AZD9496: An oral estrogen receptor inhibitor that blocks the growth
Title: AZD9496: An oral estrogen receptor inhibitor that blocks the growth of ERpositive and ESR1 mutant breast tumours in preclinical models.
Running title: Pharmacology of SERD inhibitor in breast cancer models
Hazel M. Weir,a Robert H. Bradbury,a Mandy Lawson,a Alfred A. Rabow,a David Buttar,a
Rowena J. Callis,b Jon O. Curwen,a Camila de Almeida,a Peter Ballard, a Michael Hulse,a
Craig S. Donald,a Lyman J. L. Feron,a Galith Karoutchi,a Philip MacFaul,a Thomas Moss,a
Richard A. Norman,b Stuart E. Pearson,a Michael Tonge,b Gareth Davies,a Graeme E.
Walker,b Zena Wilson,a Rachel Rowlinson,a Steve Powell,a Claire Sadler,a Graham
Richmond,a Brendon Ladd, d Ermira Pazolli,c Anne Marie Mazzola,d Celina D’Cruz,d Chris
Oncology iMed, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK;
Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG UK; c
H3 Biomedicine, Technology Sq, Cambridge, MA 02139,dOncology iMed, AstraZeneca R&D
Boston, Gatehouse Drive, Waltham, MA 02451, U.S.
Key words: SERD, estrogen receptor, metastatic breast cancer, ER mutation, ESR1 mutation.
Corresponding author: [email protected]
Disclosure of Potential Conflicts of Interest: All authors are present or past employees of
Supplementary Figures: 7
Precis: This article discloses the structure and pharmacology of an oral non-steroidal small
molecule which is potent against wild-type and mutant forms of the estrogen receptor,
producing anti-tumour efficacy either as a monotherapy or in combination with PI3K or cell
cycle inhibitors in vivo.
Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by
monthly intramuscular injection. Given its present limitations of dosing and route of
administration, a more flexible orally available compound has been sought to pursue the
potential benefits of this drug in patients with advanced metastatic disease. Here we report
the identification and characterization of AZD9496, a non-steroidal small molecule inhibitor
of ERα which is a potent and selective antagonist and down-regulator of ERα in vitro and in
vivo in ER-positive models of breast cancer. Significant tumour growth inhibition was
observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where
this effect was accompanied by a dose-dependent decrease in PR protein levels demonstrating
potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors
led to further growth inhibitory effects compared to monotherapy alone. Tumour regressions
were also seen in a long-term estrogen-deprived breast model, where significant down-
regulation of ERα protein was observed. AZD9496 bound and down-regulated clinically
relevant ESR1 mutants in vitro and inhibited tumour growth in an ESR1-mutant patient-
derived xenograft model that included a D538G mutation. Collectively, the pharmacological
evidence showed that AZD9496 is an oral, non-steroidal, selective estrogen receptor
antagonist and down-regulator in ER-positive breast cells that could provide meaningful
benefit to ER-positive breast cancer patients. AZD9496 is currently being evaluated in a
Phase 1 clinical trial.
With over 70% of breast cancers expressing the estrogen receptor alpha (ERα),
treatment with anti-hormonal therapies that directly antagonise ER function (e.g. tamoxifen)
or therapies that block the production of the ligand, estrogen (e.g. aromatase inhibitors) are
key to management of the disease both in adjuvant and metastatic settings (1, 2). Despite
initial efficacy seen with endocrine therapies, the development of acquired resistance
ultimately limits the use of these agents although the majority of tumours continue to require
ERα for growth. Through the use of pre-clinical models and cell lines, changes in growth
factor receptors have been implicated in driving resistance in cells e.g. receptor tyrosine
kinases (HER2), epidermal growth factor receptor (EGFR) and their downstream signalling
pathways, Ras/Raf/MAPK and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)
signalling (3, 4, 5). In some instances signalling from different growth factor receptor kinases
leads to phosphorylation of various proteins in the ER pathway, including ER itself, leading
to ligand–independent activation of the ER pathway (6).
