 , G. Pond2 , E. Tsiani3 , J. Wright2 ,
G. Steinberg4 , H. Skinner5 , L. Bo6 , J. Bradley7 . 1 McMaster University,
Medicine, Hamilton Ontario, Canada; 2 McMaster University, Oncology,
Hamilton Ontario, Canada; 3 Brock University, Community Health Science,
Hamilton Ontario, Canada; 4 McMaster University, Biochemistry, Hamilton
Ontario, Canada; 5 M.D. Anderson, Radiation Oncology, Houston Texas,
USA; 6 Jefferson University, Radiation Oncology, Philadelphia, USA;
7
Washington University, Radiation Oncology, St. Louis, USA
Background: In the past few years our group investigated the response of
AMP-activated kinase (AMPK) to clinical doses of radiation therapy (RT).
We found that AMPK is not only a metabolic but also a genomic stress
sensor that is activated in epithelial tumour cells downstream of Ataxia
Telengiectasia Mutated (ATM) and leads to induction of p53 and cyclin
dependent kinase inhibitors such as p21cip1 and p27kip1 . We observed that
AMPK is involved in mediation of the G2-M cycle arrest and the cytotoxicity
of RT. Knock down of AMPK in cancer cells and knockout models of
AMPK show dramatic activation of the Akt-mTOR pathway. In tumours RT
induced growth inhibition and chronic suppression of angiogenesis that
was associated with sustained activation of ATM and AMPK and inhibition
of Akt and mTOR axis. We used the anti-diabetic drug metformin (MET)
to activate AMPK in lung tumours. MET is a safe and well-tolerated agent
that has no reported toxicity when combined with RT or chemotherapy.
Materials and Methods: We performed pre-clinical evaluation of MET in
combination with RT in in-vitro and in-vivo models of lung prostate cancer.
Further, we performed a retrospective analysis of clinical outcomes in stage
III NSCLC treated with chemotherapy and radiotherapy at the Juravinski
Cancer Center over the past 14 years.
Results: MET at low microM concentrations, that are safely achieved
in the serum of diabetic patients, activated the AMPK pathway, inhibited
growth and enhanced RT responses in cancer cells and tumours. The antiproliferative and radio-sensitizing activity of MET was depended on AMPK
and led to effective inhibition of the Akt-mTOR pathway. MET increased the
pro-apoptotic and anti-angiogenic action of RT.
Retrospective analyses of clinical outcomes show that diabetic patients
with locally advanced (LA) non-small cell lung cancer (NSCLC) treated
with RT and chemotherapy have improved survival after chemotherapy or
RT treatment if they receive metformin for the treatment of diabetes.
Conclusions: Based on our pre-clinical and retrospective clinical data we
propose prospective clinical trials in LA-NSCLC. Two phase II studies of
MET in combination of chemo-RT will open to accrual soon. The Canadian
Ontario Clinical Oncology Group (OCOG) ALMERA and US NRG LU001
studies will investigate MET in combination with concurrent chemo-RT in
LA-NSCLC. LU001 will examine the pure chemo-radio-sensitizing action of
MET using the drug only concurrently with chemo-RT while ALMERA will
examine the benefits of MET when used both concurrently with chemoRT and adjuvant for 1 year. Tumour bio-specimens collected in NRGLU001 and OCOG-ALMERA will help show whether expression or mutation
status of tumour (LKB1, K-Ras, p53, EGFR and Alk) and microenvironment
(HIF1a and micro-vessel density) markers could serve as biomarkers of
MET response in NSCLC.
206
POSTER (Board P200)
Identification of novel targets for radiosensitisation of non-small cell
lung cancer by secretome analysis
A. Sharma1 , S. Bender1 , O. Riesterer1 , A. Broggini-Tenzer1 , M. Pruschy1 .
1
University Hospital Zurich, Department of Radiation Oncology, Zurich,
Switzerland
Background: The therapeutic response of ionizing radiation (IR) is
imparted by genomic instability and DNA damage. However, IR also
triggers multiple intracellular signaling processes as part of IR-induced
stress responses that lead to the secretion of various para- and autocrine
factors into the tumor microenvironment. Here we investigated treatmentdependent secretion of auto- or paracrine factors, which drive acquired
rescue mechanisms and determine the overall radiation sensitivity of the
tumor.
Material and Methods: Exhaustive large scale secretome analysis was
performed using antibody arrays for a wide range of secretory factors.
Secretion kinetics of selected factors were determined using ELISA across
different established tumor cells and in murine blood serum, derived from
irradiated tumor xenograft-carrying mice. Clonogenic survival and xenograft
tumor growth delay assays were performed in response to IR in siRNAtargeted tumor cell lines or in combination with small molecular agents.
Results: We performed an exhaustive IR-dependent secretome analysis
(>300 factors) in lung carcinoma cells and investigated IR-induced
67
expression and tumor cell secretion of the top hits, including amphiregulin,
transforming growth factor-a and ALCAM. All these factors were secreted
in a similar IR-induced time- and dose-dependent way from several nonsmall cell lung cancer (NSCLC) cell lines, indicative of a common upstream
mechanism. No changes were observed at the transcriptional level implying
potential modulation at the posttranslational level. Interestingly, irradiation
induced a dose-dependent increase in cleavage of the proform of ADAM17
(A Disintegrin and metalloprotease domain 17), which resulted in enhanced
ADAM17 activity and correlated with subsequent substrate shedding. IRinduced ADAM17 activation required both p38 mitogen-activated protein
kinases (MAPKs) and Furins. siRNA mediated silencing of ADAM17 or
targeting of ADAM17 with the small molecular inhibitor TMI-005 suppressed
IR-induced shedding of these factors, down regulated ErbB-signaling in
target cells and enhanced IR-induced cytotoxicity in vitro and in vivo.
Ex vivo substrate analysis of murine blood serum derived from irradiated
tumor xenograft-carrying mice correlate with our in vitro results.
Conclusions: Our findings demonstrate that IR significantly activates
ADAM17, which results in shedding of multiple survival factors, growth
factor pathway activation and contributes to treatment resistance in NSCLC
cells. We provide a sound rationale for positioning ADAM17 inhibitors as
radiosensitizers to improve the treatment of NSCLC.
207
POSTER (Board P201)
The enhancement of radiotherapy efficacy with docetaxel-titanate
nanotubes as a new nanohybrid for localized high risk prostate
cancer
C. Mirjolet1 , J. Boudon2 , A. Loiseau2 , S. Chevrier1 , T. Gautier2 , R. Boidot3 ,
J. Paris2 , N. Millot2 , G. Crehange1 . 1 Georges-Francois Leclerc Cancer
Centre, Radiotherapy, Dijon, France; 2 UMR 6303 CNRS U Bourgogne,
NanoScience Department MaNaPI, Dijon, France; 3 Georges-Francois
Leclerc Cancer Centre, Molecular Biology, Dijon, France
Background: From 30% to 50% of high risk prostate cancer patients
who undergo radiation therapy (RT) will have a biochemical failure. These
failures are either due to a poor local control or to distant disease which
may also be related to a local failure. Taxane-based chemotherapy has
proved to be useful in prostate cancer. Combining chemotherapy, such as
docetaxel (DXL), with RT can enhance its efficiency, however systemic
injection of the classical formulation leads to 95% uptake by healthy
tissues whereas 2−5% only reach tumors; adverse side effects are a
crucial problem. Moreover, multidrug resistance mechanisms often limit
drug efficacy by decreasing tumor cell intracellular concentration of drugs.
There is interest to develop nanocarrier of DXL to maintain drug inside
cancer cells by improving its efficacy. In a previous in vitro study, we have
highlighted that titanate nanotubes (TiONTs) which have a needle shape
can enter and stay inside cancer cells until 10 days without cytotoxic effects
induction. We suggest in this study to develop TiONt-DXL nanocarrier and
to evaluate its in vivo biodistribution as well as its efficacy in association
with RT on a prostate cancer model.
Materials and Methods: TiONts are obtained from hydrothermal synthesis.
DXL molecules were grafted on TiONts using PEG-3000 molecules
to generate the nanohybrid. In vitro cytotoxic activity of nanohybrid
was evaluated on PC-3 cell line using MTS assay. BALB/c nude mice
bearing subcutaneous PC-3 human prostate tumors were used to evaluate
nanohybrid biodistribution and efficiency after intratumoral injection.
Biodistribution analysis was performed by SPEC-CT imaging using 111Indium. To evaluate the benefit of TiONt-DXL and RT association, tumors
were irradiated using three daily fractions of 4 Gy administrated the day
after nanohybrids injection. Mice behavior, health status and tumor volume
were monitored twice a week until tumor growth recovery.
Result: TiONt-DXL in vitro cytotoxic activity was showed on PC-3 cells.
Biodistribution kinetics showed that more than 70% of nanohybrids were
localized into the tumor 96 hours after injection. Tumor growth was stopped
for mice receiving RT combined or not to TiONt or TiONt-DXL. Moreover,
mice receiving TiONT-DXL alone exhibited a decrease in tumor growth
when compared to mice receiving free DXL.
Conclusions: These results highlight that our nanocarrier improve DXL
efficacy. Local control might be improved by injecting TiONT-DXL in the
tumor during prostate brachytherapy as a boost in high risk localized
prostate cancer. A longer follow-up period will be needed to determine
if TiONt-DXL combined to RT will improve outcome.
This work was supported by the ‘Ligue Contre le Cancer du Grand Est’
(Comités Doubs and Côte d’Or).
68
208
POSTER (Board P202)
Combined treatment of a DNA-PKcs inhibitor (NU7441) and ionizing
radiation causes a differential mode of cell death in a panel of
NSCLC cell lines and exhibits robust radiosensitisation
D. Saha1 , Y. Lan1 , F. Hsu2 , V. Tumati1 , Z. Zhang1 , Y. Lin1 , B. Chen1 .
1
UT Southwestern Medical Center, Radiation Oncology, Dallas TX, USA;
2
National Taiwan University College of Medicine, Urology, Taipei, Taiwan
Background: Lung cancer is still the leading cause of cancer death
worldwide. The current standard of care for lung cancer consists of
concurrent chemotherapy and radiation. Several studies have shown direct
evidence that a novel DNA-PKcs inhibitor NU7441 is highly potent radiosensitizer in different tumor models. However, the mechanism of NU7441
induced radiosensitization has not been fully elucidated. In this study,
the combined effect of NU7441 and ionizing radiation (IR) in a panel of
non-small cell lung cancer cell lines (A549, H460 and H1299) has been
thoroughly investigated. The purpose of the study is to enhance the effect
of the radiation using a DNA-PKcs inhibitor in radio-resistant NSCLC lines
representing different genetic background.
Material and Methods: Clonogenic surviving fraction analysis, DNA double
strand break repair kinetics, cell cycle analysis, immunofluorescence,
immunoblot, apoptosis and autophagy assays were used in this study.
Results: We found that NU7441 significantly enhances the effect of
IR in all cell lines tested. The notable findings in response to this
combined treatment are (i) prolonged delay in IR induced DNADSB repair,
(ii) induced robust G2/M checkpoint in these cell lines, (iii) increase of
aberrant mitosis followed by mitotic catastrophe specifically in H1299 cells,
(iv) dramatically induced autophagy in A549 cells and (v) IR-induced
senescence specifically in H460 cells. We observed that H1299 cells show
greater G2 checkpoint adaptation after combined treatment which can be
attributed to the higher expression level of Plk1 compared to A549 and
H460. The enhanced autophagy after NU7441 treatment in A549 cells is
possibly due to the higher endogenous expression of pS6K compared to
H1299 and H460 cells.
Conclusion: Differential mode of death was noticed and that leads to
the extensive radio-sensitization in all NSCLC lines tested. The choice of
cell death pathway, in response to the combined treatment with NU7441
and IR, can be attributed to mutation status of the cells treated.
th
Plenary Session 5
08:00–10:00
PLENARY SESSION 4
Antibody-Based Therapies (ADC and
others)
209
ORAL PRESENTATION
Pre-clinical and translational pharmacology, pharmacokinetics and
pharmacodynamics for a humanized anti-OX40 antibody MOXR0916,
a T-cell agonist in the treatment of solid tumors
S. Sukumaran1 , J.M. Kim1 , M. Huseni1 , J. Ruppel1 , H. Taylor1 , K. Totpal1 ,
J. Zhu1 , C. Zhang1 , H. Chiu1 , E.G. Stefanich1 . 1 Genentech, gRED,
So San Francisco CA, USA
Background: Targeting T cell surface proteins to restore and enhance
the function of tumor-reactive T cells have been found to be an effective
way to combat cancer. OX40 is a costimulatory receptor in the TNFreceptor family that is transiently expressed on antigen-experienced T-cells.
OX40 signaling in the context of antigen recognition by the T-cell receptor
enhances the proliferation and survival of CD4+ and CD8+ effector T cells
and inhibits the suppressive function of regulatory T cells. MOXR0916 is
a humanized effector-competent IgG1 agonistic antibody against human
OX40 that is being developed as treatment for refractory solid tumors.
Methods: In vitro characterization of the effects of MOXR0916 on T cell
proliferation and interferon g (IFNg) production was performed in human
primary CD4+ memory T cells stimulated with anti-CD3. In vivo studies in
mouse syngeneic tumor models were performed with the surrogate antimouse OX40 antibody PRO307205, as MOXR0916 does not bind mouse
OX40. Dose ranging efficacy studies were performed with the EMT6 model
and relevant pharmacodynamic (PD) markers including CD4 and CD8 T cell
proliferation, IFNg production, and FOXP3 and CD8B gene expression were
measured in blood and/or tumor. Pharmacokinetics (PK) of MOXR0916
and PRO307205 were assessed in SCID mice and PK of MOXR0916 was
assessed in cynomolgus monkeys.
Results: Treatment of CD4+ memory T cells with varied concentrations
of MOXR0916 and a fixed concentration of anti-CD3 produced a
concentration-dependent increase in cell proliferation and IFNg production
with EC50 values estimated as 10 ng/mL. Both single and multiple dosing
of 0.1 to 10 mg/kg of PRO307205 treatment were found to be efficacious
in the EMT6 model, with more animals responding to treatment with higher
and more frequent dose administration. No efficacy was observed with
a 0.01 mg/kg dose. Efficacious doses were associated with evidence of
peripheral and intratumoral increase in IFNg, proliferation of effector T cells,
and reduction in number of regulatory T cells. MOXR0916 showed linear
PK with typical IgG1 clearance and distribution parameters in both mouse
and cyno. Human PK was projected based on allometric scaling of cyno
PK.
Conclusion: Based on in vitro and in vivo studies of anti-OX40 pharmacology, MOXR0916 is predicted to counteract the immunosuppressive tumor
microenvironment and promote T cell-dependent, anti-tumor immunity
via two distinct mechanisms: (1) enhancing proliferation and cytokine
production of antigen-experienced effector T cells and (2) reducing the
number and activity of intra tumoral regulatory T cells. Quantitative data
from pharmacology, PK and PD were used for predicting relevant activity
and doses of MOXR0916 for testing in clinical trials.
210
ORAL PRESENTATION
A phase 1 study of KTN3379, a human anti-ErbB3 monoclonal
antibody in patients with advanced cancers
P. LoRusso1 , T. LaVallee2 , L. Kimmel2 , C. Lubeski2 , R. Gedrich2 ,
C. Sidor2 . 1 Wayne State University, Detroit Michigan, USA; 2 Koltan
Pharmaceuticals, New Haven CT, USA
Purpose: ErbB3 is an important dimerization partner for other ErbB family
members and is emerging as a key component in the development of
resistance. KTN3379 is a human monoclonal antibody against human
epidermal growth factor receptor-3 (ErbB3 or HER3) that blocks both
ligand-dependent and independent activation of ErbB3 and is engineered
to extend half-life. The Phase 1 trial (KTN3379-CL-001) is an open
label, dose escalation and expansion study to assess the safety and
pharmacokinetics of KTN3379 in patients with advanced cancers to
determine a recommended dose for efficacy studies and to evaluate
potential biomarkers related to KTN3379 activity.
Methods: The study is being conducted in two Parts. In Part I, using
a 3 + 3 design, patients received escalating doses of KTN3379 ranging
from 5 to 20 mg/kg every 3 weeks until disease progression or intolerable
71
toxicity. In Part II, KTN3379 will be administered along with a Standard
of Care (SOC) regimen in disease specific cohorts of 6 patients. Adverse
events are assessed according to the National Cancer Institute Common
Terminology Criteria for Adverse Events and dose limiting toxicities (DLTs)
were evaluated during the initial cycle. RECIST tumor measurements were
obtained every 3rd cycle and pharmacokinetics (PK) and pharmacodynamic
(PD) assessments were evaluated during each cycle. Archival tumor tissue
was obtained if available.
Results: In Part I, 12 patients with advanced cancer (CRC, ovarian,
pancreatic, endometrial, pancreatic, HCC and germ cell cancers), median
age of 62 with ECOG performance status of 0 or 1 and multiple prior
therapies (median of 4) received at least one dose of KTN3379. There were
no DLTs or Grade 3 related events reported to date and the highest dose
administered was 20 mg/kg. The most frequent treatment related AEs were
Grade 1 and 2 diarrhea (one Grade 3), dry mouth and skin, and anemia.
PK parameters were consistent with humanized monoclonal antibodies and
could support q3 week dosing. Early PD assessments indicated elevation
of sErbB3 levels in all patients following treatment. Additional assessments
are ongoing.
Conclusions: Results suggest that doses of 20 mg/kg are safe and well
tolerated and PK supports 3-week dosing. Expansion is ongoing to assess
the safety and tolerability of KTN3379 in combination with other targeted
agents in specific cancers and to evaluate the potential for biomarkers to
identify sensitive patients.
10:30–12:30
PLENARY SESSION 5
Epigenetic Targets
211
ORAL PRESENTATION
A novel synthetic lethal interaction between the histone mark
H3K36me3 and checkpoint kinases
S.X. Pfister1 , E. Markkanen1 , Y. Jiang1 , S. Sarkar1 , V. D’Angiolella1 ,
G. Dianov1 , A.J. Ryan1 , T.C. Humphrey1 . 1 CRUK MRC Oxford Institute
for Radiation Oncology, Department of Oncology, University of Oxford,
United Kingdom
Background: Synthetic lethal interactions between cellular pathways can
be exploited to selectively kill cancer cells that carry a mutation in a gene
of one pathway by chemically inhibiting the function of a gene in a second
pathway. SETD2, a histone H3K36me3 methyltransferase, has recently
been recognised as a tumour suppressor in several cancer types, thereby
identifying SETD2 loss of function mutation as potential target for cancer
therapy. Here, we report a novel synthetic lethal interaction between SETD2
loss and inhibition of DNA replication checkpoint proteins, and provide
insights on the underlying molecular mechanisms.
Material and Methods: Growth of p53 wild type SETD2-deficient (A498,
LB996) or -proficient (RCC4, U2OS) human cancer cell lines were
measured after treatment using WEE1 (MK1775), CHK1 (LY2603618,
AZD7762, Gö6976) or ATR (VE821) kinase inhibitors. DNA replication
was analysed by DNA fibre assay and by iPOND (isolation of proteins
on nascent DNA). dNTP pools were measured by polymerase-catalyzed
incorporation of radioactive dNTP. We confirmed the in vitro efficacy of
MK1775 in vivo in SETD2-deficient A498 xenografts.
Results: Compared to wild type, SETD2-deficienct cells were more
sensitive to growth inhibition by VE821 (IC50 = 8.5 vs 3.8mM), LY2603618
(IC50 = 750 vs 374nM) or MK1775 (IC50 = 327 vs 109nM) (p < 0.0001).
Increased cell death in SETD2-deficient cells was due to DNA damage
arising from replication stress, and not premature mitosis as has
been previously reported in TP53 mutant tumour cell lines. SETD2independent methods to reduce H3K36me3 levels (overexpression KDM4A
demethylase, or expression of the dominant-negative K36M histone H3.3
variant) also significantly inhibited cell growth and increased apoptosis on
treatment with ATR, CHK1, or WEE1 inhibitors (p < 0.0001). SETD2 and
WEE1 acted on two independent pathways regulating dNTP production
during DNA replication. An in vivo experiment in established SETD2deficient A498 xenografts grown in nude mice showed that treatment
with MK1775 (60 mg/kg, po, bid, d1−12) regressed tumours, producing
a marked reduction in tumour size compared with vehicle-treated control
animals (mean tumour size = 291.2±40.0 vs 50.2±4.7 mm3 , p < 0.0001).
In addition, MK1777-treated tumours had significantly greater levels of DNA
damage as measured by gH2AX foci (p < 0.0001).
Conclusions: The findings suggest that targeted inhibition of ATR, CHK1
or WEE1 may be exploited to create novel treatments for cancers with
reduced levels of H3K36me3.
72
Plenary Session 6
212
ORAL PRESENTATION
Novel anti-tumor activity of targeted LSD1 inhibition by GSK2879552
H. Mohammad1 , K. Smitheman1 , G. Van Aller2 , M. Cusan3 ,
S. Kamat4 , Y. Liu2 , N. Johnson2 , C. Hann4 , S. Armstrong3 , R. Kruger2 .
1
GlaxoSmithKline, Cancer Epigenetics, Collegeville PA, USA;
2
GlaxoSmithKline, Cancer Epigenetics, Collegeville, USA; 3 MSKCC,
Pediatrics, New York, USA; 4 Johns Hopkins University, Oncology,
Baltimore, USA
Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase
found in various transcriptional co-repressor complexes. LSD1 mediated
H3K4 demethylation can result in repressive chromatin environment that
silences gene expression and has been shown to play a role in development
and hematopoietic differentiation. LSD1 is overexpressed in multiple tumor
types, including acute myeloid leukemia (AML). Together, these studies
suggest LSD1 is an important regulator of the epigenome that modulates
gene expression through modification of histones and its presence in
transcriptional complexes.
The current study describes the anti-tumor effects of a novel, potent,
irreversible, GSK LSD1 inhibitor (GSK2879552) in AML and small cell
lung cancer (SCLC). Screening of over 150 cancer cell lines revealed that
SCLC and AML cells have a unique requirement for LSD1. While LSD1
inhibition did not affect the global levels of H3K4me1 or H3K4me2, local
changes in these histone marks were observed near transcriptional start
sites of putative LSD1 target genes. This increase in the transcriptionally
activating histone modification correlates with increased gene expression.
Treatment of AML cells with GSK2879552 promotes the expression of
cell surface markers associated with a differentiated immunophenotype,
including CD11b and CD86. In an MV-4−11 engraftment model, increases
in CD86 and CD11b were observed as early as 8 hours post dosing.
GSK2879552 treatment resulted in a potent anti-proliferative growth effect
in a subset of SCLC cell lines tested and all AML cell lines tested. Potent
growth inhibition was also observed on AML blast colony forming ability
of bone marrow samples derived from primary AML patient samples. The
effects of LSD1 inhibition were further characterized in vivo using a mouse
model of AML induced by transduction of mouse hematopoietic progenitor
cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were
transplanted into secondary recipient mice that were treated with an LSD1
tool molecule inhibitor for 17 days. Control mice succumbed to AML by
45 days post transplant, while treated mice showed prolonged survival.
GSK2879552 treatment of mice engrafted with SCLC cell lines resulted
in greater than 80% tumor growth inhibition. Studies using patient derived
primary SCLC showed similar efficacy demonstrating the growth inhibition
of SCLC with an LSD1 inhibitor extended beyond cell lines.
Together, these data demonstrate that pharmacological inhibition of LSD1
may provide a promising treatment for AML and SCLC. A Phase I clinical
trial using GSK2879552 was initiated in March, 2014.
All studies were conducted in accordance with the GSK Policy on the
Care, Welfare and Treatment of Laboratory Animals and were reviewed
the Institutional Animal Care and Use Committee either at GSK or by the
ethical review process at the institution where the work was performed.
13:30–15:35
PLENARY SESSION 6
213
ORAL PRESENTATION
A phase I dose-finding study of BI 853520, a potent and selective
inhibitor of focal adhesion kinase (FAK), in Japanese and Taiwanese
patients with advanced or metastatic solid tumors
T. Doi1 , C.C. Lin2 , A. Ohtsu3 , J.C.H. Yang2 , K. Shitara3 , L.C. Pronk4 ,
A. Sarashina5 , A.L. Cheng2 . 1 National Cancer Center Hospital East,
Gasrtointestinal Oncology, Chiba, Japan; 2 National Taiwan University
Hospital, Department of Oncology, Taipei, Taiwan; 3 National Cancer
Center Hospital East, Department of Experimental Therapeutics,
Chiba, Japan; 4 Boehringer Ingelheim España S.A., Clinical Development
Oncology, Barcelona, Spain; 5 Nippon Boehringer Ingelheim Co. Ltd,
Clinical PK/PD department, Kobe, Japan
Background: Focal adhesion kinase (FAK) is a non-receptor tyrosine
kinase that contributes to activation of multiple downstream-signaling
pathways involved in tumor cell survival, proliferation, invasion, and
metastasis. BI 853520 is a potent and highly selective inhibitor of FAK
with an IC50 of 1nM. It has shown efficacy in multiple xenograft models of
human cancer.
Material and Methods: This is a phase I, dose-finding study of
BI 853520 given as a continuous oral dosing regimen in 28-day cycles
in a mixed population of Japanese and Taiwanese patients (pts) with
progressive, advanced or metastatic solid tumors. Endpoints include safety,
determination of maximum tolerated dose (MTD), pharmacokinetics (PK),
pharmacodynamics, and efficacy, determined according to RECIST v1.1
criteria.
Results: To date, 18 pts have received doses of 50, 100 and 200 mg qd
of which 14 pts are evaluable for dose limiting toxicity (DLT). Male/female
ratio was 12/6, median age 65 years (range, 35−77 years), ECOG PS
0/1:10/8. Primary tumor types: 4 gastric cancer, 2 colorectal cancer; 2
oesophageal cancer, and 10 others. Median number of prior systemic
treatments: 3. Drug-related adverse events (AEs) in >10% of pts included
proteinuria (44.4%), diarrhea (38.9%), nausea (22.2%), vomiting (22.2%),
inflammatory fibrous thickening in the palm of the hand (11.1%), maculopapular rash (11.1%) and decreased appetite (11.1%), all of NCI Common
Terminology Criteria for AEs (v4.03) grade 1−2 except for one patient who
had proteinuria grade 3 that occurred during cycle 2 at the 200 mg dose
and was considered DLT. So far, no DLTs have been observed during
cycle 1 and no drug-related serious AEs have been reported. The MTD
was determined as 200 mg qd and the 200 mg cohort is expanded to a
total of 12 patients to confirm the MTD. PK:Preliminary data suggest that
plasma exposure increased with increasing doses. Based on the observed
accumulation and half-life, a once-daily dosing scheme is supported.
Preliminary efficacy: Of 12 evaluable pts, 1 pt with gastric cancer achieved
a partial response (100 mg qd), 2 pts presented stable disease: 1 pt with
oesophageal cancer and 1 with urachal cancer lasting 7 and 8 cycles,
respectively (both 50 mg qd), and 9 pts progressed.
Conclusion: The safety profile of BI 853520 is favourable. PK analysis
supports the once-daily dosing schedule. Recruitment at the 200 mg dose
is ongoing to confirm the MTD. An update will be provided at the meeting.
214
ORAL PRESENTATION
Homologous recombination deficiency (HRD) score and niraparib
efficacy in high grade ovarian cancer
P. Haluska1 , K.M. Timms2 , M. AlHilli1 , Y. Wang3 , A.M. Hartman2 , J. Jones2 ,
A. Gutin2 , Z. Sangale2 , C. Neff2 , J. Lynchbury2 , L. Rudolph-Owen3 ,
M.A. Becker1 , S. Agarwal3 , K.M. Wilcoxen3 . 1 Mayo Clinic, Rochester,
USA; 2 Myriad Genetics, Salt Lake City, USA; 3 Tesaro Inc., Waltham, USA
Purpose: The therapeutic potential of PARP inhibitors is predicted to
extend beyond BRCA mutant (BRCAmut ) phenotypes to homologous
recombination deficient (HRD) cancers. An HRD assay amenable for
clinical testing was applied to treatment naı̈ve, high grade ovarian primary
tumor samples. Niraparib treated patient-derived tumorgraft models
selected from these primary ovarian tumors were utilized to evaluate the
correlation between HRD score, BRCA deficiency, platinum sensitivity and
niraparib anti-tumor response.
Methods: Utilizing patient-derived ovarian xenografts we previously
demonstrated response to niraparibin both BRCAmut and BRCA wild
type (BRCAwt ) tumors. BRCAmut status alone was neither necessary nor
sufficient to predict response to niraparib. To understand the selectivity
observed, samples from a collection of >100 high grade ovarian tumors
were subjected to HRD analysis. The HRD analysis is a DNA-based
assay that is capable of detecting homologous recombination deficiency
independent of its etiology. Genome-wide SNP data was generated from
a custom Agilent SureSelect XT2 capture followed by sequencing on an
Illumina HiSeq2500. SNP data was analyzed using all three algorithms;
Loss of Heterozygosity (LOH), Telomeric Allelic Imbalance (TAI) and
Large-scale State Transistions (LST). The final HRD score is the sum
of the LOH+TAI+LST scores with numerical outputs ranging from 0–
100. RNAseq analysis was also conducted on a subset of the ovarian
tumors. Niraparib was then evaluated as a monotherapy in a series of high
grade ovarian cancer tumorgrafts with a diverse range of HRD scores.
Intraperitoneal tumorgrafts were monitored for tumor growth with twiceweekly transabdominal ultrasound imaging. In-vivo response to niraparib
was correlated to the HRD score, BRCA status, patient’s platinum response
and RNAseq data.
Results: One hundred and six high grade ovarian tumors were evaluated
for HRD, BRCA mutation, and RNAseq analysis. Twenty one tumors (20%)
had a deleterious somatic or germline BRCAmut . BRCA1 hypermethylation
was evaluated in 98 tumors and found in 6, representing approximately 6%
of this cohort. Collectively, there was 26% BRCA deficiency in this primary
tumor collection. HRD scores ranged from 1−86 with a median score of
32 and an average score of 39. All BRCAmut tumors and 83% (5/6) of the
BRCA1 hypermethylated tumors had an HRD score of 31 or greater. The
BRCA1 hypermethylated tumor with a low HRD score does not have loss
of heterozygosity (LOH) at BRCA1, and likely retains functional BRCA1. Invivo response to niraparib monotherapy was demonstrated in BRCAmut and
Plenary Session 6
BRCAwt models with high HRD scores. In addition, niraparib resistance or
lack of in vivo efficacy was demonstrated in tumors with low HRD scores.
Conclusion: HRD testing of ovarian cancer was predictive of BRCA
deficiency. Response to niraparibwas seen in both BRCAmut and BRCAwt
tumors. Our data are supportive of the use of HRD testing to select
high grade ovarian cancer patients for the clinical evaluation of niraparib
treatment.
215
ORAL PRESENTATION
Updated clinical and preliminary correlative results of ARIEL2, a
Phase 2 study to identify ovarian cancer patients likely to respond
to rucaparib
E. Swisher1 , J. Brenton2 , S. Kaufmann3 , A. Oza4 , R.L. Coleman5 ,
D. O’Malley6 , G. Konecny7 , L. Ma8 , M. Harrell9 , D. Visscher3 ,
A.W. Hendrickson3 , K. Lin10 , M. Raponi10 , E. Mann10 , H. Giordano10 ,
L. Maloney11 , L. Rolfe12 , I. McNeish13 . 1 University of Washington
School of Medicine, Seattle WA, USA; 2 Cancer Research UK Cambridge
Institute, Oncology, Cambridge, United Kingdom; 3 Mayo Clinic, Oncology,
Rochester, USA; 4 Princess Margaret Cancer Centre, Oncology, Toronto,
Canada; 5 The University of Texas MD Anderson Cancer Center,
Oncology, Houston, USA; 6 The Ohio State University James Cancer
Center, Oncology, Columbus, USA; 7 University of California Los
Angeles (UCLA), Oncology, Los Angeles, USA; 8 Rocky Mountain Cancer
Center, Oncology, Lakewood, USA; 9 University of Washington School of
Medicine, Oncology, Seattle WA, USA; 10 Clovis Oncology, San Francisco,
USA; 11 Clovis Oncology, Boulder, USA; 12 Clovis Oncology, Cambridge,
United Kingdom; 13 Institute of Cancer Sciences University of Glasgow,
Glasgow, United Kingdom
73
Methods: Patients with recurrent gliomas (WHO grade II-IV) were
eligible for this trial. FSRT was prescribed to 30−35 Gy delivered in 10
daily fractions. Panobinostat was administrated orally once daily every
Monday, Wednesday, and Friday concurrently with radiation treatment. The
panobinostat dose was escalated from 10 to 30 mg using a 3+3 trial design.
Results: A total of 16 patients were enrolled on the trial. Four patients were
excluded from the analysis due to 1 patient failing to receive study treatment
due to rapid progression, and 3 patients having increased QTc exceeding
the protocol limit and were unable to finish protocol treatment. Of the 12
evaluable patients, 8 had recurrent GBM, and 4 had recurrent anaplastic
astrocytoma. There were 3 patients in the 10 mg panobinostat cohort, 3
patients in the 20 mg cohort, and 6 patients in the 30 mg final cohort. There
were no treatment related grade 3 or higher toxicities in the 10 mg or 20 mg
cohort. In the 30 mg panobinostat cohort, one patient developed grade 3
leukopenia and grade 4 neutropenia which was possibly related to the
treatment. There are 5 living patients in the study. The minimum follow up
of the living patients is 8.5 m. The 4-m progression survival (PFS) of all the
evaluable patients is 87%, and 6-m PFS of 56%. The medial overall survival
is 9.5 m. There is a suggestion of dose response to the panobinostat. The
4-m PFS is 100%, 100%, and 67% for 30 mg, 20 mg, and 10 mg cohort
respectively. The 6-m PFS is 83%, 33%, and 67% for 30 mg, 20 mg, and
10 mg cohort respectively. The overall survival for the 30 mg cohort is not
reached with a median follow up of 11.5 m.
