pdf LRI Scientific report 2014

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LONDON
RESEARCH
INSTITUTE
Scientific
ReporT
2014
Cover images (from the top, left to right)
Ernest Bashford OBE, ICRF Director 1902-1914.
Signalling microcluster formation in B lymphocytes
responding to membrane-tethered ligands (Image
courtesy of F Batista).
Image of stem cells forming a sprouting blood vessel
(Image courtesy of H Gerhardt).
Sir Michael Stoker, ICRF Director 1968-1979. Dendritic
cells (Image courtesy of C Reis e Sousa).
Sir Walter Bodmer, ICRF Director 1979-1991, ICRF
Director General 1991-1996.
Sir Paul Nurse, ICRF Director General 1996-2002.
Fluorescence image of the paths taken by vacciniainduced actin tails over a 5 minute period (Image
courtesy of M Way).
Tomas Lindahl, Director of the CRUK Clare Hall
Laboratories 1986-2005.
Live zebrafish embryos expressing GFP under the
control of the ptc promoter. Cells in green are those
that are responding to Hh signalling (Image courtesy
of P Ingham).
Richard Treisman, Director of the CRUK London
Research Institute 2002-2015.
Electron Microscopy of B Lymphocytes (rounded)
binding antigen on the surface of an antigenpresenting cell (Image courtesy of F Batista).
John Diffley, Director of the CRUK Clare Hall
Laboratories 2005-2015.
Scanning electron micrograph of lung cancer cells
(Image courtesy of A Weston).
SCIENTIFIC
REPORT
2014
Contents
FOREWoRD5
Research at lri laboratories – An
7
historical perspective
Origins: The Imperial Cancer Research
Fund8
LRI Laboratories Highlights 1- 20
10-48
INTRODUCTION TO THE 2014 LRI RESEARCH
REPORT50
52
Francis Crick INSTITUTE report
54
Research Highlights
Martin R Singleton
Macromolecular Structure and Function
Thomas Surrey
Lymphocyte Interaction
Axel Behrens
68
Mammalian Genetics
Dominique Bonnet
70
Haematopoietic Stem Cell
Dinis Calado
72
Immunity and Cancer
Julian Downward
74
Signal Transduction
Holger Gerhardt
76
Vascular Biology
Nathan Goehring
78
Developmental Systems
Adrian Hayday
80
Immuno Surveillance
Caroline Hill
82
Developmental Signalling
Nicholas Luscombe
Charles Swanton
Translational Cancer Therapeutics
Nicolas Tapon
Cancer Epigenetics
Sharon Tooze
Richard Treisman
Scientific report 2014 LONDON RESEARCH INSTITUTE
114
Signalling and Transcription
Frank Uhlmann
116
Chromosome Segregation
Peter Van Loo
118
Cancer Genomics
Folkert van Werven
120
Cell Fate and Gene Regulation
Michael Way
122
Cell Motility
Research Groups – Clare Hall Simon Boulton
Peter Cherepanov
90
125
126
128
Peter Karran 92
136
138
Mechanisms of Gene Transcription
Stephen C West
98
134
Cell Division and Aneuploidy
Jesper Q Svejstrup
96
132
Mammalian DNA Repair
Mark Petronczki
94
130
Chromosome Replication
140
Genetic Recombination
Hasan Yardimci
Single Molecule Imaging
2
112
Secretory Pathways
John FX Diffley
Tumour Cell Biology
Paola Scaffidi
110
Cell Regulation
Architecture and Dynamics of Macromolecular
Machines
Immunobiology
Erik Sahai Takashi Toda
88
Protein Phosphorylation
Caetano Reis e Sousa
Epithelial Biology
Alessandro Costa
Cell Cycle
Peter J Parker
108
Chromatin Structure and Mobile DNA
Structural Biology
Paul Nurse / Jacqueline Hayles
106
Apoptosis and Proliferation Control
86
Tumour Host Interaction
Neil Q McDonald 104
DNA Damage Response
84
Computational Biology
Ilaria Malanchi
102
Microtubule Cytoskeleton
Barry Thompson
Research Groups – Lincoln’s Inn FieldS 65
Facundo D Batista 66
100
142
TECHNOLOGY CORE FACILITIES
Ni
145
147
Advanced Sequencing
Aengus Stewart
148
Paul Bates
149
Biomolecular Modelling
Ruth Peat
150
Cell Services
Lucy Collinson
RESEARCH PUBLICATIONS AND THESES
RESEARCH PUBLICATIONS
THESES
163
164
186
INSTITUTE INFORMATION
ADMINISTRATION
ACADEMIC PROGRAMME
SEMINARS AND CONFERENCES
EXTERNAL FUNDING
INSTI TUTE MANAG EMEN T
CONTACT DETAILS
187
188
190
192
194
196
IBC
151
Electron Microscopy
Graham MG Clark
152
Equipment Park
Gordon Stamp
153
Experimental Histopathology
Derek Davies
154
Michael Howell
155
High-Throughput Screening
Daniel Zicha
156
Light Microscopy
Nicola O’Reilly
157
Bram Snijders
158
Protein Analysis and Proteomics
Svend Kjaer
159
P
Ian Rosewell
160
Transgenics
Ali Alidoust
161
Francois Lassailly
161
In Vivo Imaging
Stephane Mouilleron
162
Protein Structure
Anne Vaahtokari
163
CONTENTS
3
SIM image of the actin
cytoskeleton (black) in Drosophila
hemocytes lacking a functional
WAVE complex. Original image:
Sven Bogdan, University of
Münster (DE) from a collaborative
study with the Cell Motility
Group.
4
FOREWoRD
Harpal Kumar
CEO CRUK
When Sir Henry Morris, Thomas Rudd, and others decided, in 1902,
to create an institute to research the causes and treatment of cancer,
few would have imagined how profoundly important and influential
that institute would become over the ensuing years. In its various
guises and forms, what is now known as the London Research
Institute of Cancer Research UK has contributed immeasurably to
our understanding of the causes and development of cancer in
humans. Through a multitude of landmark discoveries, made by the
brightest and best scientists from around the world, the LRI has
contributed to the steady progress we have seen in recent years in
outcomes from cancer.
Over that time, the LRI has become widely known
as one of the very best biomedical research
institutes in the world, and consistently ranks in
the top five. This reputation has been propelled as
much by a constant stream of outstanding talent
as by its inspirational leaders, most latterly
including the outgoing Director Richard Treisman,
and one of his predecessors, Sir Paul Nurse, the
first Director of the Francis Crick Institute.
translation, are critical factors that have led Cancer
Research UK to conclude that the time is right for a
new institute. The Francis Crick Institute will build
from the very best that LRI has learnt and delivered
over the years. It will be a global beacon of
excellence and a magnet for the very best young
scientists from across the world. It will also inherit
the best scientists who have made the LRI what it is
today.
It is a truism of curiosity-driven fundamental
science that its directions and outputs cannot
reliably be predicted. LRI scientists, although
notionally working in a cancer institute, have made
profoundly important discoveries in a broad range
of other disease areas, including HIV/AIDS, other
infectious diseases, diabetes, and neurological
illnesses, amongst others.
I would like to offer my deepest and heartfelt
thanks to all those scientists, technicians, officers
and support staff who have, over many decades,
contributed to the development and outputs of
this great institute, the flagship institute of Cancer
Research UK.
Furthermore, as we understand more about the
drivers of cancer, we are increasingly conscious of
how much we do not, as yet, understand.
Therefore the need for an institute that carries out
discovery-oriented research has never been
stronger. However, scientific methods change, and
the need for updated facilities, as well as the
opportunities afforded by multi-disciplinarity,
cross-disease working, and an increased focus on
This scientific yearbook, the last for the LRI,
captures the extraordinary work that its latest
cohort of scientists have undertaken over the past
12 months, work that will go on to benefit
humankind in the years ahead. It is a fitting finale
to the LRI and its predecessors, and an enticing
taster of what we can expect from the Francis Crick
Institute.
Harpal Kumar
March 2015.
FOREWoRD
5
1963
1986
6
RESEARCH AT THE LRI LABORATORIES – AN
HISTORICAL PERSPECTIVE
This Report marks the end of Cancer Research UK’s operations at the
Lincoln’s Inn Fields and Clare Hall laboratories. We have therefore
devoted its first section to a review of research highlights over the
sites’ long history – Lincoln’s Inn opened in 1963, Clare Hall in 1986 –
to illustrate the achievements and introduce researchers who have
contributed to the LRI’s reputation as a leading cancer research
Institute.
Over the 50-year lifetime of the LRI’s laboratories,
our understanding of cancer and how to treat it has
undergone a revolution. We now have a broad
understanding of many of the cellular control
mechanisms that are disrupted in cancer, and
increasingly have the ability to design exquisitely
specific therapeutic reagents. Next-generation
DNA sequencing has revealed the variety of genetic
lesions in individual tumours, allowing cancer and
its evolution to be characterised in ever-increasing
detail, both during its development and its
response to therapeutic attack. We can now
appreciate the challenges posed by the
extraordinary genetic diversity of the disease: each
patient’s cancer comprises competing subclones
with distinct genotypes that we need to identify,
understand, and counter.
Patient profiling by sequencing, and the developing
‘-omics’ technologies, promises new ways to
classify individual cancers and to monitor their
response to therapy, and continues to identify new
routes for potential therapeutic development.
Increasingly, molecular analysis is complementing
traditional pathology-based tools in diagnosis, with
stratification of patients by cancer and birth
genotypes guiding the use of molecularly-targetspecific molecular therapies. Alongside this, we are
seeing improvements in our ability to image cells
and tissues in vivo, enabling us to study how cells
– both normal and cancerous – behave in the
context of the living body.
Nevertheless, in spite of our ability to identify
targets for therapeutic interference, in most cases
we do not yet know how to strike them effectively.
We must do so in such a way that cancer cells are
killed but their normal neighbours are spared, and
that the evolutionary avenues through which
therapeutic resistance develops are closed off: as
yet, however, we simply do not understand cells,
tissues and their cancerous development well
enough. Moreover, our understanding of how
cancers interact with host physiology in general,
including the immune system, remains in its
infancy, despite recent therapeutic advances. The
Francis Crick Institute will provide an unparalleled
environment to take cancer research to a new
level, integrating interdisciplinary discovery
research with the clinical and translational
opportunities available through its university
partners.
This retrospective, written by Kathleen Weston,
starts with a brief introduction to the former ICRF,
the predecessor of Cancer Research UK, and
follows with a set of twenty Highlights of research
achievements from the LRIs laboratories at
Lincoln’s Inn Fields and Clare Hall.
More detail can be found in Jane Austoker’s ‘A
History of the ICRF 1902-1986’ (Oxford Science
Publications) and Kathleen Weston’s ‘Blue Skies
and Bench Space’ (Cold Spring Harbor Laboratory
Press).
LRI Laboratories Highlights
7
Origins: The Imperial Cancer Research Fund
1901: “Little is at present known of cancer, but as
an incurable disease”
Cancer has been a scourge of humanity for
thousands of years, but at the dawn of the 20th
century, almost nothing was known about the
cause or causes of the disease. Doctors were able
to classify tumours by their appearance, and could
relate appearance to clinical outcome, but
treatments, where they existed, were brutal,
frequently agonisingly painful, and generally
useless. Cancer was seen as inexorable, relentless,
and progressive, appearing almost at random and
defying the restraints of normal growth control
mechanisms.
The advent of experimental microbiology in the
19th century led to a change in the nature of
cancer research. The notion of the ‘cancer cell’,
which multiplied to create tumours, was born, and
it became possible to start to speculate about how
cancers arose. Bacteriological research, showing
that it was possible to transmit infectious diseases
such as tuberculosis between animals, led to
successful attempts to propagate tumours in mice
and rats by analogous methods. Using such
tumours as a source of material, it was now
possible to start asking systematic scientific
questions about the origins of cancer.
Dr Ernest Bashford
8
With the methods and materials now at hand,
research laboratories dedicated to the study of
cancer emerged, with the first small laboratory
opening in Buffalo, New York, in 1897. In Britain, an
anonymous letter appeared in the St James’s
Gazette in 1901, advocating that a
subscription fund should be set up to
found a “cancer klinik”, dedicated
to studying all aspects of the
disease with a view to finding a
cure. The writer of the letter
was revealed to be Thomas
Rudd, a wealthy
businessman who had made
a fortune in South African
mining ventures. Although
Rudd became seriously ill,
and died in early 1902, his
idea was taken up by Sir
Henry Morris, a prominent
and very well-connected
surgeon. Morris persuaded the
Royal Colleges of Physicians and
Surgeons to provide laboratory
space, and a campaign to collect
subscriptions for the new Cancer Research
Fund was launched in The Times, attracting
high-profile support from the Royal Family (the
Fund was renamed the Imperial Cancer Research
Fund in 1904), the traditional aristocracy, and the
new monetary aristocracy of the City. In October
1902, the Executive Committee of the newlyfounded charity made one of the most important
and far-sighted appointments in the entire history
of the ICRF, by hiring the 29-year-old Dr Ernest
Bashford as the first General Superintendent and
Director.
Ernest Bashford and the start of research at ICRF
Ernest Bashford, an almost complete unknown,
was on the face of it an unlikely choice by the
rather crusty Executive Committee, composed as it
was of surgical grandees and Edwardian worthies.
His energetic, no-nonsense personality springs out
from his Director’s summaries of research in the
early ICRF Annual Reports, otherwise rather dusty
documents. His manifesto, a ‘Scheme for Enquiring
into the Nature, Cause, Prevention and Treatment
of Cancer’ is a model of how to approach a major
new research area, and was comprehensive,
innovative, and multi-disciplinary. In the words of
his contemporary Archibald Leitch, Director of
what is now the Institute of Cancer Research: “As a
Comet suddenly appears out of space, blazes
gloriously across the sky, and fades rapidly out of
sight, so did that forceful, brilliant and wayward
personality impress us”. Bashford in turn made an
inspired hiring decision in taking on James Murray,
who was to succeed him as the second Director in
1914, and the two of them set up together in the
new laboratories.
Bashford set about turning what had been a
haphazard, antediluvian discipline into a modern
scientifically based venture. Using a network of
suppliers comprising helpful vets, zoos, the more
enlightened hospital cancer wards, and most
spectacularly, the Medical Officers scattered
throughout India and the British Colonies and
Protectorates, he started to collect and classify
cancer samples from every source he could find. In
a few short years, he and his small group of
colleagues debunked many theories, finding
cancers in every vertebrate species, and in every
nationality. They showed that cancer was not
infectious, as had been feared, and by rational
observation, demolished many claims of miracle
cures. However, all this intensive work made one
thing alarmingly clear: cancer was not, as had been
hoped, a single disease triggered by one or a few
factors, but a many-headed hydra, with
multifactorial causes, and a huge multiplicity of
subtypes. The optimism of the founders of the
Staff of the ICRF 1909. Front row (l-r): W H Bowen, B R G Russell, M Haaland, E F Bashford, J A Murray,
C Da Fano, F Medigrazianu, W H Woglom. Middle row: W J Milton, E G Miller, M Finerty, F G Hallett,
Miss Anderson, A J Hall, A Chapman. Back row: Mrs Tuohy, W J Dunn, H L Smith, C Trott, H Thatford, L
Sawer, A Storey, A J Sheene.
Cancer Research Fund, who had hoped for a cure
within a few years, changed to a grim resolve in the
knowledge that many years of research would be
required to achieve any sort of progress.
Furthermore, decisions had to be made to prioritise
certain areas. Bashford and Murray believed
strongly that cancer had to be understood to be
cured, and the only way to do this was by
laboratory-based basic research. The focus of the
ICRF changed, moving further away from the clinic
towards finding out what caused cancers, what
made them grow, and what inherent weaknesses
might be exploited to kill them. Over the next
decades, the small band of ICRF scientists, still
numbering less than 10, produced a solid body of
research into tumour biology, and in the fields of
neoplasia and radiobiology, they made crucially
important contributions. The foundations of
modern cancer biology lie in these and other early
experiments done in a handful of pioneering labs
around the world.
Neoplasia: cancers arise from normal cells, in
stages
The concept that neoplasia, the growth of a new
cancer, begins by mutation of normal cells, and is a
multistep process, arose out of studies in a number
of labs in the first half of the 20th century, although
it was not until the 1970s that the scientific
community finally accepted the idea
wholeheartedly. Magnus Haaland, a Norwegian
working at the ICRF was one of the first to
recognise, in 1911, that “the malignant
transformation of tissue may take place by degrees
and not necessarily in one step from the normal cell
to the fully developed cancer cell” . However, his
observations were so ahead of their time that they
were forgotten for many years. Haaland aside, the
ICRF made its most important contribution to early
neoplasia research with the work of Leslie Foulds, a
pathologist appointed by James Murray in 1929 to
what must have been one of the first postdoctoral
fellowships ever offered. Foulds’s temporary
position was converted to a permanent one the
following year, and he stayed at the ICRF until 1951.
Foulds, building on the work of Haaland, Peyton
Rous and others, was the first to clearly lay out the
principles of tumour progression. He was the first to
realise that the mixed populations of cells found in
tumours were the outward sign of a dynamic
progression from normality to cancer, and that the
stages of progression were qualitatively different,
with cells mutating through precancerous stages to
become increasingly invasive and metastatic. His
book ‘Neoplastic Development’ (1969) is a classic,
describing his many decades’ worth of observations
on mouse mammary neoplasia, and is still widely
cited in today’s textbooks.
Radiosensitivity of tumours
Circumstantial evidence that ionizing radiation
might cause cancer began to accumulate in the
early years of the 20th century, and the study of
the effects of radiation on rodents, and in the
newly developed tissue culture systems, became a
worldwide theme of early cancer research. William
Cramer, a German immigrant, was the second
scientist hired by Ernest Bashford, in 1903. He
remained at the ICRF, with one short break, until
1939. In the 1930s, together with Herbert Crabtree,
who had joined the ICRF in 1923, Cramer carried
out a series of classic experiments looking at the
radiosensitivity of tumours, and how ionizing
radiation might be used as a therapeutic. Crabtree
and Cramer’s observations, that the
radiosensitivity of malignant cells was not a fixed
inherent property, but varied within wide limits,
and that, in conditions of low or absent oxygen,
cells become radioresistant, were two of the most
significant to emerge from the ICRF in the inter-war
period. Their work stimulated a flurry of
radiobiological research in the 1930s and 1940s,
which has led directly to the development of high
energy radiotherapy, still the treatment of choice
for many cancers.
Key references
J Austoker (1988). A History of the Imperial Cancer
Research Fund 1902-1986. Oxford Science
Publications
E F Bashford (1908). Draft Scheme for Enquiring
into the Nature, Cause, Prevention and Treatment
of Cancer. Scientific Report of the ICRF 3:441-7
9
LRI Laboratories Highlight 1
DNA and RNA tumour viruses
1968: Sir Michael Stoker brings virology to ICRF. In the following
decade the ICRF becomes a world-leading centre for tumour virus
research.
Background
In the late 1960s, the ICRF, whilst financially viable
and scientifically solid, was no longer the cuttingedge institution it had once been. Conflict
between the fifth Director, Guy Marrian and the
ICRF’s governing Council, mainly regarding
scientific objectives, resulted in Marrian’s forced
early retirement in 1968. The Council appointed
Sir Michael Stoker as the new Director, ushering in
a revolution in the ICRF’s scientific structure and
direction which restored the ICRF to its former
status in a remarkably short time. Stoker swept
out the old hierarchical system, replacing it with a
flat structure based on small independent
research labs, and transformed the science by
investing heavily in the new and exciting field of
the molecular biology of tumour viruses.
In 1911, Peyton Rous had shown that the chicken
virus which today, as Rous Sarcoma Virus, bears his
name, could be used to infect birds and cause
cancer. Subsequently Shope demonstrated that
rabbit papillomas (warts) were also likely
transmitted by a viral agent. There was a flurry of
interest in virally-mediated cancers, but due to
various reasons the field fell into disrepute for
some decades. Nevertheless by the early 1960s,
the idea that viruses caused cancer in many animal
Beverly Griffin
10
species was generally accepted and virology
became an important discipline in cancer research.
At the ICRF, tumour viruses became tools to try to
understand the mechanisms by which a normal cell
could turn into a tumour. Experiments were mainly
done in tissue culture (‘in vitro’), using the new
techniques developed by the fledgling molecular
biology field to look at the behaviour of the DNA,
RNA and proteins in cells. Almost all the equipment
and reagents were laboriously home made, and
methods were long-winded, temperamental and
frequently quite dangerous. However, they opened
a window into a sub-microscopic biological world
where no-one had previously explored.
The research
The ICRF labs began studying two types of tumour
virus: retroviruses, where the genetic material was
RNA, and those with a DNA viral genome. Both
camps were very successful.
The retrovirologists, under Robin Weiss,
John Wyke and Steve Martin, worked extensively
on the mechanisms by which RNA viruses were
able to infect cells, but their major contributions
were probably in the study of how retroviruses
cause cancer. In two instances, the discovery of the
Fos oncogene, and how retroviruses work as
insertional mutagens, ICRF scientists achieved
world firsts, and these are described in more detail
in Highlights 5 and 6.
The DNA tumour virologists, led by Lionel
Crawford, Mike Fried, Beverly Griffin, Bob Kamen
and Alan Smith, not to be outdone by their
colleagues, also led the world. They discovered the
p53 protein (Highlight 4), the Guardian of the
Genome, whose mutation we now know is a crucial
factor in the development of the majority of human
cancers, and showed that p53 overexpression was
characteristic of transformed cells. Ed Harlow, an
American PhD student with Lionel Crawford, later
used the methodology he learnt in the Crawford
lab to show in 1988 that the E1A transforming
protein from another DNA tumour virus,
adenovirus, bound and inactivated the
Retinoblastoma (Rb) tumour suppressor protein,
which is intimately involved in cell cycle control.
Transcription is the process whereby the DNA of
the genome is copied into RNA, which is then used
to make protein. It is responsible for turning the
DNA blueprint into the reality of a complete
organism, and as might be expected, is hugely
complicated. Today, we are still in the throes of
working out the control mechanisms that
determine which gene is transcribed when and
where, with layer upon layer of regulation still
being revealed. In the 1970s, the idea that “DNA
makes RNA makes protein” had only been accepted
for a few years, and most of what was known about
transcription came from simple bacterial systems.
Small DNA tumour viruses were seen as a way in to
studying eukaryotic transcription. Their small,
circular genomes, containing only a few genes,
made them easy to purify away from the huge
mammalian chromosomes in whose cells they
lurked, and they were lytic, meaning that they
replicated rapidly and prolifically, to give tractable
amounts of DNA: in the primitive world of early
molecular biology, they were incredibly useful
reagents.
At the ICRF, two viruses, polyoma and SV40, were
studied extensively, and together with their close
colleagues at Cold Spring Harbor Laboratory in the
US, ICRF scientists were responsible for laying
down some of the fundamental principles of
eukaryotic transcription regulation. The work at
ICRF determined the complete sequence of
polyoma virus, the structures of its transcripts, and
the proteins it encoded – only the second complete
structure of a DNA virus to be determined. It was
shown for the first time that it was possible for two
or more different mRNAs, and hence proteins, to
be made from the same piece of DNA. Different
transforming functions for the overlapping genes
of polyomavirus were identified, and they were
shown to work cooperatively in cellular
transformation, setting the scene for subsequent
discoveries about oncogene cooperation
(Highlight 6). In SV40, similar work led to the
discovery of p53 (Highlight 4). Turning their
attention to how transcripts were initiated on the
DNA, the ICRF scientists showed that transcripts
could have multiple start sites, and that there was a
faraway regulatory element in the SV40 DNA which
was needed for correct expression of the mRNA;
this was the first indication that regulation of both
transcription and DNA replication could be
facilitated by remote DNA sequences. The SV40
enhancer, as the regulatory element came to be
called, is the archetype of a universal control
mechanism.
The consequences
These experiments were of enormous significance
both for cancer biology and transcription. They
form the bedrock of our understanding of both
subjects, and are so embedded in the
consciousness of modern scientists that it is hard
to imagine a time without them.
Where are they now?
The roll call of leading scientists who have worked
in tumour virology at the ICRF is very long.
Confined solely to Institute Directors and/or Nobel
Prize Winners, the list runs as follows:
Renato Dulbecco received the Nobel Prize in 1975;
David Glover became first Director of the MRC
Laboratory of Molecular and Cell Biology,
University of London. Yoshiaki Ito is Yong Loo Lin
Professor & Director, Oncology Research Institute,
Singapore. Sir David Lane is Scientific Director of
the Ludwig Cancer Research and Chief Scientist,
A*STAR Institute, Singapore. Ron Laskey is the
Charles Darwin Chair of Animal Embryology in the
University of Cambridge, former Director of the
MRC Cancer Cell Unit in the Hutchison/MRC
Research Centre, and former Joint Director of the
Wellcome CRC Institute, Cambridge; Frank
McCormick leads the NCI RAS initiative and was
formerly Director of the UCSF Helen Diller Family
Comprehensive Cancer Center, San Francisco, USA.
Tony Pawson was a former Director, Samuel
Lunenfeld Research Center, Mount Sinai Hospital,
Toronto, and winner of the Kyoto Prize. Sir Bruce
Ponder became the first Director of the CRUK
Cambridge Institute; Alan Smith became Chief
Scientific Officer, Genzyme Corporation, US;
Richard Treisman became Director of the LRI and a
Research Director at the Francis Crick Institute;
Harold Varmus (Nobel Prize, 1989) became
Director of the US National Institutes of Health,
President of Memorial Sloane-Kettering Cancer
Center, and is now Director of US National Cancer
Institute; Robin Weiss, Professor of Viral Oncology,
UCL, is a former Director of the Institute of Cancer
Research, London; John Wyke is Director Emeritus
of the Beatson Institute, Glasgow.
11
Discovery of Fibronectin
1973: Richard Hynes and Nancy Hogg independently discover a
protein, fibronectin, which is present on normal cell surfaces, but
disappears when cells become cancerous.
2011: An entire field has grown around this discovery. The fibronectin
family, and their partners the integrin receptors, turned out to be
central to cell adhesion and migration, and many other processes.
Numerous steps in the progression of cancer, including invasion and
metastasis, involve altered adhesive properties of cells, and novel
antiadhesive therapeutics are seen as a key weapon in the fight
against cancer and other diseases.
12
Background
In the early 1970s, before the advent of the
molecular biology and genetics revolution that
would transform biological research, almost
nothing was known about the molecular structure
of a cell’s surface. However, in cancer biology,
there were hints that the surface of a cancerous
cell was different from that of its normal
counterpart in some way. Scientists reasoned that
if they could find what this difference was, they
would have the perfect marker for diagnosing
cancer, and perhaps from there would be able to
quickly find a cure. Richard Hynes, a postdoctoral
fellow in Ian Macpherson’s Tumour Virology
Laboratory, and Nancy Hogg, in Av Mitchison’s
Tumour Immunology Unit at University College,
both realised that work from other ICRF scientists
studying DNA tumour viruses could lead them to
the cancer cell marker. Their colleagues had seen
that when normal cells growing in tissue culture
were infected with tumour-causing viruses, they
were ‘transformed’. The infected cells grew faster,
heaped up into piles instead of growing in flat
monolayers, required fewer nutrients, changed
their appearance, and made tumours when
injected into mice. It seemed very likely that some
of these dramatic effects could be due to changes
in what was on the surface of the transformed
cells, and both Hynes and Hogg decided to find out
what these changes might be.
Nancy Hogg
Richard Hynes
The research
Both researchers used a biochemical technique
called lactoperoxidase labelling to tag cell surface
proteins with radioactive iodine. They took sets of
cell lines, some normal and some transformed,
labelled them up, and separated the labelled
protein products by size on fractionating gels. To
their excitement, a very large protein was present
in the normal cell controls but absent in the
transformed cells. The protein was dubbed LETS
(Large, External, Transformation-Sensitive) by
Hynes, but was subsequently renamed fibronectin
(from the Latin fibra meaning fibre, and nectere,
meaning to bind or connect).
The consequences
Although the simplistic idea of a universally
relevant cancer switch molecule was wrong, in the
search for this holy grail, Hynes and Hogg were
amongst the founders of a new field; the study of
cell adhesion. Cell adhesion is essential in all
multicellular organisms, determining how, when
and where cells move, both during embryonic
development and in normal and disease states. In
cancer, the ability of a tumour cell to grow, invade
new tissue, and metastasise all involve changes in
cell adhesion, and increasing our understanding of
this has been invaluable. Before fibronectin,
nobody had realised the importance of the
physical scaffolding, termed the extracellular
matrix, in which most cells live. Fibronectin is part
of this matrix, and studying its interactions with
other proteins led to the realisation that all cells
maintain an intimate, active conversation with
their extracellular environment, and are
exquisitively responsive to changes in it. Hynes and
others went on to discover that fibronectin and
other structural matrix proteins bind to the
integrin family of receptors, which are of huge
significance, as integrins are the main transmitters
of signals between the matrix and the internal
architecture and signalling systems of cells.
Recently, interest in fibronectin has renewed, as
certain members of the fibronectin family are
found in large amounts in the tumour vasculature,
the network of blood vessels which sustains
growing cancers. Antibodies against these
fibronectins have been used as anti-tumour and
tumour imaging reagents, and understanding the
role of fibronectins in tumour blood vessel
development will be a key requirement in the
development of anti-angiogenic cancer therapies.
What happened next?
Richard Hynes left the ICRF in 1975 to set up his
own lab at the Massachusetts Institute of
Technology (MIT), and has continued to work on
cell adhesion, making seminal contributions to the
field he founded. He is a former Head of the MIT
Biology Department and Director of the Center for
Cancer Research, and is now Daniel K Ludwig
Professor for Cancer Research at MIT. He is a
Fellow of the Royal Society, and a member of the
American Academy of Arts and Sciences, the USA
National Academy of Sciences and the Institute of
Medicine. Awards include the Gairdner Foundation
International Award, the Pasarow Medical
Research Award, a Guggenheim Fellowship and a
Research Career Development Award from the
National Institutes of Health. He sits on the Board
of Governors of the Wellcome Trust.
Nancy Hogg moved from the Tumour Immunology
Unit to the ICRF Lincoln’s Inn Fields site in 1982,
and remained there as a Principal Scientist, running
the Leukocyte Adhesion Laboratory until her
retirement in 2011. She was elected to the
Fellowship of the Academy of Medical Sciences in
2002 for her distinguished contributions to the
field of leukocyte biology.
Key references
Hynes RO (1973). Alteration of cell-surface
proteins by viral transformation and by proteolysis.
Proc Natl Acad Sci USA. 70:3170-4
Hogg N (1974). A comparison of membrane
proteins of normal and transformed cells by
lactoperoxidase labeling. Proc Natl Acad Sci USA.
71:489-92
Hynes RO (2004). The emergence of integrins: a
personal and historical perspective. Matrix
Biology. 23:333-40
13
Personalising cancer medicine
1974: Mel Greaves realises childhood leukaemias can be classified
according to their cell surface markers into different cancers of
varying prognosis, work which revolutionised the diagnosis and
treatment of childhood leukaemia and was the first step towards
personalised cancer treatment.
Background
Mel Greaves trained as an immunologist with Ivan
Roitt in the late 60s, and after some time at the
Karolinska Institute in Sweden, and then at the
National Institute for Medical Research, he wound
up working in Av Mitchison’s ICRF Tumour
Immunology Unit at University College London. He
would probably have carried on as a basic
immunologist had it not been for a life-changing
event during his time there. In Mel’s own words: “I
had a colleague working at Barts Hospital who took
me round the wards at a time when my own
children were three and four years old. I saw
children the same age stricken with leukaemia and
found it appalling. When I asked “What is
leukaemia? What is the underlying problem here?”
it was absolutely clear that ignorance was pretty
rampant. We had no idea about the nature of the
disease except that an expanding population of
cells was damaging the bone marrow and children
were dying. I felt that this must be a tractable
problem, so I started asking simple biological
questions such as “What sort of cell is involved?””
haemopoietic stem cell. Depending on cues they
receive from their location in the body, the cells
that surround them, and the demands made on
them by external events such as infection, they
mature and differentiate down particular lineages,
leading to a multiplicity of cell types, each tailored
for a particular purpose. Mel’s work in the ICRF
Tumour Immunology Unit was on two of these cell
types, the T and B white blood cells, which
recognise and respond to infection as part of a
normal immune response. As part of his work in
classifying and studying the development and
responses of T and B cells, Mel had developed a
panel of antibodies, which could be used to
specifically tag either type of cell, and could also
distinguish between different stages of their
development. By labelling his antibodies with a
coloured chemical which fluoresced when hit by a
laser beam, the cells binding the antibodies could
be automatically detected and rapidly counted by
a machine called a flow cytometer, one of the first
commercial examples of which had recently been
bought by the ICRF.
The research
All blood cells develop from a single cell type, the
The most common type of childhood leukaemia is
acute lymphoblastic leukaemia (ALL), which in the
early 70s was classed as one homogeneous
disease, with one treatment, a brutal
sledgehammer of drugs and radiation, which
effectively killed the cancer cells, but often had
dreadful side effects including severe mental and
physical impairment. From the viewpoint of a basic
immunologist such as Mel, it seemed very obvious
that the mutant ALL cells were likely to be derived
from normal T and B cells, and that finding out how
the cancers arose would be the key to studying and
treating them. Most importantly, Mel was one of
only two or three people in the world at the time
who possessed the reagents (his antibody panel)
and the technical know-how (his experience with
flow cytometry) to do anything about this
problem.
Mel Greaves
14
Using his antibody panel, Mel and his colleagues
began to analyse childhood ALLs, and as they had
expected, found that they did not all look the same.
They reclassified them into four subtypes: B-ALL;
T-ALL; pro-B ALL, and mature B-ALL. By using their
new classification to look at samples from ongoing
clinical trials, they were also able to correlate the
four types of ALL with different disease outcomes.
The rarest subtypes, pro-B and mature B-ALL, had
very poor prognoses. On the other hand, the
commonest subtype, B-ALL, had a relatively good
prognosis. Suddenly, a simple and rapid method
existed to learn exactly which type of ALL a child
had, and what their prognosis was. Each patient’s
cancer could be treated in a targeted fashion, such
that only the rare pro-B and mature B-ALLs with
poor prognoses had to be hit hard, and the others
could have less aggressive treatment.
Leukemia Foundation. He is an EMBO member, an
honorary MRCP, a Fellow of the Academy of
Medical Sciences and a Fellow of the Royal Society.
Mel’s most important work since leaving the ICRF
has been to show that childhood leukaemia begins
in the womb, and is a two-stage process. He
realised that by studying pairs of identical twins,
one with leukaemia and one without, he could look
in the healthy twin for early precancerous changes.
By this method, his lab showed that the initiating
pro-leukaemic change takes place in the womb,
and that due to shared placenta, identical twins
both have pro-leukaemic cells. The lab extended
this analysis to study how many babies were born
with the initiating change, using blood extracted
from blood spots stored on Guthrie cards (the
blood samples taken at birth from all UK-born
The consequences
This work is a translational research classic – a basic babies). They found that 1 in 20 babies are born
with a pre-leukaemic clone on board, but of these,
biologist’s view of a clinical problem provided the
new insight required for a fundamentally important only 1% go on to develop leukaemia. It is now
accepted that childhood ALL develops in two
change in diagnosis and treatment. Mel’s idea of
stages. The first change, likely spontaneous,
using panels of discriminatory antibodies coupled
happens in the womb, and is frequent. The second
to flow cytometry is now used throughout the
change triggers development of leukaemia and
world for the diagnosis and classification of
happens in early childhood, with average age of
leukaemia and many other cancers. The ICRF
became the national immuno-diagnostic service for onset 2-5 years. This second change was suggested
by Mel to be caused by an inappropriate response
childhood ALL in the United Kingdom. Diagnosis of
to infection, brought on by keeping babies in the
ALL subtype meant that for the first time,
developed world in surroundings that are too clean
personalised cancer therapy, the dream of cancer
– the hygiene hypothesis. The theory goes that the
clinicians, was possible.
immune systems of first world babies are not
sufficiently exposed to pathogens, and therefore
As well as their profound impact on the treatment
are not properly educated in the vital months after
of leukaemia, these experiments were the first
birth. The unbalanced immune system may
tentative steps taken into understanding the
biology of childhood ALL. Once cancer cells could be respond in an exaggerated way to infection,
triggering transformation of a pre-leukaemic cell
categorised, they could be compared with normal
into a leukaemia.
blood cells, and the data Mel obtained suggested
that leukaemias were a result of very immature
Mel’s current research on the genetic
blood cells somehow becoming arrested in their
heterogeneity of cancer remains at the forefront of
normal development. How this happens, what the
leukaemic cancer stem cells are, and how the roots contemporary thinking about the causes and
consequences of mutation in cancer, and how it can
of the disease can be attacked and eradicated, are
still the major preoccupations of today’s leukaemia be tackled therapeutically. His work in this field has
influenced a new generation, including Charlie
researchers.
Swanton, whose work on tumour heterogeneity
features in Highlight 20.
What happened next?
Mel Greaves left the ICRF in 1984 to become
Director of the Leukaemia Research Fund Centre at Key references
Brown G, Greaves MF, Lister TA, Rapson N,
the Institute of Cancer Research. He remains there
Papamichael M (1974). Expression of human T and
as Professor of Cell Biology. For his work on
B lymphocyte cell-surface markers on leukaemic
leukaemia, Mel has won numerous awards and
cells. Lancet. 7883:753-5
prizes, including the King Faisal International Prize
Greaves MF (2008). A Scientist’s Journey. From
for Medicine, the Gold Medal of the British Society
of Haematology and the Jose Carreras Award of the White Blood: Personal Journeys with Childhood
Leukaemia. ISBN: 978-981-279-039-2
European Hematology Association/Jose Carreras
15
Discovery of p53
1979: David Lane and Lionel Crawford show that the oncogenic viral
SV40 large T antigen binds a cellular protein, dubbed p53.
p53, the ‘guardian of the genome’, is mutated in the majority of
human cancers. Restoring normal p53 activity is the holy grail of
cancer therapeutics.
Background
In the mid 1970s, the ICRF had entered a period of
pre-eminence in virological research (Highlight 1).
Part of this endeavour was the work on the DNA
tumour virus, SV40. RNA tumour viruses, or
retroviruses, had been shown in 1976 by Varmus
and Bishop to contain genes, dubbed oncogenes,
which were hijacked, mutant versions of cellular
genes; when unleashed on a cell by viral infection,
the oncoproteins encoded by the mutant
oncogenes took over the cell’s metabolism,
allowing it to grow uncontrollably. However, the
oncogenes of DNA tumour viruses bore no
relationship to cellular genes, and their
tumourigenic mechanisms were a mystery.
16
three viral oncoproteins, unoriginally named large
T-, small T- and middle T-antigens had been found;
it seemed possible that SV40 might also have a
hitherto undetected middle T-antigen, and many
labs, including Lionel Crawford’s at the ICRF, began
work to identify this putative new oncogene. David
Lane arrived as a postdoc in the Crawford lab in
1975, fresh from a PhD with Av Mitchison at
University College London, and his skills as an
immunologist meant that he was ideally suited to a
project involving the use of antibodies to detect
novel, elusive proteins.
Upon detecting infection by a DNA tumour virus,
the host organism’s immune system was known to
react by making antibodies against the viral
proteins. Scientists realised that this antigenic
(‘antibody-making’) response could be used as a
tool to identify and monitor the viral proteins
(dubbed ‘viral tumour antigens’) in cells.
Conveniently, genetic studies showed that these
viral tumour antigens were often encoded by the
genes responsible for the oncogenic potential of
the virus. In the case of SV40, the two viral proteins
identified like this were called large T- and small
T-antigen. For polyoma virus, one of the other DNA
tumour viruses under intensive study at the time,
The research
As the antisera available at the time for detecting
the SV40 tumour antigens were of extremely
variable quality, David, in collaboration with
research assistant Alan Robbins, spent the first
part of his postdoc making antibody reagents that
were specific for large and small T, no small feat at
that time. He then used these improved antisera
for immunoprecipitation experiments in SV40transformed cells. Immunoprecipitation is
molecular fishing, using an antibody able to
specifically recognise a target protein as the hook
to fish out the protein of interest from the cellular
soup. David saw, sure enough, that his antisera
specifically brought down both large and small T,
but in addition, he always saw a third protein,
which he estimated to be about 53 kilodaltons (kD)
David Lane
Lionel Crawford
in size. The mystery protein never appeared in his
immunoprecipitates except in association with large
T, but was not virally encoded, and was not a
shortened, degraded version of either large or small
T. David was left with no option but to suggest that
the 53kD protein was virally induced, but cellular in
origin, and that it was being immunoprecipitated
because it was in a complex with large T antigen. He
presented compelling data in his paper to indicate
this was the case, and also showed that a protein,
probably the same one, could also be found at high
levels in Polyoma transformed cells. The fact that
this same protein was involved in transformation by
two separate tumour viruses led Lane and Crawford
to propose that it was likely to play a crucial role in
tumour formation. As is often the case when a
discovery is ripe to be made, it transpired that four
other groups had essentially the same observations;
papers by Lane and Crawford, Pierre May’s group in
France, Alan Smith’s group in London, and Linzer
and Levine and the Carroll group in the US, were all
published in 1979. The importance of p53 in cancer
was underscored by another publication from the
Crawford lab in 1982, showing that serum from 9%
of all breast cancer patients contained antibodies
that targetted p53. We now know this was the first
hint of the avalanche of cancers in which p53 is
found mutated.
The consequences
The first decade of p53 research was fairly
confusing due to its initial categorisation as an
oncogene in its own right. However, after the p53
gene was cloned from multiple cell lines, the field
realised that all the oncogenic versions were
actually mutated, and that normal p53 was in fact a
tumour suppressor gene. Therefore, rather than
p53 causing cancer, loss or mutation of the p53
gene was realised to be one of the contributory
factors in tumour formation. In fact, p53 turns out
to be possibly the most frequently mutated gene in
human cancer, with functional changes found in the
majority of all cases. More recent work has shown
that p53 is a transcription factor, able to directly
switch genes on and off, and that it is made by cells
when they detect they have been damaged. In
1992, it was dubbed the ‘guardian of the genome’
by David Lane, due to this ability. Induction of p53
can halt the cell cycle, so damaged DNA can be
repaired, or if the damage is too bad to be fixed, p53
forces the cell to commit suicide (apoptosis), or to
become dormant (senescence). Its pivotal role in
guarding against damage explains why it is so often
put out of action during the evolution of a cancer,
which can only grow if it overrides the normal
cellular defence mechanisms.
What happened next?
David Lane remains one of the world’s most
prominent cancer biologists, and was the second
most highly cited medical scientist in the UK in the
last decade. He has published in excess of 300
papers, and co-authored a successful
immunochemical methods handbook, ‘Antibodies’,
which has sold more than 40,000 copies. For his
efforts in cancer research, he was knighted in
2000, and he has won many awards such as the
Paul Ehrlich Prize in 1998, the Buchanan Medal in
2004, the Medal of Honor from the International
Agency for Research on Cancer in 2005 and in
2008, the Royal Medal from the Royal Society of
Edinburgh. He is a member of the European
Molecular Biology Organisation (EMBO), a Fellow
of the Royal Society and the Royal Society of
Edinburgh, and a founder member of the Academy
of Medical Sciences. Sir David was the first Chief
Scientist of Cancer Research UK, leaving that
position in 2010. After many years working in
Dundee, latterly as Director of the Cancer Research
UK Cell Transformation Research Group and
Professor of Oncology, he is currently Chief
Scientist of A*STAR in Singapore, and Scientific
Director of Ludwig Cancer Research.
Lionel Crawford worked at the ICRF labs in London
until 1988, when he moved to run the ICRF Tumour
Virus Group in the Pathology Department of the
University of Cambridge until his retirement in
1995. He was elected a Fellow of the Royal Society
of Edinburgh in 1970 and a Fellow of the Royal
Society in 1988. In 2005, in recognition of his
lifetime’s work on DNA tumour viruses, he was
awarded the Gabor Medal of the Royal Society.
Crawford was not only an internationally
recognised leader in his field, but was also much
appreciated for his extraordinarily unselfish
attitude to nurturing younger scientists in his
laboratory, many of whom besides David Lane
went on to become ‘big names’ in their own right.
In the words of a former colleague, he is “one of
the unsung heroes of British biochemistry and
molecular biology”.
Key references
Lane DP, Crawford LV (1979). T antigen is bound to
host protein in SV40-transformed cells. Nature.
278:261-3
Crawford LV, Pim DC, Bulbrook RD (1982).
Detection of antibodies against the cellular protein
p53 in sera from patients with breast cancer. Int J
Cancer. 30:403-8
Lane DP (1992). p53, Guardian of the Genome.
Nature. 358:15-16
17
Discovery of Fos oncogene
1982: Tom Curran and Natalie Teich identify the v-Fos retroviral
oncoprotein and associated protein p39 (later shown to correspond to
the Jun oncoprotein). With US collaborators they clone and sequence
the v-fos gene and show there is a cellular homologue, c-fos.
Fos and its partner Jun were the first oncoproteins shown to directly
regulate transcription, the switching on and off of genes. They are
now known to participate in controlling growth, differentiation, cell
death and activation of neurons. Their discovery led to the realisation
that upregulation and downregulation of specific crucial target genes
can cause cancer, and that all parts of the cellular machinery are
vulnerable to cancer mutations.
Background
Peyton Rous’s 1911 isolation of a cancer-causing
(oncogenic) tumour virus won him the 1966 Nobel
Prize in Physiology or Medicine, and led to the
identification of many other retroviruses with the
same ability to cause cancer. In 1976, Harold
Varmus and Mike Bishop made another Nobel
Prize-winning discovery, that Src (pronounced
‘sarc’), the cancer causing oncogene in Rous
Sarcoma Virus, had a cellular counterpart, the
c-Src proto-oncogene. Varmus and Bishop’s work
demonstrated that retroviruses could steal the
genetic matter of their hosts, and mutate it into a
superactivated form, which could then attack the
host, causing uncontrolled proliferation, and
hence cancer. Crucially, it also gave researchers a
way in to the complex problem of how to detect
other sleeping proto-oncogenes in the genome.
Tom Curran
18
Post-1976, there was a flood of research to analyse
the oncogenic proteins being made by
retroviruses, and to try to match them to proteins
in the normal genome. At the ICRF, Tom Curran, a
young Scottish student in Natalie Teich’s lab, was
given as his PhD project the task of finding the
oncogenic component of the FBJ mouse
osteogenic sarcoma retrovirus.
The research
Tom’s initial approach was to isolate the oncogenic
part of the FBJ retrovirus by making transformed
rat cells that were not also infected with a helper
retrovirus (so-called non-producer cells). He then
injected these cells under the skin of rats where
they grew to form tumours. The rats made
antibodies against the tumour cells in a vain
attempt to stop them growing. Tom then took
blood samples from the rats and used the sera to
examine cells infected with the FBJ retrovirus using
the technique of immunoprecipitation, in which an
antibody in the sera is able to specifically recognise
and capture proteins from the general cellular
soup. Tom made a stock of antiserum (a mix of
many antibodies) made from an animal inoculated
with cells containing FBJ, together with some
matching control antiserum from a normal animal.
The FBJ-specific antiserum contained antibodies
against FBJ proteins, whilst the control did not.
When he used these two antisera to analyse
multiple cell types, some infected with FBJ and
some not, he was able to show that there were two
specific and unique proteins in the FBJ-infected
cells, one 55 kilodaltons (kD) in size and one 39 kD
in size, termed p39. To find out which of these
proteins was made by the FBJ virus, Tom purified
viral RNA and used it to program a cell-free
translation system that made all the proteins
encoded by the virus. Remarkably, sera from rats
with tumors specifically recognised and captured
just the 55 kD protein showing that it was the
product of the FBJ oncogene. Tom christened the
new oncogene v-fos, (from FBJ-osteogenic
sarcoma virus). Shortly before moving to Inder
Verma’s laboratory at the Salk Institute in San
Diego for postdoctoral work, under the tutelage of
Gordon Peters (Highlight 6),Tom isolated and
purified v-fos DNA. The collaboration continued
after Tom moved to San Diego, ultimately
demonstrating, that, like all other retroviral
oncogenes, v-fos was a stolen and modifed copy of
the proto-oncogene c-fos, found in the normal
genome.
The consequences
In 1984, Tom established an independent
laboratory at the Roche Institute of Molecular
Biology. In an early collaboration with Rolf Müller
and Rodrigo Bravo at the EMBL in Heidelberg, Tom
showed that Fos was rapidly and transiently
induced (turned on) when cells are exposed to
growth factors. This work established Fos as an
archetype of the set of genes known as cellularimmediate-early genes that function in signal
transduction processes in response to extracellular
stimuli. Furthermore, Tom, together with his
long-standing collaborator Jim Morgan, showed
that this response also happened when neurons
are activated by neurotransmitters or electrical
stimulation. This finding led to the model that
changes in gene expression may underlie the long
term adaptive modifications in neurons that
contribute to learning and memory.
In 1987, Jun, another retroviral oncogene which
encoded a 39kD protein, was discovered by Peter
Vogt’s lab. Jun protein was shown to be a
component of the AP-1 transcription factor
complex. AP-1 was at that time a very hot research
topic in the transcription field, but had hitherto
only been defined as a protein complex of
unknown makeup which, in response to growth
factor signals, could bind to DNA and activate
neighbouring genes. Tom’s lab, in collaboration
with Bob Franza at Cold Spring Harbor, showed
that Fos also bound to the AP-1 DNA binding site.
Shortly afterwards, in one hectic period spanning a
few weeks in 1988, they went on to show that the
mysterious 39kD cellular partner of Fos which Tom
had defined six years before at ICRF, was actually
the Jun protein, and that Fos was the other
component of AP-1. These two oncogenes were
the first to be identified as transcription factors,
able to directly regulate gene transcription, and in
their viral form switching on genes that would only
normally be activated by strong growth signals. In
addition to this fundamental insight into cancer
biology, a flood of research into AP-1 in the next
few years established many key concepts in
transcription factor biology: the leucine zipper, a
novel structural motif which zips together the Fos
and Jun proteins to make the AP-1 complex, is now
known to be ubiquitous, and the existence of Fos
and Jun related proteins which can be mixed and
matched showed that transcription factor families
exist, and shed light on an extra level of regulation.
Perhaps most importantly, the study of how AP-1
is regulated by growth factor signals, and how the
complex can be modified to tune its activity
precisely, has been in the forefront of research
into understanding how a cell’s reaction to its
environment is translated into a change in
transcription of its genes, and hence its destiny.
What happened next?
Tom Curran is currently Deputy Scientific Director
of the Children’s Hospital of Philadelphia Research
Institute. He has won numerous awards, and is a
past President of the American Association for
Cancer Research. He was elected to the Royal
Society in 2005 and the Institute of Medicine
(IOM) of the National Academies of Science, USA
in 2009.
Natalie Teich retired from the ICRF in 1995 after 22
years as a lab head. Although her co-discovery of
v-fos was her most significant contribution to
cancer research, her work on blood cell
development, exploring the potential of stem cells,
was also influential.
Key references
Curran T, Teich NM (1982). Candidate product of
the FBJ Murine Osteosarcoma Virus Oncogene:
Characterization of a 55,000-Dalton
Phosphoprotein. Journal of Virology. 42:114-22
Curran T, Peters G, Van Beveren C, Teich NM,
Verma IM (1982). FBJ Murine Osteosarcoma Virus:
Identification and Molecular Cloning of
Biologically Active Proviral DNA. Journal of
Virology. 44:674-82
Rauscher FJ 3rd, Cohen DR, Curran T, Bos TJ,
Vogt PK, Bohmann D, Tjian R, Franza BR Jr (1988).
Fos-associated protein p39 is the product of the
jun proto-oncogene. Science. 240:1010-6
19
Insertional mutagenesis and oncogene cooperation
1983: Gordon Peters and Clive Dickson show MMTV causes tumours
by insertional mutagenesis.
1986: Peters, Lee and Dickson discover first evidence of oncogene
cooperation in an animal model.
Both these discoveries form part of the conceptual foundations of
our current understanding of cancer.
Background
Cancer-causing retroviruses are not all alike; in
addition to those that have picked up a cellular gene
and corrupted it into becoming an oncogene, there
exists a second type. These other retroviruses still
cause cancer in almost 100% of the infected hosts
but do not contain an oncogene in their genomes.
Until the 1980s, researchers were mystified as to
how they might work, but in 1981, Bill Hayward, at
the Rockefeller Institute in New York, provided one
possible answer, when he solved the puzzle of how
Avian Leukosis Virus (ALV) was able to cause
leukaemia. The answer lies in the life cycle of a
retrovirus. As the name implies, retroviruses
reproduce by copying the information in the viral
RNA into double-stranded DNA, the exact opposite
of the normal process of gene expression. The
resulting double-stranded DNA ‘provirus’ is then
inserted into the host cell’s DNA, becoming a
passenger in the genome that is copied each time
the cell divides. Each infected cell produces large
amounts of viral RNA and protein, packages it into
new retroviral shells, and dispatches it into the
extracellular soup to infect more cells.
When Hayward examined where in the genome the
ALV provirus had integrated in leukaemic cells, he
Gordon Peters (left) and Clive Dickson (right)
20
found that it had inserted itself next to a growth
controlling gene called c-Myc. c-Myc had already
been identified as the oncogene captured by
another virus, MC29, and it was later shown that
many human tumours make too much
(overexpress) c-Myc protein. This latter is exactly
what happens when the ALV proviral DNA is
inserted nearby. The viral DNA contains powerful
transcriptional control elements that are designed
to maximise viral RNA production, but their effects
can often spread to host genes that happen to be in
the vicinity of the provirus. If by chance the
adjacent gene is a potential oncogene, such as
c-Myc, its inappropriately high expression can
enable that particular cell to outgrow its
neighbours and develop into a tumour. Hayward
christened this new method of tumourigenesis
‘insertional mutagenesis’.
Clive Dickson and Gordon Peters, in Robin
Weiss’s RNA Tumour Virus section at the ICRF,
were studying a virus called Mouse Mammary
Tumour Virus (MMTV), then considered to be the
best available animal model for human breast
cancer. Like ALV, MMTV did not carry its own
oncogene. Clive and Gordon realised that
Hayward’s discovery of ALV causing cancer by
insertional mutagenesis might also apply to MMTV
- perhaps it, too, was integrating into the genome
next to a potential oncogene and switching it on.
If their hunch was right, it was likely that Hayward
had discovered a general principle of viral
tumourigenesis, and also, that new oncogenes of
great importance in breast cancer might be
discovered.
The research
Whereas the oncogene targeted by ALV was
already known and easy to recognise as c-Myc,
there were no obvious clues about the likely
targets of MMTV. However, Clive and Gordon
reasoned that if they found where the MMTV
provirus had integrated in different tumours, they
might find the same region of genomic DNA
cropping up again and again next door to the
provirus. The first step was to clone fragments of
DNA that spanned the junctions between proviral
and cellular DNA and to use these to prepare
probes that would identify the site of integration
within the genome. By using these probes to
analyse the DNA from a series of MMTV-induced
tumours, they found that in almost half of the
tumours there was a provirus in the same region of
the host genome which did not seem to
correspond to a known oncogene. At the same
time as this work was underway, Roel Nusse and
Harold Varmus in San Francisco had been
conducting similar experiments and discovered a
different MMTV integration site, which they called
int-1. Gordon and Clive’s potential oncogene was
therefore dubbed int-2. Interestingly, at both
common insertion sites, the MMTV proviruses
were clustered on either side of a host gene that
was switched off in normal mammary cells but
switched on in tumour cells by MMTV. The
following year, both groups succeeded in cloning
the respective int-1 and int-2 genes and began to
ask what these new oncogenes might do.
One of the surprises of this research was that
despite doing very similar experiments, the two
groups had discovered completely different genes.
Could there be a third or fourth gene waiting to be
discovered? If so, the obvious place to look would
be in tumours where int-1 and int-2 were not
involved but, in searching for such an example,
Gordon and Clive came upon another surprise. In
many of the tumours they analysed, proviruses had
integrated at both int-1 and int-2, not just one or the
other, and both genes were turned on. This fitted
well with the persuasive theory doing the rounds at
the time that the transformation of a normal cell
into a tumour cell requires the cooperative action
of more than one oncogene. The idea was initially
formulated from studies on mouse and rat cells
grown in tissue culture, in which the ICRF DNA
tumour virus groups had played a major role, but
Gordon and Clive’s 1986 publication in Nature was
the first to show that oncogene cooperation occurs
in real tumours in an animal model.
The consequences
As a technique for probing the molecular biology of
tumours, insertional mutagenesis has been
invaluable, for a long time remaining the tool of
choice for identifying new oncogenes, and also
for examining which oncogenes are able to
cooperate with each other. While in some cases
there is a rational explanation for co-operativity
(such as the role of c-Myc in apoptosis described in
Highlight 10), the reason for co-activation of int-1
and int-2 remains unresolved. int-1 proved to be
the prototype of the Wnt family of growth factors
that have pivotal roles in development and cancer,
and some other members of the Wnt family were
later found to be activated by MMTV insertion.
The int-2 gene product turned out to be a member
of the Fibroblast Growth Factor family, and is now
known as Fgf3. Interestingly, Fgf4 which is right
next to Fgf3 in the genome, is also activated by
MMTV, reinforcing the idea of co-operativity
between the two families of growth factors.
Following initial excitement that the human FGF3
and FGF4 genes are amplified (present in extra
copies) in many human breast cancers, Clive and
Gordon subsequently found that both genes
remain switched off in these tumours. However,
this observation suggested that there might be
another previously unknown oncogene within
the region that is co-amplified with FGF3 and
FGF4, so they set about mapping the region of
amplification. It quickly emerged that the likely
culprit was cyclin D1, a pivotal regulator of the
cell cycle.
What happened next?
Gordon Peters and Clive Dickson remained at the
LRI and continued to work closely together for
most of their careers. Gordon retired in 2013, and
Clive in 2004. Both were elected Fellows of the
Academy of Medical Sciences for their work in
cancer biology and transcription.
Key references
Peters G, Brookes S, Smith R, Dickson C (1983).
Tumorigenesis by Mouse Mammary Tumour Virus:
Evidence for a Common Region for Provirus
Integration in Mammary Tumours. Cell. 33:369-77
Dickson C, Smith R, Brookes S, Peters G (1984).
Tumorigenesis by Mouse Mammary Tumour Virus:
Proviral Activation of a Cellular Gene in the
Common Integration Region int-2. Cell. 37:529-36
Peters G, Lee AE, Dickson C (1986). Concerted
activation of two potential proto-oncogenes in
carcinomas induced by mouse mammary tumour
virus. Nature. 320:628-31
21
Growth factors and receptors can be oncogenes
1983: Mike Waterfield and colleagues show that the human gene
encoding Platelet Derived Growth Factor (PDGF), and the cancercausing sis oncogene, found in a tumour virus, are closely related.
1984: Julian Downward discovers that another tumour virus has
hijacked the human Epidermal Growth Factor Receptor (EGF-R) and
converted it into the v-erb-b oncogene.
These two discoveries showed that oncogenes can cause tumours
because they encode mutationally activated components of normal
cellular growth control mechanisms. The work transformed our
understanding of how growth is regulated in normal cells, and what
goes wrong in cancer.
22
Background
The existence of soluble proteins that could
stimulate cells to proliferate was first discovered in
the late 1950s by Rita Levi-Montalcini, Giuseppe
Levi and Stan Cohen. Levi-Montalcini and Cohen
received the 1986 Nobel Prize in Physiology or
Medicine for their discovery, which showed that
the immensely complicated process of growing a
whole organism from a single fertilised egg might
be understood by looking at what was happening
to individual cells at the molecular level. In the
years that followed, many more growth factors, as
the proteins came to be called, were discovered,
and they were shown to be the drivers of normal
cell growth, providing essential signals for growth,
differentiation (the process of developing into
functionally specialised cells) and eventually, death.
By the late 1970s, whilst it was relatively easy to
purify growth factors and find out what they did, it
was much harder to identify them as specific genes;
protein sequencing, the determination of amino
acid sequence, was extremely difficult and costly,
and databases containing sequence information
(where one could check for potential sequence
similarity to known proteins), were small and hard
to access. Mike Waterfield, however, was in a very
good position to tackle this problem. He had been a
postdoc at Caltech in the lab of Leroy Hood, a
protein sequencing pioneer, and when he set up his
own lab at ICRF, he brought with him the expertise
to build what was for a time the world’s most
sensitive protein sequencer. He decided to attempt
to sequence PDGF, a potent growth factor that
stimulates wound healing.
Mike Waterfield
Julian Downward
The research
Waterfield obtained purified human PDGF from
colleagues in Sweden and the US, and, using his
state-of-the-art protein sequencer, obtained some
partial amino acid sequences from the PDGF
protein. After some fruitless searches of public
databases, the PDGF sequence was compared to a
tape of the most up-to-date database available,
posted from San Diego by the evolutionary
biologist Russell Doolittle. There was an immediate
match, to the v-sis oncogene. V-sis, found in a
tumour virus causing sarcomas, was therefore
making a powerful growth factor normally only
induced by wounding, which meant that any cell
infected by its parent virus would immediately
start proliferating abnormally, one of the classic
hallmarks of cancer.
At the same time, Julian Downward, a graduate
student in Waterfield’s lab, and Yossi Yarden, a
visiting Israeli scientist, with collaborators Axel
Ullrich and Jossi Schlessinger, were working on a
separate project, to determine the first ever full
length nucleic acid sequence of a growth factor
receptor. To do this, they purified human
Epidermal Growth Factor Receptor (EGFR),
obtained some partial amino acid sequences, and
then used these sequences to make probes to fish
out DNA corresponding to the coding region of the
EGFR gene. Late in December 1983, Downward
compared his EGFR sequences to an in-house
database of oncogene sequences compiled by a
postdoc, Geoff Scrace, and discovered that EGFR
matched up perfectly with the viral oncogene
v-erb-B, whose host virus causes acute leukaemia.
Tumour viruses had therefore not only stolen
growth factors, but also their receptors from the
organisms in which they could cause cancer,
equipping themselves with mutant versions of the
most powerful weapons in a normal cell’s armoury.
The consequences
These findings were the first to definitively show at
the molecular level that growth factor signalling
was central to carcinogenesis. A new discipline, the
study of signal transduction, exploded into life as
many other researchers realised that it was
possible to purify, sequence and identify not only a
multitude of growth factors, but all the
components of the chain of command stretching
from the growth factor outside the cell right into
the cell’s nucleus. In cancer, there are now many
examples of mutated or overactive growth factors
and receptors causing particular human tumours,
and numerous drugs have been developed which
target such molecules. Mutations in the EGFR have
been found in lung, pancreatic and colorectal
cancers amongst others, and a number of effective
EGFR-inhibiting drugs are now in use in the clinic.
One of the EGFR’s close relatives, HER-2, is
overexpressed in about 30% of breast cancers, and
is associated with poor prognosis, but again,
herceptin, a HER-2 inhibitor, is now a standard and
effective part of first-line breast cancer treatment.
Tony Burgess, a long-time colleague of Mike
Waterfield put these findings in this context: “All
scientists strive to participate in a discovery that will
change the direction of the world. Most of us work
for a lifetime admiring the great achievements of a
few of our colleagues and occasionally we brush with
one great discovery. In the seven months from June
1983 to January 1984, Mike Waterfield led two great
teams which changed our understanding of cancer
biochemistry and biology.”
What happened next?
Mike Waterfield moved from ICRF in 1986 to head
the London University College branch of the Ludwig
Institute for Cancer Research. In 1991, he became
the Courtauld Professor of Biochemistry and
Molecular Biology at UCL, and from 1991 to 2002,
was the Head of UCL’s Department of Biochemistry
and Molecular Biology. Prof. Waterfield has also
received numerous academic awards, including
being made a Fellow of the Royal Society. His work
post-ICRF centred around the study of a family of
enzymes, the phosphoinositide-3 kinases (PI3Ks),
which are downstream components of many growth
factor signalling pathways. Alterations affecting PI3K
signalling are found in many cancers, and in 2002
Waterfield and two Cancer Research UK-funded
scientists, Peter Parker and Paul Workman, set up a
company, PIramed, to develop PI3K-targeted
therapies for cancer. The company was acquired by
Roche in 2008, and the PIramed inhibitor, GDC-0941,
is currently in early clinical trials. Mike Waterfield
retired in 2008.
After postdoctoral training with renowned cancer
researcher Bob Weinberg at the Whitehead
Institute in Boston, Julian Downward was recruited
back to the ICRF and started his own laboratory
there in 1989 (Highlight 14). He became LRI
Associate Director in 2005, and is now an Associate
Research Director at the Francis Crick Institute. He
was elected to EMBO in 1995, became a fellow of
the Royal Society in 2005, and a fellow of the
Academy of Medical Sciences in 2009.
Key references
Waterfield MD, Scrace GT, Whittle N, Stroobant P,
Johnsson A, Wasteson Å, Westermark B, Heldin
CH, Huang JS, Deuel TF (1983). Platelet-derived
growth factor is structurally related to the putative
transforming protein p28sis of simian sarcoma virus.
Nature. 304:35-9.
Downward J, Yarden Y, Mayes E, Scrace G, Totty N,
Stockwell P, Ullrich A, Schlessinger J, Waterfield MD
(1984). Close similarity of epidermal growth factor
receptor and v-erb-B oncogene protein sequences.
Nature. 307:521-7
23
Conservation of the cell cycle
1987: ICRF Cell Cycle Control laboratory, led by Paul Nurse, shows for
the first time that the cell cycle works the same way in all eukaryotic
cells, a discovery with relevance for many diseases, but especially
cancer.
2001: Paul Nurse, fellow ICRF lab head Tim Hunt, and Leland H.
Hartwell are awarded the Nobel Prize in Physiology or Medicine “for
their discoveries of key regulators of the cell cycle”.
Background
The process of reproduction is a central property
of life, and this is seen in its simplest form in single
cells. As a graduate student, Paul Nurse reasoned
that studying the cell cycle, the mechanism driving
the reproduction of cells, was important; in
particular, the control of the cell cycle would be
crucial. He realised that the best way to study such
a complex process was by using genetics in a very
simple organism. Over the next few years, Paul,
first as a post-doctoral worker and then as a lab
head in his own right, made his name by
identifying many of the key regulators of the cell
cycle in Schizosaccaramyces pombe (fission yeast).
His work was dogged by the scepticism of many
outside the yeast genetics community, who
thought that his findings were irrelevant to more
complex organisms such as mammals, but
Tim Hunt and Paul Nurse, Nobel Prize Ceremony, 2001
24
fortunately, Walter Bodmer, the then director of
the ICRF, was sufficiently farsighted that in 1984,
he recruited Paul to the Lincoln’s Inn Fields
laboratories. It was there that Paul’s lab
confounded his detractors by proving that the
fundamental components of the cell cycle were
conserved between yeast and man. in 1987, a postdoctoral fellow in his lab, Melanie Lee, showed
that one of the most important cell cycle
regulators in yeast, a gene called cdc2, had a
human counterpart, so establishing that the cell
cycle in humans was likely to be regulated in the
same way as in yeasts.
The research
Lee used a mutant version of fission yeast in which
the yeast’s own cdc2 gene could be inactivated by
raising the temperature at which the yeast was
growing. Loss of cdc2 activity is lethal, so growing
the mutant yeast at the higher temperature
resulted in its death. This strain could now be used
to hunt for genes able to substitute for cdc2 and
rescue the yeast from dying. Lee introduced into
the mutant strain a library in which all human
genes were represented; in such a screen, each
gene from the library is able to enter a small
number of cells in the total population. If the gene
is able to rescue (‘complement’) the lethal
mutation, the cell in which it resides is saved, and
keeps growing, making it easily recognisable in a
field of its dead and dying compatriots. Amazingly,
one human gene was able to save its host yeast
cell, and when its DNA was rescued and analysed,
it turned out to be very close in sequence to yeast
cdc2; it was in fact the human version of the gene,
conserved throughout millions of years of
divergent evolution between yeast and us.
The consequences
This pioneering work led to an exponential leap
forward in mammalian cell cycle research, as many
of the other genes already known to be important
in the yeast cell cycle also turned out to be
conserved. The mammalian cell cycle community
was therefore presented with a basic blueprint
showing them how cells reproduced, and saving
them years of arduous research. Lee and Nurse’s
publication laid the foundations for much of our
understanding of how the cell cycle is regulated in
both normal cells, and in diseases such as cancer
where the cell cycle is no longer controlled
properly.
Tim Hunt’s 1982 autoradiograph: the first sighting of cyclin
What happened next?
Paul Nurse became Director General of ICRF in
1996, and in 2002 oversaw its merger with the
Cancer Research Campaign to create Cancer
Research UK, the largest cancer research
organisation in the world outside of the USA. He
led Cancer Research UK as its first Chief Executive,
before moving to Rockefeller University as its
President in 2003. In 2010, he became President of
the Royal Society, and in 2011 moved back to
London as the first Chief Executive of the Francis
Crick Insitute. Knighted in 1999 for services to
cancer research, in 2010 he was voted Britain’s
most influential scientist.
Melanie Lee left the Nurse lab in 1988 for a highly
successful career in the pharmaceutical industry.
She is currently Chief Executive Officer of
NightstarRx Ltd, a company established to pursue
gene therapy treatment of the degenerative eye
disease Choroideremia. She was formerly CEO of
Syntaxin, and President of New Medicines and
Executive Vice President R&D with UCB, having
served as R&D Director at Celltech before its
merger with UCB. Melanie Lee currently chairs
Cancer Research Technology, the early drug R&D
and technology licensing company of CRUK.
Key references
Lee M G, Nurse P (1987). Complementation
used to clone a human homologue of the fission
yeast cell cycle control gene cdc2. Nature.
327:31-5
Nurse P (2010). Sir Paul Nurse – Autobiography.
http://nobelprize.org/nobel_prizes/medicine/
laureates/2001/nurse.html
25
Why can’t a woman be more like a man? The race for the male
sex-determining gene
1990: Peter Goodfellow’s lab show that the testis-determining factor,
which specifies maleness, is encoded by the SRY gene.
Background
Humans normally have 46 chromosomes,
comprising 22 pairs of autosomes and 1 pair of sex
chromosomes, XX for females, and XY for males.
Amazingly, the Y chromosome’s function as a
determinant of maleness has only been known
since 1959, when it was shown that the presence of
a Y chromosome overrides any number of X
chromosomes, and is necessary and sufficient for
testis development and maleness. But what was it
on the Y chromosome that specified maleness? By
painstaking analysis of patients with jumbled sex
chromosomes lacking pieces of Y, or where the X
and Y had managed to mix together, scientists were
able to narrow the region containing the so-called
Testis Determining Factor (TDF) to a chunk of the
Y-specific part of the Y chromosome, near the
boundary with the pseudoautosomal region (the
part of the Y chromosome which is able to pair with
the X). But this still meant that many hundreds of
thousands of base pairs of DNA had to be searched
for the TDF gene.
Peter Goodfellow, fanatical Arsenal fan, sometime
poet, and all round eccentric, was recruited by
Walter Bodmer in 1979, in his drive to bring human
genetics to the ICRF. Peter’s expertise lay in gene
mapping, pinpointing genes in the vast human
Peter Goodfellow
26
genome, at that time a complicated and esoteric
procedure. He had started his career mapping
components of the immune system as a PhD
student with Walter Bodmer in Oxford, and, after a
postdoc at Stanford (during which he attended the
last Sex Pistols concert and had the honour of being
spat on by Sid Vicious), he started a lab at the ICRF
to do the same thing for genes encoding tumourspecific antigens. By coincidence, one of these
mapped very near the putative TDF locus. The
unsolved problem of male sex determination
sparked Peter’s interest, and, equipped as he was
with the expertise to find genetic needles in
genomic haystacks, he decided to identify and
clone the elusive TDF gene.
The research
Things progressed very well to begin with. The
Goodfellow lab’s advanced methodologies allowed
them to produce a very good genetic roadmap of
the TDF region, and in 1987, they published a paper
in Nature which narrowed the region to be
searched to a manageable 50 - 150 kilobase (50,000
- 150,000 basepairs) piece of DNA abutting the
pseudoautosomal boundary. Then, what looked
like disaster struck; the nightmare of every scientist
working on a hot problem is to be scooped by the
competition, and, in a Cell paper in 1987, David
Page, at the Massachusetts Institute of Technology,
announced the cloning and sequencing of the gene
corresponding to Peter’s candidate locus. This
gene, which Page called ZFY, was claimed, with
much trumpeting, to be the long-awaited TDF.