In the metastatic setting, 500 mg fulvestrant, given as 2 x 250 mg intramuscular
injections, has been shown to offer additional benefit over the 250mg dose with improved
median overall survival (7, 8). Recent analysis of pre-surgical data from a number of trials
showed a dose-dependent effect on key biomarkers such as ER, progesterone receptor (PR)
and Ki67 labeling index (9). Although fulvestrant is clearly effective in this later treatment
setting, there are still perceived limitations in its overall clinical benefit due to very low
bioavailability, the time taken to achieve plasma steady-state of 3-6 months and ultimately
the level of ERα reduction seen in clinical samples (10, 11). It is widely believed that a
potent, orally bioavailable SERD that could achieve higher steady-state free drug levels more
rapidly would have the potential for increased receptor knock-down and lead to quicker
clinical responses, reducing the possibility of early relapses (12). Moreover, the recent
discovery of ESR1 mutations in metastatic breast cancer patients that had received prior
endocrine treatments, where the mutated receptor is active in the absence of estrogen,
highlights an important potential resistance mechanism. Pre-clinical in vitro and in vivo
studies have shown a reduction in signalling from ESR1 mutants by tamoxifen and fulvestrant
but at higher doses than required to inhibit the wild type receptor which implies that more
potent SERDs may be required to treat these patients or to used in the adjuvant setting to
limit the emergence of mutations (13, 14, 15).
Achieving oral bioavailability has remained a key challenge in the design of ER
down-regulators and until now no oral estrogen receptor down-regulators, other than GDC-
0810 and RAD1901, have progressed into clinical trials, despite encouraging reports from
others of pre-clinical activity in mouse xenograft models (16, 17, 18, 19, 20, 21). Here we
describe a chemically novel, non-steroidal ERα antagonist and down-regulator, AZD9496,
that can be administered orally and links increased tumour growth inhibition to enhanced
biomarker modulation compared to fulvestrant in a preclinical in vivo model of breast cancer.
Moreover we show that AZD9496 is a potent inhibitor of ESR1 mutant receptors and can
drive tumour growth inhibition in a patient-derived ESR1 mutant in vivo model (PDX). Hence
AZD9496 is anticipated to yield improved bioavailability and clinical benefit through
enhanced ER pathway modulation.
Cell lines and culture
All AZ cell lines were tested and authenticated by short-tandem repeat (STR) analysis. MCF-
7 was obtained from DSMZ and last STR tested February 2013. MDA-MB-361 (STR tested
October 2011), MDA-MB-134 (STR tested October 2010), T47D (STR tested June 2012),
CAMA-1 (STR tested March 2012) HCC1428 (STR tested December 2011) HCC70 (STR
tested October 2013), LnCAP (STR tested August 2011), MDA-MB-468 (STR tested March
2014), BT474 (STR tested July 2013), PC3 (STR tested October 2012), HCT116 (STR
tested August 2011) were all obtained from the ATCC. HCC1428-LTED cell line (STR
tested November 2012) was kindly provided by Carlos Arteaga (22). Cells were cultured in
RPMI-1640 media (Corning) supplemented with 10% heat-inactivated foetal calf serum
(FCS) or charcoal/dextran-treated CCS to deplete hormone and 2mmol/L L-glutamine
(Corning).. Stable MCF-7 cell lines expressing FLAG-tagged ESR1 mutant proteins in
pTRIPZ lentiviral vectors (GE Healthcare) under the control of the doxycycline inducible Tet
promoter were generated by lentiviral infection using standard techniques.
Biochemical and in vitro cell assays
Binding, ER agonism, antagonism, down-regulation and cell proliferation assays were carried
out as described previously (23). BIAcore affinity measurements and immunoblotting from
compound treated cells are described in Supplementary Methods.
Protein production, crystallization and structure determination
Protein expression, purification and crystallization of the ERα ligand-binding domain was
carried out as previously described (24). X-ray diffraction data were collected at the ESRF on
beamline ID23-1 on an ADSC detector. Data were processed using XDS (25) as implemented
within EDNA (26) and scaled and merged using SCALA (27). The structure of ERα in
complex with AZD9496 was solved by molecular replacement using AmoRE and an internal
ERα structure as the search model (28). Quality checks on the protein structures were carried
out using the validation tools in Coot (29). The final structure has been deposited in the
Protein Databank (PDB) with the ID code given in Supplementary Table S1.