Conclusions: Panobinostat administrated with FSRT is well tolerated at a
30 mg dose, which is the recommended dose for phase II trial. There is a
suggestion of a dose–response relationship with panobinostat dose. The
median overall survival is not reached with a median follow up of 11.5 m
for the 30 mg cohort. A phase II trial is warranted to confirm the efficacy of
combining panobinostat with FSRT for recurrent gliomas.
217
ORAL PRESENTATION
Results of a phase I, open-label, multicentre study to assess
the safety, tolerability, pharmacokinetics and preliminary antitumour activity of AZD9150 in patients with advanced/metastatic
hepatocellular carcinoma
Y.K. Kang1 , B.Y. Ryoo1 , T.Y. Kim2 , K.H. Lee2 , H.Y. Lim3 , S.J. Lee3 ,
M. Ikeda4 , T. Okusaka5 , S. Nadano6 , C.C. Lin7 , T.P. Poon8 , C.J. Yen9 ,
P. McCoon10 , F. Neumann10 , K. Vishwantahan10 , R. DuPont10 , P. Lyne10 .
1
Asan Medical Center, Oncology, Seoul, Korea; 2 Seoul National
University Hospital, Oncology, Seoul, Korea; 3 Samsung Medical
Center, Oncology, Seoul, Korea; 4 National Cancer Center Hospital East,
Chiba, Japan; 5 National Cancer Center Hospital, Tokyo, Japan; 6 National
Hospital Organization, Ehime, Japan; 7 National Taiwan University
Hospital, Oncology, Taipei, Taiwan; 8 Queen Mary Hospital, Surgery, Hong
Kong, Hong Kong; 9 National Cheng Kung University Hospital, Tainan,
Taiwan; 10 AstraZeneca Pharmaceuticals, Oncology IMED, Waltham, USA
216
ORAL PRESENTATION
Phase I study of panobinostat and fractionated stereotactic
re-irradiation therapy (FSRT) for recurrent high grade gliomas
W. Shi1 , Y.R. Lawrence1 , M. Werner-Wasik1 , D.W. Andrews2 , J.J. Evans2 ,
J. Glass2 , L. Kim1 , V. Bar Ad1 , Y. Moshel1 , K. Judy1 , C. Farrell1 ,
N.L. Simon1 , A.P. Dicker1 . 1 Thomas Jefferson University Hospital,
Radiation Oncology, Philadelphia PA, USA; 2 Thomas Jefferson University
Hospital, Neurological Surgery, Philadelphia PA, USA
Background: This is a phase I study to evaluate the safety, and tolerability
of oral panobinostat, an HDAC inhibitor, when combined with fractionated
stereotactic re-irradiation therapy (FSRT) for recurrent gliomas.
Background: STAT3 plays a role in tumour biology by modulating critical
functions of both tumour and stromal/immune cells. AZD9150 is a STAT3
antisense oligonucleotide (ASO) in Phase I dose expansion in two clinical
trials. We present safety, preliminary response, pharmacokinetic (PK)
and pharmacodynamic (PD) data for the dose escalation portion of a
Phase I clinical, open-label, multi-center study in advanced hepatocellular
carcinoma (HCC) patients.
Methods: Patients with advanced sorafenib relapsed/refractory HCC and
Child-Pugh A liver function were enrolled in a 3+3 dose escalation study
to determine dose limiting toxicities (DLTs), maximum tolerated dose
(MTD), safety, tolerability, PK, PD, and preliminary efficacy. AZD9150 was
administered intravenously with 3 loading doses of AZD9150 on days 1, 3,
and 5 and weekly thereafter in 28 day cycles. STAT3 RNA and protein
expression in circulating leukocytes and serum CRP were evaluated on day
8 of treatment and the first day of subsequent cycles vs. pre-treatment.
Results: Twenty-four patients were dosed in 1, 1.5, 2 and 3 mg/kg cohorts
between May 2013 and May 2014. One partial response was observed
in the 2 mg/kg cohort with 61% reduction in lung metastases and 87%
decrease in AFP. Adverse events reported in 20% patients included
AST elevation (G1/2/3 46, 8, 4%), ALT elevation (G1/2/3 35, 15, 4%),
thromobocytopenia (G1/2/3 31, 19, 4%) neutropenia (G1/2/3 0, 23, 0%)
hypertension (G1/2/3 0, 19, 4%). Transaminase elevations were mostly
mild and were reversible after interruption of AZD9150. Thrombocytopenia
was mostly mild/moderate, typically occurred after ~10 weeks of dosing,
and did not require dose modification or intervention. One patient had
G3 thrombocytopenia and transient dosing interruption. DLTs of G3
transaminase elevations occurred in 3 patients, one each in the 1.5, 2 and
3 mg cohort. 3 mg/kg was declared the MTD and the recommended dose
for subsequent studies. Maximum AZD9150 plasma concentration (Cmax)
was observed at the end of the 3 hr infusion and was dose proportional
between 1 and 3 mg/kg dose levels. There was no accumulation in mean
plasma Cmax or AUC0−24 hr after repeated 3-hr infusions. Knockdown of
74
Plenary Session 7
STAT3 RNA and protein (up to 80%) in all circulating leukocytes of patients
by day 8 of treatment was detected in the majority of samples analyzed so
far and confirms activity of the drug at the target level. Decreased serum
CRP in the majority of patients treated in the 1 and 1.5 mg/kg cohorts
and all patients in the 2 and 3 mg/kg cohorts is consistent with functional
inhibition of STAT3. Updated data will be presented.
Conclusion: Preliminary data suggest activity of AZD9150 in patients
with advanced HCC. A tolerated dose with clinical and pharmacodynamic
activity has been identified and is being explored further in dose expansion.
218
ORAL PRESENTATION
Genomic analysis identifies novel drivers and targetable pathways in
inflammatory breast cancer patient samples
D.M. Moran1 , K. Rao1 , P. Bacon-Trusk1 , K. Pry1 , V. Weigman2 ,
V. Velculescu3 , M. Cristofanilli4 , S. Bacus1 . 1 Quintiles, Translational R&D
Oncology, Westmont, USA; 2 Quintiles/Expression Analysis, Translational
Genomics, Durham, USA; 3 Johns Hopkins University, Oncology, Baltimore,
USA; 4 Thomas Jefferson University, Medical Oncology, Philadelphia, USA
Background: Inflammatory breast cancer (IBC) is a rare aggressive breast
cancer in which cancer cells block the lymph vessels in the skin of the
breast. IBC tumors are typically hormone receptor negative but have shown
a high rate of HER2 (human epidermal growth factor receptor 2) positivity
and response to HER2 targeted therapies such as lapatinib. Genomic
drivers associated with progression and drug response of IBC are not well
established. This study interrogates the mutational background of IBC to
understand drug responses in breast cancer.
Materials and Methods: A targeted NGS panel that covers whole coding
regions of 208 of the most common cancer related genes (copy numbers
and somatic mutations) and rearrangements in 17 well characterized
cancer genes was used to analyze 20 IBC patient tumor and matched
normal samples. Pathway analysis was performed on genomic variants
identified. Cell line studies were performed to understand the impact of
genomic variants on drug treatment.
Results: Intra- and inter-tumor heterogeneity was observed across the IBC
samples studied, however, common pathway motifs were also identified
among cases. Multiple variants in the HER signaling pathway were
observed including HER2 amplification (54% of samples) and a high
rate of ERBB3 mutations (26% of samples). ERBB3 point mutations
were discovered in hotspot regions in both the extracellular and kinase
domains and occurred at higher rates than previously observed in other
cancers. Genomic alterations were also identified among many genes
of the PI3K-mTOR pathway in the majority of IBC cases. Activity of the
PI3K-mTOR pathway was further confirmed by immunohistochemistry for
phosphorylated S6, a target of mTOR kinase activity.
Cell studies demonstrated potent effects of lapatinib on proliferation of IBC
cells harboring ERBB3 mutations in conjunction with HER2 amplification.
Breast cancer cells harboring PI3K mutations and HER2 amplification
were less sensitive to lapatinib but were synergistically responsive to a
combination of PI3KCA inhibitors and lapatinib.
Other frequent genomic alterations were also detected in pathways related
to chromatin modification, DNA repair, APC, JAK-STAT, KIT and Notch
signaling which may also be novel drug targets in IBC. Notably, hotspot
and/or kinase domain mutations were discovered in JAK (1 & 2) and KIT
genes in multiple IBC samples.
Conclusions: Genomic and protein analysis of IBC identified multiple
pathways that may be targetable using single and/or combination
targeted therapies in all cases studied. This study also highlighted that
drugs such as PI3K/mTOR and novel ERBB3 targeted therapies, used
alone or in combination with HER2 inhibitors, may be important in
the treatment of IBC. Co-occurrence of ERBB3 mutations and HER2
amplification/overexpression likely sensitize cancer cells to HER2 targeted
therapies and should be further explored in other HER2 positive cancers.
219
ORAL PRESENTATION
Phase I trial evaluating the antiviral agent Cidofovir in combination
with chemoradiation in cervical cancer patients: A novel approach
to treat HPV related malignancies?
E. Deutsch1 , A. Levy2 , R. Mazeron2 , A. Gazzah2 , E.A. Angevin3 ,
V. Ribrag3 , R. Balheda3 , A. Varga3 , C. Lhomme4 , C. Haie-Meder2 ,
J.C. Soria3 . 1 Institut Gustave Roussy, Villejuif, France; 2 Institut Gustave
Roussy, radiation oncology, Villejuif, France; 3 Institut Gustave Roussy,
DITEP, Villejuif, France; 4 Institut Gustave Roussy, Medicine, Villejuif,
France
16:00–17:50
PLENARY SESSION 7
Novel Mechanisms for Drug Resistance
220
ORAL PRESENTATION
Overcoming drug-resistance in multiple myeloma by XPO1 inhibitor
combination therapy
J. Turner1 , J. Dawson1 , S. Grant2 , K. Shain3 , C. Cubitt4 , Y. Dai2 ,
L. Zhoui2 , M. Kauffman5 , S. Shacham5 , D. Sullivan1 . 1 Moffitt Cancer
Center, Chemical Biology and Molecular Medicine Program, Tampa
Florida, USA; 2 Massey Cancer Center, Virginia Commonwealth
University, Richmond Virginia, USA; 3 Moffitt Cancer Center, Department
of Malignant Hematology, Tampa Florida, USA; 4 Moffitt Cancer Center,
Translational Research Laboratory, Tampa Florida, USA; 5 Karyopharm
Theraeutics, Natick Massachusetts, USA
Background: The purpose of this study was to investigate the use of XPO1
(exportin 1, CRM1) inhibitors (XPO1i) to sensitize de novo and acquired
drug resistant multiple myeloma (MM) cells to the proteosome inhibitors
(PI) bortezomib (BZ) and carfilzomib (CZ), the topoisomerase II (topo II)
inhibitor doxorubicin (DX) and the alkylating agent melphalan (ML).
Materials and Methods: Cells were treated in vitro with XPO1i (KPT-330 or
KOS-2464) +/− BZ, CZ, DX or ML. Sensitivity was measured by cell viability
assay (CellTiter-Blue). Proximity ligation assays (PLA) were performed to
assess XPO1-topoIIa binding in the presence of an XPO1i. Western blot of
proteins related to PI and XPO1i were used to determine the potential
mechanism of XPO1i-PI synergy. Comet assay for DNA damage was
performed in XPO1i/DX treated cells. Drug resistant U266 and 8226 MM
cell lines were developed by incremental exposure to BZ. Resistant and
parental MM cells were treated in vitro with XPO1i +/− MM drugs. Sensitivity
was measured by apoptosis (caspase 3). U266 resistant MM cells were
also used to challenge NOD/SCID-g mice treated with XPO1i +/− BZ or
pegylated liposomal doxorubicin (PLD). MM cells isolated from patients with
newly diagnosed, relapsed or refractory MM were treated with XPO1i +/−
BZ, CZ, DX or ML and CD138+/light chain+ MM cells assayed for apoptosis.
Results: MM cell viability was decreased synergistically by XPO1i when
used in combination with BZ, CZ, DX or ML (CI values 0.502, 0.482, 0.092
and 0.687). XPO1i prevented binding of XPO1 to topoIIa as shown by PLA,
and XPO1i increased DNA fragmentation when combined with DX (Comet
assay). Western blot showed that the XPO1i/PI combination increased IkB
and decreased NFkB in MM cells. BZ selected MM cells were found to be
resistant (>10-fold) to BZ, CZ, DX and ML when compared to parental cell
lines. Resistant MM cell lines were sensitized by the XPO1i to drugs as
shown by apoptosis assay (3 to10-fold). Drug resistant MM challenged mice
treated with XPO1i +/− BZ or PLD had increased survival when compared
to BZ or PLD alone (p0.027). CD138+/light chain+ MM cells derived from
Poster Session – Chemoprevention
newly diagnosed, relapsed and refractory MM patients were sensitized by
XPO1 inhibitors to BZ, CZ, DX and ML as shown by apoptosis.
Conclusions: XPO1i greatly improved the response of de novo and
acquired drug resistant MM to BZ, CZ, DX and ML in vitro, in vivo and
ex vivo. Combination therapies using XPO1i may significantly improve the
treatment of myeloma.
Poster Sessions
Chemoprevention
221
POSTER (Board P001)
Docosahexaenoic acid along with modulation of actin binding
proteins reduces cancer cell migration
M. Ali1 , L.K. Rogers1 . 1 Nationwide Children’s Hospital The Ohio State
University College of Medicine, The Research Institute at Nationwide
Children’s Hospital and Department of Pediatrics, Columbus Ohio, USA
Background: Cancer is the leading cause of death worldwide and all age
groups, including children, are at risk for cancer associated death. Disease
progression and metastasis are major contributors to cancer-associated
morbidity and mortality. Increased cell migration rate is characteristic
of tumor progression and metastasis. Actin binding proteins regulate
cytoskeletal remodeling at the leading edges of cancer cells, facilitate
invasive organelle (invadopodia) formation, and promote higher migration
rates. Docosahexaenoic acid (DHA), a poly unsaturated fatty acid, has
been shown to inhibit the cancer cell metastatic phenotype. Here, we
test the hypothesis that changes in actin binding proteins regulate cancer
cell migration and that supplementing cells with DHA will prevent these
changes.
Materials and Methods: Fatty acid profile of Non-cancer (MLE12) and
cancer (A549) done with mass spectrophotometry. Further cells were
treated with 8-Br-cAMP and/or DHA. F-actin content was measured using
confocal microscopy. Cell migration was estimated by wound assay and
transwell apparatus. Actin binding proteins, profilin, cofilin, vimentin and
gelsolin, were identified and quantified using confocal microscopy and
western blot to evaluate wound edges and actin co-immunoprecipitate,
respectively.
Results: F-actin content and cell migration were increased by cAMP in
association with changes in profilin, cofilin, vimentin and gelsolin levels.
DHA treatment suppressed the increase in actin content and cell migration
in cancer cells but not in non-cancer cells in association with altered levels
of actin binding proteins.
Conclusion: We postulate that DHA specifically inhibits cancer cell
migration via alterations in actin binding proteins indicating a therapeutic
potential against cancer cell metastasis. The changes in actin binding
proteins could serve as biomarkers for cancer progression and as
innovative therapeutic targets.
222
POSTER (Board P002)
Estimating predictive values of short-term morphologic assays of
cancer chemoprevention for efficacy in animal tumor assays
B. Dunn1 , V.E. Steele1 , R.M. Fagerstrom1 , C.F. Topp2 , D. Ransohoff3 ,
C. Cunningham4 , R. Lubet5 , L.G. Ford5 , B.S. Kramer5 . 1 National Cancer
Institute NIH, Division of Cancer Prevention, Bethesda Maryland, USA;
2
CCS Associates, Preclinical Research and Development Resources,
McLean Virginia, USA; 3 University of North Carolina at Chapel Hill,
Department of Medicine, Chapel Hill NC, USA; 4 Information Management
Services, Rockville MD, USA; 5 National Cancer Institute, Division of
Cancer Prevention, Bethesda MD, USA
Background: The predictive value of chemopreventive agent efficacy in
morphologic (in vitro/in vivo) assays for efficacy in in vivo tumor assays
is not well characterized. Over a 25-year period, the Chemopreventive
Agent Development Research Group in the U.S. NCI’s Division of
Cancer Prevention has tested approximately 800 agents for potential
chemopreventive activity. The current project focuses on agents tested
in both morphologic and tumor assays in order to gain a deeper
understanding of the relevant predictive values.
Materials and Methods: The early stages of the testing pathway involve
two critical steps: (1) in vitro/in vivo morphologic assays and, for agents
75
successful in these, (2) testing for tumor prevention (measured in terms
of tumor incidence and multiplicity reduction) in animal tumor assays. The
ultimate goal is to test agents that successfully decrease tumor incidence
and multiplicity in animal tumor assays in humans. In the current project
we evaluated the predictive values of our preclinical models by determining
how well the earlier-stage (morphologic) assays predict efficacy in the laterstage (animal tumor) assays. The 210 agents that were tested in both
morphologic and animal tumor assays in our program were included in our
analysis. Statistical modeling to determine how well the six most commonly
used morphologic assays predicted efficacy of the 210 tested agents in
animal tumor assays was carried out by multimodel inference applied to
ordinal logistic regression.
Results: The ability of these six morphologic assays to predict tumor
outcomes was evaluated in three statistical models, one for each animal
tumor setting: (1) all tumor types (general model), (2) colon cancer only,
and (3) mammary gland cancer only. Based on this statistical modeling,
each morphologic assay was assigned a value describing how strongly it
predicted outcomes in each of the three animal tumor assay settings.
Conclusions: These predictive models can be used to guide our future
decision-making with respect to agent selection as well as morphologic
and animal tumor assay use. The overall goal is to improve the efficiency
of the process of chemopreventive agent development.
223
POSTER (Board P003)
Preclinical assessment of nintedanib for chemoprevention in
hepatocellular carcinoma
V. Tovar1 , A. Moeini1 , S. Torrecilla1 , M. Higuera1 , J. Peix1 , I.M. Quetglas1 ,
L. Rodriguez-Carunchio1 , H. Cornella1 , M. Sole1 , J.M. Llovet1 . 1 Institut
d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), HCC
Translational Research Laboratory, BCLC group, Hospital Clı́nic, Liver
Unit − CIBEREHD, University of Barcelona, Barcelona, Spain
Background: Hepatocellular carcinoma (HCC) is the 2nd cause of cancerrelated mortality. Cirrhosis is the most common risk factor in 80−90% of
HCC patients. Angiogenesis has a pivotal role in both chronic liver disease
and hepatocarcinogenesis. Thus, early interference with anti-angiogenic
agents at cirrhotic stages may prevent HCC development. In this study,
we evaluate the chemopreventive efficacy of the angiokinase inhibitor
nintedanib (VEGFR1−3, FGFR1−3, PDGFR-a/b; Boehringer Ingelheim) in
an animal model of HCC.
Material and Methods: HCC was induced in male C57Bl/6 mice (n = 55)
by a single intraperitoneal (i.p.) injection of DEN (25 mg/kg) at day
15 postpartum followed by weekly i.p. injections of CCl4 (0.5 ml/kg)
starting at 4 weeks of age. At 12 weeks-old, mice were randomized
to receive nintedanib (50 mg/kg) (n = 29) or vehicle (n = 26). To assess
chemopreventive efficacy, mice were sacrificed at 15, 17 and 18 weeks of
age. Liver samples were collected for immunohistochemical and molecular
analysis (western blot and qRT-PCR). The primary study end-points
included HCC incidence, number and size of macroscopic tumors. Drug
tolerance was evaluated by body weight losses and plasma ALT/AST levels.
Results: Nintedanib reduced HCC incidence in treated animals compared
to placebo group at 15 weeks (13% vs 43%), 17 weeks (30% vs 89%,
p = 0.02), and 18 weeks (90% vs 100%). The number of tumor/mouse
significantly decreased at 17 weeks (0.9 vs 4.7, p = 0.003) and 18
weeks (3.5 vs 11.4, p = 0.003). Nintedanib also prevent the development
of large number of tumors (>4 tumors) at 17 weeks (p = 0.01) and
18 weeks (p = 0.008). Moreover, tumor size was significantly reduced
at 17 weeks (0.7 mm vs 6.4 mm, p = 0.01) and 18 weeks (4.8 mm vs
16.2 mm, p = 0.001). Nintedanib was well tolerated and no significant
toxicity was reported. Histological analysis and up-regulation of collagen
levels confirmed the development of HCC in a context of well-established
fibrosis. Assessment of nintedanibmechanism of action showed a trend
to reduce VEGFR-2 activation while significantly decreased downstream
activation of AKT and ERK.
Conclusion: Nintedanib decreases overall HCC incidence, number of
tumors and tumor size in a liver fibrosis-based mouse model. Nintedanib
acts by blocking the MAPK/PI3K pathway downstream of the proangiogenic receptors (VEGFR and PDGFR). Our data provides rationale
for testing nintedanib as a potential chemopreventive agent for the
development of HCC in cirrhotic patients.
76
Poster Session – Clinical Methodology
224
POSTER (Board P004)
Synergic tumor growth suppression with carbohydrate-restriction
diet and natural AMP-dependent protein kinase activators
226
POSTER (Board P006)
Efficacy of cancer preventing drugs administered by intermittent
dosing regimens
M. Choi1 , J. Lee2 . 1 Yonsei Cancer Center Yonsei University College of
Medicine, Department of Nuclear Medicine, Seoul, South Korea; 2 Yonsei
Cancer Center Yonsei University College of Medicine, Department of
Nuclear Medicine Brain Korea 21 Project for medical Sciences, Seoul,
South Korea
V.E. Steele1 , C. Grubbs2 , C.V. Rao3 , R.A. Lubet1 . 1 National Institutes of
Health/National Cancer Institute, Division of Cancer Prevention, Bethesda
MD, USA; 2 University of Alabama at Birmingham, Department of Surgery,
Birmingham AL, USA; 3 Oklahoma University Health Sciences Center,
Center for Cancer Prevention and Drug Development, Oklahoma City
OK, USA
Background: Calorie restriction (CR) or low-carbohydrate diet (LCD) can
increase life-span in normal cells but inhibit carcinogenesis. Various AMPdependent protein kinase (AMPK) activating phytochemicals also have
CR-mimetic anticancer properties. We investigated whether carbohydraterestriction diet and phytochemical supplementation induce synergic tumor
suppression.
Methods: We used a mixture of curcumin, quercetin, catechins and
resveratrol extracts for natural AMPK-activating agent. Survival analysis
was performed in B16F10 melanoma model after fed control diet, control
diet with phytochemicals (MP formula), LCD, LCD with phytochemicals
(LCDmp), moderate-carbohydrate diet (MCD), and MCD with phytochemicals (MCDmp). Tumor suppression mechanisms, especially alterations
in energy-dependent signaling pathways, histone methylation, and global
gene expression were investigated. We evaluated the role of SIRT1
under glucose-restriction condition with MTT assay. Diet-induced cancer
prevention effects were analyzed in a transgenic liver cancer model.
Results: In melanoma model, MP, LCD, or MCD intervention did
not produce survival benefit but LCDmp and MCDmp interventions increased median survival time significantly (p < 0.05). Suppression of the IGF-1R/PI3K/Akt/mTOR signaling, activation of the
AMPK/SIRT1/LKB1pathway and NF-kB suppression were the major tumor
suppression mechanisms. MTT assay showed suppressed proliferation of
the B16F10 and A375SM cells after treatment of SIRT1 activator under
low-glucose condition. Alterations in histone methylation within Pten and
FoxO3a were demonstrated in tumor tissues. In transgenic liver cancer
model, the MCDmp and LCDmp groups showed fewer tumor nodules.
Microarray analysis revealed increased PPARa with decreased IL-6 and
NF-kB after MCDmp intervention, which could be the major cancer
prevention mechanism.
Conclusion: AMPK-activating phytochemicals exert synergic anti-cancer
activities under low-carbohydrate condition, and SIRT1 induces tumor
suppression.
225
POSTER (Board P005)
Molecular targets of interest to the NCI PREVENT cancer preclinical
drug development program
R.H. Shoemaker1 , B.K. Dunn1 , C. Suen1 , R.A. Lubet1 , D.L. Boring1 ,
B.D. Klein1 , M.S. Miller1 , V.E. Steele1 . 1 National Cancer Institute Division
of Cancer Prevention, Chemopreventive Agent Development Research
Group, Bethesda MD, USA
Chemoprevention research in the Division of Cancer Prevention was
re-structured in 2011 creating the PREVENT Cancer Preclinical Drug
Development Program. This Program provides for peer-review of proposals
from the general research community for development of small molecules
or biologicals, including vaccines, for cancer prevention or biomarkers to
facilitate clinical evaluation of prevention strategies. The process for applying to the Prevent Program is described at: http://prevention.cancer.gov/
programs-resources/programs/prevent. Applications are submitted twice
yearly and reviewed by a panel of experts and scored for scientific
merit, feasibility, etc. Top scoring applications undergo secondary review
and prioritization by a panel of NIH scientists. Applications with potential
for near-term clinical translation are given highest priority. Approved
projects are implemented as Task Orders via a system of contracts with
academic and nonprofit, independent research institutions that provide
the full range of preclinical studies, including GLP toxicology, needed to
support Investigational New Drug Applications. Twenty-five Task Orders
addressing a range of molecular targets have been awarded in the first
two years of the Program. Small molecules targeting classic aspects of
inflammation as well as newer molecular-targeted agents (n = 16) are
under study. Pharmacodynamic assays are coupled to efficacy studies.
Immunoprevention strategies targeting tumor-associated antigens as well
as antigens from cancer-associated infectious agents (n = 6) are being
pursued. Preliminary data from current projects will be presented. The
PREVENT Cancer Program has engaged a broad cross-section of
investigators, supporting preclinical development of agents addressing a
wide variety of cancer prevention targets.
Background: Many potential chemopreventive drugs have adverse effects
on human subjects when given daily. One strategy to alleviate these
adverse events is to give the drugs on an intermittent dosing schedule.
Such intermittent dosing has proven effective in other scenarios to reduce
toxicity with limited effect on efficacy. Two animal models were used: the
rat methylnitrosourea (MNU)-induced mammary cancer model and the rat
hydroxy-butyl(butyl)-nitrosamine (OH-BBN)-induced urinary bladder cancer
model.
Materials and Methods: For the mammary study female Sprague-Dawley
rats were given 75 mg of MNU/kg body weight at 50 days of age and
five days later the EGFR inhibitors, erlotinib, gefitinib or lapatinib were
administered either daily or once a week at two dose levels. Multiplicity
of ER-positive mammary cancers was followed weekly for four months. For
the urinary bladder study female Fischer 344 rats were given 150 mg OHBBN/gavage twice weekly beginning at 56 days of age and continuing for
8 weeks. Two weeks after the last OH-BBN treatment the rats were given
naproxen either daily, one week on/one week off, or 3 weeks on/3 weeks off.
Bladders were excised after 8 months of naproxen treatment and bladders
with tumors were excised and weighed.
Results: EGFR inhibitors and the NSAID, naproxen, have similar efficacy
when given intermittently compared to daily. Data will be presented with two
organ specific chemoprevention animal models: ER-positive mammary and
invasive urinary bladder cancer. The EGFR antagonists, erlotinib, gefitinib
and lapatinib, decreased mammary cancer multiplicity by 90% given daily
and 75% given weekly. The NSAID, naproxen, decreased large bladder
cancers by about 65−80% given either daily, one week on/one week off or
3 weeks on/3 weeks off.
Conclusions: Intermittent dosing can be used for agents in two different
mechanistic classes in animal mammary and bladder cancer models
to lower toxicity yet show little reduction in chemoprevention efficacy
compared to daily dosing. Intermittent dosing schedules should reduce
EGFR mediated rash and NSAID induced gastric toxicity clinically while
retaining efficacy.
227
POSTER (Board P007)
Multiplexed ICE COLD-PCR: A mutation detection methodology for
achieving sensitivities of <0.01% using either Sanger or NGS
G. Wu1 , B. Legendre2 , S. Cherubin1 , C. Cubrich1 , A. Dowers2 ,
S. Jensen1 , J. Gniffke3 , A. Kruempel2 , P. Krzycki2 , E. McCutchen2 ,
E. Montagne1 , S. Peterson4 , J. Pope2 , K. Scott1 , K. Richardson5 .
1
Transgenomic, R&D, Omaha, USA; 2 Transgenomic, Biomarker
Identification, Omaha, USA; 3 Transgenomic, IT, Omaha, USA;
4
Transgenomic, CLIA, Omaha, USA; 5 Transgenomic, Omaha, USA
Background: Blood-based mutation analysis from circulating free DNA
(cfDNA) is becoming very important for molecular demographics and
diagnostics where no tumor is available as well as in the pharmacodynamic
monitoring of the patient during therapy. ICE COLD-PCR technology
is capable of high sensitivity detection for both point mutations and
insertion/deletions through unbiased enrichment of relevant gene regions.
This method preferentially amplifies low levels of mutant DNA in a sample
containing a vast excess of wild-type DNA.
Materials and Methods: In order to increase throughput as well as
address the limited amounts of DNA present from cfDNA sources, a
multiplex approach for ICE COLD-PCR has been developed. Horizon
Cell line DNA with digital PCR verified mutation percentages was first
amplified using singleplex PCR for a single region of interest or multiplex
PCR for several regions of interest. The advantage of the multiplex PCR
is to allow simultaneous amplification of all targets with the same input
DNA. The digitally-verified DNA was used for the LOD dilutions where the
starting mutation percentage was below 1%. The amplified DNA can then
be used in multiple singleplex or multiplex ICE COLD-PCR reactions. A
constraint of all ICE COLD-PCR reactions is the optimal thermal cycling
parameters needed for mutation enrichment and this critical temperature
(Tc) is dependent on the sequence context. A Veriti thermal cycler was
used for ICE COLD-PCR analysis of EGFR Exons 19, 20, and 21, KRAS
Exons 2 and 3, and NRAS Exons 2 and 3 because it can simultaneously
perform thermal cycling at 6 different Tc’s on a single 96-well plate. This
was followed by hemi-nested PCR using a single thermal cycling program,
if required, to provide sufficient sample for Sanger sequencing and NGS
using an Ion Torrent.
Results: Limits of detection experiments using the Horizon Cell Line DNA
and serial dilution of this DNA indicated that samples containing 0.01%
mutation in the starting material were easily confirmed using both Sanger
and NGS sequencing platforms. This was true for the point mutations
as well as the EGFR Exon 19 E746_A750delGGAATTAAGAGAAGC.
Concordance of cfDNA and matched FFPE tumor DNA is also presented.
Conclusion: ICE COLD-PCR can be used in a multiplex fashion for the
sensitive detection of all mutations in a region. This is important when
investigating regions with multiple mutations such as EGFR Exon 19
deletions. The sensitivities achieved indicate that ICE COLD-PCR is an
ideal tool for detection of low level mutations found in cfDNA and potentially
circulating tumor cells. The ability to confirm these mutations by either
Sanger or NGS platforms allows flexibility in rapid confirmation when few
or many gene regions need to be interrogated.
228
POSTER (Board P008)
Her2−3 heterodimer is a new and better than HER2 IHC score for
clinical outcome prognosis
G. Weitsman1 , P.R. Barber2 , K. Lawler3 , C. Gillett4 , N. Woodman4 ,
B. Kholodenko5 , L.K. Nguyen5 , T. Santra5 , B. Vojnovic2 , T. Ng1 . 1 Richard
Dimbleby Department of Cancer Research Randall Division & Division
of Cancer Studies Kings College London, Guy’s Medical School Campus,
London, United Kingdom; 2 CRUK & MRC Oxford Institute for Radiation
Oncology Gray Laboratories, Department of Oncology, Oxford, United
Kingdom; 3 Institute for Mathematical and Molecular Biomedicine Kings
College London, Guy’s Medical School Campus, London, United Kingdom;
4
Research Oncology Division of Cancer Studies King’s College London,
Guy’s Hospital, London, United Kingdom; 5 Systems Biology Ireland
at UCD Conway Institute, University College Dublin, Dublin, Ireland
The efficacy of anti-Her2 therapies in Her2-positive breast cancer
patients is proven and well documented. However, some patients with
Her2-negative tumours also benefit from the same therapies (NSABP
B-31) and there is no solid hypothesis to explain those observations.