After some time spent sitting under his desk writing
poetry and drinking too much coffee, Peter
emerged to continue working, when he realised
that the Page paper had not truly nailed the
problem. To be the TDF, ZFY had to fulfil a number
of criteria. The first, that it should be on the
Y-specific part of the Y chromosome, was clearly
fine, and that it appeared to be the only gene in a
rather empty area of DNA was also in its favour.
However, one significant problem was that the X
chromosome carried a highly homologous gene,
called ZFX. This was odd, as the two genes were so
closely related that it would be hard for them to
have different functions, as they must if ZFY, but
not ZFX, determined maleness. Secondly, both
Page and Goodfellow contacted an Australian
expert on marsupial genetics, Jenny Graves, and
asked her to probe the marsupial genome to check
that marsupial Zfy was also on the Y chromosome.
To everyone’s surprise, it was not; it lay on an
autosome, which was very unexpected, as
maleness in marsupials is also specified by the
presence of a Y chromosome. This was a significant
nail in the coffin, but ZFY’s short life as the TDF
truly ended after the Christmas 1989 edition of
Nature, which carried two papers, one from Peter’s
lab together with the Fellous lab in Paris, and one
from Peter’s collaborator Robin Lovell-Badge; the
first described a number of sex-reversed XX men
whose genomes did not contain ZFY, and the
second showed that murine Zfy was not found in
the cells specifying maleness.
more ancient specifier of maleness than SRY; it
exists in multiple non-mammalian species, in
contrast to SRY, which is solely mammalian.
The race was back on again, and this time, the
Goodfellow lab won. Andrew Sinclair, the PhD
student in Jenny Graves’s lab who had shown
that marsupial Zfy was autosomal, moved to
London to do a postdoc with Peter, and in 1990,
was first author on a Nature paper describing the
positional cloning and correct identification of SRY
as the gene for determining maleness. The SRY
gene was 150kb away from ZFY, and had been
missed before because it was tiny, a mere 1000
bases long.
What happened next?
In 1992, the flamboyance quotient of the ICRF was
reduced to a depressingly normal level when Peter
Goodfellow moved to the University of Cambridge
to become the Balfour Professor of Genetics. He
still carries the distinction of being the only head of
the Cambridge University Science Department to
have a ponytail. He left academia to become Senior
Vice President at Smith Kline Beecham in 1996, and
became Senior VP, Discovery Research upon Smith
Kline’s merger with Glaxo in 2001. He remained at
Glaxo Smith Kline until his retirement in 2006, and
now acts as a biotech consultant. His work on sex
determination won him the 1995 Louis-Jeantet
Prize, which he shared with Robin Lovell-Badge and
three others, and, with Lovell-Badge and David
Page, the 1997 Francis Amory Prize of the American
Academy of Arts and Sciences. He was elected to
the Royal Society in 1992, and in 1998, became a
founding member of the Academy of Medical
Sciences.
SRY was subjected to the same scrutiny as ZFY, but
passed with flying colours. The Goodfellow lab
published another paper using the Fellous lab’s
clinical material, showing that the three XX men
described in the 1989 paper had picked up the part
of the Y chromosome containing SRY; and as final
proof that SRY was the real deal, Peter published
yet another paper in Nature in 1991 with Robin
Lovell-Badge, showing that female mice
engineered to carry the SRY gene developed as
males. This latter paper, with its memorable front
cover image of Randy the sex-reversed mouse,
caused a media storm at the time, and with its
appearance, signalled an end to one of the highest
profile scientific races ever.
The consequences
SRY proved to be a difficult protein to study. After
many years of effort, we now know that it is a
rather weedy transcription factor, whose sole
purpose as a testis determining factor is to switch
on a second gene, SOX9 (also cloned in Peter’s
laboratory), which then does all the rest of the
work in establishing maleness. Interestingly, SOX9
is an autosomal gene, and appears to be a much
In a recent twist to the story, the long-held dogma
that femaleness was a default state, and to be male
one simply had to activate SRY and SOX9, was
overturned in 2009 by Robin Lovell-Badge in a
collaborative study which showed that in mice the
autosomal gene FoxL2 specifies femaleness, and
when present, overrides Sox9 and prevents ovaries
changing into testes. Loss of FoxL2 in adult female
mice upregulates Sox9, causing reprogramming of
some ovarian cell types to those found in testes.
Remarkably, as in life, the Sox9/FoxL2 story shows
that maleness and femaleness appear to be
established by what some might view as a
balancing act, and others as a war.
Key references
Sinclair AH, Berta P, Palmer MS, Hawkins JR,
Griffiths BL, Smith MJ, Foster JW, Frischauf AM,
Lovell-Badge R, Goodfellow PN (1990). A gene
from the human sex-determining region encodes a
protein with homology to a conserved DNAbinding motif. Nature. 346:240-4
Berta P, Hawkins JR, Sinclair AH, Taylor A,
Griffiths BL, Goodfellow PN, Fellous M (1990).
Genetic evidence equating SRY and the testisdetermining factor. Nature. 348:448-50
Koopman P, Gubbay J, Vivian N, Goodfellow P,
Lovell-Badge R (1991). Male development of
chromosomally female mice transgenic for Sry.
Nature. 351:117-21
27
Myc causes apoptosis
1992: Gerard Evan and co-workers show that the Myc oncogene is a
double-edged sword, not only instructing cells to proliferate, but also
causing programmed cell death (apoptosis).
The balance between proliferation and apoptosis is now a universally
recognised mechanism of growth control; the default pathway for all
cells is death, and cancers are as rare as they are because it is hard to
escape this default when things go wrong.
Background
As a postdoc in San Francisco in the lab of Mike
Bishop (co-winner of the 1989 Nobel Prize in
Physiology or Medicine), Gerard Evan developed
an interest in the molecular mechanisms of cell
growth and cell death, focusing particularly on the
biology of the enigmatic Myc protein, a powerful
engine of cell proliferation that is aberrantly
expressed in most human cancers. Gerard
continued to work on Myc upon his return to the
UK and, after a period in Cambridge, was recruited
to the ICRF in 1988. It had been known for some
time that when tissue culture cells were forced to
express large quantities of Myc protein, they
divided uncontrollably but, in addition to this,
researchers had noticed that there was a lot of
death in the cultures. This phenomenon, known
colloquially as ‘sick of myc’, had been dismissed
either as a tissue culture artefact, perhaps brought
on by exhaustion of the serum in the growth
medium in which the cells were bathed, or as a
Gerard Evan
28
toxic side-effect of Myc driving cells into division
without giving them time to get properly prepared.
Gerard, always the iconoclast, decided to find out
what exactly was going on.
The research
Gerard and his coworkers introduced the Myc
protein into a cell line, Rat-1 fibroblasts, and
verified that in conditions where the cells should
normally arrest their growth, too much Myc caused
the cells to continue to divide. As expected, there
was also a huge amount of cell death. To look more
carefully at the death, instead of simply counting
the cells or looking at them down a microscope,
Gerard used a different technique, time-lapse
video microscopy, which allowed the cultures to be
filmed. What he saw was entirely unexpected. The
cells were dying, not in a disorderly manner, but in
a highly stylised fashion: the cells lost contact with
their neighbours in culture; their nuclei condensed;
their outer membranes started to bulge and then
break away in small droplets (blebbing), and finally
their cytoplasms condensed and the cells blew up
spectacularly. When DNA from the dying cells was
analysed on size-fractionating gels, it ran as a
ladder of distinct bands. This sequence of
programmed cell death, christened apoptosis in
1972, had been described nearly 150 years
previously, and was well known, but in a different
context: as well as having a role in shaping
immunity, it is the main mechanism by which
embryos are refined; for example, all human
foetuses have webbed feet and hands, but the
webbing is removed in the womb through
regulated apoptosis. Gerard’s discovery that Myc,
thought to be solely a growth-promoting
oncogene, was also able to cause death as part of
its normal function, was a great surprise.
The consequences
The Myc paper, together with work on the prosurvival oncogene bcl2, was in the vanguard of an
explosion of research on apoptosis, which
previously had been considered a minority interest
even amongst immunologists and developmental
biologists. An average of about 40 papers a year on
apoptosis were published in the 20 years between
the coining of the phrase in 1972 and 1992, when
the Evan paper was published; the average is now
close to 10,000 a year. Apoptosis has been shown
to be universal, and to proceed by highly regulated
control mechanisms, which are now well
understood. The notion that cells have inbuilt
suicide genes, and that when they cannot detect
the appropriate signals for growth, the default
pathway is apoptosis, now underpins modern cell
biology.
The unmasking of Myc as a bringer of death as well
as life led to a complete reevaluation of theories as
to how cancers develop. Prior to 1992, the
prevailing view was that cancer was a disease of
uncontrolled growth but, as growth promoting
mutations are frequent, there was no good
explanation for why, if this were the case, we do
not develop many more cancers. The Evan paper
on Myc showed that if cells are forced to divide
uncontrollably by a mutated oncogene, the same
signal, by default, pushes the cells to die: cancer is
as rare as it is due to the intrinsic ability of our cells
to suppress uncontrolled growth by committing
suicide. If cancer is to develop, the default suicide
pathway has to be switched off by another,
cooperative, mutation. In more recent work, the
Evan lab has shown that in cancer cells transformed
by Myc, anti-apoptotic mutations have also
occurred and, if these mutations are reversed, the
cells die. Drugs attacking this weakness are now in
Phase III clinical trials.
What happened next?
Gerard Evan was appointed Napier Professor of the
Royal Society in 1996, whilst still at the ICRF. In
1999, upon his move to the University of California
San Francisco Cancer Center, Gerard became
Gerson and Barbara Bass Bakar Distinguished
Professor of Cancer Biology. He returned to the UK
in 2009 and is currently the Sir William Dunn
Professor and Head of the Biochemistry
Department at Cambridge University. He is an
EMBO member, and a Fellow of both the Academy
of Medical Sciences and the Royal Society. Gerard’s
current research is focused on understanding the
processes responsible for genesis and maintenance
of cancers, in particular cancers of the pancreas,
colon, brain, skin and liver. Understanding the
molecular mechanisms that underlie the cell
suicide machinery and how it can be manipulated
therapeutically are still the overarching aims of his
laboratory, and he continues to be one of the most
productive and thought-provoking cancer
biologists working today.
Key references
Evan GI, Wyllie AH, Gilbert CS, Littlewood TD,
Land H, Brooks M, Waters CM, Penn LZ,
Hancock DC (1992). Induction of Apoptosis in
Fibroblasts by c-myc Protein. Cell. 69:119-28
29
DNA repair
1980s-2000s: The Lindahl lab identifies and dissects the base and
nucleotide excision repair machinery.
1995: Rick Wood reconstitutes mammalian nucleotide excision repair
in vitro.
Background
For as long as the DNA double helix has been
around, environmental and cellular stresses have
conspired to inflict damage upon it. Heat-induced
breakages, ultraviolet light, ionizing radiation,
chemicals, and the inbuilt frailty of the DNA
molecule itself all contribute to the daily toll that
life takes upon our genetic material. So it’s no
surprise that even the simplest organisms use a
spectacular battery of error-detection and repair
mechanisms to keep their precious DNA as
fault-free as possible.
To begin with, all the work on DNA repair
happened in single-cell organisms, where one
could use biochemistry to purify components of
the repair machinery, and genetics to look at
function. However, this began to change when it
was realised that many of the ancient enzymes
discovered in bacteria were conserved in complex
multicellular organisms such as ourselves.
Furthermore, it also became clear that the DNA
repair processes in humans were clinically
important: firstly, mutations in human DNA repair
enzymes started to crop up as the causative factors
in some inherited diseases, and secondly, where
the DNA repair machinery was working efficiently,
it was opposing the action of the anti-cancer drugs
and therapies which worked by damaging tumour
cell DNA. Clearly, working on DNA repair was a
fitting topic for a cancer research institute.
Rick Wood
30
Tomas Lindahl
In the 1970s, the ICRF took its first steps into the
DNA repair field via the brilliant molecular biologist
John Cairns, who was recruited from Cold Spring
Harbor to be Director of the ICRF unit at Mill Hill.
On his departure for Harvard and a MacArthur
Genius Award in 1980, he was replaced by a major
biochemical star who, in addition to his
fundamental work on DNA repair, was to shape and
guide the ICRF Clare Hall Laboratories as their first
Director: Tomas Lindahl.
The research
As a postdoc with Jacques Fresco at Princeton in
the mid-1960s, Tomas was the first person to
realise that DNA was inherently unstable, and
could be damaged without any requirement for
exogenous mutagens. By their very nature, some of
the chemical bonds formed inside the bases, and
between the bases and the sugar-phosphate
backbone, are relatively susceptible to breakage.
Cytosine, for example, can be readily deaminated,
that is, stripped of its amino group, which turns it
into the simpler base, uracil. Uracil can pair
comfortably with adenine, rather than with
cytosine’s proper partner guanine, leading to
errors in replication and transcription similar to
those induced by chemically- and radiationinduced damage.
Base Excision Repair The discovery of DNA’s
instability defined Tomas’s career. In tracking down
how the cytosine-to-uracil error was fixed, Tomas
and his lab unmasked an entirely new class of
repair mechanism: base excision repair. Uracil is
detected and chopped out of the DNA chain by a
glycosylase, an enzyme able to cleave the bond
between the errant base and the backbone, leaving
the double-stranded backbone intact. A gang of
other enzymes then take over, chopping out the
sad remains of the original nucleotide together
with its nearest neighbours, mending the gap using
the opposite strand as a template, and finally
stitching up the break.
Over the next decades, Tomas and his lab identified
many of the weird and wonderful enzymes and
mechanisms involved in multiple types of DNA
repair, including the suicide inactivator O6methylguanine-DNA methyltransferase, which can
restore mutated O6-methylguanine bases to health by
removing the methyl groups and disposing of them by
methylating itself – the enzymatic equivalent of falling
on a live grenade (remarkably, Tomas found that
O6-methylguanine-DNA methyltransferase is also a
transcription factor, activating several repair-essential
genes, including itself). And in 1994 and 1996,
returning to Tomas’s first love, his lab achieved a
biochemical triumph: the purification and
reconstitution of base excision repair in vitro, using
respectively the E.coli and human enzymes, some of
which were remarkably conserved across the yawning
evolutionary gulf.
Xeroderma pigmentosum In 1985, Tomas hired a new
American postdoc, Rick Wood, to work on xeroderma
pigmentosum (XP). XP is a recessive autosomal
inherited disease whose sufferers exhibit extreme
sensitivity to ultraviolet light and have a strong
predisposition to skin cancer. Its genetics are complex
– there are at least ten different genes implicated – but
the root of the XP problem had been known since the
1960s: XP patients were unable to repair UV-induced
DNA damage properly. However, in the nearly 20 years
since the defect had been defined, no one had
managed to determine the mechanism involved. The
problem was the lack of a cell-free system in which to
do the fiddly biochemistry required to identify and
purify the UV-repair machinery.
Repair of UV damage in E.coli takes place via a process
called nucleotide excision repair, one of the first
repair mechanisms discovered. In a paper published in
1988, Rick, Peter Robins and Tomas showed that a
similar process was occurring in humans. Using
extracts made from human cells growing in tissue
culture, they succeeded in developing a cell-free
system capable of repairing UV-damaged DNA in a
test tube. If extract made from the cells of XP patients
was added to the assay, repair no longer happened;
the XP defect appeared to affect the detection and
nicking of damaged DNA before the repair process to
remove dud bases and replace them with the correct
ones had even kicked in.
Rick was clearly too valuable to lose, and in 1988, he
started his own laboratory at Clare Hall, to study the
biochemistry of inherited syndromes, with a particular
interest in XP. In 1995, Tomas’s faith in him paid off
when the Wood lab, together with collaborators in
France, Switzerland, Finland and the US, published a
completely reconstituted mammalian nucleotide
excision repair system. About 30 separate proteins
had been identified, purified and characterised, a
formidable achievement in the field of DNA repair.
The consequences
Correct DNA repair is the bedrock on which healthy
cells thrive. Cells have an elaborate system of
checks and balances in their transcriptional and
cell-cycle machinery to detect DNA damage, assess
its seriousness, and decide whether to fix it or to
self-distruct. The medical consequences of
defective repair are legion: for example, nucleotide
excision and mismatch repair genes are essential
for the prevention of cancer and neurological
disease. With our increasing understanding of
defects in DNA repair and checkpoint control in
cancer, there is also extensive interest in targeting
particular DNA repair and checkpoint pathways for
cancer therapy.
What happened next
Tomas Lindahl remained as Director of the Clare
Hall Laboratories until 2005, retiring in 2009. He is
still one of the world’s foremost authorities on
DNA damage. For his many landmark discoveries in
DNA repair, he has been awarded many prizes and
honours. He is an EMBO member, a Fellow of the
Royal Society and the Academy of Medical
Sciences, and has won both the Royal Society’s
Royal Medal and Copley Medal. The latter is the
oldest scientific award in the world, whose other
recipients include Charles Darwin, Michael Faraday
and Stephen Hawking.
Rick Wood returned to the US in 2001, and is
currently Grady F. Saunders Distinguished
Professor in Molecular Biology at The University of
Texas MD Anderson Cancer Center. He is a Fellow
of the Royal Society and an EMBO member, and in
1998 won the Meyenburg Prize for Cancer
Research, one of Germany’s most prestigious
science awards.
Key references
Lindahl T (1996). The Croonian Lecture, 1996.
Endogenous Damage to DNA. Phil Trans R Soc
Lond B. 351:1529-38
Wood R, Robins P, Lindahl T (1988).
Complementation of the Xeroderma Pigmentosum
DNA Repair Defect in Cell-Free Extracts. Cell.
53:97-106
Aboussekhra A, Biggerstaff M, Shivji MKK,
Vilpo JA, Moncollin V, Podust VN, Protic M,
Hubscher U, Egly J-M, Wood, RD (1995).
Mammalian DNA Nucleotide Excision Repair
Reconstituted with Purified Protein Components.
Cell. 80:859-68
31
Regulation of DNA replication
2015: John Diffley’s group reconstitutes regulated DNA replication
origin firing using purified proteins. This work marks the culmination
of over twenty five years’ study of the mechanism of replication
initiation and its control by the cell cycle and DNA damage response
machinery.
Background
Every time a cell divides, it has to make a complete
and accurate copy of its genome. If it does not, the
result is disastrous, resulting in at best, a crippled
existence likely to end in early death, and at worst,
mutation into a cellular psychopath with the
potential to cause cancer and a multitude of other
diseases. DNA replication, the process whereby the
genome is copied, is therefore one of the most
important and basic processes a cell undertakes.
The great biochemist Arthur Kornberg began the
study of DNA replication in the mid-1950s, by
looking at the enzymes required for replication in
basic organisms such as bacteria. Bacteria, with
their small genomes, start replicating from one
particular point in their DNA, known as the origin
of replication, and just continue until they have
gone full circle. This is a relatively simple process.
Eukaryotes, however, have a far larger complement
of DNA, which is broken up into separate
chromosomes, and therefore they need multiple
origins of replication in order to carry out the
process on each chromosome, and within a
reasonable timescale; in the case of mammalian
genomes, origins of replication number in the tens
of thousands. The problem of how all these origins
are able to fire just once in every cell cycle to
John Diffley
32
orchestrate the complete replication of the
genome has preoccupied researchers for the last
40 years.
In the late 70s and early 80s, Bruce Stillman’s lab at
Cold Spring Harbor, Long Island, NY, was one of the
dominant forces in the study of eukaryotic DNA
replication, using a biochemical approach to
laboriously purify and then identify the proteins
involved in viral DNA replication. John Diffley came
to the Stillman lab at Cold Spring Harbor as a
postdoc in 1985, where he initiated work on DNA
replication in yeast, the simplest eukaryotic
organism there is. Yeast is easy to grow and
manipulate, and can be used not only for
biochemistry but also for genetic studies, giving
scientists more versatility in how they approach
problems. Crucially, what holds for yeast is
frequently broadly true for higher eukaryotes, and
therefore it is a great model organism. During his
time with Stillman, John found the first ever
protein known to bind to eukaryotic replication
origins, and on the back of his outstanding
postdoctoral work, he was recruited to the Clare
Hall Laboratories of the ICRF in 1990.
The research
John Diffley studies how replication begins, the
mechanisms that ensure replication is triggered
only once during each cell cycle, and what happens
when replication is not initiated properly. His work
on all of these issues has been of seminal
importance.
ORC: After his arrival at Clare Hall, John began work
to identify the proteins bound at yeast replication
origins in a region of DNA known to be essential for
the origins to fire properly. In 1992, his lab and
Stillman’s defined these proteins as the Origin
Replication Complex (ORC), and the following year,
in collaboration with Kim Nasmyth’s lab, the Diffley
group, along with several others cloned Orc2, the
first gene of the six-member complex, and showed
by genetics that Orc2 was essential for replication.
PreRC and Licensing Factor: The identification of
ORC created a great deal of interest, but it was
unlikely to be what determined the timing of
replication during the cell cycle, as it was bound to
DNA almost all of the time. The lab decided to look
for other replication origin proteins which bound
only at particular points during the cell cycle,
reasoning that they might play a role in replication
timing. They found that one particular DNA
sequence in replication origins was only occupied
by proteins during a phase of the cell cycle called
G1, which occurs immediately before S phase, the
point where the genomic DNA is replicated. In
1994, they published evidence that this protein
complex, dubbed the Pre-Replicative Complex
(preRC), was very similar to Xenopus Licensing
Factor, an activity in frog eggs required for
initiation of replication. That similar complexes
were found in two such dissimilar organisms
suggested, much to everyone’s excitement and
relief, that the control of replication regulation
might be broadly the same in all eukaryotes, and
this has indeed turned out to be the case. In 1996,
the Diffley and Nasmyth labs again collaborated to
identify the first preRC complex protein, Cdc6, and
the Diffley lab subsequently demonstrated that
Cdc6 was required to recruit other components,
the MCM helicase proteins, into the complex. Last
year, 18 years on, they reconstituted the entire
preRC step in a test tube using purified proteins, a
scientific and technological tour de force.
DNA replication is inextricably linked to the cell
cycle: Having found that assembly of the
replication complex was cell cycle-regulated, the
next step was to find out how. In this again, the
Diffley and Nasmyth labs were, in 1996, the first to
show that the reason the preRC is only present on
DNA during G1 phase is that a class of enzymes
called cyclin dependent kinases (CDKs) modifies
and inhibits the preRC; G1 is the only time during
the cell cycle that CDKs are absent. Put together
with work from other researchers, including
Diffley’s Nobel Prize winning colleague at Clare
Hall, Tim Hunt, these results provided an elegantly
simple solution to the puzzle of how replication
only happens once during the cell cycle. First, the
preRC binds to replication origins in G1 phase,
readying the DNA for replication. Then, in S phase,
CDKs are made, and the MCM helicase is activated,
allowing the DNA to be unwound and then copied.
The CDKs switch on the initiation of DNA synthesis,
the genome is replicated, and the cells go through S
phase and into mitosis, or cell division. When G1 of
the next cell cycle comes round again, CDKs are
degraded, and the whole process repeats. In a
further flourish, the Diffley lab has shown exactly
how the CDKs inhibit the preRC, and, in 2007, that
they trigger S phase DNA replication by modifying
just two proteins, Sld2 and Sld3.
How DNA damage halts replication: The Diffley
lab has also worked extensively on how replication
in S phase is halted in response to DNA damage.
The so-called DNA damage checkpoint shuts down
the firing of late replication and this works via
modification of two key initiator proteins, Sld3 and
Dbf4. Again, CDKs turned out to be intimately
involved. There are also other mechanisms at work
too; the checkpoint can stabilise stalled replication
forks by inhibiting a crucial enzyme, the flap
endonuclease Exo1.
The ubiquity of CDKs in controlling when and
whether DNA replication occurs has major
consequences in many cancers, as CDKs are
frequently deregulated, switched on at the wrong
times, and/or overactive. Misfiring of the
replication origins leads to genome instability, and
if the normal damage limitation procedures are not
in place, the genesis of a tumour. Diffley’s work is of
extreme importance both in terms of basic science,
and also for the development of new anticancer
therapeutics targeting DNA damage checkpoints.
What happened next?
John Diffley became Deputy Director of the
London Research Institute and Director of the Clare
Hall laboratories in 2005. He is currently an
Associate Research Director of the Francis Crick
Institute. He was elected to EMBO in 1998, FRS in
2005, and elected a Fellow of the American
Association for the Advancement of Science in
2007. He won the Paul Marks Prize for Cancer
Research in 2003.
Key references
Diffley JFX, Cocker JH, Dowell SJ, Rowley A (1994).
Two steps in the Assembly of Complexes at Yeast
Replication Origins In Vivo. Cell. 78:303-16
Santocanale C, Diffley JF (1998). A Mec1- and
Rad53-dependent checkpoint controls late-firing
origins of DNA replication. Nature. 395:615-8
Tercero JA, Diffley JF (2001). Regulation of DNA
replication fork progression through damaged DNA
by the Mec1/Rad53 checkpoint. Nature. 412:553-7
Zegerman P, Diffley JF (2007). Phosphorylation of
Sld2 and Sld3 by cyclin-dependent kinases
promotes DNA replication in budding yeast.
Nature. 445:281-5
Yeeles JT, Deegan TD, Janska A, Early A, Diffley JF
(2015). Regulated eukaryotic DNA replication origin
firing with purified proteins. Nature. 519:431-5
33
Into the nucleus: regulation of transcription
1992: Identification of SAP-1, the first growth factor-regulated
transcription factor.
1995: Demonstration that proteins involved in controlling cell shape
and structure can also regulate transcription, leading to the later
demonstration that transcription can be directly regulated in
response to cytoskeletal dynamics.
Background
At the end of the 1970s, it was becoming evident
that there was a relationship between growth
factors, oncogenes, and the activation of gene
expression: cells required gene transcription to be
activated in order to respond to growth signals, so
there must be a signalling pathway leading from
outside the cell, where growth factors docked,
right across the cytoplasm and into the nucleus.
What this was, and how it caused gene
transcription, was the next big challenge in the
transcription field.
All genes are regulated by specific DNA control
sequences often found in front (upstream) of the
gene. It was clear that the secret of how signals
were transmitted to genes lay in these regulatory
sequences, but it was a mystery how this worked.
Whilst at the MRC Laboratory of Molecular Biology
in Cambridge, Richard Treisman begun working on
how the Fos gene, cloned by Tom Curran at the
ICRF (Highlight 5), was regulated. Fos is an
immediate early gene, switched on almost
instantly when a growth signal is received by a cell,
and Richard showed very quickly that this response
was controlled by a short DNA sequence, the
Serum Response Element (SRE) in the Fos
Richard Treisman
34
upstream regulatory region, to which was bound a
protein, the Serum Response Factor (SRF). After his
initial work, he and others found SREs in the control
regions of many other genes, demonstrating this
was a widespread mechanism of rapid gene
induction. However, there were still huge holes in
the pathway; although the SRE and SRF were
necessary for Fos gene induction, the method by
which the signal to fire up transcription reached
them was still unknown. In 1988, lured by the
presence of a substantial community working on
the control of eukaryotic transcription, Richard
returned to the ICRF, where he’d been a PhD
student, bringing this puzzle with him.
The research
Work on SRF continued in the Treisman lab, but
soon hit a block; SRF did not seem interested in
responding to any growth factor signals, and did
not work very well in assays where it was asked to
activate growth-factor responsive transcription by
itself. However, others in the transcription field had
descended in force on the SRE, and Peter Shaw and
Alfred Nordheim in Germany showed in 1989 that
SRF bound the SRE with a partner protein, which
they called TCF (ternary complex factor). Steve
Dalton, an Australian postdoc, used a genetic
screen to identify a protein called SAP-1 as a TCF;
and Nordheim’s group produced Elk-1, another
member of the same protein family as SAP1, as a
second TCF candidate.
But was the SAP-1 and Elk-1 family the link to
growth factor control of the SRE? While this work
was proceeding, the study of a set of enzymes in
the cell known as the MAP kinases was rapidly
merging with studies of proto-oncogenes, leading
to the discovery of a pathway whereby growth
factor receptors activated the Ras oncogene, which
then signalled to a kinase called Raf, which in turn
activated another kinase called ERK (Highlight 14).
Kinases act by putting phosphate groups onto other
proteins at particular amino acids, and excitingly,
both SAP-1 and Elk-1 contained the correct amino
acid sequences that might allow them to be direct
targets of ERK. Richard Marais, another Treisman
postdoc, spent much of the summer of 1992 in the
lab, and was rewarded for his lost holidays by data
showing definitively that ERK was able to
phosphorylate Elk-1, and that this was the
activating signal switching on SRE-regulated
transcription. For the first time, a complete
pathway leading from outside the cell into the
heart of the nucleus had been delineated.
Although it was clear that growth factors switched
on the SRE through the SRF-TCF interaction,
something about transcriptional regulation by the
SRE was still very puzzling. The Serum Response
Element, as its name suggests, is activated by
serum, a rich liquid mix of nutritional goodies used
for growing cells in tissue culture. However, there
was something in serum capable of activating the
SRE that was not a protein growth factor. Caroline
Hill, another postdoc, worked out that this extra
something was lysophosphatidic acid, or LPA, and
that for LPA induction, TCF proteins were not
required. Furthermore, the signal coming to SRF
was not transmitted by the MAPK pathway, but by
a totally different route, by Rho family proteins,
whose normal function is in remodelling the actin
cytoskeleton. The actin cytoskeleton is the main
structural framework of a cell, giving it both shape
and strength, and it must be continually
remodelled in response to the cell’s need to move,
to change shape, and to adhere to the extracellular
matrix and to other cells. The observation that the
enzymes involved in cytoskeletal control are also
activating transcription has supplied an entirely
new perspective on a cell’s ability to fine tune all its
processes in response to a stimulus.
The consequences
This work was extremely important to the nascent
field of transcriptional regulation as it laid down
some basic principles not only in terms of the
mechanics of regulation itself but also in its use of
innovative experimental techniques: Richard and
his coworkers adapted and invented methodology
which became universal tools in transcription
research. That the lab won some very hard-fought
contests in a highly competitive field is a tribute to
Richard’s unerring instinct for discerning and
pursuing what was interesting, and discarding what
was not, and his recruitment of a cohort of very
good scientists who were as unafraid of hard work
as he was himself.
The Treisman lab has continued its work on SRF and
its protein partners, and is now studying not only
basic transcriptional mechanisms but also the
biological readout of the different interactions and
signalling pathways that intersect at SRF. Their work
on how the actin cytoskeleton controls SRF is
particularly interesting. The targets of Rho family
signalling have been identified as Myocardin-related
Transcription Factors (MRTFs) called MAL and Mkl2.
MRTFs bind SRF, and the SRF-MRTF complex
performs an analogous role to SRF-TCF as the
terminus for actin cytoskeleton-regulated signalling.
Rho signalling to SRF-MRTF works by an entirely
novel mechanism; there are fluxes in the amount of
MRTF protein free to regulate transcription,
depending on how much is bound by actin, and this
itself is regulated very tightly by what the cell is
doing at any given time. Rho signalling is strongly
implicated in cancer cell invasion and metastatic
tumour spread, and the lab has now shown that
these processes require transcription regulation via
the Rho-actin-MRTF-SRF signalling pathway.
What happened next?
Richard Treisman became Director of the LRI in
2000, and is now a Research Director at the Francis
Crick Institute. He is an EMBO member, and a fellow
of the Royal Society and the Academy of Medical
Sciences. For his work on growth factor regulation
of transcription, he was awarded the 1995 EMBO
Gold Medal, and the 2002 Louis-Jeantet Prize.
Steve Dalton is Chair of Molecular Biology at the
University of Georgia. His lab works on the
molecular biology of stem cells.
Caroline Hill has been a group leader at the LRI
since 1998, working on the Transforming Growth
Factor family of signalling molecules. She was
elected an EMBO member in 2002.
Richard Marais is now Director of Cancer Research
UK’s Manchester Research Institute. He is a member
of EMBO and the Academy of Medical Sciences.
Key references
Dalton S, Treisman R (1992). Characterization of
SAP-1, a protein recruited by serum response factor
to the c-fos serum response element. Cell. 68:597612
Marais R, Wynne J, Treisman R (1993). The SRF
accessory protein Elk-1 contains a growth factorregulated transcriptional activation domain. Cell.
73:381-93
Hill CS, Wynne J, Treisman R (1995). The Rho
family GTPases RhoA, Rac1, and CDC42Hs
regulate transcriptional activation by SRF. Cell.
81:1159-70
Miralles F, Posern G, Zaromytidou A-I, Treisman R
(2003). Actin dynamics control SRF activity by
regulation of its coactivator MAL. Cell. 113:329-42
35
Upstream and downstream of the RAS oncoprotein
1990s: Julian Downward shows that signals from outside the cell can
activate the Ras oncoprotein, and goes on to demonstrate that
growth factor regulation of Ras controls the Raf-MAPK pathway.
Background
In 1981, Bob Weinberg’s lab at the Whitehead
Institute in Boston demonstrated for the first time
that there were human genes that could cause
cancer when mutated. Using the new technique of
DNA transfection, whereby foreign DNA could be
introduced directly into cells, they showed that a
fragment of DNA from a human tumour was able to
transform mouse NIH3T3 tissue culture cells. Other
groups began to see the same results using DNA
from other tumour types, and together, they
realised that the fragments of human DNA they
were isolating all contained the H-ras gene, the
cellular homologue of v-H-ras, one of two closely
related retroviral oncogenes from Rat Sarcoma
viruses. By the late 80s, Ras family proteins had
been shown to be attached to the inside of the cell
membrane, and to belong to a larger protein family,
the GTP binding proteins. Such proteins are able to
bind guanine triphosphate (GTP), a product of
cellular metabolism, and convert it to guanine
diphosphate (GDP). It was known that the
mutations that converted normal Ras proteins into
oncogenes caused them to stick in the GTP-bound
activated state, so they were permanently switched
on. However, Ras proteins existed in a knowledge
vacuum: nobody knew what proteins normally lay
upstream of them to cause the switching from GTP
to GDP binding, or what lay downstream.
Julian Downward, who as a PhD student at ICRF
had shown that the Epidermal Growth Factor
Julian Downward
36
Receptor gene EGFR had been picked up by a
retrovirus and converted into the erb-b oncogene
(Highlight 6), had gone off in 1986 to do a postdoc
in Bob Weinberg’s lab. He ended up spending quite
a lot of his time there unsuccessfully trying to work
out what the signal might be, which triggered the
activation of normal Ras proteins. In 1989, he
returned to the ICRF as a lab head, bringing with
him the problem he had been working on in
Boston.
The research
Ras can be activated by extracellular stimuli: At
the ICRF, Julian met Doreen Cantrell, a leading T
cell immunologist. As an experimental tool for
studying signal transduction, T cells are extremely
attractive, as they can be easily purified, and fooled
in vitro into activating themselves if they are fed
mock antigens. Doreen suggested to Julian that her
T cell system would be an ideal place to look at
what was activating Ras. The shift in perspective,
as can often happen in scientific research, broke
the logjam. In four incredibly productive months,
the two labs showed for the first time that the Ras
protein could be activated very rapidly in response
to extracellular signals stimulating the T cell
receptor, the cell surface molecule on T cells to
which antigen binds.
Upstream of Ras: With this fundamental
observation, that the mechanism of normal Ras
activation was via a signal from outside the cell, the
race was on to try to fit together the gaps in the
signalling pathway between the receptor on the
cell surface and Ras. After the first observation in T
cells, many other receptors were shown to respond
to extracellular stimuli by switching on Ras,
including Julian’s old friend EGFR. Julian and his
postdoc Laszlo Buday realised that work done
genetically dissecting Ras signalling in flies could be
put together with biochemical clues from
mammalian cells to produce two likely candidates
for the missing links, the Sos and Grb2 proteins. Sos
was known to act downstream of the fly EGFR gene
and upstream of fly Ras, and there was also good
reason to think that it was a guanine exchange
factor, able to catalyse rapid exchange of GDP for
GTP, and therefore a prime candidate for activating
Ras. Grb2 had been proposed to be an adaptor
protein, acting as a link between the EGFR and
intracellular signalling components, and there was
indirect evidence implicating it in Ras activation.
Julian and Laszlo used antibodies to
immunoprecipitate Ras protein along with Grb2
and Sos, and found that the interactions they
detected resulted in Ras becoming activated. Their
efforts successfully showed that the EGFR, when
activated by EGF binding, recruited Grb2 to its
intracellular tail, that Grb2 in turn bound Sos, that
Sos had hold of Ras, and that all together, this
enabled Ras to be activated. The missing upstream
links had been defined.
But what lay downstream of Ras, between it and
the genes controlling the cell cycle? Part of the
answer lay with another proto-oncogene, cRaf.
cRaf protein is a kinase (an enzyme catalysing the
addition of phosphate groups to proteins), and
there was indirect evidence linking it with
activation of Ras: disruption of cRaf activity had
been shown to block Ras action. The Downward lab
used a piece of cRaf as a biochemical bait to see
whether it was able to pull down Ras protein, and
showed that indeed, Ras and cRaf interacted, but
only when Ras was in its activated, GTP-bound
state. This was a crucial interaction to map, as
downstream of cRaf lay the mitogen-activated
protein kinase (MAPK) cascade. Kinases belonging
to the MAPK family are used throughout evolution
to control the cellular responses to external signals
such as growth factors, nutrient status, stress or
inductive signals, and, as shown in the previous year
in Richard Treisman’s lab at the ICRF, MAPK could
regulate transcription factors (Highlight 13). The
Ras signalling pathway had reached the nucleus.
There was one further twist to the Ras story, as it
became clear to Julian that not all of the things Ras
did could be explained by it simply activating the
MAPK pathway. Another protein,
phosphatidylinositol-3-kinase (PI3K), had been
shown in 1992 to interact with Ras in vitro, and PI3K
was known to be activated in response to the same
stimuli as Ras. Julian’s lab, in collaboration with his
old mentor, Mike Waterfield, demonstrated in
1994 that activated, GTP-bound Ras bound directly
to PI3K, and that this interaction was likely to result
in PI3K being activated in turn. The Downward lab’s
work had placed Ras at the apex of two
downstream signalling cascades, both of which are
vitally important for cell growth.
The consequences
The mechanisms of Ras signalling have been
fleshed out in the intervening years such that we
now have a very detailed map of signal
transduction in both normal and cancerous cells.
Pathways turn out to be interconnected, and the
molecular wiring diagrams for different cell types
are varied and complex. Scientists are turning
towards systems biology to model how
perturbations in one part of a cell’s wiring changes
outcome for the cell, and signal transduction
research is becoming rigorously quantitative.
Mutations in Ras genes account for some 20% of all
human cancers. More work from the Downward
lab has shown that mice with mutations in PI3K that
block its ability to interact with RAS are highly
resistant to lung tumours induced by KRAS, and
skin tumours induced by HRAS. Interaction of RAS
with PI3K is needed for normal growth factor
signalling and also for RAS-driven tumour
formation.
What happened next?
Julian Downward became LRI Associate Director in
2005, and is now an Associate Research Director at
the Francis Crick Institute. He was elected to EMBO
in 1995, became a fellow of the Royal Society in
2005, and a fellow of the Academy of Medical
Sciences in 2009. He is one of the top 20 most cited
European cell biologists.
Doreen Cantrell CBE moved from the LRI in 2002 to
become Professor of Cell Biology at the University
of Dundee, where she is now Vice Principal and
Head of the College of Life Sciences. She is an
EMBO member and a Fellow of the Academy of
Medical Sciences, the Royal Society of Edinburgh
and the Royal Society.
Laszlo Buday returned to his native Hungary, and is
now Director of the Institute of Enzymology of the
Hungarian Academy of Sciences in Budapest.
Pablo Rodriguez-Viciana runs a lab at the UCL
Cancer Institute, and is still working productively
on the RAS family of proteins.
Key references
Downward J, Graves JD, Warne PH, Rayter S,
Cantrell D (1990). Stimulation of p21ras upon T-cell
activation. Nature. 346:719-23
Buday L, Downward J (1993). Epidermal Growth
Factor Regulates p21ras through the Formation of a
Complex of Receptor, Grb2 Adapter Protein, and
Sos Nucleotide Exchange Factor. Cell. 73:611-20
Warne PH, Rodriguez-Viciana P, Downward J
(1993). Direct Interaction of Ras and the aminoterminal region of Raf-1 in vitro. Nature. 364:352-5
Rodriguez-Viciana P, Warne PH, Dhand R,
Vanhaesebroeck B, Gout I, Fry MJ Waterfield MD,
Downward J (1994). Phosphatidylinositol-3-OH
kinase as a direct target of Ras. Nature. 370:527-32
37
Interferon signalling and the JAK-STAT pathway
1992: Interferons are shown to signal through a novel pathway
involving the JAK and STAT protein families.
38
Background
In 1957, Alick Isaacs and Jean Lindenmann, working
at the National Institute for Medical Research
(NIMR) in Mill Hill, described a new phenomenon,
‘virus interference’. Chicken cells infected with
influenza virus produced and secreted a factor
which made non-infected cells resistant to viral
attack, not just from ‘flu viruses, but also from a
myriad of other virus types. The mysterious
antiviral factor was a small protein, which Isaacs
and Lindenmann named ‘interferon’. During the
1960s and 1970s, excitement mounted when it
became clear that interferons, by then shown to be
a family of proteins, did not just inhibit viral
infection, but also had antitumour activity. By 1981,
purified interferons were entering clinical trials as
treatments for both viral infection and cancer.
the end, from the genes being regulated in the
nucleus. George and Ian’s labs, working closely
together, went for the latter strategy, searching for
genes that were switched on when the interferon
family members IFN-α, IFN-β and IFN-γ were added
to cells. Like Richard Treisman (Highlight 13), they
reasoned that these genes must be activated by the
binding of sequence-specific, interferon-inducible
transcription factors. If they could identify the DNA
sequences to which the factors bound, they could
bootstrap their way to the factors, and thence to
the rest of the proteins in the pathway. By 1988,
they, and Jim Darnell’s lab in the US, had identified
short stretches of DNA to which interferonregulated transcription factors might bind. The
hunt to identify these transcription factors, and the
signalling cascade that led to them, was on.
In 1980, to boost its activity in the expanding
interferon field, the ICRF Director Walter Bodmer
hired a new faculty member, Ian Kerr. Ian had
made his name some years previously by
discovering one of the major antiviral defence
mechanisms activated by interferon, the 2-5A
system, and it was a major coup for the ICRF to
have bagged such a big star of the interferon field.
Matters improved still further in 1983 when Ian’s
long-time friend and collaborator, George Stark,
also moved to the ICRF, to work more closely with
Ian on the problem of exactly how interferon
signalled into cells.
To define a signalling pathway, one can start at the
beginning, at the cell surface, or work back from
The research
In 1989, Ian and George’s PhD student Trevor Dale
published a paper describing an interferoninducible DNA binding factor, dubbed ISGF3.
Attempts to purify ISGF3 in London were preempted by Jim Darnell’s lab, which, after some
heroic biochemistry, showed in 1992 that ISGF3 was
a multiprotein complex, containing three proteins
eventually named Interferon Responsive Factor 9
(IRF9), STAT1 and STAT2 (for Signal Transducer and
Activator of Transcription). Darnell’s lab also found
that in response to an interferon signal, the STAT
proteins are phosphorylated on a particular amino
acid, tyrosine. How all this fitted into a complete
pathway was unclear, though, and might have
remained so for some time but for an Italian
George Stark
Ian Kerr
postdoc in George’s lab, Sandra Pellegrini, who
used a genetic approach to solve the problem.
Sandra had arrived in George’s lab in 1986, and was
given the task of mutating cells so that they would
no longer respond to INF-α stimulation. The idea
was that once she had made a mutant cell line, she
could introduce a library of normal genes into it,
and find which one was able to rescue
(‘complement’) the mutation. Sandra developed a
cunning drug selection strategy whereby only
those mutant cells that did not respond to
interferon would survive. It took a while, and was
incredibly labour-intensive, but in the end, all the
patience and toil paid off: Sandra published a paper
in 1989 containing details of the mutant cell line,
before leaving London to set up her own lab at the
Institut Pasteur in Paris. She subsequently cloned
TYK2, a member of the Janus Kinase (JAK) family,
which phosphorylate proteins on tyrosine residues
when cells are stimulated with interferons.
Following Sandra’s success, Diane Watling, working
with Ian and George, used a cell surface marker – in
place of Sandra’s drug–based selection – to
establish that IFN-γ, of intense interest in the
immune response, also signals through a JAKdependent pathway. In all, eight cell lines defective
in interferon-inducible signalling were isolated.
They proved incredibly powerful tools; between
them, they covered the entire signalling pathway,
including the STAT proteins that the Darnell lab had
already identified, and could be used for
verification of any genes thought to encode
pathway components. The cell lines could also be
used for functional studies on the pathway
proteins, defining the domains of the proteins
required to carry out particular tasks and to
contact partners in the signalling cascade. By 1994,
when Ian, George and Jim Darnell jointly published
an influential review on the subject, the basics of
the pathway were well understood: when
interferons bound to their receptors, the JAK
kinases, which were associated with the inward
facing parts of the receptors, were activated; they
then bound and phosphorylated STAT proteins,
allowing them to move into the nucleus, find their
DNA binding sites, and fire up the interferoninducible genes. In a few hectic years, an entire
novel signalling pathway had been defined.
The consequences
Interferon’s early therapeutic promise has been
partially fulfilled: synthetic interferons are used to
treat multiple sclerosis, hepatitis B infection and
several different types of cancer, particularly
kidney cancer, malignant melanoma, multiple
myeloma and some types of leukaemia.
Interferons belong to a larger class of proteins
called cytokines, which are vitally important in cell
signalling, particularly in the immune system.
JAK-STAT pathways are used by most cytokines and
a large number of other extracellular signalling
molecules, and JAK and STAT family members are
used in a myriad of different combinations to elicit
different effects in different biological contexts.
What happened next?
Ian Kerr remained at the LRI until his retirement in
2005. He is an EMBO member, and a Fellow of the
Royal Society and the Academy of Medical
Sciences. He shared the Milstein Prize of the
International Society for Interferon and Cytokine
Research in 1993 and won it again, with George
Stark and Jim Darnell, in 1997. In 1999 the trio,
together with two others, won the William B Coley
Award for Distinguished Research in Basic and
Tumour Immunology. In 2003, Ian received the
Feldberg Foundation Prize for Outstanding Work in
Medical or Biological Sciences.
George Stark returned to the US in 1992 to become
Chair of the Lerner Research Institute at the
Cleveland Clinic Foundation. He remains there as a
Distinguished Scientist, with a joint appointment as
Emeritus Professor of Genetics at Case Western
Reserve University. In addition to the Milstein Prize
in 1997 and Coley Award in 1999 he has been
elected to the US National Academy of Sciences, to
the Royal Society, and to the US Institute of
Medicine. By 2012, on the 20th birthday of the
JAK-STAT pathway, the Kerr and Stark labs’ cell lines
had been used by 500 laboratories worldwide.
Key references
Dale TC, Imam AM, Kerr IM, Stark GR (1989). Rapid
activation by interferon alpha of a latent DNAbinding protein present in the cytoplasm of
untreated cells. Proc Nat Acad Sci USA. 86:1203-7
Pellegrini S, John J, Shearer M, Kerr IM, Stark GR
(1989). Use of a selectable marker regulated by
alpha interferon to obtain mutations in the
signaling pathway. Mol Cell Biol. 9:4605-12
Velazquez L, Fellous M, Stark GR, Pellegrini S
(1992). A protein tyrosine kinase in the interferon
alpha/beta signaling pathway. Cell. 70:313-22
Darnell JE Jr, Kerr IM, Stark GR (1994). Jak-STAT
pathways and Transcriptional Activation in
Response to IFNs and Other Extracellular Signaling
Proteins. Science. 264:1415-21
39
The Hedgehog signalling pathway
1993: Phil Ingham’s lab clones the zebrafish hedgehog homologue,
and predicts its importance as a morphogen.
The hedgehog signalling pathway is the key to development of the
vertebrate embryo. Multiple cancers exhibit hedgehog pathway
activation and drugs that inhibit signalling through the pathway are in
clinical trials.
Background
In 1980, Christiane Nüsslein-Volhard and Eric
Wieschaus published a paper in Nature detailing
the results of a screen they had devised in the fruit
fly Drosophila Melanogaster to identify every gene
required for early pattern formation in the
Drosophila embryo. This work, for which, together
with the American geneticist Ed Lewis, they were
awarded the 1995 Nobel Prize in Physiology or
Medicine, signalled the advent of modern fly
developmental biology; researchers pounced on
the many lines of mutant flies that had been
generated, knowing that each had a mutation in
just one gene, which it was now possible to isolate
and clone by the new molecular biology
techniques. Excitement spread when it was
realised that many of the genes being cloned in
Drosophila had vertebrate homologues, and that
these similar genes likely had a similar role in the
patterning of vertebrate embryos.
Phil Ingham had done a PhD with Robert Whittle in
Sussex, and by the mid-80s, was a postdoc with
David Ish-Horowicz at the ICRF labs at Mill Hill,
working, amongst other things, on fly segment
polarity genes, which set the anterior/posterior
Phil Ingham
40
(front to back) axis of each segment of a fly’s body.
In 1987, in the new ICRF Developmental Biology
Unit in Oxford, Phil started his own Molecular
Embryology lab, and in the early 90s, his interest
turned from flies towards vertebrate models,
specifically, the zebrafish Danio rerio. He began a
collaboration with Harvard scientists Andy
McMahon, a mouse geneticist, and Cliff Tabin, who
worked on chick limb development, to look at what
a segment polarity gene named hedgehog was
doing in vertebrates.
In common with all other aspects of early
development, segment polarity is achieved by a
series of inductive interactions, which are of
fundamental importance for the development of
all multicellular organisms. Inductive interactions
work rather like a very complicated domino run the preceding domino must fall correctly to trigger
the next one, and so on, until the developmental
process, such as the generation of a segment, or an
organ, or a limb, or indeed a whole organism, is
complete. They require cells to be able to signal to
each other, either by direct contact between their
proteins, or by using secreted messenger proteins
(morphogens) to move between them, sometimes
over long distances. In fly segment polarity,
hedgehog (hh) seemed very likely to be a key gene;
it was expressed in exactly the right place (the cells
at the front edge) to specify the front-to-back
organisation of each segment, and its protein
product was secreted, meaning that it could move
from one cell to another to transmit a signal.
The research
To isolate vertebrate hh, Phil and his collaborators
began by screening molecular libraries containing
all the genes from their respective organisms, and
soon found that there was a small family of three
vertebrate Hh genes which they christened Desert
hedgehog (Dhh), Indian hedgehog (Ihh), and Sonic
hedgehog (Shh). Phil’s lab showed that Shh turned
out to be the most similar to fly hh, able to
substitute for it in flies lacking their own hh gene.
Zebrafish embryo
The three collaborators then turned to analysing
where Shh was switched on (expressed) during
early vertebrate development, and discovered, to
their great excitement, that Shh’s expression
pattern made it very likely to be involved in two
processes of extreme developmental importance,
induction of floor plate, and ZPA signalling. Floor
plate induction is responsible for the patterning of
the neural tube, which eventually forms the spinal
cord, and the ZPA, or Zone of Polarising Activity, is
the region which specifies the correct development
of limbs. Further experiments forcing expression of
Shh protein in inappropriate places or times during
development confirmed that it was very likely that
Shh was the key morphogen being secreted both
from the ZPA and from the notochord, the region
responsible for inducing floor plate.
The consequences
A great deal of our knowledge of the mechanisms
of early development stem from this work. The
genes acting in both floor plate induction and the
ZPA had been an almost complete mystery which
had perplexed workers for many years, and the Shh
work was the first chink of molecular light to be
shed on the processes. In 1991, Phil had published
an important paper predicting the interactions
between Drosophila Hedgehog and its receptor,
the Patched protein, and he and others built on this
work to show that in vertebrates too, the Hh signal
is relayed into the cell by the surface proteins
Patched (PTCH) and Smoothened (SMO). In the
absence of Hh, PTCH acts as a suppressor of SMO,
and no signals are sent. Upon Hh binding, SMO is
released, and activates a transcription factor called
GLI, which moves into the nucleus and turns on Hh
target genes.
This work also has important implications for
cancer treatment. Today, we know that there are
few parts of the vertebrate body plan that are not
in some way influenced by a Hh signal, but
normally, Hh is almost completely silenced after
birth. When Hh signalling is accidentally
reactivated, it can cause cancer, sometimes by
mutations in far downstream components of the
pathway, but sometimes, in tumors such as colon,
pancreatic, ovarian and basal cell carcinomas,
because of mutation in the Hh ligands PTCH and
SMO. So, if PTCH function is lost, or SMO is mutated
in such a way that it is always on, the Hh signalling
pathway fires even in the absence of Hh protein,
causing cell overgrowth, and cancer. Basal cell
carcinomas, where loss of PTCH is common, are
highly treatable if caught early, but if not treated,
progress to an advanced metastatic stage which
has very poor prognosis and no good treatment.
Development of small molecule inhibitors of SMO
was begun by Ontogeny, a biotech start-up
company whose inception was based on licensing
the Shh patents filed by ICRF and Harvard following
the discovery of Shh. Vismodegib (Erivedge), a drug
inhibiting SMO activity developed by Curis Inc
(Ontogeny’s successor) & Genetech, was licenced
in 2012 to treat metastatic basal-cell carcinoma,
and is in clinical trials as treatment for multiple
other solid tumours.
What happened next?
Phil Ingham is currently Vice Dean, Research, and
Toh Kian Chui Distinguished Professor at the Lee
Kong Chian School of Medicine, Nanyang
Technological University, Singapore. Phil is a
member of EMBO, a Fellow of the Academy of
Medical Sciences and the Royal Society, and an
Honorary Fellow of the Royal College of Physicians.
He received the Medal of the Genetics Society of
Great Britain in 2005 and the Waddington Medal of
the British Society for Developmental Biology in
2014. He continues to work on the zebrafish
hedgehog signalling pathway and its relevance to
human disease.
Key references
Krauss S, Concordet JP, Ingham PW (1993). A
functionally conserved homolog of the Drosophila
segment polarity gene hh is expressed in tissues
with polarizing activity in zebrafish embryos. Cell.
75:1431-44
Riddle R, Johnson R, Laufer E, Tabin C (1993). Sonic
hedgehog mediates the polarizing activity of the
ZPA. Cell. 75:1401-16
Echelard Y, Epstein DJ, St-Jacques B, Shen L,
Mohler J, McMahon JA, McMahon AP (1993).
Sonic hedgehog, a member of a family of putative
signaling molecules, is implicated in the regulation
of CNS polarity. Cell. 75:1417-30
41
DNA double-strand break repair, Holliday junctions, and BRCA2
1994: Steve West and colleagues purify human RAD51, a protein that
plays a key role in DNA double strand break repair. West’s search for
the elusive human Holliday junction resolvase that processes
recombination intermediates finally succeeds in 2008, with the
identification of GEN1. West’s work has been instrumental in
understanding the implications of DNA double strand break repair
both for tumourigenesis and for cancer therapy.
Background
Chromosomal double-strand breaks (DSBs) are one
of the most dangerous forms of DNA damage. They
can be induced by external agents, such as ionizing
radiation or anti-cancer drugs, or by the cell itself,
as in the case of the damage caused by free radicals
generated from oxidative metabolism. Efficient
double-strand break repair is essential for the
survival of each cell because unrepaired breaks can
lead to chromosome fragmentation and cell death,
and improperly repaired breaks lead to mutations,
chromosomal translocations and cancer.
To preserve genomic integrity, we now know that
DNA double-strand breaks are repaired in two
ways: homologous recombination (HR) and
non-homologous end joining (NHEJ). However, 25
years ago, very little was known about how these
processes occurred in mammalian cells. In bacteria
and yeast, matters were slightly more advanced, as
it had been recognised that HR occurred, and
several of the enzymes responsible had been
identified. To study the mechanism of HR, the goal
was to purify proteins and study their activities
with DNA molecules in the test tube. To do this,
Steve West
42
biochemical expertise is a key skill. As a graduate
student in 1975 in Newcastle, Steve West identified
the bacterial E. coli RecA protein, a central player in
homologous recombination, and during his
postdoc in Yale, he produced some classic papers
on RecA, showing how it promoted the pairing of
homologous strands of DNA and subsequent DNA
strand exchange. These studies underpin much of
our present understanding of the molecular
mechanism of HR.
The research
RuvC: Steve West was recruited by Tomas Lindahl
to the brand new ICRF Clare Hall Laboratories in
1985, and on his arrival, began working to
biochemically purify the enzymes known as the
Holliday junction resolvases, the proteins that
catalyse the final resolution step in HR that allows
the separation of recombinant chromosomes. He
won the race to identify the E. coli enzymes that
process Holliday junctions, christened RuvA, RuvB
and RuvC, and characterised how they worked in a
series of in vitro experiments using purified
proteins and artificial DNA templates.
Armed with their expertise in bacterial
recombination, the focus of the West lab changed
towards addressing the far more complex puzzle of
identifying the enzymes involved in eukaryotic
recombination. In the case of RuvC, this turned out
to be a slightly knottier problem than anyone at the
time could have anticipated, and it took until 2008
to identify and clone the human and yeast versions
of RuvC, called GEN1 and Yen1 respectively.
Unfortunately for the lab members involved,
despite the functional similarity between the
bacterial and eukaryotic resolvases, there was
absolutely no sequence homology; in the case of
human GEN1, brute force biochemical techniques
had to be applied to harvest the tiny amounts of
GEN1 in each cell, whilst yeast Yen1 was finally
identified by genetic screens.
RAD51 and BRCA2: In relation to West’s PhD
project protein, bacterial RecA, things moved
rather faster, and his lab was the first to purify the
mammalian homologue of RecA, called RAD51, in
1994. Here, they found some remarkable
similarities, showing that the RAD51 recombinase
formed nucleoprotein filaments almost
indistinguishable from those made by bacterial
RecA. The subsequent discovery that RAD51 is both
positively and negatively regulated by the BRCA2
protein in response to DNA damage has brought
this work to centre stage in cancer biology. BRCA2
belongs to a class of genes known as tumour
suppressors, as its normal function is essential in
guarding the cell from possibly oncogenic
mutations. It is defective in about 10% of inherited
breast cancers, and when it is unable to work,
double strand breaks cannot be repaired by
homologous recombination, a defect that
ultimately results in tumour formation.
Biochemically, BRCA2 is a nightmare, as it is a huge
protein nearly 400kD in size, which makes working
with it very challenging due to the difficulty in
purifying the full-length protein and its propensity
to fall apart. As a result, little was known about the
precise mechanisms by which it worked. However,
a 2010 publication from the West lab, in which
BRCA2 was purified and studied in complexes by
electron microscopy has now shown that it is
responsible for controlling the ability of RAD51 to
bind DNA. These observations have at long last
provided the molecular explanation for the role of
BRCA2 in the maintenance of genome stability.
Recombination and disease: Not surprisingly,
defects in DNA repair lead to a number of rather
unpleasant and crippling diseases, and the West lab
has been at the forefront of research into the
causes of two of these, Bloom’s Syndrome and
Fanconi Anaemia. Recently, work from the West
group has defined the molecular defect associated
with a neurological disorder known as Ataxia with
Oculomotor Apraxia-1 (AOA1). AOA1 is a
progressive disease, first manifesting in apparently
healthy children at about 7 years of age. It begins
with difficulty in coordinating movement (ataxia),
and many sufferers also develop an inability to
move their eyes to look sideways (oculomotor
apraxia). Symptoms increase in severity such that
after ten years, most sufferers have atrophied
muscles and nerves, and sometimes limb
deformities, meaning that they are typically
wheelchair bound.
One gene, APTX, encoding Aprataxin, is defective in
AOA1 patients, and the West lab showed that
Aprataxin protein is the proofreader which detects
abortive DNA ligations, the process whereby two
pieces of broken DNA are sewn back together. All
cells, especially neuronal cells, are subjected to high
levels of oxidative stress resulting in the formation
of DNA strand breaks. When these breaks are
repaired by a DNA ligase, it is not uncommon for the
reaction to stall at an intermediate stage, such that
abortive ligation intermediates accumulate. It turns
out that Aprataxin specifically interacts with these
intermediates, allowing them to be resolved. When
no Aprataxin is present, unrepaired nicks
accumulate in the DNA, and eventually, there are so
many that the machinery responsible for
transcription (turning the DNA into RNA transcripts,
ready to make protein) is unable to work properly
and breaks down. Without efficient transcription,
the neuronal cells die. Interestingly, while the AOA1
defect is present in most tissues, it is only a problem
in the brain. The reason is that brain neurons are
post-mitotic, that is, they no longer replicate, and
so they lack all the replication-associated DNA
repair mechanisms which are able to sort out
stalled ligases by other means; with the loss of
Aprataxin activity, they lose their only safeguard.
What happened next?
Steve West is currently the Deputy Director of the
LRI Clare Hall Laboratories. He is an EMBO member
and a Fellow of the Royal Society and the Academy
of Medical Sciences. His awards include the 2002
Leeuwenhoek Prize of the Royal Society, the 2007
Louis-Jeantet Prize for Medicine, the 2008 Novartis
Medal and Prize from the Biochemical Society, and
the 2010 GlaxoSmithKline Medal and Prize of the
Royal Society.
Key references
Dunderdale HJ, Benson FE, Parsons CA,
Sharples GJ, Lloyd RG, West SC (1991). Formation
and resolution of recombination intermediates by
E. coli RecA and RuvC proteins. Nature.
354:506-10
Baumann P, Benson FE, West SC (1996). Human
RAD51 protein promotes ATP-dependent
homologous pairing and strand transfer reactions
in vitro. Cell. 87:757-66
Ip SCY, Rass U, Blanco MG, Flynn HR, Skehel JM,
West SC (2008). Identification of Holliday junction
resolvases from humans and yeast. Nature.
456:357-61
Thorslund T, McIlwraith MJ, Compton SA,
Lekomtsev S, Petronczki M, Griffith JD, West SC.
(2010). The breast cancer tumour suppressor
BRCA2 promotes the specific targeting of RAD51 to
single-stranded DNA. Nat Struct Mol Biol. 7:1263-5
43
Cancer genetics
1979: Walter Bodmer establishes the ICRF as a major centre for
cancer genetics.
1987: Walter Bodmer and Ellen Solomon identify the FAP locus.
2007 and 2008: Tomlinson lab publishes collaborative studies
identifying loci in genome associated with susceptibility to breast,
colorectal and prostate cancer.
44
Background
In 1979, when Sir Walter Bodmer succeeded Sir
Michael Stoker as Director of the ICRF, his
appointment signalled the start of a new initiative
at the lab, in human genetics. One of Bodmer’s
primary research interests was the study of the
genetic basis of cancer, the notion that at the
molecular level, cancer is caused by changes in
genomic DNA. These changes can be induced by
damage or mutation, but can also be hereditary;
families in which a particular sort of cancer is
common have a defect in a gene or genes making
them susceptible to the disease. Finding out what
these susceptibility genes are has been a major
cancer research goal since the 1980s. Their
identification allows for genetic testing of potential
carriers, enhances understanding of the basic
biology of cancer, and often sheds light on how
sporadic (non-hereditary) tumours form. The ICRF
has provided two particularly good examples of
this progression from gene identification to
biological understanding. Bodmer’s lab, together
with that of Ellen Solomon, mapped the location of
the gene responsible for a hereditary type of bowel
cancer, familial adenomatous polyposis (FAP), in
1987. The APC gene which lay within the FAP locus
was cloned by Kinzler and colleagues in 1991, and is
mutated in some 80% of sporadic colon cancers.
Work from many laboratories has shown that APC
lies at the centre of a network of molecules, many
of which are also implicated in colon and other
cancers. In a somewhat different arena, Peter
Goodfellow, another human geneticist recruited by
Bodmer, in 1990 identified the SRY gene (Highlight
9), which is responsible for determining the sex of
males during embryonic development.
Walter Bodmer
Ian Tomlinson
Ian Tomlinson, after postdoctoral work in the
Bodmer lab and at the Institute of Cancer
Research, started his own lab at the ICRF in 1998,
and in the next decade, was involved in a
collaborative effort searching for common genes
which increase the risk of cancer. His lab
contributed to studies to identify loci contributing
to breast and prostate cancer, and his work on
colorectal cancer produced a series of high profile
publications in 2007 and 2008.
The research
Working in a field – cancer genetics – in which big
screens for new disease genes are the norm, Ian
Tomlinson’s work required the collection of tens of
thousands of patient samples, representing huge
efforts for several members of his laboratory and
the laboratories of collaborators. The main
developments that made this venture possible
were technological and financial, owing to the
willingness of Cancer Research UK to fund what
remains very expensive work.
Ian’s group, together with colleagues at the
Institute of Cancer Research and in Edinburgh,
started searching for common alleles that
increased the risk of developing colorectal cancer,
in the hope that by fully understanding the genetic
architecture of cancer, it would be possible to
predict individual risk and thus tailor effective
cancer prevention measures to those at relatively
high risk. A secondary justification was to obtain
information about disease biology, by identifying
some of the functional pathways that might
usefully be targeted by, for example, new
chemopreventive agents. The project design was a
relatively simple association study, based on
comparing genotype frequencies in cases and
controls and searching for significant differences at
individual loci.
10 tagSNPs (genomic markers) associated with
differential risks of disease were identified. In one
case, the SNP was also associated with increased
risks of prostate and ovarian cancer, but the other
9 SNPs seemed unique to colorectal cancer. In all
cases, the effects on risk were relatively modest,
with typically a 1.1-1.3-fold differential risk per
allele (although these estimates are conservative).
These SNPs seemed to act independently of one
another, with no instances of gene-gene
interactions and little deviation from additive or
log-additive effects on risk. Perhaps most
intriguingly, there was some evidence that several
of the colorectal cancer SNPs acted in the same
functional pathway, namely bone morphogenetic
protein (BMP) signalling. Inhibition of BMP
signalling was already proposed to have an
essential function in maintaining the stem cell
niche at the bottom of the colonic crypt. If, as is
thought, cancer arises from normal stem cells, it is
therefore possible that the colorectal cancer SNPs
affected stem cell numbers, and hence the number
of cells that could potentially give rise to cancer,
thereby increasing cancer risk. Intriguingly, several
colorectal cancer SNPs lay in regions where genes
in the conventional sense were absent; thus, their
most likely function was in regulation of gene
expression, potentially over hundreds of thousands
of kilobases.
The consequences
Such genome-wide association studies have taken
genetics into new areas in which the science has
become much more similar to epidemiology than
‘classical’ genetics, and individual effort is pooled
for the good of a greater whole. Whilst intellectually
less satisfying, requiring project management
rather than purely scientific skills, such studies are
essential to make the advances in clinical research
necessary to treat many diseases. However, part of
their attraction is also to provide seedcorn for
functional studies on the genes identified, so that it
will be possible to work out exactly what the
differences are between individuals that cause
variation in disease risk, and to work out how these
differences have their effects.
What happened next?
Walter Bodmer became ICRF’s first Director
General in 1991. In 1996 he left the ICRF to become
Principal of Hertford College Oxford, from which
he retired in 2005. He is currently Head of the
Cancer and Immunogenetics Laboratory in the
Weatherall Institute of Molecular Medicine at the
University of Oxford.
Ellen Solomon moved to King’s College London in
1995, where she is Professor of Cancer Genetics in
the Division of Genetics and Molecular Medicine.
Ian Tomlinson moved to the Wellcome Trust
Centre for Human Genetics in Oxford in 2008. He
continues to work on the genetics of colorectal and
other cancers, with an emphasis on cancer
predisposition. For his contributions to the field, he
was elected to the Academy of Medical Sciences in
2009, and in 2013 won the UEG Research Prize – an
award of €100,000 for excellence in basic science
or clinical and translational research.
Key references
Tomlinson I, et al., (2007). A genome-wide
association scan of tag SNPs identifies a
susceptibility variant for colorectal cancer at
8q24.21. Nat Genet. 39:984-8
Houlston RS, et al., (2008). Meta-analysis of
genome-wide association data identifies four new
susceptibility loci for colorectal cancer. Nat Genet.
40:1426-35
Tenesa A, et al., (2008). Genome-wide association
scan identifies a colorectal cancer susceptibility
locus on 11q23 and replicates risk loci at 8q24 and
18q21. Nat Genet. 40:631-7
Tomlinson IP, et al., (2008). A genome-wide
association study identifies colorectal cancer
susceptibility loci on chromosomes 10p14 and
8q23.3. Nat Genet. 40:623-30
45
Innate immunity
Caetano Reis e Sousa has made multiple key discoveries concerning
how dendritic cells of the innate immune system recognise pathogen
associated molecular structures or normal cell constituents
generated by dying cells.
Background
The vertebrate immune system fights infection
using two closely interlinked defence networks.
Adaptive immunity, mediated by T- and
B-lymphocytes, learns from previous battles fought
with enemy antigens, honing their specificity and
response time so that the body is never taken
unawares if reinfected. However, generation of T
and B cell responses relies upon signals from the far
more ancient innate immune system, which is
found in all plants and animals, and from which
adaptive immunity evolved. Central to the whole
process are the dendritic cells, a heterogeneous
family of white blood cells that integrates innate
information and conveys it to lymphocytes.
activate the T cell branch of the adaptive immune
system. Different threats lead to differences in the
mix, producing a precisely tailored coded message
telling the microbe-specific T cells how they have to
react, or differentiate, to combat the incoming
microbes.
Infection by different types of micro-organism
creates distinctive tell-tale clues, or patterns. The
various dendritic cell subtypes work by recognising
these patterns, using a network of so-called
pattern recognition receptors and other innate
immune receptors which are programmed to
recognise deviations from cellular normality: for
example, molecules that look right, but are in the
wrong place, or dead cells cropping up where they
should not, are all detected by different classes of
dendritic cells. Once the dendritic cell has worked
out exactly what the threat is, it produces a mix of
cytokines and other factors, which are able to
The research
Right face, wrong place: Flu and other similar
viruses are recognised by dendritic cells and other
cells, which set up an antiviral response, triggering
rapid production of type 1 interferons and
activating the adaptive arm of the immune system,
as well as potentiating innate antiviral resistance.
However, both the viral clues being detected, and
the identity of the molecular sensors on the
dendritic cells were unknown. Caetano and
collaborators, in a series of papers published
between 2004 and 2010, shed light on these
mysteries, and established the concept that
mislocalisation of an ostensibly innocent molecule
is seen by dendritic cells as an extremely suspicious
event. They discovered that dendritic cells have
evolved several mechanisms for distinguishing viral
RNA from cellular RNA, relying on the fact that viral
RNA crops up in places where cellular RNA would
never normally be found. For example, two of the
detectors, the TLR7 and TLR3 receptors, live in the
endosomes, the vesicles that capture material at
the cell surface and transport it around inside the
cell. As cellular RNA is never found in endosomes,
the cell can be certain that any RNA found there is
suspect, and this provides a mechanism by which
dendritic cells can detect the presence of virally
infected cell corpses. RNA is also the key to the
third detection system, which is cytoplasmic, and
uses the RIG-I protein which recognises RNAs with
a particular modification (a 5’ triphosphate group)
46
Work from Caetano Reis e Sousa’s lab has helped
define what some of the most important microbial
signatures are, how dendritic cells recognise these
signatures, and how they then transmit this
information to the adaptive immune system. His
work has important implications for tumour
immunotherapy, where the body’s immune system
is used to destroy cancers.
at one end. Again, this mark is diagnostic of viral
RNAs and provides a way by which viral infection
can specifically activate RIG-I.
Disease and death; a Syk story: One of Caetano’s
long standing interests was to crack the cytokine
code - to work out the different mixes of cytokines
elicited by different types of microbial infection.