SILAC and mass spectrometry
SILAC assays were carried out as previously described (30) and detailed in Supplementary
methods. Compound treated samples were analysed by mass spectrometry using relative
peptide quantification by selected reaction monitoring (SRM). Degradation half-life was
measured using the one-phase exponential decay equation in GraphPad PRISM (Y=Span.e-
decreases to Plateau with a rate constant K.
+Plateau) where X is time and Y is response which starts out as Span+Plateau and
Rat Uterine and Xenograft Studies
All studies involving animals in the United Kingdom were conducted in accordance with UK
Home Office legislation, the Animal Scientific procedures Act 1986 (ASPA) and
AstraZeneca Global Bioethics policy or with US federal, state and Institutional guidelines in
an AAALAC-accredited facility. The rat uterine weight assay for the measurement of
estrogenic activity were performed as described previously (31). Details of individual
models, protein analysis of tumour fragments and pharmacokinetic analysis of plasma
samples are included in Supplementary Methods.
AZD9496 is a selective ERα antagonist, down-regulator and inhibitor of ER positive
tumour cell growth
tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid, Fig. 1A) was identified through
structure based design and iterative medicinal chemistry structure-activity-relationship (SAR)
studies from a novel ER binding motif (32). A directed ER binding screen was established to
identify novel binding motifs that could then be optimised for both cellular phenotype and
potency while maintaining good pharmacokinetic properties (23). Unlike fulvestrant, and
other well known historical modulators of the ER receptor, including hydroxy-tamoxifen
(Fig. 1B), AZD9496 lacks a phenolic moiety in which the phenol mimics the A-ring phenol
group of the endogenous ligand estradiol but gains its potency through a different series of
novel interactions with the ER protein (Fig. 1C). The indole NH picks up an interaction by
forming a hydrogen bond to the carbonyl of Leu-346, with additional lipophilic interactions
achieved by both the chiral methyl substituent, adjacent to the ring nitrogen and the N-
substituted isopropyl fluoro side-chain occupying a “lipophilic hole” in the ER protein. The
acrylic acid side chain picks up an unusual acid-acid co-localization with Asp-351 in the
Helix-12 region of the ER that has been proposed to be crucial for achieving a down-
regulator-antagonist profile (33). AZD9496 showed high oral bioavailability across three
species (F% 63, 91 and 74, rat, mouse and dog, respectively) with generally low volume and
clearance across species, albeit a higher clearance in mouse. The percent free levels in human
plasma of 0.15% were 5 fold higher than those measured for fulvestrant.
The key characteristics of a SERD is the ability to both antagonise and downregulate ER protein without inducing any partial agonist effects in breast cells. A series of in
vitro cell assays were developed specifically to measure ERα down-regulation, agonism and
antagonism through simultaneous measurement of ER and PR protein levels in cells after
treatment with AZD9496 (23). PR expression is known to be regulated by ER transcription
and was measured as a marker of ER activation in cells (34). The potency of AZD9496
approached that observed with fulvestrant in achieving pM IC50 in ERα binding, down-
regulation and antagonism and significant cell growth inhibition in hormone-depleted growth
conditions. Adding 250 nM tamoxifen significantly lowered the IC50 value, as expected, if
AZD9496 and tamoxifen are competing for binding to the LBD (Table 1A). The potent
down-regulation of ERα was also verified by immunoblotting techniques (Supplementary
To understand the binding kinetics of AZD9496 to human ERα ligand binding
domain (LBD), binding studies were performed in the presence of increasing concentrations
of AZD9496 and association (kass) and dissociation (kdiss) rate constants were calculated.