Furthermore, the definition of positive vs. negative Her2 status actually
reflects overexpression of Her2 above levels detected in normal and
non-malignant tissues. Her2 can heterodimerize with other members of
the EGFR family, regardless of expression levels, but the dimerization
is dependent upon availability of ligand(s). Her2-Her3 dimer has been
shown to drive proliferation of breast cancer cells. We have developed a
new FRET/FLIM (Förster resonance energy transfer/fluorescence-lifetime
imaging microscopy) based assay reporting protein–protein interaction
at distance below 10 nm for detection of Her2-Her3 dimer in formalinfixed paraffin embedded (FFPE) patient samples. Fluorophore labelled
antibodies against Her2 and Her3 allow us to measure a FRET signal,
which is dependent upon the number of interacting molecules. Using FFPE
samples from the METABRIC cohort, we found that the interaction between
two proteins does not correlate with expression levels of interacting
partners as judged using standard IHC scoring system. The FRET signal
measurements were evenly distributed across all samples with 0, 1, 2 and
3-plus scores for Her2 expression. Mathematical modelling suggests that
the absence of correlation between Her2 and Her3 protein and their dimer
levels, although can be quite unintuitive, is in principle possible, particularly
under scenarios when both Her2 and Her3 compete for binding with
other receptor tyrosine kinases including the other ErbB family receptors.
Interestingly, we also did not find any correlation with known genetic
signatures associated with cancer progression. However, a low FRET
signal significantly correlated with longer metastasis free survival, when
patients with metastatic events up to 10 years are considered. Our findings
may pave the way for better understanding of the biology of EGFR family
receptor’s adaptation to drug treatment, helping to predict individual patient
response to select the right patient for appropriate treatment.
229
POSTER (Board P009)
DNA methyltransferase 1 expression in human solid tumors and
lymphomas by immunohistochemistry
S.X. Yang1 , D. Nguyen1 , L. Rubinstein1 , S. Kummar1 , J.E. Tomaszewski1 ,
J.H. Doroshow1 . 1 National Cancer Institute NIH, Division of Cancer
Treatment and Diagnosis, Bethesda Maryland, USA
Background: CpG island methylation in some tumor suppressor genes
has been linked to the development or progression of certain human
77
cancers. DNA methyltransferase 1 (DNMT1) is the most abundant enzyme
among the three members of the DNMT family. Anti-tumor activity of DNMT
inhibitors has been demonstrated largely in myelodysplastic syndrome
and acute myeloid leukemia in unselected patient populations, and less
frequently in solid tumors. Currently, there are no biomarkers capable of
predicting efficacy of DNMT inhibitors and available for prospective patient
selection. This study was undertaken to investigate the expression of
DNMT1, the putative target of some DNMT inhibitors, in a spectrum of
human malignancies in an effort to identify a biomarker for efficacy.
Materials and Methods: NCI-H23 non-small cell lung cancer cells
were treated with and without a novel DNMT inhibitor, 4 -thio-5-aza-2 deoxycytidine (5-aza-T-dCyd), and changes in DNMT1 expression were
assessed by western blot and immunocytochemistry (ICC). Expression
of DNMT1 was also examined by immunohistochemistry in formalin-fixed
and paraffin-embedded primary lung and ovarian cancers as well as
lymphomas.
Results: Expression of DNMT1 in NCI-H23 cells was nearly depleted
by treatment with 1 mM aza-T-dCyd for 96 h revealed by both western
blot and ICC (mean staining index, 56.7 versus 4.3; P<0.0001; 2-sided
t test). These suggest that 5-aza-T-dCyd is a potent novel DNMT inhibitor
and the antibody used detects the difference, indicative of its specificity.
DNMT1 was primarily localized in the nucleus, and constitutively expressed
in tumor cells at intermediate and high levels in 12.5% (7/56) of lung
cancers, 7.7% (3/39) of ovarian tumors, and 15% (12/78) of lymphomas
across histologies including diffuse B-cell, Burkitt-like, T-cell, lymphocyte
predominant Hodgkin’s, or mixed cellularity Hodgkin’s lymphoma.
Conclusions: We have established a DNMT1 immunohistochemical assay
that covers a dynamic range from undetectable to weak, intermediate or
strong staining in paraffin-embedded tumor samples. The data demonstrate
that intermediate to high levels of DNMT1 expression are found in 15% or
fewer patients with either lung cancers, ovarian tumors or lymphomas. The
application of this assay holds promise for evaluating DNMT1 expression
levels as a potential pharmacodynamic and efficacy biomarker for DNMT
inhibitors.
230
POSTER (Board P010)
High correlation between clinical responses to first line AML patients
treated with cytarabine and idarubicin and their pharmacological
profiles in patient samples measured by ExviTech
J. Ballesteros1 , P. Hernandez1 , D. Primo2 , A. Robles1 , A.B. Espinosa2 ,
E. Arroyo2 , V. Garcia-Navas1 , J. Sanchez-Fenoy1 , M. Jimenez1 ,
M. Gaspar1 , J.L. Rojas1 , J. Martinez-Lopez3 , J. Gorrochategui1 . 1 Vivia
Biotech, Tres Cantos-Madrid, Spain; 2 Vivia Biotech, Salamanca, Spain;
3
Hospital 12 de Octubre, Hematology, Madrid, Spain
Background: Complete remission (CR) after induction therapy is the first
treatment goal in acute myeloid leukemia (AML) patients. The aim of this
study is to determine the ability of the Vivia’s novel ex vivo drug sensitivity
platform Exvitech to predict the CR rates after induction chemotherapy with
cytarabine (Ara-C) and idarubicin (Ida) in 1st line AML.
Material and Methods: Bone marrow samples from adult patients
diagnosed with de novo AML in Spanish centers from the PETHEMA
group were included. Whole marrow samples were incubated for 48 h in
well plates containing Ara-C, Ida, or their combination. Pharmacological
responses are calculated using pharmacokinetic population models.
Induction response was assessed according to the Cheson criteria (2003).
Patients attaining a CR/CRi were classified as responders and the
remaining as resistant.
Results: 180 patient samples were used to calculate the dose–response
(DR) curves for Ara-C alone, Ida alone, and their synergism. For clinical
correlation we used 77 patients with a median age of 55 years. Many
samples had a significant number (>20%) of resistant cells to Ara-C. This
is a strong clinical predictor of resistance because in the patient the drug
will never be present at these high doses for 48 h. The second variable that
is a good predictor of response is the synergism between these 2 drugs.
The generalized additive model identified an algebraic combination of these
2 variables that yielded the best marker to separate both groups of patients.
The probability density functions had minimal overlap. The area under the
corresponding ROC curve was 0.935 (0.872, 0.997), and the classification
probabilities for the optimal cut point, were 87% (68%to 95%) and 91%
(80% to 96%) for sensitivity and specificity, respectively. 54 patients (70.1%)
achieved CR after Ida+Ara-C, and the remaining 23 (29.9%) were resistant.
20 of the 23 (86.9%) patients who fail to achieve CR were predicted as
resistance in the ex vivo test. 49 of the 54 patients (90.74%) who achieved
CR showed good ex vivo sensitivity to Ida+Ara-C predicting for CR. When
the ex vivo test predicted a patient as sensitive it was correct in 49/52
cases (94.23%), and when it predicted resistant it was correct 20/25 cases
(80%). Overall, 69/77 patients (89.61%) had an accurate prediction of their
response to treatment.
78
Conclusions: This novel ex vivo pharmacological profile test is able to
predict the clinical response to Ida+Ara-C induction. We are increasing
the number of patients in this ongoing study, and we are planning a
personalized medicine test-adapted Clinical Trial.
231
POSTER (Board P011)
Clinical pharmacodynamic assay development for the first in class
investigational ubiquitin activating enzyme (UAE) inhibitor MLN7243
B. Bahamón1 , F. Gao2 , B. Stringer3 , Y. Yang3 , J. Shi4 , K. Burke5 ,
J. Huck4 , T. Traore4 , D. Bowman5 , H. Danaee1 , M. Millhollen5 , M. Hyer4 ,
N. Bence5 , Y. Ishii1 . 1 Takeda Pharmaceuticals International Co.,
Translational Medicine, Cambridge, USA; 2 Takeda Pharmaceuticals
International Co., Biostatistics, Cambridge, USA; 3 Takeda
Pharmaceuticals International Co., Molecular Pathology, Cambridge,
USA; 4 Takeda Pharmaceuticals International Co., Cancer Pharmacology,
Cambridge, USA; 5 Takeda Pharmaceuticals International Co., Molecular
& Cellular Oncology, Cambridge, USA
MLN7243 is a first in class investigational small molecular inhibitor of
the ubiquitin-activating enzyme (UAE) that is currently in Phase I. UAE
controls cellular ubiquitin conjugation and inhibition of UAE by MLN7243
induces cell cycle arrest, endoplasmic reticulum stress, defects in DNA
repair pathways and ultimately apoptosis. In nonclinical studies, MLN7243
behaves as a strong apoptosis inducer in vitro and also exhibited
antitumor activity against a broad range of human tumor xenograft models
representing solid and hematologic tumor types.
The first in human trial of MLN7243, a Phase I dose escalation
study in patients with advanced solid tumors is underway. Here we
describe development of pharamacodynamic (PD) biomarker assays for
clinical studies of MLN7243. PD immunohistochemistry (IHC) biomarkers
were selected to examine MLN7243 target engagement (MLN7243ubiquitin adduct) as well as pathway inhibition (cellular polyubiquitin and
monoubiquitin histone 2B (Ub-H2B)). Each IHC assay was optimized and
tested for dynamic range using a panel of xenograft samples treated
with MLN7243 for varying time points. Reproducibility and specificity were
calculated using xenograft tumor tissues of known varying expression (5
low, 5 medium and 5 high expressers for polyubiquitin or Ub-H2B). Baseline
expression of the pathway inhibition biomarkers using resected human
tumor samples from four cancer types (non-small cell lung cancer, ovarian,
colon and breast; each n = 30) was evaluated. The stained slides were
scanned using the automated Aperio System, relevant tumor area was
selected by a pathologist and analyzed by Definiens Software for positive
index (% positive) and histological score.
All three PD biomarker assays displayed a large dynamic range, small
day-to-day and operator-to-operator variation (largest CV = approx. 13%)
in both positive index (% positive) or histological scores in a xenograft
model. Power to detect 20% change in both positive index (% positive) and
histological score was calculated for each marker to determine the number
of slides needed in the clinical study (MLN7243-ubiquitin adduct = 2,
polyubiquitin = 5, Ub-H2B = 4) based on assay variability. Polyubiquitin
and Ub-H2B image analysis revealed a wide variation within each cancer
type while between cancer types variation was relatively small (similar
mean/median) with the exception of ovarian cancer. It is possible that the
baseline variation stems from differences in sample fixation, storage and
age or a biological variation in basal ubiquitin pathway flux between patient
samples. Supported by these studies, these PD biomarker IHC assays have
been implemented in the MLN7243 first in human study.
232
POSTER (Board P012)
Development of a targeted NGS assay system for patient enrollment
to the NCI-MATCH study
M. Williams1 , D. Sims1 , J. Lih1 , A. Datta1 , S. Hamilton2 , A.J. Iafrate3 ,
J. Sklar4 , S. Sadis5 , N. Takabe6 , J. Tricoli7 , J. Doroshow8 , B. Conley7 .
1
National Cancer Institute-Frederick, Molecular Charterization and Clinical
Assay Development Laboratory, Frederick, USA; 2 MD Anderson Cancer
Center, Molecular Pathology, Houston, USA; 3 Massachusetts General
Hospital, Molecular Pathology, Boston, USA; 4 Yale University Medical
Center, Molecular Pathology, New Haven, USA; 5 Life Technologies,
Compendia, Foster City, USA; 6 NCI, CTEP, Bethesda, USA; 7 NCI, CDP,
Shady Grove, USA; 8 NCI, DCTD, Bethesda, USA
Background: NGS provides a tool for multi-analyte testing and is well
suited for identifying predictive biomarkers. The NCI and ECOG-ACRIN
are leading an effort to launch a U.S. national cancer trial for the treatment
of cancer patients who have progressed after standard treatments. NCIMATCH will encompass multiple (20+) treatment arms and rely on an NGS
screening assay to identify biomarkers with sufficient levels of evidence
associated with treatment selection. This study will be open to all of the
U.S. Oncology Cooperative groups. Patient treatment will be open at all
NCTN Cancer Centers (>2,400 sites) including community cancer centers.
Four clinical laboratories have been selected and have established a clinical
laboratory network.
Material and Methods: The screening NGS assay uses the Oncomine
Cancer Panel NGS Assay which is a targeted NGS assay. Patient biopsies
will serve as the source of RNA and DNA for the assay. A single tissue preanalytics center will process all specimens prior to shipment to the clinical
laboratories. NGS data will be uploaded to a central data analysis pipeline,
where data will be mapped and variants called. Treatment actionable
variants will be identified. Upon laboratory verification of the variant calls,
the data will uploaded to a rules engine, ‘MATCHBOX’, where treatment
will be selected and the results sent to the treating physician. Details of
this process will be discussed.
Results: The NGS assay will assess 137 genes (including gain of
function and loss of function/tumor suppressors). The assay will report
out approximately 10,822 different cancer variants (SNV, small indel, large
indel, CNV and gene fusions). Details of the levels of evidence required
for selection of treatment actionable variants will be provided. Feasibility
studies will be completed and data will be presented demonstrating the
performance of the NGS assay from the four clinical laboratories. The
analytical validation plan will be discussed.
233
POSTER (Board P013)
Analytical validation and application of the MPACT assay, a next
generation sequencing based targeted mutation detection assay for
treatment selection
C. Lih1 , D.J. Sims1 , R.D. Harrington1 , E.C. Polley2 , Y. Zhao2 , R.M. Simon2 ,
M.G. Mehaffey1 , T.D. Forbes1 , W.D. Walsh1 , V. Datta1 , B.A. Conley3 ,
A.P. Chen4 , S. Kummar4 , J.H. Doroshow4 , P.M. Williams1 . 1 Leidos
Biomedical Inc. Frederick National Laboratory for Cancer Research,
Molecular Characterization Lab, Maryland, USA; 2 National Cancer
Institute, Biometric Research Branch, Maryland, USA; 3 National Cancer
Institute, Cancer Diagnosis Program, Maryland, USA; 4 National Cancer
Institute, Division of Cancer Treatment and Diagnosis, Maryland, USA
Background: Robust and analytically validated assays are essential for
development of molecular targeted cancer therapies. Here we described
the development, analytical performance and application of a clinical
diagnostic assay for MPACT (Molecular Profiling based Assignment of
Cancer Therapeutics) trial. MPACT is a pilot and randomized trial that
applies a next generation sequencing assay to select therapy.
Materials and Methods: Using Life Technologies’ Ampliseq technology
and PGM sequencer, we developed a custom NGS assay, called
the MPACT assay and assessed the performance metrics (sensitivity,
specificity, accuracy, reproducibility) for each type of actionable mutations
of interest. The assay utilizes core needle biopsies shipped and processed
as formalin fixed paraffin embedded specimens.
Results: The MPACT assay interrogates a total of 59,150 bp that
represents 391 treatment actionable variants. The rules for treatment
selection will be discussed. The analytical sensitivity study showed the
MPACT assay achieved 100% sensitivity for SNVs, SNVs at homopolymeric
region (HP), and large Insertions and deletions (large indels, >3 bp) and
83.3% for indels and 93.3% in Indels at HP. Confirmation of 39 known
variants by Sanger sequencing demonstrated 95% accuracy. The MPACT
assay achieved 100% specificity for all 5 variant types. Inter and intraoperator concordance was 100% and 97.15% respectively with greater than
0.99 R square values in allele frequency correlation for detected variants.
Analysis of 10 clinical specimens that contained 11 known mutations
identified previously by another validated sequencing assay demonstrated
the MAPCT assay identified all known mutations. By testing 10 core needle
biopsies biopsied 4 independent times by two operators, we demonstrated
the MPACT assay showed 100% reproducibility in the detected mutations
and treatment selection between all replicates. These data were submitted
to the FDA as part of the study IND. Recent upgrades of the data analysis
pipeline to TS4.0 have increased sensitivity and specificity of the assay to
100%. The MPACT Study is ongoing and data will be presented discussing
the frequency of detection of treatment actionable variants and overall
assay success rates.
Conclusions: This validation study demonstrated the MPACT assay was
well-suited for the intended investigational trial use. The MPACT Study is
open and aMOI incidence will be presented.
234
POSTER (Board P014)
Kinetic analysis of dynamic 11 C-verapamil PET study: Compartmental
v adaptive mixture models comparison
F. Hernandez1 , D. Hawe1 , S. Murphy1 , J. O’Sullivan1 , E. Wolsztynski1 ,
J. Huang1 , M. Muzi2 , J. Eary3 , K. Krohn2 , F. O’Sullivan1 . 1 University College
Cork, Statistics, Cork, Ireland; 2 University of Washington, Radiology,
Seattle, USA; 3 University of Alabama, Radiology, Birmingham, USA
Background: Temporal aspects of dynamic positron emission tomography
(PET) generally rely on compartmental models (CM), with questionable
assumptions to summarise functional kinetics of injected radio-tracers in
living tissues.
Material and Methods: The tracer signal time-course is a combination
of vascular delivery and tissue retention effects which do not always
satisfy the assumptions of tissue homogeneity and instant mixing within
compartments required by the CM. This tissue activity time-course can
typically be expressed as a convolution between the signal of the tracer in
the arterial supply and the tissue residue function. The residue represents
the amount of tracer remaining in the tissue and provides a description of
the tracer kinetics measurable by the PET scan. Thus, in statistical terms,
the residue can be thought of as a survival function for the residence of
the tracer in the tissue which does not require the physiological constraints
intrinsic in the exponential-like residue needed for CM. Accordingly more
flexible novel approaches − nonparametric and later adaptive mixture
models − to estimate the residue based on a piecewise linear form
have been developed. The new approaches have been successfully
probed on two of the most well-established PET radiotracers, 15 O-H2 O
and 18 F-fluorodeoxyglucose, used for perfusion and glucose metabolism
respectively. This study shows the extension of the new approaches to the
multiple-drug resistance transporter P-glycoprotein (P-gp) highly present at
the blood–brain barrier (BBB) using 11 C-Verapamil (Vp) in healthy humans
before and after inhibition of the P-gp by Cyclosporine (CsA) infusion.
Previous PET studies on measuring BBB activity by CsA infusion using
Vp have revealed difficulties for the CM to recover metabolic information
as they require a full understanding of the tracers metabolisation in the
tissue, which is still not certain for Vp. This problem is especially latent
when the inhibition of the P-gp allows the tracer through the BBB where it
is retained once at the brain tissue level.
Results: This work examines and evaluate adaptive mixture model as an
alternative to conventional compartment techniques in recovering metabolic
information from dynamic PET studies with less biological restrictions. Key
bioparameters such as flux, flow, blood volume and volume of distribution
from the two different approaches are compared. Cross-validation is used
to make regional comparisons and evaluate the fit of the two different
estimated residue functions to the tissue activity curves.
Conclusions: The accuracy of the standard models for Vp PET studies
can be questionable, likely because behaviour of Vp metabolites in tissue
is still unclear. Significant statistical evidence in favour of the new adaptivemixture models has been found.
235
POSTER (Board P015)
Kinetic analysis of dynamic 11 C thymidine PET imaging studies:
Compartmental and nonparametric approaches
S. Murphy1 , D. Hawe1 , F. Hernandez1 , E. Wolsztynski1 , J. Huang1 ,
J. O’Sullivan1 , M. Muzi2 , J. Eary3 , K. Krohn2 , F. O’Sullivan1 . 1 University
College Cork, Statistics, Cork, Ireland; 2 University of Washington,
Radiology, Seattle, USA; 3 University of Alabama, Radiology, Birmingham,
USA
Background: Positron emission tomography (PET) with 11 C thymidine
(TdR) as a radiotracer has been used to quantify cell proliferation and DNA
synthesis in a variety of cancer types such as malignant brain tumours.
Materials and Methods: Given time-dependent records of the tracer
activity in blood and tissue, compartment models are used to describe the
mechanisms for transfer and metabolism of the radiotracer. These models
are based on assumptions such as homogeneity of the tissue and instant
mixing within compartments, which can be difficult to justify. This may lead
to the model being a poor fit to the data and hence give misleading results.
A nonparametric approach was developed to alleviate these restricting
assumptions. This technique has been validated in the case of the 15 OH2 O and the 18 F-fluorodeoxyglucose (FDG) PET radiotracers. For TdR,
this estimation problem is more complex as the tracer is metabolised in
the body, which leads to the presence of both TdR and metabolites in
blood and tissue, with the activities of each being jointly observed by
the scanner. Therefore a multiple-input function compartment model is
required to capture and summarise metabolic activity. Calibration of the
79
multiple-input compartment model is achieved using an arterial time-course
measured by arterial sampling, which allows the activity in the arterial
blood to be separated into TdR and metabolites. An adapted version of
the nonparametric approach that is used for 15 O-H2 O and FDG is applied
to the TdR data.
Results: Simulations were undertaken to examine the efficacy of the
multiple input compartment model and also to gain an understanding of
the rates of convergence of the parameters of interest. Similar simulations
are carried out on the nonparametric model. Comparisons between the
models are generated by cross-validation. In TdR-PET studies the key
parameter of interest is the thymidine flux constant. Further numerical
analysis was carried out to examine the bias and variance of the models
for this parameter as well as others estimated in this modelling.
Conclusions: Residual analysis shows non constant variance for the
compartment model, which leads one to conclude the validity of the
model is questionable. Performance analysis of the nonparametric method
suggests it is a relevant candidate alternative for the estimation of flux as
imaged by thymidine.
236
POSTER (Board P016)
Whole genomic assay on endoscopic ultrasound-guided fine needle
aspiration samples of unresectable pancreatic cancer
J.M. Ha1 , K.H. Lee1 , J.K. Lee1 , K.T. Lee1 , W.Y. Park2 , J.S. Bae2 ,
J.K. Jung2 , D.H. Park2 , Y.K. Seong3 , E. Kim1 . 1 Samsung Medical Center,
Gastroenterology, Seoul, South Korea; 2 Samsung Medical Center,
Genomic Medicine, Seoul, South Korea; 3 Sung Kyun Kwan University
Medical School, Internal Medicine, Seoul, South Korea
Background: Targeted therapy according to an individual’s genetic profile
can improve the oncological outcomes. Endoscopic ultrasound-guided fine
needle aspiration (EUS-FNA) has been widely used to obtain tumor tissue
to diagnose unresectable pancreatic cancer. We tested the feasibility of
whole genomic assay on EUS-FNA samples for personalized therapy of
unresectable pancreatic cancer.
Material and Methods: Whole exome and whole transcriptome sequencing were performed with ten EUS-FNA samples from retrospectively
collected pancreatic cancer (9 ductal adenocarcinomas, 1 undifferentiated
carcinoma). We compared these results with the reported genomic and
transcriptomic profiles of 57 pancreatic cancer tissues in The Cancer
Genome Atlas (TCGA) to find novel mutations, differentially expressed
genes and gene fusions.
Results: Genomic profiles were successfully generated from 7 of 10
(70%) pancreatic EUS-FNA samples by whole genomic sequencing. The
frequency of observed genomic mutations was concordantly distributed
with that of TCGA; KRAS, TP53, RNF43, SMAD4, MEN1, MLL3, APC and
ARID1A, sequentially, and the cluster dendrogram of combining 7 EUSFNA samples’ profiles with TCGA data showed intergroup similarity after
TMM (trimmed mean of M-values) normalization. We also identified several
new fusion genes from analysis of our whole transcriptome dataset.
Conclusions: We were able to perform whole genomic and transcriptomic
assay on most EUS-FNA samples from pancreatic cancers and identify
that genomic alterations were well correlated with those listed in TCGA of
pancreatic cancer.
237
POSTER (Board P017)
Impact of phase 1 expansion cohorts on probability of success in
phase 2 and time-to-drug-approval: analysis of 385 new drugs in
oncology
D. Bugano1 , K. Hess2 , L.L. Siu3 , F. Meric-Bernstam4 , A.R.A. Razak3 ,
D.S. Hong4 . 1 MD Anderson Cancer Center, Hematology/Oncology
Fellowship, Houston, USA; 2 MD Anderson Cancer Center, Department of
Biostatistics Division of Quantitative Sciences, Houston, USA; 3 Princess
Margaret Cancer Centre, Drug Development Program, Toronto, Canada;
4
MD Anderson Cancer Center, Department of Investigational Cancer
Therapeutics, Houston, USA
Background: Expansion cohorts (EC) are becoming more common in the
design of oncology phase 1(P1) trials. Our objective was to determine their
impact on phase 2 (P2) trials and on time-to-drug-approval (TDA).
Material and Methods: Systematic review of MEDLINE and EMBASE for
P1 trials and their corresponding P2 trials. We included single-agent dosefinding adult oncology P1 trials published in 2006–2011. ECs were defined
as the enrollment of additional patients (pts) after identification of the P2
dose. Drug approval status and date were based on the FDA website by
04.31.2014. TDA was measured since the first publication of a P1 trial.
Positive P2 trials were those that met their primary endpoints. ‘Probability
80
Poster Session – DNA Repair Modulation (including PARP, CHK, ATR, ATM)
Table (abstract 237): Characteristics of phase 1 trials, ‘probability of positive phase 2’ and ‘time-to-drug-approval’
Targeted agent
Industry-sponsored
Multicenter
Pub >2008
Pts in dose escalation part
Tumor type
Pts in EC
Y vs N
Y vs N
Y vs N
Y vs N
21−37 vs <21
>37 vs <21
Hematologic vs solid
Hematologic + solid vs solid
Specific histology vs any solid
2−20 vs 0
21–271 vs 0
Prob Positive P2
OR (95% CI)
p
Time-to-approval
HR (95% CI)
p
0.9
1.7
0.9
1.0
0.9
1.4
4.1
0.6
1.2
2.4
3.3
0.78
0.12
0.73
0.84
0.62
0.31
0.03
0.36
0.49
0.0054
0.0008
1.0
2.1
2.4
1.0
0.9
1.4
2.4
0.6
2.1
2.1
6.6
0.95
0.33
0.17
0.94
0.88
0.46
0.17
0.62
0.066
0.14
<0.0001
(0.5; 1.6)
(0.9; 3.2)
(0.5; 1.6)
(0.7; 1.7)
(0.5; 1.5)
(1.3; 4.4)
(1.1; 14)
(0.2; 1.7)
(0.7; 2.0)
(1.3; 4.4)
(1.6; 6.6)
(0.4; 2.4)
(0.5; 9.5)
(0.7; 8.5)
(0.5; 2.2)
(0.3; 2.6)
(0.6; 3.4)
(0.7; 8.8)
(0.1; 4.6)
(1.0; 4.4)
(0.8; 5.4)
(2.9; 15)
All values are after multivariate analysis.
of positive P2’ was evaluated with logistic regression and TDA with Cox
proportional hazards regression.
Results: We identified 515 P1 and 608 P2 trials. There were 385 drugs
and 115 (30%) had at least one P1 with EC. A higher proportion of
drugs with EC moved into P2 (62% v 37%, p < 0.0001) and had at least
one randomized P2 trial (34% v 15%, p = 0.0001). We classified drugs
according to the combined number of pts enrolled in all EC for different trials
of that drug: No EC (267 drugs), EC2 − 20pts (60), EC21 − 271pts (44),
missing (14). Enrolling more than 20 pts in EC was associated with higher
probability of a positive P2 and shorter TDA (table). The probabilities of
approval at 5 years were: No EC 5%(2−8%), EC2−20 16%(4−26%), EC21–
271 31%(15−44%).
Conclusions: Expansion cohorts were associated with a higher probability
of positive phase 2 trials and shorter time-to-drug-approval. Factors
involved in the decision to open an expansion cohort might influence this
association.
DNA Repair Modulation (including
PARP, CHK, ATR, ATM)
238
POSTER (Board P018)
Reversal of primary and acquired PARP-inhibitor resistance in
BRCA-mutated triple-negative breast cancers by inhibition of
transcriptional cyclin-dependent kinases (CDKs)
S. Johnson1 , N. Johnson2 , D. Chi3 , B. Primack4 , C. Cruz5 , D. Stover1 ,
A.K. Greifenberg6 , S. Cao7 , K. O’Connor1 , J. Baselga8 , J. Balmaña5 ,
V. Serra5 , M. Geyer6 , A. D’Andrea9 , E. Lim10 , G.I. Shapiro1 .
1
Dana-Farber Cancer Institute, Department of Medical Oncology, Boston,
USA; 2 Fox Chase Cancer Center, Clinical Therapeutics, Philadelphia
Pennsylvania, USA; 3 Dana-Farber Cancer Institute, Department of
Medical Oncology, Boston MA, USA; 4 Dana-Farber Cancer Institute,
Department of Radiation Oncology, Boston MA, USA; 5 Vall d’Hebron
Institute of Oncology, Experimental Therapeutics Group, Barcelona,
Spain; 6 Max Planck Institute of Molecular Physiology, Department of
Physical Biochemistry, Bonn, Germany; 7 Dana-Farber Cancer Institute,
Medical Oncology, Boston, USA; 8 Memorial Sloan-Kettering Cancer
Center, Human Oncology & Pathogenesis Program, New York City,
USA; 9 Dana-Farber Cancer Institute, Radiation Oncology, Boston, USA;
10
Ludwig Institute of Cancer Research, Olivia Newton John Cancer
& Wellness Center, Melbourne, Australia
Background: PARP-1 is synthetically lethal with homologous recombination (HR) defects. Tumors harboring mutations in BRCA1 and BRCA2 show
sensitivity to PARP inhibition to varying degrees. Additionally, acquired
resistance to PARP inhibition occurs via emergence of reversion mutations,
re-expression of mutated BRCA proteins or alterations in expression of
negative HR regulators.
CDKs regulate cell cycle progression and transcription, and recent
evidence has shown that cyclin K-CDK12 controls transcription of HR
genes. We show that CDK12 is a target of the CDK inhibitor dinaciclib,
which causes transcriptional repression of multiple DNA damage response
and repair pathways. CDK inhibition is known to confer HR deficiency
to HR-competent tumors, rendering them PARP inhibitor sensitive.
Here, we tested whether dinaciclib-mediated transcriptional inhibition
could (1) augment the response of BRCA-mutated triple-negative breast
cancer (TNBC) cell lines and patient-derived xenograft (PDX) models
that demonstrate modest sensitivity to PARP inhibitor monotherapy; or
(2) restore PARP-inhibitor sensitivity in BRCA-mutated TNBC models that
have acquired PARP inhibitor resistance.
Results: In MDA-MB-231 cells, dinaciclib reduced phosphorylation of the
C-terminal domain of RNA polymerase II in a concentration-dependent
manner. RNA was collected from vehicle and dinaciclib-treated cells and
levels of global transcription expression change were analyzed using
Affymetrix U133A 2.0 arrays. Ingenuity Pathway Analysis (IPA) was used to
assess networks of gene transcripts significantly repressed after dinaciclib.
The top canonical pathways downregulated were comprised of DNA
damage response networks. CDK12 kinase assays using pSer7 CTD
as substrate were performed in the presence of vehicle or dinaciclib,
demonstrating potent inhibition with IC50 61.8 nM.
Dinaciclib was used in combination with veliparib or olaparib in BRCAmutated cell lines and PDX models. These combinations were synergistic
in HCC1937 and SUM149 TNBC cells (modestly sensitive to PARP
inhibitor monotherapy), as well as in a PARP inhibitor-resistant derivative
of highly sensitive MDA-MB-436 cells. Additionally, these combinations
demonstrated substantial efficacy in two PDX models with somatic BRCA1
and BRCA2 mutations, and limited de novo PARP inhibitor sensitivity, as
well as in a model derived from a BRCA1 germline carrier whose tumor
was slowly progressive on PARP inhibitor treatment.
Conclusions: PARP inhibitor monotherapy in BRCA-mutated TNBCs is
complicated by both de novo and acquired resistance. Transcriptional CDK
inhibition represents a promising strategy for augmenting responses and
reversing resistance. These results have justified clinical exploration of the
combination of dinaciclib and veliparib in both BRCA-proficient and BRCAmutated tumors (NCT NCT01434316).
239
POSTER (Board P019)
Niraparib, a selective PARP 1/2 inhibitor, is efficacious in pre-clinical
models of small-cell lung cancer
Y. Wang1 , J. Ricono2 , K. Admunson2 , S. Agarwal1 , R.J. Fram1 ,
T. Broudy2 , K.M. Wilcoxen1 . 1 Tesaro Inc., Waltham, USA; 2 Molecular
Response, San Diego, USA
Purpose: Small cell lung cancer (SCLC) is an aggressive form of cancer
that accounts for approximately 15% of all lung cancers. Treatment options
have not benefited from the development of currently approved targeted
agents, and platinum based chemotherapeutic regimens still dominate
treatment in both limited and extensive-stage disease. The PARP inhibitor
niraparib, currently in phase 3 studies in ovarian and breast cancer, was
investigated for its ability to inhibit the proliferation of SCLC in vitro and
in vivo.
Methods: Niraparib was evaluated for its ability to inhibit proliferation
and colony formation of SCLC cell lines with known deficiencies in p53,
Rb1, and PTEN as well as amplifications in FGFR and MYC genes.
Low passage SCLC patient-derived xenograft (PDX) models in mice were
utilized to evaluate the in vivo efficacy of niraparib as a monotherapy
and in combination with common first and second line therapies cisplatin/
etoposide and irinotecan. Molecular characteristics of individual tumors
were determined through RNAseq analysis and correlated with niraparib
response.
Results: Niraparib exhibited in vitro anti-proliferative effects on selected
SCLC lines with mutations in p53, Rb1, PTEN, PIK3CA and STK11.