Whilst doing this, he realised that when he gave
dendritic cells yeast as the microbial agent, they
produced cytokines as expected, but intriguingly,
did so independently of known TLR family
members. His lab began hunting for this unknown
signalling pathway, and in 2005, published a
landmark paper identifying the Syk tyrosine kinase
as the key player. Syk is activated by Dectin-1,
which recognises carbohydrates uniquely present
on fungal cell walls. The lab also showed that a
second receptor, Dectin-2, could also signal via the
Syk pathway, and that together, Dectin-1, Dectin-2
and the Syk pathway are necessary for warning the
immune system about all types of fungal infection.
Interestingly, some patients with chronic
mucocutaneous candidiasis turn out to have
mutations in components of this novel pattern
recognition pathway, highlighting its importance in
human defence from fungal infection.
The Syk pathway is important for more than just
fungal infection. Another common sign of infection
or injury is the presence of cells dying by necrosis, a
form of death occurring during trauma. Caetano’s
lab has recently characterised another C-type
lectin family member called DNGR-1 as the bridge
between recognising necrosis, and alerting the T
cells of the adaptive immune system. DNGR-1’s
discovery is provocative as it resurrects the
concept of an innate ‘danger receptor’, a molecule
able to sense abnormal cell death and trigger an
adaptive immune response, first proposed by the
American immunologist Polly Matzinger.
The consequences
Dendritic cells are a very hot topic in terms of
cancer therapeutics, as if it were possible to
harness their power to activate the adaptive
immune system, and use it to attack and destroy
tumours, the body would be able to assist in
ridding itself of cancers. The fact that DNGR1 is
only found on one particular sort of mouse
dendritic cell, the CD8a+ subtype, makes it a
potentially exciting target for cancer therapeutics,
as CD8a+ dendritic cells are very efficient at
presenting antigens to the killer T cells of the
mouse adaptive immune system. Caetano’s lab has
shown that if antigens derived from melanomas
are artificially fused to antibodies against DNGR-1,
these hybrid molecules bind to DNGR-1 on CD8a+
dendritic cells, and generate a strong response
against melanoma, shrinking hard-to-treat tumours
to almost nothing in experimental mouse models.
Vexingly, CD8a+ dendritic cells had not been
identified in species other than mouse. Caetano’s
lab recently used the specificity of DNGR-1
expression to identify the human equivalents of
mouse CD8a+ dendritic cells, bringing the
translational potential of DNGR-1 targeting one
step closer to the clinic.
What happened next?
Caetano Reis e Sousa has become a world leader in
the field of innate immunity. He was elected to
EMBO and to the Fellowship of the Academy of
Medical Sciences in 2006, won the Liliane
Bettencourt Life Sciences Award in 2008, and the
ESCI Award for Excellence in Basic/Translational
Research in 2011.
Key references
Schulz O, Diebold SS, Chen M, Naslund TI, Nolte
MA, Alexopoulou L, Azuma Y-T, Flavell RA,
Liljestrom P, Reis e Sousa C (2005). Toll-like
receptor 3 promotes crosspriming to virus-infected
cells. Nature. 433:887-92
Pichlmair A, Schulz O, Tan CP, Naslund TI,
Liljestrom P, Weber F, Reis e Sousa C (2006).
RIG-I–Mediated Antiviral Responses to SingleStranded RNA Bearing 5’-Phosphates. Science.
314:997-1001
Robinson MJ, Osorio F, Rosas M, Freitas RP,
Schweighoffer E, Gross O, SjefVerbeek J, Ruland J,
Tybulewicz V, Brown GD, Moita LF, Taylor PR,
Reis e Sousa C (2009). Dectin-2 is a Syk-coupled
pattern recognition receptor crucial for Th17
responses to fungal infection. J Exp Med.
206:2037-51
Sancho D, Joffre O, Keller A, Rogers NC, Martinez
D, Hernanz-Falcón P, Rosewell I, Reis e Sousa C
(2009). Identification of a dendritic cell receptor
that couples sensing of necrosis to immunity.
Nature. 458:899-903
Rehwinkel J, Tan CP, Goubau D, Schulz O, Pichlmair
A, Bier K, Robb N, Vreede F, Barclay W, Fodor E,
Reis e Sousa C (2010). RIG-I detects viral genomic
RNA during negative-strand RNA virus infection.
Cell. 140:397-408
Ahrens S, Zelenay S, Sancho D, Hanč P, Kjær S,
Feest C, Fletcher G, Durkin C, Postigo A, Skehel M,
Batista F, Thompson B, Way M, Reis e Sousa C,
Schulz O (2012). F-actin is an evolutionarily
conserved damage-associated molecular pattern
recognized by DNGR-1, a receptor for dead cells.
Immunity. 36:635-45
47
LRI LABORATORIES Highlight 20
Tumour heterogeneity
2012: Charlie Swanton and colleagues show that tumours are
composed of a continually evolving mixed population of cancer cells,
explaining why advanced cancer is so hard to treat.
Cancer heterogeneity arises through Darwinian evolution, and
nipping the evolutionary process in the bud by early diagnosis and
detection will be the key to successfully combatting the disease.
The background
In the first decade of this century, our
understanding of the molecular mechanisms
underlying many human cancers started a
revolution in personalised cancer treatment.
Tumours could be biopsied to determine which of
the cancer-causing oncogenes and tumour
suppressor genes were mutated, and targeted
drugs against the offending molecules could be
deployed to attack the root of the cancer. There
was a wave of optimism in the cancer community,
but very quickly, it became clear that all was not
well; whilst there were some startling successes,
with patients plucked from the jaws of death by
tailored therapies, the grim reality was that after
weeks or months, the therapies would fail as the
tumour fought back, with fatal consequences.
The question of why advanced cancers were so
lethally good at acquiring resistance to drugs was
not a new one, and an answer had in fact been
proposed in 1976 by Peter Nowell, who suggested
that cancer was an evolutionary disease: as
tumours were genetically unstable, the selective
pressure applied by aggressive therapy resulted in
subpopulations of tumour cells acquiring
resistance, thereby flourishing anew. However,
Charlie Swanton
48
whilst biopsies of advancing disease showed
correlations between new chromosomal
abnormalities and drug resistance, the limited
techniques of the time meant that finding out
what was going on at the molecular level was
impossible.
By 2010, technology had advanced enormously
and several groups, one led by former ICRF star
Mel Greaves (Highlight 3), published landmark
papers on the molecular archaeology of cancer.
Mel’s group, looking at acute leukaemia, proposed
that instead of proceeding inexorably from
normality to fatality by a series of linear
mutational events, leukaemias exhibited branched
evolution, just as in Darwin’s iconic evolutionary
tree diagram. Two other publications showed this
was also the case in solid tumours: branched
evolution meant that primary tumours and
metastases were genetically distinct. Together, the
three papers confirmed Nowell’s theory, and
suggested that tumour heterogeneity might be
more extensive than previously suspected.
Charlie Swanton’s double life as an oncology
consultant and a scientist interested in translating
research into effective anti-cancer therapeutics
meant that he was perfectly placed to tackle the
problem of tumour heterogeneity head-on.
Following his arrival as a group leader at the LRI in
2008, Charlie and a group of collaborators decided
to look at tumour evolution in unprecedented
detail. There was a real urgency for this research: if
Charlie’s experiments showed that there was
diversity between biopsies within the same
primary cancer, the implication for treatment and
predicting outcome using genomics-based
diagnosis in solid tumours was alarming: the
long-established practice of taking only a single
biopsy of a tumour, and basing therapeutic
decisions on that one sample, might mean that
clinicians were prescribing targeted drugs that
would kill only a small subset of cancer cells,
leaving the rest of the tumour unharmed.
The research
Charlie and his collaborators took multiple biopsies
from different regions of primary kidney cancers
and metastases, sequenced the tumour DNA, and
also looked at chromosomal abnormalities and
changes in gene expression. The sequencing alone
was a huge job: to ensure total accuracy, each
tumour DNA sample had to be sequenced many
times, and the LRI sequencing facility worked
solidly for 4 months to read around 140 billion
bases of DNA code.
The results exceeded everyone’s worst suspicions:
even before extensive treatment had commenced,
primary tumours were already heterogeneous; no
two samples were the same, even if they’d come
from adjacent regions of one tumour. In the
primary tumours, only about a third of the
mutations detected were common to all the
samples. Even more worryingly, genetic signatures
known to be associated with either a good or a
poor prognosis could be picked up in different
regions of the same tumour. The stark reality was
that a single biopsy would never provide a picture
of the mutational landscape of the entire tumour,
and frighteningly, could be extremely misleading.
Analysis of the mutated genes did provide one
encouraging fact; although there were only one or
two driver mutations common to all the samples,
there were quite a few examples of parallel
evolution, in which different tumour samples from
the same tumour had mutated the same genes, but
in different ways. It looked as though progression
to kidney cancer absolutely required the mutation
of certain cellular pathways, and some of these key
pathways were already therapeutic targets.
What happened next?
There was a degree of scepticism in the cancer
community following the publication of Charlie’s
paper, with some disbelief that intratumour
heterogeneity was so extreme. However, his
results have been validated in most solid tumours,
and what was once heterodox has now become an
accepted and rather obvious fact.
After the initial gloom prompted by the realisation
that their enemy was even more devious than
suspected, cancer biologists have returned to the
fray with renewed vigour and better plans. In depth
analysis of as many cancer types and tumours as
possible will eventually give the field a clear idea of
which mutations lie in the ‘trunk’ of a cancer’s
evolutionary tree, and also which pathways exhibit
parallel evolution. Once identified, therapies
directed at such driver mutations will be far more
likely to succeed than those against less ubiquitous
molecules, and more reliable prognostic tests can
also be developed, using the tens of thousands of
biomarkers already known to be associated with
cancer.
The recognition that cancers evolve, and that more
and more mutations accumulate as time goes by,
has also emphasised the importance of early
detection of disease. Catching and containing
cancer before it goes out of control is becoming a
major priority, as has defining exactly how diversity
is generated. In 2014, Charlie’s lab showed that in
colon cancer, following a genome doubling event,
tetraploid colon cancer cells became tolerant of
ongoing chromosomal instability. These tetraploid
supergenomes were strongly associated with both
poor prognosis and in the laboratory evolved much
faster than their diploid progenitors. Such
extravagantly mutated cells are an extreme
example of chromosome instability, a common and
high risk feature of cancer, and working out the
mechanisms by which the instability arises is an
ongoing preoccupation for many labs around the
world, including Charlie’s.Whilst the basic research
continues, Charlie is trying to put his findings to
clinical use by running TRACERx, a multi-millionpound multi-centre collaborative trial for lung
cancer, following 850 patients through therapy. By
sampling tumours at all stages of treatment, he can
map the ebb and flow of the different variants in
the tumours by DNA sequencing. The speed of
sampling and sequencing means that what he
learns can then be used to inform the next stage of
treatment, keeping clinicians one step ahead of the
cancer. In addition to this immediate therapeutic
benefit, Charlie will use the sequencing data from
the project to understand the evolutionary biology
of lung cancer, and to link the more common types
of therapeutic drug resistance to particular genes,
making it even faster and easier to monitor changes
in a patient’s response to treatment.
Key references
Gerlinger M, et al., (2012). Intratumor
Heterogeneity and Branched Evolution Revealed
by Multiregion Sequencing. N Eng J Med.
366:883-92
Gerlinger M, et al., (2014). Genomic architecture
and evolution of clear cell renal cell carcinomas
defined by multiregion sequencing. Nat Genet.
46:225-33
Dewhurst SM, et al., (2014). Tolerance of wholegenome doubling propagates chromosomal
instability and accelerates cancer genome
evolution. Cancer Discov. 4:175-85
49
INTRODUCTION TO THE 2014 LRI RESEARCH
REPORT
2014 saw the last full year of operation of the London Research
Institute before our coming incorporation into the Crick in 2015. The
Research Highlights section attests to how the Institute continues to
conduct its science largely as usual, but increasingly, Institute staff
have been contributing to preparations for the transfer of
operations to the new Crick laboratories.
Richard Treisman
Peter Van Loo,
Cancer Genomics
Fields to returned to his native Germany, taking up
a position at the Max Delbrück Center for
Molecular Medicine (MDC) in Berlin. The same
month, Mark Petronczki left Clare Hall to joined
Boehringer Ingelheim in Vienna, also a return to his
Austrian roots. We wish both of them all success in
the future.
Group leader recruitment to LRI has wound down
as the move to the Crick approaches, but we
continue to see both new arrivals, and departures
as established group leaders move on to take up
new research opportunities. The past year saw the
arrival of two new group leaders, hired under the
Crick’s ‘6+6’ Junior Group Leader scheme. Peter
Van Loo joined us in September from the Sanger
Institute. A bioinformatician, Peter is especially
interested in genetic variation in cancer, and
pursues genetic approaches to elucidate the
evolutionary history of tumours and their
metastases. His work will complement the
increasing interest in cancer genomics and
evolution at LRI. In contrast, Guillaume Salbreux,
who joined the Institute in February 2015, is a
soft-matter physicist. This area is particularly
relevant to understanding the movement of cells,
whether during tissue and organ development or
migration of individual cells. In September we said
farewell to Holger Gerhardt, who left Lincoln’s Inn
While these new arrivals reinforce the continued
renewal and growth of the Institute, our last year
as LRI has been also touched by the deaths of two
of its senior members, who will be sorely missed.
In April, Julian Lewis succumbed to prostate
cancer, which he had fought valiantly for several
years. Julian’s kind and modest personality was
combined with intellectual rigour, great clarity of
expression, and broad interests, both personal
and scientific. The fluency and elegance of his
writing was displayed in his beautifully written
papers, and in his contributions to ‘The Molecular
Biology of the Cell’, of which he was a long-serving
Julian Lewis
Francois Lassailly
Guillaume Salbreux,
Theoretical Physics of Biology
50
Sally Leevers,
LRI Academic Director
Donna Brown,
NIMR Director of Studies
co-author. He had continued to write following his
retirement from LRI in 2012, and his last paper
appeared only weeks before his death. Julian was
remembered by scores of colleagues and friends at
a memorial gathering held in July at Balliol College
Oxford, of which he was a fellow. As these words
were being finalised in January 2015, we were
shocked by the death of Francois Lassailly in a
traffic accident. Francois was instrumental in the
introduction of non-invasive animal imaging
methods to LRI, and was working hard to establish
the new Crick Institute animal imaging facility;
many researchers at LRI will have experienced
Francois’ good nature, enthusiasm, and skill at
first hand.
Research activity and academic life at LRI has seen
both continuity and new developments over the
last year. Outstanding research achievements by
Institute staff continue to be recognised by our
peers. Erik Sahai joined the ranks of LRI EMBO
members in Spring, while Charlie Swanton was
awarded the 2015 Laura Ziskin prize for
translational cancer research from the Stand up to
Cancer in the US. Congratulations to them both.
Marcus Wilson from Jesper Svejstrup’s group was
awarded the 2013 Pontecorvo prize for the best
CRUK-funded PhD thesis, while Andy Filby from the
Flow Cytometry facility was chosen as an Emerging
Leader by the International Society for the
Advancement of Cytometry. The last-ever LRI
retreat was held at the Oxford Examination
Schools, at which Barbara Schraml presented the
Hardimon-Redon lecture. Presentation of the 2014
Hardiman-Redon Prize will form part of the
handover ceremony on the LRI’s last day at the end
of March 2015. Finally two very enjoyable Crick
Sophie Acton, winner of the 2014
Hardiman-Redon Prize
group leader retreats introduced LRI scientists to
future colleagues, from GSK, at London Zoo in July,
and from the Wellcome Trust Sanger Institute, at
Keble College Oxford in September.
Over the past few years the profile of the Crick
amongst our junior trainees – graduate students
and postdocs – has become more and more
prominent. Development and implementation of
the Crick Graduate programme has been a major
activity for Sally Leevers and her opposite number
at NIMR, Donna Brown. Much hard work on their
part led to the formal finalisation of the regulations
governing the Crick graduate student programme
with the Crick’s University partners. As a result, the
2014 graduate student intake is the first to be
admitted entirely under Crick auspices. The Crick
held its third annual postdoc retreat at the British
Library in June, bringing together some 200
postdocs from LRI, NIMR and the Crick partners,
and featuring for an interactive day of science,
career advice and networking, including a
stimulating discussion about open-access science
publishing.
The opening of the new Lincoln’s Inn Fields
laboratories in 1963 followed an appeal for a
million pounds by the ICRF, CRUK’s predecessor. It
is sobering to note that 50 years on, the Create the
Change campaign is seeking 100 million pounds to
fund the CRUK capital contribution to the Crick
project, and as I write the campaign is well on track
towards this target. In closing I would like to thank
not only Charles Manby and the CtC Board, but also
Antonia Newman, Russell Delew and their team for
all their hard work, without which the Crick would
not have been possible.
Antonia Newman and Russell Delew, Create the Change
DIRECTOR'S INTRODUCTION
51
Francis crick institute report
During 2014, the Crick Laboratories attained their final profile, and
the main emphasis on the building project has moved towards the
fitting-out of what will be one of the most technologically complex
buildings in Europe. As the building project moved steadily closer to
completion, 2014 saw a broad range of activities aimed at
establishing the Crick as a functional research organisation in
readiness for our migration to the new building in late 2015.
David Roblin
There have been a number of important changes
in the Crick’s leadership during the year in
preparation for first year of operations, which will
be on the old LRI and NIMR laboratory sites. David
Roblin took over as COO and Director of Scientific
Translation in September, with John Cooper
moving to Projects Director. Two new committees,
the Science Strategy and Policy Committee and the
Operations Management Committee, were
established to lead on research and operational
matters respectively, chaired respectively by Paul
Nurse and David Roblin. In readiness for the
research operation, three new associate research
Directors were appointed in December: Julian
Downward, leading on STPs and BRF; John Diffley,
leading on Junior Researchers and Training; and
Anne O’Garra, leading on Group Leader
Development.
Andy Smith
Francis Crick Institute, February 2015
52
Michael Schuitevoerder and his team have worked
tirelessly during the year to develop the migration
plan for occupation of the new building. The
challenge here was to develop a robust plan by
which the science of the founding institutes can be
moved from three laboratory sites into the Crick
laboratories without significant down-time.
Following the building handover on November 3rd
2015, central support functions will move in first,
and IT and animal house facilities will be made
ready. The STPs will move over Christmas, with
some being phased to allow operations to continue
at the former Institute sites pending complete
transfer to St Pancas. Research laboratories will
move in starting early 2016, and the move will be
complete shortly after the centenary of Francis
Crick’s birth on June 8th. Alongside this planning,
work to bring the NIMR and LRI mouse populations
Francis Crick Institute atrium, February 2015
to a uniform Crick health status, guided by Kathleen
Mathers and Gary Childs, proceeded steadily
during the year. This will allow animal transfer to
the new building to occur without delay once the
new animal facilities are commissioned.
Over the year several important transitional
appointments were made, critical for the
integration of laboratory operations and science
technology platforms and migration of research
activities to the new laboratories following the
Crick’s initial period of multi-site operations. In
January Simon Caidan, David Hudson, Jo Payne and
Nigel Peat were appointed to lead on Health and
Safety, STP operations, Lab infrastructure and
logistics, and Lab Operations respectively. Later in
the year, transition science technology platform
leads were appointed from each of the two
Institutes to lead integration of support
technologies. The year’s end saw the appointment
of the Laboratory Operations Managers, who will
oversee the operation of each of the research
floors of the new laboratories, each appointee
looking after the laboratories on one sector of
each floor. At the time of writing they are working
with the transition team to finalise equipment lists
and placings, and develop the logistics of the
migration.
The nascent Crick Institute’s academic life has
continued to develop. Late in 2014 the Crick issued
a pilot call for the first wave of university
researchers to join the Crick, with a special focus on
interdisciplinarity. It is anticipated that these will
include research groups applying for secondment
to the Crick laboratories, smaller ‘satellite’ groups
that will be attached to Crick research groups for
specific collaborative work, and sabbatical
placements. These groups will join the Crick upon
the completion of the initial migration. Joining then
will be Akhilesh Reddy, a clinican scientist working
on circadian rhythm biology, who will join UCL and
the Crick from Cambridge. Ak will hold one of three
Crick Clinical Professorships at the partner
universities, in his case at UCL. Finally, a new
interim Crick Faculty Committee met for the first
time in November. This committee provides a way
for the Crick’s management to consult with the
academic staff concerning science policy,
initiatives, and strategy. It will operate in interim
form, comprising representatives of the founding
institutes until the migration is complete, when it
will be reconstituted to include members from the
Crick’s partner universities.
Finally the Crick continued to engage with its staff
and the public through its programme of Crick
scientific symposia, staff away-days and diverse
public engagement events organised by the
Comms team. Particularly notable amongst these
was a Crick-led ‘Science Museum Lates’ event in
February. The event gave visitors the chance to
mingle with researchers from the Crick’s partners
to take part in a range of experiments and
activities at the Museum. A record attendance of
6900 people took advantage of the various
opportunities, which included photographing
developing zebra fish on their smartphones,
creating and drinking a DNA cocktail, knitting a
blood vessel and meeting some of the twins taking
part in a long-term study in epigenetics.
It is now only weeks before the Crick starts
operations: our first year will be one of
tremendous excitement, with our move of our
laboratories to the new building. The following
years will surely be more exciting still as we begin
to implement the vision of what promises to be the
most significant development in UK biomedical
science in a generation.
DIRECTOR'S INTRODUCTION
53
RESEARCH HIGHLIGHTS
Every year the Report features highlights of Institute research,
summarising the findings in terms accessible to the non-specialist
scientific reader, and each year the Hardiman-Redon Prize is awarded
to a junior researcher who has made outstanding contributions to
one of these highlighted publications. Our final year has seen an
excellent output of remarkable papers, and the 2014 HardimanRedon Prize is awarded to Sophie Acton from the Immunobiology
Group headed by Caetano Reis e Sousa.
Dendritic cells control fibroblastic reticular
network tension and lymph node expansion.
Acton SE, Farrugia AJ, Astarita JL, Mourão-Sá D,
Jenkins RP, Nye E, Hooper S, van Blijswijk J,
Rogers NC, Snelgrove KJ, Rosewell I, Moita LF,
Stamp G, Turley SJ, Sahai E, Reis e Sousa C.
Nature. 2014; 14(7523):498-502
Lymph node swelling is the hallmark of an adaptive
immune response and is driven by an influx of
lymphocytes from the blood followed by their
eventual antigen-driven proliferation. Throughout
the expansion phase, the lymph node must
maintain the integrity of the stromal network that
acts as the organ’s scaffold and marks the ‘paths’
on which immune cells travel and meet one
another. In this paper, we show that a major
stromal cell type in the T cell areas of lymph nodes,
the fibroblastic reticular cells (FRCs), stretch
rapidly in response to signals from dendritic cells
(DCs) and thereby permit organ expansion driven
by lymphocyte influx. Mechanistically, DCs provide
Figure 1
Fluorescence microscope image
showing insulin-positive β cells
(red) in pancreatic ducts (green)
in mice lacking Fbw7.
54
a transmembrane protein known as CLEC-2 that
engages another known as podoplanin and which
is expressed by FRCs. Podoplanin ligation by CLEC-2
causes it to re-distribute on the FRC membrane
and stops its signalling via RhoA/C and ROCK to the
cells’ actin-based contractile machinery. The net
result is a relaxation of the FRC actomyosin
cytoskeleton that permits cell stretching. Notably,
CLEC-2 is primarily expressed at high levels on
those DCs that immigrate into lymph nodes from
sites of inflammation and deliver antigens and
co-stimulatory signals to T cells. This study
indicates that DCs additionally contribute to
adaptive immunity by delivering the key signals for
lymph node remodelling.
Loss of Fbw7 reprograms adult pancreatic
ductal cells into α, δ and β cells. Sancho R,
Gruber R, Gu G, Behrens A. Cell Stem Cell. 2014;
15(2):139-153
The hormone insulin regulates blood glucose levels
and is produced in the pancreas by the β cells. In
Type 1 diabetes, the β cells are destroyed and so
patients need to control their blood glucose levels
with insulin injections. A better approach would be
to generate replacement β cells that could be
transplanted into the patient to improve blood
glucose regulation in the long term. However, β
cells do not regenerate in the adult, and so, finding
a ‘progenitor cell’ that can produce new β cells is
an important goal of diabetes research. In this
study by Rocio Sancho, our group has shown that
rare pancreatic duct cells can be reprogrammed
into β cells in mice by inactivation of a single gene,
Fbw7. These new β cells respond to glucose and
release insulin in the same way as ordinary β cells
do. This study shows that rare duct cells can act as
pancreatic progenitor cells, raising the possibility
that they could be used to generate replacement β
cells for therapy (Figure 1).
Control mice
Usp28-deleted mice
Figure 2
Survival curve of mice showing
that Usp28 deletion in established
intestinal tumours prolongs
survival (green line).
% Survival
100
50
0
0
50
100
150
Days post Usp28 deletion
The ubiquitin protease Usp28 controls intestinal
homeostasis and promotes colorectal cancer.
Diefenbacher ME, Popov N, Blake SM,
Schülein-Völk C, Nye E, Spencer-Dene B,
Jaenicke LA, Eilers M, Behrens A. Journal of Clinical
Investigation. 2014; 124(8):3407-3418
The lining of the gut is constantly renewed by cell
division and new specialised cells mature to replace
the cells lost. These processes are normally tightly
controlled so that the gut lining is maintained in
balance. In colorectal cancer, however, this balance
goes awry and too much cell division creates a mass
of cells that becomes a tumour. In this study by
Markus Diefenbacher, our group has shown that an
enzyme called Usp28 helps to promote the division
of new cells in the healthy gut. However, Usp28 is
often present at higher levels in colorectal tumours
where it stabilises several proteins known to
promote cancer. When the gene for Usp28 is
deleted, mice genetically predisposed to develop
intestinal cancer get fewer tumours. Even in mice
with established tumours, deleting the gene for
Usp28 reduces cell division, and so, tumours grow
more slowly and the animals live longer. This study
suggests that developing drugs to inhibit Usp28
could be a promising strategy for future colorectal
cancer therapy (Figure 2).
Origin licensing requires ATP binding and
hydrolysis by the MCM replicative helicase.
Coster G, Frigola J, Beuron F, Morris EP, Diffley JFX.
Molecular Cell. 2014; 55(5):666-677
Loading of the six related Minichromosome
Maintenance (MCM) proteins as head-to-head
double hexamers at replication origins is crucial for
ensuring once-per-cell-cycle DNA replication in
eukaryotic cells. This reaction requires the Origin
Recognition Complex (ORC), Cdc6 and Cdt1. ORC,
Cdc6 and MCM are members of the AAA+ family of
ATPases, and MCM loading requires ATP hydrolysis;
but it was unclear so far which proteins need to
hydrolyse ATP. In this paper we showed that ORC
and Cdc6 mutants defective in ATP hydrolysis were
still competent for MCM loading. However, ATP
hydrolysis by Cdc6 was required for ‘proofreading’
non-productive licensing intermediates. We
showed that ATP binding stabilises the wild type
MCM hexamer. Moreover, by analysing MCM
containing mutant subunits, we showed that ATP
binding and hydrolysis by MCM are required for
Cdt1 release and double hexamer formation. This
work fundamentally changed our view of how ATP
hydrolysis promotes this key reaction.
Prereplicative complexes assembled in vitro
support origin-dependent and independent DNA
replication. On KF, Beuron F, Frith D, Snijders AP,
Morris EP, Diffley JFX. EMBO Journal. 2014;
33(6):605-620
Eukaryotic DNA replication initiates from multiple
replication origins. To ensure each origin fires just
once per cell cycle, initiation is divided into two
biochemically discrete steps: the Minichromosome
Maintenance (MCM) helicase is first loaded as an
inactive double hexamer by the origin recognition
complex (ORC), Cdt1 and Cdc6; the helicase is then
activated by a set of firing factors. We had previously
reconstituted this first reaction with purified
proteins (Remus et al., Cell. 2009; 139(4): 719-730).
In the current paper we showed that plasmids
containing MCM loaded with purified proteins
supported complete and semi-conservative
replication in extracts from budding yeast cells
overexpressing firing factors. Replication requires
cyclin-dependent kinase (CDK) and Dbf4- dependent
kinase (DDK). DDK phosphorylation of MCM did not
by itself promote separation of the double hexamer
but was required for the recruitment of firing factors
and replisome components in the extract. Plasmid
replication did not require a functional replication
origin; however, in the presence of competitor DNA
and limiting ORC concentrations, replication became
origin-dependent in this system. These experiments
showed that MCM double hexamers are precursors
of replication and provided insight into the nature of
eukaryotic DNA replication origins.
A Ctf4 trimer couples the CMG helicase to DNA
polymerase α in the eukaryotic replisome.
Simon AC, Zhou JC, Perera RL, van Deursen F,
Evrin C, Ivanova ME, Kilkenny ML, Renault L,
Kjaer S, Matak-Vinković D, Labib K, *Costa A and
*Pellegrini L. Nature. 2014; 510(7504):293-297
*Joint corresponding authors
Genome duplication requires tight coordination
between DNA unwinding and synthesis within the
RESEARCH HIGHLIGHTS
55
for multivalent interactions, illustrating a
mechanism for the concomitant recruitment of
proteins that act together at the replication fork.
Figure 3
The Ctf4 trimer can bind up to
three client proteins
concomitantly.
These findings establish the architectural
framework for further mechanistic studies of the
elongation step of DNA replication in eukaryotic
cells (Figure 3).
replisome to prevent the accumulation of
vulnerable single-stranded DNA segments and
the onset of genomic instability. In this study,
single-particle electron microscopy and
crystallography were used to establish the
architecture of the Ctf4 ‘helicase-polymerase
bridging factor’ either alone or bound to
components of the Cdc45-MCM-GINS (CMG)
helicase and the DNA Polymerase α/primase
assemblies. Ctf4 was found to form a disc-shaped
trimer, suggesting that it has the ability to link
multiple factors at replication forks. Indeed, the
Ctf4 trimer contains three docking sites that can
simultaneously bind to the GINS component of the
CMG helicase and Pol α. The helicase and
polymerase share a common mechanism of
interaction with Ctf4, with the N-terminal tails of
the catalytic subunit of Pol α and the Sld5 subunit
of GINS containing a common element that docks
onto the C-terminal extension of a Ctf4 protomer
within the trimer. Therefore, Ctf4 acts as a platform
Erlotinib + MEKi
Final 4 weeks of treatment
Before
After
MEKi
Erlotinib
Figure 4
Lung tumours in mice expressing
activated EGFR. Tumours respond
to combination therapy with EGFR
inhibitor plus MEK inhibitor even
after they have become resistant
to EGFR inhibitor alone due to
NF1 down-regulation.
56
Reduced NF1 expression confers resistance to
EGFR inhibition in lung cancer. de Bruin EC,
Cowell C, Warne PH, Jiang M, Saunders RE,
Melnick MA, Gettinger S, Walther Z, Wurtz A,
Heynen GJ, Heideman DA, Gómez-Román J,
García-Castaño A, Gong Y, Ladanyi M, Varmus H,
Bernards R, Smit EF, Politi K, Downward J. Cancer
Discovery. 2014; 4(5):606-619
Activating mutations in the EGF receptor (EGFR) are
found in about 15% of lung cancers and are
associated with clinical responsiveness to EGFR
tyrosine kinase inhibitory drugs (TKIs), such as
erlotinib and gefitinib. However, resistance to these
drugs eventually arises, often due to a second
mutation in EGFR, that prevents the drugs from
binding. Through a functional genomic screen in a
human lung cancer cell line and analysis of gene
expression of mutant EGFR-driven lung cancers in
mice, we found that erlotinib resistance was
associated with reduced expression of NF1, a
tumour suppressor that negatively regulates RAS
proteins, which are key drivers of malignant
growth. Treatment of NF1-deficient lung cancers
with a MEK inhibitory drug, which targets the RAS
pathway, restored sensitivity to erlotinib. Low
levels of NF1 expression were associated with
resistance of lung cancers to EGFR TKIs in patients.
These findings identify a subgroup of patients with
EGFR-mutant lung cancer who might benefit from
combination therapy with EGFR and MEK inhibitors
(Figure 4).
RAS interaction with PI3K p110α is required for
tumor-induced angiogenesis. Murillo MM,
Zelenay S, Nye E, Castellano E, Lassailly F, Stamp G,
Downward J. Journal of Clinical Investigations.
2014; 124(8):3601-3611
Direct interaction of RAS proteins with the lipid
kinase PI3K p110α mediates RAS-driven tumour
development. However, it is not clear how p110α/
RAS-dependent signalling mediates interactions
between tumours and host tissues. We show here
that disruption of the interaction between RAS and
p110α within host tissue reduced tumour growth
and tumour-induced angiogenesis, even when this
interaction was intact in the tumour. Functional
interaction of RAS with p110α in host tissue was
required for efficient establishment and growth of
metastatic tumours and also for efficient
angiogenesis. Additionally, disruption of the RAS
and p110α interaction altered the nature of
tumour-associated immune cells such as
macrophages, inducing expression of markers
typical for macrophage populations with reduced
tumour-promoting capacity. These results indicate
that a functional RAS interaction with PI3K p110α in
host tissue is required for the establishment of a
growth-permissive environment for the tumour,
particularly for tumour-induced angiogenesis.
Targeting the interaction of RAS with PI3K has the
potential to impair tumour formation by altering
the tumour-host relationship, in addition to
previously described tumour cell-autonomous
effects.
The role of differential VE-cadherin dynamics
in cell rearrangement during angiogenesis.
Bentley K, Franco CA, Philippides A, Blanco R,
Dierkes M, Gebala V, Stanchi F, Jones M,
Aspalter IM, Cagna G, Weström S,
Claesson-Welsh L, Vestweber D, Gerhardt H.
Nature Cell Biology. 2014;16(4):309-321
During blood vessel formation, certain molecules
and cues tell endothelial cells to take on different
characteristics, with some becoming ‘polarised tip
cells’ and other becoming ‘stalk cells’. Together,
these two cell types form new capillaries. Tip and
stalk cells were previously thought to have
established roles, with tip cells leading the way and
stalk cells following along to create a vessel tube,
but recent research has suggested that endothelial
cells are much more spontaneous and actually
undergo dynamic changes and frequently switch
positions, with stalk cells overtaking tip cells at the
leading edge of a new blood vessel.
This work proposes that when endothelial cells are
stimulated by vascular endothelial growth factor
(VEGF) – and not inhibited by Notch signalling –
they are ‘active’ and can either form a new branch
as tip cells or ‘shuffle up’ through the existing
sprout by cell rearrangement mechanisms and it
validates simulation predictions in vivo,
demonstrating that Notch regulates shuffling
movement via VE-cadherin adhesion. The team
also found that during cancer development and
progression, there is a switch: the regulation of the
adhesion between cells becomes more uniform so
that there are clustered regions of cells in an
all-active or all-inhibited shuffling state.
When simulant cells are let loose in a simple
simulated tumor environment, their collective
behavior changed dramatically. The cells go
through cyclic phases of adhesion and junctional
movements now as a group, in which they all
clamber to move at once or all remain still. In
either case they are getting nowhere as overtaking
requires differential movement of one cell
compared to its neighbors. The disrupted
rearrangement provides a new explanation for the
enlarged blood vessels we see in pathologies, such
as in mouse models of tumors or retinopathies.
Oncogenic RET kinase domain mutations perturb
the autophosphorylation trajectory by enhancing
substrate presentation in trans. Plaza-Menacho I,
Barnouin K, Goodman K, Martínez-Torres RJ,
Borg A, Murray-Rust J, Mouilleron S, Knowles P,
McDonald NQ. Molecular Cell. 2014; 53(5):738-51
Receptor tyrosine kinases (RTK) are a class of
trans-membrane proteins that are frequently
targeted for oncogenic activation, whether by
site-specific mutation, overexpression or through
fusion/translocations. Ligand-dependent RTK
activation arises through trans phosphorylation
(autophosphorylation) through allosteric or
oligomerisation mechanisms. Oncogenic
activation often arises from a dramatically
enhanced tyrosine kinase activity and subversion
of intrinsic RTK control mechanisms. In many cases
the increased tyrosine kinase activity is a
consequence of autophosphorylation of the RTK
‘activation-loop’. However, some RTKs such as the
EGFR and RET are not stimulated by activationloop phosphorylation suggesting mechanisms
distinct from most RTKs may operate. By
monitoring the appearance and timing of
autophosphorylation sites upon RET activation we
found that rapid phosphotyrosine sites are all
outside of the tyrosine kinase domain, whereas
sites within the kinase domain appear at much
later time points, consistent with a role in RET
signalling rather than activation. We then explored
the basis for oncogenic activation of RET by point
mutations found in patients with multiple
endocrine neoplasia type 2. Using biochemical,
mass spectroscopy and crystallography, we show
how such mutations alter the RET
autophosphorylation trajectory and produce a
greatly enhanced autophosphorylation substrate,
despite reducing protein stability. Our study
reveals an underappreciated role for oncogenic
RTK mutations in promoting intermolecular
autophosphorylation through an enhanced
substrate presentation.
RESEARCH HIGHLIGHTS
57
Replication origin selection regulates the
distribution of meiotic recombination. Wu PY,
Nurse P. Molecular Cell. 2014; 53(4):655-662
The pattern of origin firing during DNA replication
is altered during development and also in diseases
such as cancer, suggesting that it may play a role in
regulating biological processes. To investigate this,
we have used fission yeast meiosis as a model for a
developmental pathway and examined how the
meiotic process is altered under different patterns
of origin firing during pre-meiotic DNA replication.
We found that changing the nutritional conditions
lead to changes in the pattern of origin firing. These
changes had no overall effect on progression of
meiosis, suggesting that a particular pattern of
origin firing was not required for meiotic
progression. However, changes in the origin firing
pattern was correlated with local changes in the
binding of the recombination factor Rad51 and
meiotic recombination frequencies, with increases
in origin efficiency being associated with increased
binding of Rad51 and increased recombination
levels. Changes in the efficiency of origin firing and
Rad51 binding could be induced by directly
modulating the level of the replication factor
Cdc45, independently of the nutritional status.
Here we show that external factors such
nutritional conditions can influence the pattern of
origin firing in a cell and that this may lead directly
to changes in the pattern of Rad51 binding and
meiotic recombination.
Mitotic catenation is monitored and resolved by a
PKCε- regulated pathway. Brownlow N, Pike T,
Zicha D, Collinson L, Parker PJ. Nature
Communications. 2014; 5:5685
The metaphase to anaphase transition is a pivotal
point in cell division, where for the first time, the
newly replicated, paired sister chromatids become
physically separated. Mistakes resulting in
Figure 5
A DLD-1 cell with knockdown of
PKCε displaying a PICH positive
ultrafine bridge (green) stretched
out between separating sister
chromatids (blue) in anaphase.
These PICH positive structures are
more prevalent with the
increased metaphase catenation
triggered by the loss or inhibition
of PKCε.
58
chromosome non-disjunction are broadly
implicated in cancer and widely associated with a
poor prognosis. Using model systems, we were
able to show that a metaphase to anaphase
transition delay is triggered by the presence of
sister chromatid catenation in metaphase. This
catenation-induced delay is dependent on the
protein kinase PKCε. Under conditions of excessive
catenation, loss or acute inhibition of PKCε leads to
precocious mitotic exit and extensive cytokinesis
failure.
To determine how this delay is effected, Nicola
Brownlow monitored components of the spindle
assembly checkpoint (SAC) and found that
associated with this delay there is a PKCεdependent retention of Bub1 and BubR1 at
kinetochores but no retention of Mad2, indicative
of a partially silenced SAC. The engagement of this
metaphase pathway was found only in certain
transformed cells where the normal catenation
induced G2 checkpoint was defective. This G2
checkpoint is independent of PKCε and is lost in
various human tumours and tumour cell models,
leading to a tumour-specific dependence on the
mitotic pathway. Dissection of this mitotic
catenation monitoring system therefore presents a
potential opportunity for selective intervention in
cancer, and also has implications for our
understanding of chromosome instability in cancer
(Figure 5).
Antiviral immunity via RIG-I-mediated
recognition of RNA bearing 5’-diphosphates.
Goubau D, Schlee M, Deddouche S, Pruijssers AJ,
Zillinger T, Goldeck M, Schuberth C,
Van der Veen AG, Fujimura T, Rehwinkel J,
Iskarpatyoti JA, Barchet W, Ludwig J, Dermody TS,
Hartmann G, Reis e Sousa C. Nature. 2014;
514(7522):372-375
RIG-I is a very important innate immune sensor of
RNA viruses and can be activated by RNAs bearing
5’ tri-phosphate moieties such as found in the
genomes of influenza virus, measles virus, mumps
virus and many other human viral pathogens.
However, a few publications suggested that RNAs
lacking 5’ tri-phosphates can, in some instances,
act as RIG-I agonists and the crystal structures of
RIG-I show that the terminal gamma phosphate in
5’ ppp RNA does not make appreciable contact
with the protein. In this study, we found that RIG-I
can, in fact, respond equally well to 5’ diphosphate bearing RNA such as found in the
genome of reovirus and other viruses like the
human rotaviruses. Furthermore, the same
provides a conceptual understanding of a major
protein interaction network at microtubule ends,
which is important in order to understand the
regulation of microtubule dynamics and cargo
transport initiation in health and disease (Figure 6).
Figure 6
Fluorescence microscopy images
(top right) and a kymograph
(bottom right) showing
recruitment of the dynactin
component p150 (green) to the
growing end of a dynamic
microtubule (red) by EB1. p150 is
a key component in the +TIP
network that targets dynein to
microtubule ends (schematic,
right).
RECQL5 controls transcript elongation and
suppresses genome instability associated with
transcription stress. Saponaro M, Kantidakis T,
Mitter R, Kelly GP, Heron M, Williams H, Söding J,
Stewart A, Svejstrup JQ. Cell. 2014; 157(5):10371049
di-phosphate moieties are present in poly I:C, a
synthetic stimulus that has been used for many
decades to induce interferons. These data indicate
that the minimal determinant of RIG-I recognition
is in fact a 5’ di-phosphate-bearing blunt-ended
base-paired RNA. Thus, innate self/non-self
recognition extends to the detection of 5’ diphosphate-containing RNAs and RIG-I must
henceforth be seen as a sensor of both 5’ di- and
triphosphate RNAs.
Reconstitution of a hierarchical +TIP interaction
network controlling microtubule end tracking of
the human dynein complex. Duellberg C,
Trokter M, Jha R, Sen I, Steinmetz MO, Surrey T.
Nature Cell Biology. 2014; 16(8):804-811
Microtubules are filaments of the cytoskeleton
that constantly switch between phases of growth
and shrinkage, called dynamic instability. This
property is essential for many cellular processes
such as mitosis, migration and intracellular
transport. Microtubule ends are of particular
interest because regulation of dynamic instability
by proteins takes place directly at these ends. A
second important feature is that cargo transport
towards the cell centre is initiated directly at these
ends. Many proteins that are enriched at
microtubule end regions are known, but the
mechanism of how these so-called +TIPs get
recruited to these ends is not well understood.
Many diseases are associated with mislocalised
+TIPs. Using a bottom up approach, we have rebuilt
the recruitment of an important motor protein,
called dynein, which facilitates almost all cell
centre-directed cargo transport. We could reveal
how a hierarchically structured protein interaction
network recruits dynein in the presence of
competition for microtubule end binding. Our study
This study provides evidence that transcription
stress (RNAPII stopping and stalling in the
transcribed region of genes) can cause dramatic
genome instability, and that the tumoursuppressor RECQL5 is important to suppress it.
Interestingly, transcription stress-induced genome
instability is particularly frequent in regions that
are devoid of ‘back-up’ DNA replication origins,
causing chromosomal breakage and unscheduled/
detrimental DNA recombination in so-called
common fragile sites. Hence, when replication
stress meets transcription stress, chromosomes
break and become rearranged.
Spatial and temporal diversity in genomic
instability processes defines lung cancer
evolution. de Bruin E, McGranahan N, Mitter R,
Salm M, Wedge DC, Yates L, Jamal-Hanjani M,
Shafi S, Murugaesu N, Rowan AJ, Gronroos E,
Muhammad MA, Horswell S, Gerlinger M, Varela I,
Jones D, Marshall J, Voet T, Van Loo P, Rassl DM,
Rintoul RC, Janes SM, Lee S, Forster M, Ahmed T,
Lawrence D, Falzon M, Capitanio A, Harkins TT,
Lee CC, Tom W, Teefe E, Chen SC, Begum S,
Rabinowitz A, Phillimore B, Spencer-Dene B,
Stamp G, Szallasi Z, Matthews N, Stewart A,
Campbell P, Swanton C. Science. 2014;
346(6206):251-256
Lung cancer affects over 1.4 million people
worldwide per year and is associated with an
average survival of 18 months in advanced disease.
To gain more insights into this disease, we
performed detailed genome sequencing analyses
of seven lung tumours. By comparing the DNA
from different regions within each tumour, we
found that about one third of the DNA errors could
be found in only a subset of regions and not
throughout the whole tumour. Investigating the
mutations in DNA in more detail, we found the
typical smoking associated ‘C-to-A’ mutations in
tumours of patients that had smoked, particularly
for mutations that had occurred early in tumour
RESEARCH HIGHLIGHTS
59
Figure 7
Multiregion DNA sequencing of a
tumour allows for the analysis of
genetic intra-tumour
heterogeneity and provides
insight into the tumour’s life
history. Mutations that are
present in only one or a subset of
regions, heterogeneous
mutations, occurred at later
stages of tumour development
compared to mutations that are
present in all regions. In lung
tumours, ‘early’ mutations are
mainly caused by smoking, and
‘late’ mutations by APOBEC
enzymes.
development. We noticed different types of
mutations occurred after tumour initiation, linked
to APOBEC DNA-editing enzymes encoded by the
human genome. Normally, these enzymes are
induced by cells in response to viral infection in
order to damage viral DNA and limit their
infectious capacity. This work demonstrates a role
for this enzyme family in the initiation of mutations
later in tumour evolution that drive the expansion
of distinct cell populations within tumours,
resulting in a diversity of cancer genomes within
one patient that might have an impact on clinical
outcome and drug resistance (Figure 7).
Tolerance of whole-genome doubling propagates
chromosomal instability and accelerates cancer
genome evolution. Dewhurst SM, McGranahan N,
Burrell RA, Rowan AJ, Grönroos E, Endesfelder D,
Joshi T, Mouradov D, Gibbs P, Ward RL, Hawkins NJ,
Szallasi Z, Sieber OM, Swanton C. Cancer Discovery.
2014; 4(2):175-185
In this study we examined the impact of genome
doubling (a complete doubling of the genetic
content of a cell) in cancer evolution, a
phenomenon that occurs in up to 60% of cancers.
Using cancer cells cultured in the laboratory and
tumour data from over 500 cancer patients, we
found that genome doubling can be an important
event in tumour progression, likely driving
disordered chromosome distribution between
daughter cells (chromosomal instability), which
can contribute to cancer drug resistance and poor
patient outcome.
We developed a novel system of diploid and
genome doubled (tetraploid) colorectal cancer cell
lines that are genetically identical apart from the
amount of DNA within each cell. Growing these
cells in parallel for over two years in the laboratory
60
allowed us to investigate how they evolved over
time. We found that genome doubled cells could
tolerate chromosomal changes better than diploid
cells, and therefore evolved much more rapidly
over time. We reasoned that since genome
doubling occurred before genomic instability in our
cell lines, genome doubling might be a useful
clinical measure to identify high-risk patient groups
at an early disease stage. We used two
independent data sets comprising 539 patients
with colorectal cancer, and found that genome
doubling was predictive of poor relapse free
survival. Genome doubling has relevance as a new
means to predict outcome in colorectal cancer,
potentially allowing the escalation of therapy in
higher risk groups (Figure 8).
WIPI2 links LC3-conjungation with PI3P,
autophagosome formation, and pathogen
clearance by recruiting Atg12-5-16L1. Dooley HC,
Razi M, Polson HEJ, Girardin SE, Wilson MI,
Tooze SA. Molecular Cell. 2014; 55(2):238-252
Autophagy (self-eating) is a process that cells use to
remain healthy and survive in stressful conditions,
in particular nutrient deprivation. However,
autophagy can also be exploited by cancer cells to
survive, therefore, we aim to understand the details
of how autophagy can be controlled in human cells.
Autophagy is performed by specialised membranes,
called autophagosomes, which can be made
on-demand in response to a stress signal. A set of
proteins, so called Atg proteins, and an important
lipid, phosphatidylinositol-3-phosphate (PI3P), are
required for the formation of the autophagosome.
The PI3P involved in autophagy is a unique pool
present on the ER. LC3, a protein modified by
another lipid, phosphatidylethanolamine, during
autophagy is required for autophagosome
membrane growth and closure. However, the
specific activation of either MRTFs or TCFs can reset
the circadian clock. Many MRTF targets are
implicated in cancer invasiveness, metastasis, and
mechanosensing, suggesting that MRTF-SRF
signalling constitutes a nuclear arm of the cellular
response to matrix adhesion.
Figure 8
Clonal FISH (fluorescence in-situ
hybridisation) of a tetraploid
colorectal cancer clone derived
from HCT-116. Chromosome 2
(red) and chromosome 8 (green)
show copy number variation
between cells, indicating
chromosomal instability. Nuclei
are stained with DAPI (blue).
Biochemical reconstitution of topological DNA
binding by the cohesin ring. Murayama Y,
Uhlmann F. Nature. 2014; 505(7483):367-371
connection between Atg proteins, LC3 and PI3P,
was not known. Our publication describes the
discovery of a direct interaction between the Atg
protein WIPI2b, which binds PI3P and Atg16L1, a
component of the LC3-lipidation complex. This
discovery provides the missing link between
ER-localised production of PI3P, which is triggered
by autophagy, and recruitment of the LC3conjugation complex crucial for autophagosome
membrane formation. Furthermore, this enables us
to manipulate the process at the molecular level to
further our understanding of how autophagosomes
form and how autophagy can be regulated.
Rho-actin signaling to the MRTF coactivators
dominates the immediate transcriptional
response to serum in fibroblasts. Esnault C,
Stewart A, Gualdrini F, East P, Horswell S,
Matthews N, Treisman R. Genes and Development.
2014; 28(9):943-958
Gene transcription is a prerequisite for quiescent
cells to enter the cell cycle. Classical experiments
identified the SRF (‘Serum Response Factor’)
transcription factor as master regulator of
‘immediate-early’ genes that are induced within
minutes by mitogenic stimuli. SRF is regulated by
two families of signal-regulated cofactors. The
TCFs link SRF activity to the classical mitogenic
Ras-ERK signal pathway to control genes such as
c-fos, while the MRTFs couple SRF activity to Rho
signalling, responding to signal-induced changes in
cellular concentration of G-actin. This paper
evaluates the extent to which the SRF network
controls the immediate-early transcriptional
response, using genomic methods and pathwayspecific inhibitors. Several hundred direct targets
for MRTF-SRF and TCF-SRF signalling were
identified. MRTF targets encode regulators of the
cytoskeleton, transcription, and cell growth, and
Cohesion between replicated sister chromatids is
mediated by the chromosomal cohesin complex
and is a prerequisite for faithful chromosome
segregation in mitosis. Cohesin plays vital roles also
in DNA repair and transcriptional regulation. The
ring-shaped cohesin complex is thought to encircle
sister DNA strands, but its molecular mechanism of
action is poorly understood and the biochemical
reconstitution of cohesin activity in vitro had
remained an unattained goal. We were now
successful in reconstituting topological cohesin
loading onto DNA using purified fission yeast
cohesin and its loader complex, Scc2Mis4/Scc4Ssl3.
Surprisingly, we found that incubation of cohesin
with DNA leads to spontaneous topological loading,
but that remains inefficient. Interaction site
mapping revealed that the loader makes contact
with cohesin at multiple sites around the ring
circumference, including the hitherto enigmatic
Scc3Psc3 subunit. The loader furthermore
stimulates cohesin’s ATPase. This leads us to a first
molecular model of the cohesin loading reaction, in
which the cohesin loader acts as a template for a
conformational change in the cohesin complex that
triggers ATP hydrolysis-dependent loading. This
provides mechanistic insight into the initial steps of
establishing sister chromatid cohesion and other
chromosomal processes mediated by cohesin.
The Scc2-Scc4 complex acts in sister chromatid
cohesion and transcriptional regulation by
maintaining nucleosome-free regions.
Lopez-Serra L, Kelly G, Patel H, Stewart A,
Uhlmann F. Nature Genetics. 2014; 46(10):11471151
The cohesin complex lies at the heart of many
chromosomal activities, including sister chromatid
cohesion and transcriptional regulation. Loading of
cohesin onto chromosomes depends on the Scc2/
Scc4 cohesin loader complex, but the chromatin
features that attract Scc2/Scc4 and thereby form
cohesin loading sites remained poorly understood.
In this work, we showed that the budding yeast
Scc2/Scc4 complex is recruited to broad
RESEARCH HIGHLIGHTS
61
nucleosome-free regions in the promoters of
highly expressed genes by the RSC chromatin
remodelling complex. Unexpectedly, Scc2/Scc4
itself is required to maintain the nucleosome-free
status of its binding sites. Consequently,
inactivation of the cohesin loader or RSC complex
has similar effects on nucleosome positioning,
gene expression and sister chromatid cohesion.
These results revealed an intimate link between
local chromatin structure under control of the RSC
chromatin remodelling complex and higher order
chromosome architecture rendered by cohesin.
These findings also pertain to the similarities
between two severe human developmental
disorders, Cornelia de Lange syndrome, most
often caused by mutations in the human
Scc2NIPBL cohesin loader subunit, and Coffin-Siris
syndrome, resulting from mutations in human RSC
complex components. We suggest that both could
arise from gene misregulation due to overlapping
changes in the nucleosome landscape (Figure 9).
Ena/VASP proteins cooperate with the WAVE
complex to regulate the actin cytoskeleton.
Chen XJ, Squarr JA, Stephan R, Chen B, Higgins TE,
Barry DJ, Martin MC, Rosen MK, Bogdan S, Way M.
Developmental Cell. 2014; 30(5):569-584
Directed cell motility in response to a wide variety
of signalling events requires temporal and spatial
control of actin polymerisation at the plasma
membrane. Ena/VASP proteins and the WAVE
regulatory complex (WRC) have emerged as
important regulators of cell motility by virtue of
their ability to independently promote actin
polymerisation at the leading edge of migrating
cells. We have now demonstrated that the
N-terminal EVH1 domain of Ena/VASP proteins
Figure 9
Colocalisation of the budding
yeast cohesin loader subunit
Scc2 with the RSC chromatin
remodelling complex subunit
Sth1. The Sth1 binding profiles,
centred at each Scc2 binding site
are collated to visualise the close
correlation. Further work
showed how the cohesin loader
cooperates with RSC to maintain
nucleosome free regions,
regulate transcription from the
bound promoters and load
cohesin onto DNA.
62
interacts with an extended proline rich motif in the
Abi subunit of the WRC. In vitro, VASP cooperatively enhances the ability of the WRC to
stimulate Arp2/3 complex-induced actin assembly
in the presence of Rac1. This increased activity
depends on VASP tetramerisation and its
interaction with Abi1. In Drosophila, Ena also
interacts with the WRC via two LPPPP motifs in Abi.
Loss of this interaction in Drosophila hemocytes
results in defects in membrane protrusion and cell
spreading, as well as redistribution of Ena to the
tips of filopodia-like extensions. Rescue
experiments in an abi mutant background also
reveal a physiological requirement for the Abi:Ena
interaction in photoreceptor axon targeting and
oogenesis in vivo. Our data have demonstrated that
the activity of Ena/VASP and the WRC are
intimately linked to ensure optimal control of actin
polymerisation.
Dual control of Yen1 nuclease activity and cellular
localization by Cdk and Cdc14 prevents genome
instability. Blanco MG, Matos J, West SC. Molecular
Cell. 2014; 54(1):94-106
The careful orchestration of cellular events such as
DNA replication, repair and chromosome
segregation is essential for the equal distribution of
the duplicated genome to the two daughter cells.
To ensure that persistent recombination
intermediates are resolved prior to cell division, the
enzymes that resolve junctions need to be
activated at the appropriate time in the cell cycle.
This paper defines the mechanism by which the
Yen1 Holliday junction resolvase is regulated in
yeast. We found that Yen1 undergoes a dual mode
of regulation, by modulation of both its activity and
subcellular localisation. Cdk phosphorylation
Scc2-
inhibits Yen1 at S phase by reducing its DNA
binding affinity, whereas it is activated at anaphase
by the Cdc14 phosphatase. We find that proper
regulation is critical for chromosome segregation.
Spatial control of the GEN1 Holliday junction
resolvase ensures genome stability. Chan YW
and West SC. Nature Communications. 2014;
5:4844
Holliday junction (HJ) resolvases are necessary for
the processing of persistent recombination
intermediates before cell division. Their actions,
however, need to be restricted to the late stages of
the cell cycle to avoid the inappropriate cleavage
of replication intermediates. Control of the yeast
HJ resolvase Yen1 involves phosphorylation
changes that modulate its catalytic activity and
nuclear import. Here, we show that GEN1, the
human ortholog of Yen1, is regulated by a different
mechanism that is independent of
phosphorylation. GEN1 is controlled exclusively by
nuclear exclusion, driven by a nuclear export signal
(NES) that restricts GEN1 actions to mitosis when
the nuclear membrane breaks down. Construction
of a nuclear-localised version of GEN1 revealed
that its premature actions partially suppress
phenotypes associated with loss of BLM and
MUS81 but cause elevated crossover formation.
The spatial control of GEN1 therefore contributes
to genome stability by avoiding competition with
non-crossover promoting repair pathways.
Roles of SLX1-SLX4, MUS81-EME1 and GEN1 in
avoiding genome instability and mitotic
catastrophe. Sarbanjna S, Davies D, West SC.
Genes and Development. 2014; 28(10):1124-1136
The resolution of recombination intermediates
containing Holliday junctions (HJs) is critical for
genome maintenance and proper chromosome
segregation. Three pathways for HJ processing
exist in human cells and involve the following
enzymes/complexes: BLM–TopoIIIa–RMI1–RMI2
(BTR complex), SLX1–SLX4–MUS81–EME1 (SLX–
MUS complex), and GEN1. Cycling cells
preferentially use the BTR complex for the
removal of double HJs in S phase, with SLX–MUS
and GEN1 acting at temporally distinct phases of
the cell cycle. Cells lacking SLX–MUS and GEN1
exhibit chromosome missegregation,
micronucleus formation, and elevated levels of
53BP1-positive G1 nuclear bodies, suggesting that
defects in chromosome segregation lead to the
transmission of extensive DNA damage to daughter
cells. In addition, however, we found that the
effects of SLX4, MUS81, and GEN1 depletion
extend beyond mitosis, since genome instability is
observed throughout all phases of the cell cycle.
This is exemplified in the form of impaired
replication fork movement and S phase
progression, endogenous checkpoint activation,
chromosome segmentation, and multinucleation.
In contrast to SLX4, SLX1 (the nuclease subunit of
the SLX1–SLX4 structure-selective nuclease) plays
no role in the replication-related phenotypes
associated with SLX4/MUS81 and GEN1 depletion.
These observations demonstrate that the SLX1–
SLX4 nuclease and the SLX4 scaffold play divergent
roles in the maintenance of genome integrity in
human cells.
Structure and mechanism of action of the BRCA2
breast cancer tumour suppressor. Shaid T,
Soroka J, Kong E, Malivert L, McIlwraith MJ,
Pape T, *West SC and *Zhang X. Nature Structural
Molecular Biology. 2014; 21:962-968
*Joint corresponding authors
Mutations in BRCA2 increase susceptibility to
breast, ovarian and prostate cancers. The product
of the BRCA2 gene, BRCA2 protein, has a key role in
the repair of DNA double-strand breaks and
interstrand crosslinks by RAD51-mediated
homologous recombination. In this study, we
present a biochemical and structural
characterisation of full-length (3,418 amino acid)
BRCA2, alone and in complex with RAD51. We
found that BRCA2 facilitates nucleation of RAD51
filaments at multiple sites on single-stranded DNA.
Three-dimensional electron microscopic
reconstructions revealed that BRCA2 exists as a
dimer and that two oppositely oriented sets of
RAD51 molecules bind the dimer. Single-stranded
DNA binds along the long axis of BRCA2, such that
only one set of RAD51 monomers can form a
productive complex with DNA and establish
filament formation. Our data define the molecular
mechanism by which this tumour suppressor
facilitates RAD51-mediated homologousrecombinational repair.
RESEARCH HIGHLIGHTS
63
Drosophila wing imaginal discs
expressing DIAP1-GFP (green) and
ubi-RFP (red) and stained with
DAPI (blue). Image: Maxine
Holder, Apoptosis and
Proliferation Control Group.
64
LINCOLN'S
INN
FIELDS
The London Research Institute, Lincoln’s Inn Fields laboratories are
located in the centre of London. The research laboratories work
within the broad research themes of cellular regulatory mechanisms,
biology of tumours and tissues, and genomic integrity and cell cycle.
By carrying out basic research at the Institute we will continue to
increase the understanding of cancer biology. The researchers are
supported by an excellent range of Technology Core Facilities.
LINCOLN'S INN FIELDS
65
LYMPHOCYTE INTERACTION
www.london-research-institute.org.uk/research/facundo-batista
Group Leader
Facundo D Batista
Postdoctoral Scientists
Shweta Aggarwal
Bruno Frederico
Francesca Gasparrini
Selina Keppler
Nuria Martínez-Martín
Graduate Students
Marianne Burbage
Mauro Gaya
Carlson Tsui
Scientific Officers
Julia Coleman
Cecilia Deantonio
Christoph Feest
David Mestre
Beatriz Montaner
Paul Newman
Microscope Developer
Andreas Bruckbauer
B lymphocytes are important immune cells. They produce antigenspecific antibodies by which they can protect us against disease. In
the Lymphocyte Interaction Laboratory, we investigate the cellular
and molecular events leading to the activation of B cells and their
differentiation into antibody producing cells. We use a variety of
techniques based on genetics, biochemistry and state-of-the-art
imaging technology, both in vitro and in vivo. As examples, by tracking
single B cell receptor (BCR) particles we have previously shown that
diffusion of the BCR is restricted by an ezrin-defined actin network
(Treanor et al., 2010; Immunity. 32(2): 187-199), and that this
restriction regulates receptor signalling; or by following B cells in vivo
we can understand where and when B cell activation occurs
(Carrasco and Batista, 2007; Immunity. 27(1): 160-171). We have since
explored novel concepts of BCR signalling regulation and its
involvement in B cell activation, as well as delving deeper into our
studies of how and when B cells become activated in vivo.
The behaviour of B cells on both molecular and
cellular levels can aid our understanding of immune
responses to pathogens and disease control
We know that B cell activation is triggered by
specific recognition of antigen through the BCR
and that both positive and negative regulatory
molecules modulate this process. We have been
dissecting the detail of BCR regulation, and, in
particular, the role of the organisation and
dynamics of these regulators within the cell
membrane. Additionally, we are looking at the
behaviour and interactions of B cells with innate
immune effector cells within secondary lymphoid
organs. We have directed our focus in this respect
to the behaviour of subcapsular sinus (SCS)
macrophages during natural infections and the
resulting implications on B cell responses to
secondary pathogens. We have, therefore, been
working towards a clearer understanding of B cell
activation, antibody production and the dynamics
of lymph node organisation following infections;
knowledge that will aid us not only in the fight
against cancer but also infectious diseases.
Cdc42 organisation and dynamics
B cells recognise foreign antigens by virtue of cell
surface immunoglobulin receptors and are most
effectively activated by membrane-bound ligands.
Previously, work in the group has shown that the
early stages of this process involve a two-phase
66
response involving spreading and contraction and
that the extent of this signalling- and actindependent response determines the quantity of
antigen accumulated and, hence, the degree of B
cell activation (Fleire et al., 2006; Science.
312(5774): 738-741). Further investigation revealed
that the cortical actin cytoskeleton was involved in
the control of B cell dynamics and signalling and
that the Ezrin-Radixin-Moesin (ERM) proteins are a
crucial link in this control mechanism (Treanor et
al., 2011; J Exp Med. 208(5): 1055-1068; Treanor et
al., 2010). Delving deeper into the signalling
pathway, we found a role for the adaptor protein
Nck in BCR signalling, enabling the recruitment of
BCAP in the essential P13K-Akt pathway (Castello
et al., 2013; Nat Immunol. 14(9):966-975). Building
on this work, we demonstrated that cytoskeleton
disruption triggers B cell signalling not only
through the BCR but involves the co-receptor CD19
and tetraspanin (CD81), suggesting that receptor
compartmentalisation regulates antigen-induced
activation (Mattila et al., 2013; Immunity. 38(3):
461-474). We have most recently focused on Cdc42,
a small Rho GTPase that is known to be a regulator
of actin remodelling, so we sought to investigate
whether it was therefore also implicated in B cell
activation. In mice with Cdc42 deleted in the B cell
lineage only, we saw that antibody responses were
eliminated and the mice were incapable of forming
germinal centres or of generating plasma B cells.
3D view
Top view
3D side view
Control
CpG
Figure 1
3D multiphoton microscopy
images showing that CpG-induced
inflammation in mice leads to a
decrease in the number and
density of CD169+ macrophages
(green) in the draining lymph
nodes, along with a retraction and
change in morphology of these
cells when compared with PBS
controls. The cyan shows a
second harmonic signal generated
by collagen fibrils.
This was the case for either viral infection or for
immunisation. There were profound and multiple B
cell faults seen to be causing this severe immune
deficiency. These included early blocks during B cell
development; impaired antigen-driven BCR
signalling and actin remodelling; defective antigen
presentation and in vivo interaction with T cells;
and a severe B cell intrinsic block in plasma cell
differentiation. Our study adds a new perspective
on Cdc42 function as a master regulator of B cell
physiology (Burbage et al., 2014;doi:10.1084/
jem.20141143).
SCS macrophage disruption following infection
As well as studying lymphocyte interactions at the
micro- and nano-scale, we actively pursue the
understanding of how, where, and when B cells are
activated in vivo. The lymph node is an important
location for the presentation of antigen to B cells
and we have observed that antigen accumulates in
the macrophage-rich area at the SCS boundary,
and identified this area as a site for the initiation of
B cell responses (Carrasco and Batista, 2007). We
have also shown that SCS macrophages are
capable of presenting certain antigens to other
immune cells, including iNKT cells (Barral et al.,
2010; Nat Immunol. 11(4): 303-312). Recently, we
have focused special attention on the importance
of lymph node architecture with relation to SCS
macrophages and the implications for antigen
presentation to B cells and other immune effector
cells. In particular, we studied B cell activation in a
model of double infection. The layer of CD169+ SCS
macrophages that captures pathogens as they
enter the lymph node prevents the spread of
pathogens and triggers immune responses. So far,
the impact of infection on the organisation and
function of SCS macrophages has been largely
unexplored, so, using innovative imaging
approaches, we investigated this scenario. We
found that virus- and bacteria-induced
inflammation led to a dramatic disruption of SCS
macrophages caused in part by mature dendritic
cells entering the lymph node in response to
pathogen-associated signals. This change in the
organisation of SCS macrophages reduces their
ability to retain and present antigen in subsequent
secondary infections, leading to reduced B cell
responses. We believe that the SCS macrophage
layer acts as a kind of safety valve during infection,
which makes the lymph node temporarily unable
to fully respond to further antigenic challenge. This
observation provides an intriguing additional level
of temporal compartmentalisation during immune
responses and offers a potential mechanism for
the known phenomenon of increased susceptibility
to secondary infections. In addition, this has
important implications for our understanding of
infection and for the optimisation of vaccination
protocols (Gaya et al., 2015; Science. 347(6222):
667-672).
BliNK Therapeutics Ltd
As ever, one of the long-term objectives of our
institution is to make discoveries that can lead to
new therapies. In a good example of how our
research has translated to treatments, after the
inception and development of a novel platform by
the Lymphocyte Interactions Laboratory, BliNK
therapeutics has now been successfully launched
as an independent company operating in
Stevenage, UK and is producing therapeutic
antibodies.
Publications listed on page 164
67
MAMMALIAN GENETICS
www.london-research-institute.org.uk/research/axel-behrens
Group Leader
Axel Behrens
Atanu Chakraborty
Markus Diefenbacher
Ralph Gruber
Omar Khan
Cristina Pasi
Fabio Pucci
Eva Madi Riising
Edgar-Josue Ruiz Medina
Rocio Sancho
Antonio Tedeschi
Graduate Students
Rute Ferreira
Christopher Gribben
Hanna Halavach
Hendrik Messal
Scientific Officers
Catherine Cremona
Clive Da Costa
68
Every organ harbours adult stem cells, which have the potential for
long-term replication, together with the capacities of self-renewal
and multi-lineage differentiation. These stem cells function in tissue
homeostasis and contribute to regeneration in response to injury. In
addition, many cancers are caused by transforming mutations
occurring in tissue-specific progenitor cells. Our major focus is to
elucidate the molecular mechanisms governing stem cell function
and cancer.
Loss of Fbw7 reprograms adult pancreatic ductal
cells into α, δ, and β cells
The adult pancreas is capable of limited
regeneration after injury, but has no defined stem
cell population. The cell types and molecular signals
that govern the production of new pancreatic tissue
are not well understood. Previous studies from our
laboratory and others have shown that Fbw7, the
substrate recognition component of an SCF-type E3
ubiquitin ligase, controls the stability of several key
cell fate determinants and oncoproteins. Loss of
Fbw7 alters the balance of stem and progenitor
cells in tissues such as the nervous system and
intestine. Surprisingly, we found that inactivation of
Fbw7 in the adult pancreas induces a subset of
pancreatic ductal cells to reprogram into β cells,
and to a lesser extent α and δ cells. Loss of Fbw7
stabilised the transcription factor Ngn3, a key
regulator of endocrine cell differentiation.
Moreover, expression of a stable form of Ngn3 in
pancreatic ductal cells induced a similar frequency
of reprogramming to β cells. The induced β cells
resemble islet β cells in morphology and histology
and express a comprehensive panel of β cell
markers. A frequent stumbling block in previous
models inducing cell reprogramming has been the
functionality of the newly formed β cells. In
contrast, Fbw7-mutant induced β cells secrete
comparable amounts of insulin after glucose
challenge as bona fide β cells isolated from
pancreatic islets, suggesting that they are
functional. Thus, loss of Fbw7 appears to reawaken
an endocrine developmental differentiation
program in adult pancreatic ductal cells, identifying
Fbw7 as a master regulator of cell fate decisions in
the pancreas. Our study highlights the plasticity of
seemingly differentiated adult cells and reveals
adult pancreatic duct cells as a latent multipotent
cell type (Sancho et al., 2014; Cell Stem Cell.
15(2):139-153) (Figure 1).
The deubiquitinase USP28 controls intestinal
homeostasis and promotes colorectal cancer
Colorectal cancer is the third most common cancer
worldwide. Although the transcription factor
c-MYC is misregulated in the majority of colorectal
tumours, it is difficult to inhibit it directly.
Researchers are therefore looking for alternative
ways to target c-MYC, for example by altering its
stability. The deubiquitinase USP28 stabilises
c-MYC as well as other oncogenic factors, but its
role in tumourigenesis and in the intestine was
unknown.
Using murine genetic models, we determined that
USP28 antagonises the ubiquitin-dependent
degradation of c-MYC, as well as 2 additional
oncogenic factors, c-JUN and NOTCH1, in the
intestine. Mice lacking USP28 were healthy, but
showed reduced intestinal proliferation and
increased differentiation of secretory lineage cells.
In a murine model of colorectal cancer, mice
harbouring Usp28 deletion developed fewer
intestinal tumours. More importantly, even in mice
with established tumours, deleting Usp28 reduced
tumour size and dramatically increased lifespan.
USP28 deficiency promoted tumour cell
differentiation accompanied by decreased
proliferation, suggesting that USP28 acts similarly
in intestinal homeostasis and colorectal cancer
models. Moreover, we identified Usp28 as a c-MYC
target gene highly expressed in murine and human
intestinal cancers. USP28 and c-MYC form a
positive feedback loop that maintains high c-MYC
protein levels in tumours. As deubiquitinases
similar to USP28 have been successfully inhibited
by small molecules, inhibition of USP28’s
enzymatic activity may be a promising strategy for
cancer therapy (Diefenbacher et al., 2014; J Clin
Invest. 124(8):3407-3418).