AZD9496 was calculated as having an apparent dissociation rate constant (kdiss) for ERα
LBD of 0.00043 sec-1, at 25 ºC equating to a half-life (t ½) of 27 ± 2 min and the affinity
derived from the ratio of the rate constants gave a KD of 0.33 nM (Supplementary Table
S2). This is in good agreement with the binding IC50 of 0.82 nM and correlates well with
previously published data on estrogen and tamoxifen binding to ER LBD (35). AZD9496
showed pM equipotent binding to both ERα and ERβ isoforms as expected given their high
sequence homology and similarity in tertiary structure in their LBDs (36) and highly selective
binding compared to progesterone (~650 fold), glucocorticoid (~11223 fold) and androgen
(~36375 fold) receptor LBDs (Supplementary Table S3). Selectivity for ER was also
evident by measuring cell growth inhibition in a panel of ER +ve and ER -ve cell lines
(Supplementary Table S4). No significant growth inhibition was seen in any ER -ve cell
lines (EC50 >10 μM) in contrast to the low nM activity seen in a range of ER +ve cell lines.
AZD9496 directly targets ERα for down-regulation in vitro
To measure the rate at which AZD9496 down-regulated ERα, stable isotope
labelling of amino acids (SILAC) experiments were performed in MCF-7 cells using mass
spectroscopy analysis to detect ERα protein levels after treatment with compounds. Addition
of AZD9496 or fulvestrant increased the rate of degradation of the isotope-labelled ERα
compared to DMSO control. Levels of newly synthesised ERα peptide were also reduced
following AZD9496 and fulvestrant treatment, presumably due to on-going degradation of
newly-synthesised ERα protein by compound present, whereas ERα protein levels continued
to increase over time in the presence of tamoxifen or DMSO (Fig 2). The t½ of ERα was
decreased from 3 hours, in the presence of DMSO, to 0.75 hours with 100 nM AZD9496 and
0.6 hours with 100 nM fulvestrant. (Supplementary Fig. S2). Down-regulation of ER occurs
through the 26S proteosomal pathway as no decrease in ERα protein level in MCF-7 cells
was seen in the presence of the proteosome inhibitor MG132 (Supplementary Fig. S3).
Additionally, ERα down-regulation was shown to be reversible in compound wash-out
experiments, where ERα levels increased in a time-dependent manner back to basal levels
over a 48 hour period following compound removal. (Supplementary Fig. S4).
Agonism of ERα in human breast and uterine cells
In contrast to an AstraZeneca reference compound (AZ10557841) which has known ERα
agonist activity in breast cells, AZD9496 was shown to cause no increase in PR protein levels
but rather a slight decrease below the detected basal levels (Fig. 3A). Some endocrine
therapies, such as tamoxifen, can act as partial ER agonists in certain tissues e.g.
endometrium, due to conformational changes that take place at the ER and the resulting effect
on co-factor recruitment and transcriptional gene activation from the tamoxifen-bound
receptor (37). To test if AZD9496 could act as a partial agonist in other tissues, agonist
effects were measured in vivo in a previously validated immature female rat model designed
to detect agonistic properties of test compounds by measuring increases in uterine weight
(31). AZD9496, given once daily orally at 5 and 25 mg/kg produced statistically significant
increases in uterine weight compared to the fulvestrant control (p<0.001) but significantly
lower than tamoxifen (p=0.001) (Fig. 3B). Histological staining of uterine tissue samples
also showed that the lengthening of the endometrial cells in the rat uteri appeared to have
decreased compared to tamoxifen (Fig. 3C). In further studies, ERα protein levels were
reduced with fulvestrant and AZD9496, but not tamoxifen, compared to relevant vehicle
controls, whereas PR levels were significantly increased with tamoxifen alone (Fig. 3D).