However, niraparib activity in these cells was not definitively correlated
with a known mutationsor platinum sensitivity. In a SCLC PDX model
derived from a metastatic tumor, daily 50 mg/kg niraparib demonstrated
antitumor activity in combination with both irinotecan and cisplatin/
etoposide. A cohort treated with a single cycle of etoposide (8 mg/kg,
days 1−3) and cisplatin (4 mg/kg, day 1) and 4 weeks of niraparib
(50 mg/kg, QD) showed significant tumor growth inhibition. Upon rechallenge with niraparib (75 mg/kg) after a dosing holiday and observable
tumor regrowth, tumor growth inhibition was again observed despite the
presence of large tumors (>500 mm3 ). Additional SCLC PDX models were
selected for evaluation of niraparib efficacy based on in vitro sensitivity
to platinum agents. Tumor growth inhibition and/or regression during
treatment of PDX mice with niraparib (100 mg/kg, QD) was observed in
75% of evaluable models. The determination of niraparib efficacy in SCLC
platinum responsive maintenance treatment and molecular characterization
of niraparib sensitive models is ongoing. Results will be discussed in the
context of developing translational tumor marker classifier candidates for
niraparib sensitivity in clinical studies of SCLC.
Conclusion: Niraparib exhibited anti-cancer activity against SCLC as an
in vitro and in vivo as a monotherapy and in combination with standard of
care agents. These results support the clinical investigation of niraparib in
SCLC.
240
POSTER (Board P020)
The combination of Chk-1 and ATR inhibitor synergistically kills
cancer cells
K. Sanjiv1 , A. Hagenkort1 , P.M. Reaper2 , T. Koolmeister1 , O. Mortusewicz1 ,
N. Schultz1 , M. Scobie1 , U.W. Berglund1 , P.A. Charlton2 , J.R. Pollard2 ,
T. Helleday1 . 1 Science for Life Laboratory Karolinska Institutet, Division of
Translational medicine and Chemical Biology MBB, Stockholm, Sweden;
2
Vertex Pharmaceuticals, (Europe), Abingdon Oxfordshire, United Kingdom
Inhibition of Chk1 has been reported to lead to phosphorylation of a series
of ATR substrates. We therefore hypothesized that a combination of ATR
and Chk1 inhibition may provide benefit. Herein we show that treatment
of cancer cells with the Chk-1 inhibitor, AZD7762, led to replication stress,
which was converted to replication catastrophe and apoptosis when cells
were co-treated with the ATR inhibitor VE-821. Synergism was observed in
a variety of cancer cell lines but not in normal fibroblast cells. In contrast to
previous studies that have shown ATR or Chk-1 inhibitors are most effective
in cells defective for p53 function, we observed that the combination of ATR
and Chk1 inhibition was equally effective in cancer cells with or without
a functional p53 response. Co-treatment with AZD7762 and VE-821 in
U2OS cancer cells induced massive loading of RPA onto chromatin and
pan nuclear gH2AX, indicative of high levels of ssDNA formation and severe
DNA damage. This was associated with stalled replication fork progression,
S-phase arrest and cell death. In contrast, non-cancer fibroblast VH-10
cells tolerated the combination well with no DNA damage or cell death
observed. Under conditions of induced replication stress, by treatment with
hydroxy urea, AZD7762 plus VE-821 caused marked nuclear fragmentation
and early onset of apoptosis in the U2OS cancer cells but not in normal
fibroblast cells. In mouse xenograft models of lung and breast cancer,
treatment with VE-822 (an analog of VE-821) in combination with AZD7762
significantly delayed tumor growth and increased overall survival when
compared with mice treated with vehicle or either agent alone. These data
support the clinical development of ATR and Chk-1 inhibitor combinations
to complement existing DNA damage based chemotherapy.
241
POSTER (Board P021)
Epigenetic loss-of-function BRCA1 mediates tumor cure by
single dose radiotherapy
C. Campagne1 , T.H. Thin1 , J.D. Fuller2 , K. Manova-Todorova3 ,
A. Haimovitz-Friedman1 , S.N. Powell1 , R.N. Kolesnick2 , Z. Fuks1 .
1
Memorial Sloan-Kettering Cancer Center, Radiation Oncology, New York,
USA; 2 Memorial Sloan-Kettering Cancer Center, Molecular Pharmacology
and Chemistry, New York, USA; 3 Memorial Sloan-Kettering Cancer
Center, Molecular Cytology, New York, USA
Background: The mechanism of tumor cure by ionizing radiation is
regarded tumor cell autonomous, effected by misrepair of radiation-induced
DNA double strand breaks (DSBs) via the function of error prone nonhomologous end joining (NHEJ). This model prevails at the low dose range
(8 Gy), with cure depending on tumor propensity for NHEJ misrepair,
requiring repeated exposures for tumor ablation. Here we report high
(>10 Gy) single dose radiotherapy (SDRT) engages an alternative dual
target model.
Material and Methods: DSB repair was analyzed in situ by quantitative
assessment of the time-dependent buildup and resolution of ionizing
radiation-induced foci (IRIF) of specific NHEJ or homology-driven repair
(HDR) mediators. Effect of SDRT on the tumor microvasculature was
assessed by dynamic contrast-enhanced magnetic resonance imaging
(DCE-MRI). Engagement of microvascular dysfunction in DSB repair
was assessed using acid sphingomyelinase (ASMase)-deficient mice,
refractory to vascular endothelial injury. Western blot analysis of Small
Ubiquitin-like Modifiers (SUMO) in tumor extracts and studies of SUMO
conjugating enzymes IRIF in situ were used to evaluate effects of SDRT
on SUMOylation.
81
Results: SDRT concomitantly induces DSBs in tumor cells and an early
wave of ASMase-mediated microcirculatory ischemia/reperfusion (I/R).
Reactive oxygen species (ROS) generated therein in parenchymal tumor
cells oxidize and dysfunction SUMO conjugating enzymes, leading to
catastrophic reprograming of DSB repair. Ku- and Tumor Suppressor p53Binding Protein 1 (53BP1)-mediated NHEJ are not affected, although
53BP1 resolution is delayed. In contrast, HDR is aborted, as SUMO dysfunction impairs recruitment of Receptor-Associated Protein 80 (RAP80),
Breast Cancer 1 protein (BRCA1), Replication Protein A (RPA) and RAD51
recombinase into DSB repair foci, reversible by genetic I/R inhibition
or pharmacological post-I/R ROS scavenging. The epigenetic loss-offunction BRCA1, coupled with abnormally persistent 53BP1 function,
diverts DSB repair to an aberrant 53BP1-dependent lethal pathway,
mediating tumor cure.
Conclusions: We present a new mechanism of tumor cure by ionizing
radiation, in which high SDRT engages a co-dependent dual target model,
involving both DNA DSBs and a transient tumor microvasculature injury. It
represents an alternative to the classical single target mechanism operating
at the low dose range, and provides new targets for modulation of the
radiation response, with a potential for yielding new cures in cancer.
242
POSTER (Board P022)
BMN 673 as single agent and in combination with temozolomide or
PI3K pathway inhibitors in small cell lung cancer and gastric cancer
models
Y. Feng1 , L.E. Post1 , R. Cardnell2 , L.A. Byers2 , B. Wang1 , Y. Shen1 .
1
BioMarin Pharmaceutical Inc., Research and Drug Discovery, Novato
CA, USA; 2 MD Anderson Cancer Center, Thoracic/Head & Neck Medical
Oncology, Houston TX, USA
BMN 673 is a potent, specific PARP1/2 inhibitor with an antitumor cytotoxic
mechanism that includes efficient trapping of PARP-DNA complexes.
BMN 673 shows significant clinical activity in patients with germline
BRCA mutation ovarian and breast cancer; partial responses were also
reported in non-BRCA small cell lung cancer (SCLC) patients treated with
BMN 673 (ASCO 2014). To explore BMN 673 anti-tumor activity beyond
BRCA, we further investigated the combination potential of BMN 673 with
temozolomide (TMZ) or PI3K pathway inhibitors in SCLC and gastric cancer
(GC) models.
TMZ potentiates the activity of PARP inhibitors including BMN 673 in
various tumor models and has shown single-agent activity in SCLC
patients. Here we evaluated the combination of BMN 673 with TMZ
using human SCLC cell-derived xenograft models. In NCI-H209 xenografts,
BMN 673 (0.25 mg/kg, qd×4) plus TMZ at much reduced dosage (3 mg/kg,
qd×4) resulted in tumor shrinkage, while each drug alone had no inhibitory
effect on tumor growth under the same dosage and schedule.
Recent studies show that PARP inhibitor treatment activates the
PI3K/mTOR pathway in SCLC cell lines and animal models (CSHL, 2014).
We therefore assessed anti-tumor activity of BMN 673 in combination
with several PI3K pathway inhibitors including Pan-PI3K, isoform-specific
PI3K, and Akt inhibitors in SCLC cells. Additive or weak synergistic effect
was observed in different lines based on combination index (CI) value at
ED50. In vivo studies are currently being conducted to confirm the in vitro
observation.
Beyond BRCA1/2 mutations, defects in other components of homologous
recombination DNA repair pathway could induce sensitivity to PARP
inhibition. Report has suggested that low ATM expression in some GC
cell lines may contribute to Olaparib sensitivity in vitro. We examined 14
GC cell lines with various mutational backgrounds and found many of them
are highly sensitive to BMN 673 with IC50 values similar to those of BRCA
mutant cell lines. Western analysis of protein markers in these cell lines
suggests a correlation of BMN 673 sensitivity with reduced ATM protein
level and activity (p-ATM). Consistent with the SCLC findings, in vitro
assays also demonstrated a synergistic activity of BMN 673 and PI3K
pathway inhibitors in some of the GC lines.
Together, these findings suggest that combination of BMN 673 with TMZ
or PI3K inhibitor(s) have better anti-tumor activity than either single agent
alone in SCLC and GC models.
243
POSTER (Board P023)
Modulation of PI3K/mTOR pathway following PARP inhibition in
small cell lung cancer
R.J. Cardnell1 , Y. Feng2 , L. Diao3 , Y. Fan1 , F. Masrorpour1 ,
S. Mukherjee1 , J. Shen2 , J. Wang3 , L.A. Byers1 . 1 MD Anderson Cancer
Center, Thoracic/Head & Neck Medical Oncology, Houston TX, USA;
2
BioMarin Pharmaceutical Inc., Novato CA, USA; 3 MD Anderson Cancer
Center, Bioinformatics & Computational Biology, Houston TX, USA
Background: Small cell lung cancer (SCLC) is the most aggressive form
of lung cancer, accounting for 13% of new lung cancers in the US. We have
82
previously shown that PARP1 is expressed at high levels in SCLC and that
preclinical models of SCLC are sensitive to PARP inhibition. Based on this
work, clinical trials of single agent BMN 673 are ongoing and have shown
partial responses in a subset of SCLC patients (Wainberg, Z., et al., ASCO
2014). Recently, we also identified an association between higher baseline
activation of the PI3K pathway and PARP inhibitor resistance. Here, we
investigate changes in PI3K/mTOR pathway signaling in SCLC following
PARP inhibition and the impact of combined PARP−PI3K targeting.
Material and Methods: >140 total and phospho-proteins were measured
by reverse phase protein array (RPPA) pre- and post-treatment with three
PARP inhibitors (olaparib, rucaparib, BMN 673) in cell lines and xenograft
tumors.
Results: In cell lines, treatment with PARP inhibitors caused a significant
increase in several phosphorylated proteins in the PI3K/mTOR pathway,
including p-mTOR, pAKT and pS6 (p 0.02). This observation was
recapitulated in H1048 xenografts treated with BMN 673, with increases
in p-mTOR and p-AKT (p 0.02). In contrast, the LKB1 pathway was
down-regulated after treatment with decreased levels of LKB1, p-AMPK,
and p-TSC (p 0.04). Because LKB1 negatively regulates PI3K/mTOR,
reduced levels of LKB1 may play a role in PI3K/mTOR activation. On the
basis of these results, we then tested the combination of BMN 673 with the
PI3K inhibitor BKM120. Preliminary in vitro analyses show an additive effect
of BKM120, with a greater decrease in SCLC proliferation from combination
treatment vs either drug alone.
Conclusions: In conclusion, we have demonstrated activation of the
PI3K/mTOR pathway in response to treatment with multiple PARP inhibitors
in SCLC. These results, along with our published work showing greater
PARP inhibitor resistance in SCLC with baseline PI3K/mTOR activity,
suggest a potential role of this pathway in both primary and acquired PARP
inhibitor resistance. We propose that PI3K inhibition may increase clinical
activity of PARP inhibitors in SCLC. Based on these results, in vivo testing
of PI3K−PARP targeting is ongoing.
244
POSTER (Board P024)
Development of xenoimplants from germline BRCA1/2 mutant
breast cancer (BC) for the identification of predictive biomarkers,
mechanisms of resistance against poly(ADP-ribose) polymerase
(PARP) inhibitors and evaluation of novel therapies
C. Cruz1 , Y. Ibrahim2 , B. Morancho3 , P. Anton2 , J. Grueso2 , P. Cozar2 ,
M. GuzmÁn2 , P.M. Avilés4 , M.J. Guillén4 , C. Galmarini4 , J. Arribas3 ,
J. Baselga5 , J. Balmaña1 , V. Serra2 . 1 Vall d’Hebron Institute of Oncology,
High Risk Cancer Prevention Group, Barcelona, Spain; 2 Vall d’Hebron
Institute of Oncology, Experimental Therapeutics Group, Barcelona,
Spain; 3 Vall d’Hebron Institute of Oncology, Growth Factors Group,
Barcelona, Spain; 4 PharmaMar S.A., Non Clinical Pharmacology and
Toxicology Department, Colmenar Viejo Madrid, Spain; 5 Memorial Sloan
Kettering Cancer Center, NY, New York, USA
BRCA1/2-mutant BC is characterized by deficient homologous recombination (HR) DNA repair, resulting in synthetic lethality upon treatment with
inhibitors of the single-strand DNA repair enzyme PARP and remarkable
clinical responses. Nevertheless, PARP inhibitors (PARPi) have failed to
show primary efficacy and/or durable responses in a subset of patients.
Several mechanisms of resistance to PARPi have been described both
in vitro and in vivo, but their significance in the clinic and their impact
on subsequent treatments is unknown. In order to improve current
treatment strategies for individual BRCA1/2-mutation carriers, clinically
relevant preclinical models are required, which allow the study of PARPiresistance mechanisms, the identification of predictive biomarkers and
the assessment of novel therapies. PM01183 is a novel transcription
inhibitor with promising activity in BRCA1/2-mutated BC that is currently
in phase II clinical trials, and a potential therapeutic strategy for PARPiresistant tumors.
Material and Methods: BRCA1/2 mutation carriers with BC or ovarian
cancer (OvCa) treated at our institution for primary surgery or metastatic
disease were selected for the study and signed the corresponding IRBapproved informed consent. Primary or metastatic tumor samples from
these patients were implanted in immunosupressed mice to establish
patient-derived xenograft models (PDXs). To characterize PDX sensitivity
to the PARPi olaparib, tumor-bearing mice were treated with olaparib
50 mg/kg po 6 days per week or vehicle; bi-weekly tumor growth
measurements were performed. Olaparib treatment was maintained in
olaparib-sensitive models to generate PDX models with acquired resistance
to the PARPi. PM01183 antitumor activity was tested in an olaparibresistant model (PDX196) derived from a BRCA1-mutated OvCa (PM01183
0.18 mg/kg iv per week).
Results: Ten PDX models were obtained from BRCA1/2-related cancer
patients. Six PDX models were tested for PARPi response with good
correlation with the corresponding patient response. PM01183 showed
antitumor activity in an olaparib-resistant PDX.
Conclusions: PM01183 might show efficacy in patients with BRCA1/2mutant metastatic disease resistant to PARPi.
245
POSTER (Board P025)
Chk1 is a potential novel therapeutic target that regulates cell
survival and potentiates chemotherapy in osteosarcoma (OS) models
S.J. Strauss1 , P. Mistry1 , A. Mendoza1 , M. Robson1 , H. Holme1 ,
P. Nandabhiwat1 , B. Kwok2 , M. Qadir2 , R.B. Pedley1 , J.S. Whelan3 ,
P.H.B. Sorensen2 . 1 UCL Cancer Institute, Dept of Oncology, London,
United Kingdom; 2 BC Cancer Research Centre, Dept of Molecular
Oncology, Vancouver, Canada; 3 University College Hospital, Dept of
Medical Oncology, London, United Kingdom
Background: Outcome for patients with osteosarcoma (OS) is improved
when chemotherapy is given to treat micrometastatic disease. However,
micrometastatic cells differ from those of the primary tumour and appear
more resistant to chemotherapy. The aim of this study was to identify
potential novel therapeutic targets through the use of genetic loss of
function screens in primary and ‘micrometastatic’ in vitro OS tumour
models.
Methods: An siRNA kinome screen was performed in an OS cell
line, MNNG grown as monolayers (M) and ‘micrometastatic’ anchorageindependent cultures (S). Genes that significantly reduced cell survival in
both conditions were validated using a panel of OS cell lines and specific
inhibitors.
Results: Silencing of the cell cycle checkpoint kinase 1 (Chk1) had the
most significant effects in M and S cultures inhibiting growth by 60%
in 5 of 6 OS cell lines tested. Chk1 is a serine/threonine-protein kinase
that regulates S and G2/M phases of the cell cycle. It plays a role in
regulation of the DNA damage response and is highly expressed in OS
tumours with a poor response to neo-adjuvant chemotherapy (Man et al.,
Cancer Res, 2005). Here, Chk1 protein was expressed in all OS cell lines.
OS cell lines were sensitive to the Chk inhibitor LY2606368 mesylate
monohydrate (hereafter LY2606368) with IC50 concentrations in the low
nanomolar range (3.4−9.3nM). Chk1 siRNA and LY2606368 caused cell
cycle arrest in G1/S by 24 hours, and caspase 3 cleavage indicative of
apoptosis. Sub-toxic concentrations of LY2606368 (0.31−5nM) potentiated
the effect of methotrexate and doxorubicin in M and S cells. Synergistic
effects were most marked when the Chk1 inhibitor was administered
24 hours after the cytotoxic agent and with antimetabolite methotrexate
(CI 0.0001–0.03). Combining LY2606368 and doxorubicin or methotrexate
resulted in significantly greater gH2AX formation than either drug alone
demonstrating increased DNA damage (p < 0.0001). Single agent in vivo
activity of LY2606368 was demonstrated in a subcutaneous MNNG OS
mouse model with administration of 4 mg/kg twice daily for 4 of 7 days for
3 weeks, significantly delaying tumour growth (p < 0.01) with no significant
weight loss.
Conclusions: Inhibition of Chk1 is a potentially valuable therapeutic
strategy in OS and warrants further validation.
246
POSTER (Board P026)
PARP inhibitors trap PARP1 onto damaged DNA via catalytic
inhibition and not by an allosteric mechanism
T. Hopkins1 , L. Solomon1 , Y. Shi1 , L. Rodriguez1 , C. Donawho1 ,
E. DiGiammarino1 , S. Panchal1 , A. Olson1 , D. Stolarik1 , D. Osterling1 ,
W. Gao1 , E. Johnson1 , D. Maag1 . 1 AbbVie, Oncology Discovery, North
Chicago IL, USA
Background: PARP inhibitors potentiate the cytotoxicity of DNA alkylating
agents in vitro and in vivo. Trapping of PARP1 onto single-strand breaks in
the presence of PARP inhibitors appears to be related to this increased
cytotoxicity of PARP inhibitor-alkylator combinations. Two models have
been proposed to explain this effect. In the first, catalytic inhibition of
PARP1 prevents auto-PARylation, which is known to promote dissociation
of PARP1 from DNA. In the second, PARP inhibitors allosterically enhance
the affinity of PARP1 for damaged DNA. Direct evidence of allostery has yet
to be reported. The aim of this study was to probe the relative contributions
of allostery and catalytic inhibition to the trapping phenomenon.
Methods: In vivo efficacy and pharmacokinetics were evaluated in HeyA8
xenograft tumor-bearing SCID mice. PARP1 trapping was evaluated by
cellular fractionation, TR-FRET and BLI.
Results: Veliparib markedly potentiates the activity of TMZ in vivo in a
HeyA8 xenograft model. PARP inhibitors (veliparib, olaparib and BMN-673)
all potentiate the activity of MMS and TMZ and trap PARP1 in HeyA8 cells
in vitro. PARP inhibitors do not directly stabilize the PARP1-DNA complex.
Complex stabilization is achieved by inhibition of auto-PARylation in the
presence of NAD+ . In HeyA8 cells, NAD+ depletion via NAMPT inhibition
traps PARP1 as effectively as PARP inhibitors. PARP inhibitors do not
enhance PARP1 trapping after NAD+ depletion.
Conclusions: These studies reveal no evidence for an allosteric trapping
mechanism and indicate that all PARP inhibitors examined trap PARP1
via catalytic inhibition. The potency of PARP inhibitors with respect
to trapping and catalytic inhibition is linearly correlated in biochemical
systems. In cells, trapping potency is related to concentrations required for
potentiation in animal models. Detection of PARP trapping in cells requires
supraphysiologic conditions that exhaust cellular NAD+ and PAR, exceed
concentrations required to elicit synergism and are not tolerated in vivo. In
addition trapping potency appears to be inversely correlated with tolerability.
Quantitation of the degree of trapping that is tolerable and is required for
therapeutic benefit is under active investigation.
Disclosures: All authors are employees of AbbVie. The design, study
conduct, and financial support for this research were provided by AbbVie.
AbbVie participated in the interpretation of data, review, and approval of
the publication.
247
POSTER (Board P027)
Selective inhibitors of nuclear export (SINE) block the expression
of DNA damage repair proteins and sensitize cancer cells to DNA
damage therapeutic agents
T. Kashyap1 , M. Crochiere1 , S. Friedlander1 , B. Klebanov1 ,
W. Senapedis1 , E. Baloglu1 , D. del Alamo1 , S. Tamir1 , T. Rashal1 ,
D. McCauley1 , R. Carlson1 , M. Kauffman1 , S. Shacham1 , Y. Landesman1 .
1
Karyopharm Therapeutics, Natick MA, USA
Background: SINE is a family of small-molecule drugs that inhibit
Exportin 1 (XPO1/CRM1) mediated nuclear export, resulting in retention of
major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IkB
and subsequently in specific cancer cell death. Selinexor is the clinical SINE
compound currently in human phase I/II clinical trials in patients with solid
and hematological malignancies. The goal of this study was to evaluate the
effects of selinexor on DNA repair mechanisms and to test the cytotoxic
effects of combining selinexor with DNA damaging agent on hematological
and solid tumor.
Methods: Whole protein cell lysates from solid and hematological cancer
cell lines treated with selinexor with or without agents that induce
DNA damage were analyzed in Reverse Phase Protein Arrays (RPPA),
immunoblots and quantitative PCR. Selinexor treated cells from solid
and hematological cancer lines were analyzed by immunofluorescence
to evaluate DNA damage. Non-small cell lung cancer A549 Xenografts
were treated with the selinexor (5 mg/kg) and radiation (3 Gy) alone or in
combination and tumor growth was evaluated for 28 days.
Results: Treatment of solid and hematological cancer cell lines with
selinexor did not induce DNA damage in cancer cells but reduced the
expression of DNA damage repair proteins: MSH2, MSH6, PMS2, MLH1,
Rad51, CHK1 and FOXM1. Selienxor regulates the expression of CHK1,
RAD51, MSH2, MSH6 and MLH1 on the transcriptional levels and PMS2
expression on the posttranslational level. There was a trend between the
degree of DNA-damage-repair-protein reduction to selinexor sensitivity.
Knock down of Chk1 alone, induced cytotoxicity whereas silencing of the
other DNA repair proteins did not affect cell viability. Selinexor treatment
following exposure to DNA damaging agents like doxorubicin and idarubicin
inhibited the repair mechanism of DNA damage caused by these agents
and resulted in synergistic cell killing as measured by induction of PARP
and Caspase 3 cleavage. In vivo, selinexor (5 mg/kg) and radiation (3 Gy)
decreased xenograft tumor size of the non-small cell lung cancer A549
by 15% and 43% respectively, relative to vehicle whereas combination of
selinexor and radiation resulted in a 96% tumor decrease.
Conclusion: Selinexor reduce the DNA repair mechanism in solid and
hematological cancer cell lines and combination of selinexor with agents
that cause DNA damage induces cancer cell death that is superior to each
therapy alone. These data suggest that such a combination treatment is
predicted to result with synergistic therapeutic outcome in cancer patients.
248
POSTER (Board P028)
Use of ATR inhibitor in combination with topoisomerase I inhibitor
kills cancer cells by disabling DNA replication initiation and fork
elongation
R. Jossé1 , S.E. Martin2 , R. Guha2 , P. Ormanoglu2 , T. Pfister3 , J. Morris4 ,
J. Doroshow4 , Y. Pommier1 . 1 NCI, Developmental Therapeutic Branch,
Bethesda MD, USA; 2 NCATS, Division of Preclinical Innovation, Rockville
MD, USA; 3 Leidos Biomedical Research Inc, Laboratory of Human
Toxicology and Pharmacology, Frederick MD, USA; 4 DTP-DCTD, Drug
synthesis and Chemistry Branch, Bethesda MD, USA
Camptothecin and its derivatives, topotecan and irinotecan are specific
topoisomerase I inhibitors and potent anticancer drugs. These agents
produce well-characterized double-strand breaks upon collision of replication forks with topoisomerase I cleavage complexes. In an attempt to
83
develop novel drugs combinations, we conducted a synthetic lethal siRNA
screening using a library that targets nearly 7000 human genes. Depletion
of ATR, the main transducer of replication stress-induced DNA damage
response came at the top candidate gene with synthetic lethality with
camptothecin. Validation studies showed that ATR siRNA exacerbated
cytotoxic response to both camptothecin and the indenoisoquinoline LMP400 (indotecan), a novel topoisomerase inhibitor in clinical trial. Inhibition of
ATR by the recently developed specific inhibitor VE-821 induced synergistic
antiproliferative activity when combined with either topoisomerase inhibitor.
Cytotoxicity induced by the combination with LMP-400 was greater than
with camptothecin. Using single cell analysis and DNA fiber spread, we
show that VE-821 abrogates the S-phase checkpoint, and restores origin
firing and replication fork progression in cells treated with camptothecin
or LMP-400. Moreover, the combination of topoisomerase inhibitors with
VE-821 inhibited the phosphorylation of ATR and ATR-mediated Chk1
phosphorylation, while strongly inducing gH2AX. Single cell analysis
revealed that the gH2AX pattern changed over time from well-defined focus
to a pan-nuclear staining. The change in gH2AX pattern can be useful
as a predictive biomarker to evaluate the efficacy of therapy. The key
implication of our work is the mechanistic rationale it provides to evaluate
the combination of topoisomerase I inhibitors with ATR inhibitors.
249
POSTER (Board P029)
Preclinical efficacy of the PARP inhibitor rucaparib (CO338/AG014699/PF-01367338) in pancreatic cancer models with
homologous recombination deficiencies (HRD)
L. Robillard1 , K. Lin1 , P.P. Lopez-Casas2 , M. Hidalgo2 , T.C. Harding1 .
1
Clovis Oncology, San Francisco CA, USA; 2 CNIO, Madrid, Spain
Rucaparib is an oral, potent, small molecule inhibitor of poly (ADP-ribose)
polymerase (PARP) being developed for the maintenance treatment of
platinum-sensitive ovarian cancer in patients with homologous recombination deficient (HRD) tumors, including those with BRCA1 and BRCA2
mutations. Mutations in BRCA and other homologous recombination HR
pathway genes are frequently observed in other tumor types including
breast and pancreatic (Alexandrov et al., 2013; Nature). Here, we
investigated the efficacy of rucaparib as single agent in pancreatic cancer
(PC) cell lines and xenograft models with HRD. Sensitivity to rucaparib
was determined in a panel of PC lines (SU86.86, Panc-1, MIA PaCa-2,
Panc 10.05) following siRNA knock-down of HR genes (ATM, ATR,
BRCA1, BRCA2, PALB2 and RAD51C) to model gene mutations frequently
observed in PC. BRCA1, BRCA2, PALB2 and RAD51C siRNA knockdownshowed synthetic lethality (30% of control GI50 ) in the majority of PC
cell lines examined and correlated with the induction of HRD as assessed
by gH2AX and RAD51 foci formation. As a complement to the cell line data
we examined the efficacy of rucaparib in 3 BRCA2 mutant patient-derived
xenograft (PDX) PC models (Oncotest, GmbH): PAXF_1876 (BRAF, PTEN,
HRAS), PAXF_2005 (KRAS, TP53) and PAXF_2094 (KRAS). Models were
selected on BRCA2 frameshift or nonsense mutations that should be
functionally deleterious. Rucaparib administration to animals bearing preestablished tumors at 150 mg/kg/day BID (modeled Phase 2/3 dose in
mice) resulted in significant monotherapy activity in all models examined
with a 44, 96 and 67% reduction in tumor growth at 28 days post-dosing
for PAXF_1876, 2005 and 2094, respectively. Expansion of rucaparib
preclinical efficacy data beyond BRCA2 mutated PDX models is currently
on-going in a PALB2 mutated PDX PC model (JH033; Villarroel et al.,
2010; Mol. Ca. Ther.). In addition, the activity of rucaparib was examined
in a BRCA2 mutant pancreatic ductal adenocarcinoma patient following
FOLFIRINOX progression in a Phase I study (NCT01482715) was shown to
be consistent with preclinical observations; a 56% reduction in the patient’s
target lesions and a PFS of 6.4 months was observed with no significant
toxicity. A Phase 2 study of rucaparib in patients with pancreatic cancer and
a known deleterious BRCA mutation is currently on-going (NCT02042378).
These findings support the hypothesis that pancreatic cancers exhibiting
HRD are sensitive to rucaparib inhibition.
84
250
POSTER (Board P030)
Comprehensive genomic profiling of pancreatic acinar cell
carcinomas identifies recurrent RAF fusions and frequent
inactivation of DNA repair genes
J. Chmielecki1 , K.E. Hutchinson2 , G.M. Frampton3 , Z.R. Chalmers3 ,
A. Johnson3 , C. Shi4 , J. Elvin5 , S.M. Ali5 , J.S. Ross5 , O. Basturk6 ,
S. Balsubramanian7 , D. Lipson3 , R. Yelensky3 , W. Pao2 , V.A. Miller8 ,
D.S. Klimstra6 , P.J. Stephens9 . 1 Foundation Medicine, Cambridge, USA;
2
Vanderbilt University, Cancer Biology, Nashville, USA; 3 Foundation
Medicine, Computational Biology, Cambridge, USA; 4 Vanderbilt
University, Pathology, Nashville, USA; 5 Foundation Medicine, Pathology,
Cambridge, USA; 6 Memorial Sloan Kettering Cancer Center, Pathology,
New York, USA; 7 Foundation Medicine, Strategic Alliances, Cambridge,
USA; 8 Foundation Medicine, Medical Affairs, Cambridge, USA;
9
Foundation Medicine, Cancer Genomics, Cambridge, USA
Background: Pancreatic acinar cell carcinomas (PACCs) account for ~1%
(~500 cases) of pancreatic cancer diagnoses annually in the United States.
Oncogenic therapuetic targets have proven elusive in this disease, and
chemotherapy and radiation have demonstrated limited efficacy against
these tumors.
Materials and Methods: We performed comprehensive genomic profiling
of a large series of PACCs (n = 44), including closely related mixed
acinar carcinomas (16 pure PACC, 14 mixed acinar/neuroendocrine, 6
mixed acinar/ductal, 2 mixed acinar/neuroendocrine/ductal, and 6 samples
with incomplete histological analysis), using FoundationOne® , a nextgeneration sequencing (NGS)-based platform. DNA was analyzed for base
substitutions, insertions/deletions, copy number alterations, and select
rearrangements; eleven samples had sufficient material for broad fusion
detection using targeted RNA-sequencing.
Results: Recurrent rearrangements involving BRAF and RAF1 (CRAF)
were observed in 10 samples (23%) of mixed and pure histology, and
were mutually exclusive with other known driver events. Biochemical
characterization of the most prevalent fusion, SND1-BRAF (n = 5), resulted
in activation of the mitogen activated protein kinase (MAPK) pathway which
could be abrogated with MEK inhibition. SND1-BRAF was transforming,
and cells dependent on this fusion were sensitive to treatment with the MEK
inhibitor, trametinib. Broad analysis of recurrent cancer-related genomic
alterations in PACC revealed a unique genomic landscape compared
to other subtypes of pancreatic cancer. Notably, we observed lower
frequencies of KRAS and NF1 alterations compared to pancreatic ductal
adenocarcinoma and neuroendocrine tumors, respectively. Inactivating
alterations in DNA repair genes were observed in 45% of PACCs, including
mixed and pure histologies, and were mutually exclusive with RAF genomic
alterations.
Conclusions: These findings have immediate clinical impact for PACC
patients. RAF fusions in other diseases have demonstrated clinical
sensitivity to targeted inhibitors; these agents may represent potential
treatment options for the 23% of PACCs driven by these fusions. To our
knowledge, this is the first report of RAF fusions in any form of pancreatic
cancer. DNA repair deficiencies (45% of PACCs) are associated with
sensitivity to platinum-based therapies and may also predict susceptibility
to PARP inhibitors currently in late-stage clinical development. Although
these alterations have been implicated in other forms of pancreatic cancer,
they have been described only rarely in PACC. Collectively, these data
suggest multiple potential therapeutic options for over two-thirds of PACC
patients, and provide a rationale for using personalized therapies in this
disease.