UBR5-mediated ubiquitination of ATMIN is
required for IR-induced ATM signalling and
function
The checkpoint kinase ATM directs the cellular
response to ionising radiation (IR) by localising to
DNA damage sites and actively phosphorylating
proteins involved in repair and survival. ATM is
recruited and activated at damage sites via an
interaction with the Mre11/Rad50/NBS1 (MRN)
complex. An alternative ATM binding partner,
ATMIN, is not involved in the response to IR but
mediates ATM kinase signalling in response to
chromatin changes. The molecular mechanism
favouring either MRN or ATMIN in response to
specific stimuli is enigmatic. We have previously
shown that ATMIN competitively inhibits ATM’s
interaction with the NBS1 subunit of MRN,
suggesting that there must be a mechanism
preventing ATMIN from disrupting ATM signalling
in IR conditions. We have now identified the E3
ubiquitin ligase UBR5 as a key component of ATM
activation in response to IR. We discovered that
UBR5 ubiquitinates ATMIN, which favours its
dissociation from ATM, freeing ATM to interact
with NBS1. This mechanism allows efficient ATM
activation at damage sites and promotes cell
survival after irradiation. UBR5 interacts with
ATMIN and catalyses ubiquitination of ATMIN at
lysine 238 in an IR-stimulated manner. We showed
that UBR5 deficiency, or mutation of ATMIN lysine
238, prevents ATMIN dissociation from ATM and
inhibits ATM and NBS1 foci formation after IR. This
reduction in ATM signalling impairs checkpoint
activation and increases radiosensitivity. Thus,
UBR5-mediated ATMIN ubiquitination is a vital
event for ATM pathway selection and activation in
response to DNA damage (Zhang et al., 2014; Proc
Natl Acad Sci U S A. 111(33):12091-12096).
Figure 1
Fbw7 maintains pancreatic ductal
cell fate.
Tamoxifen-inducible inactivation
of Fbw7 in adult pancreatic ductal
cells (expressing CK19) results in
an increase in the endocrine
transcription factor Ngn3 and
emergence of insulin-positive β
cells within the ducts. Ngn3 is a
direct target of ubiquitination by
the Fbw7-containing SCFFbw7 E3
ubiquitin ligase complex, which in
wild type ducts marks Ngn3 for
degradation. In the Fbw7
knockout (KO) ducts, Ngn3
protein is stabilised, favouring
transdifferentiation of selected
ductal cells to an endocrine fate.
69
HAEMATOPOIETIC STEM CELL
www.london-research-institute.org.uk/research/dominique-bonnet
Group Leader
Dominique Bonnet
Ander Abarrategi
Alessandro Di Tullio
Katie Foster
Ashley Hamilton
Diana Passaro
Kevin Rouault-Pierre
Graduate Students
Alessandra Audia
Amy Bradburn
Alexander Waclawiczek
Scientific Officers
Linda Ariza-McNaughton
Erin Currie
Fernando dos Anjos Afonso
Jashu Patel
Our group is interested in studying human normal haematopoietic
stem cells (HSCs) and leukaemic stem cells (LSCs). We are at present
investigating the relationship between normal HSCs, LSCs and their
microenvironment. For that we have developed in vivo imaging
techniques allowing us to visualise and define the HSC and LSC niche
in vivo. We have also developed a 3D scaffold system where human
stroma cells could be co-cultured in vivo with HSCs or LSCs to study
the effect of the interaction, and the cross-talk between stroma and
HSCs/LSCs. These projects should shed light into pathways or
interactions that are more specifically used by LSCs and where
therapeutic intervention might be developed.
Maintenance of LSCs ex vivo
In Acute Myloid Leukemia (AML), we know that
LSCs cannot be maintained ex vivo without the
addition of stromal support indicating that LSCs
are dependent on their microenvironment for their
survival/maintenance. By modelling key elements
of the bone marrow niche using different stromal
feeder layers and hypoxic culture conditions, we
recently demonstrated that we can maintain LSCs
over at least three weeks and support their
self-renewal properties. This culture system
can be used as an in vitro surrogate for
xenotransplantation and has the potential to
dramatically increase the throughput of the
investigation about LSCs. This would further
provide the means by which to identify and target
the functionality of the different signalling
pathways involved in the maintenance and
resistance of LSCs to improve AML treatments
(Griessinger et al., 2014; Stem Cell Trans Med. 3(4):
520-529).
LSC niche(s) and cross-talk between LSCs and
their microenvironment
Understanding the crosstalk between LSCs and
their microenvironment is crucial to better
understand the dependency of LSCs to their
microenvironment. We thus started a project
trying to better define the factors involved in the
crosstalk between AML and their stroma. AML
samples were co-cultured ex vivo with
mesenchymal stroma cells (MSCs) and after one
week, microarray analysis was performed on
sorted MCSs. Using pathway analysis and the Gene
Go program, we built a network of the combined
datasets. Further studies will be crucial to evaluate
the effect of the different keys factors. If any of
70
these factors affect the growth, differentiation or
apoptosis of AML, we will further investigate one or
more pathway(s) in more detail in vitro but also in
vivo using knock-down approaches.
HSC and LSC niche(s)
In order to better characterise the bone marrow
microenvironment, stem cells and their niches, we
have developed different technologies for in vivo
contrasting procedures as well as for tracking
normal and leukaemic cells in vivo, combining
whole body near infrared fluorescence,
bioluminescence imaging, intravital microscopy of
intact live bone marrow as well as histology and
flow cytometry. We believe that the combined use
of advanced multimodal and multiscale analysis of
the bone marrow will very likely contribute to shed
new light on our understanding of haematopoietic
stem cells and their niches in health and disease
(Lassailly et al., 2010; Blood. 115(26): 5347-5354;
Lassailly et al., 2013; Blood. 122(10): 1730-1740).
Using this technique on live animals adding
time-lapse imaging, we visualised the arrival of
human HSCs in the bone marrow and their
behaviour over time. This should allow us to better
define the normal HSC niche (Foster et al., under
revision). We are now also starting to look into how
leukaemia develops in vivo and visualise its invasion
(Figure 1).
Efficacy of immunotherapy against LSCs using
CAR-T cells
As significant numbers of AML patients are still
refractory to conventional therapies or experience
relapses, immunotherapy using T cells expressing
chimeric antigen receptors (CARs) might represent
A
Figure 1
Seeing leukaemia development:
Visualisation of the expansion
and invasion of leukaemia over
time (A: day 8; B: day 15 and C:
day 21) using intravital
non-invasive imaging of the bone
marrow cavities of the calvaria of
this mouse. HL-60 AML cells
transduced with GFP (green)
were injected into
immunodeficient mice.
Red: blood vessels contrasted
with a blood pool agent.
B
a valid treatment option. AML cells frequently
overexpress the myeloid antigens CD33 and CD123,
for which specific CARs can be generated. However,
CD33 is also expressed on normal haematopoietic
stem/progenitor cells (HSPCs), and its targeting
could potentially impair normal haematopoiesis. In
contrast, CD123 is widely expressed by AML, while
low expression is detected in HSPCs, making it a
much more attractive target. In a recent study we
demonstrated the in vivo efficacy and safety of
using cytokine-induced killer (CIK) T cells
genetically modified to express anti-CD33 or
C
anti-CD123 CAR to target AML. We showed that
both these modified T cells are very efficient in
reducing leukaemia burden in vivo, but only the
anti-CD123 CAR has limited killing in normal HSPCs,
thus making it a very attractive immunotherapeutic
tool for AML treatment (Pizzitola et al., 2014;
Leukemia. 28(8): 1596-1605).
71
Immunity and Cancer
www.london-research-institute.org.uk/research/dinis-calado
Group Leader
Dinis Calado
Rita Barbosa
Giulia Morlino
Nikolay Popov
Andrea Taddei
Graduate Students
Djamil Damry
Magdalena Gabrysiak
Amparo Toboso-Navasa
Scientific Officer
Nora-Ann McFadden
Our immune system is remarkable. It is composed of a network of
cells including T and B cells, which work together to protect our body
from pathogens such as bacteria, viruses and parasites. During an
immune response to infection, B cells undergo a T cell-dependent
maturation stage called germinal centre (GC) within secondary
lymphoid organs in order to become activated and produce high
affinity antibodies against the invading pathogen. During this
maturation stage, B cells undergo processes of DNA mutation and
recombination while rapidly dividing. Infidelities in these processes
may lead to oncogenic DNA lesions. Indeed, the most common types
of haematological malignancies in adults such as Hodgkin and nonHodgkin lymphomas as well as multiple myeloma originate from GC
or post-GC B cells emphasising the importance of the maturation
events within the GC microenvironment for lymphomagenesis.
Interplay between immunity and cancer
development
A major interest of the Immunity and Cancer group
is to understand how healthy cells become
cancerous. To address this question we study B
cells as they provide an ideal model system: firstly,
their normal development is well defined;
secondly, most common types of human
haematological malignancies are derived from the
transformation of healthy B cells into cancer cells.
Although cancer cells may arise from healthy B
cells at several stages of development, lymphomas
derived from GC or post-GC B cells account for the
most common haematological malignancies in
adults, including the vast majority of Hodgkin and
non-Hodgkin lymphomas (e.g. Diffuse Large B Cell
Lymphoma (DLBCL) and Burkitt Lymphoma) and
multiple myeloma.
Therefore, using an integrative approach to
immunology and cancer biology our group aims to
understand the mechanisms by which healthy B
cells within the GC become cancerous. For that, we
primarily perform studies on the following
complementary research themes:
Identification and characterisation of specific
subpopulations within healthy GC or post-GC B
cells with high potential of becoming cancer cells,
forming the source of the so-called cancer’s ‘cells
of origin’
An example of such work is the identification of
c-Myc+ B cell subpopulations in immature and
72
mature GCs, playing indispensable roles in the
formation and maintenance of GCs (Calado et al.,
2012; Nat Immunol. 13(11): 1092-1100). The
identification of these functionally critical cellular
subsets has important implications for human B
cell lymphomagenesis, given that it frequently
involves MYC chromosomal translocations. As
these translocations are generally dependent on
transcription of the recombining partner loci, the
c-Myc+ GC subpopulations may be at a particularly
high risk for malignant transformation.
Investigation of causative mutations responsible
for the transformation of normal B cells into
cancer cells
High-throughput sequencing of human cancer
genomes, including those of haematological
malignancies, has produced large datasets.
However, in human cancers the driver mutations
are sometimes masked by their inclusion on large
amplifications or deletions or by other so-called
passenger/bystander mutations that do not
contribute to the phenotype or progression of the
cancer. Strategies aiming to discriminate these
mutations are required to prioritise further
functional validation. If truly important causative
genes and their associated mechanisms are
evolutionary conserved, mouse models of human
cancers can be employed. Mouse versus human
inter-species oncogenomic comparisons may serve
as a very powerful tool to identify causal mutations
and to interrogate their role in a defined genetic
context.
A
100
80
Percent survival
60
40
Cγ1-cre e YFP stopFL (n=27, Med. surv: und)
20
Cγ1-cre IKK2castopFL (n=16, Med. surv: und)
**
Cγ1-cre Blimp1FF eYFPstopFL (n=50, Med. surv: und)
Cγ1-cre Blimp1FF IKK2castopFL (n=24, Med. surv: 466 days)
0
0
B
C
100
300
Time (days)
400
***
500
Cγ1-cre Blimp1FF Cγ1-cre Blimp1FF
eYFP stopFL
IKK2castopFL
Control
H&E
200
ns
Control
IRF4/Mum1 Control
Cγ1-cre Blimp1FFeYFP stopFL
Cγ1-cre Blimp1FFeYFP stopFL
Cγ1-cre Blimp1FFIKK2castopFL
Cγ1-cre Blimp1FFIKK2castopFL
Expressed in ABC-DLBCL
Study the processes of cancer cell evasion from
the immune system through analysis of the
interplay between a developing B cell lymphoma
and immune cells
Host immunity plays a fundamental role in tumour
formation and progression and cancer immune
escape is an emerging ‘Hallmark of Cancer’. For
that reason, the study of cancer cannot be
confined to the intrinsic cellular mechanisms of
oncogenic transformation, but needs to be
expanded to involve interactions of (pre-) cancer
cells with their microenvironment. However, most
of the current mouse models do not fulfil the
requirements to study these interactions as the
introduction of oncogenic mutations is neither
tissue-specific nor can it be temporally or spatially
regulated, targeting only a small subset of cells. We
are currently developing novel genetic approaches
in vivo, which allow us to introduce oncogenic
mutations in a small fraction of GC B cells,
mimicking the sporadic nature of the cancer
initiating cells. Furthermore, we employ an
inducible gene modification strategy, which allows
us to trace mutant cells during cancer
development and progression.
Non NFκB targets
NFκB targets
Expressed in
GC DLBCL
D
As an example of such work, it was known that
constitutive NF-κB activity and abrogation of
terminal B cell differentiation through Blimp1
disruption are two recurrent genetic events in
human DLBCL. In order to investigate if these two
events were causal for DLBCL, an in vivo mouse
model targeting specifically GC B cells was
generated. Not only did the combination of these
events lead to lymphomagenesis; these
lymphomas also recapitulated many of the
features of the most aggressive DLBCL subtype, the
so-called Activated B Cell DLBCL (ABC-DLBCL),
which has a poorer clinical prognosis (Calado et al.,
2010; Cancer Cell. 18(6): 580-589). Thus, this work
suggests that both NF-κB signalling as an oncogenic
event and Blimp1 as a tumour suppressor, play
causal roles in the pathogenesis of human ABCDLBCL, and illustrates well the relevance of mouse
models in the understanding of cancer
pathogenesis (Figure 1).
Figure 1
Constitutive canonical NF-κB
activation cooperates with
disruption of Blimp1 in the
pathogenesis of activated B
cell-like diffuse large cell
lymphoma.
A. Kaplan-Meier survival plots of
mice carrying the Cγ1-cre
transgene (for GC B cell-specific
Cre expression) together with a
conditional Blimp1 allele
(Blimp1FF) and/or a conditional
R26 allele expressing a
constitutively active IKK2 kinase
(IKK2castopFL) leading to NF-κB
activation. Mice carrying the
Cγ1-cre transgene together with a
conditional R26 allele expressing
eYFP (eYFPstopFL) served as
controls. Med. surv, median
survival; und, undefined.
B. Representative pictures of
spleens from mutant and control
mice.
C. Left panel, representative H&E
staining of spleens from mutant
and control mice.
Right panel, representative
immunohistochemical staining
for MUM1/IRF4 (characteristic of
the ABC-DLBCL subtype but not of
the GC-DLBCL subtype) of spleens
from mutant and control mice.
Scale bar, 1000 μm; inset, 100 μm.
D. Heat map showing the relative
transcript levels of candidate
genes in lymphoma samples
compared to normal GC B cells.
Heat map represents Hprtnormalised value of each
candidate gene normalised to
equal a mean of 0 and variance
of 1. Adapted from Calado et al.,
2010.
GC : Germinal Center B-cells
L1/L2/L3: Cγ1-cre Blimp1FFeYFPstopFL lymphomas
L4/L5/L6: Cγ1-cre Blimp1FFIKK2castopFL lymphomas
73
SIGNAL TRANSDUCTION
www.london-research-institute.org.uk/research/julian-downward
The Signal Transduction Laboratory is interested in the mechanisms
by which cancer cells become addicted to growth and survival signals
generated by activated oncogenes and loss of tumour suppressor
genes. We particularly focus on identifying unique dependencies of
oncogene addicted cancer cells that might be targetable in the
therapy of human cancer.
Group leader
Julian Downward
Associate Scientist
David Hancock
Ralph Fritsch
Madhu Kumar
Miriam Molina Arcas
Clare Sheridan
Davide Zecchin
Alice Zhou
Graduate Students
Matthew Coelho
Daniël Lionarons
Heike Miess
Clinical Research Fellow
Alexandra Pender
Scientific Officers
Lai-Kay Cheung
Christopher Moore
Patricia Warne
Masters Student
Nadia Lima
74
Investigation of mechanisms of oncogene driven
transformation and drug resistance
Much of our work has focused on the RAS family of
oncogenes and the signalling pathways that they
control. RAS genes are activated by point mutation
in some 20% of all human tumours and are known
to play a key role in the establishment of the
transformed phenotype. While the signalling
pathways activated by RAS are well characterised,
it remains a major challenge to identify what
proteins are selectively important in the
establishment and maintenance of the RAS
transformed phenotype and may therefore act as
potential therapeutic targets for cancer treatment.
When cells become progressively transformed
during the evolution of cancer, they suffer stresses
that are not seen by normal cells and become
increasingly dependent on stress management
pathways. This means that the tumour cells show a
unique set of dependencies, both on the oncogenic
drivers and also on stress handling pathways,
termed oncogene addiction and non-oncogene
addiction, respectively. We have investigated
these dependencies by functional genomic
screening using RNA interference, comparing a
cancer cell line containing an activated KRAS allele
with a normal (‘isogenic’) derivative in which this
has been removed (Wang et al., 2010; Oncogene.
29(33): 4658-4670), and also using a panel of thirty
or so cancer cell lines, half of which were mutant
and half wild type for KRAS (Steckel et al., 2012; Cell
Res. 22(8): 1227-1245). This approach uncovered
proteins whose therapeutic targeting might be
expected to provide differential toxicity towards
KRAS mutant tumour cells. One example of a
determinant of non-oncogene addiction found in
this way, the transcription factor GATA2, has been
investigated in detail using genetic mouse models.
The development and continued maintenance of
KRAS-induced lung cancer is highly dependent on
the expression of GATA2 (Kumar et al., 2012; Cell.
149(3): 642-655).
We have also used similar genome-wide screening
approaches to investigate how tumours develop
resistance to targeted therapies, for example the
EGFR receptor tyrosine kinase inhibitor erlotinib in
the case of EGFR mutant lung cancer. This has
revealed a number of genes whose loss of function
can cause resistance to erlotinib in vitro.
Comparison of these hits with expression profiles
of erlotinib resistant and sensitive tumours from a
mouse model of EGFR-driven lung cancer reveals a
number of genes that are under-expressed in the
resistant tumours. One of these is NF1, a negative
regulator of RAS, and we find evidence in erlotinib
resistant tumours that this can lead to endogenous
RAS becoming activated independent of EGFR,
providing an alternative mechanism for erlotinib
resistance in addition to the well-documented
T790M gatekeeper mutation in EGFR itself (de
Bruin et al., 2014; Cancer Discov. 4(5): 606-619).
The role of phosphatidylinositol 3-kinase in
RAS-driven oncogenesis
RAS proteins signal through direct interaction with
a number of effector enzymes, including type I
phosphatidylinositol 3-kinases (PI3Ks). Mice with
mutations in the RAS binding domain (RBD) of the
Pik3ca gene encoding the PI3K catalytic p110α
isoform are highly resistant to endogenous KRAS
oncogene induced lung tumourigenesis and HRAS
oncogene induced skin carcinogenesis (Gupta et
al., 2007; Cell. 129(5): 957-968). The interaction of
RAS with p110α is thus required in vivo for
RAS-driven tumour formation. We have also
generated mice with inducible expression of the
inactivating mutation in the RBD of p110α, so that
the requirement of this interaction for
maintenance of established tumours can be
assessed. Blocking the RAS/p110α interaction
causes partial regression and stasis of tumours,
although more complete regression requires
coordinate inhibition of the MEK pathway
(Castellano et al., 2013 Cancer Cell. 24(5): 617-630).
However, not all of these effects are tumour cell
erlotinib
erlotinib + MEKi
final 4-weeks treatment
before
after
MEKi
Figure 1
Effectiveness of combination
treatment of an erlotinib
resistant EGFR driven lung cancer
mouse model with a combination
of EGFR and MEK inhibitors.
Erlotinib resistant lung cancers in
mice expressing L858R mutant
EGFR in the lung were treated
with erlotinib and/or the MEK
inhibitor trametinib over four
weeks. Tumour size before and
after treatment was determined
by micro CT scanning. For details,
see de Bruin et al., 2014.
autonomous: using a murine tumour cell
transplantation model, we have demonstrated that
disruption of the interaction between RAS and
p110α within host tissue reduced tumour growth
and tumour-induced angiogenesis, leading to
improved survival of tumour-bearing mice, even
when this interaction was intact within the
transferred tumour. Furthermore, functional
interaction of RAS with p110α in host tissue was
required for efficient establishment and growth of
metastatic tumours with normal PI3K, indicating
that a functional RAS interaction with PI3K p110α is
required in host tissue, as well as in the tumour, for
the establishment of a growth-permissive
environment (Murillo et al., 2014; J Clin Invest.
124(8): 3601-3611)
While combined inhibition of the RAS effector
pathways MEK and PI3K can cause impressive
tumour regression, this combination has high
toxicity that may be problematic in the clinic. We
have sought to find less toxic ways of inhibiting
PI3K in KRAS-mutant lung cancer cells using a drug
library screen and have found that inhibition of
IGF1 receptor allows this. It appears that the
activation of PI3K by mutant KRAS in lung cancer
cells is dependent on basal signalling by IGF1
receptor. The combination of MEK and IGF1
receptor inhibition shows potential in preclinical
models of KRAS-mutant lung cancer (Molina-Arcas
et al., 2013; Cancer Discov. 3(5): 548-563).
We have also created a mouse with inactivating
mutations in the RAS binding domain of p110β, the
other ubiquitously expressed PI3K catalytic subunit
isoform. Unexpectedly, we have found that the
p110β isoform is not controlled by direct
interaction with RAS, unlike p110α, γ and δ, but
rather that the RBD of p110β interacts directly with
a number of other small GTPases with distinct
biological function – the RAC and CDC42 proteins.
This has led us to a significantly revised model of
how extracellular stimuli, especially those
signalling through G protein coupled receptors,
activate the PI3K activity of p110β, and the
importance of this mechanism in cancer metastasis
and also fibrosis (Fritsch et al., 2013 Cell. 153(5):
1050-1063).
75
VASCULAR BIOLOGY
www.london-research-institute.org.uk/research/holger-gerhardt
Group Leader
Holger Gerhardt
Raquel Blanco
Claudio Franco
Martin Jones
Andrea Taddei
Anne-Clemence Vion
Graduate Students
Irene Aspalter
Véronique Gebala
Filipa Neto
Benedetta Ubezio
Scientific Officers
Russell Collins
Anan Ragab
The Vascular Biology Laboratory aims to unravel the fundamental
cellular principles and the molecular control of blood vessel
patterning in development and disease. Blood vessels are critical for
tissue growth and healthy organ function. Effective blood vessel
function requires that the endothelial cells lining the vessel assemble
a patterned network of interconnected tubes with adequate
diameter and branching frequency to support blood flow. As
different organs serve distinct functions, and possess different
metabolic requirements, blood vessel patterning bears organ
specific characteristics.
We use a cell biology approach in various model systems in vivo and
in vitro, in combination with computational modelling, to investigate
how endothelial cells respond to signals from the tissue and
communicate with each other in order to orchestrate cell behaviour
leading to functional network formation.
Our work over the past decade established some of
the fundamental cellular and molecular principles
of vascular patterning through sprouting
angiogenesis. Endothelial cells activated by
hypoxia-regulated VEGF-A are specified into tip
cells spearheading new vessel growth, and stalk
cells, which proliferate and form vascular lumen.
VEGF-A activity drives both tip cell migration and
stalk cell proliferation, but Dll4/Notch signalling
establishes the differential specification of tip and
stalk cells, and thereby also the differential
functional response of the endothelial cells. Our
work identified that endothelial cells dynamically
compete for the tip cell position by means of VEGF
receptor (VEGFR) regulation. Dll4/Notch lateralinhibition appears to function as amplifier of
stochastic variations in VEGF receptor levels,
ultimately leading to two populations of cells:
some which possess more of the signalling
receptors VEGFR2 and VEGFR3 and less of the
decoy/sink receptor VEGFR1 – these are tip cells;
and some which possess more of the sink receptor
and less of the signalling receptors – these will be
stalk cells. Although it has become clear that this
feedback loop operates to generate the differential
pattern and thus regulates vascular branching
frequency, we currently know little about the true
downstream effectors of Notch.
In a study focussing on a VEGF co-receptor,
Neuropilin 1 (Nrp1), we tested its role in tip cell
formation using mosaic loss-of-function in a 3D
76
mouse embryonic stem cell sprouting assay as well
as in vivo (collaboration with Ian Rosewell, LRI
Transgenic Services). To our surprise we found that
heterozygous cells lacking just one allele of Nrp1
are incapable of forming tip cells when competing
with wild type cells (Figure 1), and this holds true
even when Notch is inactive. This is in marked
contrast to all previous examples as Notch
inhibition would normally lead to dramatic
hypersprouting by driving endothelial cells to their
default tip-cell response to VEGF-A stimulation.
For Nrp1 loss or inhibition, this is uniquely
different. We also found that Notch differentially
regulates Nrp1 levels in the endothelium, and
these relative differences are most fundamental in
establishing the ability of cells to form tip cells. In
short, these findings identified Nrp1 as the first and
critical downstream effector of Notch signalling in
establishing tip and stalk cells in angiogenesis.
How would Nrp1, a cell surface molecule with
diverse functions, achieve this?
In search for the mechanism, we considered a
possible cross-talk with a pathway recently shown
to also impact on the ability of cells to form tip
cells, that is the BMP/Smad signalling pathway.
BMP9 and BMP10 have been shown to supress
sprouting and tip cell formation through activation
of Alk1, and downstream effectors Smad1/5. Also,
TGF-β has been shown in vitro to limit sprouting by
activating Alk5. Using cell culture models and Nrp1
depletion or overexpression, we found that the
levels of Nrp1 affect activation of Smad2/3 when
Figure 1
Nrp1-heterozygous cells do not
form tip cells when competing
with wild type cells in vivo.
Chimeric retinal vessels derived
from blastocyst injection of a wild
type host with Nrp1LacZ/+
embryonic stem cells, assayed at
post natal day P5. The segmented
image shows the overall
vasculature stained with Pecam
(blue) and wild type cells,
expressing a DsRED marker (red).
The nuclei of the two respective
genotypes were segmented using
Erg staining and displayed in
pseudo-colours; wild type nuclei
in white and nuclei from Nrp1LacZ/+
cells in green.
cells are stimulated with TGF-β. Surprisingly,
although Nrp1 has been proposed to bind TGF-β
and increase its activity on lymphocytes, Nrp1
expression reduced Smad2/3 activation in
endothelial cells.
In a series of genetic compound experiments in
collaboration with the team of Anne Eichmann
(Yale, USA) and drug treatments we discovered that
reducing Alk1 or Alk5 expression or activity in
Nrp1-deficient cells rescued their ability to form tip
cells. Also, unlike Notch inhibition, inhibiting TGF-β
signalling restored sprouting in Nrp1 deficient
vessels, illustrating that overactivation of the
TGF-β/Smad2/3 pathway is responsible for the
sprouting defects in Nrp1 mutants.
More work is required to unravel precisely how
BMP and TGF-β signalling interact and jointly affect
vascular sprouting and what the signalling effectors
of Smad2/3 in the endothelium are that mediate
this effect. However, given that the TGF-β pathway
is genetically involved in a number of
vasculopathies including hereditary hemorrhagic
telangiectasia (HHT) and arterio-venous
malformations (AVM), the finding that Nrp1 levels
quantitatively regulate this signalling axis may turn
out to be therapeutically meaningful.
From the basic angiogenesis angle, our work
challenged previous concepts that posit that
endothelial tip cells are a default response of the
endothelium to VEGF activation, whereas the stalk
cell phenotype is acquired through Notch. Instead,
it appears that Nrp1 expression, which is highest in
tip cells, functions to actively suppress the stalk cell
phenotype by limiting Smad2/3 signalling. These
findings open new avenues and directions to
investigate mechanisms of vascular patterning in
various organs in physiology, as well as in
pathological conditions.
Acknowledgement: We would like to thank Ian
Rosewell and his team for enormous support
generating chimeric experimental mouse cohorts.
We are grateful to Caroline Hill and team members,
who shared tools and experience/insight into the
intricacies of TGF-β signalling.
77
www.london-research-institute.org.uk/research/nathan-goehring
Group Leader
Nathan Goehring
Florent Peglion
Nelio Rodrigues
Graduate Students
Lars Hubatsch
Jacob Reich
Scientific Officer
Nisha Hirani
Pattern-forming systems provide essential spatial cues to guide the
complex multi-dimensional puzzle that is organismal development.
For many of these systems, we have identified the key molecules
involved; yet we are only beginning to understand how the collective
activities of these molecules give rise to spatiotemporal patterns at
the cell and tissue scale. How are pattern boundaries established?
What sets the scale of patterns? What are the properties of networks
that permit pattern formation? How do pattern-forming networks
adapt during development?
Our group takes an integrative approach to these
questions, combining genetics, pharmacology,
quantitative imaging and mathematical modelling
to identify the core design principles of these
systems. We are currently exploring these
questions in the context of symmetry-breaking in
polarised cells, a process that is essential for animal
development and is commonly affected in cancer.
The PAR polarity network
The PAR cell polarity network is an intracellular
pattern-generating system that regulates
asymmetry in polarised animal cells. PAR polarity is
intricately tied to the establishment of cell form,
fate, and function. It plays a key role in processes
as diverse as cell migration, the orientation of cells
with respect to their environment, cell fate
specification during asymmetric stem cell-like cell
divisions, and the generation of complex cell
morphologies. Not surprisingly, failure of this
system is associated with developmental defects
and cancer.
The embryo of the nematode worm,
Caenorhabditis elegans, is a near unmatched
system for quantitative, imaging-based analysis of
PAR polarity network behaviour in a living system:
symmetry-breaking by the PAR proteins is highly
stereotyped and rapid; there is a robust genetic
toolkit to identify, perturb and manipulate the
molecules involved; and embryos are transparent
and highly amenable to quantitative microscopy.
To date, our primary focus has been to develop
quantitative models for polarity establishment in
the one-cell embryo. Kinetic measurements of the
behaviour of core network components as well
as cytoskeletal disruption experiments have led
us to propose that PAR proteins comprise a
78
self-organising pattern-forming system.
Mathematical modelling confirms that a minimal
reaction-diffusion scheme based on measured
behaviours reproduces observed patterns of PAR
polarity seen in the embryo. Notably, this model
can account for symmetry-breaking in the PAR
network in response to cytoplasmic fluid flows,
demonstrating, in principle, how a purely
mechanical cue can trigger pattern formation by
this biochemical network.
Going forward, our focus will be several aspects of
this pattern forming system, including defining the
core network features of the PAR network that
enable pattern formation, identifying the detailed
mechanisms that govern PAR protein mobility on
cellular membranes, and examining how PAR
network behaviour adapts to and is affected by
changing developmental contexts.
Defining core features of the PAR network
At the core of the PAR polarity network is a
mutually antagonistic feedback loop between two
groups of membrane-associated PAR proteins.
Data suggests that these two groups, anterior PARs
(aPARs) and posterior PARs (pPARs), reciprocally
exclude one another from the plasma membrane
(Figure 1). Although we have some insight into the
molecular activities responsible for mutual
exclusion, the lack of mechanistic details or
information regarding reaction kinetics makes
formation of explicit mathematical models
problematic. Indeed, existing models require
assumptions about the nature of PAR feedback
that remain to be tested. We are developing tools
to enable acute, quantitative perturbation of
network connections within the embryo, which we
hope will allow us to better define core design
features of this essential polarity network.
Figure 1
A simplified, two-component
reaction-diffusion model
reproduces symmetry-breaking of
the PAR polarity network in the
one-cell C. elegans embryo. Here,
two mutually antagonistic
components exchange between
membrane and cytoplasmic
states. When membraneassociated, each component can
displace the opposing component
from the membrane such that
co-existence of the two
components at any given space is
strongly disfavoured. This model
yields a multi-stable system,
capable of supporting either a
stable unpolarised or a stable
polarised state, reflecting the
state of the embryo before and
after symmetry-breaking. In the
embryo, switching between these
two states is triggered by cortical
flows, which induce an asymmetry
in membrane-associated anterior
PARs (red), thereby allowing
loading of posterior PARs (blue)
onto the posterior membrane.
Figure 2
Polarisation of the polarity
protein PAR-2 during early C.
elegans development.
PAR-2 is segregated
asymmetrically in a series of 4
polarised cell divisions before
being partitioned equally into
two germ line stem cells.
Regulation of PAR membrane association
PAR polarity is ultimately a question of membrane
dynamics. Although PAR proteins actively exchange
between membrane and cytoplasmic pools, and
PAR polarity is ultimately responsible for setting up
cytoplasmic gradients of downstream effectors,
spatial segregation of PAR proteins occurs only on
the membrane. Rapid diffusion and mixing of
soluble PAR protein in the cytoplasm ensures that
no significant spatial asymmetries arise. A key aim
of our current work is to define the molecular
determinants that govern association with and
mobility of PAR protein on the plasma membrane,
as well as how association and mobility is regulated
in time and space, potentially through interactions
with other members of the PAR network.
Ultimately, it is these kinetic behaviours, such as
membrane binding rates and diffusion coefficients
that define both the ability of reaction-diffusion
systems to form patterns and the characteristics of
the patterns that result.
PAR network behaviour in the establishment of a
developmental lineage
PAR proteins are responsible for the polarity of
diverse cell types that vary distinctly in size, shape,
and context. We are particularly interested in
exploring whether physical limits impact
mechanisms of symmetry-breaking in different cell
types, and whether strategies have evolved to
cope with such physical limits, e.g. by the addition
of alternative molecular players. The early cell
divisions of the C. elegans embryo provide a
simplified model system to begin to explore these
issues. Beginning with the division of the one-cell
embryo, PAR proteins regulate a series of four
consecutive asymmetric divisions during C. elegans
development that are critical for restricting germ
cell determinants to a pair of germ-line stem cells
(Figure 2). In each of these divisions, the PAR
proteins are polarised and asymmetrically
distributed between the two daughter cells.
However, both the size of these cells and their
arrangement with respect to neighbours differ
significantly, thus, providing an ideal system for
examining how properties of the PAR system adapt
over time in a developmental lineage. We are using
our quantitative models as a starting point for
testing predictions for how a minimal PAR system
could adapt to this changing environment.
79
Immuno Surveillance
www.london-research-institute.org.uk/research/adrian-hayday
Group Leader
Adrian Hayday
Lucie Abeler-Dorner
Sara Cipolat
Livija Deban
Deena Gibbons
Yasmin Haque
Rosie Hart
Marialuisa Iannitto
Fernanda Kyle
Adam Laing
Olga Sobolev
Pierre Vantourout
Martin Woodward
Graduate Students
Deborah Enting
Rafael di Marco Barros
Bodhi Hunt
Sean O’Farrell
Rick Woolf
Scientific Officer
Anett Jandke
Figure 1
The murine small intestinal
epithelium is normally enriched in
γδ T cells, relative to αβ T cells
(green vs red; left-hand panel)
viewed by confocal microscopy of
the gut. Conversely, mice mutant
in a single intestinal-specific
member of the B7 superfamily
(right-hand panel) show a severe
reduction in γδ T cells because of
the selective loss of cells
expressing Vγ7, the signatory T
cell receptor of the small
intestine. Moreover, most
residual γδ T cells express lower
levels of the TCR, and hence show
less bright green staining (image
courtesy of Rosie Hart).
80
Infections occur largely at body surfaces, and it is therefore not
surprising that many organs, including skin, intestine and
reproductive tract harbour major immune compartments. Such
tissue-resident immune cells may play key roles in the initiation and
progression of solid tumours, many of which develop at body
surfaces. Ironically, tissue-resident immune cells have received little
attention relative to their circulating counterparts. In seeking to
redress this balance, we hypothesised that epithelial cells in discrete
anatomical sites express molecules that determine the unique
composition of their associated T cell compartments. Such molecules
may thereby play key roles in cancer immunosurveillance. Supporting
our hypothesis, we identified Skint1 as a novel gene regulating the
signatory T cell compartment of the murine epidermis. We have now
obtained data suggesting the generality of this principle, and showing
that molecules such as Skint1 constitutively communicate to local T
cells whether a tissue is normal or dysregulated.
Epithelial orchestration of immune compartments
An iconic, 20-year-old picture in immunology
depicts the association of discrete sets of T cells
with different organs. Thus, γδ T cells expressing
Vγ5+ T cell receptors (TCRs) compose the signatory
epidermal γδ T cell compartment; Vγ6+ T cells
populate the reproductive tissues; while Vγ7+ T
cells populate the small intestine. These
associations are significant, since mice in which we
replaced skin Vγ5+ cells with other γδ T cells
became hypersensitive to skin irritants and
carcinogens (Strid et al., 2008; Nat Immunol. 9(2):
146-154). However neither the biological rationale
nor the molecular basis for organ-specific T cell
associations is known. We considered that
epithelial cells in different organs might express
unique ‘address-ligands’ that selectively engage T
cells specifically associated with the respective
organs. Supporting this, we and our collaborators
identified Skint1, a novel immunoglobulin
superfamily gene expressed exclusively by thymic
epithelial cells and keratinocytes and upon which
the normal development of skin Vγ5+ T cells
depends (Turchinovich and Hayday, 2011;
Immunity. 35(1): 59-68). Whereas initial studies of
Skint1 related to its developmental regulation
(‘selection’) of thymic Vγ5+ T cell progenitors, our
studies throughout 2012-2014 established that
Skint1 regulates steady-state contacts of mature
Vγ5+ T cells with suprabasal keratinocytes, thereby
maintaining an appropriate state of peripheral
immune regulation. Our ongoing studies focus on
the nature of those Skint1-dependent contacts: do
they include direct Skint1-TCR interactions, or do
they feature novel, organ-specific checkpoint
regulators akin to the activity of PD1 on T cells that
infiltrate tissues from the systemic compartment?
Novel epithelial T cell regulators
Skint1 provides a prototype for how the epithelium
determines the composition and status of an
organ-specific T cell compartment. Skint1 is
distantly related to B7 genes that include CD80,
B7H4, and PD-L1 that are profoundly effective,
clinically significant T cell regulators. Thus, other
B7-like, Skint1-related genes might encode
epithelial regulators of other T cell compartments.
Indeed, we have identified mice mutant in a B7-like
gene, expressed specifically in small intestinal
epithelial cells, and in which intestinal T cells
expressing the signatory Vγ7+ TCR are almost
completely absent (Figure 1). However, unlike
yet unknown, tissue-specific B7-like factors that we
now seek (Figure 2). We are likewise investigating
the pathophysiologic roles played by human
innate-like γδ T cells, and developing clinically
applicable methods to regulate tissue-associated
γδ T cell activities within the context of human
tumour immunotherapy.
Figure 2
Schematic representation of
members of the extended
B7-supergene family that includes
epithelial regulators of discrete γδ
T cell compartments. (IgV; IgC
refer to domains similar to
Immunoglobulin Variable and
Constant regions, respectively;
B30.2 denotes a domain unique to
BTN/Btnl and TRIM proteins;
diagonal line denotes membranespanning region; upper case type
denotes a human protein; lower
case denotes murine proteins)
thymic, Skint1-dependent regulation of epidermal
T cells, epithelial ‘selection’ of Vγ7+ T cells occurs
exclusively in the gut, perhaps consistent with the
required turnover of intestinal γδ T cells
throughout life, even after thymic involution. The
biology and underpinning mechanism of this first
example of intestinal T cell repertoire selection is a
prime focus of ongoing studies.
Figure 3
T lymphocytes in newborn babies
display a novel functional
potential to produce IL-8 (CXCL8),
which correlates with increases in
CRP, commonly diagnostic of
neonatal sepsis. No such
correlation is shown by
conventional T cell cytokines, e.g.
interferon-gamma (IFNγ) or
interleukin-17 (IL-17). Thus, CD4 + T
cell production of CXCL8 may
prove a useful new neonatal
biomarker, and offer functional
insight into human T cell
mediated immunosurveillance.
Human tissue T cells orchestrate immune
responses
We first showed that epidermal T cells can respond
in vivo to innate ‘alarmins’, rather than require
specific TCR-dependent antigen stimulation. These
findings, since validated by many other
laboratories, fuelled the notion of ‘innate-like’ T
cells that orchestrate downstream immune
responses following their rapid activation by tissue
dysregulation. Such tissue-resident, innate-like T
cells might form a first-line of defence to infections
and/or early stages of tumourigenesis. To assess
whether humans harbour such innate-like T cells,
our clinical research team at Guy’s Hospital has
refined methods for T cell isolation, permitting us
to confirm that human skin and gut each harbour
vast lymphocyte compartments comprising many
different T cell types. Among those, we have
identified the first examples of human tissueresident, innate-like γδ T cells specifically
associated with either skin or gut. Moreover, some
similarities of those cells with their mouse
counterparts are so striking as to fuel the
hypothesis that they, too, may be regulated by as
In the very young
Although the young are most vulnerable to
infection, there has been little study of T cells in
human neonates. Moreover, such data as exist
show an inability of neonates’ T cells to produce
cytokines such as interferon-gamma, underpinning
the view that neonates are immunodeficient.
Based on our elaboration of innate-like T cell
responses, we asked whether T cells in neonates
– rather than lacking function – perform functions
more commonly associated with innate cells, e.g.
monocytes. Indeed, we provided the first evidence
that the major effector potential of neonates’ CD4+
T cells is production of interleukin-8 (IL-8), a major
activator of neutrophils with the potential to
protect babies against relentless exposure to
bacteria (Gibbons et al., 2014; Nat Med. 20(10):
12060-1210) (Figure 3). We are now asking
whether IL-8-producing T cells are limited to
childhood, or are implicated in immunoprotection
and/or immunopathologies in adults.
Big Science
We further built up ‘3i’, a mutli-centre consortium
aiming to comprehensively immunophenotype
>800 gene knockout mouse strains over five years.
After only 18 months of unprecedentedly highthroughput immunological analysis, 3i is already
providing substantial new insight into genes
regulating immune cell development and function,
including within tissues, thereby germinating many
new projects.
In parallel, our comprehensive immunemonitoring of human responses to vaccination has
been expanded via collaboration with Momenta
Pharmaceuticals (Cambridge, MA) with whom we
are developing novel ways to analyse and
disseminate huge human immunology data-sets. A
key goal is to improve immunological assessment
of cancer patients, thereby guiding clinical
decision-making in relation to immunotherapies.
81
DEVELOPMENTAL SIGNALLING
www.london-research-institute.org.uk/research/caroline-hill
Group Leader
Caroline Hill
John Chesebro
Claire Heliot
Annasuya Ramachandran
Antonius van Boxtel
Graduate Students
Davide Coda
Tessa Gaarenstroom
Daniel Miller
Scientific Officers
Debipriya Das
Ilaria Gori
Work in the Developmental Signalling group focuses on the signalling
mechanism and function of Transforming Growth Factor β (TGF-β)
superfamily ligands, which include the TGF-βs, Activins, Nodals, BMPs
and GDFs. Many of these ligands play fundamental roles in early
vertebrate development, acting as morphogens in the specification
and patterning of the germ layers, and aberrant TGF-β, BMP and
Nodal signalling is implicated in many different types of cancer. A
major goal of the lab is to understand at the molecular level how
these ligands signal from the plasma membrane to the nucleus and
how they regulate transcription of target genes. We want to
determine how they function and are regulated in embryonic
development, using zebrafish as a model system, and how these
signalling pathways are hijacked by tumour cells to promote growth
of primary tumours and metastasis to distant sites. The highlights this
year have been our genome-wide analyses addressing how activated
Smads induce transcriptional activation via chromatin, our
mechanistic work focused on signalling dynamics, and our in vivo
studies on Nodal signalling in zebrafish.
Regulation of transcription by activated Smad
complexes
The main function of the TGF-β superfamily/Smad
pathways is to induce new programmes of gene
expression, but apart from previous work from my
group showing that Smads activate transcription
by remodelling the chromatin template, little is
understood about how exactly they achieve this.
Our work in this area is focused on Nodal signalling,
which is not only critical for embryonic
development, and for maintaining pluripotency of
human embryonic stem (ES) cells, but is exploited
by cancer cells to promote tumour progression and
metastasis. We are using the embryonic carcinoma
cell line, P19, which responds to Nodal and Activin
both acutely and chronically as a model system. As
well as being a model for cancer, these cells
express a combination of pluripotency factors and
mesendoderm markers in response to Activin/
Nodal. To characterise the transcriptional
responses to Activin we performed RNA-seq on
P19 cells in the non-signalling state, in cells treated
with Activin for short (1 h) or extended (8 h) times,
and in the untreated state (autocrine signalling).
This has allowed us to define four main classes of
response genes (transiently induced; induced
sustained; delayed induced; repressed), which is
82
enabling us to define different classes of enhancers
that are co-regulated. In the same conditions we
have also performed ChIP-seq analysis for Smad2,
for total histone H3, for two different histone
modifications characteristic of active chromatin
(H3K27ac and H3K9ac), and for two different forms
of RNA Polymerase II (initiating state
phosphorylated at Ser 5, and elongating state
phosphorylated at Ser 2). Ongoing work integrating
the RNA-seq data with all of these ChIP-seq
datasets is revealing exciting and novel insights
into the mechanism whereby activated Smad2containing complexes find their targets in
chromatin and activate transcription. Motif
enrichment analysis is being used to define what
other cofactors bind with Smad2 at enhancers of
Activin-responsive genes, distinguishing between
those that bind with Smad2 1 h after Activin
stimulation and those that are synthesised in
response to Activin and bind with Smad2 at later
time points. This is yielding a number of interesting
candidates, which are being experimentally
verified.
The dynamics of TGF-β signalling
Understanding how cells respond to ligands in
complex physiological and pathological contexts in
vivo requires knowledge of the dynamics of
signalling in addition to an understanding of the
molecular wiring of the pathway. This year we have
been building on our analysis of the dynamics of
TGF-β signalling, which indicated that an acute
TGF-β stimulation resulted in refractory behaviour
as a result of rapid internalisation of activated
receptors from the cell surface, and their very slow
recovery. We have now performed several whole
genome siRNA screens to identify new regulators
of TGF-β signalling dynamics and are building
receptor biosensors to track the trafficking route of
activated receptors. We are also extending this
analysis to other TGF-β superfamily signalling
pathways and have shown that neither BMP, nor
Activin or Nodal stimulation triggers refractory
behaviour. In these cases, cell surface receptors
appear to continuously monitor ligand in the
extracellular milieu. We are investigating
mechanistically how this occurs.
Spatial regulation of Nodal signalling
To understand how TGF-β superfamily ligands
function in vivo, we are using early zebrafish
embryos as a model system. We want to determine
how ligand activity is regulated, how the ligands
function in a dose-dependent manner and how
they contribute to tissue specification. This year we
have been focusing on spatial and temporal control
of Nodal signalling. To visualise Nodal signalling in
vivo, we generated a transgenic zebrafish line in
which an EGFP reporter is controlled by 3 copies of
the Activin-responsive enhancer (ARE), which binds
a complex of activated Smad2–Smad4 with the
transcription factor FoxH1. Using EGFP mRNA as a
readout we can track active Nodal signalling in a
developing embryo, and have made some
surprising discoveries. Shortly, after Nodal
signalling is initiated, it is visible in the embryo
margin in a shallow dorsal-ventral gradient, but
contrary to long standing assumptions in the field,
it extends only four or five cell diameters towards
the animal pole, coincident with the expression
domains of cyclops and squint (the fish Nodal
ligands), and lefty1/2 (the antagonists), and is not
graded in this direction. The predominant model to
explain the generation of domains of Nodal
signalling is the reaction-diffusion model, which is
based on the assumption that a highly diffusible
inhibitor (in this case Lefty1/2) and a less diffusible
activator (Nodal ligands) can create a network as a
result of short-range activation and long-range
inhibition. Our findings do not support this model.
Instead, our results are leading us to an alternative
model, whereby a temporal delay in the translation
of the ligand antagonists Lefty1/2 allows Nodal
signalling to become established in four to five cell
tiers at the margin, at which point Lefty protein
levels reach a sufficiently high threshold to prevent
further spread of Nodal signalling. We are
determining the mechanism underlying this
behaviour. Our data is suggesting that rather than a
spatial gradient of Nodal signalling in the early
embryo, the gradient may be temporal.
Figure 1
Regulation of transcription by
activated Smad complexes.
Schematic indicating the molecular
events that occur to enable
activated Smad2–Smad4
complexes to regulate
transcription. The Smad complexes
bind to enhancer DNA through
interactions with other
transcription factors, such as
FoxH1, as well as other cofactors.
Smad binding induces chromatin
remodelling through the SWI/SNF
complex and via histone
modification, which may include
demethylation of H3K27 and H3K9,
acetylation via p300 of multiple
lysines on H3 including H3K27 and
H3K9 (depicted as AcH3) and
trimethylation of H3K4. Chromatin
looping results in recruitment of
mediator and RNA polymerase II
(Pol II) at the transcription start
site. Pol II becomes
phosphorylated initially at serine 5
and subsequently at serine 2,
which is its elongating form.
83
COMPUTATIONAL BIOLOGY
www.london-research-institute.org.uk/research/nicholas-luscombe
Group Leader
Nicholas Luscombe
Federico Agostini
Aylin Cakiroglu
Elodie Darbo
Borbala Gerle
Raphaelle Luisier
Anna Poetsch
Andrew Steele
Alessandra Vigilante
Katharina Zarnack
Graduate Students
Filipe Cadete
George Cresswell
Arsham Ghahramani
Robert Sugar
Cellular life must recognise and respond appropriately to diverse
internal and external stimuli. By ensuring the correct expression of
specific genes at the appropriate times, the transcriptional regulatory
system plays a central role in controlling many biological processes:
these range from cell cycle progression and maintenance of
intracellular metabolic and physiological balance, to cellular
differentiation and developmental time-courses. Numerous diseases
result from a breakdown in the regulatory system and a third of
human developmental disorders have been attributed to
dysfunctional transcription factors (TFs). Furthermore, alterations in
the activity and regulatory specificity of TFs are now established as
major sources for species diversity and evolutionary adaptation.
Indeed, increased sophistication of the regulatory system appears to
have been a principal requirement for the emergence of metazoan life.
Much of our basic knowledge of transcriptional
regulation has derived from molecular biological
and genetic investigation. In the past decade, the
availability of genome sequences and development
of new laboratory techniques have generated (and
continue to generate) information describing the
function and organisation of regulatory systems on
an unprecedented scale. Genomic studies now
allow us to examine regulatory systems from a
whole-organism perspective; on the other hand
however, many observations made with these data
are unexpected and appear to complicate our view
of gene expression control.
The continued flood of biological data means that
many interesting questions require the application
of computational methods to answer them. The
combination of computational biology and
genomics enables us to uncover general principles
that apply to many different biological systems;
any unique features of individual systems can then
be understood within this broader context.
The Computational Biology Group applies
computational and genomic methods to answer
three main questions:
(i) How is gene expression regulated?
(ii) How do these mechanisms control interesting
biological behaviours?
(iii) How does gene regulation interact with
evolutionary processes?
Much of our work until now has been purely
computational, either analysing publicly available
84
data or in collaboration with experimental
laboratories performing functional genomic
investigations.
Research highlights
• Prevention of aberrant exonisation of Alu
elements
In collaboration with Jernej Ule’s group (UCL ,
Institute of Neurology) we developed
nucleotide-resolution, genome-wide techniques
to identify protein-RNA interactions (Konig et al.,
2010; Nat Struct Mol Biol. 17(7):909-15). We
demonstrated how hnRNP C binds to enhanced
and repressed splice sites. Recently, we
discovered how competitive binding between
U2AF65 and hnRNP C protects the transcriptome
from the detrimental exonisation of thousands of
Alu elements (Zarnack et al., 2013; Cell.
152(3):453-66).
• Statistical models of gene expression in fly
development
Using compiled in situ hybridisation images from
the Virtual Embryo dataset, we developed
statistical models that for the first time accurately
reproduce even skipped expression. Importantly,
the models precisely forecast behaviours beyond
the training data, making them truly predictive
(e.g. effects of regulatory perturbations). The
study generated experimentally testable
hypotheses and provided new insights into the
underlying mechanisms of transcriptional
regulation (Ilsley et al., 2013; Elife. 2:e00522)
Figure 1
Schematic explaining how hnRNP
C binding to pre-mRNAs aids
accurate splicing, whereas its loss
leads to aberrant exonisation.
Future work
Nuclear organisation of chromosomes
It is increasingly appreciated that the spatial
organisation of chromosomes profoundly
influences gene expression; however the details of
how this is achieved are poorly understood. We will
build on our successful collaborations with the
Akhtar group (Max Planck Institute of
Immunobiology and Epigenetics, Freiburg, DE) to
study the effects of X-chromosomal positioning on
dosage compensation. Excitingly, we recently
initiated collaborations with Peter Fraser
(Babraham Institute, Cambridge), a world-expert
on ChIA-PET and Hi-C, to investigate nuclear
organisation in mammalian cells.
Gene regulation in disease states
We will apply our basic knowledge of gene
regulation to disease systems. There are
indications that bacterial infections cause changes
to the host’s regulatory system, so affecting
expression patterns. We have initiated
Figure 2
A. Predicted expression of
even-skipped 2 (eve 2) wild-type
Drosophila embryos.
B. Predicted eve 2 expression in
giant (gt) mutant Drosophila
embryos.
C. in situ hybridisation of gt
mutant from Small et al., 1992:
EMBO J. 11(11):4047-57.
A
B
collaborations with Richard Hayward (University
College London) to apply genomic techniques to
investigate the prevalence of these effects, and the
influence they have on the progression of bacterial
infections.
Gene regulation and DNA-damage repair
A major implication of our mutation rate study is
that highly expressed genes are preferentially
protected from DNA damage; however
mechanisms such as transcription-coupled repair
do not explain our observations. There are early
indications that similar mechanisms operate in
cancer. DNA damage repair is traditionally studied
from a molecular perspective: we have initiated
collaborations with the Mammalian Genetics
Group to examine this phenomenon from a
genomic viewpoint, too. This will dramatically
improve understanding of how DNA damage repair
operates on a genome-wide scale.
C
85
TUMOUR HOST INTERACTION
www.london-research-institute.org.uk/research/ilaria-malanchi
Group Leader
Ilaria Malanchi
Laurie Gay
Luigi Ombrato
Graduate Students
Yaiza del Pozo Martin
Sathya Muralidhar
Misa Ogura
Stefanie Wculek
Scientific Officer
Robert Moore
Figure 1
A. Schematic representation and
immune-fluorescent images of
cancer cells (green) within the
lung during metastatic
colonisation and
B. during micro-metastatic
establishment. Modifications
within the metastatic tissue are
detected by Smooth Muscle Actin
staining (red).
86
A cancer cell surrounded directly by the ‘normal’ host environment
of a tissue would be unable to develop into a tumour. Only by
modifying their environment, cancer-initiating cells survive,
proliferate and build a supportive stromal structure consisting of
tumour associated host cells locally or systemically recruited. The aim
of our group is to understand the crosstalk that tumour cells establish
with the host organism to allow both tumour onset and metastatic
progression.
Tumours are composed by a growing mass of
cancer cells embedded into a heterogeneous
assembly of tissue-derived cells. These cells are
collectively referred to as tumour-associated
stroma. Cancer cell growth is always accompanied
by a concomitant modification in the surrounding
host tissue and these two components co-evolve
during tumour progression. Within the cancer cell
compartment, cancer cells are very heterogeneous
in their intrinsic tumourigenic potential and only a
small fraction of cells, termed Cancer Stem Cells
(CSCs), retain the potential of sustaining long-term
growth. CSCs are the precursor of all cancer cells
and they can replace part or the entire tumour if
required. The relevance of CSCs’ intrinsic potential
is maintained during metastatic progression. We
have previously provided direct evidence that a
sub-pool of cells (metastatic CSCs) identified within
A
Metastatic Colony
B
Micro-metastasis
the primary tumour, drive metastatic colonisation
(Malanchi et al., 2011; Nature. 481(7379): 85-89).
Importantly, metastatic CSCs also rely on signals
derived from their local microenvironment,
referred to as ‘niche’, for controlling their
behaviour and maintenance. We aim to clarify how
the metastatic cells create their niche, as well as
the crosstalk they establish with it to allow
metastatic outgrowth.
Tumour-host interactions extend well beyond the
local tissue microenvironment and tumours not
only respond to, but also actively perturb host
organs at distant anatomic sites. For instance,
some inflammatory cells, once assumed to
collectively act only to attenuate tumour
development, clearly play a role in tumour
promotion and malignancy. Indeed, inflammatory
Figure 2
Metastasis from ‘niche-labelling
cancer cells’.
A. Immunofluorescence of
metastasis from niche-labelling
cells expressing both GFP and
CHERRY proteins. Niche cells are
the tissue cells retaining only
CHERRY protein. White lines
outline the borders between
cancer and niche cells, and which
are removed in the inset.
B. FACS analysis of lungs injected
with niche-labelling cancer cells.
Unstained cells represent lung
tissue cells. GFP-CHERRY double
positive cells represent the cancer
cells growing in the lung. CHERRY
single positive cells are the subset
of lung tissue cells labelled by the
cancer cells and which represent
the metastatic niche.
A
B
Metastatic
niche
conditions are able to increase the speed of tumour
progression and chronic inflammation is the
best-known critical carcinogenic risk factor. By
focussing on a particular immune cell
compartment, the neutrophils, which play a
fundamental role in inflammatory responses, we
investigate the interaction of tumour and
inflammatory cells in the context of primary
tumour formation and metastatic onset.
Cancer stem cells and their metastatic potential
What defines a cancer cell’s metastatic potential?
To answer this question, we consider two facts:
firstly, cells capable of metastasising belong to a
sub-pool of cancer cells that retain the intrinsic
tumour initiation potential (stemness) of
reconstituting a cancer cells mass. Secondly, during
early metastatic colonisation, cancer cells change
their microenviroment to create their
niche (Figure 1). Therefore, the ability of cancer
cells to initiate an extrinsic niche will also impact
metastatic competence. We have previously
identified a sub-pool of metastatic CSCs (mCSCs)
within primary tumours of the MMTVpyMT mouse
model for their exclusive metastatic initiation
potential. Therefore, studying the characteristics of
those mCSCs in comparison to the rest of the
cancer cells is guiding us to identify specific intrinsic
and extrinsic metastatic potential.
Despite its crucial function, metastatic niche
composition and its evolution during the different
phases of metastatic progression, is yet to be
understood. To help characterise the niche, we
have generated a unique tool capable of labelling in
vivo tissue cells coming in close proximity with
metastatic cancer cells. This has not been achieved
to date. A specially designed expression tool
integrated in the genome of cancer cell lines allows
for expression of two fluorescent proteins: a green
fluorescent protein (GFP), which is retained within
the cell and a red fluorescent protein (CHERRY),
which is secreted by the cell and can be taken up by
neighbouring cells (Figure 2). This unique tool
allows us to identify niche cells surrounding the
cancer cells during the different phases of
metastatic establishment: from colonisation to
first micro- and then macro-metastases.
Finally, by combining the expression profile
changes occurring in both cancer and niche cells
during early and late stages of metastasis, we aim
to clarify the signals of this crucial crosstalk with
the final aim to develop blocking strategies for
clinical applications.
Pro-tumourigenic activity of neutrophils
The pro-tumourigenic and pro-inflammatory
activity of some innate immune cells has been
characterised, however, few studies have
concentrated on neutrophils. Neutrophils are the
first cells migrating towards the inflammatory site
and are crucial to amplify and control the
inflammatory response. We found these cells
consistently present during initiation of both
primary tumours as well as metastases.
Remarkably, lack of neutrophils reduces the
efficiency of both tumourigenesis and metastatic
progression. Using genetic and chemical strategies
to block neutrophils or a certain neutrophil
activity, we aim to clarify their tumour promoting
functions during initiation of both primary tumour
and metastases. These approaches will be clinically
relevant to antagonise tumour onset and
metastasis formation.
87
STRUCTURAL BIOLOGY
www.london-research-institute.org.uk/research/neil-mcdonald
Group Leader
Neil Q McDonald
Rakhee Chauhan
Julio Martinez-Torres
Agata Nawrotek
Ivan Plaza-Menacho
Peter Saiu
Graduate Students
Emily Burns
Robert Constable
Marina Ivanova
Scientific Officers
Maureen Bowles
Phillip Knowles
John Lally
Andrew Purkiss
Growth factor signalling deregulation is a hallmark of cancer and is
frequently associated with altered catalytic functions of pathway
components. These components form molecular complexes that
transduce signals across or within the cell membrane and can
combine allosteric, catalytic or recruitment mechanisms to
propagate growth factor-initiated signals. Our primary research aim
is to understand the assembly, activation and deregulation of growth
factor activated complexes that influence spatial signalling. Our
group uses structural methods (crystallography, electron microscopy
and SAXS), biophysical methods (calorimetry and fluorescence
polarisation), enzymatic and cell-based assays, to elucidate signalling
mechanisms.
Cell surface receptor activation and oncogenic
deregulation
Receptor tyrosine kinases (RTKs) respond to
extracellular ligands received at the cell membrane
and undergo ligand-dependent activation triggering
intracellular signalling pathway activation. We study
the RET receptor as a model RTK to understand how
ligands are recognised and how this interaction
drives RET tyrosine kinase activation by allosteric
or/and clustering mechanisms. This is important as
RET signalling is crucial for both embryonic and
adult development, whilst RET missense mutations
underlie at least three human diseases
(Hirschsprung’s disease, kidney agenesis and
cancer). The RET receptor is activated by a group of
GDNF family ligands (GFLs) that belong to the
cystine knot growth factor superfamily. GFLs can
only engage RET if presented by a GFRα co-receptor.
The co-receptor is linked to the cell surface by a
GPI-linkage but a soluble form can also bind GFLs
and stimulate RET activation in trans.
To characterise the interaction between RET
extracellular domain (ECD) and its bipartite
GDNF-GFRα ligand, we reconstituted two
vertebrate ternary complexes (TC) containing RET
ECD and a GDNF-GFRα ligand. We used a hybrid
structural approach to define the RET ECD
organisation by low-angle X-ray scattering and
determined negative-stain electron microscopy
reconstructions for both vertebrate RET ternary
complexes (Goodman et al., 2014; Cell Rep. 8(6):
1894-1904). The RET ternary complex structures
indicate that RET contacts are primarily driven by
co-receptor interaction. Dimeric GDNF primarily
serves as a co-receptor cross-linking function. It
88
establishes that RET has an unusual composite
binding site closer to cytokine receptor assemblies,
with discrete contacts and large gaps between the
bipartite ligand and the RET ECD. Another
important feature of the RET–ligand complex is a
homotypic RET cysteine-rich region (CRD) C-tail
interaction. Deletion of this region enhances
ligand affinity for RET ECD suggesting that ligandbinding forces an unfavourable membrane
proximal self-association of the CRD C-tail. This is
highly relevant for multiple endocrine neoplasia
type 2A (MEN2A) cancer patients with missense
mutations within the CRD C-tail generating a
disulphide-crosslinked and constitutively activated
form of RET.
One downstream consequence of RET-ECD
interaction with GDNF-GFRα ligand is the
generation of highly specific phospho-tyrosine
sites within the RET intracellular region. These sites
are generated in trans by activation of the RET
tyrosine kinase to phosphorylate a neighbouring
RET molecule. We found that regions flanking the
tyrosine kinase domain are targeted much earlier
than the canonical RTK ‘activation’ loop sites.
Comparing tyrosine phosphorylation site kinetics
by label-free mass spectrometry revealed that
aggressive RET tyrosine kinase domain mutations
found in MEN2B patients present a better autophosphorylation substrate than wild type RET
(Plaza-Menacho et al., 2014; Mol Cell. 53(5):738751). We are continuing to characterise how
regions flanking the core RET kinase domain
contribute to ligand-dependent activation as well
as how they are perturbed in an oncogenic
context.
Figure 1
Recognition of a bipartite
GDNF-GFRa1 ligand by the RET
extracellular domain (RET-ECD).
A. Reconstitution of recombinant
RET-ECD complex containing a
GDNF-GFRa1 ligand.
B. Electron microscopy
reconstruction and fitted
structural model for the
RET-ECD-GDNF-GFRa1 complex.
C. Location of a crucial receptorligand interaction ‘hotspot’
centred on site III.
D. Quantification of RET-ECD
binding to immobilised
GDNF-GFRa1 mutant ligands. Site
III mutant contained a triple
mutation within GFRa1.
A
B
Our earlier structural work on full length RET
identified a folding bottleneck that could be
eliminated by substitution of just two amino acids.
Removing these residues in RET Hirschsprung’s
(HSCR) disease mutants restored cell surface
localisation overcoming a maturation block that
otherwise left these RET missense mutants trapped
in the ER as immature forms. To uncover pathways
unique to RET maturation, we recently carried out
a biased siRNA knockdown screen to identify
components that restored RET HSCR mutant cell
surface location and ligand-dependent signalling.
Several putative hits identified were amenable to
selective chemical inhibition allowing us to rapidly
validate our initial screen. Current efforts are
characterising these hits to demonstrate RET
signalling can also be restored. Understanding
pathways and uncovering new components that
control RET maturation and prevent RET HSCR
mutant export may present new drug targets to
modulate RET signalling and novel approaches to
treat HSCR disease.
Polarity signalling assemblies at the plasma
membrane
A second major project of our group investigates
the structure, activation and substrate recruitment
mechanisms of the atypical protein kinase C (aPKC)
isoforms iota and zeta (PKCι and PKCζ). These
serine/threonine kinases play important roles in
many types of cell polarity and asymmetric cell
division. They have less well understood functions
in cell proliferation as well as in Ras-dependent
transformation. The aPKC kinase is a component of
a larger assembly called the Par complex that
contains the binding partner Par6, the small
GTPase Cdc42 and a transiently associated Par3
PDZ-containing protein. We have reconstituted the
C
D
Par complex to investigate its biochemical
properties, to define the functional role of each
component and to initiate structural studies.
Recent results for Par3 interaction with aPKC
suggest two discrete binding modes. We have
collaborated with the Protein Phosphorylation
Laboratory and Epithelial Biology Laboratory to
validate our biochemical and structural findings in
drosophila and mammalian cell lines.
The Par complex is localised to the apical
membranes of epithelial cells where it targets a
specialised set of membrane-associated
substrates. Once phosphorylated, Par complex
substrates alter their subcellular localisation,
frequently being displaced from the apical
membrane to other membrane domains. We are
interested in substrate recruitment mechanisms
used by the Par complex. Validated polarity
substrates such as Par1, Par3, LLGL2 and Kibra all
have basic residues flanking a phospho-acceptor
site. However, short peptides (4-14 residues)
derived from these proteins are often poor
surrogates for intact protein substrate
counterparts suggesting recognition elements may
exist remote to the phospho-acceptor site. In
collaboration with the Protein Phosphorylation
Laboratory, we recently uncovered a motif used by
aPKC to engage a subset of PKCι substrates
including the tumour suppressor LLGL2 (Linch et
al., 2013; Sci Signal. 6(293): ra82). Surprisingly, PKCι
missense mutants associated with human cancer
map within this motif highlighting the importance
of PKCι substrate recruitment. We continue to
investigate the structural basis for the recruitment
of these PKCι substrates to the Par complex.
89
CELL CYCLE
www.london-research-institute.org.uk/research/paul-nurse
We use the simple eukaryote Schizosaccharomyces pombe (S. pombe)
as a model for understanding processes that are important for
eukaryotic cell biology. S. pombe is a single celled rod-shaped
eukaryote that grows by tip elongation and divides by medial fission.
It has a typical eukaryotic cell cycle and genome organisation, with
around 70% of its genes conserved in humans. It thus provides a
simple system to study complex problems of eukaryotic cell biology.
Our current research consists of four main areas: (i) The mitotic and
meiotic cell cycles, (ii) Entry into the meiotic cell cycle, (iii) Size control
and (iv) Chemical biology.
Group Leaders
Paul Nurse
Jacqueline Hayles
Research Co-ordinator
Ryoko Mandeville
Pilar Gutierrez-Escribano
Francisco Rivero Navarro
Graduate Students
Martina Begnis
Helena Cantwell
James Patterson
Matthew Swaffer
Elizabeth Wood
Scientific Officers
Jessica Greenwood
Linda Jeffery
Andrew Jones
Richard Lewis
Juan-Juan Li
90
The mitotic cell cycle
Matthew Swaffer is using the bipartite CDK-CyclinB
complex, which brings about an ordered
progression of the eukaryotic cell division cycle to
investigate how activity of a single CDK can
differentially initiate S phase and mitosis. Using
phospho-proteomics (with Andrew Jones in the
Protein Analysis and Proteomics Facility), and
chemical manipulation of CDK activity, Matthew
has defined CDK-dependent phosphorylation
events in S phase and mitosis, of both known and
novel CDK substrates. Around 10% of the phosphoproteome in mitosis appears to be dependent on
CDK activity with around 10-fold more CDKdependent phosphorylation events in mitosis than
S phase. CDK-dependent phosphorylation events
that first appear in S phase are sustained across the
cell cycle. In contrast, mitotic phosphorylation
events increase dramatically upon mitotic entry.
Both S phase and mitotic substrates are
dephosphorylated with similar kinetics at mitotic
exit. These experiments are consistent with low
CDK activity phosphorylating S phase substrates
and higher kinase activity being required for
mitotic substrate phosphorylation.
Eukaryotes duplicate their genomes using multiple
replication origins. Atanas Kaykov has investigated
the organisation of origin firing along
chromosomes, using a genome-wide analysis on
single DNA molecules around the length of a fission
yeast chromosome (Figure 1). He has shown that at
S phase onset origins fire randomly and sparsely
throughout the genome, whereas later in S phase
clusters of fired origins appear in these sparser
regions and form nuclear replication foci. The rate
of origin firing during S phase gradually increases,
peaking just before mid S phase. Towards the end
of S phase nearly all unfired available origins fire
within the unreplicated regions, contributing to
the timely completion of genome replication. The
data show that origin firing is mostly random and
that the majority of origins do not fire as a part of a
deterministic programme.
To examine origin firing during the meiotic cell
cycle Jenny Wu showed that local changes in origin
efficiencies, led to changes in meiotic
recombination frequencies due to an increase in
the binding of Rad51 recombination factor. This
suggests that origin selection determines
recombination frequencies, and shows that
modifications in the DNA replication programme
can modulate cellular physiology (Wu and Nurse,
2014; Mol Cell. 53(4): 655-662).
Wild type cells progressing through the meiotic cell
cycle require both mitotic cyclins and meiosis
specific cyclins. Pilar Gutierrez-Escribano has
analysed the requirements for these different
cyclins and shown that the bipartite CDK-CyclinB
complex, when moderately overexpressed, allows
cells to undergo a near normal meiotic cell cycle in
the absence of other cyclins. This shows that
qualitatively different CDK complexes are not
required for cell cycle progression either during the
meiotic or mitotic cycles.
To identify gene products important for the timing
of progression through the cell cycle, Linda Jeffery
identified 17 genes that are haploinsufficient for
cell cycle progress. These include cdc2, cdc25 and
wee1, previously shown to be haploinsufficient for
mitotic entry. Other genes that regulate CDK1
activity at the G2/M transition, as well as genes
involved in nuclear cytoplasmic transport, and
protein translation, were also identified as
haploinsufficient.
Recently, Jessica Greenwood and James Patterson
have initiated new projects. Jessica is using
Figure 1
A combed single 5.6Mb DNA
molecule approximately the
length of chromosome I. Yellow,
newly synthesised DNA; red,
unreplicated DNA.
genome-wide ChIP-seq to investigate chromatin
localisation of Cdc2 and Cdc13 throughout the cell
cycle, and James is investigating how the variability
in the number of molecules governs cellular
processes and influences cell cycle progression.
Entry into the meiotic cell cycle
Deletion of the zfs1 gene affects timing of the
mitotic cell cycle G2/M transition independently of
CDK Tyr15 phosphorylation. Francisco Navarro
identified a further role for Zfs1 at the transition
between the mitotic and meiotic cell cycles. Zfs1 is
a CCCH-type tandem zinc finger RNA binding
protein and sequencing of Zfs1-associated RNAs
identified transcripts, both protein coding and
non-coding, as potential targets of Zfs1 regulation.