AZD9496 is a potent, oral inhibitor of breast tumour growth in vivo
The effect of chronic, oral dosing of AZD9496 was explored in MCF7 human breast
xenografts, as a representative ER+/PR+/HER2+ breast cancer model. Good bioavailability
and high clearance gave a terminal t½ of 5-6 hours after oral dosing in the mouse and resulted
in significant dose-dependent tumour growth inhibition with 96% inhibition at 50 mg/kg and
no toxicity or weight loss relative to the vehicle control group (Fig. 4A). To confirm that
AZD9496 was targeting the ER pathway, PR protein levels were measured in tumour samples
taken at the end of the study and a significant reduction in PR was seen which correlated with
tumour growth inhibition. A >90% reduction in PR was seen with both 10 and 50 mg/kg
doses and a 75% decrease even with the 0.5 mg/kg dose, demonstrating that AZD9496 can
clearly antagonise the ER pathway (Fig. 4B). Dosing 5 mg/kg of AZD9496, the minimal
dose required to see significant tumour inhibition, gave greater tumour growth inhibition
compared to 5 mg/mouse fulvestrant given 3x weekly and tamoxifen given 10 mg/kg orally,
daily (Fig. 4C). A series of pharmacodynamic in vivo studies were conducted to measure
time taken to reach maximal inhibition of PR levels and time taken to recover back to basal
levels. Three days of dosing with 5 mg/kg of AZD9496 gave 98% reduction of PR protein
and continued to suppress protein levels at 48 hours (Fig. 4D) with full recovery by 72 hours
(data not shown) which indicates a long pharmacological half life in vivo. Fulvestrant, given
as 3 x 5 mg/mouse doses over one week, gave a 60% reduction in PR protein over a
prolonged period with measured plasma levels ~8-fold higher than those achieved clinically,
at steady state, with 500 mg fulvestrant (Fig. 4E). As estrogen itself is known to down-
regulate ER protein (38, 39), we were unable to detect further decreases in ERα protein
compared to control animals with AZD9496 or fulvestrant in the MCF-7 model presumably
due to the high circulating plasma levels of estrogen from implanted pellets at the time
tumour samples were taken (Supplementary Fig. S5). A mouse specific metabolite of
AZD9496 was detected in circulating plasma at similar levels to AZD9496 and showed a
similar pharmacokinetic profile. Testing this metabolite in the in vitro MCF-7 assays resulted
in ~5 fold lower ERα antagonism activity and 7 fold lower ERα down-regulation activity
than AZD9496 (data not shown). Using a PK/PD model based on PR inhibition data, at the 5
mg/kg dose in vivo, which gives 98 % inhibition of PR, the inhibitory activity that could be
attributed to the parent compound alone was 85% when the activity of the metabolite was
To explore mechanistic effects on ER pathway regulation further, a gene
expression analysis study was performed using tumour samples dosed at 10 mg/kg for 3 days
with AZD9496 or tamoxifen and fulvestrant given as a single dose 5 mg dose
subcutaneously. Tumours were taken 24 hours after the last dose of AZD9496 and tamoxifen
and mRNA levels measured using a Human Transcriptome Array (HTA 2.0). A number of
known ER regulated genes were examined to see if AZD9496 could antagonise these genes
in a similar manner to Fulvestrant and tamoxifen (40-42). The heap map shows that
AZD9496 could indeed down-regulate the mRNA levels of estrogen responsive genes
(Supplementary Fig. S6A). As differences in the levels of mRNA down-regulation in vivo
could be due to different PK profiles of the molecules, the effects of compounds on the
mRNA levels of a subset of estrogen regulated genes were also measured in MCF-7 cells that
had been treated with or without estrogen (Supplementary Fig. 6 B-E). For two of these
genes, TFF1 and AREG, tamoxifen was shown to increase mRNA levels in the absence of
estrogen but not with either fulvestrant or AZD9496 (Supplementary Fig. 6 D, E). Where
transcript levels were increased to varying levels in the presence of estrogen, fulvestrant and
AZD9496 inhibited transcript levels in a dose dependent fashion compared to tamoxifen. No
significant effects were seen in ESR1 mRNA levels in vitro indicating that the protein down-
regulation described previously is due to down-regulation of the protein and not a decrease in
transcript levels per se (Supplementary Fig. 6F).