251
POSTER (Board P031)
A combined in vitro and mathematical modelling approach for
understanding the impact of an inhibitor of ATR on DNA damage and
repair after ionising radiation
J. Yates1 , S. Checkley2 , L. MacCallum2 , R. Odedra1 , J. Barnes3 ,
A. Lau1 . 1 AstraZeneca, iMED Oncology, Macclesfield, United Kingdom;
2
AstraZeneca, Discovery Sciences, Macclesfield, United Kingdom;
3
AstraZeneca, Drug Safety and Metabolism, Macclesfield, United Kingdom
Background: AZD6738 is a potent specific inhibitor of ATR. As part
of clinical development it is planned to investigate the combination of
AZD6738 with ionising radiation (IR) in head and neck cancer patients.
We have developed a novel cell cycle model to predict cellular responses
to combination AZD6738/IR treatment.
Materials and Methods: A simple mathematical model of the cell cycle,
incorporating DNA damage and repair, was proposed. The model was
formulated so that AZD6738 was assumed to inhibit the repair of replication
stress induced damage during S-phase of the cell cycle. The model was
calibrated using in vitro dose–response data generated using a colon
carcinoma cell line. gH2AX biomarker data was used to measure DNA
damage, with cell count as the indicator of tumour proliferation. The
in vitro calibrated model was incorporated into a solid tumour growth model
and AZD6738 time-varying concentration informed by observed plasma
pharmacokinetics in the mouse. Validation of model predictions was against
gH2AX changes over time in the same tumour cell line xenografted in mice
in vivo and the resulting efficacy after repeated doses of AZD6738 and IR
Results: The model was successfully parameterised using in vitro data
generated at a range of concentrations of AZD6738 as well as after
replenishing with AZD6738 free media to simulate drug washout. The
resulting in vivo tumour growth model was capable of accurately predicting
in vivo mouse xenograft data, without requiring any additional modification
of model parameters.
Conclusions: Our prediction of AZD6738/IR combination efficacy has
informed on the minimum dosing levels required in the clinic to be
pharmacologically active. Minimum efficacious dose will minimize the risk
of overdosing and so toxicological effects such as mucositis. The model
also predicts drug efficacy and tumour proliferation rates in response to
intermittent dose schedules, thus optimizing drug exposure for tumour
regression. The model provides a framework that can be extended across
other targetted therapy classes, supplementing mathematical models of low
throughput in vivo data with high throughput in vitro assays.
252
POSTER (Board P032)
The DNA damage response gene Schlafen 11 (SLFN11) is a
transcriptional target of ETS transcription factors in Ewing’s sarcoma
and other cancers
Y. Pommier1 , S.W. Bilke2 , F. Sousa3 , M. Yamade3 , J. Murai3 ,
V. Rajapakse3 , L. Helman4 , P. Meltzer2 . 1 National Cancer Institute,
Laboratory of Molecular Pharmacology, Bethesda, USA; 2 National
Cancer Institute, Genetics Branch, Bethesda, USA; 3 National Cancer
Institute, Developmental Therapeutics Branch, Bethesda, USA; 4 National
Cancer Institute, Pediatric Oncology Branch, Bethesda, USA
SLFN11 is a critical determinant of response to DNA targeted therapies
including topoisomerase I and II inhibitors (camptothecins, etoposide,
doxorubicin) and cisplatin. Ewing’s sarcoma (EWS), which is characterized
by expression of the chimeric transcription factor EWS-FLI1, has notably
high SLFN11 expression. This led us to investigate whether EWSFLI1 is causative for elevated SLFN11 expression. ChIP-Seq analysis of
EWS-FLI1 in A673 EWS cells showed that EWS-FLI1 binds near the
transcription start site of SLFN11. We further demonstrate that EWSFLI1 is a positive transcriptional regulator for SLFN11 and that EWS-FLI1mediated SLFN11 overexpression is responsible for high sensitivity of EWS
to the topoisomerase I inhibitor camptothecin. The correlated expression
between SLFN11 and FLI1 extends to leukemia, pediatric, breast and
prostate cancers. These analyses suggest that, in addition to FLI1, several
ETS members, including ETS1 regulate SLFN11 expression. Together, our
results suggest the emerging relevance of SLFN11 for therapeutic response
to DNA damaging agents in ETS-activated cancers.
253
POSTER (Board P033)
Phase 1 correlative study of ARQ761, a b-lapachone analogue that
promotes NQ01-mediated programmed cancer cell necrosis
D. Gerber1 , Y. Arriaga1 , M.S. Beg1 , J.E. Dowell1 , J.H. Schiller1 ,
A.E. Frankel1 , R. Leff2 , C. Meek2 , J. Bolluyt3 , O. Fatunde3 , R.T. Martinez3 ,
P. Vo4 , F. Fattah4 , V. Sarode5 , Y. Zhou6 , Y. Xie6 , M. McLeod7 ,
B. Schwartz7 , D.A. Boothman4 . 1 University of Texas Southwestern
Medical Center, Hematology-Oncology, Dallas Texas, USA; 2 Texas Tech
University, School of Pharmacy, Dallas Texas, USA; 3 University of Texas
Southwestern Medical Center, Hematology-Oncology, Dallas Texas, USA;
4
University of Texas Southwestern Medical Center, Harold C. Simmons
Cancer Center, Dallas Texas, USA; 5 University of Texas Southwestern
Medical Center, Pathology, Dallas Texas, USA; 6 University of Texas
Southwestern Medical Center, Clinical Sciences, Dallas Texas, USA;
7
ArQule Inc., Woburn Massachusetts, USA
Background: NAD(P)H:quinone oxidoreductase 1 (NQO1) is a twoelectron oxidoreductase expressed in multiple tumor types at levels 5- to
200-fold above normal tissue. ARQ761 (ArQule, Woburn, MA, USA) is
a highly soluble intermediate b-lapachone hydroquinone analogue, complexed in hydroxypropyl-b-cyclodextrin, that exploits the unique elevation of
NQO1 found in solid tumors to cause tumor-specific cell death by eliciting
a futile redox cycle generating high levels of reactive oxygen species and
ultimately PARP1 hyperactivation-dependent cell death.
Materials and Methods: We initiated a 3+3 dose escalation study of 3
schedules (weekly, every other week, 2/3 weeks) of ARQ761 monotherapy
as a 1-hr or 2-hr infusion. Eligible patients had refractory advanced solid
Poster Session – Drug Delivery
tumors, ECOG 0−1, adequate organ function, and central venous access.
Patient blood samples were analyzed for ARQ761 levels and NQO1
polymorphisms. Archival tumor tissue was analyzed for NQO1 staining
intensity and prevalence.
Results: As of May 2014, a total of 31 patients were enrolled and had
received at least one regimen of ARQ761. For weekly administration, the
maximum tolerated dose was 195 mg/m2 . For every other week and 2/3
week administration, dosing at 540 mg/m2 is ongoing. Mean ARQ761 halflife was approximately 50 minutes. Among 23 patients with response data
available, best response was stable disease in 5 patients. Two patients
had minor responses, including one heavily pretreated patient with bladder
cancer with >50% shrinkage of lung metastases at the 195 mg/m2 dose
level. Tissue biomarker analysis was performed for 20 cases. Clinical
benefit was correlated with tissue NQO1 expression: disease control
rate was 65% in NQO1-positive tumors and 18% in NQO1-negative
tumors (P = 0.06). No patients were homozygous for *3 and/or *2 NQO1
polymorphisms. Hemolytic anemia and apparent methemoglobinemia
(transient hypoxemia observed by peripheral saturation monitor but not
confirmed by arterial blood gas) were the principal toxicities
Conclusions: ARQ761 has clinical activity in NQO1-positive tumors.
Principal toxicities include hemolytic anemia and methemoglobinemia.
Monotherapy and combination studies in NQO1-positive tumors are
underway.
254
POSTER (Board P034)
Molecular analysis in breast cancer: correlation with
immunohistochemical classification and pathologic complete
response (pCR) to neoadjuvant chemotherapy (NAC)
S. Baulies1 , M. Gonzalez-Cao2 , N. Karachaliou2 , A. Rodriguez Capitan2 ,
M.A. Molina-Vila2 , M.T. Cusido3 , C. Teixido2 , S. Viteri2 , R. Fabregas3 ,
X. Gonzalez2 , R. Rosell2 . 1 Institut Universitari Dexeus Quiron, Barcelona,
Spain; 2 Institut Universitari Dexeus Quiron, Oncology, Barcelona, Spain;
3
Institut Universitari Dexeus Quiron, Gynecology, Barcelona, Spain
Background: Breast cancer is a heterogeneous disease. Dysregulated
genes, via expression alterations, affect cellular signalling pathways,
leading to chemoresistance. In the present study, we analysed expression
levels of a panel of genes implicated in molecular pathways that could
have a role in chemoresistance. High expression levels of RTKs as AXL
(and its ligand GAS6), and FGFR1 have been described as a poor
prognostic factors, although they are mainly expressed in HR+ breast
cancer. PTPN12 tumor supresor gene inhibits expression of erb2 or EGFR,
and it has been described as frequently downregulated in Triple Negative
(TN) tumors. BRCA1 and RAP80 genes are implicated in DNA repair
pathway and they are downregulated in TN breast cancer conferring
enhanced chemosensitivity. Lower expression levels of BIM (a proapoptotic
protein) can be found after response to chemotherapy. As described in TN,
EZH2 marks poor prognosis. BTRCP leds to EZH2 degradation through
ubiquitination. ZNF217 is a transcription factor that confers poor prognosis
in all breast cancer subtypes. YAP/TAZ is implicated in Hippo pathway and
it has a role in breast cancer proliferation.
Patients and Methods: Gene expression analysis was performed in
pretreatment samples of 92 breast cancer patients treated with NAC.
Expression levels of AXL/GAS6, FGFR1, PTPN12, BRCA1, RAP80, Bim,
EZH2, BTRCP, YAP and ZNF217 genes were analysed by RT-PCR. We
explored the correlation of molecular markers with immunohistochemical
classification and with pCR.
Results: 53 patients were classified as hormone receptor positive (HR+)
(57%), 28 HER2+ (31%) and 11 patients as TN (12%). HR+ tumors
had higher levels of AXL (p = 0.006), GAS6 (p = 0.05), FGFR1 (p = 0.006),
RAP80 (p = 0.007), BTRCP (p = 0.003), PTPN12 (p = 0.023) and ZNF217
(p = 0.005). TN tumors had lower levels of BRCA1 (p = 0.18) and Bim
(p = 0.05).
Predictor of response: High levels of Bim (p = 0.014), or low levels
of AXL (p = 0.003), low FGFR1 (p = 0.014), low YAP (p = 0.010), low
BRCA1 (p = 0.008) and PI3K mutations (p = 0.031) were predictive of pCR.
Interestingly, patients with high levels of FGFR1 had an OS at 10 years of
0% vs 78% (p = 0.051).
Conclusions: Gene expression profile varies by breast cancer subtype.
Chemosensitivity is higher in tumors with high levels of Bim or low levels
of FGFR1, BRCA1, AXL and YAP.
85
Drug Delivery
255
POSTER (Board P035)
Pre-clinical in vivo characterization of MLN7243, an investigational
ubiquitin activating enzyme inhibitor, in solid tumor models
T. Traore1 , J.H. Huck1 , J.S. Shi1 , D.S. Sappal1 , J.D. Duffey1 , Y.Y. Yang1 ,
E.K. Kadakia1 , A.C. Chakravarty1 , B.S. Stringer1 , Y.I. Ishii1 , R.G. Griffin1 ,
C.X. Xia1 , M.M. Milhollen1 , J.C. Ciavarri1 , P.F. Fleming1 , N.B. Bence1 ,
M.H. Hyer1 . 1 Takeda Pharmaceuticals International Company, Oncology,
Cambridge, USA
Clinical results of VELCADE (bortezomib) For Injection have prompted
evaluation of other enzymes within the ubiquitin proteasome system (UPS)
as druggable targets for human cancer. We have identified a first in
class investigational drug (MLN7243) which targets the ubiquitin activating
enzyme, UAE (aka UBA1), an essential cellular enzyme responsible for
activating >99% of all cellular ubiquitin. Ubiquitin is involved in multiple
cellular processes including, but not limited to: ubiquitin-dependent protein
turnover, cell cycle progression, regulation of apoptosis, protein localization
and response to DNA damage. In vitro, MLN7243 has single digit
nanomolar potency against the UAE enzyme and double digit nanomolar
potency in cell-based viability assays. In vivo, MLN7243 administration
leads to anti-tumor activity in a wide variety of cell-line-derived and primary
human xenograft tumor models grown in mice. Pharmacokinetic analysis
in mice indicates MLN7243 is quickly cleared from plasma but maintains a
long half-life in xenograft tumor tissues. MLN7243, once in cells, binds to
UAE and forms a MLN7243-ubiquitin adduct; this adduct species inhibits
the UAE enzyme and remains detectable in tumors for days after the
compound has cleared from plasma. A series of tumor biomarkers have
been utilized to directly track MLN7243 target engagement (MLN7243ubiquitin adduct), measure global cellular polyubiquitin levels, measure
mono-ubiquitinated Histone 2B and measure tumor cell apoptosis (cleaved
caspase-3). Pre-clinical PK/PD/EF relationships have been established to
help guide clinical decisions. MLN7243 is currently being evaluated in a
phase I clinical trial that will evaluate safety, tolerability, pharmacokinetics,
pharmacodynamics, and anti-tumor activity in patients with advanced solid
tumors.
ClinicalTrials.gov identifier: NCT02045095.
256
POSTER (Board P036)
Minicell packaged targeted delivery of shRNA to cancer cells
M. Jivrajani1 , N. Shrivastava2 , M. Nivsarkar1 . 1 B.V. Patel Pharmaceutical
Education and Research Development (PERD) Centre, Pharmacology
and Toxicology, Ahmedabad, India; 2 B.V. Patel Pharmaceutical Education
and Research Development (PERD) Centre, Pharmacognosy and
Phytochemistry, Ahmedabad, India
Background: The genetic nature of cancer provides solid support for the
rationale of si/shRNA-mediated gene therapy. However, a major hurdle to
develop RNAi as cancer therapeutics is successful in vivo delivery. The
aim of this study was to explore the efficacy of folate-conjugated bacterial
minicells as a novel targeted delivery system for shRNA.
Materials and Methods: In this work, plasmid based shRNA against
VEGF A (psNIPERDU6A2) was packaged in minicells. These minicells
were linked with folic acid for active targeting of tumor cells via folate receptor in vitro and in vivo. Cancer cell lines, LNCaP, HeLa and KB have been
selected as positive control whereas A549 was selected as negative control
in terms of folate receptor overexpression. In vitro delivery was studied
by delivering 109 minicells from each the group, (1) FA minicellspSUPERneo ,
(2) minicellspsNIPERDU6A2 , (3) FA minicellspsNIPERDU6A2 in selected cell lines
and expression of VEGF A was analysed by RT-PCR. Tumor xenograft
of A549, LNCaP and KB cells were developed in immunosuppressed
C57BL/6 mice, where the animals of each xenograft group were treated
with (1) saline, and 109 of respective minicells, i.e. (2) FA minicellspSUPERneo ,
(3) minicellspsNIPERDU6A2 , (4) FA minicellsPSNIPERDU6A2 intravenously to study
in vivo delivery. Tumor volume (mm3 ) was measured in each group
every week during the study. At the end of study, tumor was excised
to observed relative angiogenesis. Subsequently, expression of VEGF A
was analyzed from tumor by RT-PCR. Eventually, In vivo biodistribution of
FA
minicellspsNIPERDU6A2 was studied.
Results: Expression of VEGF A did not change in any of the groups in
A549 cell line. In contrast, expression of VEGF A reduced significantly
in FA minicellspsNIPERDU6A2 treated group when compared with other two
groups in positive control cell lines. There was a gradual increase in
the tumor volume till the end of treatment in all four treatment groups of
A549 xenograft. Whereas in case of LNCaP and KB xenograft, there was
86
a significant decrease in tumor volume inFA minicellspsNIPERDU6A2 treated
group as compared to other groups. Relative angiogenesis was decreased
significantly in FA minicellspsNIPERDU6A2 treated LNCaP and KB xenograft as
compared to A549 xenograft. Similarly, expression of VEGF A was found
to be same in all the groups when compared with the saline treated group
in A549 xenograft. On the contrary, significant downregulation of VEGF A
was found in FA minicellsPSNIPERDU6A2 treated LNCaP and KB xenograft.
In vivo biodistribution study revealed that majority of FA minicellspsNIPERDU6A2
localized in the tumor followed by liver and heart.
Conclusions: FA minicellspsNIPERDU6A2 have delivered psNIPERDU6A2
effectively in vitro and in vivo in folate receptor overexpressing cancer cells
through receptor mediated endocytosis which was confirmed by reduced
gene expression of VEGF A and tumor regression.
257
POSTER (Board P037)
Development of peptide-mediated drug delivery systems for colon
cancer targeted imaging and therapy
H.C. Wu1 , C.H. Wu1 , Y.H. Kuo1 . 1 Academia Sinica, Institute of Cellular
and Organismic Biology, Taipei, Taiwan
Colorectal cancer is one of the most common diagnosed cancers and
a leading cause of cancer mortality worldwide. Lack of tumor specificity
remains a major problem for chemotherapies in which side effects prevent
the delivery of the drug dosages needed to eliminate the majority
of cancer cells. Thus, targeted drug delivery system is necessary to
effectively deliver the anticancer drugs to the tumor. In this study, we have
successfully identified specific peptides binding to the human colorectal
carcinoma (hCRC) cells through in vitro biopanning using phage-displayed
peptide library. Three high affinity phage clones to colorectal carcinoma
were identified, and their binding activities were confirmed by cellular
ELISA and flow cytometry. The hCRC-targeted phages recognized five
colorectal carcinoma cell lines and surgical specimens from colorectal
cancer patients. The tumor homing ability of hCRC-targeted phages was
confirmed by xenograft model in vivo. To investigate whether hCRCtargeted peptides could be used to enhance the therapeutic efficacy of anticancer drugs, we synthesized the peptide-mediated liposome encapsulated
anti-cancer drugs. Notably, hCRC-targeted peptides conjugated liposomal
drugs markedly inhibited hCRC tumor growth in mouse xenograft models.
Combination treatment of peptide-mediated targeting liposomes was able
to completely eradicate tumors in three-sixth of the total number of
tumor-bearing mice without any signs of recurrence. Targeting liposomes
improved the therapeutic index by enhancing therapeutic efficacy, reducing
side effects, and increasing the survival rate of tumor-bearing mice in
orthotopic animal model. Biodistribution studies in tumor-bearing mice
indicated that chemotherapeutic drugs were localized in tumor tissue
following administration of hCRC targeting peptides conjugated liposomal
doxorubicin. Our findings indicate that hCRC-targeted peptides have great
potential to be developed into targeted drug delivery systems and imaging
for colorectal carcinoma.
258
POSTER (Board P038)
Pancreatic cancer cells expressing the OATP1B3 transporter show
promising sensitivity to the highly cytotoxic microcystin-LR molecule
V. Kounnis1 , G. Chondrogiannis2 , M.D. Mantzaris3 , D. Fokas4 ,
N.A. Papanikolaou3 , I. Sainis1 , E. Briasoulis1 . 1 University of Ioannina,
Cancer Biobank Center, Ioannina, Greece; 2 University of Ioannina,
Department of Anatomy-Histology-Embryology Medical School, Ioannina,
Greece; 3 University of Ioannina, Laboratory of Biological Chemistry
Medical School, Ioannina, Greece; 4 University of Ioannina, Department
of Materials Science and Engineering, Ioannina, Greece
Background: Microcystins are natural cyclopeptides synthesized mainly
by cyanobacteria and are considered health hazardous due to their toxic
effects after acute consumption or chronic exposure.
From a pharmacologic point of view, microcystins are extremely stable,
hydrophilic cyclic heptapeptides with the ability to cause extensive
cell damage once uptaken by the specific Organic Anion Transporting
Polypeptides (OATP).
Their main toxicity mechanisms are the inhibition of the protein phosphatases PP1 and 2A, the reduction of glutathione levels and the
increase of Reactive Oxygen Species (ROS), ultimately leading to global
hyperphosphorylation and increased cellular oxidative stress.
The present study focused on the investigation of microcystin-LR as a
potential anticancer compound to target pancreatic cancer cell lines proved
to express the OATP1B3.
Materials and Methods: For the purposes of this study, we used the
characterized cell lines Bx-PC3, MIA PaCa-2 and CHO. The expression
of OATP1B3 on the protein level was assessed by Western Blotting and
the monoclonal antibody mMDQ, whereas its endogenous expression
was assessed by real time PCR and specifically designed primers.
xCELLigence and flow cytometry (Annexin/PI) methods were used to study
the cell toxicity of microcystin-LR, Gemcitabine and their combination.
Results: Data resulted from the experimental procedures confirmed the
expression of OATP1B3 in both cancer cell lines Bx-PC3 and MIA PaCa-2
with the first showing more prone expression by 38% on the protein and
2.4 fold on the mRNA level.
In addition the two pancreatic cancer cell lines, showed a relative
proportional − to the transporter expression − sensitivity to microcystin-LR
with IC50 of 83.5nM and 2.1mM respectively.
Of interest, microcystin LR surpassed the toxic effect of the approved
chemotherapeutic agent gemcitabine for the Bx-PC3 cell line, which IC50
was 226nM.
Furthermore the combination of microcystin-LR at low micromolar concentrations and gemcitabine at upper nanomolar concentrations showed
an increased cytotoxic effect for both cell lines when compared to that of
each agent alone with relevant IC50 of 2.9nM and 4.4nM for Bx-PC3 and
MIA PaCa-2. Annexin/PI flow cytometry confirmed the proportional − to the
transporter expression − dose response for microcystin-LR.
Conclusions: The expression of OATP1B3 in cancer cells, could be
considered as a candidate therapeutic target. We propose that structural
modification of the highly cytotoxic MC-LR molecule − especially its
conjugation with N-acetylcysteine, could result in the development of novel
compounds with activity against pancreatic cancers that express OATP1B3.
259
POSTER (Board P039)
Factors that limit delivery of Cdk4/6 inhibitor palbociclib to GBM
K. Parrish1 , J. Pokorny2 , R. Mittapalli1 , K. Bakken2 , J. Sarkaria2 ,
W. Elmquist1 . 1 University of Minnesota Medical School, Minneapolis,
USA; 2 University of Minnesota, Pharmaceutics, Minneapolis, USA
Background: Developing effective therapies for glioblastoma multiforme
(GBM) is significantly hampered by the blood–brain barrier (BBB),
which limits delivery of many potentially effective anti-cancer agents to
infiltrative tumor cells. The cyclin-dependent kinase 4 (Cdk4) pathway is
hyperactivated in approximately 75% of GBM tumors in association with
homozygous deletion of p16 (52%) and amplification of Cdk4 (18%) and
Cdk6 (1%). Palbociclib (PD0332991) is a potent Cdk4/6 inhibitor that has
shown efficacy in treating some non-brain tumors. The purpose of this study
is to determine the mechanisms limiting delivery and efficacy of palbociclib
therapy in an orthotopic xenograft model of patient-derived GBM.
Methods: Palbociclib distribution to the brain was examined in FVB wildtype (WT), and triple-knockout (TKO; Mdr1a/b(−/−) Bcrp1(−/−) ) mice following
escalating oral doses (10, 50, 100 or 150 mg/kg). Concentrations of
palbociclib in plasma and brain were determined by LC-MS/MS. Survival
studies were conducted in patient-derived primary GBM22 xenograft model
in athymic nu/nu mice, and additional drug distribution studies were
conducted in these tumor-bearing mice to correlate delivery with efficacy.
Results: The brain exposure of palbociclib in TKO mice following a
10 mg/kg oral dose (AUCbrain-to-AUCplasma ratio) was 150-fold higher
than in WT mice [WT: 0.044; TKO: 6.24]. Two hours post-dose, the brainto-plasma ratio was constant in WT and TKO mice over a dose range of
10 to 150 mg/kg [brain-to-plasma ratio (10 mg/kg: WT: 0.15±0.06; TKO:
10.3±1.7), (50 mg/kg: WT: 0.19±0.06; TKO: 8.5±3.6), (100 mg/kg: WT:
0.17±0.02; TKO: 4.5±1.7), (150 mg/kg: WT: 0.17±0.03; TKO: 7.8±0.52)].
For efficacy studies, palbociclib was dosed at 150 mg/kg/day continuously.
Consistent with sub-therapeutic delivery across the BBB, palbociclib
did not prolong the median survival of an orthotopic GBM22 xenograft
model. Conversely, treatment of GBM22 xenografts grown as flank tumors
resulted in a significant (45 day) survival benefit. Additionally, the brain
concentrations following a 150 mg/kg dose were intentionally comparable to
the flank tumor concentrations following a 10 mg/kg dose [770±230 ng/mL
and 730±510 ng/mL, respectively], and neither provided a therapeutic
response.
Conclusion: These data suggest that efflux transport at the BBB limit the
brain delivery of palbociclib and hence, efficacy, of this Cdk4/6 inhibitor in
the treatment of GBM. This has important translational implications in the
use of palbociclib in either mono or combination therapies for either primary
or secondary brain tumors.
260
POSTER (Board P040)
Hsp90 Inhibitor Drug Conjugates (HDC): Payloads and possibilities
D. Chimmanamada1 , W. Ying2 , J. Zhang2 , D. Proia3 , T. Przewloka1 ,
J. Jiang1 , D. Vutukuri1 , G. Lu1 , S. Osman1 , S. Chen1 , J. Chu3 ,
P. Rao3 , D. Zhou3 , T. Inoue4 , L. Shin Ogawa4 , R. Singh4 , N. Tatsuta4 ,
A. Sonderfan4 , C. Cortis5 . 1 Synta Pharmaceuticals, Chemistry,
Lexington MA, USA; 2 Synta Pharmaceuticals, Chemistry, Lexington
MA, USA; 3 Synta Pharmaceuticals, Biology, Lexington MA, USA; 4 Synta
Pharmaceuticals, DMPK, Lexington MA, USA; 5 Synta Pharmaceuticals,
Business Development, Lexington MA, USA
Background: Despite the emergence of various therapies, chemotherapy
still plays a significant role in the treatment of cancer. Though toxic,
the potency and broad applicability of chemotherapeutic drugs make
compelling rationale for using them as payloads in drug delivery systems.
Several recent technologies, especially antibody drug conjugates (ADC)
have been very successful, however for narrow sets of indications. We have
recently disclosed the HSP90 inhibitor Drug Conjugate (HDC) platform,
which utilizes the unique pharmacokinetic property of HSP90 inhibitors,
where the drug is selectively retained in tumor in high concentrations while
clearing from plasma and normal tissues relatively quickly. By combining
the overexpression of HSP90 and longer residency of its inhibitors in
tumor, we conceived an idea to conjugate small molecule anticancer drugs
(payload) to HSP90 inhibitor in such a way that the payload is released
selectively and gradually in tumor having a prolonged exposure.
Results: In this presentation, the rationale for payload selection, their
applicability and preliminary assessments will be discussed. Though
virtually any small molecule anticancer agent qualifies as payload for HDC
technology, the expensive drug development paradigm calls for a strong
rationale every time. It has been shown by multiple groups including us
that a combination therapy of an HSP90 inhibitor with a suitable drug,
such as docetaxel is superior compared to the single agent. Hence, the
HDC of taxanes make sense. Other categories include but not limited to,
are the drugs that have poor solid tumor penetration such as proteasome
inhibitors, drugs that have poor pharmacokinetics (highly metabolized) such
as gemcitabine and other antimetabolites, drugs that develop resistance
due to efflux (e.g., pgp) from cancer cells such as camptothecins all make
good payloads. Several cell cycle inhibitors, such as pan-CDK inhibitors
and pan-PI3K inhibitors (such as staurorporine) are also considered.
A lead SN-38 conjugate for which detailed evaluations has been done
showed that the payload is released in tumor for a prolonged period of
time (0.22uM and 0.38uM at 24 h and72 h respectively in tumor against
30nM at 24 h and no drug quantified at 72 h for Irinotecan, at their
maximum tolerated doses (MTDs). In vivo efficacy in multiple tumor models
also exhibited remarkable activity for the HDC compared to Irinotecan.
Toxicological evaluations also revealed that the HDC is safer than Irinotecan
at their efficacious doses.
Conclusion: The HDC platform has the potential to utilize a number of
commonly used small molecule anticancer agents as payloads; expanding
applicability through improved efficacy and toxicity profiles. It also has the
potential to resuscitate the development of drugs that were abandoned due
to toxicity.
261
POSTER (Board P041)
Lesion characterization with ferumoxytol MRI in patients with
advanced solid tumors and correlation with treatment response to
MM-398, nanoliposomal irinotecan (nal-IRI)
R.K. Ramanathan1 , R.L. Korn2 , J.C. Sachdev1 , G.J. Fetterly3 ,
G. Jameson1 , K. Marceau1 , V. Marsh1 , N. Raghunand4 , J. Prey3 ,
S.G. Klinz5 , J. Kim5 , E. Bayever5 , J.B. Fitzgerald5 . 1 Scottsdale
Healthcare/TGen, Virginia Piper Cancer Center, Scottsdale AZ, USA;
2
Imaging Endpoints, Scottsdale AZ, USA; 3 Roswell Park Cancer Institute,
Buffalo NY, USA; 4 Arizona Cancer Center, Translational Cancer Imaging,
Tucson AZ, USA; 5 Merrimack Pharmaceuticals, Inc., Cambridge MA, USA
Introduction: MM-398, a nanoliposomal irinotecan (nal-IRI), is designed to
exploit leaky vasculature for enhanced drug delivery to tumors. Deposition
of nal-IRI and subsequent conversion to SN-38 in both neoplastic cells
and tumor associated macrophages (TAM) may positively correlate with
activity. Predictive biomarkers to measure tumor deposition could identify
patients likely to benefit from nal-IRI. Ferumoxytol (FMX), a 30 nm ironoxide nanoparticle with MRI contrast properties, is taken up by TAMs and
has similar biodistribution patterns to nal-IRI in preclinical models. We
have shown the feasibility of quantitative FMX MRI of tumor lesions and
report here the correlation of FMX levels with nal-IRI activity in the study
population.
Patients and Methods: Eligible patients (n = 15; 4M, 11F; median age
58 [28−80] years) with refractory solid tumors had FMX MRI scans prior
87
to and following (1, 24, 72 h) FMX infusion. T2* signal was used to
calculate FMX levels in plasma and lesions by comparison to a standard
curve. A mechanistic PK model built on these values indicated that tissue
permeability to FMX contributed to early FMX MRI signals at 1 h and 24 h,
while FMX binding contributed at 72 h. Patients continued on nal-IRI at
80 mg/m2 q2w until progression. Core biopsies were obtained 72 h after
both FMX and nal-IRI infusions. RECIST v1.1 evaluation was done by CT
every 8 weeks.
Results: 13/15 (87%) patients received nal-IRI, and 11/13 (85%) were
evaluated for response with 1 (7%) partial response (PR), 5 (38%) stable
disease and 5 (38%) progressive disease. FMX levels were measured
in 31 lesions from 9 patients and compared to lesion size changes
after nal-IRI treatment. Median FMX levels in tumor lesions at 1 h and
24 h were 34.1 and 33.6 mcg/mL, respectively. FMX levels above the
median were significantly associated with better lesion responses as
measured by change in lesion size (p < 0.001 at 1 h; p < 0.003 at 24 h);
no relationship was observed at 72 h. Receiver operating characteristics
for lesion classification according to PR or lesion size reduction had an
AUC 0.8 for early FMX measurements. Levels of irinotecan and SN-38
averaged 3.73 mcg/g [0.13–12.75 mcg/g] and 14.67 ng/g [1.2–64.0 ng/g],
respectively, at 72 h. SN-38 levels in biopsies were 5-fold higher than
in plasma at 72 h (p = 0.013). Prussian Blue staining of ferumoxytol in
biopsies was predominately observed at the stroma-tumor interface where
it colocalized with TAMs.
Conclusions: Clinical activity of nal-IRI was observed in refractory solid
tumors. Our findings are consistent with local activation of MM-398 in the
tumor. The relationship between FMX levels in tumor lesions and nal-IRI
activity suggests that lesion permeability to FMX may be a useful biomarker
for tumor response to nal-IRI in patients with solid tumors.
262
POSTER (Board P042)
Improved cytotoxic activity of Nor-b-lapachone-loaded PLGA
microcapsules in PC3M prostate cancer cell line
C. Pessoa1 , A.C.S. Feitosa1 , M.P. Costa1 , F.C. Evangelista1 ,
F.A.M. Sales1 , I.S. Bomfim1 , E.N. Silva Júnior2 , G.G. Dias2 , V.N. Freire3 ,
W.S. Caetano4 . 1 Ceara Federal University, Pharmacology and Physiology,
Fortaleza, Brazil; 2 Minas Gerais Federal University, Chemistry, Fortaleza,
Brazil; 3 Ceara Federal University, Physics, Fortaleza, Brazil; 4 Instituto
Federal do Ceará, Physics, Fortaleza, Brazil
Background: b-lapachone is one of the most widely studied naphthoquinones. However, several efforts have been made to find new lapachone
analogues, more potent and less toxic. Nor-b-lapachone (NbL), a
semisynthetic naphthoquinone derivative from b-Lapachone, is a cytotoxic
agent against several cancer cell lines.