The mRNA of the G1 cyclin Puc1 is a target of Zfs1,
which has a destabilising effect on the puc1
transcript. Reduction in levels of Puc1 is necessary
for entry into the meiotic programme, suggesting
that the effect of Zfs1 on Puc1 levels is decisive for
this transition.
Size control
Fission yeast shows a tight control over cell size at
entry into mitosis, with wild type cells dividing at
around 14µm. Lizzie Wood identified mutants that
have increased variability in cell size at division in
order to understand the underlying mechanism(s)
affecting size control. She has identified three
genes encoding, a regulator of lipid metabolism, a
polyA polymerase and a sequence orphan, and is
analysing how they affect cell size variability.
We are also investigating how cells regulate the
size of membrane bound organelles. There is a
near constant nuclear-cytoplasmic ratio (N/C ratio)
in fission yeast, which is not directly dependent on
DNA content. Helena Cantwell is investigating
whether an active mechanism corrects
perturbations in N/C ratios. A pom1 deletion strain
that divides asymmetrically generates cells with
varied N/C ratios. Helena found that cells with low
or high N/C ratios could recover to the wild type
value within one cell cycle, whilst those with high
N/C ratios recovered at a slower rate.
Chemical biology
Richard Lewis and Juan-Juan Li have identified
several bacterial strains, which produce potentially
new biologically important molecules affecting cell
cycle progression, causing cells to become highly
elongated or to misplace the nucleus and division
septum. An alternative approach by Jun Funabiki,
uses small molecule libraries to identify inhibitors
of AAA ATPases, in collaboration with Tarun
Kapoor’s Group (The Rockefeller University).
91
PROTEIN PHOSPHORYLATION
www.london-research-institute.org.uk/research/peter-parker
Group Leader
Peter Parker
Nicola Brownlow
Katharina Deiss
Tanya Pike
Philippe Riou
Frances Willenbrock
Graduate Students
Jeroen Claus
Khalil Davis
Joanna Kelly
Yixiao Zhang
Julien de Naurois
Scientific Officers
Veronique Calleja
Jacqueline Marshall
Narendra Suryavanshi
Masters Student
Alexander Wallroth
Figure 1
PKCε loss of function is associated
with PICH-positive ultrafine
bridges in anaphase.
HeLa cells (control or knocked
down for PKCε as indicated) were
grown on cover slips, fixed and
stained for DNA (DAPI; blue) and
the helicase PICH (green). Cells in
anaphase were selected for
imaging. The control cell image
illustrates an apparently normal
anaphase with no evidence of
chromosome bridges or
PICH-positive ultrafine bridges
(only 25% of control cells show
any aberrations of this type). In
PKCε knockdown cells, there is
consistent evidence of nondisjunction illustrated here by a
PICH positive ultrafine bridge
running between the two
presumptive daughters (>65% of
cells show such aberrations).
Scale bar = 5μM.
92
It has long been understood that aberrations in normal cellular
control hierarchies underpin the pathophysiology of cancer. The
nature of individual dysfunctions and the way in which these lead to
pathological properties and emergent tumour autonomous and
tumour host dependencies remain areas of enormous interest and
activity. Dissection of these processes in any given tumour setting
provides both markers relevant to prognosis and targets for
intervention. As pleiotropic cellular regulators, it is not surprising that
protein kinases feature prominently as targets in the expanding
portfolio of approved cancer treatments. The PKC subfamily of the
AGC kinase arm of the human kinome remain central to our work,
which addresses the potential of these as targets in cancer.
An emergent PKCε dependency of
transformed cells
We have continued to dissect the underlying
mechanisms associated with the cell cycle actions
of PKCε that are particularly manifested in certain
transformed cell models. This has led to the
conclusion that PKCε is involved in a conditional
pathway that triggers both a delay in the
metaphase-anaphase transition and resolution of
catenated DNA. The trigger for PKCε engagement
is the sensing of catenation specifically in
metaphase.
Following DNA replication, the catenation of sister
chromatids requires resolution, a process that is
normally completed through S phase and G2. When
cells are transiting G2, incomplete decatenation
triggers a checkpoint arrest enabling elimination of
catenation before completion of G2. Some
transformed cells by-pass this G2 checkpoint and
consequently enter metaphase faster but with
DMSO
siPKCε
retained catenation. This is not compatible with
effective metaphase exit/anaphase entry where
there needs to be complete disjunction of sister
chromatids in order to permit chromosomal
segregation into daughter cells.
It is this catenation-dependent delay to metaphase
exit that we have shown is under the control of
PKCε. Notably the earlier catenation checkpoint
that operates in G2 is not influenced at all by PKCε
and hence normal cells that retain their G2
checkpoint do not require the metaphase PKCεcontrolled pathway to effect segregation.
However, the subset of tumour cells that have lost
their G2 response to catenation are particularly
dependent on the PKCε-mediated response in
mitosis to prevent non-disjunction in anaphase
(Figure 1). This distinct behaviour of tumour cells is
a vulnerability providing an opportunity for
intervention with an expectation of a high
therapeutic index.
When and how is PKCε engaged in cell cycle
control?
In the context of the cell cycle, defining when PKCε
intervention might usefully be deployed, requires
specific biomarkers informing on the loss of the G2
catenation checkpoint and/or defining the
circumstances surrounding the engagement of the
PKCε pathway.
To provide insight into the key players in the G2
catenation checkpoint and so enable scrutiny of
loss-of-function/expression patterns in tumour
settings, we have completed a whole genome
screen for gene products that when lost cause a
by-pass of the checkpoint (collaboration with Dr
Mike Howell, LRI). A series of ~80 genes have been
identified through multiple rounds of screening and
library deconvolution; we are currently pursuing
these to (in)validate them as G2 checkpoint players
and candidate biomarkers. In parallel, we are
investigating the mechanisms involved in the
triggering of PKCε action in response to metaphase
catenation. This has also produced promising
insights that inform on mechanism and may serve
the purpose of marking mitotic catenation, and
hence predict PKCε inhibitor sensitivity.
Aberrant proliferation, polarity and targeting
aPKCι
aPKCι is an established regulator of cellular polarity
and proliferation. We have defined aPKCι as a
selective driver in certain oncogenic backgrounds
and established models, which are highly
dependent on aPKCι for both proliferation and
polarity.
Our studies on this multifunctional kinase have
been divided between: (i) a drug development
programme executed through CRT (Drs Jon Roffey
and Christian Dillon) and a collaborating
commercial activity, and (ii) mechanistic studies
directed at understanding how mutant Ras
influences aPKCι pathways and how aPKCι
downstream targets define polarising and/or
proliferative actions.
The drug development programme has made
excellent progress and now sits at a very late
pre-clinical stage. Our particular input into this
programme has centred upon the identification of
substrates whose phosphorylation may serve as
pharmacodynamic markers and/or as biomarkers
for patient stratification. This has entailed the
development of a substrate screening platform – a
method we have termed KIPS for Kinase Proximal
Substrate screening.
Addressing more directly the pathways involved in
promoting aberrant proliferation and polarity
(including migration; collaboration with Dr Erik
Sahai, LRI), we have focused on a Ras-pathway link
to aPKCι and on specific partner proteins that are
recruited through the ‘RIPR’ motif, a substrate
recruitment surface that has been found mutated
rarely but repeatedly in tumours (collaboration
with Dr Neil McDonald, LRI). The change of function
associated with mutations of this RIPR motif,
appears to selectively disable polarising capabilities
of aPKCι. How specific partner proteins contribute
to aPKC-dependent polarity is currently the subject
of ongoing work. Directly related to the
proliferative programme in which aPKCι can also
sit, we have identified aPKCι-dependent proximal
functions that are sensitive to oncogenic Ras.
Understanding the underlying mechanisms will
prove important in relating aPKCι action with
intervention in Ras-driven tumours.
Emerging properties; PKNs and pseudokinases
The PKN members of the PKC family remain
perhaps the least well characterised. We have
continued to elaborate functions for this family
from a genetic perspective and have evidence that
PKN is involved in regulation of various aspects of
cellular migration. In parallel, we have pursued
their roles in in vivo models, particularly in prostate
cancer (collaboration with Dr Angus Cameron,
QMUL). From a mechanistic, substrate perspective,
we have also initiated studies on downstream
screening employing the KIPS approach
successfully deployed for aPKCι.
Extrapolating our work on nucleotide-driven
allostery defined in PKC family members, we have
investigated how the nucleotide binding pocket of
the pseudokinase HER3 is involved in signalling
through heterodimerisation with HER2 and also
how this influences behaviour of HER3 in the
context of models of drug resistance. This has in
particular addressed the underlying organisational
changes associated with the receptor and its
heterodimer partner as a function of inhibitor
treatment (collaboration with Prof. Tony Ng, KCL
and Dr Marisa Martin-Fernandez, STFC).
Importantly, the evidence indicates that nucleotide
binding pocket occupation has a profound effect on
signalling through this inactive kinase and
highlights HER3 as both an opportunity and a
liability for targeted interventions.
93
IMMUNOBIOLOGY
www.london-research-institute.org.uk/research/caetano-reis-e-sousa
Group Leader
Sophie Acton
Jan Boettcher
Safia Deddouche
Julie Helft
Jatta Houtari
Pierre Maillard
Barbara Schraml
Naren Srinivasan
Annemarthe van der Veen
Paul Whitney
Santiago Zelenay
Graduate Students
Susan Ahrens
Oliver Gordon
Pavel Hanč
Janneke van Blijswijk
Scientific Officers
Aaron Farrugia
Delphine Goubau
Sonia Lee
Neil Rogers
Oliver Schulz
Kathryn Snelgrove
Dendritic Cells (DCs) are thought to function as antigen-presenting
cells for priming T cell responses. Over the last year, we have found
that DCs in fact perform a broader array of functions. We
uncovered a key role for DCs in orchestrating innate resistance to
systemic fungal infection and we have found that signals from DCs
allow for stretching of lymph node stromal cells and permit acute
lymph node expansion at the outset of an immune response. In
parallel, we have continued our work on receptors used by DC and
other cell types to detect viral invaders and thereby ensure host
protection from infection and from viral oncogenesis. Our work
adds to our understanding of the role of DCs and other cells in
detecting infection and cancer and induction of responses aimed at
restoring homeostasis.
Cell-intrinsic detection of viruses
Antiviral defence in vertebrates is orchestrated by
type I interferons, cytokines that can be
synthesised by all cells in response to triggering of
viral sensors present in the cytosol (Goubau et al.,
2013; Immunity. 38(5): 855–869). The latter include
the RIG-I-like receptors (RLRs), RIG-I, LGP2, and
MDA5, which detect atypical RNAs associated with
viral presence. To identify RNAs that trigger MDA5
activation in infected cells, we purified and
characterised LGP2/RNA complexes from cells
infected with encephalomyocarditis virus (EMCV),
a picornavirus detected by MDA5 and LGP2 but not
RIG-I (Deddouche et al., 2014; Elife. 3:e01535). We
found that those complexes were highly enriched
for MDA5-stimulatory activity and for a specific
sequence corresponding to the L region of the
EMCV antisense RNA (Deddouche et al., 2014).
Genomic deletion of the L region in EMCV
generated viruses that were less potent at
stimulating MDA5-dependent IFN production
(Deddouche et al., 2014). Thus, the L region
antisense RNA of EMCV is a key determinant of
innate immunity to the virus and represents the
first natural RNA isolated from virally-infected cells
that activates MDA5.
RIG-I and MDA5 are activated by distinct viral RNA
structures. Earlier evidence from our group and
others indicated that RIG-I selectively responds to
RNAs bearing a triphosphate (ppp) moiety at the
5’-end (Pichlmair et al., 2006; Science. 314(5801):
997-1001, Hornung et al., 2006; Science. 314(5801):
994-997). This year we showed that RIG-I also
mediates antiviral responses to RNAs bearing
94
5’-diphosphates (5’pp) (Goubau et al., 2014;
Nature. 514(7522): 372–375). Genomes from
mammalian reoviruses with 5’pp termini, 5’pp-RNA
isolated from yeast L-A virus, and base-paired
5’pp-RNAs made by in vitro transcription or
chemical synthesis, the latter in collaboration with
Gunther Hartmann’s group in Bonn, all bound to
RIG-I and served as RIG-I agonists (Goubau et al.,
2014). Furthermore, a RIG-I-dependent response
to 5’pp-RNA was essential for controlling reovirus
infection in cultured cells and in mice (Goubau et
al., 2014). Thus, the minimal determinant for RIG-I
recognition is an RNA with 5’pp. Such RNAs are
found in some viruses but not in uninfected cells,
indicating that recognition of 5’pp-RNA, like that of
5’ppp-RNA, acts as a means of self/non-self
discrimination by the innate immune system.
Syk function in DCs and innate resistance to
fungal infection
Earlier work from our group and others indicated
that protection from fungal infection ensues from
the activity of Syk-coupled C-type lectin receptors
and MyD88-coupled toll-like receptors in myeloid
cells, including neutrophils, macrophages and DCs
(Osorio et al., 2011; Immunity. 34(5): 651-664).
Given the multitude of cell types and receptors
involved, elimination of a single pathway for fungal
recognition in a single cell type such as DCs,
primarily known for their ability to prime T cell
responses, would be expected to have little effect
on innate resistance to fungal infection (Osorio et
al., 2011). Earlier this year we reported that this is
surprisingly not the case and that selective loss of
Syk but not MyD88 in DCs abrogates innate
Figure 1
Paraffin embedded section of
inguinal lymph node from
PDGFRα-H2B-GFP mouse showing
nuclei of fibroblastic reticular cells
in brown and podoplanin staining
in pink. Surrounding leukocytes
are shown in blue.
resistance to acute systemic Candida albicans
infection in mice (Whitney et al., 2014; PLoS
Pathog. 10(7): e1004276). In collaboration with
Salomé LeibundGut-Landmann (a former lab
member, now independent in Zürich), we showed
that Syk expression by DCs is necessary for IL-23p19
production in response to C. albicans, which is
essential to transiently induce GM-CSF secretion by
NK cells that are recruited to the site of fungal
replication (Whitney et al., 2014). NK cell-derivedGM-CSF in turn sustains the anti-microbial activity
of neutrophils, the main candidicidal effectors (Bär
et al., 2014; Immunity. 40(1): 117–127). Thus, the
activity of a single kinase in a single myeloid cell
type orchestrates a complex series of molecular
and cellular events that underlies innate resistance
to fungal sepsis.
DC control of lymph node expansion
Lymph nodes (LNs) are essential meeting places for
T lymphocytes and DCs. T cell and DC interactions
within LNs are supported by fibroblastic reticular
cells (FRCs), a complex interconnected stromal cell
network that produces and ensheathes dense
bundles of collagen and other extracellular matrix
components (Katakai et al., 2004; Int Immunol.
16(8): 1133–1142). FRC networks additionally
provide key physical routes for leukocyte traffic, as
well as act as a source of chemoattractants for T
cells and DCs. Notably, contact with FRCs promotes
chemokinesis in immigrant DCs to facilitate their
migration within lymph nodes (Acton et al., 2012;
Immunity. 37(2): 276-89). This is in part due to
cytoskeletal changes that take place in DCs in
response to signalling by the Syk-coupled C-type
lectin CLEC-2 upon engagement by its ligand, the
glycoprotein podoplanin (PDPN), expressed by
FRCs (Acton et al., 2012). Interestingly, we had
previously noticed that CLEC-2 expression by DCs is
upregulated in inflammatory conditions (MourãoSá et al., 2011; Eur J Immunol. 41(10): 3040–3053).
We therefore wondered whether, in addition to
signalling to help promote DC movement along
FRCs, CLEC-2 might also work in reverse to
modulate PDPN function and alter the properties
of the FRC network. In collaboration with the group
of Erik Sahai (Tumour Cell Biology Group, LRI), we
found that PDPN positively regulates actomyosin
contractility in FRCs via activation of RhoA/C and
the downstream effector Rho-kinase (Acton et al.,
2014; Nature. 514(7523): 498–502). PDPN
engagement by CLEC-2 causes clustering of PDPN
and rapidly and robustly uncouples PDPN from
RhoA/C activation, which relaxes the actomyosin
cytoskeleton, permitting FRC stretching (Acton et
al., 2014). Notably, we found that LN expansion
upon immunisation was significantly constrained
in mice lacking CLEC-2 expression in DCs but this
could be reversed by administering recombinant
protein (Acton et al., 2014). Thus, the same DCs
that initiate immunity by presenting antigens to
lymphocytes also initiate remodelling of LNs by
delivering CLEC-2 to FRCs and permitting FRC
network stretching. CLEC-2 modulation of PDPN
signalling allows for the rapid LN expansion driven
by lymphocyte influx and proliferation that is the
critical hallmark of adaptive immunity.
95
TUMOUR CELL BIOLOGY
www.london-research-institute.org.uk/research/erik-sahai
Group Leader
Erik Sahai
Esther Arwert
Eishu Hirata
Marco Montagner
Danielle Park
Graduate Students
Stefanie Derzsi
Anna Dowbaj
Nil Ege
Alexander Kuznetsov
Scientific Officers
Steven Hooper
Robert Jenkins
96
The Tumour Cell Biology group aims to understand how cancer cells
move through the body and their interactions with other
components of the tumour microenvironment. In particular, we are
interested in the cross-talk between cancer cells and a type of noncancerous cells that is found in most solid tumours: cancer-associated
fibroblasts (CAFs). These non-cancerous cells can promote the
invasion of cancer cells both by producing soluble factors and altering
the composition and structure of the extracellular matrix. By
understanding the interaction of cancer cells with their environment
we hope to gain information that can be used to reduce the spread of
cancer and improve responses to chemotherapies.
We use a range of different approaches to learn
about how cancer cells might interact with the
tumour microenvironment. At one end of the
spectrum, Rob Jenkins has been implementing
computational modelling approaches to explore
how varying the properties of cancer cells and
CAFs leads to different patterns of cancer invasion.
Ideas from the modelling are being tested
experimentally by Takuya Kato, a year-long visitor
as part of an Anglo-Japanese collaborative grant.
Steve Hooper, Alex Kuznetsov and Danielle Park are
exploring cancer cell invasion in 3D culture systems
that recreate many features of physiological tissue.
Nil Ege is trying to understand how both cancer
cells and CAFs might sense the physical properties
of the extracellular matrix. Anna Dowbaj has
recently joined our group and is attempting to use
light to control cell signalling in both cancer cells
and CAFs. We believe that this will be a powerful
tool to understand how changes in the behaviour
of either a single cell, or a small group of cells,
affect neighbouring cells. Marco Montagner,
Esther Arwert, and Eishu Hirata are all using a
combination of in vitro and in vivo approaches to
learn how communication between cancer cells
and non-cancerous enables the most lifethreatening aspects of cancer. Marco is studying
how cancer cells arriving at metastatic sites
manage to alter the behaviour of cells at their new
location and thereby facilitate the outgrowth of
metastases. Esther is trying to understand how the
wound healing microenvironment after tumours
excision affects residual disease, either locally or in
distant organs. Finally, Eishu has been using
intravital imaging to investigate why targeted
therapies against oncogenic kinases might fail. He
has been exploring the hypothesis that CAFs may
provide signals that are able to compensate for
inhibition of the oncogenic kinases. In particular,
he has implemented methods for live imaging of
the activity of ERK/MAP kinase signalling in
tumours before and after the administration of
targeted therapies.
2014 has also seen the award of a PhD to Stefanie
Derzsi for her work into heterotypic cell-cell
contacts between cancer cells and CAFs, and the
completion of two projects initiated by former lab
members, Chris Madsen and Cerys Manning (these
are described in detail below).
Cancer cell migration is necessary for tumours to
spread through the body. As cancer cells transit
from their site of origin to other locations in the
body they encounter a diverse range of
environments with varying matrix composition,
stiffness, and geometry. To understand how cells
adapt to enable migration through changing
environments we have continued our collaboration
with Paul Bates (Biomolecular Modelling, LRI). This
analysis has revealed that localised coordination of
the actomyosin cytoskeleton and coupling of the
actin network to the plasma membrane is critical
for cells to adapt to changes in matrix geometry.
However, the molecular mechanisms that might
achieve this coordination are not well understood.
We hypothesised that the migration of cells during
development might provide clues to this
mechanism. In collaboration with Barry
Thompson’s group (Epithelial Biology Laboratory,
LRI), we screened for genes that play a role in both
developmental cell migration in Drosophila and
cancer cells. This led us to focus on the STRIPAK
complex that includes both PP2A subunits and
MST3&4 kinases. The MST3&4 kinases are able to
found in tumours prevent negative regulation
of MST3&4. This work has led us to propose
that the STRIPAK complex is a key enabler of
cancer cells being able to adapt their
migratory behaviour depending on their
environment and thereby metastasise.
Figure 1
Image shows two breast cancer
cells transfected with the
phosphatase regulatory subunit
PPP1R14C (blue) and stained for
F-actin (red) 80x80microns.
(Adapted from Madsen et al.,
2015; Nat Cell Biol. 17(1): 68-80)
coordinate the regulation of the actomyosin
network with its coupling to the plasma
membrane. They achieve this by directly
phosphorylating the Ezrin/Radixin/Moesin family
of proteins that link the actin network to the
plasma membrane and by phosphorylating the
PPP1R14A-D family of PP1 regulators. The
phosphorylation of PPP1R14 proteins lead to
reduced dephosphorylation of MLC, and
consequently increased actomyosin contractility. If
this mechanism is perturbed by depleting MST3&4
then cancer cells are unable to squeeze through
small gaps, although they remain competent at
migration of flat surfaces. This lack of adaptability
ultimately leads to reduced metastatic efficiency.
Excitingly, we have also been able to show that
truncations of the STRIPAK component FAM40B
In addition to our interest in the mechanistic
aspects of how cancer cells move, we are also
interested in what makes cancer cells invasive.
Previous intravital imaging studies from our
group and others have shown that only a small
minority of cells are motile in tumours. To
learn more about these cells we introduced
fluorescent reporter constructs into
melanoma cells that enable us to monitor the
activity of SRF and Notch signalling. Intravital
imaging of tumours generated from these
melanoma cells revealed that both of these
signalling pathways are activated in the motile
sub-population of cells. Further analysis of
transcription changes associated with the
activation of these pathways lead us to
identify an overlapping set of genes
associated with motile melanoma cells. We
identified the histone trimethylase EZH2 as a
critical regulator of this set of genes. Not only
is EZH2 required for the expression of this
genes, it is also required for the invasive
behaviour of melanoma cells and their ability
to form lung metastases.
97
CANCER EPIGENETICS
www.london-research-institute.org.uk/research/paola-scaffidi
The Cancer Epigenetics group is interested in uncovering
fundamental principles of cancer development, with particular
emphasis on the role of epigenetics in determining malignant cellular
features. By focussing on cancer stem cells (CSCs), the cells that fuel
the long-term growth of tumours, we aim to identify novel
therapeutic targets and promote the design of more effective
strategies to treat the disease.
Group Leader
Paola Scaffidi
Graduate Students
Josep Monserrat Sanchez
Thomas Mortimer
Scientific Officer
Christina Morales Torres
Masters Student
Tristan Henser-Brownhill
Epigenetic mechanisms regulating
tumour maintenance
Cancer is a clonal disease originating from a single
cell. Yet, most human cancers are characterised by
astounding intra-tumour heterogeneity and
comprise various subpopulations of cells with
distinct phenotypes and biological properties.
Even neighbouring cells within a tumour may have
different morphologies, express differential
transcriptional programs and display specific
repertoires of surface molecules (Figure 1). Most
importantly, not all cancer cells possess the same
proliferative potential and in most cancers only a
subset of cells is truly immortal. These cells act as
CSCs and are responsible for maintaining the
long-term growth of the tumour. We are interested
in understanding how epigenetic mechanisms
involving chromatin and DNA methylation
contribute to intra-tumour heterogeneity and how
they affect CSC function.
Findings over the past ten years have strongly
implicated deregulation of epigenetic instructions
in cancer. Epigenetic alterations in chromatin and
DNA methylation are universal features of
neoplasia and recurrent mutations in proteins
involved in epigenetic control are increasingly
identified in various cancers. Although
comprehensive epigenetic profiles of various
cancer types can now be generated, discriminating
Figure 1
Phenotypic intratumour
heterogeneity.
Immunofluoresce microscopy of a
breast cancer section showing
highly heterogeneous patterns of
methylated histone H3 (green)
and histone H1.0 (red).
98
between ‘driver’ epigenetic alterations, which play
critical roles in cancer development, and
‘passenger’ epigenetic changes, which simply
occur as consequence of altered cell function,
remains often challenging. This is partially due to
the fact that traditional bulk approaches analyse
tumours in their entirety and disregard
intratumour heterogeneity.
CSCs form in vivo heterogeneous and
hierarchically-organised tumours in which only a
small subset of cells has unlimited proliferative
potential and maintains tumour growth. The
molecular basis underlying functionally distinct
subpopulations of cells in a tumour are at present
unclear. Since CSCs generate their nontumourigenic progeny through a differentiation
process, epigenetic mechanisms are likely to play a
critical role in defining the malignant phenotype of
a cancer cell within a growing tumour (Figure 2).
We are employing genome-wide mapping
approaches (ChIP-Seq and DHS-seq) to
characterise the chromatin landscape of CSCs and
identify epigenetic features, which distinguish
them from the rest of the tumour. By combining
these studies with in vivo gain- and loss-of function
experiments, we aim to identify epigenetic
features that are critical for CSC function and can
be modulated for therapeutic purposes. Using
these approaches we have recently identified the
differentiation-related histone variant H1.0 as an
important regulator of CSC function. We have
found that H1.0 is downregulated in a variety of
cancers, including glioblastoma, breast cancer,
kidney cancer and melanoma, and that its
downregulation is required for CSC self-renewal.
H1.0 loss in CSCs leads to genome-wide activation
of oncogenic transcriptional programs by altering
the higher-order structure of chromatin and
inducing coordinated changes in gene expression
in large chromosomal domains. We are currently
trying to understand how we can modulate H1.0
expression, in order to inhibit CSC self-renewal and
drive their terminal differentiation.
Figure 2
Epigenetic and functional
heterogeneity of cancer cells.
Tumours comprise functionally
distinct subpopulations of cells.
Histone modifications, DNA
methylation, and higher-order
chromatin structure define the
epigenetic status of CSCs and
contribute to the maintenance of
CSC properties. The bulk of the
tumour arises through
differentiation of CSCs into
heterogeneous cell types,
characterised by various
epigenetic states that activate or
silence distinct sets of genes.
Changes in the epigenetic
landscape occur during
differentiation and are associated
with loss of self-renewal and
tumourigenicity.
Extracellular signalling and cancer cell plasticity
What drives epigenetic heterogeneity within
tumours? While many epigenetic abnormalities in
cancer are the consequence of mutations, some
epigenetic changes are reversible and, as such,
cannot be the result of genetic changes.
Interactions of cancer cells with the tumour
microenvironment strongly affect cancer
development. These interactions are based on
extracellular signalling and they are likely to result
in epigenetic changes that affect the differentiation
status of cancer cells and, as a consequence, their
proliferative potential. We are developing novel
microscopy-based tools, which will allow
assessment of the epigenetic status of CSCs and
their plasticity in response to extracellular
signalling in intact tumours. These tools will also
allow visualisation of CSCs in living animals and the
analysis of CSC dynamics. It is at present unclear
whether tumour-maintaining cells are a stable
subpopulation of cells within tumours or whether
cells can fluctuate between a CSC and a non-CSC
state as a result of reversible epigenetic changes.
This has major implications for the design of
targeted therapeutic strategies. We aim to
understand how stable the CSC phenotype is and
whether it can be modulated by interfering with
chromatin-based mechanisms.
Oncogenic reprogramming and CSC formation
In many cancers, CSCs arise through
reprogramming of committed cells that lose their
cellular identity and acquire self-renewal ability.
Thus, alterations in proteins involved in
maintenance of epigenetic memory are likely
players in the genesis of CSCs. Indeed, we have
recently identified a tumour-suppressive
mechanism that utilises the chromatin-binding
protein BRD4 to inhibit oncogenic reprogramming
of committed cells into CSCs. We have discovered
that BRD4, a histone acetylation ‘reader’,
maintains a gene expression programme, which
protects normal cells from de-differentiation in
response to oncogenic stimuli. We found evidence
for this tumour protective mechanism in lung and
breast tissues. Interestingly, the same protein is
critically important for disease maintenance in
various haematological cancers, suggesting that
BRD4 can exert a tumour-protective or a tumourpromoting function, depending on the cellular
context (Fernandez et al., 2014; Cell Rep. 9(1):
248-260). Extending these studies, we are
currently performing unbiased, CRISPR/Cas9based, loss-of-function screens targeting proteins
involved in the establishment and recognition of
DNA methylation patterns, writer, readers and
erasers of chromatin marks, chromatin
remodellers and proteins controlling the highorder structure of chromatin, in order to identify
other epigenetic regulators which either prevent
(tumour suppressors) or promote (therapeutic
targets) the appearance of CSCs.
99
MACROMOLECULAR STRUCTURE AND FUNCTION
www.london-research-institute.org.uk/research/martin-singleton
Group Leader
Martin R Singleton
William Chao
Stephan Lefevre
Silva Zakian
Graduate Students
Vera Leber
Thibaud Perriches
Ben Wade
Scientific Officer
Xiao Hu
Our group studies the molecular mechanisms of eukaryotic
chromosome segregation, with a particular focus on the
macromolecular complexes involved in attaching replicated sister
chromatids to each other and to spindle microtubules. We are
interested in determining the atomic structures of these complexes,
and understanding how their overall architecture and function aids
the cell in bringing about the rapid and accurate dissemination of the
genome. We employ a combination of biophysical and biochemical
techniques to address these questions, primarily those of X-ray
crystallography for high-resolution studies and electron microscopy
to analyse larger complexes.
Regulation of kinetochore formation
Kinetochores are structures that attach replicated
sister chromatids to spindle poles via microtubules.
There is considerable complexity involved in the
formation of an attachment that is both strong
enough to carry spindle forces, yet at the same
time remain bound to the dynamically unstable
microtubule. In addition, the kinetochore must
allow for rapid dissolution of erroneous
attachments that might result in chromosome
missegregation. These requirements have lead to
the evolution of a large, modular structure, with
distinct functionality associated with each
collection of sub-complexes. Two key elements are
the so-called CCAN (constitutive centromere
associated network) and the KMN (Knl1, Mis12,
Ndc80) network (Figure 1a), which interact with
centromeric chromatin and spindle microtubules
respectively. The kinetochore does not remain
attached to the underlying chromatin throughout
the cell cycle, rather it appears to be assembled in
a step-wise fashion after DNA replication, with the
exact timing of events varying between organisms.
The mechanisms controlling this assembly process
have yet to be fully understood, but perhaps
unsurprisingly, reversible phosphorylation of
multiple kinetochore proteins are thought to
be involved.
In collaboration with David Glover’s group in the
Department of Genetics, Cambridge, we have been
characterising a novel phosphatase-kinetochore
interaction. A proteomic analysis of centromereassociated factors in Drosophila revealed that the
targeting subunit of the protein phosphatase 4
(PP4) complex, known as Falafel (Flfl) co-purifies
with the CCAN constituent CENP-C. Unlike the
100
better-studied protein phosphatase 2A (PP2A),
little is known about PP4 targets and activity. Like
PP2A, it is a multi-subunit enzyme, comprising a
catalytic subunit (which is extremely similar to the
PP2A equivalent) and two other variable subunits,
one of which, R3 is presumed to be the regulatory
subunit, responsible for substrate recognition.
Falafel is the Drosophila homolog of the human R3
protein, and the interaction with CENP-C provides
a suitable model to better understand PP4 activity.
The Glover group has demonstrated that a direct
interaction between CENP-C and Falafel exists, and
that the catalytic activity of PP4 is required for
correct mitotic progression. Depletion or
inactivation of the enzyme results in mislocalisation of CENP-C as well as outer kinetochore
components at metaphase. Studies using
truncation mutants demonstrated that the
N-terminal of Falafel is required for the interaction
with CENP-C.
Using peptide array analysis (provided by the LRI
Peptide Chemistry Facility) we were able to map
the exact binding site on CENP-C, and define a
minimal construct of Falafel required for the
interaction. This corresponds to the N-terminal
domain that had been previously predicted to
adopt a PH-like fold. We solved the structure of
this domain bound to the cognate peptide from
CENP-C at 1.5Å resolution (Figure 1b). Structural
homology analysis showed that the fold is closely
related to the EVH1 domain, as typified by the Ena/
Vasp family of proteins. These usually bind a
proline-rich sequence in a left-handed helical (PPII)
configuration. However, in our structure, the
recognition motif is FKKP, with the phenylalanine
and proline making the key conserved contacts
Figure 1
A. Schematic diagram of the
kinetochore-centromere
interface showing the two main
conserved complexes, the CCAN
and KMN network.
B. Structure of the Falafel
N-terminal domain bound with
the target peptide from CENP-C.
The critical interacting
phenylalanine and proline
residues are depicted in green.
A
B
with Falafel. Despite this sequence variance, the
peptide still adopts a classic PPII helical form, and
provides the first insights into the molecular basis
of substrate recognition by the PP4 family of
enzymes. We believe that recruitment of PP4
family phosphatases to the inner centromere is
likely to a conserved phenomenon, and it will be
interesting to delineate how the process occurs in
other eukaryotes.
Sister chromatid cohesion and DNA replication
The establishment of persistent sister chromatid
cohesion requires the action of replication fork
associated proteins, presumably to ensure that
inter-sister rather than intra-chromatid linkages
are maintained. A key reaction is the acetylation of
the Smc3 subunit of cohesin by the
acetyltransferase Eco1, which is thought to
antagonise cohesin destabilisation by Wapl. In
addition to this pathway, a number of other
proteins are thought to contribute to cohesion
establishment in both Eco1-depedent and
independent manners. We have recently
started to carry out structural and
biochemical studies on some of these
proteins, in order to understand their
recruitment and function at the replication
fork, and how these activities impinge on the
process of sister chromatid separation. In
addition, Eco1 has been proposed to play a
role in double strand break repair via a
replication-independent pathway, and it will
be of great interest to understand how
differential targeting of the enzyme is
achieved.
101
MICROTUBULE CYTOSKELETON
www.london-research-institute.org.uk/research/thomas-surrey
Group Leader
Thomas Surrey
Jayant Asthana
Todd Fallesen
Franck Fourniol
Johanna Roostalu
Einat Schnur
Graduate Students
Hella Baumann
Tanja Consolati
Jonathon Hannabuss
Rupam Jha
Scientific Officers
Nicholas Cade
Christian Duelberg
Iris Lueke
Claire Thomas
Masters Student
Stefan Keller
Microtubules are polymers with a variety of essential functions in all
eukaryotic cells. The microtubule cytoskeleton forms a scaffold for
the internal organisation of cells, it provides tracks for molecular
motor transport and - during cell division - it forms the mitotic spindle
that segregates the chromosomes. Our research aims at a better
understanding of the molecular mechanisms that govern how key
proteins regulate cytoskeleton organisation, dynamics and function,
which is of crucial importance to maintain cells in a healthy state. To
reach this goal, we study the proteins that control microtubule
dynamics that give localised functionality, and that organise
microtubules in space. We use biochemistry and cell biology
approaches, in combination with advanced fluorescence microscopy.
Local nanoscale conformational transitions at the
ends of growing microtubules
The dynamic properties of microtubules depend
on complex structural rearrangements in their end
regions. Members of the EB1 protein family
interact autonomously with microtubule end
regions and recruit several other proteins to
localise their activities to specific sites in the cell.
The autonomous end binding property of
fluorescently labelled EB1 proteins can also be
used to monitor nanoscale conformational
transitions in microtubule end regions by
fluorescence microscopy (Maurer et al., 2014; Curr
Biol. 24(4): 372-84). Such analysis revealed two
consecutive conformational transitions that freshly
microtubule-incorporated tubulins undergo as
microtubules grow. This shows how growing
microtubule ends mature at the nanoscale. Further
analysis revealed an important role of the detected
conformational states for the control of the
stability of microtubules. These results advance
our understanding of the molecular mechanism
that controls the fundamental characteristics of
microtubule dynamics.
A hierarchical protein interaction network
controls microtubule end tracking of dynein
Microtubule ends are hubs of protein activities,
because a variety of proteins accumulate there in
order to regulate microtubule dynamics and
facilitate interactions of microtubules with cellular
targets. Another function of end accumulation is
motor protein loading and transport initiation. The
major minus-end directed motor, dynein, is
recruited to microtubule ends by several proteins;
this is needed to initiate cargo transport along the
102
microtubule away from its end, typically towards
the cell centre or - in mitosis - to the spindle poles.
These dynein recruiting proteins form dynamic
interaction networks, whose behaviour depends
on a number of potentially competitive interaction
modes. The rules that determine which of the
various proteins are recruited to the limited
number of available binding sites at microtubule
ends remain elusive. In collaboration with the
laboratory of Michel Steinmetz, PSI Villigen,
Switzerland, we have examined how human dynein
is targeted to growing microtubule ends in the
presence of competing proteins (Duellberg et al.,
2014; Nature Cell Biol. 16(8): 804-11). Using in vitro
reconstitutions and fluorescence microscopy, we
found that a hierarchical recruitment mode is
needed to overcome competition. These
results highlight how the connectivity and
hierarchy within a localised protein interaction
network is orchestrated.
Motor-mediated microtubule organisation in
lipid-monolayered micro-compartments
The correct spatial organisation of microtubules is
important for the establishment and maintenance
of the internal architecture of eukaryotic cells.
Microtubules are arranged in space by a multitude
of biochemical activities and by spatial constraints
imposed by the cell boundary. The principles
governing the generation of distinct intracellular
architectures are only poorly understood. We have
studied the consequences of spatial confinement
on the self-organisation of purified
microtubules and motor proteins that were
encapsulated in lipid-monolayered droplets in oil,
using in vitro reconstitutions
Figure 1
Microtubule end tracking of a
dynactin and dynein.
A fluorescence microscopy image
sequence (left) and a kymograph
(space-time plot, middle) of a
dynamic microtubule (red) with
the dynactin component p150 Glued
(green) is shown. This is part of the
hierarchical protein interaction
network that recruits dynein to
growing microtubule plus-ends
(schematic, right). (Modified from
Duellberg et al., 2014).
(Baumann et al., 2014; J Biol Chem. 289(32):
22524-35). We varied the diameter of these
micro-containers from five to a hundred
micrometres, which covers the size range of typical
cell bodies. We found that container size had a
major organising influence. The presence of a
microtubule-crosslinking motor protein decreased
the number of accessible types of microtubule
organisations. Depending on the degree of spatial
confinement, the presence of the motor caused
either the formation of a cortical array of bent
microtubule bundles or the generation of single
microtubule asters inside the droplets. These are
two of the most prominent forms of microtubule
arrangements in plant and metazoan cells. These
results provide insight into how the combined
organising influence of spatial constraints and
crosslinking motor activities determines distinct
microtubule architectures in a minimal biomimetic
system.
Figure 2
Self-organisation of motors and
microtubules in microcompartments.
Microtubule-crosslinking motors
can organise microtubules either
into astral (top) or cortical
(bottom arrays) inside lipidmonolayered droplets.
Scale bars: 20 μm. (Modified from
Baumann et al., 2014).
103
TRANSLATIONAL CANCER THERAPEUTICS
www.london-research-institute.org.uk/research/charles-swanton
The Translational Cancer Therapeutics group focuses on mechanisms
generating cancer genetic diversity and its consequences on clinical
outcome. Our group and others, through next generation sequencing
studies have demonstrated that the principles of Darwinian evolution
apply to the growth and adaptation of human tumours (Gerlinger et
al., 2012; N Engl J Med. 366(10): 883-892, Gerlinger et al., 2014; Nat
Genet. 46(3): 225-233, Nowell PC, 1976; Science. 194(4260): 23-28).
Group Leader
Charles Swanton
Clinical Scientists
Crispin Hile
Mark Stares
Samra Turajilic
Nicolai Birkbak
Sebastijan Hobor
Laurent L’Epicier-Sansregret
Carlos Lopez-Garcia
Carolina Navas
Gareth Wilson
Panos Zalmas
Graduate Students
Andrew Crockford
Sally Dewhurst
Nicholas McGranahan
Tom Watkins
Tom Webber
Scientific Officers
Eva Gronroos
Andrew Rowan
The causes and consequences of cancer diversity
Our group has demonstrated that intratumour
heterogeneity, through tumour sampling bias,
impacts upon our ability to successfully qualify
cancer biomarkers for clinical use (Gulati et al.,
2014; Eur Urol. 66:936-48). We have also found
evidence for extensive parallel evolution in human
tumours, with multiple spatially separated
subclones acquiring distinct mutations in the same
gene, protein complex or signal transduction
pathway, suggesting profound constraints to
tumour evolution that might be exploited for
therapeutic benefit (Gerlinger et al., 2012;
Gerlinger et al., 2014) (Figure 1). Finally, we are
building on recent findings from our group that
DNA replication stress (Burrel et al., 2013; Nature.
494(7438): 492-496) and genome doubling events
(Figure 2) (Dewhurst et al., 2014; Cancer Discov.
4(2): 175-185) appear to contribute to
chromosomal instability and accelerate cancer
evolution in order to develop deeper insight into
how patterns of cancer diversity may be limited for
patient benefit (Burrell et al., 2013; Nature.
501(7467): 338-345).
Through the integration of our work with the UCL
Cancer Trials Centre and UCL Cancer Institute, we
are recruiting into the TRACERx study to decipher
evolutionary processes in non-small cell lung
cancer, and the DARWIN (Deciphering Anti Tumour
response With INtratumour heterogeneity) trial
program aimed at targeting clonally dominant
driver events and deciphering how branched
heterogeneous driver events contribute to drug
resistance and treatment failure (Yap et al., 2012;
Sci Transl Med. 4(127): 127ps10).
TRAcking Cancer Evolution through Therapy/Rx
(TRACERx) Clinical Study
Our group, together with the UCL Cancer Trials
Centre has initiated the TRACERx 842 patient
clinical study that aims to decipher tumour
evolutionary trajectories in early non-small cell
104
lung cancer. Through multi-region sequencing
analysis of primary tumours and recurrent
metastatic biopsies we will attempt to address the
origins of the lethal tumour subclone, distinguish
the changing patterns of tumour evolution over
time, the associations of intratumour
heterogeneity with disease outcome and the host
immune response, and the impact of cancer upon
the emergent subclonal genetic landscape at
relapse. Through computational approaches we
hope to identify epistatic relationships that dictate
constraints to tumour evolution that might be
exploitable for therapeutic benefit.
Tumour genomic instability mechanisms are
spatially and temporally heterogeneous
Our early data from the TRACERx study has
revealed that mutational processes shaping the
lung cancer genome are dynamic over both time
and space in early stage non-small cell lung cancer.
Firstly, our work has shown that even in early stage
lung cancer, multi-region sequencing reveals that
genetic driver events may be missed through the
analysis of a single biopsy. Secondly, we find
evidence for an illusion of clonality, where genetic
events within one biopsy may appear fully clonal,
that are subsequently revealed to be branched
heterogeneous events upon deeper genomic
analysis of spatially separated tumour regions.
Thirdly, we have found evidence that APOBEC
mutational processes appear to be enriched at
later time points following branched evolution of
lung adenocarcinomas, even in current smokers.
APOBEC mutational processes result in mutations
in key driver genes that appear to generate the
substrate of genetic diversity subject to further
evolutionary selection and subclade expansion.
Finally we have found early evidence suggesting
that lung cancer evolutionary histories are
prolonged with long latency periods between the
development of mutations in key driver genes and
clinical presentation, providing added support for
lung cancer screening strategies.
Figure 1
Multi-region exome sequencing
of a clear cell renal cancer
reveals evidence of parallel
evolution with distinct somatic
mutations affecting different
members of the SWI/SNF
complex in different regions of
the primary tumour (Gerlinger et
al., 2014).
Parallel evolution of cancer subclones
Our work in clear cell renal cell carcinoma of the
kidney has found evidence for parallel evolution of
tumour subclones (Figure 1). We find multiple
spatially distinct mutations in SETD2, KDM5C,
BAP1, PBRM1, PTEN and PIK3CA occurring within
distinct spatially separated sites of the same
tumour. We also find evidence that components of
the same protein complex, SWI/SNF, may be
subject to mutations in different regions of the
same tumour (Figure 1). Similarly, we find evidence
from an analysis of independent tumours within
the same patient, developing from a germline VHL
mutant background, converge upon activation of
the mTOR signal transduction pathway, suggesting
that despite the semblance of genomic chaos
within these tumours, there are major constraints
to the evolutionary histories of solid tumours
(Fisher et al., 2014; Genome Biol. 15(8): 433). Our
work continues to attempt to decipher such
evolutionary constraints in solid tumours through
longitudinal analysis of cancer genomes.
Figure 2
Chromosomal Instability Index for
diploid and genome doubled
colorectal cancer cell line clones
over time.
Weeks in culture is indicated in the
key. Over time diploid clones
remain chromosomally stable,
whereas genome doubled clones
have higher chromosomal
instability indices, which tend to
increase over time. Genome
doubled clones also have scores
higher than 0.2 (dotted line) a
threshold which in colorectal
cancer patients separates highly
chromosomal unstable anueploid
tumours from diploid
chromosomally stable tumours
(Dewhurst et al., 2014).
Identifying drivers of cancer diversity and cancer
evolution
We have recently found that cellular survival
following a genome doubling event in colorectal
cancer results in the tolerance and propagation of
chromosomal instability in subsequent daughter
cells. This results in accelerated cancer genomic
evolution with tetraploid cells developing extensive
chromosomal instability in contrast to their diploid
progenitors which remain genomically stable
throughout the 18 month time course of the
experiment (Figure 2) (Dewhurst et al., 2014).
Intriguingly, as well as becoming increasingly
chromosomal unstable, genome doubled cells also
drift towards triploid, similar to observations in
colorectal cancers in vivo. Consistent with this
observation, genome doubled/tetraploid early
stage colorectal cancers have a significantly worse
disease-free survival outcome than their diploid
counterparts in multivariate analysis. Our current
work is investigating the cellular mechanisms that
lead to tolerance of an aneuploid or tetraploid
genomic state.
Our work has recently implicated loss of
chromosome 18q in the initiation of chromosomal
instability through loss of three CIN suppressor
genes, MEX3C, ZNF516 and PIGN. Loss of each one
of these genes generates both structural and
numerical chromosomal instability and
intercellular heterogeneity, a substrate for cancer
evolution (Burrell et al., 2013). These data
implicate replication stress in the generation of
structural and numerical CIN and intratumour
heterogeneity, and provide evidence that
combining tumour bioinformatics approaches with
intricate functional genomics analysis can reveal
novel mechanisms contributing to intratumour
heterogeneity. The TCT group is building on the
experimental frameworks established through this
approach to identify mechanisms generating
genomic instability and addressing whether
replication stress is a common contributor to
chromosomal instability in other tumour types.
105
APOPTOSIS AND PROLIFERATION CONTROL
www.london-research-institute.org.uk/research/nicolas-tapon
Group Leader
Nicolas Tapon
Billel Benmimoun
Teresa Bertra
John Davis
Ieva Gailite
Graduate Students
Anna Ainslie
Jennifer Banerjee
Nicola Brindle
Yanxiang Zhou
Scientific Officers
Birgit Aerne
Maxine Holder
Masters Student
Annabel Ebbing
Our work is aimed at understanding how tissue size is specified during
development, which remains one of the most challenging questions
in biology. In order to achieve consistent organ and body size in
individuals of the same species, cell growth and cell number must be
tightly controlled, not only during development, but also to prevent
tumour formation during adult homeostasis. Genetic screens in
Drosophila have identified the Hippo (Hpo) pathway as a determinant
of tissue size control. The Hpo pathway restricts tissue and organ size
by both inhibiting cell proliferation and promoting apoptosis.
Subsequent studies in mammals have shown that this growth control
function is conserved and that Hpo signalling is dysregulated in many
types of cancer.
The core of the Hpo pathway is a kinase cascade
comprising the Ste20-related kinase Hpo and the
Dbf2-related kinase Warts (Wts). Upon Hpo
activation, the downstream kinase Wts
phosphorylates and inhibits the pro-growth
transcriptional co-activator Yorkie (Yki). Hpo
signalling has been proposed to sense various local
cues relating to cell density (contact inhibition of
growth and mechanical tissue properties),
patterning (morphogen gradients) or nutrition, and
translate these cues into a growth arrest signal
once an individual tissue has reached its
appropriate size.
Several lines of evidence suggest that Hpo pathway
activity is tightly coupled to epithelial architecture
(Genevet et al., 2011; Biochem J. 436(2): 213-224).
Firstly, Yki/YAP transcriptional activity has been
shown to depend on the structure of the actin
cytoskeleton, with F-actin promoting YAP nuclear
translocation, though the precise mechanisms and
the involvement of the core kinase cascade in this
process remain unclear. Secondly, the basolateral
polarity proteins Scribble and Lethal(2) giant larvae
(Lgl) have been shown to promote Hpo pathway
activity. Thirdly, the adherence junction protein
α-catenin has been proposed to function as a
membrane tether for YAP in keratinocytes. Finally,
the apical protein Crumbs (Crb) antagonises Yki/
YAP activity, both in Drosophila and mammals.
Crb is a transmembrane protein that contains
multiple EGF repeats in its large extracellular
domain. Crb is a key apical polarity determinant
that recruits other polarity proteins through its
short 37 amino acid intracellular domain. Beside its
106
well-documented role in polarity, Crb is also
required for normal growth control, since loss of
Crb function leads to tissue overgrowth. This has
been ascribed to a role in both Notch and Hpo
signalling. The function of Crb in Hpo signalling is
thought to involve the recruitment of the FERM
domain protein Expanded (Ex) to the apical
membrane. Indeed, the FERM domain of Ex can
bind the Crb FBM in vitro. Once apically localised,
Ex forms a complex with the scaffold proteins Kibra
and Merlin (Mer), which promotes inhibitory
phosphorylation of Yki by Wts. In addition, Ex is
thought to act as an apical tether for Yki by binding
the Yki WW domains through its Pro-Pro-X-Tyr (PY)
motifs.
In agreement with a proposed role for Crb as a
transmembrane receptor for the Hpo pathway,
loss of crb promotes expression of Yki target genes.
However, paradoxically, overexpression of the
intracellular domain of Crb (Crbintra) leads to strong
tissue overgrowth and Yki-target gene derepression. Although this could be due to a
dominant-negative effect, it is important to note
that Crbintra overexpression leads to loss of apical Ex
in developing wings and eyes, while co-expression
of Crbintra and Ex in cell culture leads to Ex
phosphorylation and reduced expression.
We recently reconciled these findings by showing
that Crb is not only required for Ex tethering at the
apical membrane but also to promote its
degradation via the SCFSlimb/β-TrCP E3 ubiquitin ligase
(Ribeiro et al., 2014; PNAS. 111(19): E1980-9).
Indeed, immediately downstream of its FERM
domain, Ex contains a sequence that conforms
Figure 1
Depletion of slmb using two
different dsRNAs leads to a
stabilisation of Ex in the presence
of Crb. Compare lane 1 with lanes
3 and 4. Expression of Ex with a
Crb form lacking the FERM
binding domain does not affect Ex
stability. Western blot from S2
cell lysates expressing the
indicated constructs.
to the D/S/TSGφXS consensus sequence for
canonical Slimb (Slmb) targets, which is conserved
in Ex orthologues from arthropod species but
absent from related FERM domain proteins such as
Moe and Mer. In addition, Slmb depletion prevents
Crbintra-induced Ex degradation in cell culture
(Figure 1), while loss of Slmb increases Ex levels in
vivo (Figure 2). Thus, in crb mutants, Ex no longer
reaches the apical membrane, and is protected
from degradation in the cytoplasm, where it
accumulates but is presumably unable to repress
Yki. When Crbintra is overexpressed, Ex turnover at
the membrane (or in an endocytic compartment if
Ex degradation occurs after Crb internalisation) is
accelerated, leading to its depletion and
consequent Yki activation. Therefore, in both
cases, the outcome is Yki de-repression, albeit for
different reasons.
Our work indicates that Crb fulfils a dual function in
Hpo signalling, both recruiting Ex apically to
repress Yki activity and promoting its turnover
through phosphorylation and Slmb-dependent
degradation. This mechanism would ensure
constant turnover of Ex at the apical membrane,
allowing Yki activity to rapidly respond to changing
environmental conditions. This dynamic
equilibrium could be particularly important to
promote fast tissue regeneration upon injury.
Figure 2
Elevated Ex and Ci levels in hsFLP/
FRT generated slmb mutant
clones in the Drosophila wing
imaginal disc. Confocal
micrographs of a wing imaginal
disc stained with anti-Ex (A) and
anti-Ci155 (B). The mutant tissue is
negative for GFP (C). Merged
image is shown in (D).
Scale bar = 10 μm.
107
EPITHELIAL BIOLOGY
www.london-research-institute.org.uk/research/barry-thompson
Group Leader
Barry Thompson
Mario Aguilar
Ahmed Elbediwy
Graduate Students
Graham Bell
Mariana Campos
Ichha Khanal
Zoe Vincent
Scientific Officer
Georgina Fletcher
Robert Ray
We are interested in the biology of epithelial cells, the cell-type of
origin for most human cancers. Epithelial cells form tissue layers and
tubes by connecting with neighbours via adherens junctions. In
tumours, epithelial tissue structure becomes disrupted, enabling
groups of cells to become invasive. We are particularly interested in
how the behaviour of epithelial cells is normally controlled, how they
become polarised, and how they know to divide within the plane of
the epithelium.
Recent work from a PhD student, Graham Bell, has
addressed how the mitotic spindle is oriented
within the plane of the epithelium. Normally,
spindles align with cell-cell junctions so that the
two daughter cells will remain within the
epithelium. Misorientation of the mitotic spindle is
thought to be one possible mechanism by which
epithelial cells can escape the epithelium to form
invasive tumours.
Graham has discovered a new role for the Lethal
Giant Larvae (Lgl) protein in spindle orientation in
the fruit fly Drosophila. Lgl is normally found at the
lateral and basal sides of the cell, where it overlaps
with a septate junction protein called Discs-Large
(Dlg). Graham observed that in mitosis, Lgl
Figure 1
Aurora kinases phosphorylate Lgl
to induce mitotic spindle
orientation in Drosophila.
108
relocalises to the cytoplasm. He showed that this
change is caused by activation of the Aurora A and
B kinases, which can directly phosphorylate the Lgl
protein to disrupt its association with the plasma
membrane. This loss of Lgl from the membrane
then allows other proteins, such as the mitotic
spindle orientation factor Pins (called LGN in mice
and GPSM in humans), to bind to Dlg and thus align
the mitotic spindle with septate junctions.
When Aurora-mediated phosphorylation of Lgl
was blocked by specific mutation of the phosphosites in Lgl, the entire process of mitotic spindle
orientation was completely disrupted. As a result,
spindles oriented randomly at mitosis instead of
adopting their normal orientation within the plane
Figure 2
Planar polarisation of the atypical
myosin Dachs around a clone of
cells expressing Fat. FbxL7 and
Dachs localise to opposite sides
of the cell.
of the epithelium. Since both Lgl and Dlg are
tumour suppressors in Drosophila, it could be that
misorientation of the mitotic spindle is a possible
tumour-initiating event. Graham is now exploring
this possibility in more detail.
Another PhD student in the lab, Mariana Campos,
has examined how the mitotic spindle, once
oriented in the plane of the epithelium, becomes
further oriented in a planar-polarised fashion.
Planar polarity is an asymmetry that appears at
cell-cell junctions and is a form of polarity that is
orthogonal to the normal apical-basal axis of
epithelial cells. Mariana has identified a novel
protein called FbxL7 that is essential to regulate
this process. She has shown that FbxL7 acts as a
ubiquitin ligase to control the planar polarisation
of the Dachsous-Fat-Dachs system in Drosophila.
The key effector of this system is the Dachs protein,
which encodes an atypical myosin that localises to
the distal side of cell-cell junctions. Mariana has
found that FbxL7 localises to the proximal side of
junctions, opposite to Dachs. In the absence of
FbxL7, Dachs accumulates abnormally all around
cell junctions, indicating that FbxL7 normally acts
to remove Dachs from the proximal side of the cell.
When FbxL7 is overexpressed, Dachs is degraded
everywhere. Either loss or gain-of-function of
FbxL7 is therefore associated with misorganisation
of spindle orientation and tissue shape. In addition,
tissue size is affected due to a role for Dachs in
regulating the Hippo signalling pathway. Thus,
FbxL7 is a novel regulator of planar polarity and
tissue growth.
109
CELL REGULATION
www.london-research-institute.org.uk/research/takashi-toda
Group Leader
Takashi Toda
Takayuki Koyano
Yuzy Matsuo
Akiko Nishi
Graduate Students
Aldona Chmielewska
Corinne Pinder
Ngang Heok Tang
Scientific Officer
Hirohisa Masuda
Masters Student
Agathe Morand
Visiting Scientist
Masashi Yukawa
Impeccable chromosome segregation during mitosis underlies
genome stability and integrity. Any errors in this process would result
in miscarriage, birth defects and/or aneuploidy, the hallmark of
human cancers. Segregating each sister chromatid towards opposite
poles is implemented by the mitotic spindle, a dynamic ensemble of
microtubules, microtubule-associated proteins (MAPs) and motor
proteins. Nucleation of microtubules in vivo does not occur
spontaneously; instead specialised structures called microtubule
organising centres (MTOCs) are required, in which the minus-end of
microtubules is embedded. In animal cells, the centrosome comprises
a major MTOC, whilst in fungi the spindle pole body (SPB) plays an
analogous role.
Our group has been uncovering the principles of
microtubule structure, function and regulation
using the genetically amenable model system,
fission yeast (Schizosaccharomyces pombe). More
recently we have been using zebrafish and human
culture cells to scrutinise the evolutionary
conservation of our findings obtained from work in
fission yeast. The long-term goal of our research is
simple: to understand the molecular mechanisms
of how the mitotic spindle ensures faithful
chromosome segregation. During 2014, we have
made seminal progress in the following two areas:
First, we have uncovered the conserved molecular
mechanism by which the minus end of
microtubules is tethered to the centrosome and
further identified the physiological defects when
this process is perturbed. Second, we have
determined the molecular pathway leading to the
recruitment of the conserved MAP complex, Alp7/
TACC-Alp14/TOG, to the SPB upon mitotic entry
and shown that this process is critical for mitotic
spindle assembly.
The conserved Msd1 protein family plays a
ubiquitous role in the anchoring of the
microtubule to the centrosome
The centrosome plays multi-layered roles in both
yeasts and vertebrates. These include the
canonical role in microtubule nucleation as the
MTOC, cell cycle transition as a structural hub
integrating cell cycle regulators and ciliogenesis as
the basal body. In addition, there is at least one
more critical role, i.e. anchoring of the microtubule
minus-end. This mechanism ensures that
interphase microtubule arrays emanate from the
centrosome and that structure and orientation of
110
mitotic spindles are safeguarded. However, our
knowledge of microtubule anchoring to this
organelle remains surprisingly limited at the
molecular level.
We previously showed that in fission yeast, a
mitosis-specific SPB component, mitotic spindle
disanchored 1 (Msd1), is required for anchoring the
minus-end of spindle microtubules to the SPB
(Toya et al., 2007; Nat Cell Biol. 9(6): 646-653).
Now, we have identified the human and zebrafish
Msd1 orthologues (Figure 1A) and characterised
their roles in microtubule anchoring. We have
unveiled that the human Msd1 (hMsd1) protein is
delivered to the centrosome in a centriolar
satellite- and dynein-dependent manner, wherein
it physically binds the microtubule-nucleating
γ-tubulin complex (Figure 1B). siRNA-mediated
hMsd1 knockdown results in disorganised
interphase microtubules due to the inability of the
microtubule to be tethered to the centrosome
(Figure 1C). During mitosis, mitotic spindles
become abnormally tilted and misoriented, which
is attributed to the release of astral microtubules
from the centrosome, leading to a faulty
interaction between microtubules and the cell
cortex (Figure 1C).
Moreover, we have found that in both humans and
zebrafish, the Msd1 orthologues are critical for
ciliogenesis. Remarkably, the loss of Msd1 leads
zebrafish embryos to left-right asymmetry defects
(Hori et al., 2014; EMBO Rep. 15(2): 175-184).
Collectively, the Msd1 proteins are the first
molecules to be identified as the conserved
microtubule-anchoring factors.
Figure 1
The Msd1 family comprises the
conserved microtubuleanchoring factors.
A. Phylogenetic dendrogram
showing the evolutionary
relationship among Msd1
orthologues. Msd1 orthologues in
human, mouse and Aspergillus
nidulans are also called, SSX2IP,
ADIP and TINA respectively.
B. A model of hMsd1-mediated
microtubule anchoring to the
centrosome. hMsd1 (red) is
transported to the centrosome
accompanied by centriolar
satellites (C.S., light blue) and the
dynein motor (grey) along
microtubules (thick pink lines).
Upon delivery to the
pericentriolar region, hMsd1
interacts with the γ-tubulin
complex (not shown), thereby
directly anchoring microtubule
minus-ends to the pericentriolar
material. Paired centrioles each
containing procentrioles are
shown in green.
C. Disorganised microtubule
morphologies upon hMsd1
depletion in U2OS cells.
Microtubule structures during
interphase (far-left panels) and
mitosis (second panels) in control
(top row) and hMsd1-depleted
cells (bottom row) are shown.
Schematic microtubule structures
showing spindle (mis)orientation
in relation to the substratum
(coverslip) are depicted on the
right-hand two panels (top and
side views). Note that spindle
microtubules are rotated
randomly upon hMsd1 depletion,
as astral microtubules become
unstable, by which they cannot
interact with the cell cortex to
maintain horizontal spindle
positioning. Scale bar = 5μm.
A
B
C
The conserved Alp7/TAC-Alp14/TOG microtubule
associated protein complex is targeted to the SPB
via the pericentrin-like molecule
The Transforming Acidic Coiled Coil (TACC) family
proteins were originally identified as a group of
proteins implicated in human cancers, and the
family is conserved throughout evolution. TACC
orthologues in various organisms localise to the
centrosome. These proteins form a stable complex
with another conserved TOG/Dis1/XMAP215 MAPs
in virtually all organisms examined, which is
Figure 2
Recruitment of the Alp7-Alp14
complex to the SPB via Pcp1 and
establishment of proper
kinetochore-microtubule
attachment.
The Alp7-Alp14 complex is loaded
on the SPB by directly binding to
pericentrin-like Pcp1 (top). The
SPB-localising complex is
essential for mitotic spindle
assembly. Upon recruitment to
the SPB, the Alp7-Alp14 complex
promotes spindle microtubule
polymerisation towards the
kinetochore, in which Alp7 binds
to the outer kinetochore
component Ndc80, thereby
establishing proper microtubulekinetochore attachment. Dis1 is
another TOG/XMAP215 protein
that also binds to Ndc80.
essential for spindle assembly and proper spindle
microtubule-kinetochore attachment. In human
cells, either downregulation or upregulation of the
TACC proteins is intimately linked to
tumourigenesis, and their expression profiles are
used for cancer prognosis. In fact, centrosome
abnormalities associated with defective spindle
assembly are one of the hallmarks of cancer. The
molecular understanding of the mechanisms
underlying centrosomal recruitment of the TACC
proteins, therefore, is of critical importance for the
aetiology of cancer and other human diseases.
However, at the moment, it remains elusive as of
which molecule(s) localising to the centrosome/
SPB is/are responsible for the recruitment of the
TACC-TOG complex, i.e. the receptor for TACC at
the centrosome.
In fission yeast, Alp7/TACC and Alp14/TOG also
form a stable complex, which is critical for mitotic
and meiotic spindle assembly and proper
chromosome segregation (Sato et al., 2007; Nature.
447(7142): 334-337; Kakui et al., 2013; Nat Cell Biol.
15(7): 786-796). Now we have determined five
amino acid residues clustered within the TACC
domain of Alp7 required for SPB localisation.
Critically, these sequences are essential for the
functions of Alp7, including proper spindle
formation and mitotic progression. Moreover, we
have identified pericentrin-like Pcp1 as a receptor
for Alp7 loading on the mitotic SPB (Figure 2).
The pericentrin family consists of the conserved
centrosomal/SPB component proteins and is
required for centrosome biogenesis and
maturation. However, it is not known whether
human pericentrin is involved in the recruitment of
TACC proteins. It would be of great interest to
explore whether human pericentrin is responsible
for TACCs recruitment to the centrosome, and if so,
what is the physiological consequence when this
interaction is disrupted.
111
SECRETORY PATHWAYS
www.london-research-institute.org.uk/research/sharon-tooze
Group Leader
Sharon A Tooze
Delphine Judith
Christopher Lamb
Maria New
Tim van Acker
Martina Wirth
Graduate Students
Hannah Dooley
Andrea Gubas
Justin Joachim
Scientific Officers
Harold Jefferies
Minoo Razi
Autophagy is a highly conserved, homeostatic membrane-mediated
pathway that delivers cytoplasmic components to the lysosome.
Once delivered to the lysosome the material is degraded and
recycled for re-use. Autophagy maintains cell health by targeting
damaged proteins and organelles for degradation, and restoring
amino acid pools during starvation through recycling by the
lysosome. Furthermore, autophagy plays an essential role in
infectious diseases, and in pathological conditions, such as
neurodegeneration and tumorigenesis. A further understanding of
the molecular mechanisms underlying the process is essential to
exploit the potential benefit of manipulating autophagy to treat
disease. Autophagy requires intracellular membrane compartments,
such as the endoplasmic reticulum, Golgi complex, endosomes and
lysosomes, alongside dedicated protein machinery, the autophagy
related (Atg) proteins. We study how Atg proteins, novel autophagy
regulators and trafficking proteins function during acute amino-acid
withdrawal to elucidate the molecular basis of autophagy.
Macroautophagy (here referred to as autophagy:
self-eating) is the non-selective engulfment and
removal of cytoplasmic proteins and organelles.
Autophagy occurs at basal levels in all eukaryotic
cells but is upregulated during amino acid
deprivation, which increases the flux through the
autophagosome to the degradative autolysosome.
From the degradative autolysosome the cytosolic
pool of amino acids, lipids and macromolecules can
be replenished. Formation of autophagosomes
occurs within minutes of amino acid deprivation in a
now well defined pathway initiated at the preautophagosomal structure (PAS) located at
specialised sites on the ER known as omegasomes,
from which the phagophore forms. The phagophore
then closes to form an autophagosome. The
formation of autophagosomes utilise 18 out of the
36 Atg proteins identified in yeast. The ULK complex,
the most upstream complex in the pathway, is
negatively regulated by the master growth and
energy sensors mTORC1 and AMPK. Inhibition of
mTORC1, and activation of AMPK triggers activation
of the ULK complex, and the class III
phosphatidylinositol 3-kinase (PI3K) complex and
their translocation to the phagophore. Downstream
effectors of the ULK and PI3K complex include WIPI
proteins that bind PI3-phosphate (PI3P), and the
ubiquitin-like conjugation systems, Atg12-Atg5-
112
Atg16 which produce LC3-PE (LC3-II). While the Atg
protein machinery is now known, many questions
remain unanswered about the regulation of
membrane dynamics, the source of the rapidly
expanding autophagosomal membrane, and the
interaction between the autophagosome and the
other subcellular compartments.
WIPI2 links PI3P to Atg12-5-16
Upon induction of autophagy, the class III PI3K
complex produces a specific pool of PI3P on the
omegasome. The autophagy-specific PI3P
effectors, the WIPI proteins, are then recruited to
the nascent phagophore. WIPI proteins are
seven-beta propeller domain proteins, which are
essential for autophagy, and through a
comparative analysis of WIPI1 and WIPI2 we
discovered WIPI2 functions to recruit the
ubiquitin-like conjugates Atg12-5-16 and
subsequently LC3-II. Hannah Dooley, in
collaboration with Michael Wilson at the
Babraham Institute, Cambridge UK, showed
WIPI2b directly binds Atg16L1 (Dooley et al., 2014;
Mol Cell. 55(2): 238-252). By mapping the
reciprocal binding site on each protein, WIPI2 and
Atg16L1 (Figure 1), we identified a single pair of
amino acids, which mediate binding. WIPI2b
binding to Atg16L1 is necessary and sufficient for
structures adjacent to endosomes and forming
phagophores lined up along the ER (Figure 2).
Figure 1
Structural model of the region of
Atg16L1 207-246 and residues
that interact with WIPI2b (E226
and E230) and FIP200 (E235-E239)
modelled ab initio using I-TASSER.
See Dooley et al., 2014 for details.
starvation-induced autophagy, ectopic LC3-II
lipidation and for Salmonella targeting to
autophagy.
Atg9, trafficking and Rab effectors
Phagophore and autophagosome formation both
depend on Atg9, a multi-spanning membrane
protein. We have shown that Atg9 traffics in small,
mobile vesicles between multiple organelles,
including recycling endosomes, late endosomes,
and the Golgi compartment. Atg9 is also found in a
unique conserved vesicular-vacuolar compartment
called the ‘Atg9 compartment’, which is adjacent to
forming phagophores. The control of Atg9
trafficking is likely to involve a complex set of
regulatory proteins and machinery, including ones
responsible for trafficking under normal growth
conditions as well as under nutrient-deprived
conditions. The composition of this compartment
is being studied by Delphine Judith who, in
collaboration with Bram Snijders and the Protein
Analysis and Proteomics facility at LRI, is analysing
the vesicular pathway taken by Atg9 in mammalian
cells at a proteomic level.
Figure 2
A panel of images from the
cryo-CLXM analysis of HEK293
cells expressing mRFP-Atg9 and
GFP-LC3. Left, cryo-fluorscence,
middle reconstructed tomoX
stack, and right, tomogram of the
Atg9-compartment (red arrows)
adjacent to endosomes (yellow)
and GFP-LC3-positive forming
phagophores (green arrows),
which are seen on ER membranes
(blue). White/black arrows
indicate gold particles used for
orientation. See Duke et al., 2014
for details.
One of the strengths of our work on autophagy is
the morphological techniques we use to place our
biochemical work in a cellular context. In
collaboration with Lucy Collinson, head of the
Electron Microscopy facility at LRI, and Elizabeth
Duke (Diamond, Oxford), Minoo Razi has studied
the morphology of the Atg9 compartment after
using cryo-soft X-ray and correlative light
microscopy (Duke et al., 2014; Ultramicroscopy.
143: 77-87). Under these near-native imaging
conditions, Atg9 was seen in distinct vacuolar
While recent data supports the role of the ER in the
formation of the phagophore, it is equally clear
that Golgi and other compartments may provide
additional membranes. To understand the
contributions of other membranes, we focused on
Rab proteins, GTPases that mediate vesicular
fusion, and RabGAPs, GTPase activating proteins
that inactivate Rabs, called TBC proteins. TBC1D14,
a Rab11 effector, robustly co-localises with ULK1
and is present on Golgi and recycling endosomes
(Longatti et al., 2012; J. Cell Biol. 197(5): 659-675).
Chris Lamb is exploring how TBC1D14 coordinates
recycling endosome traffic to autophagosomes
and the machinery, including the Rabs, which link
TBC1D14 to autophagosome formation.
Novel regulators of autophagy
Our genome-wide siRNA screen under amino acid
starvation identified several putative candidates
that our group continues to investigate. SCOC, a
short coiled-coil protein, is a Golgi-localised
protein that interacts with ULK1 and UVRAG,
dependent on FEZ1 (McKnight et al., 2012; EMBO J.
31(8): 1931-1946). Given that ULK1 and UVRAG (a
subunit of the endosomal PI3K complex) act
sequentially in autophagy, we are testing the
hypothesis that SCOC may regulate the progression
of the autophagosome membrane and maturation.
Martina Wirth in collaboration with Stéphane
Mouilleron (Structural Biology facility, LRI) are
using a structure-function approach to understand
the function of SCOC. Martina is complementing
these studies by developing zebrafish models to
study SCOC and other autophagy regulators. WAC,
a WW domain-containing adaptor with coiled-coil
protein, is another autophagy regulator identified
in our genome-wide siRNA screen. WAC is found in
both a nuclear and cytoplasmic pool, and Justin
Joachim is dissecting the function of WAC in these
two compartments, in particular the interaction of
WAC with the Golgi complex.