AZD9496 causes tumour regressions in combination with PI3K pathway and CDK4/6
inhibitors and in an estrogen deprived ER +ve model of resistance
Acquired resistance to aromatase inhibitors and antiestrogens is driven through
a variety of mechanisms which include effects on the ER pathway itself i.e. down-regulation
of ER expression and altered expression of ER co-regulators, as well as activation of
alternative pro-survival or proliferative pathways and cross-talk between growth factor
receptors and ER pathways. This has led to a number of clinical trials with PIK3, mTOR,
AKT inhibitors, as well as CDK4/6 inhibitors in combination with aromatase inhibitors or
fulvestrant and early results have showed increased progression-free survival (PFS) in many
cases (43). Combinations of AZD9496 with AZD2014 (dual mTORC1/2), AZD8835
(PIK3Cα/δ) and Palbociclib (CDK4/6) inhibitors were tested in the MCF-7 in vivo model and
in all cases tumour regressions seen with the combination arms alone compared to tumour
stasis with monotherapy (Fig. 5A-C). AZD9496 was also tested in a long-term estrogen
deprived model (LTED), using the HCC-1428 LTED cell line that was previously adapted to
grow in the absence of estrogen and as such represents a model of aromatase inhibitor
resistance (22). Tumour regressions were seen with both AZD9496 and fulvestrant (Fig. 5D)
and both compounds completed ablated ER protein levels in the end of study tumours
(Supplementary Fig. S7).
AZD9496 antagonises and down-regulates mutant ER in vitro and in vivo
The recent identification of ESR1 mutations in patients that had received one or more
endocrine therapy treatments has led to the discovery that these mutations can drive cell
proliferation in the absence of estrogen and may constitute a resistance mechanism to some
endocrine agents. In vitro binding studies were performed using wt, D538G and Y537S
LBDs and both AZD9496 and fulvestrant were able to bind to mutant LBD with nM potency
although 2-3 fold reduced compared to wt (Table 1B). The different IC50 values obtained for
binding of AZD9496 and fulvestrant to wt ERα compared to the value in Table 1A is likely
due to different sources of protein used in the Lanthascreen kit assays versus the laboratory
derived wt and ESR1 mutant proteins. Down-regulation of ER mutant proteins was measured
using MCF-7 doxycycline inducible stable cell lines in vitro. ER protein levels increased on
induction with 1 μg/ml doxycyline and led to increased levels of PR in the absence of
estrogen. AZD9496 and fulvestrant were able to down-regulate all mutant ER proteins and
decreased PR levels whereas tamoxifen appeared to stabilise ER protein levels and reduced
PR levels to a lesser extent (Fig. 6A). To explore activity against an ESR1 mutant in vivo,
tumour growth inhibition studies were performed in a patient-derived xenograft (PDX)
model, CTC-174, which harbours a D538G mutation in the LBD and constituted 31% of the
transcripts variants identified by RNA-seq analysis. Tumours were grown from implanted
tumour fragments either with or without estrogen and in both cases viable tumours grew.
Given published data that higher doses of SERM/SERDs may be needed to inhibit ESR1
mutants (14, 15), 25mg/kg of AZD9496 was used alongside 5mg/mouse of fulvestrant which
was known to achieve higher PK levels in mouse than the current clinical dose. AZD9496
and fulvestrant inhibited tumour growth by 66% and 59% respectively, compared to
tamoxifen which gave 28% inhibition. Efficacy correlated with antagonism of the pathway
with AZD9496 giving a 94% decrease in PR levels compared to 63% with fulvestrant (Fig.
6B and C). In the absence of estrogen, and given the significant PR changes seen with
25mg/kg, the dose of AZD9496 was lowered but a 5 mg/kg dose still achieved growth
inhibition of ~70% and led to a significant decrease of 73% in ERα protein levels at the end
of study (Fig. 6D and E). Given the similarities in growth inhibition either with or without
estrogen, residual tumour growth is likely to be due to either additional mutations (e.g.
PIKCA N345K) and/or growth factor signalling pathways involved in driving tumour growth
in addition to the ER pathway.