Material and Methods: To overcome its liposolubility and non-specific
toxicity, this drug was formulated in Poly(d,l)-lactide-co-glycolide (PLGA)
microparticles by the emulsion-solvent evaporation technique. Surface
morphology, particle size distribution, zeta potential, Raman spectroscopy,
Fourier transform infrared (FT-IR) spectroscopy, drug encapsulation
efficiency, drug release kinetics and in vitro cytotoxicity of the drug-loaded
microcapsules were determined.
Results: Spherical microcapsules with a size range of 1.03±0.46 mm
were obtained. The drug encapsulation efficiency was approximately 19%.
The NbL-loaded PLGA microcapsules exhibited a pronounced initial
burst release. After the in vitro treatment with the NbL-loaded PLGA
microcapsules, a clearly phagocytosis of the spheres was observed in a
few minutes. The cytotoxic activity of NbL against PC3M cells was greater
when delivered by PLGA microcapsules compared to the free drug. After
incubation for 72 h, the IC50 values of the free and encapsulated forms of
NbL were 2.045 (1.97–2.12) and 1.046 (0.82–1.34) mg·mL−1 , respectively.
We suggest that PLGA microcapsules containing NbL could be a promising
drug delivery system to be studied using in vivo models of prostate cancer.
Conclusions: The results suggest that NbL-loaded PLGA microcapsules
could be used as a promising delivery system for NbL administration in
in vivo tests of prostate cancer.
263
POSTER (Board P043)
Controlled release of cisplatin using hyaluronic oligosaccharidescoated gold nanoparticles as an efficient delivery system applied to
the treatment of pancreatic tumors
H. Parkkola1 , L. Sobrevals Amieva1 , L. Vivero2 , R. Miñana2 ,
J. Sendra3 . 1 Endor Nanotechnologies, Preclinical, Barcelona, Spain;
2
Endor Nanotechnologies, Nanomaterials, Barcelona, Spain; 3 Endor
Nanotechnologies, R&d Director, Barcelona, Spain
Many chemotherapeutic agents currently used in oncology can produce
toxicity inducing many side effects, even at the optimal therapeutic doses.
88
This problem could be addressed by targeting cellular differences between
cancer and normal cells and controlling the delivery of cancer drugs. One
such difference is the selective overexpression of cell surface receptors
in tumor cells that can act as specific targets for the delivery of a
drug. It is well known that CD44, the main receptor for hyaluronic
acid (HA), is overexpressed in a wide range of malignancies and in
cancer tumor initiating cells, playing an important role in promoting cancer
cell proliferation, migration, invasion, and angiogenesis. The aim of our
therapeutic approach is based on the need to develop new therapies
that can efficiently target tumors, reducing the associated side effects and
increasing the antitumor efficacy. In this line, the use of engineered HAcoated gold nanosystem for the delivery of chemotherapeutics is eligible
to achieve those objectives. Advanced gold nanosystem uptake by tumoral
cells was shown to be CD44-dependent, this was proven by studies with cell
lines that exhibit differing CD44 expression levels as well as downregulating
the expression of the HA receptor in a cell line that expresses high levels
of CD44. Moreover, in vitro studies showed no signs of genotoxicity or
oxidative damage and even though very high concentrations showed a
slow decrease in cell viability. Advanced gold nanosystem carrying cisplatin
was stable in solution and indicated a gradual drug release. Conjugation of
cisplatin did not prevent the drug from performing its action in vitro, the drug
showed slightly higher toxicity when encapsulated compared to the free
drug. In vivo studies conducted in pancreatic cancer xenografts showed
that the antitumoral effect was increased compared to free cisplatin,
however we detected also increased toxicity when higher doses were
administered, suggesting a fine tight therapeutic dosage schedule. Highest
gold accumulation was in liver, followed by skin and tumor in pancreatic
cancer xenografts. Therefore our data provide evidences of the relevance
of using advanced drug delivery nanosystem for the treatment of pancreatic
cancer.
264
POSTER (Board P044)
Vasculogenic mimicry in small cell lung cancer
F. Trapani1 , R.L. Metcalf1 , R. Polanski1 , A. Fusi2 , C. Hodgkinson1 ,
D. Nonaka3 , M.J. Hendrix4 , C. Morrrow1 , F. Blackhall3 , K.L. Simpson1 ,
C. Dive1 . 1 Paterson Institute for Cancer Research, Clinical and
Experimental Pharmacology, Manchester, United Kingdom; 2 Christie
NHS Foundation Trust, Manchester, United Kingdom; 3 Institute of Cancer
Studies University of Manchester, Manchester, United Kingdom; 4 Cancer
Biology and Epigenomics Program Ann and Robert H. Lurie Children’s
Hospital of Chicago Research Center Robert H. Lurie Comprehensive
Cancer Center Northwestern University Feinberg School of Medicine,
Chicago, USA
Introduction: Small cell lung cancer (SCLC) causes 25% of lung cancerrelated deaths worldwide. Despite initial responses to chemotherapy,
most patients relapse with drug resistant disease. A minority of SCLC
patients are inherently chemorefractory. Improved treatments are urgently
required. Targeting tumour vasculature in SCLC with anti-angiogenic
drugs produced disappointing results. Angiogenesis-independent tumour
vascularisation pathways, including vasculogenic mimicry (VM), warrant
further investigation. Aggressive cancer cells displaying VM mimic
characteristics of host endothelial cells forming fluid conducting vascular
channel-like structures. We sought to determine the prevalence of VM in
SCLC and explore any associations of VM with chemotherapy sensitivity
and patient survival.
Methods: VM was evaluated using CD31/periodic acid-Schiff (PAS)
staining in a tissue micro-array (TMA) from 41 limited stage SCLC chemonaive patients and in tumours from 2 chemosensitive and 2 chemorefractory Circulating Tumour Cell (CTC) Derived Explant (CDX) models
that mimic donor patient responses to platinum/etoposide chemotherapy
(Hodgkinson et al Nature Medicine, 2014). The relative abundance of
VM channels (CD31-ve/PAS+ve) compared to host derived blood vessels
(CD31+ve/PAS+ve), (VM/total vessels) in the TMA was compared to patient
overall survival (OS) and in CDX models, to chemotherapy sensitivity.
Results: In the TMA, a VM/Total Vessels score >16% was a poor
prognostic factor for OS by univariate (p = 0.011) and multivariate
(p = 0.014) analyses. In CDX models, chemo-sensitive tumours had a more
VM structures than chemo-refractory tumours.
Conclusion: We present the first evidence of VM in SCLC patient biopsies
and of the VM phenotype in our unique CDX models. The correlation
between high VM in patient biopsies and poorer OS corresponds with
findings in other cancers notably melanoma, and may reflect a ‘cancer
stem cell’ phenotype linked to VM. We are pursuing the testable hypothesis
that high versus low VM levels in chemo-sensitive versus chemo-refractory
CDX respectively could affect tumour drug delivery.
265
POSTER (Board P045)
Pretargeted nanoparticles to deliver both chemotherapeutics and
radiation for the treatment of lymphoma
C. Fang1 , J.C. Jones1 , S.M. Frayo1 , M.H. Hylarides1 , M. Zhang2 ,
O.W. Press1 . 1 Fred Hutchinson Cancer Research Center, Clinical
Research Division, Seattle WA, USA; 2 University of Washington,
Materials Science & Engineering, Seattle WA, USA
Background: We developed a new generation of therapeutic nanoparticles
(NPs) that can deliver both chemotherapeutics and radionuclides specifically to non-Hodgkin lymphoma (NHL) cells. Only one third of NHL patients
are presently cured with chemotherapy and antibody (Abs) treatments.
Radioimmunotherapy (RIT) delivers radiation specifically to tumor cells
using Abs conjugated to a radioisotope that can be therapeutically effective
against resistance to chemotherapy. However, the therapeutic window of
RIT is limited by slow clearance of unbound radiolabeled Abs from the
circulation, resulting in substantial radioactivity in normal tissues. Codelivery of both chemotherapeutics and radiation will result in therapeutic
synergism that will maximize tumor cell killing while reducing resistance to
single-agent therapy. Targeting NPs to the CD20 or CD19 antigen that is
widely expressed on the NHL cell surface will result in effective delivery of
the therapeutic payloads and thus maximize cell killing.
Material and Methods: We use lymphoma xenograft athymic mouse model
to demonstrate the efficacy of NPs. The NPs are based on multifunctional
biodegradable chitosan polymer with well-characterized molecular weight
and composition. Chemotherapeutic drug monomethyl auristatin (MMAE) is
incorporated to polymer via disulfide linkages while therapeutic radionuclide
yttrium-90 (90 Y) is attached via chelating agents. The size and zeta
potential of NPs were characterized by dynamic light scattering. We
administrate the NPs utilizing ‘pre-targeted’ delivery strategy by inject
antibody-streptavidin conjugates (Ab-SA) first, then clearing agents to
remove Ab-SA from circulation, and then inject NPs 24 h after. The
pharmacokinetics (PK), biodistribution (BD), toxicity and therapeutic effects
of NPs are examined.
Results: The NPs were monodisperse and with neutral surface charge with
sufficient drug loading. BD and PK studies showed rapid clearance of NPs
in circulation and preferential accumulation of NPs in tumor (5% I.D./g) of
xenograft mice. Pretargeted approach improves the tumor-to-normal organ
ratio to over 1 for all clearance organs. The NPs show no sign of toxicity in
terms of circulating blood cell analysis and blood chemistry panel. To date,
the NPs with both chemo and radiation eliminated tumor at lower dose than
the effective dose of single agents.
Conclusions: The pretargeted NPs co-delivering MMAE and Y-90 to the
antigens that are widely expressed on the NHL cell surface results in
effective internalization of the therapeutic payloads and maximize tumor
cell killing with minimum side effects.
Data table
Name
Unit
Data
Hydrodynamic size
Polydispersity Index
Zeta Potential
MMAE Loading
nm
27.9
0.150
1.56
8.2
Figure: Structure.
mV
(w/w)%
Poster Session – Drug Design
266
POSTER (Board P046)
Auxiliar treatment by targeting the extracellular matrix to improve
drug delivery and therapeutic response
E. Henke1 , F. Roehrig1 , H. Hoffmann1 , F. Escorcia2 , M. Wartenberg3 ,
S. Volova4 , S. Gaetzner5 , A. Rosenwald3 , S. Erguen6 , D.A. Scheinberg2 ,
Z.V.I. Granot7 . 1 Institute for Anatomy and Cell Biology, Universität
Würzburg, Würzburg, Germany; 2 Molecular Pharmacology, Memorial
Sloan-Kettering Cancer Center, New York, USA; 3 Institute for Pathology,
Universität Würzburg, Würzburg, Germany; 4 Institut for Clinical
Biochemistry, Universitätsklinikum Würzburg, Würzburg, Germany;
5
Institute for Tissue Engineering, Universität Würzburg, Würzburg,
Germany; 6 Institute for Anatomy and Cell Biology II, Universität
Würzburg, Würzburg, Germany; 7 Developmental Biology and Cancer
Research, Hebrew University, Jerusalem, Israel
During the formation of a new tumor, tumor cells do not only proliferate,
but also form their own microenvironment. The newly established
microenvironment not only supports the further growth and proliferation
of the tumor cells, but it also protects the tumor from the effects of
therapeutic agents. An important part of the tumor microenvironment is
the extracellular matrix (ECM). Besides providing structural support, the
ECM is also directly involved in regulating cell behavior like motility and
invasiveness. Lysyl oxidases are a family of enzymes that catalyze the
cross linking of proximal fibrous protein strands. As a result, the ECM
becomes more rigid. Lysyl oxidases are up-regulated in tumors and it has
been shown that the resulting increased rigidity of the tumor tissuedirectly
contributes to invasiveness and metastatic behavior.
We found recently that crosslinking catalyzed by lysyl oxidases also
increases the physical barrier function of ECM proteins. Lysyl oxidase
activity reduces interstitial diffusion, thereby protecting tumor cells from
exposure to drugs. Inhibition of lysyl oxidases can improve drug transport
within the tumor and dramatically enhance treatment efficacy. We treated
early lung metastasis in mouse models with the pan lysyl oxidase inhibitor
3-aminopropionitrile (BAPN). Lysyl oxidase inhibition reduced build-up
of fibrous matrix and left the lesions in a more immature state. Drug
diffusion and oxygenation was significantly improved. Adding lysyl oxidase
inhibition to chemotherapeutic treatment strongly improved efficacy of the
cytotoxic drugs both in primary tumors and in established metastatic
disease. Impotantly, BAPN-treatment reversed the negative impact of
antiangiogenic therapy on drug delivery in an anti-VEGF refractory tumor
model, synergistically improving drug transport and treatment efficacy in
a study that combined lysyl oxidase inhibition with VEGF-ablation and
cytotoxic treatment.
Our results indicate that interfering with the formation of the supportive
tumor microenvironment might be an effective way to slow improve drug
delivery and response. Especially, in combination with anti-angiogenic
treatment it has the potential to significantly improve the effficacy of the
therapeutics already at our disposal to combat cancer.
267
POSTER (Board P047)
Novel adjuvant therapy with leptin peptide receptor antagonist-2
conjugated to nanoparticles (IONP-LPrA2) to minimize
chemoresistance in triple negative breast cancer
T. Harmon1 , A. Harbuzariu2 , L. Yang3 , R.R. Gonzalez-Perez1 .
1
Morehouse School of Medicine, Atlanta GA, USA; 2 Morehouse School
of Medicine, MBI, Atlanta GA, USA; 3 Emory University, Atlanta GA, USA
Background: Triple negative breast cancer (TNBC) is a life-threatening
disease with no targeted therapy, which is linked to obesity incidence.
Leptin, a cytokine produced by adipose tissue and overexpressed in breast
cancer, induces TNBC growth and survival. Leptin binds to its receptor,
OB-R, inducing expression of Notch and breast cancer stem cell (BCSC)
genes. Notch is a pro-angiogenic factor, which interacts with leptin and
IL-1 signaling crosstalk (NILCO). NILCO induces the expression of several
pathways involved in cell proliferation, metastasis, and angiogenesis; notably vascular endothelial growth factor (VEGF/VEGFR2). We have created
leptin peptide receptor antagonists linked to iron oxide nanoparticles which
will inhibit leptin signaling, thus decreasing the expression of Notch and
BCSC genes and proteins.
Methods: Several human TNBC cell lines were treated with the
chemotherapeutics (IC50 doses: taxanes, doxorubicin, cyclophosphamide
and anti-angiogenic/chemotherapeutic, sunitinib) combined with IONPLPrA2 for 24−72 h. Cell viability, apoptosis rate and the expression levels
of NILCO molecules were analyzed using the Cellometer Vision Image
Cytometer® .
Results: IONP-LPrA2 improved the effects of chemotherapeutics on cell
viability and apoptosis, which could allow the reduction of effective dosage.
89
The expression of several NILCO components was significantly reduced by
IONP-LPrA2 alone and in combination with chemotherapeutics.
Conclusion: These findings suggest that IONP-LPrA2 may be useful
as an adjuvant therapy in the treatment of TNBC. Results also indicate
that IONP-LPrA2 may work in concert with chemotherapeutics and antiangiogenic drugs in vivo. Present findings suggest that IONP-LPrA2
adjuvant therapy could be a novel way to specifically target TNBC, and
to reduce chemoresistance as well as the undesired side effects of
chemotherapeutics. This may be particularly important for obese patients
that show a higher incidence and poorest prognosis of TNBC.
Acknowledgements: This work was partially supported by the National
Institutes of Health and National Cancer Institute Grant 1SC1CA138658−05
and U54 CA118638, and DOD Idea Award BC 123427 to RRGP; and
facilities, and support services at Morehouse School of Medicine (NIH
RR03034 and 1C06 RR18386) and NIH/NCRR grant 1G12RR026250−03.
Drug Design
268
POSTER (Board P048)
A potent and highly efficacious bivalent Smac Mimetic APG-1387 in
Phase I clinical development
J. Lu1 , S. Rong1 , H. Sun1 , L. Liu1 , D. McEachern1 , G. Wang2 , J. Wen2 ,
Y. Zhai2 , M. Guo2 , D. Yang2 , S. Wang1 . 1 University of Michigan,
Comprehensive Cancer Center, Ann Arbor MI, USA; 2 Ascentage Pharma
Group, Preclinical development, Taizhou Jiangsu, China
Background and Aim: Inhibitors of apoptosis proteins (cIAP1/2 and
XIAP) are key regulators of apoptosis and are attractive cancer therapeutic
targets. Targeting IAPs with small molecules represents a promising new
strategy for cancer treatment. APG-1387 was designed a potent smallmolecule inhibitors that mimic the natural dimeric Smac protein. We report
the design and preclinical characterization of APG-1387 for its anticancer
activity and mechanism of action.
Methods and Results: Using a structure-based computational drug design
method, we designed and synthesized SM-1387 (renamed as APG-1387
by Ascentage Pharma) as a bivalent Smac mimetic and a potent inhibitor
of cIAP1, cIAP2 and XIAP. Biochemical assays showed that APG-1387
binds to XIAP, cIAP1, cIAP2 proteins with Ki values of 1, 30 and 20 nM,
respectively. APG-1387 potently antagonizes XIAP proteins containing both
the BIR2 and BIR3 domains and induces cIAP-1 degradation at 1−10 nM in
human cancer cell lines. Cell growth assays showed that APG-1387 inhibits
cell growth in a subset of human cancer cell lines and achieves IC50 values
of 10 and 23 nM, respectively, in MDA-MB-231 human breast cancer and
SK-OV-3 ovarian cancer cell lines. Flow cytometry and western blotting
assays showed that APG-1387 at 10–100 nM induces profound apoptosis
in the MDA-MB-231 and SK-OV-3 cell lines. In vivo study showed that APG1387 is capable of inducing tumor regression even at weekly dosing in
the MDA-MB-231 xenograft model. APG-1387 shows a strong combination
synergy with docetaxel and achieves tumor regression in the HCC1954
triple negative breast cancer model with no signs of toxicity.
Conclusion: Our preclinical data suggest that APG-1387 is a promising
anticancer agent. A phase I clinical trial of APG-1387 is ongoing to evaluate
its safety, pharmacokinetics, PD biomarkers and preliminary efficacy in
patients with advanced cancers.
269
POSTER (Board P049)
The NCI-60 as an effective tool for scaffold hopping: A phenotypic
systems-based approach to the design of novel chemotherapeutics
D.G. Wishka1 , V. Kumar2 , B. Teicher3 , G. Kaur4 , B. Fang5 , P. Risbood6 ,
M. Hollingshead7 , J. Zais7 , J. Morris6 . 1 National Cancer Institute,
Drug Synthesis and Chemistry Branch, Frederick MD, USA; 2 Leidos
Biomedical Research, Drug Synthesis and Chemistry Branch, Frederick
MD, USA; 3 National Cancer Institute, Molecular Pharmacology Branch,
Rockville MD, USA; 4 National Cancer Institute, Molecular Pharmacology
Branch, Frederick MD, USA; 5 M.D. Anderson Cancer Center, Houston
TX, USA; 6 National Cancer Institute, Drug Synthesis and Chemistry
Branch, Rockville MD, USA; 7 National Cancer Institute, Biological Testing
Branch, Frederick MD, USA
The indole-3-carbinol NSC-743380 [(1-(3-chlorobenzyl)-1H-indol-3-yl)methanol] causes regression in the mouse A498 renal xenograft model.
Although its mechanism of action is not well understood, NSC-743380
produces a distinct growth inhibition pattern in the NCI-60 cell line
screen. NSC-743380 acts very rapidly in cell culture, with changes
observed in multiple signaling pathways within 5 minutes of exposure.
90
Using cell lines derived from the NCI-60 panel, a search for a second
xenograft model sensitive to NSC-743380 was unsuccessful. In addition,
a significant medicinal chemistry effort examining modifications to the
indole-3-carbinol structure of NSC-743380 failed to produce a suitable
alternative. The COMPARE algorithm is a powerful tool for identifying
compounds that share common mechanisms of action by matching their
respective growth inhibition patterns in the NCI-60 cell panel. Running a
COMPARE analysis on the existing NCI-60 data for the NCI compound
collection with NSC-743380 as the exemplar identified a number of
alternative chemotypes, including one represented by the mustard, NSC101490 [(E)-N -(4-(bis(2-chloroethyl)amino)-2-chlorobenzylidene)nicotinohydrazide]. Using the NCI-60 growth inhibition patterns as a guide to
overlay of the SAR features from indole-3-carbinol series of NSC-743380
with that of the mustard series of NSC-101490 provided the basis for the
scaffold hop to the new Benzyl-Amino-Benzyl-Alcohol (BABA) class of
chemotherapeutics. Herein, we report the identification of a potent subtype
of the BABA chemotype that shares with NSC-743380 the ability to cause
regression in the A498 xenograft model as well as its distinctive NCI60 growth inhibition pattern. In addition, evidence will be presented to
show that sulfotransferase activation may be a component involved in the
antiproliferative activity of the BABA series against sensitive cell lines.
270
POSTER (Board P050)
Discovery, development and optimization of low molecular weight
EPH−ephrin protein–protein inhibitors
M. Tognolini1 , C. Giorgio1 , I. Hassan-Mohamed1 , E. Barocelli1 , M. Mor1 ,
D. Pala1 , S. Russo1 , M. Incerti1 , A. Lodola1 . 1 University of Parma,
Pharmacy, Parma, Italy
Background: The EPH−ephrin system plays a key role in tumorigenesis
and its de-regulation correlates with a poor clinical prognosis in many solid
tumors. Agents targeting the EPH−ephrin system could be potentially useful
for the inhibition of different facets of cancer progression. However, even
though some classes of small molecules targeting EPH−ephrin interactions
have been reported, their use is hampered by poor chemical stability
and low potency. Stable and potent ligands are essential to obtain strong
pharmacological data.
Materials and Methods: We recently identified lithocholic acid (LCA)
as a competitive and reversible compound able to disrupt EPH−ephrin
interaction showing a Ki value of 49 mM. Using LCA scaffold as a reference
structure and starting from a docking model of the EPHA2−LCA complex
we designed and synthetized a series of amino acid conjugates of
lithocholic acid and the new compounds were characterized for their
ability to disrupt the EPHA2–ephrin-A1 interaction by means of an ELISA
binding assay. The most promising compounds were further characterized
for their cellular functional properties including: aspecific cytotoxicity,
antiproliferative effect, cell cycle, inhibition of Eph kinase phosphorylation,
induction of morphological changes, angiogenesis and migration.
In order to check for the overall selectivity of the new compounds selected
molecules were tested for their ability to interact with physiological-targets
of bile acids (FXR, TGR5) as well as some other kinases (EGFR, VEGFR).
Results: The L-homo-Trp-conjugated of LCA (UniPR129) emerged as a
potent and selective antagonist of EPH receptors, inactive on the FXR,
TGR5, EGF and VEGF receptors. UniPR129 reversibly and competitively
disrupted EPHA2–ephrin-A1 interaction with Ki = 370 nM in an ELISA
binding assay and it showed low micromolar potency in cellular functional
assays, including inhibition of EPHA2 activation, cell migration and cell
rounding and disruption of in vitro angiogenesis without cytotoxic effects.
Structure of UniPR129 was modified, with the aim to improve its
pharmacokinetic properties, leading to the identification of a new orally
bioavailable compound.
Conclusions: The discovery of UniPR129 represents not only a major
advance in potency compared to the existing EPH−ephrin antagonists but
also an improvement in terms of cytotoxicity, making this molecule a useful
pharmacological tool and a promising lead compound. Moreover, steroid
scaffold can be modified in order to obtain potent, selective, and soluble
bioavailable compounds.
271
POSTER (Board P051)
Data integration and graph analysis for cancer genomics and drug
discovery
B. Bernard1 , M. Miller1 , H. Rovira1 , I. Shmulevich1 . 1 Institute for Systems
Biology, Seattle, USA
Overview: Each tumor type being studied by TCGA consists of tens of
thousands of molecular data points derived from thousands of primary
human tumor samples, from which we identify millions of statistically
significant molecular associations. From these data, we know that each
tumor harbors numerous somatic mutations and copy number aberrations
that contribute to loss and gain of function in the cancer cells. While certain
genes are more frequently aberrated than others, each tumor sample has
a unique mutation and copy number profile that should inform patient
stratification, treatment planning, novel target identification, and drug
repurposing for personalized therapeutic strategies and drug discovery.
The search space across millions of associations for tens of thousands of
genes and thousands of potential drugs is tremendously underexplored.
Consequently, promising targets and therapies are not identified or
evaluated. Approaches to integrate and query molecular associations from
heterogeneous sources of data pose a significant research and computational challenge in drug discovery. To this end, we have integrated statistical
analysis of molecular data from thousands of primary tumor samples in
TCGA with a large and heterogeneous cancer knowledge base, including
cell line screening, protein–inhibitor interactions, semantic-based information in the literature, and protein interaction databases. We model these
heterogeneous relationships in a graph database, and query this large
graph to generate novel and testable predictions for target identification,
drug repurposing, and tumor type prioritization for therapeutic intervention.
Example 1. Identification of novel targets in primary human tumors:
We have combined the Cancer Cell Line Encyclopedia (CCLE) and Achilles
shRNA screening projects to infer synthetic lethal (SL) relationships in
cell lines with characterized mutation profiles. Integration of these SL
relationships with molecular associations in TCGA enables the identification
of graph patterns and subsequent prioritization of potential cross-cancer
therapeutic targets with direct evidence for SL relationships in both cell line
and primary human tumor samples.
Example 2. Patient stratification and prioritization of tumor types
for clinical trials: Analysis of targeted drug screening in cell lines reveals
that the sensitizing genes (i.e., those genes harboring mutations and copy
number aberrations in drug-sensitive cell lines) are invariably not limited
to the drug target itself. For example, AKT1 inhibition is most effective
in cell lines with PTEN mutations. By analyzing the integration of cell
line screening hits with accompanying relationships derived from primary
tumor sample data in TCGA, we can stratify patients based on their
aberration profiles and prioritize clinical trials for tumor types with evidence
for relationships between the drug target and sensitizing aberrations.
272
POSTER (Board P052)
8-(1-Anilino)ethyl)-2-morpholino-4-oxo-4H-chromene-6-carboxamides
as PI3Kbeta/delta inhibitors: structure–activity relationships and
identification of AZD8186, a clinical candidate for the treatment of
PTEN deficient tumours
B. Barlaam1 , S. Cosulich1 , S. Degorce1 , M. Fitzek1 , S. Green1 ,
U. Hancox1 , C. Lambert-van der Brempt1 , J.J. Lohmann1 , M. Maudet1 ,
R. Morgentin1 , M.J. Pasquest1 , A. Peru1 , P. Ple1 , T. Saleh1 , M. Vautier1 ,
M. Walker1 , L. Ward1 , N. Warin1 . 1 AstraZeneca, Oncology, Macclesfield,
United Kingdom
Several studies have highlighted the dependency of PTEN deficient
tumours to PI3Kb activity and specific inhibition of PI3Kd has been shown
clinical efficacy against human B-cell cancers. We previously reported
the identification of a new series of pyrido[1,2-a]pyrimidin-4-ones and our
efforts1−2 to optimize this series as orally active PI3Kb/d inhibitors, leading
to compounds 1 and 2. Combining high potency, high solubility and high
metabolic stability was a significant challenge in the pyrido[1,2-a]pyrimidin4-one series, possibly because of the intrinsic lipophilicity of the
pyrido[1,2-a]pyrimidin-4-one core. In this poster, we describe the discovery
and optimization of a new series of 8-(1-anilino)ethyl)-2-morpholino-4oxo-4H-chromene-6-carboxamides as PI3Kb/d inhibitors, leading to the
identification of the clinical candidate AZD8186, a potent inhibitor of p-Akt in
cells sensitive to PI3Kb inhibition (IC50 0.003 uM (+/− SEM: 0.001) in PTEN
null breast adenocarcinoma MDA-MB-468 cells) and in cells sensitive to
PI3Kd inhibition (IC50 0.017 uM (+/− SEM: 0.004) in Jeko B cell line), but
not to cells sensitive to PI3Ka inhibition (IC50 0.752 uM (+/− SEM: 0.293)
in PIK3CA mutant human breast ductal carcinoma BT474). On the basis of
the lower lipophilicity of the chromen-4-one core compared to the previously
utilised pyrido[1,2-a]pyrimid-4-one core, this series of compounds (and
AZD8186 in particular) displayed high metabolic stability and suitable
physical properties for oral administration. AZD8186 showed profound
pharmacodynamic modulation of p-Akt in PTEN-null PC3 prostate tumour
bearing mice after oral administration and showed complete inhibition of
tumour growth in the mouse PTEN-deficient PC3 prostate tumour xenograft
model. AZD8186 was selected as a clinical candidate for treatment of
PTEN-dependent cancers and has recently entered phase I clinical trials.
91
cell growth of HCT116 cells and also significantly suppressed tumor growth
in the HCT116 xenograft model. Those results suggest that the STLC
derivatives with two linked phenyl rings could be a novel lead compound to
design further clinical candidates of the next generation of KSP inhibitors
for antitumor chemotherapies.
273
POSTER (Board P053)
Significance of serine-167 and cysteine-129 residues in the active site
of the immune-suppressive enzyme indoleamine 2,3-dioxygenase 1
(IDO1) for the binding of novel inhibitors
P. Tomek1 , B.D. Palmer1 , J.U. Flanagan1 , L. Ching1 . 1 Auckland Cancer
Society Research Centre, The University of Auckland, Auckland, New
Zealand
Background: IDO1 is an enzyme expressed by a broad range of cancers
to suppress the host’s immune system. It is a validated target for cancer
therapy, with two IDO1 inhibitors already in human clinical trials. The
IDO1 active site amino acids Ser-167 and Cys-129 are predicted to form
hydrogen bond interactions with inhibitors. We have screened for inhibitors
to probe the importance of these residues in the IDO1 active site for ligandbinding and functional activity of the enzyme.
Material and Methods: The National Cancer Institute Diversity Set III
library (1597 compounds) was screened for inhibition of IDO1 enzymatic
activity using our recently developed fluorescence assay automated with
a JANUS robotic workstation. Mutant IDO1 proteins were prepared using
Stratagene QuikChange® Site-Directed Mutagenesis Kit.
Results: The NCI library was screened against wild-type IDO1 and
alanine replacement mutants of serine-167 (S167A) and cysteine-129
(C129A). This identified three new classes of IDO1 inhibitors (pyrimidinones, phenanthroimidazoles and benzoxadiazoles). Compounds from
the phenanthroimidazoles and benzoxadiazoles classes exhibited a tightbinding, reversible mode of inhibition that was strikingly different to that of a
well-characterised IDO1 inhibitor, 4-phenyl-1H-imidazole. When screened
against the S167A mutant, 18 of the 69 compounds that inhibited wildtype IDO1 (>25% at 20 mM) exhibited decreased activity against the
S167A mutant. Structure–activity studies around two of these compounds,
2-(1H-phenanthro[9,10-d]imidazol-2-yl)phenol and 6-carboxy-5-((4-iodo-2methylphenyl)amino)benzo[c][1,2,5]oxadiazole 1-oxide, were performed,
and showed that the hydroxy and N-oxide substituents on these two
molecules respectively, were related to the decreased activity. When
screened against C129A mutant, no compounds in the library showed
decreased activity compared to wild-type IDO1, however three compounds
showed increased inhibition.
Conclusions: Three IDO1 inhibitors identified in this study displayed
tight-binding reversible mode of inhibition, one of the more desirable
mechanisms of action for therapeutic agents. The hydroxyl group of serine167, but not the thiol of cysteine-129 in the IDO1 active site appears to be
important for interactions with a range of different inhibitors.
274
POSTER (Board P054)
Novel cysteine derivatives for the next generation anticancer agents
acting on KSP
N. Ogo1 , J. Sawada1 , Y. Ishikawa1 , K. Matsuno1 , A. Hashimoto2 ,
A. Asai1 . 1 University of Shizuoka, Graduate School of Pharmaceutical
Sciences, Shizuoka, Japan; 2 Taiho Pharmaceutical Co. Ltd., Tsukuba
Research Center, Ibaraki, Japan
Kinesin spindle protein (KSP), known as human Eg5, plays an important
role in the early stages of mitosis. It is responsible for the formation
and maintenance of the bipolar spindle. Inhibition of KSP leads to cell
cycle arrest during mitosis and causes cells with a monopolar spindle
followed by cell death. A large number of KSP inhibitors have been reported
and some of them entered into clinical trials. Although clinical efficacy of
these inhibitors has been limited to date, recently better results have been
obtained in the treatment of hematological malignancies with thiadiazole
based KSP inhibitors such as ARRY-520. We previously identified S-tritylL -cysteine (STLC) as a selective KSP inhibitor from our HTS campaign
and reported that several STLC derivatives induced M-phase accumulation
and subsequent apoptosis in leukemic cells (BMCL 2007, 17, 3921–3924,
BMCL 2010, 20, 1578–1580, Cancer Lett. 2010, 298, 99–106).
The objective of this study is to improve the potency of these leads and to
confirm the antitumor efficacy in vivo. As a result of further structure–activity
relationship studies, we identified novel derivatives with two fused phenyl
rings which showed remarkably potent KSP inhibitory activity with IC50
values in nanomolar ranges. Docking simulation of these novel derivatives
using AMBER12 indicated that cross-linking of the phenyl rings in the trityl
group allowed for better binding by occupying a hydrophobic pocket in the
L5 allosteric binding site. The representative derivatives potently inhibited
Figure: Novel cysteine derivatives.