113
SIGNALLING AND TRANSCRIPTION
www.london-research-institute.org.uk/research/richard-treisman
Extracellular stimuli such as growth factors and mitogens act through
signal transduction processes to induce alterations in cell proliferation,
differentiation and mechanical activities including motility and
adhesion. Our group focuses on analysis of the serum response factor
(SRF) transcription factor network, which controls transcription of
many genes involved in these processes.
Group Leader
Richard Treisman
Associate Scientist
Patrick Costello
Laura Collard
Jessica Diring
Cyril Esnault
Charles Foster
Anastasia Mylona
Graduate Students
Sofie Eriksson
Francesco Gualdrini
Magdalena Kratochvilova
Richard Panayiotou
Cynthia Yu-Wai-Man
Scientific Officers
Diane Maurice De Coulon
Mathew Sargent
SRF activity is regulated by two families of signalregulated cofactors. The myocardin-related
transcription factors (MRTFs) sense changes in
cellular G-actin concentration associated with
altered Rho GTPase activity. We study both the
MRTFs and other proteins subject to this novel
form of regulation. In contrast, the ternary
complex factors (TCFs) are classical ERK
phosphorylation substrates downstream of Ras.
Both Rho and Ras signalling are implicated in
transformation, invasion and metastasis, but the
role of the SRF network in these processes remains
to be elucidated.
Rho-actin signalling and the MRTF pathway
A central focus of current work is the mechanism
by which Rho GTPase signalling controls cell
behaviour. The MRTFs are novel G-actin binding
proteins, binding G-actin through an N-terminal
regulatory domain containing three copies of the
RPEL G-actin binding motif. Three other families of
RPEL proteins have been identified: the Phactr
proteins bind the PP1 catalytic subunit and
represent a novel family of putative PP1 regulators;
in addition, our recent studies have identified two
protein families of cytoskeletal regulators. While
G-actin-controlled nucleocytoplasmic shuttling
represents a major mode of regulation for MRTF-A,
MRTF-B, and Phactr1, our data shows that G-actin
also regulates the MRTFs at other levels, and other
regulatory mechanisms must clearly operate on
non-shuttling RPEL proteins.
Our previous work with the MRTFs identified a
nuclear import signal with the RPEL domain whose
ability to recruit importin a-b heterodimers is
antagonised by G-actin binding. A similar
mechanism operates in the case of the Phactr1
protein, which also shuttles to the nucleus in
response to Rho GTPase activation. Structural
studies of the N-terminal Phactr1 NLS-RPEL motif
by Stéphane Mouilleron (Protein Structure, LRI)
show that actin binding physically occludes
sequences required for membrane association of
Phactr3 and Phactr4, so Magdalena Kratochvilova
114
is studying whether G-actin also affects this
interaction. Magdalena has demonstrated that the
Phactr1-PP1 complex does not contain other
cofactors, and with Stéphane, has shown that
Phactr1 binding does not occlude the PP1 substrate
binding site. Magdalena is now using proteomic and
affinity-labelling approaches to identify potential
Phactr1-PP1 substrates.
The Phactr1-PP1 and MRTF-importin interaction
studies demonstrate a common mechanism for
G-actin regulation by direct competition for
overlapping binding sites. Jessica Diring has now
shown that a related mechanism operates in the
case of the RPEL cytoskeletal regulatory proteins.
These contain a catalytic domain adjacent to a
single RPEL motif, and form a stable 1:1 complex
with actin. Catalytic activity is inhibited in the
complex, and Jessica finds that mutations that
prevent actin binding relieve this inhibition.
Structural studies conducted with Stéphane show
that in the complex, G-actin makes additional
specific contacts with the catalytic domain, and
Jessica has shown that ablation of these contacts
also relieves inhibition.
Richard Panayiotou has continued his investigation
of MRTF nuclear export, which is dependent on the
Crm1 exportin and the integrity of the N-terminal
RPEL motifs. Richard has mapped a Crm1 binding
site in the MRTF N-terminal sequences,
demonstrating that its activity is potentiated by a
nearby phosphorylation event, and showing that it
can confer regulated export on the Phactr1 protein,
whose export is normally independent of Crm1.
With data from others, these observations suggest
that regulated MRTF localisation requires
cooperation between multiple independent Crm1
binding sequences, together with G-actindependent occlusion of the importin binding site.
Cyril Esnault and Francesco Gualdrini have
continued studies on the regulation of MRTFs by
G-actin. Francesco has continued the analysis of
MRTF regulation by nuclear actin using genomic
approaches to show that targeting of MRTFs to the
Figure 1
Haematopoietic stem/progenitor
cells (HSC/Ps) lacking SRF exhibit
defective adhesive and
polarisation responses to SDF-1.
HSC/P cells (CFSE: green) were
plated on monolayers of
endothelial cells (SNARF: red)
above a source of SDF-1 and
imaged after 45 minutes. Cells
lacking SRF failed to flatten and
move into the endothelial
monolayer toward the source
of SDF-1.
nucleus in the absence of G-actin depletion is
insufficient to induce transcription. This appears to
reflect the operation of two novel MRTF regulatory
pathways controlled by G-actin through the
N-terminal regulatory domain. One pathway acts
to control MRTF-SRF interaction, and Francesco is
using biochemical approaches to investigate the
MRTF sequences involved, complementing
Anastasia Mylona’s structural work on the MRTFSRF interaction. The other pathway appears to
regulate RNA Polymerase II, and genomic data
suggest that it prevents productive transcription by
disruption of CTD phosphorylation.
A central question is how these regulatory
phenomena relate to the natural physiology of
MRTF-SRF regulation. MRTFs are nuclear under
resting conditions in certain cell types or
environments. Cynthia Yu-Wai Man is investigating
human Tenon’s fibroblasts, where MRTFs are
nuclear under resting conditions, while Charlie
Foster is looking at mouse carcinoma-associated
fibroblasts, both of which appear to be
‘myofibroblast’-like. Given that Cyril Esnault’s
genomic analysis of the SRF network identified a
strong link between adhesion signalling and MRTF
activation, Cynthia and Charlie are investigating the
relation between MRTF nuclear accumulation and
matrix stiffness, and in related work, Laura Collard
is pursuing the relationship between adhesion,
steady state G-actin levels and MRTF activation.
Patrick Costello and Mathew Sargent have
completed our analysis of the role of the SRF
network in murine haematopoiesis. Patrick
previously found that hematopoietic stem cells fail
to colonise the foetal bone marrow, although other
tissues remain normal. With Mathew, he extended
this to show that this
reflects uncoupling of SRF
from MRTF activity, and
that MRTF-A and MRTF-B
function redundantly in
this system. With Cyril
Esnault, Patrick
performed a
transcriptome analysis,
demonstrating that as in
the fibroblast system,
SRF-dependent
transcripts are highly
enriched in those
encoding cytoskeletal
structural genes and
regulators. Curiously,
however, many of these
genes are specific to the stem cells, showing that
cell context is an important determinant of
specificity for the SRF-MRTF system.
Regulatory properties of the TCF-SRF network
Although SRF was first identified in studies of the
fibroblast response to serum mitogens over two
decades ago, its role in this response has remained
unclear. Patrick Costello and Diane Maurice found
that TCF-SRF signalling is important for both acute
TCR-stimulated and homeostatic proliferation of
CD4 T cells. They are using RNAseq to characterise
the transcriptional responses involved. Diane has
now also characterised the role of SAP-1 and SRF in
T cell differentiation and the response to infectious
challenge. Diane found that SRF is required for the
initial proliferative expansion of short-lived
effector cells and simultaneously suppresses early
memory precursor differentiation.
The TCFs are phosphorylated at multiple sites.
Anastasia Mylona has shown that these exhibit
variable phosphorylation kinetics, and with Charlie
Foster showed these act both positively and
negatively on mediator recruitment. Cyril Esnault
and Francesco Gualdrini are investigating the
relationship between ERK signalling, TCF
phosphorylation, chromatin modifications and
transcription. Using phorbol esters to activate ERK
signalling at model TCF-dependent genes, they
found that unphosphorylated Elk-1 is sufficient to
induce certain chromatin modifications, while
others require phosphorylation but not
transcription, and others reflect an RNA
polymerase II recruitment. They are now seeking
to extend these studies genome-wide.
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CHROMOSOME SEGREGATION
www.london-research-institute.org.uk/research/frank-uhlmann
Group Leader
Frank Uhlmann
Yasutaka Kakui
Thomas Kuilman
Lidia Lopez Serra
Yasuto Murayama
Catarina Samora
Graduate Students
Molly Godfrey
Meghna Kataria
Ainhoa Mariezcurrena
Rahul Thadani
Scientific Officers
Celine Bouchoux
Maria Ocampo-Hafalla
Aneuploidy, i.e. missing or supernumerary chromosomes, is a
hallmark of malignant tumour progression. A large number of genes
that orchestrate faithful chromosome segregation during mitotic cell
divisions are tumour suppressors or turn into potent oncogenes if
misregulated. The aim of the Chromosome Segregation group is to
investigate the function of these genes and the cellular mechanisms
that safeguard accurate chromosome segregation. In particular, we
are investigating the contribution of structural chromosomal proteins
to sister chromatid cohesion and chromosome condensation,
processes that ensure faithful segregation of centimetre-long
chromosomal DNA molecules within micrometre-sized cells.
We also investigate how the kinases and phosphatases of the cell
division cycle machinery bring about ordered completion of
chromosome segregation.
Chromosome cohesion and tumourigenesis
The cohesin complex is a central player in
chromosome biology. Defects in cohesin and its
regulators are responsible for chromosome
missegregation in many human malignancies. They
are also the cause for Cornelia de Lange syndrome,
a severe developmental disorder.
The genomic DNA that makes up the chromosomes
is replicated during S phase of the eukaryotic cell
cycle. After replication, the two newly synthesised
sister chromatids remain connected with each
other by the chromosomal cohesin complex. Our
group has contributed over the years to
understand how this ring-shaped multi-subunit
protein complex works to build sister chromatid
cohesion. Sister chromatid cohesion forms the
basis for the pairwise alignment of DNA replication
products on the spindle apparatus in mitosis, to
allow their faithful segregation into daughter cells.
Cells defective in sister chromatid cohesion make
errors in chromosome segregation, giving rise to
aneuploid cells that lack or contain extra copies of
chromosomes. Aneuploidy is a hallmark of
malignant tumour progression. Human heritable
mutations that compromise the fidelity of
chromosome segregation are inevitably linked to
early onset tumourigenesis. This year, we have
made progress towards understanding how
cohesin works as a fascinating molecular machine
that holds sister chromatids together.
116
Biochemical reconstitution of topological DNA
binding by the cohesin ring
The cohesin complex consists of at least four
subunits that together form a large proteinaceous
ring. It is thought that cohesin holds together sister
chromatids by topologically embracing them.
While the embrace model provides an important
conceptual framework for sister chromatid
cohesion, it leaves many fundamental question
wide open. If cohesin topologically embraces DNA,
how does the DNA get into the ring and where on
chromosomes can this reaction happen? Equally,
how does DNA come out of the ring again during
cohesin’s dynamic DNA binding cycle? Finally,
cohesin incorporates an ABC-type ATPase that is
required for its function, so how does the ATPase
fuel cohesin’s activities? Definitive answers to
these questions require that we can study
cohesin’s behaviour in vitro, however, the
biochemical reconstitution of cohesin activity has
remained an unattained goal.
We were now successful in expressing and
purifying the fission yeast cohesin complex, as well
as its Mis4/Ssl3 cohesin loading factor, which is
essential for cohesin function in vivo (Figure 1).
Incubation of cohesin with DNA led to spontaneous
topological loading of cohesin onto DNA, in an ATP
hydrolysis-dependent fashion, but this reaction
remained inefficient. Addition of the cohesin
loader stimulated ATP hydrolysis and cohesin
Figure 1
Biochemical reconstitution of
topological cohesin loading onto
DNA.
A. Purification of fission yeast
cohesin and the cohesin loader
complex after overexpression of
their respective subunits. The
final gel filtration steps of the
purification are shown. Fractions
were analysed by SDS
polyacrylamide electrophoresis
followed by Coomassie blue
staining.
B. Schematic of the cohesin
loading reaction.
C. A circular DNA substrate is
required for the loading reaction,
hinting at the topological nature
of cohesin binding. The input and
bead-bound fractions following
the loading reaction are shown
and were quantified. The mean
and standard deviation of
three independent experiments
are shown.
B
A
C
loading onto DNA. We found that the cohesin
loader contacts cohesin at multiple sites around
the ring circumference. One of these contacts lays
on cohesin’s Psc3 subunit, an essential yet hitherto
enigmatic part of the cohesin complex. Using
mutational analysis and peptide competition
experiments, we showed that at least three loader
contacts along the cohesin ring coordinatedly
stimulate the cohesin loading reaction. Considering
the large dimensions of the cohesin ring, it is likely
that a conformational rearrangement must take
place to accommodate three simultaneous
contacts of the loader with cohesin. The cohesin
loader can thus be thought of as a template or
mould onto which cohesin holds onto to facilitate
the loading reaction.
Our in vitro reconstitution of cohesin loading onto
DNA provides mechanistic insight into the initial
steps of the establishment of sister chromatid
cohesion and other chromosomal processes
mediated by cohesin. The results are important not
only to understand cohesin, but also the ubiquitous
family of chromosomal structural maintenance of
chromosomes (SMC) complexes, of which cohesin
is a member. SMC complexes share essential
functions in various chromosomal activities in all
organisms from bacteria to humans.
Outlook
Now that we have gained the ability to investigate
cohesin behaviour in vitro, we would like to directly
observe cohesin’s loading onto DNA. We will use a
combination of biochemical, structural, single
molecule and imaging approaches to do this. In
particular, single molecule FRET-based assays and
electron microscopy have the potential to shed
unprecedented insight into both the cohesin
loading reaction as well as the final product of the
reaction, the cohesin ring on DNA. Once cohesin is
loaded onto DNA, the probably most exciting time
during its residence on chromosomes comes
during DNA replication in S phase, when the sister
chromatid is synthesised and cohesin will ensure to
hold the two together. We will extend our
biochemical assays to address how cohesin
identifies the two replication products and
establishes linkages between them.
117
CANCER GENOMICS
www.london-research-institute.org.uk/research/peter-van-loo
Group Leader
Peter Van Loo
Graduate Student
Stefan Dentro
The advent and exponential cost decrease of massively parallel
sequencing technologies over the past years has enabled sequencing
entire cancer genomes. This resulted in unique opportunities for
cancer research. Large-scale consortia (The Cancer Genome Atlas
(TCGA) and the International Cancer Genome Consortium (ICGC))
have now produced whole genome sequences of thousands of
cancer genomes, and are making their data available to the
community. I argue that we have so far only skimmed the surface of
what can be learned from this unprecedented wealth of data.
Therefore, there is a clear need for in-depth large-scale pan-cancer
analyses. Our group, which started in October 2014, focuses on
integrative analyses of large-scale public ‘omics’ data, leveraging the
wealth of cancer genomics data into large-scale pan-cancer analyses
to understand carcinogenesis and cancer evolution.
Characterising the landscape of tumour
suppressor genes
Many cancer genes are somatically altered in only
a very low proportion of tumours, providing a clear
rationale for large-scale pan-cancer analyses of
driver mutations. We are applying approaches
centred on copy number analysis to characterise
the landscape of tumour suppressors. Tumour
suppressors can be inactivated by a combination of
a deleterious germline variant, combined with
somatic loss-of-heterozygosity (LOH) of the other
allele. We are performing a large-scale pan-cancer
screen for this combination of events aiming to
identify new tumour suppressors.
Many tumour suppressors are targeted by
homozygous deletions, removing both parental
copies. Because any homozygous deletion that
includes a gene that confers a survival advantage is
eliminated by negative selection, homozygous
deletions are rare and often focal. Admixture of
normal cells in tumour samples has historically
hindered the reliable identification of homozygous
deletions. We previously developed ASCAT
(Allele-Specific Copy number Analysis of Tumours,
Van Loo et al., 2010; Proc Natl Acad Sci U S A.
107(39): 16910-16915), a method to derive copy
number profiles of tumour cells accounting for
normal cell admixture and tumour aneuploidy.
Methods such as ASCAT can now effectively
deconvolute copy number profiles of tumour cells
from those of admixed normal cells and reliably
identify homozygous deletions in tumour samples.
118
We are applying ASCAT to thousands of samples
across cancer types, to screen for tumour
suppressors through recurrent homozygous
deletions. For a subset of these cases, point
mutation data, gene expression data and/or DNA
methylation data will also be available, which we
will correlate with detected homozygous (and
hemizygous) deletions, allowing us to more clearly
delineate target genes within regions of
homozygous deletions. Through this screen, we
aim to characterise the landscape of tumour
suppressors and, particularly, identify rare tumour
suppressors.
Molecular archaeology of cancer: inferring
timelines of cancer development and evolution
The cancer genome contains within it an
archaeological record about its past, and we
previously pioneered methods to disentangle a
cancer’s life story from sequencing data (Nik-Zainal
et al., 2012; Cell 149(5): 994-1007) (Figure 1). We
anticipate that a large-scale pan-cancer approach
to obtain detailed evolutionary histories of
tumours would give profound insights into
carcinogenesis and cancer evolution.
We can construct life histories of thousands of
tumours from their genome sequences, using both
driver and passenger mutations. By obtaining
detailed timelines of many cancers’ evolutionary
histories that include driver mutations, copy
number changes, rearrangements and mutational
processes, we aim to identify the initiating events
transcriptome is confounded by expression signals
originating from admixed normal cells. Gene
expression analysis by massively parallel
sequencing (RNAseq) allows allele-specific
expression measurements. It can be shown that
the expression in tumour cells can be separated
from that in normal cells, given the fraction of
tumour cells, the allele-specific copy number
profiles of tumour cells, and applying a few
reasonable hypotheses (Figure 2). We aim to
develop such bioinformatics approaches to
deconvolute the tumour cell transcriptomes from
transcriptomes of admixed normal cells.
Figure 1
Molecular archaeology of cancer:
an example.
From the picture on the right, one
can infer that the purple
mutations happened first, then
the blue chromosome duplicated,
and then the yellow mutations
occurred. In addition, from the
relative numbers of yellow and
purple mutations, one can infer
when in the tumour’s lifetime the
blue chromosome duplicated.
of cancer development, and the events that are
selected for later in a cancer’s lifetime, including
those that drive late clonal expansions and that
may play a role in tumour malignancy. In addition,
these analyses will allow blueprints of the
subclonal architecture across cancer types in
unprecedented detail and on an unprecedented
number of cases, allowing a glimpse into a
tumour’s future.
Complementary to this, we are performing
smaller-scale collaborative studies of tumour bulk
sequencing, in combination with single-cell and
multi-sample sequencing of primary tumours,
metastases and circulating and disseminated
tumour cells, aiming to gain insight into tumour
evolution and metastasis.
Deconvoluting expression in tumour and
normal cells
We aim to understand how changes to the genome
lead to transcriptomic changes to eventually cause
cancer. Deep understanding of the cancer
Figure 2
Principle of a method to
deconvolute the tumour cell
transcriptomes from
transcriptomes of admixed
normal cells, using copy number
data and (allele-specific)
expression from RNAseq.
We will apply these methods to large pan-cancer
RNAseq datasets, allowing a transcriptome-wide
view of cancer across cancer types. We expect
these tumour cell-specific expression profiles will
result in a better taxonomy of cancer than mixed
cell population expression profiles. Finally,
expression profiles of admixed normal cells will
allow insight into the cellular composition and
transcriptional state of the tumour stroma.
In the longer term, we aim to develop integrative
genomics-transcriptomics approaches that study
the influence of point mutations, copy number
changes and structural variants on transcription at
the gene or transcript level and at the
transcriptome level, and to apply these
approaches in a large-scale pan-cancer setting to
understand the basic principles of cancer
development and cancer evolution within and
across tumour types.
Copy number profile
Expression in
tumour samples
Samples
Genes
Samples
Genes
Genes
Samples
Inferred
expression in
tumour cells
Inferred
expression in
normal cells
119
CELL FATE AND GENE REGULATION
www.london-research-institute.org.uk/research/folkert-van-werven
Group Leader
Folkert van Werven
Postdoctoral Scientist
Fabien Moretto
Graduate Student
Minghao Chia
Scientific Officer
Gianpiero Spedale
Masters Student
Natalia Robert
Figure 1
Schematic overview of how the
mating type signal controls entry
into gametogenesis.
The aim of our research group is to elucidate the molecular
mechanisms by which the cell integrates multiple signals to achieve a
binary cell fate decision – whether or not to differentiate. Unfolding
these mechanisms is critical for the understanding of how cell
specialisation leads to multi-cellularity during development, and how
impaired signalling can cause abnormal development and diseases
such as cancer. The budding yeast S. cerevisiae is an ideal model
system to study this problem. In response to a combination of
extracellular and intracellular cues budding yeast undergoes a highly
conserved cell differentiation programme called gametogenesis.
Since entry into gametogenesis is controlled by only two master
regulators in this model organism, there is the unique opportunity to
study the molecular and quantitative aspects of this cell fate.
Transcription of two long noncoding RNAs
controls the cell fate decision leading to
gametogenesis
Expression of the master regulatory genes, IME1
and IME4, drive the cell fate decision leading to
gametogenesis in budding yeast. This cell fate is
also controlled by the mating-type locus. In order
to initiate gametogenesis, diploid yeast cells need
to express both mating type genes, MATa and
MATα. The combined gene product of MATα and
MATa, the a1-α2 heterodimer, inhibits RME1
transcription in diploid cells (Figure 1A), but how in
A
B
120
haploid cells Rme1 represses IME1 transcription
was never understood. We discovered that in cells
with the haploid mating-type, expression of IME1
is inhibited by a long noncoding RNA (lncRNA)
called IRT1 that is located in the IME1 promoter
and induced by the Rme1 transcription factor
(Figure 1B). Transcription of this lncRNA recruits
the Set2 histone methyltransferase and Set3
histone deacetylase complex to establish
repressive chromatin in the IME1 promoter
(Figure 1B).
Figure 2
A. Overview of the signals
controlling the IME1 promoter.
B. Annotation of two lncRNAs in
the IME1 promoter.
C. Model describing the
mechanism by which the lncRNA
IRT1 represses IME1.
A
B
C
The chromatin remodelling enzymes Set2 and Set3
are highly conserved from yeast to human, and
lncRNAs are also abundant in higher eukaryotes.
This raises the interesting possibility that lncRNAs
repress gene expression by a conserved
mechanism. In addition to IME1, expression of the
master regulator IME4 is antagonised by an
antisense transcript in haploid cells (Figure 1B).
This antisense transcript in turn is repressed by
a1-α2 allowing expression of IME4 in diploid cells
(Figure 1B). The mating-type dependence of IME4
expression led to a second key discovery from this
study. When the expression of both the lncRNA in
the IME1 promoter and the IME4 antisense
transcript is inhibited, cells expressing the haploid
mating-type enter gametogenesis with kinetics
that are indistinguishable from cells expressing
both mating-types (Figure 1). Thus, transcription of
two lncRNAs governs mating-type control of
gametogenesis in yeast.
Current Research directions
The promoter of the master transcription factor for
entry into gametogenesis, IME1, integrates
nutrient and mating-type signals to make the
binary cell fate decision (Figure 2A). Whole
genome transcriptome analysis identified two
lncRNAs in the IME1 promoter (Figure 2B). In cells
of the haploid mating-type, IME1 is repressed by
the lncRNA IRT1 (Figure 2C). Induction of IRT1 by
the transcription factor Rme1 recruits the
methyltransferases Set1 and Set2 to methylate
histone H3 at lysine 4 and 36. These marks are
recognised by the histone deacetylase complexes
Set3C and Rpd3(S) and establish a repressive
chromatin state in the IME1 promoter (Figure 2C).
The mechanism of cell fate control described here
provides a starting point for further investigations.
The goals are: Firstly, screen for novel factors
required for gene repression by lncRNAs and,
secondly, investigate how widespread gene
repression by lncRNAs is across the genome.
How do master regulatory genes integrate
multiple signals?
Multiple signals are required to initiate
gametogenesis in budding yeast: nutrients such as
nitrogen compounds and glucose need to be
absent from the medium, cells need to respire (use
a non-fermentable carbon source) and must be
diploid. These signals drive entry into
gametogenesis and all converge on the IME1
promoter (Figure 2A). This promoter is one of the
largest and highly regulated promoters in budding
yeast. To understand how these signals are
integrated to make a binary cell fate decision, it is
essential to identify all the molecular players and
pathways involved in acting at the IME1 promoter.
The lab will use proteomic and genetic approaches
to identify these factors.
Perspective
The aims outlined here form the beginning of a
systematic investigation on how multiple signals
are integrated to drive cell fate decisions. It is
often not well understood how, in higher
eukaryotes, master regulatory genes make binary
decisions that are important for development of
an organism. Aberrant expression of these master
genes due to impaired signalling can cause
abnormal development and diseases such as
cancer. The IME1 promoter serves as a basic model
system for studying signal integration at master
regulatory genes. The findings from these studies
could shed light on how complex promoters are
regulated in higher eukaryotes.
121
CELL MOTILITY
www.london-research-institute.org.uk/research/michael-way
Group Leader
Michael Way
Jasmine Abella
David Barry
Joseph Cockburn
Flavia Leite
Caitlin Tolbert
Graduate Students
Chiara Galloni
Julia Pfanzelter
Xenia Snetkov
Scientific Officers
Theresa Higgins
Antonio Postigo
Cell adhesion and motility play a critical role during the development
and throughout the lifetime of multi-cellular organisms.
Unfortunately, deregulation of these two fundamental cellular
processes frequently occurs during pathological situations such as
tumour cell metastasis. Our research focuses on understanding how
signalling networks and the cytoskeleton regulate cytoplasmic
transport, as well as cell adhesion and migration. We use a
combination of quantitative imaging and biochemical approaches to
study vaccinia virus as a model system to interrogate the regulation
and function of Src and Rho GTPase signalling, actin and
microtubule-based transport as well as cell migration. Outside the
context of vaccinia infection, we also investigate the cellular function
of Tes, a tumour suppressor that negatively regulates Menadependent cell migration, as well as the mechanisms regulating the
assembly and function of invadopodia.
Ena/VASP proteins regulate cell migration by
promoting actin polymerisation at the plasma
membrane by antagonising actin filament capping
and acting as processive actin polymerases. The
intracellular targeting of Ena/VASP proteins is
mediated through the interaction of their
N-terminal EVH1 domain with ‘FPPPP’ sequence
motifs found in a variety of cytoskeletal proteins
including lamellipodin and zyxin. Of all the proteins
interacting with the EVH1 domain of Ena/VASP
proteins, Tes, a tumour suppressor and focal
adhesion protein, stands out as the only one
known to lack an ‘FPPPP’ motif. Tes interacts with
Mena via its C-terminal LIM3 domain and is also
unique in being the only protein shown to bind a
single Ena/VASP family member. Furthermore, Tes
inhibits Mena-dependent cell migration by
negatively regulating the localisation of Mena at
focal adhesions and the leading edge.
Given the interaction of Tes with Mena, we sought
to identify additional Ena/VASP EVH1 binding
partners lacking ‘FPPPP’ motifs. Using a
combination of biochemical approaches and mass
spectrometry, we found the EVH1 domain
interacts directly with Abi in the WAVE regulatory
complex (WRC), even though it lacks an ‘FPPPP’
motif. The WRC plays an essential role in
promoting Arp2/3 complex-dependent actin
polymerisation in response to Rac signalling. Using
a Far-western approach, we found that the EVH1
domain interacts with residues 352-394 in the
122
proline rich region of human Abi1. With this
information, we were able to generate an Abi
mutant (termed AbiΔEVH1) that incorporates into
the WAVE complex but cannot interact with Ena/
VASP proteins. When expressed in cells, GFPtagged AbiΔEVH1 was recruited to plasma
membrane but was less effective than Abi at
promoting cell migration.
Given this phenotype, we collaborated with
Michael Rosen (Howard Hughes Medical Institute
and Department of Biophysics, UT Southwestern
Medical Center, Dallas, USA) to investigate
whether VASP modulates the ability of the WRC to
promote Arp2/3-dependent actin polymerisation
in vitro. We found that VASP increases the extent
of actin polymerisation induced by Rac1-activated
WRC. Importantly, this stimulation, which is over
and above the intrinsic actin filament elongation
activity of VASP, depends on the proline rich region
of Abi. In contrast to the tetrameric full-length
protein, monomeric VASP or its isolated EVH1
domain does not activate the WRC to stimulate
Arp2/3-mediated actin polymerisation even at
high concentrations. Loss of the VASP F-actin
binding motif, which is essential for its actin
filament elongation activity, also completely
abolishes the ability of VASP to enhance actin
assembly by the WRC. It is possible that the
simultaneous engagement of a VASP tetramer with
Abi and the ‘LPPPP’ motif in WAVE increases the
activity of the WRC. However, oligomerisation
interaction in Drosophila, we collaborated with
Sven Bogdan (Institute of Neurobiology, University
of Münster, Germany). Drosophila larval
macrophages adhering to a substrate have a
polarised actin cytoskeleton with a broad
lamellipodial cell front (Figure 1). In contrast, abi
mutant macrophages have a ‘spiky’ morphology.
Re-expression of dAbi but not dAbi∆Ena rescues
this defect in cell morphology. Live cell imaging
also reveals that Abi∆Ena macrophages have a
reduced rate of membrane protrusion compared
to dAbi macrophages. Furthermore, Ena is no
longer at the leading edge of lamellipodial
protrusions but becomes re-localised to the
tips of filopodia-like protrusions when it cannot
bind dAbi.
Figure 1
Maximum intensity projection
SIM image of the actin
cytoskeleton (black) in
Drosophila hemocytes
alone cannot account for our observations, since
mutating the F-actin binding motif of VASP
abrogates activity. While not definitive, our data
are most consistent with a model in which VASP
binds Rac-activated WRC with high affinity based
on tetramerisation-mediated avidity as well as
actin filaments, thus increasing the association of
the WRC with filaments.
To facilitate analysis of the physiological role of the
Ena/VASP – WRC interaction, we switched to
Drosophila, as it only has a single isoform for each
protein. Drosophila Abi (dAbi) and the EVH1
domain of Ena have 38 and 72 % sequence identity
to their respective human counterparts.
Nevertheless, the EVH1 domain of Ena still binds
dAbi. Using a Far-western approach, we found that
Ena interacts with two ‘LPPPP’ motifs in dAbi.
Mutation of both ‘LPPPP’ motifs does not affect the
ability of dAbi (dAbi∆Ena) to incorporate into the
WRC or co-localise with WAVE at the plasma
membrane of Drosophila S2 cells. To investigate the
consequences of the loss of the dAbi-Ena
In vivo, dAbi and a functional WRC are required in
the Drosophila larval visual system for the correct
axonal targeting of photoreceptor neurons (R-cells)
to their respective optic ganglions in the fly brain.
We found that the loss of the ability of dAbi to
interact with Ena resulted in a similar defect in
R-cell targeting as the complete loss of dAbi. Ena
and the WRC are also essential for normal
Drosophila egg development, while the loss of abi
in the germline results in small and abnormally
shaped eggs. Re-expression of wild type Abi
rescues the egg morphology defects and female
sterility of abi mutant flies. In contrast, abi mutant
flies expressing dAbi∆Ena are still completely
sterile, containing smaller and abnormally round
eggs. Late stage Abi∆Ena mutant egg chambers
also have defects in nurse cell cortical actin
integrity resulting in detached cytoplasmic actin
bundles and ring canals. In summary, our in vitro
analysis has now demonstrated that Ena/VASP
proteins directly impact on the activity of the
WAVE complex, while our observations in
Drosophila have revealed that the function and
activity of Ena/VASP proteins and the WAVE
complex are intimately linked.
123
Sub-nanometre resolution
structure of a replicative helicase.
Image: Alessandro Costa,
Architecture and Dynamics of
Macromolecular Machines Group.
124
CLARE
HALL
The London Research Institute Clare Hall Laboratories are located
North of London. The main focus of the research for the laboratories
housed on the Clare Hall campus is genome integrity; including DNA
repair, recombination and replication, cell cycle control and
transcription. The researchers are supported by an excellent range
of Technology Core Facilities.
CLARE HALL
125
DNA DAMAGE RESPONSE
www.london-research-institute.org.uk/research/simon-boulton
Group Leader
Simon Boulton
Carrie Adelman
Sara Garcia-Gomez
Ana Maria Leon Ortiz
Pol Margalef
Paulina Marzac
Kenichiro Matsuzaki
Stephanie Panier
Grzegorz Sarek
Julien Stingele
Jennifer Svendsen
Jean-Baptiste Vannier
Graduate Students
Rafal Lolo
Martin Taylor
Scientific Officers
Valerie Borel-Vannier
Zuzana Licenikova-Horejsi
Julie Martin
Mark Petalcorin
Tohru Takaki
DNA is highly susceptible to damage and must be repaired correctly
to prevent genome instability. Failure to correctly repair DNA damage
is the underlying cause of a number of hereditary cancer
predisposition syndromes such as Fanconi Anemia and Blooms. The
long-term aim of my group is to understand how DNA double-strand
break (DSB) repair pathways, such as non-homologous end joining
(NHEJ) and homologous recombination (HR), are regulated in mitotic
cells and during meiosis. We also have an active interest in
understanding how these pathways impact on human diseases such
as cancer.
Phosphorylation-dependent PIH1D1 interactions
define substrate specificity of the R2TP
co-chaperone complex
Molecular chaperones facilitate the folding and
unfolding of polypeptides and are essential for the
assembly of large protein complexes. The human
R2TP complex consists of four subunits: RUVBL1,
RUVBL2, PIH1D1 and RPAP3 and is essential for the
assembly of a number of multi-subunit molecular
machines including small nucleolar
ribonucleoproteins (snoRNPs), spliceosomal
snRNP U4, RNA polymerase II and mTORC1 and
SMG1 complexes. However, the molecular basis of
substrate recognition by the R2TP complex
was unclear.
Our previous study revealed that CK2
phosphorylation of the co-chaperone TEL2 is
essential for direct binding to PIH1D1 and its
disruption leads to destabilisation of mTOR and
SMG1 and to a lesser extent ATM, ATR and
DNA-PKcs. As PIH1D1 is not predicted to contain
any of the known phospho-binding domains such
as 14-3-3, FHA, BRCT, WD40, WW and Polo-box
domains, it was unclear how it recognises
phosphorylated TEL2. Furthermore, whether
phosphorylation-dependent binding represents a
universal substrate recognition mechanism for the
R2TP complex had not previously been explored.
In recent work in collaboration with the group of
Steve Smerdon at Mill Hill (NIMR), we demonstrate
that the N-terminal PIH1D1 region PIH-N is a novel
phospho-binding domain required for recognition
of phosphorylated substrates, while the C-terminal
region of PIH1D1 binds to the other components of
the R2TP complex. The crystal structure of PIH-N
domain fragment bound to the phosphorylated
TEL2 peptide revealed a highly specific phosphopeptide recognition mechanism in which Lys 57
126
and 64 in PIH1D1, together with a conserved
DpSDD phospho-motif within TEL2, are
essential and sufficient for binding. Proteomic
analysis of PIH1D1 interactors identified R2TP
complex substrates that are recruited by the PIH-N
domain in a sequence-specific and
phosphorylation-dependent manner suggestive of
a common mechanism of substrate recognition.
We proposed that protein complexes assembled
by the R2TP complex are defined by
phosphorylation of a specific motif and
recognition by the PIH1D1 subunit.
RTEL1
RTEL1 is a helicase that was originally identified by
mapping of loci that control telomere length
differences between M. musculus and M. spretus.
RTEL1 plays a critical role in genome stability as
knockout mice are embryonic lethal and cells
derived from these mice exhibit telomere fragility
and loss. We previously identified RTEL1 as a key
regulator of homologous recombination (HR) in a
genetic screen for anti-recombinases and
biochemical studies revealed that human RTEL1
promotes the disassembly of D loop recombination
intermediates in vitro. Based on our original study
of RTEL1 we speculated that its role at telomeres
might reflect a need to regulate HR. Indeed, we
previously established that RTEL1 performs two
distinct functions at vertebrate telomeres in
promoting t-loop unwinding (requiring its D-loop
disrupting activity) and counteracting the
formation of telomeric G4-DNA structures to
facilitate telomere replication (Vannier et al., 2012;
Cell 149(4): 795-806). We proceeded to show that
binding of RTEL1 to proliferating cell nuclear
antigen (PCNA) is critical for unwinding telomeric
G4-DNA structure but was found to be dispensable
for t-loop disassembly (Vannier et al., 2013; Science
342(6155): 239-242). While it was known that
RTEL1 transiently localises to telomeres during the
cell cycle, the mechanism by which RTEL1 is
recruited to telomeres to promote t-loop
disassembly is totally unclear.
In our most recent study, we make the unexpected
discovery that a single telomere binding protein,
TRF2, is responsible for both the assembly and
S-phase specific disassembly of the t-loop, which is
necessary to prevent catastrophic t-loop resolution
by the SLX1/4 nuclease complex. We demonstrate
that the ability of TRF2 to coordinate t-loop
disassembly strictly depends on its ability to bind
and recruit RTEL1 to telomeres in S-phase. We
show that the TRF2-RTEL1 interaction requires a
previously uncharacterised metal-coordinating
C4C4 motif in RTEL1. Genetic studies in mouse cells
revealed that the C4C4 mutant is a classic
separation of function mutation, which uncouples
the two distinct functions for RTEL1 at telomeres in
t-loop disassembly and suppression of telomere
fragility: the C4C4 domain (mediates the RTEL1-
TRF2 interaction) is required for t-loop unwinding
but is dispensable for suppressing telomere
fragility. Thus, the PIP-box and C4C4 motifs in
RTEL1 convey distinct and separable functions,
which impact on the targeting of RTEL1 to
replication forks and telomeres, respectively. The
clinical importance of our work was highlighted by
the fact that the TRF2-RTEL1 interaction is
abolished by mutations in RTEL1 that are causal for
Hoyeraal-Hreidarsson syndrome (HHS), a severe
form of Dyskeratosis congenita. Specifically, the
RTEL1 pR1264H mutation is causal for HHS and has
a carrier frequency of 1% within the Ashkenazi
Orthodox Jewish population and 0.45% in the
general Ashkenazi Jewish population. We
established that the pR1264H mutation, which
resides within the C4C4, specifically disrupts the
TRF2-RTEL1 interaction. These findings have major
implications for the understanding of telomere
function, genome instability and human disease.
Figure 1
PIH1D1 and its phospho-binding
PIH domain mediated substrate
recognition for assembly by the
R2TP chaperon complex.
Clare Hall
127
CHROMATIN STRUCTURE AND MOBILE DNA
www.london-research-institute.org.uk/research/peter-cherepanov
Group Leader
Peter Cherepanov
Jun He
Paul Lesbats
Daniel Maskell
Graduate Student
Samual Dick
Scientific Officers
Nicola Cook
Valerie Pye
Our research focuses on the structural biology of chromatin function
and its interactions with the retroviral DNA integration machinery.
Using X-ray crystallography and complementary approaches we aim
to elucidate three-dimensional structures and mechanisms of
biological machineries involved in regulation of gene expression, DNA
replication and retroviral integration. This year we made substantial
headway in several of our main projects, including the crystallography
of human Cdc7 kinase and of the archaeal Orc1-DNA complex, which
are described below. We have also made progress towards
understanding how the retroviral integration machinery interfaces
with chromatin.
Structure of the essential S-phase kinase Cdc7
Eukaryotic chromosomal DNA replication is
initiated at multiple origins at the onset of and
throughout S phase. The replisome assembly and
initiation of DNA synthesis at individual origins
critically depend on activities of S phase cyclindependent kinases and Cdc7. Both types of kinases
are regulated by their respective activating
subunits (cyclins and Dbf4, respectively).
Phosphorylation of MCM2-7 by Cdc7 allows
recruitment of essential replication initiation
factors en route to the replisome assembly. Cdc7 is
overexpressed in many cancers and tumour cell
lines. Due to its pivotal role in cell proliferation,
this S phase kinase is emerging as a target for the
development of cancer therapeutics.
Two years ago we reported the first crystal
structure of Cdc7, which revealed how Dbf4
activates the kinase (Hughes et al., 2012; Nat Struct
Mol Biol. 19(11): 1101-1107). However, the
crystallised Cdc7-Dbf4 construct was only partially
active, and the initial structure did not explain how
the kinase recognises its target substrates. The
observed drop in activity could be explained by
mutations that had to be introduced into the
kinase construct in order to obtain well-ordered
crystals. Looking closely at the amino acid
sequence of Cdc7, in particular at the regions that
were affected by the mutations in our initial
structure, we identified a cluster of highly
conserved Cys residues abutting of the activation
loop of Cdc7. The presence of this region was
required to restore activity of our recombinant
kinase preparations. Following extensive
screening, we were able to crystallise a fully active
heterodimeric Cdc7-Dbf4 construct and refined the
128
structure to a resolution of 1.4 Å. The structure
revealed a novel zinc-binding domain within the
kinase insert 2 that pins the beginning of the
activation loop to the motif-M of Dbf4 and the
C-lobe of Cdc7 (Figure 1A). Importantly, these
interactions lead to complete ordering of the
activation loop, which is consistent with the
recovery of the kinase activity. The breakthrough
enabled us for the first time to crystallise the
kinase bound to a substrate peptide. The co-crystal
structure containing an MCM2-derived peptide,
refined to a resolution of 1.7 Å, explained the
known specificity of the kinase for Ser or Thr
residues followed by a negatively-charged residue
(Glu, Asp, or phospho-Ser). The new structure also
revealed an intriguing possibility that Cdc7 may be
programmed to recognise sites primed by
phosphorylation with CDK2, whose target site
consensus includes an Arg or a Lys residue at the
P+3 position. We are now focusing on the
mechanisms of regulation of Cdc7 activity
throughout the cell cycle. Our results will aid in the
development of more potent and specific
inhibitors of Cdc7 kinase.
Structure of an archaeal Orc1 in the
ATP-bound state
The molecular machinery involved in the initiation
of DNA replication in archaea shares many
common features with that of eukaryotes.
Archaeal chromosomes replicate from multiple
origins, which are marked by binding of conserved
Orc/Cdc6 AAA+ proteins that load the hexameric
MCM DNA helicase. The archaeal Orc/Cdc6
proteins display extremely tight nucleotide
binding, and are invariably isolated in their
ADP-bound states. Their active ATP-bound forms
Figure 1
A. Crystal structure of Cdc7 kinase
with fully ordered activation loop.
The activation loop and the
zinc-binding domain found within
the kinase insert 2 (KI2) are shown
in orange. The rest of Cdc7 and
Dbf4 (motifs C and M) are shown
in green and blue, respectively.
B. Crystal structure of Sulfolobus
islandicus Orc1-1 bound to an ATP
mimetic and cognate DNA (ORB).
The winged helix domain (WHD)
and the initiator specific motif
(ISM, magenta) are indicated.
Weighted 2Fo-Fc electron
density map for the bound
nucleotide and the associated
metal atom is contoured at 2σ
(blue chicken wire).
A
B
are thought to be short-lived, likely contributing to
the temporal control of initiation of DNA
replication by restricting it to a very narrow time
window. Following a single round of MCM loading
and ATP hydrolysis, the initiator would remain in an
inactive state until the next round of cell division,
to be replaced by the de novo synthetised ATPbound form. The structural rearrangements
within Orc/Cdc6 proteins associated with ATP
hydrolysis are therefore of particular interest. Our
goals in this project are to characterise
structural rearrangements in archaeal Orc1
associated with ATP hydrolysis and to determine
the structural basis for MCM loading by archaeal
Orc/Cdc6 proteins.
We were able to optimise production of the apo
form of Sulfolobus islandicus Orc1-1, which allowed
us to crystallise and determine the structure of this
protein bound to an ATP mimetic and its cognate
DNA element. The structure has been refined to a
resolution of 2.7 Å, and the nucleotide is defined
very well in the electron density map (Figure 1B).
Intriguingly, the overall conformation of the
protein in our structure is substantially different
from those in previously reported ADP-bound
forms. We are now investigating the functional
consequences of the conformational
rearrangements revealed by our crystal structure.
Clare Hall
129
ARCHITECTURE AND DYNAMICS OF
MACROMOLECULAR MACHINES
www.london-research-institute.org.uk/research/alessandro-costa
Group Leader
Alessandro Costa
Panchali Goswami
Jin Chuan Zhou
Graduate Students
Ferdos Abid Ali
Paolo Swuec
Scientific Officers
Adelina Davies
Julian Gannon
Ludovic Renault
Errors in the mechanisms that maintain gene copy number give rise
to genomic instability, which is a hallmark of cancer cells. Our
research aims to understand how the biological nanomachines
involved in chromosome replication function to maintain genome
integrity. To this end, we employ single-particle cryo-electron
microscopy and biochemistry to study how DNA molecules are
duplicated and how this process is coordinated with DNA repair and
other nucleic acid transactions. By describing the architecture and
dynamics of the DNA replication machinery and its interactors we
seek to establish a molecular framework to explain how eukaryotic
cells respond to DNA damage and how cell proliferation is regulated
to avoid tumourigenesis.
DNA replication – Structure of the translocating
eukaryotic replicative helicase
The Cdc45/Mcm2-7/GINS (CMG) helicase unwinds
the DNA double helix during replication in
eukaryotes. How the CMG is assembled and how it
engages DNA substrates remains only partially
understood. Using negative-stain electron
microscopy, we have determined the structure of
the CMG (Figure 1) in the presence of a slowly
hydrolysable ATP analogue and a DNA duplex
substrate with a 3’ single-stranded tail. The
structure shows that the Mcm motor subunits of
the CMG bind single- and not double-stranded
DNA, supporting a steric exclusion mechanism for
replication fork unwinding. We used biotinstreptavidin labelling to establish the polarity by
which DNA enters into the Mcm2-7 channel, and
elucidate how Cdc45 keeps the helicase
topologically linked to the translocation strand
during DNA unwinding. The Mcm2-7 motor
Figure 1
Architecture of the Cdc45/
Mcm2-7/GINS (CMG) complex.
Adapted from Costa et al., 2014;
eLife 3: e03273.
130
subcomplex forms a right-handed spiral when
DNA-bound, revealing unexpected similarities
between the CMG and other hexameric ATPases
such as the bacterial DnaB helicase and the Rpt1-6
AAA+ motor of the eukaryotic proteasome. We
identified a subpopulation of dimeric CMGs, which
allowed us to establish the subunit register of
Mcm2-7 double hexamers assembled onto DNA
before replication origin firing. Altogether, our
results provide novel important insights into the
nucleoprotein architecture of the replication fork.
We are now interested in describing the detailed
molecular mechanism of DNA translocation by the
activated Mcm2-7 AAA+ motor. To this end, we
have optimised preparations of substrate-bound
CMG molecules embedded in vitreous ice for
high-resolution cryo-electron microscopy.
By imaging our nucleoprotein preparations using a
direct electron detector on a 300 kV electron
These findings establish the architectural
framework for further mechanistic studies of the
elongation step of DNA replication in eukaryotic
cells (Figure 3).
Figure 2
The Ctf4 helicase-polymerase link
can bind up to three client
proteins concomitantly. Adapted
from Simon et al., 2014; Nature.
510(7504): 293-97.
We are currently working on reconstituting a larger
protein assembly, where Ctf4 is linked to the CMG
helicase as well as the Pol α/primase holo-enzyme.
Describing the structure and function of a full
helicase-polymerase super-complex will help us
elucidate the mechanism of coupling DNA
unwinding and synthesis in the eukaryotic
replisome.
microscopy instrument, we aim at determining the
near-atomic resolution structure of the
translocating CMG helicase in various stages of the
ATP hydrolysis cycle. Our results will inform us on
the mechanism of ATPase cycling and nucleic acid
translocation by a hetero-hexameric motor.
DNA replication – Helicase/polymerase coupling
Genome duplication requires tight coordination
between parental duplex-DNA unwinding and
daughter-strand synthesis within the replication
machinery, to prevent the accumulation of
vulnerable single-stranded DNA segments and the
onset of genomic instability. We recently employed
single-particle electron microscopy coupled with
biochemistry and crystallography (in collaboration
with Luca Pellegrini at the University of
Cambridge), to describe the architecture of the Ctf4
‘helicase-polymerase bridging factor’, either alone
or bound to components of the CMG helicase and
the DNA Polymerase α/primase assemblies (Figure
2). We showed that budding yeast Ctf4 forms a
homo-trimeric disk, suggesting that it has the
ability to link multiple factors at replication forks.
Indeed, the Ctf4 trimer contains three docking sites
that recognise a conserved motif mapping within
the Pol α catalytic subunit as well as one of the four
GINS subunits of the CMG helicase. Importantly, we
showed that the Ctf4 trimer is capable of
simultaneously binding to GINS and the amino
terminus of the Pol α catalytic subunit.
Figure 3
Speculative representation of
the helicase-primosome
super-assembly in the eukaryotic
replisome.
Homologous Recombination
Double-strand breaks can be repaired by
homologous recombination in eukaryotic cells. At
the end of this process Holliday Junctions, covalent
linkages between donor sequences, can be
resolved by the action of a nuclease. This event
results in the exchange of genetic information
between two DNA segments, which can lead to the
rise of deleterious mutations. To prevent this,
eukaryotes have developed a strategy to dissolve
homologous recombination intermediates back to
their pre-recombination state (DNA-crossover
suppression). The key player in this process is the
four-member dissolvasome complex, comprising
Topoisomerase IIIα, the RMI1/2 factors and the
BLM helicase, whose mutation is linked to genetic
disease and cancer development (reviewed in
Swuec and Costa, 2014; Cell Biosc. 4: 36). While the
mechanism of Holliday Junction dissolution is still
unclear, a wealth of information is available on
various orthologs of the dissolvasome assembly.
For example, Topoisomerase IIIα belongs to the
type-IA class of topoisomerases, pad-lock shaped
enzymes that effect changes in DNA topology
through a ‘strand-passage’ mechanism. RMI1 and
RMI2, OB-fold containing factors, have been
implicated in nucleic acid engagement or in
protein-protein interactions. BLM contains a
RecQ-type DNA helicase domain, whose ATPase
function is required for DNA opening and
translocation. Many issues remain unresolved and
a mechanistic understanding of Holliday Junction
dissolution is still lacking. Using an integrated
approach that combines biochemistry,
crystallography and electron microscopy we are
working to determine the architecture of the
dissolvasome complex, to elucidate the molecular
basis of double Holliday junction dissolution.
Clare Hall
131
CHROMOSOME REPLICATION
www.london-research-institute.org.uk/research/john-diffley
Group Leader
John FX Diffley
Corella Casas-Delucchi
Gideon Coster
Max Douglas
Belén Gómez-González
Stephanie Hills
Kerstin Kinkelin
Christoph Kurat
Joseph Yeeles
Mona Yekezare
Graduate Students
Tom Deegan
Mohammed Raihaan Hassan
Jake Hill
Agnieszka Janska
Scientific Officers
Dominik Boos
Lucy Drury
Anne Early
Jordi Frigola
Khalid Siddiqui
We each synthesise roughly 5x1011 metres of DNA every day – more
than the distance from the earth to the sun and back. Despite this
scale, and the large number of tumour suppressor genes and
potential oncogenes in our genomes, 2/3 of the UK population will
live cancer-free lives. Thus, DNA replication and the quality control
mechanisms associated with it are remarkably efficient. Once cells
become cancerous, however, genome instability becomes the norm,
which can drive intratumour heterogeneity and tumour evolution.
Oncogenes induce replicative stress, but we still do not understand
the nature of this stress. Our goal is to understand how DNA
replication initiates and how it is controlled, which will help us
understand how high fidelity DNA replication is ensured in normal
cells. This is of fundamental importance for understanding cell
proliferation, and is also a prerequisite to understanding and
exploiting the subversion of this process in cancer. This year we made
significant progress in understanding some of the key biochemical
reactions that underpin this process.
Roles for ATP hydrolysis in origin licensing
DNA replication in eukaryotes initiates from
multiple chromosomal locations termed origins,
and the stability of the genome is dependent
upon each origin firing once and only once per
cell cycle. This is achieved by the temporal
separation of replication initiation into two
distinct steps. The first step, origin licensing,
involves the loading of the hexameric MCM
helicase comprising the six related Mcm2-7
subunits into pre-replicative complexes (pre-RCs).
This occurs during late mitosis and G1 phase. The
second step, origin firing, involves the conversion
of the inactive MCM double hexamer into two
functional replisomes during S phase.
Origin licensing occurs in an ordered fashion.
First, the Origin Recognition Complex (ORC) binds to
origin DNA. Origin binding requires ORC binding to
(but not hydrolising) ATP. Cdc6 is then recruited to
form an ORC/Cdc6 complex on origin DNA, which
requires ATP binding by Cdc6. The budding yeast
MCM forms a complex with the Cdt1 protein and
this MCM/Cdt1 complex is recruited to ORC/Cdc6
via an essential C-terminal domain in Mcm3. The
loading of MCM into salt-resistant double hexamers
bound around double stranded DNA requires ATP
and is not supported by the ATP analogue ATPγS,
indicating a requirement for ATP hydrolysis. Once
Figure 1
Roles of ATP binding and
hydrolysis in MCM loading.
Details are described in the text.
ATP
C
132
MCM is loaded, it no longer requires ORC, Cdc6 or
Cdt1 to maintain its origin association.
Using purified yeast proteins, we systematically
analysed the role of ATP binding and hydrolysis in
origin licensing. We found that ORC and Cdc6
mutants defective in ATP hydrolysis are competent
for origin licensing, even when combined. This has
allowed us to uncover a novel role for ATP
hydrolysis by Cdc6 in release of non-productive
loading intermediates. Cdc6 mutants that cannot
hydrolyse ATP are lethal in vivo, suggesting that this
‘proofreading’ activity is an essential function of
Cdc6. Surprisingly, we found that ATP binding and
hydrolysis by MCM subunits play distinct and
essential roles during pre-RC assembly: ATP binding
is required for stability of the MCM complex under
licensing reaction conditions and ATP hydrolysis is
required for MCM loading and Cdt1 release. These
results are summarised in Figure 1.
The MCM double hexamer is a precursor for DNA
replication
Upon entry into S phase, increase in the activities
of cyclin dependent kinase (CDK) and Dbf4
dependent kinase (DDK) promotes the activation of
the inactive MCM double hexamers. These kinases,
together with other firing factors, including
Sld2,3,7, Dpb11, GINS, Mcm10 and DNA
polymerase ε, convert each inactive MCM double
hexamer into two Cdc45/MCM/GINS (CMG)
Figure 2
Bidirectional DNA replication in
vitro.
Replication reactions were
sequentially labelled with BrdU
and biotin-dUTP. Reactions were
stopped and spread on
microscope coverslips by
molecular combing. BrdU (red)
was detected with anti-BrdU;
biotin (green) was detected with
fluorescently labelled
streptavidin. Several
representative images are shown.
complexes each containing a hexamer of MCM. To
accomplish this, the double hexamer must
separate, origin DNA must melt, the MCM ring
must open, the lagging strand template must be
extruded and the MCM ring, along with Cdc45 and
GINS must close around the leading strand
template. Some firing factors, including Dpb11 and
the key CDK substrates Sld2 and Sld3, are required
for initiation but not elongation, whilst others, like
Cdc45, GINS and the leading strand DNA
polymerase ε, are required for initiation and then
form part of the elongation machinery.
We have developed a soluble, cell-free DNA
replication system to study the initiation of DNA
replication from MCM double hexamers loaded
with purified proteins. In this system, a subset of
firing factors (Dpb11, Sld2, Sld3, Sld7 and Cdc45)
was overexpressed from galactose-inducible
promoters. These cells, which harbour a cdc7
temperature-sensitive mutation, were first
synchronised in G1 phase with α-factor and then
released from α-factor at 37°C, inducing arrest of
cells in an S phase-like state. MCM double
hexamers loaded onto plasmids with purified
proteins were then incubated in this extract with
labelled nucleotide and products examined by gel
electrophoresis. DNA replication of plasmids in
these extracts is semiconservative, requires
pre-assembly of pre-RCs and results in the
formation of fully replicated, covalently closed
circular product. Moreover, as shown in Figure 2,
replication is bidirectional. Consequently, factors
required for elongation and termination do not
appear to be lacking from this extract. Using a
proteomic approach, all three replicative
polymerases as well as virtually all of the other
identified components of the previously described
replisome progression complex were found
specifically associated with template DNA in a
DDK-dependent manner suggesting that our
extracts assemble replisomes similar to those
generated in vivo. Surprisingly few proteins other
than known replisome components and firing
factors were found associated in a DDK-dependent
manner, suggesting that most or all core factors
involved in initiation and elongation have been
identified. If true, it should be possible to
reconstitute this process with this set of purified
proteins in the future.
Clare Hall
133
MAMMALIAN DNA REPAIR
www.london-research-institute.org.uk/research/peter-karran
Group Leader
Peter Karran
Postdoctoral Scientist
Elizabeth McAdam
Graduate Students
Melisa Guven
Matt Peacock
Scientific Officers
Reto Brem
Peter MacPherson
Figure 1
Protein oxidation and NER
inhibition by a fluoroquinolone
and UVA.
A. Protein carbonylation.
CCRF-CEM cells were treated
with ciprofloxacin for 1h at the
concentrations shown. After
washing, they were irradiated
with UVA (20 kJ/m2). Cell extracts
were treated with AlexaFluor 647
Maleimide to derivatise
carbonyls. Proteins were
separated by PAGE. Green
fluorescence is from derivatised
protein carbonyls. Red
fluorescence is Sypro Ruby stain
for total protein.
B. NER inhibition.
Extracts prepared from cells
treated with ciprofloxacin and
UVA (20 kJ/m2) as indicated were
incubated with circular DNA
substrates containing a single
site-specific NER substrate
lesion. NER activity is monitored
by the formation of excision
products (27-31 nt
oligonucleotides) that are
radiolabelled and separated by
PAGE.
134
Therapy-related cancer is a significant clinical problem. The
approximately 100-fold increased skin cancer incidence in
immunosuppressed organ transplant patients is an extreme example
of cancer risk associated with medication. Sun exposure is a cofactor
in this risk. Previous work in the Mammalian DNA Repair Laboratory
has linked the photochemical properties of azathioprine, one of the
most widely-prescribed immunosuppressants, to skin cancer.
Azathioprine causes skin photosensitivity.
Photosensitivity is a common side-effect of drug
treatment and is often associated with a known, or
suspected, increased risk of skin cancer. Based on
our extensive studies of the photochemical effects
of azathioprine, the group is investigating the
mechanisms by which other drugs cause
photosensitivity and whether this might be related
to an increased skin cancer risk. These studies have
led us to examine how different wavelengths of
solar radiation might contribute to the
development of skin cancer.
Photosensitisation and DNA repair
Work from the group previously demonstrated
that azathioprine causes photosensitivity by
embedding the thiopurine 6-thioguanine (6-TG) in
patients’ DNA. DNA 6-TG is a photosensitiser
because, unlike normal DNA bases, it can absorb
energy from the UVA in sunlight. (UVA is generally
considered to be relatively harmless but it
comprises ≥ 90% of the UV radiation that we are
exposed to). The UVA energy absorbed by DNA
6-TG is transferred to oxygen to generate singlet
A
oxygen (1O2), a highly reactive and damaging form
of oxygen. We have identified numerous forms of
damage to DNA and protein caused by the 1O2
generated from the DNA 6-TG/UVA interaction.
Cellular proteins are particularly vulnerable to
damage by 1O2. This protein oxidation reduces the
efficiency of DNA repair, including nucleotide
excision repair (NER), the main protection against
mutation and cancer in the skin. One particularly
hazardous product of oxidation is the crosslinking
of proteins to DNA. Working with the Protein
Analysis and Proteomics Laboratory, Melisa Guven
has used SILAC to investigate whether important
DNA repair factors are sequestered by covalent
linkage to DNA under the oxidative conditions
generated by combined DNA 6-TG and UVA. This
analysis reveals that key proteins from all the major
DNA repair pathways (NER, DNA double strand
break rejoining, DNA mismatch repair and base
excision repair) become crosslinked to DNA under
these conditions.
Many commonly prescribed drugs are
photosensitisers and UVA chromophores. Unlike
6-TG, most do not become incorporated into DNA.
Nevertheless, their effects can mirror those of DNA
6-TG/UVA. Matt Peacock has shown that the 1O2
that is generated when cells are treated with
fluoroquinolone antibiotics and irradiated with
UVA causes extensive protein oxidation. UVA
irradiation of fluoroquinolone-treated cells impairs
their ability to remove canonical sunlight-induced
B
Figure 2
Effects of combined UVA on NER
and mutation.
A. NER in vivo.
HaCaT cells were irradiated with
UVC to induce canonical UV DNA
photoproducts. Half the cells also
received a non-toxic dose of UVA
(100 kJ/m2). Excision of
UVC-induced NER substrates (6-4
Py:Py photoproducts) was
measuredby ELISA.
B. NER in vivo.
Extracts prepared from HeLa cells
that had been irradiated with UVA
(left panel) or approximately
equitoxic doses of UVA, UVB or
UVC (right panel) were assayed
for the ability to carry out NER on
a circular DNA substrate that
contained a single NER.
C. Mutation induction.
The frequency of TK mutation
was measured in TK6 cells
irradiated with UVB and/or UVA
as indicated. White bars are the
observed mutation frequencies
induced by combined UVA and
UVB treatment. Red bars are the
expected values for mutation
frequency if UVA and UVB
treatment were simply additive.
Means ± SD of three
determinations.
A
B
C
mutagenic DNA lesions by NER. Together with Peter
MacPherson, Matt showed that the effect on NER
reflects damage to proteins, and extracts of
treated cells have low levels of NER activity
(Figure 1). He also showed that damage to DNA
repair proteins is not confined to NER; other DNA
repair pathways are also inhibited by oxidation of
participating proteins.
Sunlight, protein oxidation and skin cancer
Sun exposure is a known risk factor for skin cancer.
This is because the UVB (wavelengths 280-320 nm)
in sunlight damages DNA. UVB-introduced DNA
lesions are firmly implicated in skin cancer
development; skin tumour genomes are dominated
by mutations that bear the hallmark of UVBinduced damage. Paradoxically, UVB phototherapy
for chronic skin conditions, which involves multiple
rounds of DNA-damaging UVB treatment, is not
associated with an increased skin cancer incidence.
This surprising observation suggests that the UVA
(wavelengths 320-400 nm) radiation that
comprises ≥ 95% of the UV in incident sunlight is
important for mutation and skin cancer
development. Although it is generally accepted
that UVA does contribute to cancer risk, how it
does this is not understood. To investigate how
UVB and UVA radiation might interact to influence
mutation and cancer risk, Lizzy McAdam is
comparing the effects of UVB alone and in
combination with UVA in cultured human cells. She
has confirmed that UVA irradiation of cultured cells
generates 1O2 and increases their levels of oxidised
proteins (carbonyls). The increased protein
carbonylation is associated with less efficient NER
and the removal by NER of canonical UV-induced
DNA lesions is slower in cells irradiated with UVA
(Figure 2A). Extracts prepared from UVA-irradiated
cells are less efficient at NER, implicating damage
to NER proteins in the reduced repair efficiency.
The effects are specific for UVA and exposure of
cells to UVB or UVC radiation does not affect NER
efficiency (Figure 2B). The effect of combining UVB
and UVA radiation on mutation has been studied in
the well-characterised TK6 mutation system that
detects mutational inactivation of the single TK
locus in a cultured human cell line. Measurements
of UVB and UVA-induced TK mutations in TK6 cells
suggest that their mutagenic effects are partly
synergistic. When cells are irradiated with a low
dose of UVB together with UVA, the frequency of
induced TK mutations is higher than the frequency
that would be expected if UVA and UVB were
contributing separately to mutation induction
(Figure 2C). The effect of protein oxidation on
human genome stability has not been extensively
studied. In simpler organisms, protein oxidation is
an important influence on survival and mutation.
Certain extremophiles are protected against the
lethal and mutagenic effects of radiation by highly
efficient antioxidant systems that prevent protein
oxidation. In addition, there are indications that
the level of protein carbonyls directly influences
both spontaneous and induced mutation in
bacteria. Our findings are compatible with a similar
relationship in human skin. They suggest that
sunlight UVA contributes to skin cancer risk by
increasing the level of protein oxidation. Because
they point to an important role for UVA in mutation
by sunlight, these findings also have implications
for recommendations for appropriate sunscreen
formulation.
Clare Hall
135
CELL DIVISION AND ANEUPLOIDY
www.london-research-institute.org.uk/research/mark-petronczki
Graduate Students
Anna Dowbaj
Kristyna Kotynková
Scientific Officer
Tohru Takaki
Figure 1
Depletion of splicing factors
abrogates sister chromatid
cohesion and the stable
association of cohesion with
chromatin.
A. Transfection of HeLa Kyoto
cells with siRNAs against splicing
factors leads to a premature loss
of sister chromatid cohesion.
Images of DAPI stained
chromosome spreads from cells
reveal that, while chromosomes
from control depleted cells
display the characteristic ‘X’
shape, cells depleted of splicing
factors have lost the connections
between sister chromatids.
B. Fluorescence recovery after
photobleaching (FRAP) in HeLa
Kyoto cells stably expressing a
core cohesin subunit, SMC1-EGFP,
reveal enhanced dissociation of
cohesin from chromatin in
MFAP1- depleted cells. The
nuclear SMC1-EGFP fluorescence
was repeatedly bleached in
approximately half of the nucleus
and the loss of fluorescent
intensity in the unbleached area
was plotted over time. These
experiments demonstrate that
depletion of splicing factors
weakened the stability of
cohesion-chromatin interaction
in interphase cells.
136
The process of cell division is indispensable for life
to flourish and diversify. It ensures equal
segregation of cellular content including DNA to
the two nascent daughter cells. Accurate
segregation of chromosomes in eukaryotic
organisms relies on connections between
replicated sister chromatids, a phenomenon
known as sister chromatid cohesion. Sister
chromatid cohesion is mediated by a conserved
ring-like protein complex, cohesin, that
topologically entraps the sister chromatids until
they are ready to be segregated. Cohesin lies at the
heart of chromosome biology. It is essential for
chromosome segregation, the proper repair of
DNA lesions and important for gene regulation.
Recent cancer genome studies have concluded
that cohesin subunits are frequently mutated in
human malignancies. Defects in cohesin function
can give rise to aneuploidy and genome instability
and thereby contribute to tumourigenesis in
A A
somatic cells or developmental defects and
infertility in the germline.
Using available functional genomic and proteomic
data sets along with a focused RNAi approach, we
identified a set of factors involved in pre-mRNA
splicing whose depletion prevented successful cell
division in human cells. Loss of these splicing
factors abrogated chromosome alignment and
trapped cells in mitosis. This mitotic phenotype
was accompanied by a dramatic loss of sister
chromatid cohesion that occurred soon after DNA
replication (Figure 1A). While depletion of premRNA splicing mediators had no striking effect on
bulk loading of cohesin onto chromatin,
fluorescence recovery after photobleaching
experiments (FRAP) revealed that depletion of
splicing factors prevented the stable association of
cohesin with chromatin in post-replicative cells
(Figure 1B).
Splicing factor depletion
Control siRNA
MFAP1 siRNA
14%
B B
SART1 siRNA
NHP2L1 siRNA
98%
99%
% spreads with split sister chromatids
81%
Time (min)
SMC1-EGFP
Control
Laurent L’Epicier-Sansregret
Murielle Serres
Antonio Tedeschi
Maria Dolores Vázquez Novelle
0
siRNA
Mark Petronczki
MFAP1
Group Leader
Every second several hundred thousand cells in our body duplicate
themselves through a process known as cell division. To generate
healthy and viable cells the division process has to accurately
partition all 46 chromosomes to daughter cells. Our group uses
animal cell systems to investigate the molecular mechanisms
underlying cell division and the consequences of genomic imbalances
caused by cell division errors. We have recently discovered that
pre-mRNA splicing is essential for sister chromatid cohesion in human
cells (Sundaramoorthy et al., 2014; EMBO J. 33(22): 2623-2642).
Photobleach
2
10
30
60
120
Figure 2
Model for the role of splicing
factors in Sororin-mediated sister
chromatid cohesion.
In control cells (left), pre-mRNA
splicing of Sororin ensures the
presence of functional Sororin
protein at the S phase of every
cell cycle to counteract the
destabilising activity of WAPL.
Compromised splicing (right)
leads to a sharp decline in levels
of Sororin protein. The lack of
Sororin, a factor necessary for
cohesion maintenance leads to a
reduced association between
cohesin and chromatin and
thereby to a premature loss of
sister chromatid cohesion.
Immunoblotting revealed that the depletion of
splicing factors caused a 5-fold reduction in the
protein levels of Sororin, a protein required for
stable association of cohesin with chromatin.
Bolstering our observation, erroneous splicing of
Sororin pre-mRNA was detected upon depletion of
splicing factors. Importantly, the sister chromatid
cohesion loss caused by depletion of splicing
factors could be suppressed by a Sororin transgene
that does not depend on pre-mRNA splicing for its
expression. Furthermore, we found that depletion
of the cohesion release factor WAPL that is
antagonised by Sororin also restores sister
chromatid cohesion in cells lacking spliceosome
components. Thus, the pre-mRNA splicing of
Sororin is a rate-limiting step in the maintenance of
sister chromatid cohesion in human cells (Figure 2).
Our results highlight the loss of cohesion as an
early cellular consequence of compromised
splicing. Our work linking splicing and sister
chromatid cohesion has potential implications for
the pathology of Chronic Lymphocytic Leukemia
(CLL). One of the splicing factors that we implicate
in sister chromatid cohesion, SF3B1, is a gene that
is one of the most frequently mutated genetic
drivers found in CLL patients.
Clare Hall
137
MECHANISMs OF GENE TRANSCRIPTION
www.london-research-institute.org.uk/research/jesper-svejstrup
Group Leader
Jesper Q Svejstrup
Stefan Boeing
Andreas Ehrensberger
Lea Gregersen
Theo Kantidakis
Michael Ranes
Marco Saponaro
Yuming Wang
Hannah Williams
Laura Williamson
Graduate Students
Konstantin Fritz
Michael Lim
Kotryna Temcinaite
Diana Zatreanu
Scientific Officers
Barbara Dirac-Svejstrup
Michelle Harreman
Jane Walker
Maintaining genome integrity is of utmost importance for the longterm survival of cells and organisms. However, the key immediate
cellular response to genotoxic insult is arguably to maintain gene
expression. Indeed, without transcription, cells cannot proceed
through the cell cycle, and even non-dividing cells will perish. The
overall aim of our research is to understand the basic mechanisms
underlying RNA polymerase II (RNAPII) transcript elongation, but in
particular how elongation interfaces with other processes on DNA,
such as DNA repair, replication and recombination. We also
investigate the cellular and molecular consequences of transcription
stress, such as that caused by DNA damage. We believe that a
detailed insight into the cellular responses to transcription elongation
impediments will make it possible for us to understand certain
human diseases, and cancer in particular, and thereby eventually how
to treat them.
We use biochemical, genetic, and cell biological
approaches, often combined with modern ‘omics’
technologies to understand the process of
transcript elongation and its interface with other
DNA-related processes such as DNA replication
and repair. Our published work in 2014 included
studies of the tumour suppressor RECQL5 and its
effect on transcription stress, and on Cockayne
Syndrome B (CSB) – the basis for Cockayne
syndrome (CS).
RECQL5 suppresses the genome destablising
effects of transcription stress
It has become increasingly evident that
transcription is closely integrated with other
DNA-related processes, such as DNA replication
and repair. Indeed, the process of expressing genes
comes at a cost: the movement of RNA
polymerases through DNA is associated with
genome instability (Helmrich et al., 2013; Nat
Struct Mol Biol. 20(4): 412-418), and RNAPII
stalling, pausing, arrest, and/or backtracking
(collectively referred to as transcription stress)
generates a cellular response akin to the DNA
damage response (Wilson et al., 2013; Biochem
Biophys Acta. 1829(1): 151-157). Transcribing
polymerases are also potent modulators of
DNA-related processes such as DNA replication.