Despite the enhanced clinical benefits of directly antagonising and down-regulating ER and
thereby targeting any ligand-independent ER driven pathways, the pharmacokinetic and IHC
biomarker data for ER, PR and Ki67 from clinical trials with fulvestrant would suggest that a
plateau of effect has not been reached, and there is scope to further improve on the 50%
reduction in ER levels seen after 6 months of treatment (8, 12). As one of the potential draw-
backs associated with fulvestrant is the very low bioavailability which is seen both in patients
and in pre-clinical animal models, a potent, orally bioavailable SERD could have the
potential to significantly antagonise ER activity and increase ERα receptor knockdown
versus fulvestrant due to higher exposures. Our approach focused on identifying novel ER
motifs with established drug-like properties that could then be optimized for cellular
phenotype and potency and led to the identification of AZD9496 which showed pM potency
in vitro in ER+ve cell lines only. Previous structural analysis of a number of SERMs with
ERα LBD identified key interactions with Asp 351 which was shown to be critical for the
antagonist activity of anti-estrogens such as raloxifene, lasofoxifene and GW5638 through
shielding or neutralisation of the negative charge. Unlike tamoxifen however, GW5638
repositioned residues Helix-12 through its acrylate side chain, increasing the exposed
hydrophobic surface of ERα resulting in destabilisation and ultimately 26S proteosomal
degradation of the receptor. The acrylic acid moiety of AZD9496 is co-localized with the Asp 351
residue in the helix-12 region of ER, in an unusual acid-acid interaction and it is therefore proposed
that AZD9496 also repositions residues associated with the helix-12 via specific contacts with the N-
terminus region resulting in a mixed antagonist and down-regulator profile (33, 44). Evidence that
AZD9496 was directly binding and down-regulating the receptor at the same site as other
anti-estrogens was seen by the reduced potency observed on prior addition of tamoxifen to
the down-regulation assay (Table 1A). Mechanistically we have shown that AZD9496 has
very similar effects on antagonism, ERα down-regulation and agonism to fulvestrant. Even
where ERα was over-expressed in MCF-7 cells, unlike tamoxifen, AZD9496 was able to
down-regulate and antagonise the over-expressed receptor (Fig 6A). Our transcriptional
studies in both MCF-7 cells and the in vivo model also demonstrated antagonism of the
transcriptional activity of the receptor as seen by decreased mRNA levels of ER-activated
genes in a dose dependent manner (Supplementary Fig. 6).
Unlike fulvestrant, AZD9496 did show some degree of agonism in endometrial
tissue (Fig. 3). This effect was dose independent and would indicate that there are subtle
differences in how AZD9496 interacts with ERα compared to tamoxifen and fulvestrant and
therefore the extent to which tissue specific co-activators can interact with the receptor.
Given the widespread use of tamoxifen in the adjuvant setting for 5-10 years, the low level of
agonism seen with AZD9496 is not viewed as a deterrent for clinical development. A number
of studies have looked at the incidence of endometrial cancer in patients taking tamoxifen
over a number of years and despite the small increase in the incidence of endometrial cancer,
the benefits of tamoxifen greatly outweigh any risks of developing uterine malignancy in
post-menopausal patients (45, 46).
A PK-PD-efficacy model was established, integrating diverse endpoints such as
drug exposure, PR modulation and inhibition of tumour growth (data not shown). The model
reflected relative contributions of AZD9496 and its active mouse metabolite to the overall
biomarker modulation and efficacy. Given the half life of the parent drug and metabolite are
around 5 hours, to explain the prolonged PD effect observed with AZD9496 at 48 hours, an
indirect response model for PR was developed which estimated a degradation rate constant of
around 23 hours for PR. This modulation in PR was linked directly to tumour growth
inhibition by a proportional decrease of the tumour growth constant, and correlated well with
the wide range of measured efficacies.
We consistently saw enhanced tumour growth inhibition of AZD9496
compared to fulvestrant in the MCF-7 in vivo model (Fig. 4). This model contains high
circulating levels of plasma estradiol over the dosing period of the study as a result of the
estrogen pellets used to drive growth of the MCF-7 cells as a xenograft. With plasma levels
between 750 and 1000 pg/ml over day 21-35, this model is more representative of the pre-
menopausal setting where estrogen levels can vary between 20 – 400 pg/ml (47). As the
receptor binding data does not suggest a significant difference in affinity for the receptor
between AZD9496 and fulvestrant, increased efficacy is likely due to the higher free drug
levels of AZD9496 versus fulvestrant in this model. It would therefore be interesting to test
the activity of potent oral SERDs, such as AZD9496, in the pre-menopausal setting where
there is no enhanced risk of endometrial cancer observed for tamoxifen.