275
POSTER (Board P055)
Modulation of PIP2 levels through small molecule inhibition of PIP5K
D. Andrews1 , S. Cosulich2 , N. Divecha3 , D. Fitzgerald3 , V. Flemington2 ,
C. Jones1 , D. Jones3 , O. Kern4 , E. MacDonald4 , S. Maman4 , J. McKelvie5 ,
K. Pike1 , M. Riddick6 , G. Robb1 , K. Roberts2 , J. Smith4 , M. Swarbrick4 ,
I. Treinies7 , M. Waring1 , R. Wood6 . 1 AstraZeneca, Chemistry, Macclesfield,
United Kingdom; 2 AstraZeneca, Bioscience, Macclesfield, United Kingdom;
3
Cancer Research UK, Inositide Laboratory, Manchester, United Kingdom;
4
Cancer Research Technology, Chemistry, Cambridge, United Kingdom;
5
Cancer Research Technology, Chemistry, London, United Kingdom;
6
Cancer Research Technology, Bioscience, Cambridge, United Kingdom;
7
Cancer Research Technology, Bioscience, London, United Kingdom
Background: Phosphatidyl inositol (4,5)-bisphosphate (PIP2 ) is a key
phospholipid signalling molecule, involved in cellular processes such
as cell proliferation and survival. Cellular PI(4,5)P2 is synthesised by
phosphorylation of PI(4)P on the D-5 position of the inositol head group by
phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks). The family of PIP5K
is comprised of 3 isoforms a, b and g regulated by membrane receptors,
phosphorylation and small GTPases of the Rho and ARF family. Activation
of this pathway is known to promote growth and invasion of cancer cells,
rendering PIP5K an attractive therapeutic target for antitumour therapies
Material and Methods: Two separate focussed screening campaigns
identified a number of hit series. Structure Activity Relationships (SAR)
was built around three chemotypes, (A, B & C) with biochemical assays
routinely run against all three isoforms of PIP5K, two isoforms of PI4K and
PI3Ka. Compounds were also routinely assayed cellular assays, measuring
surrogate markers of cellular PIP2 levels.
Results: Potent, selective inhibitors (pIC50 >8), of PIP5K kinases have
been developed in three chemical series. The SAR of series B will be
described in detail. Live cell imaging results will be presented.
Conclusions: Cell data demonstrate that PIP5K inhibition slows the rate
of PIP2 synthesis.
PIP5Ka (pIC50 )
PIP5Kb (pIC50 )
PIP5Kg (pIC50 )
PI3Ka (pIC50 )
PI4Ka (pIC50 )
PI4Kb (pIC50 )
PIP2 inhib @ 3mM
Series A
Series B
Series C
5.8
7.2
7.2
5.3
5.1
5.1
3.7%
7.8
8.6
8.9
4.5
4.8
5.4
21%
9.3
8.8
8.9
<4.0
4.0
4.0
17%
276
POSTER (Board P056)
An X-ray crystal structure-based understanding of the inhibition of
the MDM2−p53 protein–protein interaction by isoindolinones
B. Anil1 , E. Blackburn1 , T. Blackburn2 , S. Cully2 , J. Liu3 , C.J. Drummond2 ,
J.A. Endicott1 , B.T. Golding4 , R.J. Griffin2 , K. Haggerty2 , J. Lunec2 ,
D.R. Newell2 , C.H. Revill2 , C. Riedinger1 , A.F. Watson2 , Q. Xu2 , Y. Zhao2 ,
I.R. Hardcastle2 , M.E.M. Noble1 . 1 University of Oxford, Department of
Biochemistry, Oxford, United Kingdom; 2 Newcastle University, Northern
Institute for Cancer Research, Newcastle upon Tyne, United Kingdom;
3
Newcastle University, Northern Istitute for Cancer Research, Newcastle
upon Tyne, United Kingdom; 4 Newcastle University, School of Chemistry,
Newcastle upon Tyne, United Kingdom
The p53 tumor suppressor plays a pivotal role in responding to cellular
stress. Activation of p53 protein results in the transcription of a number of
92
genes that govern progression through the cell cycle, the initiation of DNA
repair, and apoptosis. The activity of p53 is tightly regulated by the MDM2
protein, in an auto-regulatory feedback loop. Inhibition of the MDM2−p53
protein–protein complex by small molecule inhibitors has been shown to
reactivate normal p53 pathways in cells overexpressing MDM2, and so
exert an anti-cancer effect. A number of series of potent small-molecule
inhibitors have been developed as far as clinical trials. Understanding of
the structural basis for inhibition from ligand-bond protein X-ray structures
has been important in the optimisation of most of these series.
We have developed a class of inhibitors of the MDM2−p53 interaction, based on an isoindolinone scaffold [J. Med. Chem. 2006,
49, 6209–6221, Bioorg. Med. Chem. Letters 2011, 21, 5916–
5919]. NU8406A, (R)-4-chloro-3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (IC50 = 43.8±6.2
nM) shows comparable in vitro activity to Nutlin-3a. Recently, we reported
the MDM2 co-crystal structure of Nutlin-3a using a surface-entropy
reduction mutant [Acta Cryst 2013, D69, 1358–1366]. We have applied
these MDM2 mutants to solve the co-crystal structure of NU8406A. The
structure shows that the binding mode of the isoindolinone is comparable
to that for Nutlin-3a, and confirms the (R)-stereochemistry of the ligand.
Preliminary structure–activity studies have revealed significant differences
between the two series in the way that the Phe19 pocket is occupied.
277
POSTER (Board P057)
The discovery and pre-clinical development of the first clinical stage
EZH2-inhibitor, EPZ-6438 (E7438)
K. Kuntz1 , H. Keilhack2 , R. Pollock2 , S. Knutson2 , N. Warholic2 , V. Richon2 ,
R. Chesworth1 , R. Copeland1 , M. Porter-Scott1 , C. Sneeringer1 ,
T. Wigle1 . 1 Epizyme Inc, Molecular Discovery, Cambridge Massachusetts,
USA; 2 Epizyme Inc, Biology, Cambridge Massachusetts, USA
Mutations within the catalytic domain of the histone methyltransferase
EZH2 have been identified in subsets of patients with non-Hodgkin
lymphoma (NHL). Cancers harboring these genetic alterations are
dependent on EZH2 enzymatic activity for cellular survival. Here, we
disclose the discovery of EPZ-6438 (E7438), as a potent, selective and
oral bioavailable small molecule inhibitor of EZH2 in preclinical models of
NHL. Previously we have disclosed the properties of EPZ005687, a tool
compound useful for exploring the in vitro biology of EZH2 inhibition. Multiparametric optimization of the potency, pharmokinetics, oral biovailability
and safety properties of this series led to the discovery of the clinical
compound, EPZ-6438. Modulation of the log P was required to reach the
optimal balance between clearance and bioavailability while maintaining
the requisite potency. EPZ-6438 selectively inhibits intracellular lysine 27
of histone H3 (H3K27) methylation in a concentration- and time-dependent
manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition
of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of
human lymphoma cell lines bearing EZH2 catalytic domain point mutations.
Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438
causes dose-dependent tumor growth inhibition, including complete and
sustained tumor regressions with correlative diminution of H3K27Me3
levels in tumors and selected normal tissues. EPZ-6438 recently entered
clinical testing as E7438 in a dose escalation phase 1 trial in relapsed or
refractory malignancies.
278
POSTER (Board P058)
Sentinel lymph nodes mapping of macrophage targeted mannosyl
human serum albumin-indocyanine detected by combined color and
near infrared fluorescence imaging system in esophagus
Y. Quan1 , Y. Oh2 , J.I.H.O. Park3 , J. Park4 , J. Jeong4 , B. Kim5 ,
H. Kim1 . 1 Korea University Guro Hospital, Department of Thoracic
Cardiovascular Surgery, Seoul, Korea; 2 Korea University, Department of
Bio-Convergence Engineering, Seoul, Korea; 3 Korea Advanced Institute
of Science and Technology, Department of Bio and Brain Engineering,
Seoul, Korea; 4 Seoul National University, Department of Transdisciplinary
Studies Program in Biomedical Radiation Science, Seoul, Korea; 5 Korea
University, Department of Biomedical Engineering, Seoul, Korea
Objective: Esophageal cancer is one of the most poor prognosis diseases.
Therefore, 3-field lymph node dissection is performed for surgically curable
esophageal cancer. However these extended lymphadenectomy is leads
to high morbidity. Thus, sentinel lymph node (SLN) concept is essential
point for esophageal cancer surgery to avoid unnecessary lymph node
dissection. There are some pre-clinical trials use fluorescence indocyanine
green (ICG) or radioisotope to selective removing SLN. In this study, we
used mannosyl human serum albumin (MSA)-ICG as a new SLN targeting
tracer to investigate esophageal SLN mapping using intraoperative color
and fluorescence merged imaging system (ICFIS).
Methods: We activated macrophage U937 cells and treated ICG or MSAICG respectively. The distributions of ICG and MSA-ICG in cells were
investigated by Infrared Imaging System. In-vivo study, the ICG or MSAICG was injected into footpad of nude mice and then the lymphatic flow
was investigated by Infrared Imaging System. Additionally, 5 Yorkshire pigs
underwent thoracotomy and received submucosal injection of MSA-ICG
with endoscopic needle through an esophagoscope. The distribution of
MSA-ICG in SLN of pig esophagus was investigated by ICFIS.
Results: Our study found that the MSA-ICG were specific bound to
macrophage than free ICG, and also the detected lymph node imaging
more distinguishable than free ICG in mice. The pig esophageal SLN which
received MSA-ICG submucosal injection were detectable from 15 min. In 3
of 5, two SLN were identified, however in 2 of 5, one SLN was detected.
Discussion: In fluorescence guided esophageal surgery, the MSA-ICG
provided precise real-time imaging of SLN. Therefore the MSA-ICG may
be a reliable tracer for SLN identification in esophagus cancer.
279
POSTER (Board P059)
Discovery of multiple kinases inhibitors, DBPR114, as the novel
anti-cancer agent
H.P. Hsieh1 , C.C. Kuo2 , J.J. Chiu2 , T.A. Hsu1 , T.K. Yeh1 , C.T. Chen1 .
1
National Health Research Institutes, Institute of Biotechnology and
Pharmaceutical Research, Zhunan Miaoli County, Taiwan; 2 National
Health Research Institutes, Institute of Cellular and Systems Medicine,
Zhunan Miaoli County, Taiwan
Purpose: The goal of targeted therapy is to identify the suitable target
molecules to be inhibited, in order to achieve the best antitumor effect. The
classical viewpoint is that inhibitor acting on single target allows selectivity
and less adverse effect. However, clinical experience revealed that almost
relapsed cancer patients developed drug resistance, often due to the
activation or development of alternative signaling pathways or mutations
that single target drugs are unable to effectively inhibit them. A current trend
in the development of kinase inhibitors is the assumption that multi targeted
therapy, which targets at several signaling pathways simultaneously, is more
effective than single targeted therapy. Thus, paradigm in designing new
anticancer drug is shifted: The drugs that act on multiple targets might
have a better chance of inhibiting cancer cell proliferation than drugs that
act on a single target. The objective of this research is aimed at identifying
a multi-targeted kinase inhibitor as a development candidate for cancer
therapy.
Material and Method: We established Structure–Activity Relationship
(S.A.R.) study based on 800 compounds that were synthesized by High
Throughput Parallel Synthesis. Scaffold hopping approach by changing
pharmacophore moiety had led us to discover DBPR114 as a multipletargeted kinase inhibitor.
Results: We discovered a novel multiple-targeted kinase inhibitor,
DBPR114, that effectively inhibited 15 kinases in a panel of 57 oncogenic
related kinases profiling, particularly against to Aurora kinase A and
B, FLT-1, FLT-3 and c-Met in nanomolar range. In pharmacological
study, DBPR114 significantly shrank tumor in 8 different xenograft
models including Mia-Paca2, AsPC-1 (pancreatic carcinoma), Hep3B
(hepatocellular carcinoma), MKN-45 (gastric carcinoma), MOLM-13 and
MV4;11 (acute myeloid leukemia), NTUB-1 (bladder cancer), and Colo205 (colorectal carcinoma) at a dose of 3 to 20 mg/kg by intravenous
administration without significant adverse effect in most group of in vivo
evaluations.
Conclusion: This multiple targeting inhibitory properties plays a major role
to shrinks tumor growth of various cancer cells in vivo and in vivo such as
MOLM-13, MV4;11, MKN45, Colo-205, Mia-Paca2 and NTUB-1. The broad
spectrum of anti-tumor activity of BPR1K0871 provides a great potential to
become a promising multi-targeted kinase inhibitors as next generation of
anti-cancer drug.
280
POSTER (Board P060)
A hybrid drug design approach to overcome imatinib resistance for
treating leukemia
Y.M. Wei1 , K.K.W. To2 , S.C.F. Au-Yeung1 . 1 The Chinese University of Hong
Kong, Department of Chemistry, New Territories, Hong Kong; 2 The Chinese
University of Hong Kong, School of Pharmacy, New Territories, Hong Kong
Background: Imatinib, a multi-targeted tyrosine kinase inhibitor, exhibits
potent anticancer activity against leukemia harboring the bcr-abl oncogene
and some solid tumors overexpressing c-kit and PDGFR. However, its
clinical efficacy is severely hindered by the emergence of resistance
primarily due to acquired mutations in the bcr-abl kinase domain.
Material and Methods: By applying our established ‘Platinum (Pt) drug −
Bioactive ligand’ drug design platform, novel Pt-imatinib hybrid compounds
were synthesized and fully characterized by 1 H-NMR and ESI+ MS. In silico
docking analysis was performed to investigate the interaction of the
new compounds with wild-type bcr-abl and its various resistance-causing
mutants. Anticancer activity was evaluated by the colorimetric MTT assay.
Kinase inhibition profiling was performed to assess the specificity of the new
compounds on various receptor tyrosine kinases. Cell cycle regulation and
apoptosis were studied by propidium iodide and Annexin V-7AAD staining,
respectively. Reaction between the new compounds and DNA was studied
by NMR using 5 -GMP as a model nucleotide.
Results: The new Pt-imatinib hybrid compounds were found to be highly
effective against the leukemia cell line K562 harboring bcr-abl, but not
in another non-bcr-abl expressing leukemia RPMI8226 and some other
solid tumors. Importantly, the new compounds maintained their anticancer
activity in an imatinib-selected resistant K562 subline. Moreover, the new
compounds did not appreciably affect cell growth of normal HEK293 and
LLC-PK1 cell lines. Kinase profiling revealed that the new compounds
maintain selectivity towards bcr-abl, c-kit and PDGFR, similar to imatinib.
By docking simulation, the new compounds were predicted to interact with
both wild-type bcr-abl and some of its resistance-mediating mutants. This
was verified by biochemical kinase inhibition assay on selected bcr-abl
mutants. In K562 cells, the new compounds produced G1 phase arrest,
in contrast to G2/M arrest mediated by classical Pt drugs. Unlike classical
Pt compounds, the Pt-imatinib hybrids only form monofunctional Pt-DNA
adducts, presumably contributing to the minimal toxicity in normal cells.
Conclusions: New lead Pt-imatinib hybrid compounds were found to
be specific towards bcr-abl-expressing leukemia cells and capable of
circumventing resistance mediated by clinically identified mutations. Their
good safety profile and unique mechanisms advocate further clinical
development.
Figure: General formula of the designed compounds.
281
POSTER (Board P061)
Novel hybrid drug design strategy to circumvent erlotinib resistance
and to optimize its pharmacokinetic properties for treating lung
cancer
D.C.S. Poon1 , S.C.F. Au-Yeung2 , K.K.W. To1 . 1 The Chinese University
of Hong Kong, School of Pharmacy, New Territories, Hong Kong;
2
The Chinese University of Hong Kong, Department of Chemistry, New
Territories, Hong Kong
Background: Erlotinib is an EGFR tyrosine kinase inhibitor (TKI) highly
potent towards lung cancer carrying the sensitizing EGFR mutations.
However, its usefulness is severely compromized by resistance mediated
primarily by the secondary EGFR T790M mutation. Most EGFR TKIs are
substrates of the efflux transporters Pgp and ABCG2. Induction of these
93
transporters can remarkably decrease cellular accumulation of erlotinib,
thereby conferring drug resistance. Moreover, EGFR TKIs are not effective
for brain metastasis from primary lung cancer because of poor drug
penetration restricted by Pgp and ABCG2 at the blood–brain barrier.
Material and Methods: Novel platinum (Pt)–erlotinib hybrid compounds
were synthesized by our established ‘Pt-bioactive ligand’ drug design
approach. They were fully characterized by 1 H-NMR and ESI+ MS.
Anticancer activity in a panel of non-small cell lung cancer cell lines was
evaluated by sulforhodamine B assay. Docking simulation was performed
to investigate the interaction of the new compounds with EGFR harboring
different mutations. Inhibition of different EGFR mutant kinases was
evaluated by biochemical kinase assay. Drug uptake and transport across
cell monolayer specifically overexpressing P-gp/ABCG2 were studied to
examine whether the new compounds are substrate of the transporters.
Results: Similar to erlotinib, new Pt-erlotinib hybrid compounds were
found to be highly effective against HCC827 harboring the sensitizing
EGFR L858R mutation (IC50 : 0.006–0.16 mM) but become inactive in H520
with minimal EGFR expression (IC50 ~15 mM). Importantly, they were
much less affected than erlotinib by acquired resistance mediated by the
secondary EGFR T790M mutation in H1975 cells (fold resistance: ~200
versus ~12,000 for erlotinib). By docking simulation, the new compounds
were predicted to maintain their binding with the EGFR T790M mutant.
Results from biochemical kinase assay reveal that the new compounds
inhibited EGFR in an ATP-competitive manner and that the inhibition was
only minimally reduced by the EGFR T790M mutation. Unlike erlotinib,
cellular uptake and transport of the hybrid compounds were not affected
by Pgp and ABCG2 overexpression.
Conclusions: Novel Pt-erlotinib hybrids were found to circumvent
resistance mediated by the acquired EGFR T790M mutation. By escaping
P-gp/ABCG2-mediated efflux, the new compounds may be effective for
treating metastatic brain cancer. Further development of the hybrid
compounds is warranted.
282
POSTER (Board P062)
Membrane anchorage of Stat3 via artificial protein lipidation
M. Avadisian1 , S. Fletcher1 , B. Liu1 , W. Zhao2 , J. Turkson2 , C. Gradinaru1 ,
P. Gunning1 . 1 University of Toronto, Chemistry and Physical Sciences,
Mississauga, Canada; 2 University of Central Florida, College of Medicine,
Orlando, USA
Lipidation localizes cytosolic proteins within cellular membranes through
a covalent attachment of sterol or lipid groups. This post-translational
modification restricts the motility of otherwise soluble proteins to the
cell membrane. Using artificial lipidation, we propose to mimic Nature
by artificially inducing protein–membrane anchorage through the use
of a rationally designed Protein–Membrane Anchor (PMA) to inhibit a
protein’s motility and function within the cell. We hypothesized that induced
membrane anchorage of proteins can hold significant therapeutic value
when applied to cancer-promoting cell-signaling proteins.
To demonstrate the protein–membrane anchorage strategy, we choose to
target Stat3 because of its constitutive activity in a number of cancers.
Thus, our goal was to develop an inhibitor that could sequester Stat3, a
93 kDa protein, at the plasma membrane and suppress its motility through
PMA-induced protein–membrane association. Our proto-type PMA 1 was
composed of two binding modules: a recognition motif to bind the protein
and an anchor to sequester the protein complex to the membrane.
PMA 1 was composed of a potent Stat3 inhibitor covalently attached to
a cholesterol membrane anchor.
PMA 1 was able to anchor Stat3 to the cell membrane in MDA-MB-231
breast cancer cells that are known to have constitutively-active Stat3. Most
excitingly, in the presence of 25mM concentration PMA 1, we observed
complete sequestration of Stat3 to the cell membrane through PMA−Stat3
association. To improve metabolic stability, we synthesized a new library
of PMAs with varying Stat3 binding and membrane anchoring moieties.
Currently, we are conducting biophysical and immunofluorescent studies
on the new PMAs.
94
283
POSTER (Board P063)
The development of the first selective inhibitors of the UBA5 enzyme
to probe for E1 activity in diseased cells
S.R. da Silva1 , S.L. Paiva1 , M. Bancerz2 , M. Geletu2 , A.M. Lewis2 ,
J. Chen3 , Y. Cai3 , H. Li3 , P.T. Gunning1 . 1 University of Toronto, Chemistry,
Toronto, Canada; 2 University of Toronto Mississauga, Chemical and
Physical Sciences, Mississauga, Canada; 3 Georgia Regents University,
Biochemistry and Molecular Biology, Augusta, USA
Cancer cells produce and degrade proteins more rapidly than most normal
cells, and are in turn more sensitive to changes in protein regulation.
Similarly to secretory cells, cancer cells have the propensity to undergo
endoplasmic reticulum (ER) stress due to higher protein turnover that, if
left uncorrected, can result in apoptosis. To avoid these fates, cells have
developed support systems such as the conjugation of the ubiquitin-fold
modifier 1 (UFM1) ubiquitin-like protein to other protein targets, which
has been implicated in counteracting apoptotic ER stress in pancreatic
secretory cells.
Our research has recently focused on the development of the first inhibitor
of the UFM1 pathway by targeting its E1 enzyme, UBA5, in order to
counteract the anti-apoptotic effects of this system and to further study the
role of UFM1 conjugation in cancer cells. We have successfully identified
a lead inhibitor, 5C-Z, which incorporates an adenosine moiety coupled
to a zinc(II) polyazamacrocylic coordination complex. Through enzymatic
assays that evaluate the transthiolation ability of UBA5 in the first step
of UFM1 activation, our inhibitor exhibits low micromolar activity against
UBA5 while demonstrating potent selectivity over other E1 enzymes (>20fold). Kinetic assays reveal that 5C-Z acts non-competitively on UBA5,
indicating that the compound could possibly be binding to and inhibiting
the “inactive” subunit of the UBA5 homodimer. Furthermore, treatment of
lung and leukemia cells that exhibit high levels of UBA5 protein expression
leads to a decrease in cell proliferation yet does not induce cell death in
diseased or healthy cells, up to 200 mM.
This novel strategy of inhibiting UBA5 and UFMylation would make cancer
cells that are highly dependent on this system more susceptible to
treatment regimens of principle drugs, which could lead to the use of milder
drug dosing strategies. Our novel inhibitors produced from this research
can be used as a probe to further investigate the role of E1 activating
enzymes in cancer progression.
Figure: General formula of the compounds.
284
POSTER (Board P064)
Poly(ADP-ribose) glycohydrolase (PARG) inhibitors increase nuclear
poly(ADP-ribose) after methylating DNA damage
A. Jordan1 , B. Acton1 , E. Fairweather1 , N. Hamilton1 , S. Holt1 , J. Hitchin1 ,
C. Hutton1 , D. James1 , S. Jones1 , A. McGonagle1 , H. Small1 , K. Smith1 ,
A. Stowell1 , I. Waddell1 , B. Waszkowycz1 , D. Ogilvie1 . 1 Cancer Research
UK Manchester Institute, Drug Discovery, Manchester, United Kingdom
Background: DNA single strand breaks (SSBs) are the most common type
of damage occurring in cells. Poly(ADP ribose) polymerase (PARP) binds
to SSBs and auto-ribosylates itself using NAD+ as a substrate. This creates
chains of poly ADP ribose (PAR) which provide a signal for other proteins
to repair the lesion. Sequential removal of the PAR chains is accomplished
by Poly(ADP-ribose) glycohydrolase (PARG). Failure to complete the DNA
repair process, either by inhibition of PARP (e.g. with olaparib) or inhibition
of PARG (with shRNA), can lead to cell death.
PARG is the only enzyme known to efficiently catalyse the hydrolysis of
O-glycosidic linkages of ADP-ribose polymers and exists (unlike PARP) as
a single gene. We therefore sought to exploit this vulnerability and as part
of a fostering agreement with AstraZeneca have developed novel small
molecule inhibitors of PARG activity.
Methods: A screening cascade for small molecule inhibition of PARG
was developed. Biochemical inhibition of PARG was measured using a
bespoke HTRF assay. In addition, selected PARG inhibitors were tested
against other enzyme substrates (ARH3, PARP1) to assay for selectivity.
Active compounds were then taken forward and tested in cells for PAR
chain persistence and for cytotoxicity using a 3-day HeLa assay. Suitable
compounds were then evaluated for their physico-chemical properties and
their in vivo PK profiles determined.
Results: Early PARG inhibitors increased nuclear PAR chain levels in cells
after 1 h treatment with the methylating agent methyl methanesulfonate
(MMS) but also displayed off-target cytotoxicity in the absence of MMS.
Applying computational chemistry enabled us to discover novel series of
compounds with sub-micromolar potency with a wide differential to acute
cytotoxicity. Compounds were also highly selective against PARP1 and
ARH3 in vitro.
PAR chain persistence in cells was also maintained after using the more
clinically-relevant methylating agent temozolomide (TMZ). PARG inhibitors
displayed satisfactory in vitro and in vivo PK profiles.
Conclusions: We have developed PARG inhibitors that block the
breakdown of PAR chains in cells after exogenous DNA damage by
methylating agents. These tool compounds are potent and selective and
enable us to both explore in more detail the cellular mode of action and
investigate pre-clinical in vivo models for PARG inhibition.
285
POSTER (Board P065)
A nanomolar-potency small molecule inhibitor of the STAT5 protein
A.A. Cumaraswamy1 , A. Lewis2 , M. Geletu2 , A. Todic2 , D.B. Diaz2 ,
X.R. Cheng3 , C.E. Brown2 , R. Laister4 , D. Muench5 , K. Kerman3 ,
H.L. Grimes5 , M.D. Minden4 , P.T. Gunning1 . 1 University of Toronto,
Department of Chemistry, Mississauga ON, Canada; 2 University of
Toronto, Chemistry, Mississauga ON, Canada; 3 University of Toronto,
Department of Physical & Environmental Sciences, Scarborough ON,
Canada; 4 Princess Margaret Cancer Institute, Ontario Cancer Institute,
Toronto ON, Canada; 5 Cincinnati Children’s Hospital Medical Centre,
Division of Experimental Hematology, Cincinnati OH, USA
Signal Transducer and Activator of Transcription 5 (STAT5) protein has
gained notoriety for its aberrant role in many human cancers. In contrast
to normal cells where STAT5 activity is rapid and transient, in cancer
cells, including leukemias, STAT5 activity is routinely hyper-activated,
conferring resistance to cell death and driving excessive expression
of proto-oncogenes. Despite significant evidence showing STAT5’s role
in human cancers, there has been little progress in developing direct
inhibitors of STAT5 function. Potent and selective small molecule inhibitors
of STAT5 will be effective therapeutics for treatment of hematological
cancers and overcoming the side effects of current treatments. STAT5
is activated extracellularly by ligand-receptor binding, which results in
STAT5 recruitment to intracellular receptor sites via their Src Homology 2
(SH2) domain. STAT5 is then phosphorylated, facilitating STAT5-STAT5
dimerization via reciprocal phosphotyrosine-SH2 interactions. The dimer
shuttles to the nucleus and induces target gene transcription. In cancer
cells, STAT5 is persistently activated, leading to the aberrant expression
of STAT5 target genes that promote cancer cell survival and prevent cell
death. To achieve this goal, we have employed a structure-based drug
design strategy using computational analysis, medicinal chemistry and
synthetic methods amenable to generating a diverse library. Herein, we
report the first nanomolar, STAT5-selective inhibitor, AC-3-019, possessing
a phosphotyrosyl-mimicking salicylic acid group, which potently and
selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain
complexation events in vitro, silences activated STAT5 in leukemic cells,
as well as STAT5’s downstream transcriptional targets, including MYC and
MCL1, and as a result, leads to apoptosis. We believe AC-3-019 represents
a useful probe for interrogating STAT5 function in cells as well as being a
potential candidate for advanced preclinical trials.
286
POSTER (Board P066)
Potent and selective non-sulfamate-containing small molecule
inhibitors of the ubiquitin activating enzyme
S. Paiva1 , S.R. da Silva1 , M. Bancerz1 , H. Quereshi1 , G.W. Xu2 ,
A.D. Schimmer2 , P.T. Gunning1 . 1 University of Toronto, Chemical and
Physical Sciences, Mississauga, Canada; 2 University Health Network,
Princess Margaret Cancer Centre, Toronto, Canada
Within the cell, many essential regulatory functions including protein
recycling, translocation and cell signaling are mediated by the conjugation
of ubiquitin (Ub) and ubiquitin-like (Ubl) proteins to target substrates. Ub/Ubl
protein conjugation occurs through a cascade of events involving three
enzymes: the ubiquitin activating enzyme (E1), the ubiquitin conjugating
enzyme (E2) and the ubiquitin ligase (E3). In cancer cells, many of these
systems are hyperactive making them attractive targets for the design of
novel cancer therapeutics. More specifically, in recent years there has
been a plethora of interest in the development of strategies that focus on
designing inhibitors specific for different E1 isoforms. To this end, there has
been some success, namely the mechanistic inhibitor MLN4924 and PYZD4409 that target the NEDD8 and Ub activating enzymes, respectively (NAE
and UAE). MLN4924 is a sub-micromolar NAE inhibitor currently in Phase I
clinical trials for the treatment of leukemia, lymphoma, multiple myeloma
and solid tumours, while PYZD-4409 is a micromolar UAE inhibitor that
promotes disease regression in leukemia and myeloma models. Given
the great success of MLN4924 and the promising results of PYZD-4409,
targeting E1s is an excellent strategy for controlling cancer cell growth and
disease progression. However, the design of an inhibitor with potent and
selective UAE activity still remains an elusive goal in this field of research.
Our research has primarily focused on the rational design of selective and
potent non-mechanistic UAE inhibitors by targeting a novel druggable site
on the UAE protein using a two-prong approach. We have successfully
developed inhibitors that display low nanomolar inhibition of UAE in purified
enzymatic based assays (IC50 ~120–500 nM), as well as exhibiting >10fold selectivity for UAE over other E1 enzymes. To date, these are the most
potent small molecule non-sulfamate-based inhibitors of UAE reported.
287
POSTER (Board P067)
The discovery and optimization of small molecule antagonists of the
WDR5−MLL interaction
R. Al-Awar1 , R.S. Al-Awar1 , M. Getlik1 , D. Smil2 , Y. Bolshan2 ,
G. Poda1 , G. Senisterra2 , H. Wu2 , A. Allali-Hassani2 , G.A. Wasney2 ,
D. Barsyte-Lovejoy2 , L. Dombrovski2 , A. Dong2 , H. He2 , A. Seitova2 ,
I. Chau2 , F. Li2 , J.F. Couture2 , E. Kuznetsova2 , R. Marcellus1 . 1 Ontario
Institute for Cancer Research, Drug Discovery, Toronto, Canada;
2
Structural Genomics Consortium, University of Toronto, Toronto, Canada
ABSTRACT. Among other cellular processes, gene expression is regulated
by epigenetic histone modifications. Histone methyltransferases catalyze
the transfer of the methyl group from S-adenosylmethionine to specific
lysine residues on histones. Mixed lineage leukemia 1 (MLL1) is a
methyltransferase that methylates lysine 4 on histone H3 (H3K4me3) and
is an important regulator of the haemopoietic system. Deregulation of
MLL1 is often associated with acute myeloid and lymphoid leukemias
and was proposed as a novel therapeutic target. WD40 repeat protein 5
(WDR5) is a component of the multiprotein MLL1 complex that is essential
for its methyltransferase activity. Thus, therapeutic intervention of the
WDR5/MLL1 interaction may lead to possible novel therapeutic agents for
MLL-dependent leukemias. Using a structure-based design approach our
structure activity relationship studies identified selective and cell-permeable
compounds that bind to WDR5 with low nanomolar affinities. In future
chemical biology studies these molecules will serve as valuable molecular
probes to dissect the biological role of WDR5.
288
POSTER (Board P068)
Synthetic isomalyngamide A analogs that inhibit breast cancer
migration
W. Li1 , T. Chang1 , C. Hung1 , C. Chen1 , S. Jao2 . 1 Academia Sinica,
Institute of Chemistry, Taipei City, Taiwan; 2 Academia Sinica, Institute
of Biological Chemistry, Taipei City, Taiwan
Background: Because of frequent relapses in patients due to drug
resistance and characteristically aggressive in very young women, breast
95
cancers trigger enormous burden in health system, especially in the family
of patient. Considerable progress has been made in the development
of synthetic glycosylated isomalyngamide A analogs-related antitumor
agents for medicinal interest. Based on the previous observations, we
hypothesized that the isomalyngamide A analog might be a perfect
substance with which to examine the role played by glycosylation and
various linkers on antimigratory properties.
Material and Methods: A series of synthetic glycosylated isomalyngamide A analogs with different linkers were prepared using the method
of solution-phase synthesis and evaluated for their biological activities in
human breast cancer cells. The effects of synthetic isomalyngamide A
analogs on cell viabilities were determined by using the MTT assay.
The inhibition of in vitro migration and invasion activities of synthetic
isomalyngamide A analogs were examined by using a transwell assay.
The potency in inhibition of cancer cell adhesion were studied by using
the adhesion assay. Furthermore, the complete molecular mechanism for
synthetic isomalyngamide A analog-mediated antimigratory property was
explored using western blot analysis.