Indeed, transcription-associated DNA
recombination involves clashes between
transcription and replication, and transcription is
138
associated with mutagenesis and contraction of
CAG repeats, as well as breaks at chromosome
fragile sites (Helmrich et al., 2013). However, the
mechanisms underlying transcription-associated
genome instability remain largely obscure, and
little is known about factors that might have
evolved to counteract it.
The RECQ proteins constitute a family of conserved
DNA helicases that are important for maintaining
genome stability from bacteria to humans (Chu
and Hickson, 2009; Nat Rev Cancer. 9(9): 644-654).
RECQL5 is unique among this family by interacting
with RNAPII, and it harbours two RNAPII
interaction domains which are relevant for the
genome instability phenotypes of cells lacking
RECQL5 (Aygün and Svejstrup, 2010; DNA Repair
(Amst). 9(3): 345-353). Intriguingly, transcription
reactions reconstituted with pure transcription
factors and RNAPII suggested that RECQL5 acts as
an inhibitor of transcription (Aygün et al., 2009;
J Biol Chem. 284(35): 23197-23203). However, the
physiological relevance, if any, of this observation
was not investigated. Likewise, the mechanistic
foundation for the well-known destabilising effects
of RECQL5 mutation on genome integrity has also
remained unclear.
We investigated the effect of RECQL5 on transcript
elongation and its role in suppressing genome
instability (Saponaro et al., 2014; Cell. 157(5):
Figure 1
Model for RECQL5 function.
RECQL5 ‘dampens’ transcript
elongation causing a slow down of
overall average elongation rates
while simultaneously avoiding
excessive transcription stalling
and arrest (i.e. transcription
stress), which can otherwise lead
to genome instability and,
eventually, to cancer.
1037-1049). Our results indicate that RECQL5 is a
general elongation factor, important for preserving
genome stability during transcription. Depletion or
overexpression of RECQL5 resulted in
corresponding shifts in the genome-wide RNAPII
density profile. Transcript elongation was
particularly affected across most genes, with
RECQL5 depletion causing a striking increase in the
average elongation rate. Concurrently, increased
transcription stress was observed in coding
regions, together indicating that RECQL5 controls
the movement of RNAPII across genes. Importantly,
loss of RECQL5 also resulted in the loss or gain of
genomic regions, with the breakpoints of lost
regions located in genes and common fragile sites.
Intriguingly, the chromosomal breakpoints
overlapped with areas of elevated RNAPII
transcription stress, suggesting that RECQL5
suppresses the detrimental effects of such stress,
and thereby prevents genome instability in the
transcribed region of genes (Saponaro et al., 2014).
The molecular basis of Cockayne syndrome
CS is an autosomal-recessive, multi-system
disorder, characterised by severe neurologic
disease, growth failure, developmental
abnormalities, photosensitivity, and degeneration
of different organ systems (Brooks, 2013; DNA
Repair (Amst.). 12(8): 656-671). The majority of CS
patients carry mutations in the gene encoding the
DNA translocase CSB/ERCC6 (~80% of patients), or
the gene encoding ubiquitin ligase-associated CSA/
ERCC8. These proteins are best known for their role
in transcription-coupled nucleotide excision repair
(TC-NER), a process whereby bulky DNA lesions,
such as those generated by UV-irradiation, are
preferentially removed from the transcribed strand
of active genes. CS is thus frequently referred to as
a TC-NER disease. However, CS cells are sensitive to
a number of additional DNA-damaging agents, and
oxidative damage in particular, implicating the CS
proteins in other repair pathways as well. Indeed,
the idea that CS results from inefficient repair of
oxidative DNA damage has gained momentum over
the last decade. Finally, studies from Weiner, Egly
and other groups have reported evidence of a role
for CSB in gene regulation, which might provide an
alternative explanation for CS (Brooks, 2013).
However, the relationship between deficiencies in
molecular pathways affected by CS mutation and
patient disease symptoms have generally remained
tenuous, or unexplored.
We investigated the connection between the
neuropathology of CS and dysregulation of gene
expression (Wang et al., 2014; Proc Natl Acad Sci
USA. 111(40): 14454-14459). Transcriptome
analysis of human fibroblasts revealed that, even in
the absence of DNA damage, CSB affects the
expression of thousands of genes, many of which
are neuronal genes. CSB is present at a significant
subset of these genes, suggesting that regulation is
direct. Importantly, cellular reprogramming of CS
fibroblasts to neuron-like cells was defective
unless an exogenous CSB gene was introduced.
Moreover, neuroblastoma cells from which CSB
was depleted showed defects in gene expression
programmes required for neuronal differentiation
and failed to differentiate and extend neurites.
Likewise, neuron-like cells could not be maintained
without CSB. Finally, a number of disease
symptoms may be explained by the marked gene
expression changes observed in the brains of
diseased CS patients. Together, our data point to
dysregulation of gene regulatory networks, rather
than DNA repair defects, as the cause of the
neurological symptoms in CS (Wang et al., 2014).
Clare Hall
139
GENETIC RECOMBINATION
www.london-research-institute.org.uk/research/stephen-west
Group Leader
Stephen C West
Gary Chan
Miguel Gonzalez Blanco
Gregoriy Dokshin
Kasper Fugger
Maria Jose Martin Pereira
Marieke Peuscher
Kanagaraj Radhakrishnan
Shriparna Sarbajna
Joanna Soroka
Haley Wyatt
Graduate Student
Kristyna Kotynkova
Scientific Officers
Michael McIlwraith
Rajvee Shah
Mammalian cells possess a large repertoire of DNA repair processes
that maintain the integrity of our genetic material. Some individuals,
however, carry mutations in genes required for DNA repair, and this
often leads to inheritable disease. An important repair process
involves recombination, and defects in this process are linked with
cancer predisposition, in particular breast cancers caused by
mutation of the BRCA2 gene, acute leukemias associated with
Fanconi Anemia, and a wide range of cancers found in individuals with
the chromosome instability disorder known as Bloom’s Syndrome.
The focus of our research is to determine the
molecular mechanisms of recombinational repair,
and to define why defects in these processes cause
cancer.
Our genetic material (DNA) is continually subjected
to damage, either from endogenous sources such
as reactive oxygen species produced as byproducts of oxidative metabolism, from the
breakdown of replication forks during cell growth,
or by agents in the environment such as ionising
radiation or carcinogenic chemicals. To cope with
such damage, cells employ elaborate and effective
repair processes that are each specialised to
recognise different types of lesions in DNA. These
repair systems are essential for the maintenance of
genome integrity. Some individuals, however, are
genetically predisposed to crippling diseases or
cancers that are the direct result of mutations in
genes involved in the DNA damage response.
The breast cancer tumour suppressor BRCA2
For several years we have been interested in the
mechanisms of homologous recombination, how
they contribute to the repair of DNA double-strand
breaks, and how they promote genome stability.
Many of the proteins required for recombination
have been purified in this laboratory, and we use
biochemical and molecular and cell biological
approaches to understand how they bring about
the repair of DNA breaks. One of these proteins is
the BRCA2 breast cancer tumour suppressor.
Mutations in the BRCA2 gene lead to a greatly
increased risk of breast and ovarian cancers, and
biallelic BRCA2 mutations have been associated
with the cancer-predisposition syndrome Fanconi
Anemia (FA) sub-type FA-D1. We therefore want to
understand the precise role of the product of this
gene, the BRCA2 tumour suppressor, in DNA repair
140
mediated by recombination. Previous work from
our group has shown that BRCA2 is required for the
assembly of nucleoprotein filaments formed by the
RAD51 recombinase, and in particular for the
delivery of RAD51 to sites of DNA damage. But until
recently the exact mechanism of how BRCA2
coordinates the RAD51-mediated steps of
recombinational repair remained elusive.
In new work, we obtained the first structural and
mechanistic insights into the role that BRCA2 plays
in promoting RAD51 nucleoprotein filament
formation. In collaboration with Professor
Xiaodong Zhang’s group at Imperial College
London, we visualised full-length BRCA2 protein
and the BRCA2-RAD51 complex using negative
stain electron microscopy combined with single
particle techniques to generate a threedimensional (3D) reconstruction of the protein’s
structure (Shahid et al., 2014; Nat Struct Mol Biol.
21(11): 962-968). We found that BRCA2 forms
dimers and that single-stranded DNA (ssDNA)
binds across long axis of the dimeric protein. Our
structural analysis of the BRCA2-RAD51 complex
revealed that BRCA2 remains in a dimeric form
upon binding to RAD51, and delivers RAD51 to the
ssDNA thereby increasing the number of RAD51
filaments. Two 3D views of BRCA2 modelled into
the structure of the BRCA2-RAD51 complex are
shown in Figure 1. Further analysis of BRCA2RAD51-ssDNA complexes revealed that BRCA2 is
present at one end of the RAD51 filament and
directs unidirectional growth of the filaments
along the ssDNA. In essence, it acts as a
molecular chaperone. Together, our data has
uncovered novel molecular insights into the
mechanistic aspects of BRCA2 action in
homologous recombination (HR) and have
potential implications for designing more efficient
therapeutic intervention of cancer.
Figure 1
Two views of the BRCA2 dimer
(the two subunits are coloured
yellow and cyan) modelled into
the BRCA2-RAD51 complex (pink
mesh). Four RAD51 monomers are
shown on each side of BRCA2. This
BRCA2-RAD51 complex plays a key
role in the initiation of DNA repair
by recombination.
Structure-selective nucleases resolve
chromatid bridges to ensure chromatid
segregation at mitosis
Recombinational repair requires a reciprocal
exchange of DNA strands between sister
chromatids or homologous chromosomes, leading
to the formation of DNA intermediates, such as
Holliday junctions (HJs), in which the two interacting
DNAs become covalently interlinked. The efficient
processing of these joint molecules is essential for
chromosome segregation at cell division, and is also
important in determining the outcome of
recombination: for example, crossovers (COs)
between homologous chromosomes are required
for meiotic division, whereas non-crossovers
(NCOs) are favoured in mitotic cells in order to avoid
‘loss of heterozygosity’ (LOH), a known driver of
tumourigenesis.
The importance that the cell places on the efficient
and timely processing of these intermediates is
clear from our discovery of the involvement of
three distinct pathways of HJ resolution, and the
way that these pathways are regulated throughout
Figure 2
The sub-cellular localisation of the
GEN1 Holliday junction resolvase
is directed by its nuclear export
sequence (NES). In normal cells,
GEN1 is cytoplasmic and can only
resolve recombination
intermediates when the nuclear
envelope breaks down at mitosis.
Mutation of the NES can be used
to target GEN1 into the nucleus,
but its presence causes increased
sister chromatid exchanges (SCEs).
The cytoplasmic localisation is
therefore important to control the
nuclease, and yet facilitate
chromosome segregation in
mitosis.
the cell cycle (Matos et al., 2011; Cell. 147(1):
158-172, Wechsler et al., 2011; Nature. 471(7340):
642-646, Sarbajna et al., 2014; Genes Dev. 28(10):
1124-1136). Firstly, the human BTR complex
(BLM-TOPIIIα-RMI1-RMI2) plays a major role in the
removal of double HJs to generate NCO products.
The preferential formation of NCOs is important to
avoid LOH. Secondly, at G2/M phase of the cell
cycle, our cells create a novel endonuclease that is
capable of resolving persistent recombination
intermediates that have escaped the attention of
BTR. This nuclease, which forms in response to
CDK/PLK1-dependent phosphorylation events,
involves interactions between two structureselective endonucleases, MUS81-EME1 and
SLX1-SLX4 (Wyatt et al., 2013; Mol Cell. 52(2):
234-247). Finally, at mitosis, any remaining
intermediates are acted upon by GEN1, a classical
Holiday resolvase (Chan et al., 2014; Nat Comm. 5:
4844). The MUS81-EME1-SLX1-SLX4 and GEN1
nucleases are potentially dangerous in S phase,
since they generate COs (which can be measured by
an increased frequency of sister chromatid
exchanges) providing a good rationale why their
activities are held in check until late in the cell cycle.
Our recent work uncovered the mechanisms by
which GEN1 (and it’s yeast ortholog Yen1) are
regulated throughout the cell cycle in order to
avoid CO formation and LOH. In yeast, Yen1 is held
in an inactive state in the cytoplasm by virtue of its
phosphorylation by Cdk. During mitosis, however,
the protein is dephosphorylated by Cdc14, leading
to its nuclear entry and activation (Blanco and
West, 2014; Mol Cell. 54(1): 94-106). In human
cells, the situation is similar, although
mechanistically different from that in yeast: GEN1
is exported to the cytoplasm by a strong Nuclear
Export Sequence (NES), and only gains access to
the chromosomal DNA upon nuclear envelope
breakdown at mitosis (Chan and West, 2014; Nat
Commun. 5: 4844). Artificial localisation into the
nucleus (by NES mutation and addition of nuclear
localisation sequences) leads to inappropriate
cleavage of replication forks and causes increased
sister chromosome exchange (SCE) formation
(Figure 2). The regulation of HJ processing
pathways is therefore important for the
maintenance of genome stability, both by limiting
CO formation and for ensuring proper
chromosome segregation.
Clare Hall
141
SINGLE MOLECULE IMAGING
www.london-research-institute.org.uk/research/hasan-yardimci
Group Leader
Hasan Yardimci
Daniel Burnham
Melania Strycharska
Graduate Student
Hazal Busra Kose
Scientific Officers
Sherry Xie
Sevim Yardimci
Before a cell divides it has to duplicate its genome so that two
identical copies of its DNA content can be partitioned into daughter
cells. In eukaryotic cells, replication is initiated at thousands of origins
on the DNA, each resulting in the assembly of two replisomes that
travel away from the initiation site in opposite directions. Complete
and high-fidelity duplication of the genome is essential for faithful
transmission of genetic information. When DNA replication goes
awry, the result could be cells with mutations, missing or extra
genetic material, a hallmark of the genomic instability seen in most
cancers. Our group aims to investigate processes involved in
eukaryotic replication using a combination of conventional
biochemistry and single-molecule imaging tools.
Helicase mechanisms
To copy their DNA in preparation for cell division,
cells must separate the two strands of the double
helix. All cells contain a ring-shaped hexameric
DNA helicase, which performs this task. Bacteria
use DnaB, whereas eukaryotes use the MCM2-7
complex. In G1 phase, two MCM2-7 hexamers
assemble around double-stranded DNA (dsDNA) at
each origin of replication in a head-to-head fashion
(Figure 1). This structure, known as the prereplication complex (pre-RC), remains idle in the
G1 phase. In S phase, MCMs are activated through
the action of a number of proteins including Cdc45
and GINS, and multiple kinases to subsequently
unwind DNA at the replication fork.
Figure 1
Eukaryotic DNA replication.
In G1 phase, double hexamers of
the MCM2-7 complex are loaded
onto origin DNA by ORC, Cdc6,
and Cdt1. MCMs are activated in
S phase via the action of a number
of factors to initiate unwinding.
Finally, polymerases replicate
unwound DNA.
142
To understand the molecular mechanism by which
MCM2-7 functions, we developed a single
molecule assay in which a DNA template
immobilised on the surface of a microfluidic flow
cell is efficiently replicated in soluble extracts
derived from Xenopus laevis eggs (Figure 2). Using
this system, we showed that MCM2-7 complexes,
which initially load as double hexamers on DNA,
can physically uncouple and function as single
hexamers during replication (Yardimci et al., 2012;
Mol Cell. 40(5): 834-840), in contrast to some
models. We also showed that MCM2-7 translocates
along the leading strand template to unwind DNA,
suggesting that the helicase goes through a
conformational change during activation and
transitions from a dsDNA to a single-stranded DNA
(ssDNA) binding mode (Fu et al., 2011; Cell. 146(6):
931-941). In the future, we aim to gain an in depth
understanding of the MCM2-7 dynamics through
real-time visualisation of the helicase at the single
molecular level.
Simian Virus 40 (SV40), a mammalian DNA tumour
virus, has served as a robust model system for
investigating the mechanism of eukaryotic
replication for several decades. The virus encodes
its own replicative helicase, Large T-antigen (T-ag),
which utilises host cell factors for replication of its
genome. Our work indicated that T-ag activated at
an origin functions as a single hexamer and
translocates along ssDNA, similar to the MCM2-7
complex (Yardimci et al., 2012; Nature. 492(7428):
205-209). Importantly, we also discovered a
surprising new property of T-ag. We found that
T-ag can efficiently bypass a protein adduct
covalently cross-linked to the translocation strand.
Figure 2
Single molecule visualisation of
eukaryotic replication.
λ DNA was stretched and
immobilised at both 3’ ends on
the streptavidin-functionalised
surface of a microfluidic flow cell.
Immobilised DNA was exposed to
Xenopus egg extracts to initiate
MCM2-7-dependent replication.
Finally, a second extract
containing digoxigenin-modified
dUTP (dig-dUTP) was withdrawn
into the flow cell to confirm
bidirectional replication. Extracts
were removed via SDS containing
buffer, dsDNA was labelled with
SYTOX Orange and dig-dUTP was
labelled with a fluorescent
anti-dig antibody. A stretched λ
DNA molecule that underwent
replication in extracts (bottom).
High intensity SYTOX tract
corresponds to a replication
bubble. Anti-dig tracts coincide
with both ends of the bubble
indicating bidirectional
replication.
This remarkable plasticity of T-ag may help the
SV40 replisome overcome bulky barriers such as
DNA-protein cross-links. Current work is focused
on defining the molecular mechanism by which a
ring-shaped hexamer can overcome large obstacles
during translocation.
Architecture and dynamics of the eukaryotic
replication machinery
An essential component of the replisome complex
is the polymerase, which synthesises new DNA on
unwound strands. First, a primase complex
associates with unwound DNA and synthesises
DNA/RNA primers. At the leading strand, the DNA
primer is extended continuously the lagging strand
is synthesised discontinuously as Okazaki
fragments (Figure 1). It is important to understand
how different polymerases coordinate DNA
synthesis for accurate replication. Live cell imaging
in bacteria showed that a single replisome contains
three polymerases, one acting on the leading
strand and two on the lagging as opposed to the
previous assumption that there is one polymerase
acting on each strand. The presence of two lagging
strand polymerases was shown to be important for
processive lagging strand synthesis.
Unlike prokaryotes, eukaryotes employ different
polymerases to synthesise leading and lagging
strands. Upon priming by polymerase alphaprimase complex (Pol α), the leading strand is
replicated by polymerase epsilon (Pol ε) while the
lagging strand is replicated by the action of
polymerase delta (Pol δ) (Figure 1). Currently,
little is known about the stoichiometry and
dynamics of eukaryotic replisome components
including polymerases. How long does Pol α
remain on DNA before Pol ε or Pol δ takes over?
How many Pol ε and Pol δ molecules are associated
with individual replisomes? How often do
polymerases exchange at the fork while
synthesising the leading and lagging strands? To
address these questions, we will visualise
individual molecules in real time during replication
of stretched DNA molecules. Our work will also
provide important insight into how the replication
machinery acts upon encountering different types
of DNA damage.
Clare Hall
143
False coloured SEM of a human blood
sample showing red blood cells and a
single white blood cell surrounded by
a mesh of fibrin. Image: Electron
Microscopy, Technology Core
Facility, LRI.
144
TECHNOLOGY
CORE
FACILITIES
The London Research Institute benefits from access to a wide range
of high quality research services. Scientific support for researchers at
the LRI is provided by some nineteen core facility groups of various
sizes run by the Institute. These aim to provide state of the art
facilities for LRI researchers that are proactive in enabling the
research groups to carry out world leading science.
145
The London Research Institute has benefited from access to a wide
range of high quality research services. Scientific support for
researchers at the LRI is provided by core facility laboratories of
various sizes run by the Institute. These aim to provide state of the art
facilities for LRI researchers that are proactive in enabling the
research groups to carry out world leading science.
Julian Downward
Associate Director, LRI
The past year has seen the final details of the
planning of the space for the core facility labs at
the Francis Crick Institute, where they will be
named Science Technology Platforms (STPs). Plans
have been made for combining the core facilities
from the LRI and NIMR and transition leads for
each facility were appointed in the spring. The
move of STPs to the Crick building will start at the
end of 2015 and will be arranged to minimise
disruption to users during the transition period. At
Crick we will maintain and build on the fantastic
quality of the LRI core facilities, providing
researchers with access to unparalleled
technologies to pursue outstanding science.
The quality and development of LRI core facilities
has been driven by user committees, made up of
representatives of the service provider, users and
management, which provide advice on technical
advances, prioritise projects when facilities are
limited, and act as a focal point for interactions
with researchers. In addition, a programme of
review by external experts every three to four
years has ensured that the LRI core technology
facilities have remained cutting edge. Similar
mechanisms will be put in place at the Crick to
ensure the continued excellence of the STPs.
Technology core facilities provided centrally at the
LRI allow access to cutting edge equipment and
instruction in its correct and effective usage. Within
these facilities, services may either be run by
dedicated staff or by researchers themselves with
appropriate training. LRI core facilities include the
following: Light Microscopy provides conventional,
confocal, multiphoton and automated microscopy,
time-lapse video and microinjection services.
Electron Microscopy has field emission and serial
block face scanning electron microscopes and
transmission electron microscopes. The FACS
Facility provides a comprehensive flow cytometry
service, with several analysers and sorters,
including a FlowSight imaging flow cytometer.
Experimental Histopathology provide expertise in
mouse and human histopathology, in situ
146
hybridisation techniques, laser capture
microdissection and automated image acquisition.
The Equipment Park provides DNA Sanger
sequencing, robotic nucleic acid preparation,
quantitative PCR, gel imaging systems and HPLC
micro-purification. Advanced Sequencing provides
next generation DNA sequencing with three
Illumina HiSEQ 2000s and an Ion Torrent PGM. The
High Throughput Screening Facility brings together
the equipment, personnel and expertise needed to
carry out and interpret large scale screening assays.
It has recently acquired an Echo acoustic dispenser,
enabling accurate automated delivery of nanolitre
volumes. The Protein Purification Facility
specialises in the production of pure recombinant
proteins for structural studies, using baculoviral,
bacterial and mammalian tissue culture systems.
Bioinformatics and Biostatistics provide support for
all the Institute’s bioinformatics needs, ranging
from high throughput sequencing data analysis and
high throughput screen interpretation to global
gene expression analysis. Cell Services provides a
wide range of quality controlled cells and media, as
well as the production of monoclonal antibodies
from hybridoma lines. The Peptide Chemistry
Facility provides custom made peptides, peptide
arrays and cross-linking reagents. Transgenics
provide the latest methodology for the generation
of genetically modified mice.
The In Vivo Imaging facility was established over
the past five years by Francois Lassailly to support
visualisation of animal cancer models by high
resolution ultrasound, micro CT, bioluminescence
imaging and intravital multiphoton microscopy.
The whole institute has been deeply saddened by
the sudden and unexpected death of Francois in
January 2015. We will all hugely miss his
inspirational leadership, intelligence and charm.
After nine years of overseeing the core technology
facilities at LRI, I look forward to helping to
establish the Science Technology Platforms at the
Crick as world leading in their fields.
ADVANCED SEQUENCING
www.london-research-institute.org.uk/technologies/advanced-sequencing
The past five years saw a significant increase in Next Generation
Sequencing (NGS) activity at the LRI. The number of samples being
analysed per project has increased from tens to thousands in some
cases, supported by continued investment in technologies and
protocols.
Head
Nik Matthews
Staff
Sharmin Begum
Jennifer Biggs
Ben Phillimore
Adam Rabinowitz
Figure 1
HiSeq sequencing report (near
perfect) for a 101x2bp Paired End
run with 7bp of index sequence in
the centre. The figure shows the
flowcell representation on the
left.
The other four diagrams are the
cycle by cycle intensities (top left),
cluster densities per lane (bottom
left), passed filter Q-scored (top
right) and Q-score heat map
(bottom right) for the run.
The Advanced Sequencing Facility (ASF) has three
Illumina HiSeq systems and one Ion Torrent PGM
system. As early adopters of these technologies the
ASF has pushed the limits of the systems to include
low concentration samples (down to single cell)
and fragmented RNA. The facility offers four main
methodologies (each with multiple protocols):
– ChIP-seq
– Paired End sequencing (e.g. whole genome,
amplicon-seq)
– RNA-seq (mRNA and total RNA)
– Bait Targeted Enrichment
ChIP-seq
ChIP-seq is the evolution of ChIP-ChIP and involves
the study of DNA-associated proteins. This
technique can be used to study heritable
information and epigenetic patterns in cancer cells
and help validate RNA-seq experiments. This
methodology includes also 4C and ChIA-PET.
Paired End sequencing
Paired End sequencing on the HiSeq is a method
that makes it possible to sequence genomes of all
model organisms to a high coverage. The HiSeq can
be used to sequence these model organisms
genomes, including human, to a level of accuracy
essential to the study of rare variants.
RNA-seq
Unlike the genome, which is rather static apart
from mutations, the transcriptome is very fluid and
can change with internal and/or external
conditions. An RNA-seq method can look simply at
these expressed genes or more in-depth at e.g.
splice variants. Other aspects of RNA biology can
also be studied using shRNA-seq, RIP-seq and
GRO-seq.
Bait Target Enrichment
Bait Target Enrichment is similar in many ways to
amplicon-seq but is not a PCR method. Target
enrichment is a method of pulling out specific
contiguous or non-contiguous areas within any
genome, e.g. exome, using complimentary oligo
baits. This is a good alternative to larger whole
genome sequencing as it allows reduction in costs
and machine time. Figure 1 shows a whole exome
targeted enrichment HiSeq sequencing report.
In the time the ASF has worked at the LRI we have
worked alongside some great PhD students,
postdocs and group leaders on some fantastic
studies and publications. We look forward to this
continuing at the Crick.
147
BIOINFOMATICS AND BIOSTATISTICS
www.london-research-institute.org.uk/technologies/bioinfomatics-and-biostatistics
Head
Aengus Stewart
Staff
Probir Chakravarty
Philip East
Mickael Escudero
Stuart Horswell
Gavin Kelly
Anna Lobley
Richard Mitter
Harshil Patel
Adam Rabinowitz
Max Salm
Figure 1
Circos plots depicting inter- and
intrachromosomal translocations,
as well as deletions and insertions
for tumour regions R1 and R3 for
samples L002 and L008; shared
events are indicated in blue,
events private to region R1 are
indicated in red, and private to
region R3 in green. The outer
circle represents the integer copy
number data for R1 and the inner
circle for R3 for each tumor
sample; copy number segments
with an integer value greater than
mean ploidy are in red and those
less than mean ploidy in blue.
148
The Bioinformatics and Biostatistics Facility (BABS) collaborates with
groups that require the analysis of large or complex biological
datasets. The majority of this data is derived from genomic
experiments that use sequence data as their readout and the
application list is continually growing, e.g. transcript isoform profiling
(TIF-Seq). After processing, the data is put in a biological context by
combining statistical and mathematical methods for identifying
significance with annotation and other biological datasets to provide
informed insight.
VarSLR – algorithm for assessing mutation calling
in clinical samples
Accurate mutation calling in clinical tumour
samples remains a formidable challenge,
confounded by sample complexity, experimental
artefacts and algorithmic constraints. In particular,
high sequencing error rates (~0.1-1x10-2 per base)
entail costly manual review of putative mutations
followed by orthogonal validation. Efficient
filtering is currently required, given that most
mutation callers identify many thousands (in
exome sequencing), if not millions (in whole
genome sequencing), of candidate mutations per
experiment. To aid in this process, we developed
the open-source VarSLR R package to identify
somatic nucleotide and insertion-deletion
mutations that are likely to be sequencing
artefacts. The algorithm incorporates putative
confounders of call accuracy (Koboldt et al., 2012;
Genome Res. 22(3):568-576) into stepwise logistic
regression models and subsequently classifies
variants within a simple, 4-tiered quality schema.
VarSLR is highly scalable and designed to be run in
an ‘embarrassingly parallel’ fashion, thus
benefiting from the LRI’s high-performance
computing facility. Moreover, VarSLR performed
with high precision when tested with synthetic and
experimental data, and has been successfully
applied to numerous projects (e.g. de Bruin EC et
al., 2014; Science. 346(6206):251-256).
Identifying genes involved in drug resistance
using a genome transposon screen
Given the correct sequence architecture it is
possible to insert promoter containing DNA
elements using transposons into the human
genome. If inserted in the correct orientation and
close to a gene’s transcription start site the
inserted promoter can activate transcription. This
provides a mechanism for a genome wide positive
screen using elements inserted into randomly
selected transposon sites within a population of
cells. The transcriptional effect of a given insert on
cell survival within the context of different
environmental conditions such as drug treatment
can then be assayed. To determine the specific
genes involved, we first need to map the insertion
sites within the population of cells before and after
treatment. The inserts are sequenced within their
genomic context and with software developed by
BABS we remove adapter from within the
sequenced reads, identify and map the genomic
component of the read, orientate the insert,
quantify the insertion event via the number of
reads, cluster insertion events that lie in close
proximity to one another and finally associate the
insertion events to genes. The aim is to identify
genes involved in drug resistance but it is also
hoped that this powerful screening technique will
be made more widely available to Crick Institute
scientists once established. This work has been
carried out in
collaboration with Su Kit
Chew and Charles
Swanton.
Publications listed on
page 177
www.london-research-institute.org.uk/research/paul-bates
Group Leader
Paul Bates
Staff
Raphael Chaleil
Tammy Cheng
Mieczyslaw Torchala
Graduate Students
Rudi Agius
Sakshi Gulati
Erik Pfeiffenberger
We study fundamental and challenging problems in both structural
and systems biology; in particular, how macromolecules interact at
the atomic level to facilitate cellular events. Much of the work
involves the design of novel computer algorithms that are based upon
the principles of physics and evolutionary biology. These simulations
are proving to be important in helping to interpret experimental data
and suggest further experiments to probe complex molecular
systems. Outlined below are two systems currently under
investigation.
Validating biomarkers for clear cell renal cell
carcinoma (ccRCC)
Candidate biomarkers have been identified for
ccRCC patients, but most have not been validated.
In collaboration with Charles Swanton (Translational
Cancer Therapeutics) we have analysed 28 genetic
or transcriptional biomarkers in 350 ccRCC patients
in terms of cancer-specific survival (CSS). Our
conclusion from the study is that only one
biomarker, a gene expression set called ccB, could
be considered to be an independent prognostic
biomarker for CSS in ccRCC (Gulati et al., 2014; Eur
Urol. 66: 936-948).
Mapping the shape of protein-protein binding
funnels with SwarmDock
Predicting the effects of mutations on the kinetic
rate constants of protein-protein interactions is
Figure 1
Schematic diagram of the binding
funnel between two proteins,
actin and one of its binding
partners, a vitamin D-binding
protein.
The left hand panel shows the
complete search space between
the two proteins as a connected
graph of conformational states.
Larger nodes represent more
stable protein conformations.
The highly connected set of
nodes represents the true
binding funnel.
The right-hand panel shows an
exploded view of the docking
funnel, actin in magenta, and in
green the final conformation of
the vitamin D-binding protein.
Moving from A (edge of the
binding funnel) to D (near the
final bound state) stabilising
interactions between the two
proteins can be seen to increase.
important to both the modelling of complex
diseases, such as cancer, and the design of effective
protein inhibitors. To facilitate our understanding
of how mutations affect binding kinetics we are
mapping the conformational shapes of binding
funnels for wild type and mutated binding
partners. We are developing software to display
the output of our publicly available
macromolecular docking program SwarmDock
(bmm.cancerresearchuk.org/~SwarmDock) to
interpret these binding funnel shapes (Figure 1).
Details of how to effectively use our docking
program are given in a recent publication (Torchala
and Bates, 2014; Methods Mol Biol. 1137:181-197).
Transition Probility
0
1
Equilibrium Population
0
B
A
15
D
B
A
C
D
C
149
CELL SERVICES
www.london-research-institute.org.uk/technologies/cell-services
Our dedicated team provides LRI research groups with all their cell
culture needs. We work together to fulfil cell culture requirements,
purified antibody, cell authentication, customised media/plates and
mycoplasma screening requests as well as providing washroom
services at Clare Hall. We continue to offer the London Research
Institute scientific community a high level of practical and advisory
support.
Head
Ruth Peat
Staff
Susan Capon
Trevor Cooper
Warren Cooper
Darren Haines
Darren Harvey
Marley Holding-Pillai
Rachel Horton-Harpin
Spencer Horton-Harpin
Samantha Kenton
Julie Morrin
Christine Saunders
Martin Saunders
Robert Saunders
Debbie Schofield
Sonal Sheth
Karen Stoughton
Mark Thorlby
Paul Willis
Preparations for the transfer to the Crick Institute
As we are preparing our own cell stocks for
transition to the new Institute, we have
undertaken to grow, mycoplasma screen and
validate cell lines for groups within both the LRI
and NIMR, this follows the cell survey carried out in
2013. The project will enable our scientists to have
access to pre-prepared, clean, cell stocks and
minimise ‘down-time’ during migration to the
Crick. Currently we have processed over 500 cell
lines.
Mycoplasma screening developments
We have recently changed our PCR-based
mycoplasma screening kit for one supplied by the
ATCC. This offers us the same sensitivity and
reliability as before, at a much-reduced cost. We
use this particular test if there is a disparity
between our two regular screening processes
(Agar culture and Fluorescence) and also to
provide a quicker result, usually within 24 hours,
for people waiting to use cells in the Containment
Level 2 facility.
Containment Level 2. Currently Cell Services are
unable to grow these lines in our facility. Using our
new ATCC Mycoplasma Screening kit, we are able
to test the genomic DNA from Containment Level 2
lines, to ascertain the mycoplasma status – all we
need is approximately 1μg of DNA to run on our
test.
FTA cards for STR Profiling
We continually look for ways to improve all of our
existing techniques. Currently we are investigating
the use of FTA cards as an alternative to genomic
DNA preparation for use in short tandem repeat
(STR) Profiling. Our initial tests are very positive
and if this method is fully adopted it will allow us to
store DNA at room temperature. In addition, it
makes the process of collecting DNA from Human
cell lines much simpler – adjust the cell volume and
drop a certain number of cells onto the card. We
hope that this innovation will make it easier for our
scientists to give us DNA prior to, during and after
their experiments, enabling us to provide them
with the STR profile data, so often required before
journal publication.
150
Mycoplasma - ve control
Mycoplasma + ve control
Mycoplasma + ve
Mycoplasma + ve
Mycoplasma + ve
Mycoplasma + ve
Mycoplasma + ve
Mycoplasma - ve
Figure 1
ATCC Mycoplasma gel result
showing both positive and
negative results, along with
controls. A positive result reveals
a band between 434-468 bp.
100 bp Ladder
For some time we have wanted to offer
mycoplasma screening for lines that are
ELECTRON MICROSCOPY
www.london-research-institute.org.uk/technologies/electron-microscopy
Head
Lucy Collinson
Staff
Elisabeth Brama
Raffaella Carzaniga
Marie-Charlotte Domart
Martin Jones
Christopher Peddie
Matthew Russell
Anne Weston
The Electron Microscopy Unit (EMU) is a Core Technology Facility
providing the equipment and expertise necessary to image the
structure of molecules, cells and tissues at high resolution. The main
core of the EMU team consists of six experienced post-doctoral
electron microscopists working closely with research groups at the
LRI to plan, optimise and implement high-resolution imaging
experiments. In addition, two post-doctoral scientists with image
analysis and laser physics expertise joined the team this year, with a
remit to develop and build new integrated light and electron
microscopes for biomedical applications.
The EMU has expertise in preparing, imaging and
interpreting a wide range of samples, including:
• Proteins, DNA, and protein:DNA complexes
• Yeast
• Viruses and virus-infected cells
• Cultured cell lines
• Tissues
• Model organisms (fruit flies, worms and
zebrafish)
Figure 1
GFP-C1 fluorescence and HeLa
cell ultrastructure, imaged from a
single 200nm In-Resin
Fluorescence (IRF) section in an
integrated light and scanning
electron microscope.
We use a wide range of sample preparation and
imaging techniques, including:
• Negative staining and low angle rotary shadowing
• Resin embedding and ultramicrotomy
• Cryosectioning and immunolabelling
• Plunge freezing
• High pressure freezing and freeze substitution
• Correlative light and electron microscopy (CLEM)
• Transmission EM and Scanning EM
• Electron tomography
• Volume electron microscopy (Serial Block Face
SEM, Focused Ion Beam SEM)
• Integrated light and electron microscopy (ILEM)
• Cryo-fluorescence microscopy
• Cryo-soft X-ray tomography (at Diamond, ALBA
and BESSY II synchrotrons)
Figure 2
TEM micrograph of a HeLa cell
prepared in one day: from living
to imaging.
The facility currently houses four electron
microscopes, including:
• FEI Tecnai Twin 120kV TEM
1
2
• FEI Tecnai BioTwin 120kV TEM with iCorrTM
integrated light microscope
• Zeiss Sigma SEM with 3View for automated 3D
EM
• FEI Quanta SEM with a DELMIC SECOM
integrated light microscope
New in 2014
This year we have continued to push the limits of
electron microscopy…
• We are now able to collect thousands of images
from cells and tissues automatically using our
3View Serial Block Face SEM. We have adapted
this technique for 3D correlative imaging of cells
expressing fluorescent protein constructs.
• We have developed and published a method for
maintaining GFP fluorescence in cells and tissues
prepared for EM (Figure 1). This means that we
can perform in situ correlative imaging in an
integrated light and electron microscope. We are
the only site worldwide to have three integrated
light and electron imaging systems (TEM, SEM
and benchtop SEM). We are now developing
integrated super-resolution light and electron
microscopy for nanoscale accuracy in protein
localisation.
• We are one of ten sites worldwide to beta-test a
new automated ultramicrotome called the
ATUMtome, developed in Jeff Lichtman’s lab at
Harvard, which can cut thousands of resin
sections onto tape for array tomography in an
SEM.
• We are starting to apply new protocols that
allow us to prepare cells and tissues extremely
quickly, going from a live sample to TEM imaging
in one day (as opposed to one week or longer)
(Figure 2).
151
EQUIPMENT PARK
www.london-research-institute.org.uk/technologies/equipment-park
The Equipment Park provides access to state of the art molecular
biology instrumentation and offers instruction in the correct and
efficient use of the technologies involved. The range of equipment is
constantly reviewed and specific requests from laboratory heads are
encouraged.
Head
Graham MG Clark
Staff
Vicky Dearing
Olga O’Neil
David Philips
Ramin Sadri
The technologies include:
Real-Time PCR quantitation – we have seven
systems (4x96 well and 3x384 well format)
designed to detect fluorescence during the
thermal cycling of PCR. By plotting the increase in
fluorescence versus the cycle number, the system
produces amplification plots that provide a more
complete picture of the PCR.
PHERASTARPlus micro-plate reader – multidetection HTS micro-plate reader with
Simultaneous Dual Emission in all modes. The
reader is able to perform all leading non-isotopic
detection technologies including:
• Fluorescence Intensity
• Fluorescence Polarisation
• Time-Resolved Fluorescence
• TR-FRET
• Luminescence
• Absorbance UV/Vis
The QIAgility is a bench-top instrument for
automated setup of PCR reactions that is able to
handle a wide variety of tube and plate formats.
Figure 1
LC3 lipidation assay to analyse
autophagic flux.
Lipidated LC3 (LC3 II) associates
with autophagosomes and
migrates faster in SDS-PAGE.
Starvation (SM) induces protein
degradation with autophagy and
leads to an increase in LC3 II.
Inhibition of lysosomal
Bafilomycin A1 increases LC3 II
under basal (FMB) and starvation
(SMB) conditions.
A stable inducible GFP-SCOC HeLa
cell line was used for this
experiment.
Image courtesy of Dr Martina
Wirth, Secretory Pathways
Laboratory.
152
The system performs the preparation of master
mix from individual reaction components and
dilutions of standard series. Optionally available
UV light and HEPA filter help to reduce the risk of
sample carry-over.
In addition to PCR setup, the flexibility of the
system allows high-precision pipetting applications
including:
• Normalisation of DNA and RNA concentration
• Transfer of liquid samples from one tube format
to another
• Serial dilutions with variable dilution ratios
• Sample pooling
Biomek FX Robot – advanced liquid-handling
applications include:
• High-density replication
• Assay plate set-up (PCR, quantitative PCR (384
well), ELISA, kinase etc.)
• PCR and sequencing reaction clean-up using
paramagnetic technology.
ImageQuant LAS 4000 – a digital (CCD) imaging
system for sensitive, quantitative imaging of gels
and blots without film, by chemiluminescence and
fluorescence. The high sensitivity and wide
dynamic range is designed to capture the signals
from ECL western blotting reagents. A wide range
of visible fluorescent dyes can also be imaged via
red, green and blue epi-illumination.
Li-COR Bioscience Odyssey Infrared Imager – the
imaging system offers a different way to analyse
blots and gels. Odyssey is uniquely equipped with
two infrared channels for direct fluorescence
detection enabling simultaneous probing of two
separate targets on the same gel, e.g. Western
Blots (Figure 1).
EXPERIMENTAL HISTOPATHOLOGY
www.london-research-institute.org.uk/technologies/experimental-histopathology
The Experimental Histopathology (EHP) Facility provides advice,
training and expertise in a range of techniques to analyse cells and
tissues from experimental models and human tissue banks.
Head
Gordon Stamp
Staff
Janni Bertelsen
Tamara Bunting
Kornelia Fritsch
Emma Nye
Bradley Spencer-Dene
Richard Stone
Figure 1
Positive specific staining
(brown) for glucagon mRNA, a
secreted hormone found only
in α-cells in the periphery of
adult mouse pancreatic islets.
Figure 2
Positive staining for ZFP36/
tristetraprolin in human lung
tumour and adjacent stroma.
1
Technical procedures performed include:
• Human & rodent histopathology:
• MicroPix image capture teaching/discussion
platform
• Optimal handling/fixation of fresh tissue
• Adult & embryonic mouse dissection
• Mouse developmental analysis, including
embryonic lethal phenotypes
• Histological sectioning/staining of frozen/fixed
tissue
• High resolution photomicroscopy using NIS
Elements platform with semi-automated
morphometric analysis for object classification/
measurement
• Immunohistochemistry (IHC) against >350
mouse orientated antibodies plus novel antibody
optimisation (automated stainer for high
throughput)
• Non isotopic in situ hybridisation (ISH) on slides
and whole-mount embryos and organs
• 3D volume rendered reconstruction
• Laser Capture Microdissection
• Ariol SL50 Scanning system with morphometric
software modules;
– IHC: for quantitating membrane, nuclear and
cytoplasmic expression
– FISH and immunofluorescent capture
– Tissue Microarray
– General Morphometric Image Analysis module
(ploidy, angiogenesis, area/volume etc.)
EHP encourages participation by graduate students
and postgraduate scientists, and we provide
individual training and expert advice of laboratory
techniques.
2
Technology Highlight
RNAscope, now available in the EHP Facility, is an
innovative non-radioisotopic in situ hybridisation
assay for visualising the expression of any gene
within any tissue to extremely low abundance
mRNA. Results generated have provided highly
specific detection of mRNA in cells in routinely
fixed tissues. This provides a solution to
demonstrating gene expression in tissues where
there is no suitable antibody for IHC.
RNAscope uses a methodology similar to FRET
whereby two independent (double Z) probes must
hybridise to the target sequence in tandem so that
signal amplification occurs.
Key features of the assay include:
• Single RNA detection limit – most sensitive in situ
assay. Unique ‘ZZ probe’ design for very high
specificity
• Manual workflow: 8 hours from slide to image
analysis. 3 weeks from target sequence to probe
for any gene, in any tissue, in any species
• Integrated QC assay to confirm presence of
detectable mRNA and degree of fixation
• Duplex chromogenic and multiplex fluorescence
readouts are available
• Fully automated option available on the EHP
lab’s Ventana Discovery ULTRA
To date we have successfully localised mRNA in
human cancer TMAs, formalin-fixed paraffin
embedded tissues, and cell pellets, with various
applications such as:
• Demonstration of LOXL2 gene expression
heterogeneity in human renal cancer
• Determining specific location of low abundance
biomarkers PDL-1 and ZFP36 in human lung
cancers
• Localising secreted proteins/transcription
factors, e.g. Ngn3 and differentiation markers,
e.g. glucagon, in mouse pancreas as well as
cytokines in mouse models of lung cancer
153
FLUORESCENCE ACTIVATED CELL SORTING
www.london-research-institute.org.uk/technologies/facs
Head
Derek Davies
The FACS Facility at the London Research Institute is a dedicated
scientific service offering an extensive flow cytometry analysis and
sorting facility. Flow cytometry is a sophisticated form of
fluorescence microscopy where cells in suspension pass one by one
through a laser beam and emitted fluorescence can be captured and
measured. Any part of a cell or any function of a cell that can be
tagged by a fluorochrome may be measured by flow cytometry,
which makes it an essential technique in many biological applications.
Staff
Laura Bazley
Julfa Begum
Joana Cerveira
Andy Filby
Carl Henderson
Sukhveer Purewal
Kirsty Sharrock
Equipment
Analytical cytometers
The FACS Facility has 9 analytical cytometers
including one plate-reading cytometer. These may
all be user-operated and we offer a one to one
training for all new users of the facility.
Masters Student
Thomas Scott
• FACS Calibur: 4 fluorescence detectors, 2 lasers
(488nm and 633nm)
• LSRII: 13 fluorescence detectors, 4 lasers
Cell sorters
These are able to retrieve up to six specifically
defined populations so that cells may be recovered
for re-culture, functional assays or RNA or DNA
recovery. Only members of the FACS Laboratory
operate the sorters but experiments are scheduled
and planned in close collaboration with our users.
Three of the sorters are housed in Class 2
Microbiological Safety Cabinets.
(355nm, 405nm, 488nm, 633nm)
• LSRII-SORP: 16 fluorescence detectors, 4 lasers
(405nm, 488nm, 561nm, 648nm)
• LSR Fortessa: 15 fluorescence detectors, 5 lasers
(355nm, 405nm, 488nm, 561nm, 638nm)
• Two LSR Fortessas: 18 fluorescence detectors,
5 lasers (355nm, 405nm, 488nm, 561nm, 638nm)
• MACSQuant VYB: 8 fluorescence detectors,
3 lasers (405nm, 488nm, 561nm)
• FlowSight Imaging flow cytometer, 10 colour
detection, 4 lasers (405nm, 488nm, 561nm,
640nm)
• Image Stream X Imaging flow cytometer: 12
channel detection with 4 lasers (405nm, 488nm,
561nm, 640nm)
Figure 1
The Amnis ImageStreamX Imaging
Flow cytometer, which allows
quantitation of fluorescence in
conjunction with cell imagery.
• MoFlo XDP: 9 fluorescence detectors, 4 lasers
(355nm, 488nm, 561nm or 648nm)
• FACS Aria III: 13 fluorescence detectors, 4 lasers
(405nm, 488nm, 561nm, 640nm)
• Influx: 14 fluorescence detectors, 4 lasers
(405nm, 488nm, 561nm, 640nm)
• FACS Aria Fusion: 13 fluorescence detectors,
4 lasers (405nm, 488nm, 561nm, 640nm)
Other services
The members of the FACS Facility are available to
provide advice on the design of experiments,
sourcing and supply of reagents, data analysis,
presentation and interpretation as well as
troubleshooting machines and experiments. We
also develop and introduce new techniques and
technologies that would be useful to our users. We
collaborate closely with the facility’s users and this
has led to several recent publications particularly
with groups involved in stem cell investigation,
imaging flow cytometry or where DNA analysis and
cell kinetic information is required.
154
HIGH THROUGHPUT SCREENING
www.london-research-institute.org.uk/technologies/high-throughput-screening
The High Throughput Screening Facility enables research groups to
access large-scale screening technologies. Primarily this takes the
form of genome-wide siRNA screens although other types of
screening are increasingly popular particularly screening with our
bespoke collection of well-characterised small molecule modulators.
Head
Michael Howell
Staff
Rachael Instrell
Ming Jiang
Rossella Rispoli
Becky Saunders
Summer Student
Silvia Benito
Last year we reported upgrades to our machinery
aimed at increasing our capacity and throughput.
Until now large-scale screening experiments have
been conducted using 96- or 384-well plate
formats. Even when using the 384-well format, a
genome-wide siRNA screen requires 200 such
plates and remains expensive. Although our
improved machinery makes it possible to utilise the
newer 1536-well format, there is still a question as
to whether some biological responses could be
compromised by this very small scale (Figure 1A).
This year we tested this format in a real world
screening setting.
We have previously conducted a genome-wide
siRNA screen in 384-well format for the
Developmental Signalling Group aimed at
identifying regulators of the TGF-β signalling
pathway where the activity of the pathway can be
monitored by the level of Smad2 accumulation in
the nucleus (Figure 1B). We repeated this screen in
the 1536-well plate format to see whether we
could identify any siRNA reagents that inhibited
the nuclear accumulation of Smad2 in response to
A
1536well
C
More importantly, there was a 50% overlap
between siRNA reagents significantly affecting
Smad2 accumulation in the two formats (Figure
1D). This degree of overlap is similar to what we,
and others, have observed when identical screens
in the same format, but conducted on separate
occasions, are compared. We conclude therefore
that this new format does not inherently
compromise the biology under observation.
Moreover, this format offers a viable approach to
screening cells with limited availability e.g. stem
cells, or conducting screens across many related
lines at reduced cost thereby opening up whole
new research possibilities.
B
384well
96well
2
R ~0.7
Raw cell no. replicate 2
Figure 1
A. The relative sizes of an
individual well from a 96-, 384and 1536-well plate.
B. Nuclear accumulation of Smad2
in HaCat cells in response to
TGF-β monitored by
immunofluorescence. From these
images, parameters such as cell
number per well (a measure of
viability) and Smad2 nuclear
intensity (a measure of Smad2
accumulation) are calculated.
C. Scatter plot comparing raw ‘cell
number per well’ measures for
each well across two replicates
from the 1536 genome screen.
The correlation coefficient of 0.7
indicates a satisfactory degree of
reproducibility.
D. Venn-diagram indicating the
overlap between siRNA reagents
identified as significantly
inhibiting TGF-β signalling (more
than 3 S.D.) in either the 1536- or
384-well format screens.
TGF-β in this format and if so whether hits from
such a screen bore any similarity to those
previously identified. When analysed in isolation,
the 1536-well screen data was internally consistent
and reproducible with replicates showing good
correlation with each other (Figure 1C).
Untreated
D
1536-well screen
209
Raw cell no. replicate 1
TGF-β treated
384-well screen
208
206
Number of siRNA reagents
inhibing TGF-β signalling
155
LIGHT MICROSCOPY
www.london-research-institute.org.uk/technologies/light-microscopy
We provide services in multi-dimensional imaging with fixed and live
specimens using confocal microscopy and low-light-level imaging.
Support is also available for image processing and motion analysis. In
addition, we pursue collaborative research in application and
development of light microscopy techniques.
Head
Daniel Zicha
Staff
Dominic Alibhai
Deborah Aubyn
Trevor Duhig
Peter Jordan
Alastair Nicol
Matthew Renshaw
Imaging technology overview
Laser scanning confocal microscopes
• LSM 880 (Zeiss)
• LSM 780s (Zeiss) including a multiphoton system
with a tuneable Chameleon Ultra II laser
(Coherent)
• LSM 710s (Zeiss)
• SP5 (Leica)
• LSM 510s (Zeiss)
• Swept Field Confocal microscope Opterra
(Bruker)
• UltraVIEW spinning-disk confocal imaging
system (PerkinElmer)
• low-light-level imaging systems based on
Metamorph software (Molecular Devices)
including a confocal high content screening
system Discovery 1 (Molecular Devices) and a
microinjection system (Eppendorf)
have been configured for contrast enhancement,
high resolution 3D and dynamic imaging of
biological specimens in multiple fluorescence
channels with optical sectioning using motorised
focus at multiple fields. Detailed information on
functionality of individual imaging systems is
presented on the Intranet. Image processing and
statistical analysis can be employed for
deconvolution, co-localisation, automatic or
interactive segmentation of cells and intracellular
structures, morphometry and tracking using
Figure 1
Images of Biotium CF TM 488A
labelled microtubules in type II
pneumocytes fixed with 4% PFA
produced with LSM 510 using a
plan apochromat 63x/1.4
objective lens. Time-lapse
sequence of Z-stacks was
acquired while temperature in
the microscope’s environmental
incubator was increasing. XY and
XZ projections from raw stacks
showed spatial drift, which was
corrected using the Huygens
Object Stabilizer. Red arrows
show positions of identical
structures before and after
correction. Plots of detected drift
were automatically produced
during the processing. Stack size
is 20 x 21 x 6 μm.
156
Huygens (SVI), Volocity (Improvision), Imaris
(Bitplane), AQM/ iQ (Kinetic Imaging/ Andor),
Metamorph (Universal Imaging), Fiji-ImageJ,
Mathematica (Wolfram Research), MATLAB
(MathWorks) and C.
New confocal imaging system
This year we introduced a state of the art inverted
laser scanning confocal microscope LSM 880 from
Zeiss. We are the first laboratory in the UK with this
technology. The LSM 880 has improved sensitivity
and speed due to a water cooling system and
upgraded electronics. Its updated software ZEN 2
features new flexible Experiment Designer.
New stabiliser software
This year we also introduced a new Huygens Object
Stabilizer from Scientific Volume Imaging (Figure
1). A range of algorithms suitable for different
situations is available.
Collaborative research highlights
We have developed bespoke software for
quantitative analysis of Fluorescence Loss In
Photobleaching (FLIP) for the specific requirements
in a collaborative project (Brownlow et al., 2014;
Nat Commun. 5:5658).
PEPTIDE CHEMISTRY
www.london-research-institute.org.uk/technologies/peptide-chemistry
Head
Nicola O’Reilly
Staff
Ganka Bineva-Todd
Stefania Federico
Dhira Joshi
The Peptide Chemistry Facility provides peptides and peptide arrays
to LRI scientists. We make peptides of many lengths and
modifications including biotin addition, dye labels, phosphorylation,
methylation, acetylation, isoprenylation, branched peptides and
peptides linked by disulphide bridges. We are keen to make unusual
peptides or peptide-based reagents, which can enable scientists to
further their research. We can aid in design of peptides and
conjugation and immunisation strategies of peptides for antibody
generation. We have around 20 peptides in stock that are used to
elute proteins from columns, prime immune cells and synchronise
yeast. This year we have synthesised the yeast mating factor M-factor.
Synthesis of M-factor
Conjugation in Schizosaccharomyces pombe is
controlled by the reciprocal action of mating
pheromones, p-factor and M-factor. M-factor,
released by cells of mating type Minus, is a
nonapeptide in which the C-terminal cysteine
residue is carboxyl-methylated and S-farnesylated.
M-factor was synthesised following the method of
synthesis for a-factor (O’Reilly et al., 2012; Yeast.
29:233-40).
Method: The synthesis of M-factor was performed
on a 433A synthesiser using Fmoc solid phase
peptide chemistry on a 2-Chlorotrityl resin. The
N-terminal amino acid was protected with a Boc
Figure 1
Schematic representation of the
synthesis of M-factor.
Figure 2
FACS data on the M-factor of
Schizosaccharomyces pombe cells
shows that even at the lower
concentration tested (0.1mg/ml)
the pheromone induced G1 arrest
(1c). In collaboration with
Francisco Navarro (Cell Cycle
Group).
1
group. The peptide was cleaved from the resin
using 1% TFA in DCM whilst all side chains and the
N-terminal were still protected. Next, the
C-terminal end of the peptide was methylated
using trimethylsilyldiazomethane. Following this,
side chain and N-terminal protecting groups were
removed using 95% TFA - 2.5% H2O - 2.5% TIS.
Finally the peptide was farnesylated using
farnesyl bromide then purified by RP-HPLC. A
schematic representation of the synthesis is
shown (Figure 1).
2
Boc-Tyr(OtBu)-Thr(OtBu)-Pro-Lys(tBoc)-Val-ProTyr(OtBu)-Met-Cys(Trt)-Cltrt Resin
1% TFA in DCM
Boc-Tyr(OtBu)-Thr(OtBu)-Pro-Lys(tBoc)-Val-ProTyr(OtBu)-Met-Cys(Trt)-OH
Trimethylsilyldiazomethane
Boc-Tyr(OtBu)-Thr(OtBu)-Pro-Lys(tBoc)-Val-ProTyr(OtBu)-Met-Cys(Trt)-OCH3
95% TFA - 2.5% H2O - 2.5% TIS
NH2-Tyr-Thr-Pro-Lys-Val-Pro-Tyr-Met-Cys-OCH3
Farnesyl Bromide
NH2-Tyr-Thr-Pro-Lys-Val-Pro-Tyr-Met-Cys(SFarnesyl)-OCH3
157
PROTEIN ANALYSIS AND PROTEOMICS
www.london-research-institute.org.uk/technologies/protein-analysis-and-proteomics
Head
Bram Snijders
Staff
Karin Barnouin
Vesela Encheva-Yokoya
Helen Flynn
David Frith
Andrew Jones
The Protein Analysis and Proteomics (PAP) Facility at Clare Hall
provides a wide variety of workflows for the analysis of proteins,
peptides and their post-translational modifications. The laboratory
houses two LTQ-Orbitrap instruments, a Q-Exactive and a 5800 Maldi
TOF/TOF instrument. We specialise in the following key areas:
1) Interaction proteomics, 2) Post-translational modifications,
3) Global and subcellular proteomics, 4) Targeted approaches,
5) Computational proteomics. In order to extract the maximum
amount of information from your sample we provide expertise in all
key workflow aspects including analytical design, wet lab procedures,
computational and statistical procedures.
Post-translational modifications
We have established workflows for the
investigation of post-translational modifications in
complex cell lysates or on purified proteins.
Typically, these workflows require PTM
enrichment steps using TiO2 or PTM specific
antibodies. Depending on the desired result we are
also able to employ fractionation strategies such as
strong-cation-exchange (SCX) chromatography or
off-line reverse phase chromatography. The
resulting GBs of data can contain hundreds of
thousands of spectra that are then subjected to
high performance computation and rigorous
statistical analysis for identification, quantification,
PTM localisation and FDR control.
Targeted approaches
Once peptide properties such as transitions and
retention times are known they can be used for
confirmation or follow-up experiments through
targeted SRM and PRM approaches. In 2014 PAP
increased its capabilities in this area through an
increased usage of Skyline software and the
implementation of Panorama server for storage of
spectral and chromatogram libraries. Further,
Figure 1
Log2 Ratio vs intensity plot of the
phosphoproteome of activated
raw cells. Erk phosphorylation
sites indicated in pink. Total
number of quantified sites is
14922. Plots generated using
Persues software.
1
Figure 2
Quantification of CDC7 peptides
with attomole sensitivity as
measured by a SIM/PRM scan.
158
targeted and multiplexed scans on the Q-Exactive
instrument demonstrated sensitivities and
specificities typical for western blot analysis
without the requirement for affinity reagents.
Interaction proteomics
Co-immunoprecipitation of proteins followed by
their identification by mass spectrometry is a
powerful strategy for the discovery of novel protein
complexes. Despite this, it can be challenging to
recognise bona fide interactors and absence/
presence of protein identification criterion alone.
Depending on the bait and the outcome of
preliminary experiments we apply quantitative
approaches based on chromatogram peak areas
(iBAQ, Silac) or spectral counting. Further, we
maintain databases of common contaminants that
provide an additional quality filter.
2014 also saw an increased integration with NIMR
and the development strategic model in
preparation for relocation of the facility to the
Crick Institute.
2
PROTEIN PURIFICATION
www.london-research-institute.org.uk/technologies/protein-purification
The Protein Purification Facility (PPF) supports and collaborates with
LRI-scientists in numerous aspects of research involving expression
and characterisation of recombinant proteins. These activities span
from early-stage project discussion relating to expression and
purification strategies through to hands-on pilot and productionscale involvement and, finally, generation of biophysical data.
Head
Svend Kjær
Staff
Annabel Borg
Roger Rajesh George
Sara Kisakye-Nambozo
Figure 1
Crystal structure of anti-RET Fab
fragment. Collaboration with Neil
McDonald and Andrew Purkiss.
PPF specialises in high-end expression
technologies such as baculovirus (BV)-mediated
protein expression in silkworm-derived insect cells
as well as transient transfection of suspension
culture HEK293 cells. Additionally, an S2 cell-line
(Drosophila melanogaster-derived) expression
system has been added recently. The S2 expression
system is particularly suited for secreted proteins.
For large-scale expression, two Minifors
bioreactors can be deployed. The purification
process has been advanced through
implementation of two AKTA Pure state of the art
chromatography workstations, which accelerate
workflows through automated and sequentially
linked multi-column setups. To identify buffer
conditions or small molecule additives, which
stabilise the purified proteins, PPF now routinely
applies the Thermofluor technology.
Subsequently, the PPF can either educate users in,
and/or perform various protein characterisation
techniques such as Multi-Angle Light Scattering
(MALS) for molecular weight determination,
Isothermal Calorimetry (ITC), Biolayer
Interferometry (BLI) and most recently Microscale
Thermophoresis (MST) as various means of
determining affinities of macromolecular
interactions.
The year 2014 at the PPF
In line with previous years, 2014, the final year as
PPF in its current incarnation, was very busy with
the demand for PPF main deliverable – BV-related
work – reaching 120-130 virus, including several
MultiBac projects. Additionally, the trimming of
certain upstream workflows has liberated
resources for purification and protein
characterisation and hence, the yearly output of
well-characterised proteins for downstream
applications such as crystallography, activity
measurements and small molecule screens was
unprecedented. Finally, the ‘Mab-to-Fab’ –
pipeline was finalised with crystallisation and
structural determination of an anti-RET Fab
fragment (expressed in S2 cells) in collaboration
with Neil McDonald (Figure 1).
PPF at the Francis Crick Institute
As of 1st of April 2015, the LRI becomes part of
the Francis Crick Institute and the PPF will join the
Structural Biology Science Technology Platform.
The platform will provide support to the structural
biology community and engage with nonstructural biologists in a variety of aspects of
biochemical and biophysical characterisation of
proteins.
159
TRANSGENICS
www.london-research-institute.org.uk/technologies/transgenics
Transgenic Services are a Core Facility of seven people providing the
skills, services and techniques relevant to supporting mouse genetics
at the institute. This can involve genetic modification techniques or
involve cryopreservation and rederivation to optimise the use of
available space and resource for this work.
Head
Ian Rosewell
Staff
Jessica Gruninger
Mary Ann Haskings
Natalia Karzakova
Natalia Moncaut
Jaroslaw Narloch
Sunita Varsani-Brown
Genetic modification has traditionally involved two
routes. One, the injection of the pronucleus of a
one cell stage embryo with DNA solution, providing
for random integration of sequence. Then,
targeted modifications have been possible via the
embryonic stem (ES) cell route that adds the
possibility of screening multiple clones to isolate
relatively rare homologous recombinants.
In the last two years genetic modification via
targeted delivery of nucleases has added a further
option. Nucleases can create double strand breaks
(DSB) in a pre-determined DNA sequence that
could for example remove gene expression. If the
DSB is repaired from a co-injected repair template,
scope for all forms of complex genetic
modifications become possible.
This is an exciting development that is already in
use across many research laboratories within the
LRI. In particular the use of CRISPR/Cas9 to deliver
nucleases to the genome suggests significant
advantages over current technologies and we have
been keen to advance its use with mice at the LRI.
Figure 1
Cytoplasmic injection of a one cell
stage fertilised mouse embryo to
achieve CRISPR/Cas9 gene editing.
Last year in collaboration with the Translational
Cancer Therapeutics laboratory a gene targeting
project in ES cells was the first to use CRISPR/Cas9
for generating a gene-targeted mouse. This year
has seen the first mice created by direct injection
of CRISPR/Cas9 RNA into the cytoplasm of a one
cell fertilised mouse embryo creating a ‘knockout’
mouse. Several further projects, for example to
create conditional knockout mice, are underway.
Another strand of development was prompted by a
seminar given in 2012 at Lincolns Inn Fields. Dr Jos
Jonkers from the Netherlands Cancer Institute
presented work that involved the generation of
‘chimera cohorts’. This work is highly relevant to
the use of mouse cancer model research at the LRI.
Where using such animal models would normally
have involved breeding multiple alleles into a
background strain requiring extensive, time
consuming breeding programmes. The alternative
is to use ES cells derived from strains bearing
complex allele combinations, to generate
experimental cohorts of chimeric mice from
blastocyst injection. This approach builds on our
expertise in generating and further modifying ES
cells, enabling experimental cohorts to be
produced with the potential for repeated cohorts.
An archive and discussion of such cell lines is
available at www.infrafrontier.eu.
160
FERMENTATION
IN VIVO IMAGING
www.london-research-institute.org.uk/
technologies/in-vivo-imaging
Fermentation
Alireza Alidoust
Namita Patel
The Fermentation Service is based at Clare Hall
Laboratories and provides pilot-plant scale
production of microbiological organisms for LRI
scientists. Batches of 10-100 L of yeast, bacteria or
nematodes can be produced under stringently
controlled conditions using four state of the art
fermenters. It is possible to run batch or fed-batch
cultures where additional nutrient or induction
chemicals can be added at any point during the
run. Computer control of the main growth
parameters (pH, temperature, dissolved oxygen,
aeration and agitation) allows a wide spectrum of
growth conditions and reproducibility.
The types of organisms we grow routinely are
yeasts (Saccharomyces cerevisiae,
Schizosaccharomyces pombe and Pichia pastoris),
the bacteria Escherichia coli and the nematode
Caenorhabditis elegans for whole cell production
or protein purification.
The service also offers breakage of cells using one
of three available methods:
• cell disrupter
• ball mill
• freezer mill
Structural, functional, cellular and molecular
imaging technologies have become pivotal to
better understand the development and
homeostatic regulation of normal and diseased
tissues and to assess the efficacy of new
therapeutic strategies in the context of
longitudinal studies. This is particularly true in
oncology but also applies to virtually any
biomedical research field.
The facility is equipped with 5 cutting edge imaging
modalities providing complementary information:
• Whole body bioluminescence and fluorescence
(IVIS Spectrum; PerkinElmer)
• Hybrid multiphoton/confocal microscope
(LSM710-NLO; Zeiss)
• X-ray micro-computed tomography (micro-CT;
Skyscan1176; Bruker)
• High resolution ultrasound (US; Vevo2100;
Visualsonics)
A number of exciting projects have developed this
year to image different types of cancer models as
well as new collaborations (Figure 1).
We are delighted to announce the establishment
of a partnership with the UCL Centre for Advanced
Biomedical Imaging (CABI) for the development of
the In Vivo Imaging capabilities of the Francis Crick
Institute. The CABI is a world class imaging centre,
which will contribute its expertise, notably to
establish MRI and nuclear imaging at the Francis
Crick Institute.
In Vivo Imaging
Francois Lassailly
May Zaw Thin
Fermentation Figure 1
New Brunswick fermenter
In Vivo Imagining Figure 1
A. Visualisation of individual human acute myeloid leukaemia
(AML) cells (green) developing around blood vessels (red)
within an intact bone (cyan) thanks to intravital microscopy.
Collaboration with Diana Passaro, Haematopoietic Stem Cell.
B. Image-guided injection for precise delivery of cells or (bio)
chemicals at a specific anatomical localisation using
ultrasound imaging.
Collaboration with Rute Ferreira, Mammalian Genetics.
C. Micro-CT phenotyping of pups (left: whole body; right:
calcified tissues (bones), where bone density has been
pseudocoloured).
Collaboration with Prof. Paul Gissen – GOSH/ICH-UCL.
D. A pilot micro-CT study performed in collaboration with
Owen Arthurs (GOSH and ICH-UCL) demonstrated the use of
new technology to improve perinatal, post-mortem clinical
diagnostic practice, showing detailed views of the morphology
of the heart chambers and great vessels.
A
C
B
D
161
PROTEIN STRUCTURE
technologies/protein-structure
X-ray crystallography can provide highly detailed
molecular structure of large molecular assemblies.
Elucidating the atomic details of molecular
interactions is particularly important to
understand the biological function of a protein/
enzyme at an atomic level.
Protein Structure
Stéphane Mouilleron
The Protein Structure Facility provides to any
biology group from the LRI the opportunity to
undertake structural studies of their favourite
proteins in-house at the LRI. This allows groups,
collaborative access to the state of the art high
throughput crystallisation robots and regular
access to the ESRF (Grenoble, France) and Diamond
synchrotron (Oxford, UK) to collect high quality
data from protein crystals.
We are currently collaborating with five LRI groups
and, so far, four of those collaborations lead to the
resolution of one or more crystal structures.
SUPER-RESOLUTION
MICROSCOPY
technologies/super-resolution-microscopy
Super-resolution microscopy continued to be an
important tool for research projects that study
how chromosomes segregate during cell division.
Errors in this process lead to too few or too many
chromosomes in daughter cells, which is a hallmark
of cancer progression in humans. Some of these
studies are done using yeast cells. Yeast is an
important model system. However, due to the
small size of yeast cells, it is difficult to image
molecules in these cells using conventional,
diffraction-limited microscopes. Super-resolution
microscopy has been used e.g. to measure the
amount of native chromatin condensation in yeast
cells. This is not possible using a confocal
microscope.
One of the most recent super-resolution
microscopy projects studies centrosomal proteins
such as Msd1 and Msd2. Anomalous centrosomes
are often present in tumour cells so it is important
to better understand the structure and function of
centrosomes. High-resolution imaging has
revealed new features in how the centrosome is
organised at the molecular level.
Super-Resolution Microscopy
Anne Vaahtokari
Protein Structure Figure 1
Protein crystal structures solved by the Protein Structure
Facility.
Super-Resolution Microscopy Figure 1
Centrosomes of asynchronously growing HeLa cells stained
with the indicated combinations of antibodies and visualised
using either a structured illumination super-resolution (OMX)
or deconvolution (DeltaVision) microscope. Scale bar, 1 μm.
Courtesy of Akiko Nishi, Cell Regulation Laboratory.
162
RESEARCH
PUBLICATIONS
THESES SUBMITTED 2014
163
Facundo Batista (page 66)
Lymphocyte Interaction
Primary Research Paper
Burbage M, Keppler SJ, Gasparrini F,
Martinez-Martín N, Gaya M, Feest C, Domart MC,
Brakebusch C, Collinson L, Bruckbauer A, Batista FD.
Cdc42 is a key regulator of B cell differentiation and is
required for antiviral humoral immunity. J Exp Med.
2014;doi:10.1084/jem.20141143
Schülein-Völk C, Wolf E, Jing Zhu, Xu W, Taranets L,
Hellmann A, Jänicke LA, Diefenbacher ME,
Behrens A, Eilers M, Popov N. Dual regulation of
Fbw7 function and oncogenic transformation by
Usp28. Cell Rep. 2014;9(3):1099-109
Zhang T, Cronshaw J, Kanu N, Snijders AP, Behrens A.
UBR5-mediated ubiquitination of ATMIN is required
for ionizing radiation-induced ATM signaling and
function. Proc Natl Acad Sci USA.
2014;111(33):12091-6
Other Publication
Feest C, Bruckbauer A, Batista FD. B Cell Receptor
Signaling. Cell Membrane Nanodomains: From
Biochemistry to Nanoscopy by Cambi A and Lidke DS.
CRC Press. 2014;ISBN 9781482209891
Other Publication
Behrens A, van Deursen JM, Rudolph KL,
Schumacher B. Impact of genomic damage and ageing
on stem cell function. Nat Cell Biol. 2014;16(3):201-7
Axel Behrens (page 68)
Dominique Bonnet (page 70)
Mammalian Genetics
Primary Research Papers
Schülein-Völk C, Nye E, Spencer-Dene B, Jaenicke LA,
Eilers M, Behrens A. The deubiquitinase USP28
controls intestinal homeostasis and promotes
colorectal cancer. J Clin Invest. 2014;124(8):3407-18
Griessinger E, Anjos-Afonso F, Pizzitola I,
Rouault-Pierre K, Vargaftig J, Taussig D, Gribben J,
Lassailly F, Bonnet D. A niche-like culture system
allowing the maintenance of primary human acute
myeloid leukemia-initiating cells: A new tool to
decipher their chemoresistance and self-renewal
mechanisms. Stem Cells Transl Med. 2014;3(4):520-9
Sancho R, Gruber R, Gu G, Behrens A. Loss of Fbw7
reprograms adult pancreatic ductal cells into α, δ, and
β cells. Cell Stem Cell. 2014;15(2):139-53
Pizzitola I, Anjos-Afonso F, Rouault-Pierre K,
Lassailly F, Tettamanti S, Spinelli O, Biondi A, Biagi E,
Bonnet D. Chimeric Antigen Receptors against CD33/
CD123 antigens efficiently target primary Acute
Myeloid Leukemia cells in vivo. Leukemia.
2014;28(8):1596-605
Other Publications
Anjos-Afonso F, Bonnet D. Forgotten gems: human
CD34(-) hematopoietic stem cells. Cell Cycle.
2014;3(4):503-4
Tettamanti S, Biondi A, Biagi E, Bonnet D. CD123 AML
targeting by chimeric antigen receptors: A novel
magic bullet for AML therapeutics? Oncoimmunology.
2014;3:e28835
Zhao W, Phinney DG, Bonnet D, Dominici M,
Krampera M. Mesenchymal stem cell biodistribution,
migration, and homing in vivo. Stem Cells Int.
2014;2014;292109
Co-staining for CK19 (red) and insulin (green) in an Fbw7-deficient
pancreatic duct.
164
Julian Downward (page 74)
Signal Transduction
de Bruin EC, Cowell C, Warne PH, Jiang M,
Saunders RE, Melnick MA, Gettinger S, Walther Z,
Wurtz A, Heynen GJ, Heideman DA, Gómez-Román J,
Bernards R, Smit EF, Politi K, Downward J. Reduced
NF1 expression confers resistance to EGFR inhibition
in lung cancer. Cancer Discov. 2014;4(5)606-19
Endesfelder D, Burrell R, Kanu N, McGranahan N,
Howell M, Parker PJ, Downward J, Swanton C,
Kschischo M. Chromosomal instability selects gene
copy number variants encoding core regulators of
proliferation in ER+ breast cancer. Cancer Res.
2014;74(17):4853-63
Murillo MM, Zelenay S, Nye E, Castellano E,
Lassailly F, Stamp G, Downward J. RAS interaction
with PI3K p110α is required for tumor-induced
angiogenesis. J Clin Invest. 2014;124(8):3601-11
Wang Y, Bu F, Royer C, Serres S, Larkin JR, Soto MS,
Sibson NR, Salter V, Fritzsche F, Turnquist C, Koch S,
Zak J, Zhong S, Wu G, Liang A, Olofsen PA, Moch H,
Hancock DC, Downward J, Goldin RD, Zhao J, Tong X,
Guo Y, Lu X. ASPP2 controls epithelial plasticity and
inhibits metastasis through β-catenin-dependent
regulation of ZEB1. Nat Cell Biol. 2014;16(11):1092-104
Other Publications
Downward J. RAS’s cloak of invincibility slips at last?
Cancer Cell. 2014;25(1):5-6
Geraghty RJ, Capes-Davis A, Davis JM, Downward J,
Freshney RI, Knezevic I, Lovell-Badge R, Masters JR,
Meredith J, Stacey GN, Thraves P, Vias M. Guidelines
for the use of cell lines in biomedical research. Br J
Cancer. 2014;111(6):1021-46
Holger Gerhardt (page 76)
Vascular Biology
Bentley K, Franco CA, Philippides A, Blanco R,
Dierkes M, Gebala V, Stanchi F, Jones M, Aspalter IM,
Cagna G, Weström S, Claesson-Welsh L,
Vestweber D, Gerhardt H. The role of differential
VE-cadherin dynamics in cell rearrangement during
angiogenesis. Nat Cell Biol. 2014;16(4):309-21
Bernabeu MO, Jones ML, Nielsen JH, Krüger T,
Nash RW, Groen D, Schmieschek S, Hetherington J,
Gerhardt H, Franco CA, Coveney PV. Computer
simulations reveal complex distribution of
haemodynamic forces in a mouse retina model of
angiogenesis. J R Soc Interface. 2014;11(99):20140543
Fan J, Ponferrada VG, Sato T, Vemaraju S, Fruttiger M,
Gerhardt H, Ferrara N, Lang RA. Crim1 maintains
retinal vascular stability during development by
regulating endothelial cell Vegfa autocrine signaling.
Development. 2014;141(2):448-59
Maes H, Kuchnio A, Peric A, Moens S, Nys K,
De Bock K, Quaegebeur A, Schoors S, Georgiadou M,
Wouters J, Vinckier S, Vankelecom H, Garmyn M,
Vion AC, Radtke F, Boulanger C, Gerhardt H,
Dejana E, Dewerchin M, Ghesquière B, Annaert W,
Agostinis P, Carmeliet P. Tumor vessel normalization
by chloroquine independent of autophagy. Cancer
Cell. 2014;26(2):190-206
Mleynek TM, Chan AC, Redd M, Gibson CC, Davis CT,
Shi DS, Chen T, Carter KL, Ling J, Blanco R,
Gerhardt H, Whitehead K, Li DY. Lack of CCM1 induces
hypersprouting and impairs response to flow. Hum
Mol Genet. 2014;23(23):6223-34
Schoors S, De Bock K, Cantelmo AR, Georgiadou M,
Ghesquière B, Cauwenberghs S, Kuchnio A,
Wong BW, Quaegebeur A, Goveia J, Bifari F, Wang X,
Blanco R, Tembuyser B, Cornelissen I, Bouché A,
Vinckier S, Diaz-Moralli S, Gerhardt H, Telang S,
Cascante M, Chesney J, Dewerchin M, Carmeliet P.
Partial and transient reduction of glycolysis by PFKFB3
blockade reduces pathological angiogenesis. Cell
Metab. 2014;19(1):37-48
Wälchli T, Mateos JM, Weinman O, Babic D, Regli L,
Hoerstrup SP, Gerhardt H, Schwab ME, Vogel J.
Quantitative assessment of angiogenesis,perfused
blood vessel and endothelial tip cells in postnatal
mouse brain. Nat Protoc. 2014;doi: 10.1038/
nprot.2015.002
Other Publication
Wacker A, Gerhardt H, Phng LK. Tissue guidance
without filopodia. Commun Integr Biol. 2014;7:e28820
165
Nathan Goehring (page 78)
Nicholas Luscombe (page 84)
Computational Biology
Trong PK, Nicola EM, Goehring NW, Kumar KV,
Grill SW. Parameter-space topology of models for cell
polarity. New J Phys. 2014;16:065009
Castelnuovo M, Zaugg JB, Guffanti E, Maffioletti A,
Camblong J, Xu Z, Clauder-Münster S, Steinmetz LM,
Luscombe NM, Stutz F. Role of histone modifications
and early termination in pervasive transcription and
antisense-mediated gene silencing in yeast. Nucleic
Acids Res. 2014;42(7):4348-62
Other Publication
Goehring NW. PAR polarity: From complexity to
design principles. Exp Cell Res. 2014;328(2):258-66
Adrian Hayday (page 80)
Ilaria Malanchi (page 86)
Tumour Host Interaction
Immuno Surveillance
Gibbons D, Fleming P, Virasami A, Michel ML,
Sebire NJ, Costeloe K, Carr R, Klein N, Hayday A.
Interleukin-8 (CXCL8) production is a signatory T cell
effector function of human newborn infants. Nat
Med. 2014;20(10):1206-10
Other Publication
Ombrato L, Malanchi I. The EMT universe: Space
between cancer cell dissemination and metastasis
initiation. Crit Rev Oncog. 2014;19(5):349-61
Vantourout P, Willcox C, Turner A, Swanson CM,
Haque Y, Sobolev O, Grigoriadis A, Tutt A, Hayday A.
Immunological visibility: posttranscriptional
regulation of human NKG2D ligands by the EGF
receptor pathway. Sci Transl Med.
2014;6(231):231ra49
Other Publications
Hayday A, Vantourout P. A long-playing CD about the
γδ TCR repertoire. Immunity. 2014;39(6):994-6
Vantourout P, Hayday A. Regulation of immunological
visibility by the EGF receptor. Med Sci (Paris).
2014;30(8-9):742-4
Caroline Hill (page 82)
Developmental Signalling
Other Publications
Gaarenstroom T, Hill CS. TGF-β signaling to
chromatin: How Smads regulate transcription during
self-renewal and differentiation. Semin Cell Dev Biol.
2014;32:107-18
Vizán P, Miller DSJ, Schmierer B, Hill CS. Response to
comment on ‘Controlling long-term signaling:
Receptor dynamics determine attenuation and
refractory behavior of the TGF-β pathway’-Smad2/3
activity does not predict the dynamics of
transcription. Sci Signal. 2014;7(344):lc2
166
The murine small intestinal epithelium is normally rich in γδ T cells
expressing T cell receptors (TCRs) composed of Vγ7Vδ4 chains not
found anywhere else in the body and detected by confocal
microscopy of the gut (pink-stained cells; top panel). Conversely,
an almost total loss of such signatory intestinal T cells (lower
panel) in mice mutant in a single intestinal epithelium-specific
member of the B7 supergene family, permits the identification of
the first, epithelial regulator of a gut-specific T cell compartment
potentially involved in immune-surveillance.
Key: pink – Vγ7+Vδ4+ cells; orange – Vγ7-Vδ4+ cells;
blue – Vγ7+Vδ4- cells; white – Vγ7-Vδ4- γδ cells; green – αβ T cells
Neil McDonald (page 88)
Structural Biology
Peter Parker (page 92)
Fisher R, Horswell S, Rowan A, Salm MP, de Bruin EC,
Gulati S, McGranahan N, Stares M, Gerlinger M,
Varela I, Crockford A, Favero F, Quidville V, André F,
Navas C, Grönroos E, Nicol D, Hazell S, Hrouda D,
O Brien T, Matthews N, Phillimore B, Begum S,
Rabinowitz A, Biggs J, Bates PA, McDonald NQ,
Stamp G, Spencer-Dene B, Hsieh JJ, Xu J, Pickering L,
Gore M, Larkin J, Swanton C. Development of
synchronous VHL syndrome tumors reveals
contingencies and constraints to tumor evolution.
Genome Biol. 2014;15(8):433
Brownlow N, Pike T, Zicha D, Collinson L, Parker PJ.
PKCepsilon-regulated pathway. Nat Commun.
2014; 5:5685
Goodman KM, Kjær S, Beuron F, Knowles PP,
Nawrotek A, Burns EM, Purkiss AG, George R,
Santoro M, Morris EP, McDonald NQ. RET recognition
of GDNF-GFRα1 ligand by a composite binding site
promotes membrane-proximal self-association. Cell
Rep. 2014;8(6):1894-904
Gatliff J, East D, Crosby J, Abeti R, Harvey R,
Craigen W, Parker PJ, Campanella M. TSPO interacts
with VDAC1 and triggers a ROS-mediated inhibition of
mitochondrial quality control. Autophagy.
2014;10(12):2279-96
Plaza-Menacho I, Barnouin K, Goodman K,
Martínez-Torres RJ, Borg A, Murray-Rust J,
Mouilleron S, Knowles P, McDonald NQ. Oncogenic
RET kinase domain mutations perturb the
autophosphorylation trajectory by enhancing
substrate presentation in trans. Mol Cell.
2014;53(5):738-51
Other Publications
Linch M, Riou P, Claus J, Cameron AJ, de Naurois J,
Larijani B, Ng T, McDonald NQ, Parker PJ. Functional
implications of assigned, assumed and assembled PKC
structures. Biochem Soc Trans. 2014;42(1):35-41
Plaza-Menacho I, Mologni L, McDonald NQ.
Mechanisms of RET signaling in cancer: current and
future implications for targeted therapy. Cell Signal.
2014;26(8):1743-52
Paul Nurse/Jacqueline Hayles (page 90)
Cell Cycle
Wu PY, Nurse P. Replication origin selection regulates
the distribution of meiotic recombination. Mol Cell.
2014;53(4):655-62
Other Publication
Nurse P. EMBO at 50. Science. 2014;343(6167):117
Endesfelder D, Burrell RA, Kanu N, McGranahan N,
copy-number variants encoding core regulators of
2014;74(17):4853-63
Kiuchi T, Kiuchi T, Ortiz-Zapater E, Monypenny J,
Matthews DR, Nguyen LK, Barbeau J, Coban O,
Lawler K, Burford B, Rolfe DJ, de Rinaldis E, Dafou D,
Simpson MA, Woodman N, Pinder S, Gillett CE,
Devauges V, Poland SP, Fruhwirth G, Marra P,
Boersma YL, Plückthun A, Gullick WJ, Yarden Y,
Santis G, Winn M, Kholodenko BN,
Martin-Fernandez ML, Parker PJ, Tutt A,
Ameer-Beg SM, Ng T. The ErbB4 CYT2 variant protects
EGFR from ligand-induced degradation to enhance
cancer cell motility. Sci Signal. 2014; 7(339): ra78
Ménard L, Parker PJ, Kermorgant S. Receptor
tyrosine kinase c-Met controls the cytoskeleton from
different endosomes via different pathways. Nat
Commun. 2014;5:3907
Rossé C, Lodillinsky C, Fuhrmann L, Nourieh M,
Monteiro P, Irondelle M, Lagoutte E, Vacher S,
Waharte F, Paul-Gilloteaux P, Romao M,
Sengmanivong L, Linch M, van Lint J, Raposo G,
Vincent-Salomon A, Bièche I, Parker PJ, Chavrier P.
Control of MT1-MMP transport by atypical PKC during
breast-cancer progression. Proc Natl Acad Sci USA.
2014;111(18):E1872-9
Veeriah S, Leboucher P, de Naurois J, Jethwa N,
Nye E, Bunting T, Stone R, Stamp G, Calleja V,
Jeffrey SS, Parker PJ, Larijani B. High-throughput
time-resolved FRET reveals Akt/PKB activation as a
poor prognostic marker in breast cancer. Cancer Res.
2014;74(18):4983-95
167
Other Publications
Brownlow N, Pike T, Crossland V, Claus J, Parker PJ.
Regulation of the cytokinesis cleavage furrow by
PKCepsilon. Biochem Soc Trans. 2014;42(6):1534-37
Calleja V, Laguerre M, de Las Heras-Martinez G,
Parker PJ, Requejo-Isidro J, Larijani B. Acute
regulation of PDK1 by a complex interplay of
molecular switches. Biochem Soc Trans.
2014;42(5):1435-40
Claus J, Patel G, Ng T, Parker PJ. A role for the
pseudokinase HER3 in the acquired resistance against
EGFR- and HER2-directed targeted therapy. Biochem
Soc Trans. 2014;42(4):831-6
Martin-Liberal J, Cameron AJ, Claus J, Judson IR,
Parker PJ, Linch M. Targeting protein kinase C in
sarcoma. Biochim Biophys Acta. 2014;1846(2):547-59
Parker PJ, Justilien V, Riou P, Linch M, Fields AP.
Atypical protein kinase Cι as a human oncogene and
therapeutic target. Biochem Pharmacol.
2014;88(1):1-11
Caetano Reis e Sousa (page 94)
Immunobiology
Dendritic cells control fibroblastic reticular network
tension and lymph node expansion. Nature.
2014;14(7523):498-502
Bär E, Whitney PG, Moor K, Reis e Sousa C,
LeibundGut-Landmann S. IL-17 regulates systemic
fungal immunity by controlling the functional
competence of NK cells. Immunity. 2014;40(1):
117-27
Deddouche S, Goubau D, Rehwinkel J, Chakravarty P,
Begum S, Maillard PV, Borg AP, Matthews N, Feng Q,
van Kuppeveld FJM, Reis e Sousa C. Identification of
an LGP2-associated MDA5 agonist in picornavirusinfected cells. Elife. 2014;3:e01535
Goubau D, Schlee M, Deddouche S, Pruijssers AJ,
Zillinger T, Goldeck M, Schuberth C,
Van der Veen AG, Fujimura T, Rehwinkel J,
Iskarpatyoti JA, Barchet W, Ludwig J, Dermody TS,
Hartmann G, Reis e Sousa C. Antiviral immunity via
RIG-I-mediated recognition of RNA bearing
5’-diphosphates. Nature. 2014;514(7522):372-5
Li J, Ahmet F, Sullivan LC, Brooks AG, Kent SJ,
De Rose R, Salazar AM, Reis e Sousa C, Shortman K,
Lahoud MH, Heath WR, Caminschi I. Antibodies
targeting Clec9A promote strong humoral immunity
without adjuvant in mice and non-human primates.
Eur J Immunol. 2014;doi: 10.1002/eji.201445127
van Blijswijk J, Schraml BU, Rogers NC, Whitney PG,
Zelenay S, Acton SE, Reis e Sousa C. Altered lymph
node composition in diphtheria toxin receptor-based
mouse models to ablate dendritic cells. J Immunol.
2014;doi: 10.4049/jimmunol.1401999
Whitney PG, Bär E, Osorio F, Rogers NC, Schraml BU,
Deddouche S, LeibundGut-Landmann S,
Reis e Sousa C. Syk signaling in dendritic cells
orchestrates innate resistance to systemic fungal
infection. PLoS Pathog. 2014;10(7):e1004276
Breast cancer (bottom right area) and adjacent normal tissue
(top left).
168
Other Publications
Reis e Sousa C, Unanue ER. Antigen processing.
Curr Opin Immunol. 2014;26:138-9
Martin Singleton (page 100)
Schraml BU, Reis e Sousa C. Defining dendritic cells.
Curr Opin Immunol. 2014;doi: 10.1016/j.
coi.2014.11.001
Drechsler H, McHugh T, Singleton MR, Carter NJ,
McAinsh AD. The Kinesin-12 Kif15 is a processive
track-switching tetramer. Elife. 2014;3,e01724
Erik Sahai (page 96)
Thomas Surrey (page 102)
Tumour Cell Biology
2014;514(7523):498-502
Manning CS, Hooper S, Sahai EA. Intravital imaging of
SRF and Notch signalling identifies a key role for EZH2
in invasive melanoma cells. Oncogene.
2014;doi:10.1038/onc.2014.362
Other Publications
Charras G, Sahai E. Physical influences of the
extracellular environment on cell migration. Nat Rev
Mol Cell Biol. 2014;15(12):813-24
Hirata E, Park D, Sahai E. Retrograde flow of
cadherins in collective cell migration. Nat Cell Biol.
2014;16(7):621-3
Huttenlocher A, Sahai E. Editorial overview: cell
adhesion and migration. Curr Opin Cell Biol. 2014;
30:v-vi
Baumann H, Surrey T. Motor-mediated cortical versus
astral microtubule organisation in lipid-monolayered
droplets. J Biol Chem. 2014;89(32):22524-35
Duellberg C, Trokter M, Jha R, Sen I, Steinmetz MO,
Surrey T. Reconstitution of a hierarchical +TIP
interaction network controlling microtubule end
tracking of the human dynein complex. Nat Cell Biol.
2014;16(8):804-11
Maurer SP, Cade NI, Bohner G, Gustafsson N,
Boutant E, Surrey T. EB1 accelerates two
conformational transitions important for
microtubule maturation and dynamics. Curr Biol.
2014;24(4):372-84
Other Publications
Fourniol FJ, Li TD, Bieling P, Mullins RD, Fletcher DA,
Surrey T. Micropattern-guided assembly of
overlapping pairs of dynamic microtubules. Methods
Enzymol. 2014;540:339-60
Maurer SP, Fourniol FJ, Hoenger A, Surrey T. Seeded
microtubule growth for cryoelectron microscopy of
end-binding proteins. Methods Mol Biol.
2014;1136:247-60
Paola Scaffadi (page 98)
Cancer Epigenetics
Ben-David U, Biran A, Scaffidi P, Herold-Mende C,
Boehringer M, Meshorer E, Benvenisty N. Elimination
of undifferentiated cancer cells by pluripotent stem
cell inhibitors. J Mol Cell Biol. 2014;6(3):267-9
Fernandez P, Scaffidi P, Markert E, Lee J, Rane R,
Misteli T. Transformation resistance in a premature
aging disorder identifies a tumor-protective function
of BRD4. Cell Rep. 2014;9(1):248-60
169
Charles Swanton (page 104)
Translational Cancer Therapeutics
de Bruin E, McGranahan N, Mitter R, Salm M,
Wedge DC, Yates L, Jamal-Hanjani M, Shafi S,
Murugaesu N, Rowan AJ, Gronroos E,
Campbell P, Swanton C. Spatial and temporal diversity
in genomic instability processes defines lung cancer
evolution. Science. 2014; 346(6206):251-6
Dewhurst SM, McGranahan N, Burrell RA, Rowan AJ,
Grönroos E, Endesfelder D, Joshi T, Mouradov D,
Gibbs P, Ward RL, Hawkins NJ, Szallasi Z, Sieber OM,
Swanton C. Tolerance of whole-genome doubling
propagates chromosomal instability and accelerates
cancer genome evolution. Cancer Discov.
2014;4(2):175-85
2014;74(17):4853-63
Genome Biol. 2014;15(8):433
Gerlinger M, Horswell S, Larkin J, Rowan AJ,
Salm MP, Varela I, Fisher R, McGranahan N,
Matthews N, Santos CR, Martinez P, Phillimore B,
Begum S, Rabinowitz A, Spencer-Dene B, Gulati S,
Bates PA, Stamp G, Pickering L, Gore M, Nicol DL,
Hazell S, Futreal PA, Stewart A, Swanton C. Genomic
architecture and evolution of clear cell renal cell
carcinomas defined by multiregion sequencing. Nat
Genet. 2014;46(3):225-33
170
Gulati S, Martinez P, Joshi T, Birkbak N, Santos CR,
Rowlan AJ, Pickering L, Gore M, Larkin J, Szallasi Z,
Bates PA, Swanton C, Gerlinger M. Systematic
evaluation of the prognostic impact and intratumour
heterogeneity of clear cell renal cell carcinoma
biomarkers. Eur Urol. 2014;66(5):936-48
Lönnstedt IM, Caramia F, Li J, Fumagalli D, Salgado R,
Rowan A, Salm M, Kanu N, Savas P, Horswell S,
Gade S, Loibl S, Neven P, Sotiriou C, Swanton C, Loi S,
Speed TP. Deciphering clonality in aneuploid breast
tumors using SNP array and sequencing data. Genome
Biol. 2014;15(9):470.
Molnár J, Póti A, Pipek O, Krzystanek M, Kanu N,
Swanton C, Tusnády GE, Szállási Z, Csabai I, Szüts D.
The genome of the chicken DT40 bursal lymphoma cell
line. G3 (Bethesda). 2014;4(11):2231-40
Ng CK, Weigelt B, A’Hern R, Bidard FC, Lemetre C,
Swanton C, Shen R, Reis-Filho JS. Predictive
performance of microarray gene signatures: impact of
tumor heterogeneity and multiple mechanisms of
drug resistance. Cancer Res. 2014;74(11):2946-61
Roylance R, Endesfelder D, Jamal-Hanjani M,
Burrell RA, Gorman P, Sander J, Murphy N,
Birkbak NJ, Hanby AM, Speirs V, Johnston SR,
Kschischo M, Swanton C. Expression of regulators of
mitotic fidelity are associated with intercellular
heterogeneity and chromosomal instability in primary
breast cancer. Breast Cancer Res Treat.
2014;148(1):221-9
Staples CJ, Myers KN, Beveridge RD, Patil AA,
Howard AE, Barone G, Lee AJ, Swanton C, Howell M,
Maslen S, Skehel JM, Boulton SJ, Collis SJ. Ccdc13 is a
novel human centriolar satellite protein required for
ciliogenesis and genome stability. J Cell Sci.
2014;127(Pt 13):2910-9
Other Publications
Bakhoum SF, Swanton C. Chromosomal instability,
aneuploidy, and cancer. Front Oncol. 2014;4:161
Burrell RA, McClelland SE, Bartek J, Swanton C.
Response to Bakhoum et al. Curr Biol. 2014;24(4):R150
Burrell RA, Swanton C. The evolution of the unstable
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Davies H, Denison A, Djearman M, Goldman J,
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Electron Microscopy (page 151)
Lucy Collinson
PKCε-regulated pathway. Nature Commun.
2014;5:5685
Burbage M, Keppler SJ, Gasparrini F,
Martinez-Martín N, Gaya M, Feest C, Domart MC,
Brakebusch C, Collinson L, Bruckbauer A, Batista FD.
Cdc42 is a key regulator of B cell differentiation and is
required for antiviral humoral immunity. J Exp Med.
2014;doi: 10.1084/jem.20141143
Bush M, Ghosh T, Sawicka M, Moal IH, Bates PA,
Dixon R, Zhang X. The structural basis for enhancerdependent assembly and activation of the AAA
transcriptional activator NorR. Mol Microbiol. 2014;
doi: 10.1111/mmi.12844
Genome Biol. 2014;15(8):433
False coloured SEM of cell cultured lung cancer cells.
179
Peddie CJ, Blight K, Wilson E, Melia C, Marrison J,
Carzaniga R, Domart MC, O’Toole P, Larijani B,
Collinson LM. Correlative and integrated light and
electron microscopy of in-resin GFP fluorescence,
used to localise diacylglycerol in mammalian cells.
Russell MRG, West M, Peddie CJ, Collinson LM.
3D electron microscopy of cells across scales: electron
tomography and serial block face scanning electron
microscopy. Cell Imaging: Methods Express. Ed.
Stephens, Scion Publishing. 2014.
Simão D, Pinto C, Piersanti S, Weston A, Peddie CJ,
Bastos AE, Licursi V, Schwarz SC, Collinson L,
Salinas S, Serra M, Teixeira AP, Saggio I,Lima PA,
Kremer EJ, Schiavo G, Brito C, Alves PM. Modeling
human neural functionality in vitro: 3D culture for
dopaminergic differentiation. Tissue Eng Part A.
2014;doi:10.1089/ten.tea.2014.0079
Experimental Histopathology (page 153)
Terenzio M, Golding M, Russell MRG, Wicher K,
Rosewell I, Spencer-Dene B, Ish-Horowicz D,
Schiavo G. Bicaudal-D1 regulates the intracellular
sorting and signalling of neurotrophin receptors.
EMBO J. 2014;33(14):1582-98
Other Publications
Carzaniga R, Domart MC, Duke E, Collinson LM.
Correlative cryo-fluorescence and cryo-soft X-ray
tomogarphy of adherent cells at European
synchrotrons. Methods Cell Biol. 2014;124:151-78
Duke E, Dent K, Razi M, Collinson LM. Biological
applications of cryo-soft X-ray tomography. J Microsc.
2014;255(2):65-7
Larijani B, Hamati F, Kundu A, Chung GC, Domart MC,
Collinson LM, Poccia DL. Principle of duality in
phospholipids: regulators of membrane morphology
and dynamics. Biochem Soc Trans. 2014;42(5):1335-42
Patwardhan A, Ashton A, Brandt R, Butcher S,
Carzaniga R, Chiu W, Collinson LM, Doux P, Duke E,
Ellisman M, Franken E, Grünewald K, Heriche JK,
Koster A, Kühlbrandt W, Lagerstedt I, Larabell C,
Lawson CL, Saibil HR, Sanz-García E, Subramaniam S,
Verkade P, Swedlow JR, and Kleywegt GJ. A 3D
cellular context for the macromolecular world. Nat
Struct Mol Biol. 2014;21(10):841-5
Peddie CJ, Liv N, Hoogenboom JP, Collinson LM.
Integrated light and scanning electron microscopy
of GFP-expressing cells. Methods Cell Biol.
2014;124:363-89
Peddie CJ, Collinson LM. Exploring the third
dimension: volume electron microscopy comes of
age. Micron. 2014;61:9-19
180
Gordon Stamp
2014;514(7523):498-502
cancer evolution. Science. 2014; 346(6206):251-6
Schülein-Völk C, Nye E, Spencer-Dene B,
Jaenicke LA, Eilers M, Behrens A. The deubiquitinase
USP28 controls intestinal homeostasis and promotes
colorectal cancer. J Clin Invest. 2014;124(8):3407-18
Genome Biol. 2014;15(8):433
Genet. 2014;46(3):225-33
Terenzio M, Golding M, Russell MR, Wicher KB,
EMBO J. 2014;33(14):1582-98
Jeffrey SS, Parker PJ, Larijani B. High throughput
time-resolved-FRET reveals Akt/PKB activation as a
2014;74(18):4983-95
Zhuang Z, Frerich JM, Huntoon K, Yang C, Merrill MJ,
Abdullaev Z, Pack SD, Shively SB, Stamp G,
Lonser RR. Tumor derived vasculogenesis in von
Hippel-Lindau disease-associated tumors. Sci Rep.
2014;4:4102
Fluorescence-Activated Cell
Sorting (page 154)
Derek Davies
Filby A. “Mega” cytometry for a “mega” challenging
cell type. Cytometry A. 2014;85(4):289-91
Sage EK, Kolluri KK, McNulty K, Lourenco Sda S,
Kalber TL, Ordidge KL, Davies D, Gary Lee YC,
Giangreco A, Janes SM. Systemic but not topical
TRAIL-expressing mesenchymal stem cells reduce
tumour growth in malignant mesothelioma. Thorax.
2014;69(7):638-47
Sarbajna S, Davies D, West SC. Roles of SLX1-SLX4,
MUS81-EME1, and GEN1 in avoiding genome
instability and mitotic catastrophe. Genes Dev.
2014;28(10):1124-36
Drosophila wing imaginal discs expressing expanded-GFP (green)
and ubi-RFP (red) and stained with DAPI (blue).
High Throughput Screening (page 155)
Michael Howell
de Bruin EC, Cowell C, Warne PH, Jiang M,
Saunders RE, Melnick MA, Gettinger S, Walther Z,
Wurtz A, Heynen GJ, Heideman DA, Gómez-Román J,
Bernards R, Smit EF, Politi K, Downward J. Reduced
NF1 expression confers resistance to EGFR inhibition
in lung cancer. Cancer Discov. 2014;4(5):606-19
2014;74(17):4853-63
Staples CJ, Myers KN, Beveridge RD, Patil AA,
Howard AE, Barone G, Lee AJ, Swanton C, Howell M,
Maslen S, Skehel JM, Boulton SJ, Collis SJ. Ccdc13 is a
novel human centriolar satellite protein required for
ciliogenesis and genome stability. J Cell Sci.
2014;127(Pt 13):2910-9
Sundaramoorthy S, Vázquez-Novelle MD,
Lekomtsev S, Howell M, Petronczki M. Functional
genomics identifies a requirement of pre-mRNA
splicing factors for sister chromatid cohesion. EMBO J.
2014;33(22):2623-42
181
Luxton HJ, Barnouin K, Kelly G, Hanrahan S, Totty N,
Neal DE, Whitaker HC. Regulation of the localisation
and function of the oncogene LYRIC/AEG-1 by
ubiquitination at K486 and K491. Mol Oncol.
2014;(3):633-41
Maertens GN, Cook NJ, Wang W, Hare S, Gupta SS,
Öztop I, Lee K, Pye VE, Cosnefroy O, Snijders AP,
KewalRamani VN, Fassati A, Engelman A,
Cherepanov P. Structural basis for nuclear import of
splicing factors by human Transportin 3. Proc Natl
Acad Sci USA. 2014;111(7):2728-33
On KF, Beuron F, Frith D, Snijders AP, Morris EP,
Diffley JF. Prereplicative complexes assembled in vitro
replication. EMBO J. 2014;33(6):605-20
Drosophila early larval central nervous system marge with GFP
(green), c855a (red) and DAPI (blue).
Light Microscopy (page 156)
Daniel Zicha
PKCε-regulated pathway. Nature Commun.
2014;5:5685
Peptide Chemistry (page 157)
Nicola O’Reilly
Skehel JM, O’Reilly NJ, Ogrodowicz RW, Smerdon SJ,
cochaperone complex. Cell Rep. 2014;7(1):19-26
Protein Analysis and
Proteomics (page 158)
Bram Snijders
Skehel JM, O’Reilly NJ, Ogrodowicz RW, Smerdon SJ,
cochaperone complex. Cell Rep. 2014;7(1):19-26
182
Pitcher DS, de Mattos-Shipley K, Wang Z, Tzortzis K,
Goudevenou K, Flynn H, Bohn G, Rahemtulla A,
Roberts I, Snijders AP, Karadimitris A, Kleijnen MF.
Nuclear proteasomes carry a constitutive
posttranslational modification which derails SDSPAGE (but not CTAB-PAGE). Biochim Biophys Acta.
2014;1844(12):2222-8
2014;53(5):738-51
Ribeiro P, Holder M, Frith D, Snijders AP, Tapon N.
Crumbs promotes expanded recognition and
degradation by the SCF(Slimb/β-TrCP) ubiquitin ligase.
Proc Natl Acad Sci USA. 2014;111(19):E1980-9
Swarts DC, Jore MM, Westra ER, Zhu Y, Janssen JH,
Snijders AP, Wang Y, Patel DJ, Berenguer J, Brouns SJ,
van der Oost J. DNA-guided DNA interference by
a prokaryotic Argonaute. Nature.
2014;507(7491):258-61
Zhang T, Cronshaw J, Kanu N, Snijders AP,
Behrens A. UBR5-mediated ubiquitination of ATMIN is
required for ionizing radiation-induced ATM signaling
and function. Proc Natl Acad Sci USA.
2014;111(33):12091-6
Protein Purification (page 159)
Svend Kjær
Ben-Addi A, Mambole-Dema A, Brender C,
Martin SR, Janzen J, Kjær S, Smerdon SJ, Ley SC.
IκB kinase-induced interaction of TPL-2 kinase with
14-3-3 is essential for Toll-like receptor activation of
ERK-1 and -2 MAP kinases. Proc Natl Acad Sci USA.
2014;111(23):E2394-403
Begum S, Maillard PV, Borg A, Matthews N, Feng Q,
van Kuppeveld FJ, Reis e Sousa C. Identification of an
LGP2-associated MDA5 agonist in picornavirusinfected cells. Elife. 2014;3:e01535
Goodman KM, Kjær S, Beuron F, Knowles PP,
Nawrotek A, Burns EM, Purkiss AG, George R,
Santoro M, Morris EP, McDonald NQ. RET
recognition of GDNF-GFRα1 ligand by a composite
binding site promotes membrane-proximal selfassociation. Cell Rep. 2014;8(6):1894-904
EMBO J. 2014;33(14):1582-98
In vivo imaging (page 161)
Francois Lassailly
Griessinger E, Anjos-Afonso F, Pizzitola I,
Rouault-Pierre K, Vargaftig J, Taussig D, Gribben J,
Lassailly F, Bonnet D. A niche-like culture system
allowing the maintenance of primary human acute
myeloid leukemia-initiating cells: a new tool to
decipher their chemoresistance and self-renewal
mechanisms. Stem Cells Transl Med. 2014;(4):520-9
2014;53(5):738-51
Pizzitola I, Anjos-Afonso F, Rouault-Pierre K,
Lassailly F, Tettamanti S, Spinelli O, Biondi A, Biagi E,
Bonnet D. Chimeric antigen receptors against CD33/
CD123 antigens efficiently target primary acute
myeloid leukemia cells in vivo. Leukemia.
2014;28(8):1596-605
Simon AC, Zhou JC, Perera RL, van Deursen F, Evrin C,
Ivanova ME, Kilkenny ML, Renault L, Kjær S,
Matak-Vinković D, Labib K, Costa A, Pellegrini L.
polymerase α in the eukaryotic replisome. Nature.
2014;510(7504):293-7
Protein Structure (page 162)
Transgenics (page 160)
Ian Rosewell
Stephane Mouilleron
2014;53(5):738-51
Stamp G, Turley SJ, Sahai E, Reis e Sousa C. Dendritic
cells control fibroblastic reticular network tension
and lymph node expansion. Nature.
2014;514(7523):498-502
183
Other Research Papers
Vincenzo Costanzo
Bellelli R, Castellone MD, Guida T, Limongello R,
Dathan NA, Merolla F, Cirafici AM, Affuso A, Masai H,
Costanzo V, Grieco D, Fusco A, Santoro M,
Carlomagno F. NCOA4 transcriptional coactivator
inhibits activation of DNA replication origins. Mol Cell.
2014;55(1):123-37.
Errico A, Aze A, Costanzo V. Mta2 promotes Tipindependent maintenance of replication fork integrity.
Cell Cycle. 2014;13(13):2120-8
Nancy Hogg
De Filippo K, Neill DR, Mathies M, Bangert M,
McNeill E, Kadioglu A, Hogg N. A new protective role
for S100A9 in regulation of neutrophil recruitment
during invasive pneumococcal pneumonia. FASEB J.
2014;28(8):3600-8
McNeill E, Hogg N. S100A9 has a protective role in
inflammation-induced skin carcinogenesis. Int J Cancer.
2014;135(4):798-808
Wang Y, Fang C, Gao H, Bilodeau ML, Zhang Z,
Croce K, Liu S, Morooka T, Sakuma M, Nakajima K,
Yoneda S, Shi C, Zidar D, Andre P, Stephens G,
Silverstein RL, Hogg N, Schmaier AH, Simon DI.
Platelet-derived S100 family member myeloid-related
protein-14 regulates thrombosis. J Clin Invest.
2014;124(5):2160-71
David Ish-Horowicz
Hayashi R, Wainwright SM, Liddell SJ, Pinchin SM,
Horswell S, Ish-Horowicz D. A genetic screen based on
in vivo RNA imaging reveals centrosome-independent
mechanisms for localizing gurken transcripts in
Drosophila. G3 (Bethesda). 2014;4(4):749-60
Soza-Ried C, Öztürk E, Ish-Horowicz D, Lewis J.
Pulses of Notch activation synchronise oscillating
somite cells and entrain the zebrafish segmentation
clock. Development. 2014;141(8):1780-8
EMBO J. 2014;33(14):1582-98
184
Banafshe Larijani
Byrne RD, Veeriah S, Applebee CJ, Larijani B.
Conservation of proteo-lipid nuclear membrane
fusion machinery during early embryogenesis.
Nucleus. 2014;5(5):441-8
Calleja V, Laguerre M, de Las Heras-Martinez G,
Parker PJ, Requejo-Isidro J, Larijani B. Acute
regulation of PDK1 by a complex interplay of
molecular switches. Biochem Soc Trans.
2014;42(5):1435-40
Larijani B, Hamati F, Kundu A, Chung GC,
Domart MC, Collinson L, Poccia DL. Principle of
duality in phospholipids: regulators of membrane
morphology and dynamics. Biochem Soc Trans.
2014;42(5):1335-42
Peddie CJ, Blight K, Wilson E, Melia C, Marrison J,
Carzaniga R, Domart MC, O’Toole P, Larijani B,
Collinson LM. Correlative and integrated light and
electron microscopy of in-resin GFP fluorescence,
used to localise diacylglycerol in mammalian cells.
Jeffrey SS, Parker PJ, Larijani B. High-throughput
time-resolved FRET reveals Akt/PKB activation as a
2014;74(18):4983-95
Zhang H, Cohen AL, Krishnakumar S, Wapnir IL,
Veeriah S, Deng G, Coram MA, Piskun CM,
Longacre TA, Herrler M, Frimannsson DO, Telli ML,
Dirbas FM, Matin AC, Dairkee SH, Larijani B,
Glinsky GV, Bild AH, Jeffrey SS. Patient-derived
xenografts of triple-negative breast cancer reproduce
molecular features of patient tumors and respond to
mTOR inhibition. Breast Cancer Res. 2014;16(2):R36
Julian Lewis
Soza-Ried C, Öztürk E, Ish-Horowicz D, Lewis J. Pulses
of Notch activation synchronise oscillating somite
cells and entrain the zebrafish segmentation clock.
Development. 2014;141(8):1780-8
Taija Makinen
Lutter S, Makinen T. Regulation of lymphatic
vasculature by extracellular matrix. Adv Anat Embryol
Cell Biol. 2014;214:55-65
Gordon Peters
proteins in the DNA damage response - a
Mikawa T, Maruyama T, Okamoto K, Nakagama H,
Lleonart ME, Tsusaka T, Hori K, Murakami I, Izumi T,
Takaori-Kondo A, Yokode M, Peters G, Beach D,
Kondoh H. Senescence-inducing stress promotes
proteolysis of phosphoglycerate mutase via ubiquitin
ligase Mdm2. J Cell Biol. 2014;204(5):729-45
O’Loghlen A, Martin N, Krusche B, Pemberton H,
Alonso MM, Chandler H, Brookes S, Parrinello S,
Peters G, Gil J. The nuclear receptor NR2E1/TLX
controls senescence. Oncogene. 2014;doi: 10.1038/
onc.2014.335
Giampietro Schiavo
Bercsenyi K, Schmieg N, Bryson JB, Wallace M,
Caccin P, Golding M, Zanotti G, Greensmith L,
Nischt R, Schiavo G. Tetanus toxin entry. Nidogens
are therapeutic targets for the prevention of tetanus.
Science. 2014;346(6213):1118-23
Hislop JN, Islam TA, Eleftheriadou I, Carpentier DC,
Trabalza A, Parkinson M, Schiavo G, Mazarakis ND.
Rabies virus envelope glycoprotein targets lentiviral
vectors to the axonal retrograde pathway in motor
neurons. J Biol Chem. 2014; 289(23):16148-63
Schmieg N, Menendez G, Schiavo G, Terenzio M.
Signalling endosomes in axonal transport: travel
updates on the molecular highway. Semin Cell Dev
Biol. 2014;27:32-43
Simão D, Pinto C, Piersanti S, Weston A, Peddie CJ,
Bastos AE, Licursi V, Schwarz SC, Collinson LM,
Salinas S, Serra M, Teixeira AP, Saggio I, Lima PA,
Kremer EJ, Schiavo G, Brito C, Alves PM. Modeling
human neural functionality in vitro: three-dimensional
culture for dopaminergic differentiation. Tissue Eng
Part A. 2014;doi: 10.1089/ten.TEA.2014.0079
EMBO J. 2014;33(14):1582-98
Terenzio M, Golding M, Schiavo G. siRNA screen of ES
cell-derived motor neurons identifies novel regulators
of tetanus toxin and neurotrophin receptor trafficking.
Front Cell Neuroscience. 2014;8:140
Wang Y, Chakravarty P, Ranes M, Kelly G, Brooks PJ,
Neilan E, Stewart A, Schiavo G, Svejstrup JQ.
Acad Sci U S A. 2014;111(40):14454-9
Almut Schulze
Bensaad K, Favaro E, Lewis CA, Peck B, Lord S,
Collins JM, Pinnick KE, Wigfield S, Buffa FM, Li JL,
Zhang Q, Wakelam M, Karpe F, Schulze A, Harris AL.
Fatty acid uptake and lipid storage induced by HIF-1α
contribute to cell growth and survival after hypoxiareoxygenation. Cell Rep. 2014;9(1):349-65
Peck B, Schulze A. Cholesteryl esters: fueling the fury
of prostate cancer. Cell Metab. 2014;19(3):350-2
Yizhak K, Devedec SE, Rogkoti VM, Baenke F,
de Boer VC, Frezza C, Schulze A, van de Water B,
Ruppin E. A computational study of the Warburg
effect identifies metabolic targets inhibiting cancer
migration. Mol Syst Biol. 2014;10:744
Helen Walden
Hodson C, Purkiss A, Miles JA, Walden H.
Structure of the human FANCL RING-Ube2T complex
reveals determinants of cognate E3-E2 selection.
Structure. 2014;22(2):337-44
185
THESES
Rudi Agius
Understanding the Stability of Protein-Protein
Complexes
Susan Ahrens
Immunobiology
Extracellular actin in innate immunity
Graham Bell
Alessandra Audia
In vitro model to study the role of Notch pathway
in the interaction between Haematopoietic Stem
Cells (HSCs) and their microenvironment.
Hella Baumann
In vitro reconstruction of confined microtubule
cytoskeleton self-organisation
Emily Burns
Graham Bell
Epithelial Biology
The roles and regulation of the Drosophila Lgl
tumour suppressor in cell division
Kinga Bercsenyi
Molecular Neuropathobiology
Nidogens are essential for the entry of tetanus
toxins into motor neurons
Mariana Campos
Emily Burns
Structural Biology
Structural and functional aspects of RET receptor
tyrosine kinase maturation, signalling and chemical
inhibition
Mariana Campos
Epithelial Biology
Bul and Kul are novel components of the DachsousFat planar polarity system
Heike Miess
Rahul Thadani
Gary Hong Chun Chung
Cell Biophysics
The role of fusogenic vesicles in the regulation of
nuclear envelope assembly
Alex Fennell
Telomere Biology
Centromeres and telomeres display unanticipated
and interchangeable roles in promoting nuclear
division in fission yeast
Francesco Gualdrini
Mechanisms of transcriptional regulation by SRF
co-factors
Heike Miess
Gene Expression Analysis
Identification of metabolic genes essential for
proliferation of clear cell Renal Cell Carcinoma
(ccRCC) cells
Richard Panayiotou
Phosphorylation mediated regulation of MRTF-A
Matt Peacock
UVA Photosensitisers, Protein Oxidation and DNA
Repair
Thibaud Perriches
The CBF3 complex structure and function during
point centromere establishment
Ngang Heok Tang
Cell Regulation
A study on Kinetochore-Spindle Microtubule
Attachment: Ndc80 and TACC-TOG/MAPs
Martin Taylor
DNA Damage Response
Mechanism of action of Rad51 paralogs
Jeroen Claus
The pseudokinase HER3: Structure/function
relationships and inhibitor-induced signalling
Rahul Thadani
The condensin ATPase: towards a mechanistic view
of chromosome condensation
Tom Deegan
Investigating the Mechanism of Activation of the
Mcm2-7 Replicative Helicase
Martin Wallace
Molecular Neuropathobiology
Phosphorylation of Rab7 at serine 72 and its role in
the regulation of the late endocytic pathway
Stefanie Derzsi
Tumour Cell Biology
Investigation of heterotypic interactions between
cancer cells and cancer-associated fibroblasts
Tianyi Zhang
Mammalian Genetics
The role of ATMIN in regulating ATM signalling
Hannah Dooley
Secretory Pathways
Investigation of the role of WIPI2 in
autophagosome formation: Functional
characterisation of the WIPI2-Atg16L1 interaction
186
Christian Duellberg
Mechanism and control of microtubule dynamic
instability probed by in vitro reconstitutions and
microfluidics approaches
Yanxiang Zhou
Developmental functions of Drosophilia ASPP and
RASSF8
INSTITUTE
INFORMATION
Administration
Academic Programme
Seminars and Conferences
External Funding
Institute Management
INSTITUTE INFORMATION
187
ADMINISTRATION
Director of Operations
Ava Yeo PhD
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Charis Ashton
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188
LRI administration
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team led by the LRI Director of Operations is
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This year saw the launch of the new Crick PhD
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Postdoc Retreat. Postdocs from the LRI, NIMR,
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Group leaders receive comprehensive secretarial
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Laboratory management services
The Laboratory Services and Support team works
closely with the research laboratories and Core
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The team liaises closely with both the Health and
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ensuring co-operation across all areas at the LRI.
This is particularly important when setting up new
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been refurbished for incoming group leaders.
We help with the running of laboratories, provide
advice about equipment repairs and maintenance
as well as other technical support. We also support
the Purchase to Pay (P2P) system and are a first
point of contact for questions Scientific Officers
(SOs) may have regarding the placing of orders. In
addition, this year saw the involvement of many
SOs with the Crick Quadrant Working Groups, an
exercise undertaken to help plan the new
laboratory space at the Crick with our colleagues
from the NIMR. In order to prepare for sharing
more communal space at the Crick, the team has
also instigated an institute-wide laboratory clear
out to try and dispose of any unwanted reagents,
equipment and paper before the move.
IT
Surinder Dio
Claire Brewer
Marion Edwards*
Andy Foster
Jacki Goldman
Simon Grierson
Ellen Gyapong
Mat Hillyard
Andrew Jordan
Chris Manser
Santosh Nittala
Wing Poon
Phil Spratt
Mark Tomlinson*
Lab Aides
Susan Hill
Ian Morris
Chris Coomber
Kim Crane
Mark Dalton
Gareth Dineen
Carol Du Preez
Brenda Foran
Annette Pereira
Lucia Scalco
Jill Sheehan
Patricia Smith-Carington
Susan Smith-Carington
Michelle Wood
Laboratory Management Team
Nigel Peat
Mark Johnson
Hans Nicolai
Fiona Johnson
Elizabeth Li
Kathryn Snelgrove
Reception, Clare Hall
Susan Hughes
Janet Almond
Research Administrators
Jessica Adams*
Katherine Ames
Aleksandra Banasiak
Helen Batley
Nicola Howes
Sophie Kidane*
Sophie Kontakkis
Jackie Martinez
Aileen Nelson
Mary Nicolaou
Anastasia Photiou
Stores
David Hawkes
Phillip O’Brien
Andy Bendon
Anthony Crane
Lee Goldstein
Ronald Main
Gary Martin
Paul Riley
Michael Wilkins
Washroom
Jayson Webb
Wayne Bushell
Yaw Sarpong
Andy Perry
Paul Chambers
*= Part year.
Health and Safety
The Health and Safety team at the LRI provides
advice, training and support in all aspects of
welfare and safety throughout the institute,
whether for scientific, administrative or
maintenance work. This year the team have been
working with Property Services to implement
changes to the way waste is recycled across the
institute and in the processing of laboratory waste.
This has led to a saving of £30,000 over the year.
The Health and Safety team have also been working
hard in planning for the safe decommissioning of 44
LIF and Clare Hall prior to the move to the Crick.
IT
The LRI IT team have been involved with:
• An expansion of the archive solution to allow for
a multi-tiering storage solution, that
automatically migrates files older than a
specified date to be migrated to slower storage
• The implementation of a soft quota’s system to
allow for monitoring and reporting of storage
both at user and group level
• Continued expansion of the network
• Increased activity in Crick-related work
• Piloting of the EduRoam wifi service
• Improvements on the security of network
services with the deployment of a new system
for hosting externally facing web sites
• Improvements to the overall service delivery of
IT within the LRI
• Improved disaster recovery for the virtual
infrastructure with replication across LIF and CH
of essential services
Outreach
2014 has been a busy year for the LRI’s outreach
programme, which aims to engage the public,
especially school children, in our research. We
encourage all staff to contribute to these activities.
• Code Breaking: Reading the Genetic Code with
Raspberry Pi
What is the genome, how can we ‘read’ it, and
why do ‘mistakes’ in the genome cause cancer?
This autumn we explored these questions with
12-15 year-old students from the Maria Fidelis
School in Camden to highlight the role of
computers in cancer research. In the third
iteration of this programme, LRI scientists helped
students write programs to ‘decode’ DNA in
search of potential cancer-causing mutations. By
providing course materials adapted to
inexpensive technologies, such as the Raspberry
Pi computer and open source software, we are
doing our part to help schools bring
programming into the classroom.
• ‘Exploring the Microworld’ Workshop
In this CREST Accredited project, LRI scientists
led short activities for 12-17 year-old students
aimed at demystifying the microscope. Students
built £10 microscopes, learned about using
inexpensive microscopes for field diagnosis, and
got the chance to use various microscopes to
examine fruit flies, zebrafish and nematode
worms to learn how these model organisms help
us answer big questions in cancer biology. The
workshop was featured at the Royal College of
Pathologists, Guildhall and a local Camden
school.
• Science Museum Lates
LRI scientists played a big part at this Crick-led
event, which attracted approximately 7000
visitors. Groups from the LRI discussed the
power of electron microscopy, the role of the
immune system in cancer and designer drugs.
• Work Experience
Finally, we continued the LRI Schools Work
Experience Scheme at both Clare Hall and
Lincoln’s Inn Fields, allowing students to spend
time in both Core Technology Facilities and
research laboratories. Additionally two Nuffield
Science Bursary places were offered to 6th form
students in the Macromolecular Structure and
Function Laboratory and Light Microscopy
Facility.
Crick Transition
During 2013-14 we have continued our work to
prepare for the LRI’s transition to the Francis Crick
Institute. As the transfer approaches we have
undertaken detailed planning activity to ensure all
elements of the transfer are understood and the
required work to ensure a smooth transition is
underway.
The LRI Transition Core Team, made up of the
Workstream Leads and the Transition Project
team, meet regularly to review progress and
discuss key issues. The Core Team reports monthly
to the LRI Transition Steering Group. LRI transition
is governed by the CRUK Crick Programme Board
chaired by CRUK’s Chief Executive, Harpal Kumar.
The priority for the next year will be to execute the
legal transfer of the LRI to the Francis Crick
Institute and to continue working on the
preparation for the physical move and building
closedown activity of 44LIF and Clare Hall.
Successful transition will require strong
partnership working between the LRI, CRUK and
the Crick.
ADMINISTRATION
189
ACADEMIC PROGRAMME
At any one time the LRI has approximately 100 graduate students and
nearly 180 postdoctoral fellows carrying out their research and
participating in scientific training programmes designed to develop
their skills and lay the groundwork for their future careers. The
students and postdocs form the core of the institute’s scientific
community, carrying out high quality research, as evidenced in the
research highlights and publications sections of this report.
Graduate Students
Our graduate students are recruited via a highly
competitive annual selection process designed to
identify outstanding students who are passionate
about carrying out research leading to a PhD. In
2014 we recruited the first cohort of students that
will follow the new Crick PhD programme. 27
Crick-LRI students were selected to join the
programme in September 2014 alongside 20
Crick-NIMR PhD students. Nine of these students
are jointly funded by the Crick and either Imperial
College London (Imperial) or King’s College London
(King’s). These joint students, with an institute
supervisor and Imperial or King’s supervisor, will
carry out collaborative and interdisciplinary
research projects spanning both supervisor’s
research groups. The students contribute to the
international flavour of our institute, with 30% of
the 2014 intake coming from the rest of Europe
and 20% from outside the EU.
A key feature of both the LRI PhD programme and
the new Crick PhD programme is the cohesion of
the student community, which provides a strong
peer-to-peer support network and can lead to
highly fruitful scientific collaborations. This
network is fostered from the very beginning of the
PhD programme when new students attend a
4-day induction. The September 2014 Crick PhD
student induction programme included sessions to
introduce students to the Crick and its founding
institutes and university partners, to prepare them
for undertaking their PhD, and to help them to get
to know each other. Topics covered via talks and
interactive sessions included organising yourself
and your research, keeping on top of the literature,
effective experiment design, and introductions to
the Core Technology Facilities. The week ended
with a student-organised symposium named after
Francis Crick’s book on scientific discovery, ‘What
Mad Pursuit’. Two students from each institute, in
each year of their PhD, gave short presentations
about their research project and provided some
advice for new students embarking on their PhDs.
190
The new Crick-LRI students are registered for their
PhDs with one of the Crick partner universities,
Imperial, King’s or University College London (UCL).
The universities provide an important source of
additional training, facilities and support, which
complements those provided within the institute.
In addition to guidance from their primary
supervisor and other research group members,
students receive scientific advice from their thesis
committee, which they meet with at key
‘progression points’ during their PhD. Thesis
committees are made up of three senior
researchers including one faculty member from
the university that the student has registered with
for their PhD. An exciting new activity in 2014 was
the development and introduction of the new
‘Crick Grad Log’ – an online system to record and
monitor students’ progress and training. Students
submit their research reports, upload talks, log
training and receive feedback from their
supervisors and thesis committee via the Log.
Further support for PhD students throughout their
time at the institute is provided by the LRI student
admin team: Sally Leevers, LRI Academic Director,
Andrew Brown, LRI Research Manager for
Graduate Studies and Sabina Ebbols, LRI Academic
HEI Liaison Manager, and by their NIMR and Crick
colleagues.
While the 2014 PhD students are following the
new Crick PhD Programme, the LRI PhD
Programme continues for the institute’s second,
third and fourth year students. For example, the
second year students participated in CRUK’s
course on communicating science to scientific and
non-scientific audiences prior to giving their
second year seminars, while the third year
students attended the National Cancer Research
Institute annual conference, where many of them
gave poster presentations on their research. Other
student activities included attending the
International PhD Student Cancer Conference at
the DKFZ in Heidelberg, the student-organised
summer event and Christmas lecture as well as
Intake of Crick 2014 PhD Students
contributing to Cancer Research UK fundraising by
giving talks to supporters and other activities.
Postdoctoral Fellows
The LRI postdocs are a vibrant and diverse
community – about 20% are British, 60% are from
other European countries, and 20% from further
afield. Approximately half are funded by Cancer
Research UK postdoctoral fellowships, some are
supported by external grants awarded to their
group leaders, and many of them secure their own
personal fellowships from external agencies such
as the European Molecular Biology Organisation,
the Human Frontiers Science Programme and
Marie Skłodowska-Curie Actions.
LRI Postdocs can access a training programme that
was developed collaboratively by postdocs, group
leaders and the Academic Director. The
programme starts with an induction to orient
postdocs and make them aware of the facilities and
activities available, and to introduce them to the
Core Technology Facilities that will facilitate their
research. Soon after starting, postdocs submit a
project proposal and throughout their programme,
they have annual career development reviews with
their group leaders. These reviews focus on the
postdocs’ science, taking a broader perspective
than normal day-to-day conversations, and
provide structure and focus for postdocs’ scientific
and career development to aid them in their future
career. Postdocs also present seminars about 18
months and 3 years after starting at the LRI. Their
second seminar is followed by a Postdoc Career
Development Discussion. Postdocs select and
invite several group leaders to join them for these
discussions about their scientific progress,
publication strategy and future career plans. About
quarter of the postdocs leaving the LRI take up
independent positions to establish their own
research groups, about half go to a second
postdoctoral position, and others move into areas
such as pharma, biotech, science communication,
publishing, and clinical trails.
Postdocs are invited to attend Postdoc Consultative
Meetings (PDCMs), which provide an important
opportunity for postdocs to discuss issues that
affect them within the LRI and to communicate
with LRI Administration and Management. In
addition, postdoc representatives attend the LRI
Scientific Staff Meeting and the Fellowships
Committee to feedback to the PDCM what was
discussed at these meetings.
The 2014 Crick Postdoc Retreat, organised by a
committee of postdocs from the LRI and NIMR, was
held in June at the British Library, next to the
Francis Crick Institute site (see the Seminars and
Conferences section of this report). More than 150
postdocs from LRI, NIMR and the Crick’s university
partners attended the stimulating and interactive
day with a theme of ‘Inspiring Science’. Speakers
from academia and pharma gave presentations on
their science and careers, and there was a
discussion session on open access publishing.
Other highlights included a postdoc networking
nexus, and an orienteering trail from the British
Library to the evening venue, which provided an
opportunity to explore the area around the Crick.
Joint Crick 2014 Post Doc retreat at The British Library
ACADEMIC PROGRAMME
191
The London Research Institute hosts a Special Seminar Series to invite
external speakers from around the world to present their work,
covering a broad spectrum of cutting-edge topics within the areas of
genome integrity, signal transduction, structural biology,
developmental biology and immunology. There are also a number of
Special Interest Groups within the different areas of interest within
the institute, which are open to external visitors to attend, providing
a unique networking opportunity to encourage collaboration within
London and the surrounding area. A selection from this year’s
programme is listed below:
Special Seminars
Judith Campisi, The Buck Institute, USA
Cancer and aging: Rival demons?
Aaron Straight, Stanford University, USA
Turning on the genome: mechanisms of zygotic
genome activation
Advanced Bioimaging
Kishan Dholakia, St Andrews University, UK
Shaping the future of imaging for biomedicine
Rainer Heintzmann, University of Jena, Germany
Structured Illumination and the Analysis of Single
Molecules in Cells
Computational and Mathematical Biology
Franca Fraternali, Kings College London, UK
A Multiscale view of Protein-Protein Interactions
Trevor Graham, Barts Cancer Institute, UK
Quantifying the evolution of human intestinal stem
cells
Developmental Biology
Immunology
Shane Crotty, La Jolla Institute, USA
T follicular helper cell (Tfh) differentiation and
genetics
Antal Rot, University of Birmingham, UK
New pathophysiological roles of atypical
chemokine receptors.
Molecular Medicines & Therapeutics
Sandra Misale, Istituto di Candiolo, Turin, Italy
Vertical suppression of the EGFR pathway to
overcome acquired resistance to anti-EGFR therapy
in colorectal cancer
Andy West, GlaxoSmithKline, UK
Imaging drug distribution using mass spectrometry
– from tissues to cells
Signalling
Katsuhiko Shirahige, University of Tokyo, Japan
Transcriptional regulation by Cohesin loader
NIPBL-Mau2 complex
Oriol Casanovas, Catalan Institute of Oncology,
Barcelona, Spain
Anti-angiogenic Therapies: Learning from their
Limitations
Alison Woollard, Biochemistry, University of
Oxford, UK
C. elegans development: getting the seams right
Victoria Sanz Moreno, King’s College London, UK
Signalling pathways controlling amoeboid tumour
dissemination
Genes to Cells
Kazuhiro Maeshima, National Institute of
Genetics, Japan
Chromatin fluctuation in live mammalian cells
192
Roop Mallik, Tata Institute of Fundamental
Research, Mumbai, India
Biophysics of Motor protein driven transport in
Phagosome maturation
Conferences
31st March - 1 April
LRI Retreat
The last LRI Retreat took place at the University of
Oxford from 31st March - 1 April. 360 delegates
from Lincoln’s Inn Fields and Clare Hall enjoyed a
packed couple of days in the city of Oxford. The
Retreat began with talks from newer group leaders
who have not attended the LRI Retreats before:
Nate Goehring, Paola Scaffidi and Alessandro
Costa, along with Borbala Mifsud who spoke on
behalf of Nick Luscombe. There was also a slot
dedicated to the Create the Change Campaign,
CRUK’s campaign to raise money for the Francis
Crick Institute. These talks were followed by a busy
and successful poster session. The next day saw a
busy schedule of talks from more group leaders
and Core Technology Facilities heads, (Dinis Calado,
Peter Cherepanov, Hasan Yardimci, Francois
Lassailly, Bram Snijders) and a few postdocs who
had been nominated to speak - including Marco
Sapanaro from the Mechanisms of Gene
Transcription Group, and Sophie Acton from the
Immunobiology Group. Barbara Schraml
(Immunobiology Group), winner of the 2013
Hardiman-Redon Prize also gave a talk.
Poster session at the last LRI retreat, Oxford, April 2014
23rd June
Postdoc Retreat
The 3rd Francis Crick Institute Postdoc Retreat was
held on Monday 23rd June 2014 in association with
the British Library at their conference centre. The
theme of the event was ‘Inspiring Science’ and
consisted of speakers from academia and industry
offering a diverse range of talks. The Retreat was
opened by the Francis Crick Institute Research
Directors Richard Treisman (LRI Director) and Jim
Smith (NIMR Director and MRC Deputy Chief
Executive and Director of Strategy), and Roly
Keating, Chief Executive of the British Library. The
speakers were at various stages in their career with
inspirational stories to tell about how they got
there. Speakers included: Professor Mark Lythgoe,
Founder and Director of the UCL Centre for
Advanced Biomedical Imaging (CABI); Dr Claire
Spottiswoode, Research Fellow at the Department
of Zoology, University of Cambridge; Dr Nessa
Carey, Senior Director in External R&D Innovation
at Pfizer; Professor Julian Parkhill, Head of
Pathogen Genomics at the Wellcome Trust Sanger
Institute and Professor Olivier Voinnet leading
expert in the field of small RNAs. All the speakers
were very well received and their talks stimulated
lively discussions.
More tan 150 Postdocs from the Francis Crick
Institute’s partner organisations attended and
were given the opportunity to interact in a
‘Postdoc nexus’ networking session, and at the
evening social event.
More than 90% of attendees rated the retreat as
‘very good’ or ‘excellent’ and indicated that they
would attend future Francis Crick Institute Postdoc
Retreats.
The Retreat was organised by a committee
consisting of postdocs from the LRI and NIMR.
11th - 13th June
International Graduate Student Conference
This year’s International PhD Student Cancer
Conference, was hosted and organised by students
from the German Cancer Research Center (DKFZ) in
Heidelberg. This annual two and a half day
conference is held with other Cancer Research UK
funded institutes (Beatson, Paterson, LRI and the
Oxford Institute for Radiation Oncology) as well as
the Netherlands Cancer Institute (NKI), the DKFZ,
the European School of Molecular Medicine in
Milan (SEMM: IFOM-IEO), the Vita-Salute San
Raffaele University also in Milan (DIBT), and the
Spanish National Cancer Centre (CNIO) in Madrid.
All attendees presented their research - which
covered many topics related to cancer, from basic
biology to clinical aspects of the disease. The
scientific talks and poster sessions were mixed
with social activities.
Lutz Gissman, Professor of Genome Modifications
and Carcinogenesis at DKFZ, and Gottfried Schatz,
Emeritus Professor of Biochemistry at the
University of Basel gave highly engaging plenary
lectures, and faced numerous excellent questions
from the audience.
193
EXTERNAL FUNDING
Awards and Grants
Association for International Cancer Research
Axel Behrens – Mammalian Genetics
Julian Downward – Signal Transduction
Astellas
Sharon Tooze – Secretory Pathways
Medical Research Council
Lucy Collinson – Electron Microscopy
Dinis Calado – Immunity and Cancer
Breast Cancer Campaign
Mark Petronczki – Cell Division & Aneuploidy
Erik Sahai – Tumour Cell Biology
National Institute of Health
Peter Cherepanov – Chromatin Structure and
Mobile DNA
British Council
Holger Gerhardt – Vascular Biology
Pfizer
Charles Swanton – Translational Cancer
Therapeutics
Cephalon
Peter Parker – Protein Phosphorylation
Cancer Research UK Travel Award
Erik Sahai – Tumour Cell Biology
European Molecular Biology Organisation
(EMBO Young Investigator Prize)
Mark Petronczki – Cell Division & Aneuploidy
Barry Thompson – Epithelial Biology
European Commission
Axel Behrens – Mammalian Genetics
Simon Boulton – DNA Damage Response
Peter Cherepanov – Chromatin Structure and
Mobile DNA
John Diffley – Chromosome Replication
Julian Downward – Signal Transduction
Caroline Hill – Developmental Signalling
Caetano Reis e Sousa – Immunobiology
Jesper Svejstrup – Mechanisms of Gene
Transcription
Thomas Surrey – Microtubule Cytoskeleton
Therapeutics
Richard Treisman – Signalling and Transcription
Frank Uhlmann – Chromosome Segregation
Stephen West – Genetic Recombination
Genetech
Dominique Bonnet – Haematopoietic Stem Cell
Facundo Batista – Lymphocyte Interaction
Leducq
Leukaemia and Lymphoma Research
Dominique Bonnet – Haematopoietic Stem Cell
194
Louis Jeantet
Paul Nurse – Cell Cycle
Richard Triesman – Signalling and Transcription
Steve West – Genetic Recombination
Rosetrees Trust
Therapeutics
Cancer Research Technology Reward to Inventors
Peter Parker – Protein Phosphorylation
Neil McDonald – Structural Biology
The Scripps Research Institute
Facundo Batista – Lymphocyte Interaction
Unity through Knowledge Fund
Adrian Hayday – Immuno Surveillance
Weizmann Institute
Giampietro Schiavo – Molecular
Neuropathobiology
Wellcome Trust
Paul Nurse/Jacqueline Hayles – Cell Cycle
Barry Thompson – Epithelial Biology
Fellowships
Canadian Institute of Health Research
Jasmine Abella – Cell Motility
Laurent L’Epicier-Sansregret – Cell Division and
Aneuploidy/Translational Cancer Therapeutics
Cancer Research UK
Samra Turajlic – Translational Cancer Therapeutics
European Commission – Marie Skłodowska-Curie
actions
Corella Casas Delucci – Chromosome Replication
Ieva Gailite – Apoptosis and Proliferation Control
Eishu Hirata – Tumour Cell Biology
Jatta Huotari – Immunobiology
Christoph Kurat – Chromosome Replication
Nuria Martinez – Lymphocyte Interaction
Marco Montagner – Tumour Cell Biology
Maria Jose Martin Pereira – Genetic Recombination
Claire Sheridan – Signal Transduction
Joanna Soroka – Genetic Recombination
Martina Wirth – Secretory Pathways
FEBS Return-to-Europe-Fellowship
Joseph Yeels – Chromosome Replication
Fondazione Veronesi Italy
Davide Zecchin – Signal Transduction
German Academy of Sciences
Katharina Deiss – Protein Phosphorylation
Human Frontier Science Project
Madhu Kumar – Signal Transduction
Japan Society for the Promotion of Science
Yasutaka Kakui – Chromosome Segregation
Takayuki Koyano – Cell Regulation
Sir Henry Wellcome Fellowship
Sophie Acton – Immunobiology
Esther Arwert – Tumour Cell Biology
Hanna Mischo – Mechanisms of Gene Transcription
Johanna Roostalu – Microtubule Cytoskeleton
Patrycja Kozik – Immunobiology
Frances Willenbrock – Protein Phosphorylation
Swedish Research Council
Omar Khan – Mammalian Genetics
Swiss National Science Foundation
Pierre Maillard – Immunobiology
Kanagaraj Radhakrishnan – Genetic Recombination
Student Fellowships
Agency for Science, Technology and Research
Minghao Chia – Cell Fate and Gene Regulation
Boehringer Ingelheim Fund
Agnieszka Janska – Chromosome Replication
James Patterson – Cell Cycle
Janneke Van Blijswijk – Immunobiology
EC Marie Curie Initial Training Network
Tom Watkins – Translational Cancer Therapeutics
Fundação para a Ciência e a Tecnologia
Filipa Neto – Vascular Biology
Overseas Research Scholarship - UCL
Djamil Damry – Immunity and Cancer
Melisa Guven – Mammalian DNA Repair
Marina Ivanova – Structural Biology
Carlson Tsui – Lymphocyte Interaction
Rafael Di Marco Barros – Immuno Surveillance
Marco Gerlinger – Translational Cancer
Therapeutics
Ministerio de Ecomomia y Competitividad
Ander Abarrategi – Haematopoietic Stem Cell
Netherlands Organisation for Scientific Research
Annemarthe van der Veen – Immunobiology
EXTERNAL FUNDING
195
Institute management
London Research Institute Management
Committee
Richard Treisman PhD FRS (Chair)
Julian Downward PhD FRS
John Diffley PhD FRS
Erik Sahai PhD
Michael Way PhD
Stephen West PhD FRS
Ava Yeo PhD
London Research Institute Faculty Committee
Richard Treisman PhD FRS (Chair)
Michael Way PhD (Co-Chair)
Simon Boulton PhD
Holger Gerhardt PhD
Caroline Hill PhD
Neil McDonald PhD
Erik Sahai PhD
Jesper Svejstrup PHD FRS
Frank Uhlmann PhD
Ava Yeo PhD (in attendance)
Julian Downward PhD FRS (ex officio)
John Diffley PhD FRS (ex officio)
London Research Institute Fellowships
Committee
John Diffley PhD FRS (Chair)
Nic Tapon PhD (Deputy Chair)
Simon Boulton PhD
Adrian Hayday PhD
Ilaria Malanchi PhD
Mark Petronczki PhD
Neil McDonald PhD
Peter Parker PhD FRS
Sharon Tooze PhD
Takashi Toda PhD
Sally Leevers PhD (ex officio)
196
London Research Institute Graduate Students
Advisors Committee
Sally Leevers PhD (Chair)
Peter Cherepanov PhD
Caroline Hill PhD
Thomas Surrey PhD
Jesper Svejstrup PhD
Charles Swanton PhD
Nic Tapon PhD
Kaila Srai PhD (UCL)
Andrew Brown (in attendance)
Sabina Ebbols (in attendance)
CONTACT DETAILS
London Research Institute
Scientific Report 2014
Kings Cross
St Pancras
Lincoln’s Inn Fields Laboratories
44 Lincoln’s Inn Fields
London
WC2A 3LY
ON
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Euston
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AY
Clare Hall Laboratories
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Cancer Research UK
Registered charity in England & Wales (1089464),
Scotland (SC041666) and the Isle of Man (1103).
A company limited by guarantee.
Registered as a company in England and Wales
(4325234) and the Isle of Man (5713F).
Registered address: Angel Building, 407 St John Street,
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An electronic version of this report can be
found at:
http://crick.ac.uk/news/publications/
Waterloo
ISSN 1479-0378
Copyright © 2014 Cancer Research UK
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