Results: The findings show that the flexible rather than the rigid linkers
to isomalyngamide A backbone more critically affect cell migration and
invasive ability. Two synthetic isomalyngamide A analogs were observed
to suppress migration, invasion and adhesion events in human breast
adenocarcinoma MDA-MB-231 cells. Evidence has been achieved for a
mechanism for inhibition of metastatic activities in MDA-MB-231 cells
by synthetic isomalyngamide A analogs through the integrin-mediated
antimetastatic pathway, suppression of the expression of p-FAK and
paxillin.
Conclusions: The observations made in this study demonstrate how the
impact of the flexible and the rigid linker upon backbone of molecule affects
the ability of synthetic isomalyngamide A analogs to modulate motility,
migration, invasive capacity and adhesive property. In addition, the results
of a complete western blot analysis of the in vitro antimigratory activities
of synthetic isomalyngamide A analogs, show that these substances are
potential antimetastatic agents for the treatment of breast cancer.
289
POSTER (Board P069)
Progress in drugging CYP1A1, 1B1 and CYP2W1 overexpressed in
cancer
K. Pors1 , V. Le Morvan2 , S. Travica3 , S.D. Shnyder1 , M. Sutherland1 ,
H.M. Sheldrake1 , M. Searcey4 , I. Johansson3 , S. Mkrtchian3 ,
P.M. Loadman1 , J. Robert5 , M. Ingelman-Sundberg3 , L.H. Patterson1 .
1
University of Bradford, Institute of Cancer Therapeutics, Bradford, United
Kingdom; 2 University of Bordeaux, Institut Bergonié, Bradford, France;
3
Karolinska Institute, Stockholm, Sweden; 4 University of East Anglia,
School of Chemical Sciences and Pharmacy, Norwich, United Kingdom;
5
University of Bordeaux, Institut Bergonié, Bordeaux, France
Background: The cytochrome P450 (CYP) enzymes are responsible for
the oxidation of a diverse range of xenobiotic and endogenous compounds.
Although CYPs operate mainly to detoxify xenobiotics and endogenous
molecules, members of the CYP1 family are also known to catalyse one of
the first steps in the metabolism of carcinogens originating from exposure to
xenobiotics with the risk of developing cancer. Another member of the CYP
family, CYP2W1, has been found to be expressed in adrenal gland, gastric,
lung, rhabdomyosarcoma and colon cancer while the expression in adult
non-transformed tissues remains absent. The high expression of CYP1A1,
1B1 and 2W1 in tumour tissue and surrounding stroma compared to nearby
normal tissue provides an opportunity for development of selective cancer
therapeutics.
Materials and Methods: Briefly, these include synthetic chemistry
necessary for synthesis of duocarmycin bioprecursors, use of recombinant
CYP bactosomes for metabolite identification using LC/MS, transfected
CYP1A1, 1B1 and 2W1 cancer cell lines and analysis of primary tissues.
Results: At this meeting we will present both published and unpublished
work, which is focussed on re-engineering the duocarmycin family of natural
products for tumour-selective therapy. Novel data include progress on using
ICT2726 as a biomarker to detect CYP2W1 functional activity and a range
of agents that are activated in CYP1B1-transfed cell lines (Cal27 and
Cal33). We are also disclosing information on our ongoing research of
R and S-enantiomers of several duocarmycin bioprecursor compounds in
both in vitro and in vivo models.
Conclusions: Our findings reveal opportunities in targeting CYP1A1, 1B1
and 2W1 with personalized molecular cancer therapeutics based on the
duocarmycin pharmacophore.
96
Poster Session – Molecular Targeted Agents I
290
POSTER (Board P070)
Exosome analysis in cancer patients: From the preclinical towards
the clinical application: Trial design
I. Mertens1 , M. Castiglia2 , A.P. Carreca2 , G. Baggertman1 , M. Peeters3 ,
P. Pauwels2 , C. Rolfo4 . 1 University Antwerp − VITO, Center for
Proteomics − CFP-CeProma, Antwerp, Belgium; 2 Antwerp University
Hospital, Molecular Pathology Unit Department of Pathology, Antwerp,
Belgium; 3 Antwerp University Hospital, Oncology Department, Antwerp,
Belgium; 4 Antwerp University Hospital, Phase I − Early Clinical Trials
Unit Oncology Department, Antwerp, Belgium
Background: Cancer cells produce a heterogeneous mixture of vesicular,
organelle-like structures (extracellular vesicles, EVs) into their surroundings
including blood and other body fluids. Exosomes are small (40 to 100 nm)
membrane derived vesicles that develop from exophytic budding of the
cellular membrane after the fusion of multivesicular bodies or mature
endosomes with the cellular membrane. It has been shown that tumour
cells exposed to hypoxia secrete exosomes with enhanced angiogenic and
metastatic potential. Thus exosomes might be involved in tumor progression and they can potentially be used for prognosis and therapy selection,
as they contain a variety of molecules such as signal proteins and/or peptides, microRNAs, mRNAs and lipids, which could be potential biomarkers.
Materials and Methods: To evaluate the biomarker potential of the
exosome derived RNA and protein content, we first optimized an extraction
protocol for exosomes in plasma based on the Optiprep density gradient
protocol. After purification, the exosomes are characterized and quantified
using Western Blot and Nanosight analysis. Later, the RNA and protein
fraction of the extracted exosomes from 60 NSCLC, 60 pancreatic cancer
and 60 colorectal cancer patients is compared to 60 healthy controls. The
proteome content is evaluated using mass spectrometry based quantitative
shotgun proteomics. The RNA profiles are generated using next generation
sequencing. After profound bioinformatics analysis, the potential of the RNA
and protein profiles will be evaluated for diagnostic, prognostic and therapy
purposes.
Results: We are able to purify and characterize exosome material from
plasma samples derived from patients. The data from nanosight analysis
and Western blot indicate that we are able to work with very pure exosome
samples for RNA and protein axtraction. Currently, RNA and protein profiles
from different cancer types are being compared to healthy controls.
Conclusion: The first step in bringing exosome analysis to the clinic is
optimized: exosome purification and characterization. In a next step, RNA
and protein profiles are being evaluated as potential biomarkers.
291
POSTER (Board P071)
Aflibercept has anti-tumor activity in bevacizumab-escaping tumors
of colorectal cancer: Molecular profiles and mechanisms
1
2
2
2
3
4
C. Dib , R.G. Bagley , P. Mankoo , J. Pollard , J. Watters , M. Chiron .
1
Sanofi R&D, Oncology, Vitry-Sur-Seine, France; 2 Sanofi R&D, Oncology,
Cambridge, Usa; 3 Sanofi R&D, Oncology, Vitry-Sur-Seine, France;
4
Oncotest Gmbh, Oncology, Freiburg, Germany
Background: Aflibercept (Afl, ziv-aflibercept in the US) is a potent multiple
angiogenic factor trap that prevents VEGF-A, VEGF-B, and placental
growth factor (PlGF) from activating the native receptors. Based on an
OS benefit in the phase 3 VELOUR trial, Afl + FOLFIRI was approved
to treat metastatic colorectal cancer (mCRC) following a prior oxaliplatincontaining regimen. In prior preclinical studies, Afl demonstrated superior
anti-tumor activity relative to bevacizumab (Bev) in 39/48 CRC patientderived xenografts (PDX). The PDX were profiled for KRAS status and
genomic analysis to elucidate mechanisms and potential biomarkers
behind the differential activity.
Methods: Primary or metastatic CRC tumors were engrafted and passaged
subcutaneously into NMRI nude mice (Chiron et al., Mol Cancer Ther,
2014). In 3 PDX models showing some response to Bev, mice were
subsequently randomized to either continue treatment with Bev or switched
to Afl treatment. Tumors collected 1 day after last treatment were profiled
by RNA sequencing. An informatics workflow was devised to associate
RNA expression changes to either the human tumors or the mouse
stroma, and differentially expressed genes were identified in each of the
two treatments (fold change magnitude of 2 by Wilcoxon test and BH
corrected P-value <0.05). Profiling for KRAS status and genomic analysis
was done by whole exome sequencing and/or Sanger technique.
Results: In 48 CRC PDX models tested, the differential activity of Afl and
Bev showed no association with KRAS mutation status (Fisher-exact test
p-value >0.05): 24 were KRAS mutant and 24 were wild type. In 3 PDX
models where treatment was either switched to Afl or maintained with Bev,
Afl demonstrated greater anti-tumor activity than Bev (t-test: P < 0.0001, 2
being KRAS mutant and 1 KRAS WT). RNA sequence analysis revealed
differential effects between Afl and Bev in both the murine host (643
genes, 374 up-regulated Afl vs. Bev) and human tumors (82 genes, 19 upregulated Afl vs. Bev). A number of angiogenesis-promoting genes were
down-regulated by Afl vs. Bev including: VEGF receptor genes (FLT1, FLT4,
KDR), Notch pathway genes involved in vessel formation control (Dll4,
Notch 3 & 4), the Ephrin family that regulate cell migration (Epha2, Ephb1),
and the matrix metalloproteinases (MMP10, 13, 15, 17, 28).
Conclusions: The superior anti-tumor activity of Afl vs. Bev in CRC PDX
was independent of KRAS status. Gene expression profiling of stroma and
tumor genes of CRC PDX tumors showed clearly different modulation
patterns of both stroma and tumor between Afl and Bev. The results
suggest that increased control of expression of angiogenesis-promoting
genes by Afl may be associated with its superior anti-tumor activity relative
to Bev in CRC PDX.
292
POSTER (Board P072)
Salmonella typhimurium A1-R decoys quiescent cancer cells to cycle
rendering them chemosensitive
S. Yano1 , Y. Zhang1 , M. Zhao1 , Y. Hiroshima1 , S. Miwa1 ,
F. Uehara1 , H. Kishimoto2 , H. Tazawa3 , T. Fujiwara2 , R.M. Hoffman4 .
1
AntiCancer Inc., San Diego California, USA; 2 Okayama University
Graduate School of Medicine Dentistry and Pharmaceutical Sciences,
Department of Gastroenterological Surgery, Okayama, Japan; 3 Okayama
University Hospital, Center for Innovative Clinical Medicine, Okayama,
Japan; 4 University of California San Diego, Department of Surgery,
San Diego California, USA
Background: A major problem with solid tumors is that most cancer
cells within them are not cycling and therefore resistant to currently-used
chemotherapy.
Material and Methods: Tumor sphere models as well as tumors in vivo
were infected with tumor-targeting Salmonella typhimurium A1-R (A1-R)
and the cell cycle phase and drug sensitivity of the cancer cells within the
spheres or tumors were determined.
Results: Fluorescent ubiquitination-based cell cycle indicator (FUCCI)expressing cancer cells in tumor spheres in serum-free medium were
quiescent. After A1-R infection, the cancer cells in the spheres entered
S-phase and became sensitive to cisplatinum (CDDP) to which they
were resistant before A1-R infection. A1-R infection of FUCCI-expressing
subcutaneous tumors also induced them to cycle. The combination of A1-R
followed by CDDP reduced tumor size compared with A1-R monotherapy
or CDDP alone.
Conclusions: This study demonstrates that A1-R decoys quiescent cancer
cells to cycle and thereby become sensitive to conventional chemotherapy.
293
POSTER (Board P073)
Inhibition of the cell cycle regulated Cdc7 kinase pathway is an
efficacious therapeutic approach for hematologic malignancies and
solid tumors
R. Santos1 , D. Shum2 , D. Carrillo1 , R. Zhang1 , M. Churchill1 ,
S. Mukherjee1 , R. Brentjens3 , C. Radu2 , T.J. Kelly4 , H. Djaballah2 ,
M.G. Frattini1 . 1 Columbia University Medical Center, Medicine, New
York NY, USA; 2 Memorial Sloan Kettering Cancer Center, Molecular
Pharmacology and Chemistry, New York NY, USA; 3 Memorial Sloan
Kettering Cancer Center, Medicine, New York NY, USA; 4 Memorial Sloan
Kettering Cancer Center, Molecular Biology, New York NY, USA
Background: Despite extensive drug discovery efforts, drug-candidate
failure and patients relapsing in the clinic remain as persistent problems
likely secondary to escape mechanisms focused on the control of both
DNA replication and DNA repair pathways. Cdc7 is a cell cycle regulated
serine/threonine kinase whose activity is required for the initiation of
DNA replication and has also been implicated in the control of the DNA
damage response, and therefore linked to the maintenance of genomic
integrity. Both Cdc7 and its known substrate, the mini-chromosome
maintenance (MCM) complex that functions as the replicative DNA
helicase, are overexpressed in the majority of solid tumors, leukemias, and
lymphomas making Cdc7 kinase activity a potential therapeutic target in
both hematologic malignancies and solid tumors.
Materials and Methods: In order to identify a small molecule inhibitor of
the Cdc7 kinase, we performed a high throughput screen for inhibitors
of Cdc7 kinase activity. Chemo-informatic analysis of the hits revealed
enrichment in one chemical cluster made up of several naturally occurring
allosteric inhibitory compounds, that we termed MSK-747 and MSK-777.
Results: MSK-747 and 777 cytotoxic activities were assessed against
panels of cancer cell lines and primary patient samples representing
the majority of solid and liquid tumors and demonstrated efficacy, with
potencies (EC50) in the low nanomolar range. We have shown that both
747 and 777 are effective against cell lines harboring over-expression
of the multidrug resistance (MDR) efflux pump, demonstrating that Cdc7
inhibition can overcome a major mechanism of chemotherapy resistance
in human tumor cells. In addition, cell lines and patient samples harboring
high-risk structural and molecular mutations and samples from relapsed
and/or chemotherapy refractory patients were also found to be susceptible
to Cdc7 kinase inhibition. Cell cycle analysis in both cancer cell lines and
primary patient samples exposed to the compounds revealed an S phase
arrest, cell cycle dependent caspase-3 activation, and apoptotic cell death.
In vivo dose-dependent anti-tumor activity of 747 and 777 was shown
in mouse models of leukemias (ALL and AML), NSCLC, melanoma, and
ovarian carcinoma. In addition, biomarker identification and the mechansim
of cancer cell selectivity over normal cells with these compounds will be
discussed. In all studies, MSK-777 was found to be more efficacious and
therefore was chosen as the lead candidate to continue preclinical and
clinical development.
Conclusions: Cdc7 kinase inhibition is a novel and efficacious mode of
therapy for both hematologic malignancies and solid tumors. MSK-777, a
naturally occurring allosteric inhibitor of this kinase, is scheduled to enter
Phase I clinical trials in early 2015.
294
POSTER (Board P074)
Synthetic lethal screen identifies Aurora A as a selective target in
HPV driven cervical cancer
B. Gabrielli1 , F. Bokhari2 , M. Ranall2 , A. Stevenson2 , M. Murell3 ,
M. Kelly3 , S. McKee2 , G. Leggatt2 , T. Gonda4 , N. McMillan3 . 1 University
of Queensland, Diamantina Institute for Cancer Immunology and
Metabolic Medicine, Brisbane Qld, Australia; 2 University of Queensland,
Diamantina Institute, Brisbane Qld, Australia; 3 Griffith University, Griffith
Health Institute, Gold Coast Qld, Australia; 4 University of Queensland,
School of Pharmacy, Brisbane Qld, Australia
HPV has been identified as the definitive agent in cancers of the cervix,
penis, vulva, vagina, anus, skin, eye, and head and neck, and is responsible
for 5% of the total cancer burden worldwide. HPV oncogenes disable a
number of tumour suppressor pathways, including p53 and Rb, contributing
to the transformed phenotype. We have performed an siRNA screen using
the kinome (779 genes) library to identify genes that when depleted are
synthetically lethal with HPV transformation. The primary and validations
screens have confirmed Aurora A kinase (AURKA) as a potential synthetic
lethal target selective for HPV transformed cells. In vitro research using
the investigational selective small molecule AURKA inhibitor alisertib found
alisertib to be significantly more potent towards the HPV transformed
cells, and selectively promoted apoptosis in the HPV cancers. The drug
was shown to target the HPVE7 oncogene, the level of expression of
this oncogene possibly influencing sensitivity. Apoptosis was sensitive
to Mcl-1 but not Bcl-2 over expression, indicating that the mechanism
is associated with the proteolytic destruction of Mcl-1 in the extended
mitosis in the alisertib treated HPV cancer lines. Xenograft experiments
with cervical cancer cell lines showed alisertib inhibited growth of HPV
and non-HPV xenografts during treatment. The non-HPV cancer growth
was delayed, but in two separate HPV cancers models, regression and
no resumption of growth was detected at even 50 days post-treatment.
A second transgenic model of premalignant disease driven solely by
HPVE7 similarly demonstrated sensitivity to drug treatment. These findings
provide preclinical evidnce that alisertib warrants evaluation as a potential
targeted compound with activity in HPV-transformed cervical cancer and
premalignant disease that may have application to other HPV driven
cancers.
295
POSTER (Board P075)
Combining forces: Study of the cytotoxic effect of the MDM2 inhibitor
Nutlin-3 in combination with CDDP in non-small cell lung cancer cell
lines
C. Deben1 , C. Rolfo2 , V. Deschoolmeester1 , A. Wouters1 , M. Peeters3 ,
I. Gil-Bazo4 , F. Lardon1 , P. Pauwels5 . 1 University Antwerp, Center for
Oncological Research, Antwerp, Belgium; 2 Antwerp University Hospital,
Phase I − Early Clinical Trials Unit Oncology Department, Antwerp,
Belgium; 3 Antwerp University Hospital, Oncology Department, Antwerp,
Belgium; 4 Clı́nica Universidad de Navarra, Oncology Department,
Pamplona, Spain; 5 Antwerp University Hospital, Molecular Pathology
Unit Department of Pathology, Antwerp, Belgium
Background: The p53/MDM2 interaction has been a well-studied target for
new drug design leading to the development of, among others, the small
97
molecule inhibitor Nutlin-3. Clinical phase 1 and 2 studies show promising
results for drugs of the Nutlin-3 family administrated as monotherapy
in several tumor types. Furthermore, combining targeted therapies with
conventional treatments has received much attention in the past few years.
Materials and Methods: Therefore, we combined Nutlin-3 with the
chemotherapeutic drug CDDP, a known activator of the p53 pathway, in a
series of non-small cell lung cancer cell lines with different p53 background.
We focused on the importance of the dosing regime and the role of wild
type p53. Thus we used the commonly used NSCLC cell line A549 as
wild type p53 cell line and used a p53 shRNA lentiviral vector to obtain
a p53 deficient sub cell line. In addition we used CRL-5908, harboring a
p53 mutant genotype (R273H). Cells were treated with both CDDP and
Nutlin-3 or CDDP followed by Nutlin-3 for 24 hours. The SRB-assay was
used to determine the cytotoxic effect of both mono- and combination
therapies after which possible synergism was calculated using the Chou–
Talalay method, both under normoxic and hypoxic conditions. In addition we
determined the p53 protein levels as well as the mRNA and protein levels of
its main transcription targets MDM2, p21, PUMA and BAX. The induction of
apoptosis and cell cycle arrest were determined by flowcytometric analysis.
Results: The strongest synergistic effect was observed in the p53 wild type
cell lines when treatment with CDDP (IC20 : 2mM) was followed by Nutlin-3
treatment under normoxic (CI: 0.486±0.138, 5mM Nutlin-3), and hypoxic
conditions (CI: 0.625±0.082, 5mM Nutlin-3). However, when administrated
simultaneously, this effect was only present at very low concentration
of CDDP and induced an average additive effect under normoxic (CI:
0.990±0.082, 5mM Nutlin-3) and hypoxic conditions (CI: 1.068±0.361,
5mM Nutlin-3). After sequential treatment, the mRNA and protein levels
of p53’s transcription targets MDM2, p21, PUMA, and BAX were markedly
increased compared to CDDP monotherapy, as for the number of apoptotic
cells (27.37±7.97% Ann V positive cells vs. 9.36±2.93%) and cells in G2/M
phase arrest (66.67±0.64% vs. 18.15±7.89%). These findings were not
observed in the p53 mutant and deficient cell lines, confirming the role of
p53 in this synergistic effect.
Conclusions: Our results point towards a promising combination therapy,
being the induction of DNA damage by CDDP, followed by an increase in
p53 levels by Nutlin-3, leading to a synergistic cytotoxic effect in a wild type
p53 NSCLC cell line. Translationally, a lower dose of CDDP could be used
in combination with Nutlin-3, potentially reducing side effects for NSCLC
patients.
296
POSTER (Board P076)
A first-in-Asian phase I dose escalation study to evaluate the safety
and pharmacokinetics of VS-6063 (defactinib), a focal adhesion
kinase inhibitor in subjects with non-hematologic malignancies
T. Shimizu1 , H. Aida2 , J. Horobin3 , M. Keegan3 , M. Padval3 , A. Poli3 ,
C. Hashii2 , K. Nakagawa1 . 1 Kinki University Faculty of Medicine, Phase I
Unit at Department of Medical Oncology, Osaka, Japan; 2 Japan Clinical
Research Operations (JCRO), Tokyo, Japan; 3 Verastem Inc., Cambridge
MA, USA
Background: Defactinib (VS-6063) has been shown to be a potent,
reversible inhibitor of focal adhesion kinase (FAK) and proline-rich tyrosinekinase-2. Blockade of FAK reduces tumor growth and metastasis through
inhibition of tumor cell survival, proliferation and invasion as well as tumor
angiogenesis. Treatment with FAK inhibitors has been demonstrated to
reduce the proportion of cancer stem cells (CSCs) in a dose dependent
manner while chemotherapy standard of care agents (SOC) enrich for
CSCs. The ability of CSCs to survive exposure to chemotherapy but
remain susceptible to novel drugs suggests a unique therapeutic approach
whereby SOC may be combined or sequenced with targeted drugs to
kill surviving CSCs, prevent tumor recurrence and metastasis. This is a
single-center, phase 1, open-label, dose-escalation study to investigate the
safety and pharmacokinetics (PK) of defactinib in first-in-Asian (Japanese)
subjects.
Methods: Subjects with no further standard of care options and a life
expectancy of 3 months with advanced non-hematologic malignancies
were enrolled. Defactinib was administered continuously at a starting
dose of 200 mg BID, escalated to 400 mg BID and then to 600 mg BID.
Patients continued treatment with defactinib until disease progression.
Pharmacokinetics were collected on Day 1 and 15 during cycle 1.
Results: Nine subjects were enrolled (n = 3 each; 200, 400 and 600 mg
cohorts): median age was 60 years (38−75); ECOG PS was 0 or 1.
Defactinib was well tolerated and no dose limiting toxicities were observed
at any of the 200 mg, 400 mg or 600 mg dose levels. Common toxicities
included: Fatigue, headache, increased bilirubin and diarrhea. PK analyses
confirmed the exposure at the recommended phase 2 dose (RP2D) of
400 mg BID was comparable to that previously reported in non-Japanese
subjects. Durable stable disease (SD) 24 weeks was confirmed in two
patients (malignant mesothelioma and colon cancer) with improvement of
clinical symptoms.
98
Conclusions: Overall PK and AE profiles of Japanese subjects are
consistent with those previously reported in non-Japanese subjects. Data
from this study supports the entry of Japanese subjects at the RP2D
to the ongoing multinational trial (COMMAND) of defactinib in malignant
mesothelioma patients.
Clinical Trial Information: NCT01943292
297
POSTER (Board P077)
Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis
in NSCLC
J. Chen1 , K. Amato2 , S. Wang1 , V. Youngblood2 , D. Brantley-Sieders1 ,
R. Cook2 , L. Tan3 , N. Gray3 . 1 Vanderbilt University Medical Center,
Medicine, Nashville TN, USA; 2 Vanderbilt University Medical Center,
Cancer Biology, Nashville TN, USA; 3 Dana Farber Cancer Institute,
Biochemistry & Molecular Pharmacology, Boston MA, USA
Background: Genome-wide analyses determined previously that the
receptor tyrosine kinase (RTK) EPHA2 is commonly overexpressed in nonsmall cell lung cancers (NSCLCs). EPHA2 overexpression is associated
with poor clinical outcomes; therefore, EPHA2 may represent a promising
therapeutic target for patients with NSCLC.
Material and Methods: The EphA2 knockout mouse model was used to
determine lung cancer progression in Kras-mutant knockin mice. RNAimediated depletion or pharmacologic inhibition of EPHA2 was employed
to determine cell viability and signaling in human lung cancer cell lines
in vitro and tumor growth in vivo. Tumor cell proliferation was measured by
BrdU incorporation ELISA or PCNA immunohistochemistry and apoptosis
by TUNEL assay or Cell Death ELISA. We assessed tumor growth in vivo
using H358 human lung cancer cells xenografts in mice.
Results: Targeted disruption of EphA2 in a murine model of aggressive
Kras-mutant NSCLC impairs tumor growth. Knockdown of EPHA2 in human
NSCLC cell lines reduced cell growth and viability, confirming the epithelial
cell autonomous requirements for EPHA2 in NSCLCs. Targeting EPHA2 in
NSCLCs decreased S6K1-mediated phosphorylation of cell death agonist
BAD and induced apoptosis. Furthermore, an ATP-competitive EPHA2 RTK
inhibitor, ALW-II-41−27, reduced the number of viable NSCLC cells in a
time-dependent and dose-dependent manner in vitro and induced tumor
regression in human NSCLC xenografts in vivo.
Conclusion: Collectively, these data demonstrate a role for EPHA2 in the
maintenance and progression of NSCLCs and provide evidence that ALWII-41−27 effectively inhibits EPHA2-mediated tumor growth in preclinical
models of NSCLC.
298
POSTER (Board P078)
Met degradation by SAIT301, a Met monoclonal antibody, reduces the
invasion and migration of nasopharyngeal cancer cells via inhibition
of EGR-1 expression
C. Kim1 , B.S. Lee1 , K.A. Kim2 , Y.J. Song2 , K.H. Cheong2 . 1 Ajou Univ.
Hospital, Otolaryngology, Suwon, South Korea; 2 Samsung Advanced
Institute of Technology (SAIT)/Samsung Electronics Co., Bio Therapeutics
Lab, Suwon, South Korea
Nasopharyngeal carcinoma (NPC) is a common malignant tumor with high
invasive and metastatic potential. The hepatocyte growth factor (HGF)-Met
signaling pathway has a critical role in mediating the invasive growth of
many different types of cancer, including head and neck squamous cell
carcinoma. HGF also stimulates NPC cell growth and invasion in the cell
line model.
In this study, we determined the inhibitory effect of Met, using a Mettargeting monoclonal antibody (SAIT301), on the invasive and growth
potential of NPC cell lines. Met inhibition by SAIT301 resulted in highly
significant inhibition of cell migration and invasion in both the HONE1
and HNE1 cell lines. In addition, we also found that co-treatment of
SAIT301 and HGF decreased the anchorage-independent growth induced
by HGF in HNE1 cell lines. After SAIT301 treatment, Met, together with
its downstream signaling proteins, showed downregulation of p-Met and
p-ERK, but not p-AKT, in both HONE1 and HNE1 cell lines. Interestingly, we
found that HGF treatment of NPC cell lines induced early growth response
protein (EGR-1) expression, which is involved in cell migration and invasion.
In addition, co-treatment with SAIT301 and HGF inhibited the HGF-induced
expression of EGR-1. Next, knockdown of EGR-1 using small-interfering
RNA inhibited HGF-induced cell invasion in NPC cell lines, suggesting that
the expression level of EGR-1 is important in HGF-induced cell invasion
of NPC cells. Therefore, the results support that SAIT301 inhibited Met
activation as well as the downstream EGR-1 expression and could have
therapeutic potential in NPC. Taken together, we suggest that Met is an
anticancer therapeutic target for NPC that warrants further investigation
and clinical trials and SAIT301 may be a promising tool for NPC therapy.
299
Tyk2-src dependence of kidney cancer
POSTER (Board P079)
B. Krishnan1 , S.C. Hanna2 , H.L. Wilson1 , S.T. Bailey1 , J.S. Damrauer1 ,
T. Simamura3 , R.L. Levine4 , K.K. Wong5 , G.L. Johnson2 , W.Y. Kim1 .
1
University of North Carolina Chapel Hill NC USA, Lineberger
Comprehensive Cancer Center, Chapel Hill NC, USA; 2 University of
North Carolina Chapel Hill NC USA, Department of Pharmacology,
Chapel Hill NC, USA; 3 Loyola University Chicago, Oncology Institute
Department of Molecular Pharmacology and Therapeutics, Maywood
IL, USA; 4 Memorial Sloan-Kettering Cancer Center, Human Oncology
and Pathogenesis Program Leukemia Service Department of Medicine,
New York NY, USA; 5 Dana-Farber/Harvard Cancer Center, Department
of Medicine Harvard Medical School Department of Medical Oncology
and Lowe Center for Thoracic Oncology, Boston MA, USA
Renal cell carcinoma (RCC) accounts for 209,000 new cases and 102,000
deaths worldwide each year. Cytotoxic chemotherapy and radiotherapy
are largely ineffective, especially for metastatic disease, where the 5-year
survival is only 10%. This represents an important need to develop better
therapies against advanced RCC. Inhibitors of the vascular endothelial
growth factor receptor (VEGFR) tyrosine kinase and the mammalian target
of rapamycin (mTOR) delay the progression of advanced renal tumors.
Given these therapeutic successes and that targeting kinase signaling
cascades in cancer remains a promising avenue for drug development, we
performed a high-throughput siRNA screen of the kinome to identify novel
kinase targets for therapy. RNAi of the Janus kinase (JAK) family member
tyrosine kinase 2 (TYK2) was highly lethal to RCC cells. Both shRNA
as well as pharmacologic TYK2 inhibition were effective in decreasing
in vitro and in vivo RCC tumor growth. Additionally RCC tumors were
significantly enriched for the GNF2_TYK2 genes compared to adjacent
normal kidney in 2 independent datasets (TCGA & GSE33093), also
TYK2 is activated in a subset of RCC tumors; this increased activation
correlates with poor overall survival of patients (TCGA). Moreover, we noted
that mTORC1 inhibition resulted in IL6 mediated induction of pTYK2 and
that dual inhibition of mTORC1 and TYK2 significantly reduced RCC cell
growth over either alone, suggesting that TYK2 can mediate mTOR inhibitor
resistance. We looked to further elucidate the effects of TYK2 knockdown
on the reprogramming of RCC kinome. To this end, the phosphorylation
status of 43 phospho-kinases was simultaneously assessed by phosphokinase arrays in RCC cells stably expressing shRNA’s to NS or TYK2.
Of the probed phospho kinases, 4 of the 8 SRC family kinases (SFK)
were significantly decreased relative to shNS. Furthermore treatment with
rapamycin induced SFK activation as did knock-down of raptor. Given
the lack of a clinically available selective TYK2 inhibitor, we explored
the effect of combined mTOR and SRC inhibition in vivo; SRC + mTOR
inhibition significantly decreased tumor growth over single agent treatment.
In summary, our studies provide evidence that TYK2 is activated in
RCC, demonstrate that TYK2 signals to regulate pSFK, and show that
the TYK2−SRC axis is upregulated in the face of mTOR inhibition. In
aggregate, we have uncovered a TYK2 and SRC dependence of RCC
with important implications for therapy.
300
POSTER (Board P080)
Preclinical pharmacologic characterization of GSK2849330, a
®
monoclonal AccretaMab antibody with optimized ADCC and CDC
activity directed against HER3
N. Clarke1 , C. Hopson2 , A. Hahn2 , K. Sully3 , F. Germaschewski3 ,
J. Yates1 , C. Akinseye1 , B. Mangatt2 , Z. Jonak4 , C. Matheny4 .
1
GlaxoSmithKline, Biopharm Discovery, Stevenage Herts, United
Kingdom; 2 GlaxoSmithKline, Oncology R&D, Collegeville, USA;
3
GlaxoSmithKline, Biomarker Discovery, Stevenage Herts, United
Kingdom; 4 GlaxoSmithKline, Biopharm R&D, Upper Merion PA, USA
Background: HER3 is expressed in a broad range of solid tumors where its
signaling is important in tumorigenesis and drug resistance. GSK2849330
is an IgG1/Ig3 chimeric, glycoengineered humanized monoclonal antibody
directed against HER3 with enhanced potency to mediate antibodydependent cell cytotoxicity (ADCC) and complement dependent cytoxicity
(CDC), resulting in three potential modes of anti-tumor activity.
Methods: Various in vitro and in vivo studies were conducted in tumor
cell lines and tumor xenograft models to characterize GSK2849330
pharmacology.
Results: GSK2849330 selectively bound HER3 at extracellular Domain
III with ~2 nM binding affinity, blocked ligand (heregulin) binding, receptor
dimerization and activation. In tumor cell lines, GSK2849330 antagonized heregulin-induced phosphorylation of the HER3 receptor and its
downstream effector, AKT, and inhibited heregulin-induced proliferation.
GSK2849330 dose-dependently inhibited tumor growth in a variety of
mouse xenograft models.
Compared with the non-ADCC/CDC enhanced antibody, GSK2849330 had
a ~17-fold greater binding affinity to human FcgRIIIA (initiation of ADCC)
and to human complement protein C1q (initiation of CDC). In ADCC assays
using human leukocytes as effector cells and various tumor target cells,
GSK2849330 was up to two orders of magnitude more potent than the
WT antibody. GSK2849330 caused complement-mediated lysis of HEK293
cells expressing HER3, which was proportional to HER3 protein expression
on the cell surface.
Conclusions: GSK2849330 inhibits HER3 signal transduction in vi

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