We were unable to detect a reduction in ER protein levels in the MCF-7 in vivo
model with AZD9496 at any dose tested (data not shown). Having shown in vitro that
estradiol can efficiently down-regulate ERα protein, we postulate that down-regulation of
ERα by estradiol is already taking place in vivo given the high levels of circulating estrogen
and that any additional changes by addition of a SERD are difficult to detect using the
current methods. This was supported by measuring ERα down-regulation in in vivo models,
such as CTC-174 and HCC1428LTED, not requiring estrogen supplements, and showing
potent down-regulation of ERα by AZD9496 (Fig. 6E, Supplementary Fig. S7). The level of
ERα reduction seen in the HCC1428 model is greater than seen clinically, with PK levels 5
fold higher than steady state plasma levels of 500 mg fulvestrant, and supports the concept
that if increased plasma levels of an active SERD agent could be achieved clinically, greater
ERα knock-down may be possible. Pre-clinically, this appears to translate to enhanced
tumour growth inhibition.
Achieving increased serum levels of a biologically active SERD could be
particularly beneficial in the recently identified ESR1 mutant patient setting. Pre-clinical in
vivo studies using PDX models derived from ESR1 mutant patients grown in the absence of
estrogen supplementation have shown that high doses of fulvestrant (>500 mg/kg) can lead to
tumour regression and significant decreases in ER protein levels but at significantly higher
doses than could be achieved clinically (48). Our preclinical data would indicate that both
AZD9496 and fulvestrant can potently bind and down-regulate D538G and Y537C/N/S ERα
proteins in vitro and significant tumour growth inhibition rather than regression seen in vivo.
This is in contrast to published work showing relatively little down-regulation of Y537N
mutant protein in vitro with increasing doses of fulvestrant (15) but could be attributed to
different expression systems and cell lines used for the analysis. It is notable that the Dox-
inducible mutant receptors do not appear to be constitutively down-regulated in a similar
manner to wt ER exposed to estradiol (Supplementary Fig. S1). This may due to the level of
over-expression of the receptor in each stable cell line and it will be interesting to compare
these findings from engineered expression systems to those from genetically altered mutant
cell lines where expression is under control of the native promoter. Although very high doses
of both compounds were not tested in the PDX CTC-174 ESR1 mutant model, the lack of any
significant dose response might indicate the need to inhibit other growth factor activated
pathways in addition to the ER pathway to achieve significant anti-tumour growth. As trials
commence to explore treatment of ESR1 mutant patients with new oral SERDs, data will
hopefully emerge that will indicate whether lack of response is due to inability to fully inhibit
mutated ER receptors or whether in these advanced metastatic patients, other mutations and
pathways are or become dominant in driving tumour growth.
In metastatic breast cancer the median survival time is still around two years as
a result of acquired endocrine resistance, highlighting the need for additional therapies (43).
This has led to a number of clinical trials using PI3K-Akt-mTOR pathway inhibitors in
combination with AIs as well as cyclin-dependent kinase inhibitors such as palbociclib which
was recently approved for combination treatment with letrozole in the metastatic breast
cancer. Results from Phase III BOLERO-2 trial also demonstrated the PFS benefits of a
steroidal AI plus everolimus in patients that had progressed on a non-steroidal AI (49). More
recently further trials have been initiated using fulvestrant as the combination endocrine
partner which will allow analysis and understanding of any additional benefit from
combination with a SERD versus AIs in this setting. Our data clearly demonstrated enhanced
combination effects on tumour growth of an oral SERD agent with either dual mTORC1/2,
PIKCα/δ or CDK4/6 inhibitors and furthermore opens up the possibility of using a combined
endocrine and targeted inhibitor approach with longer term dosing in the adjuvant setting,
assuming emerging data from on-going metastatic trials supports the testing of combinations
in early breast disease. The identification of a potent, orally bioavailable compound such as
AZD9496 is a step forward in the next generation of SERD agents to undergo clinical
evaluation as a future monotherapy or combination partner of choice.
The authors would like to thank Elaine Hurt, Haifeng Bao, Sanjoo Jalla for access to the
CTC-174 in vivo model developed at MedImmune, Gaithersburg MD 20878.The authors
thank Paul Hemsley and Emma Linney for providing ER mutant binding assay data, Helen
Gingell for supporting the crystallography work and Zara Ghazoui and Henry Brown for the
transcript data and analysis at AstraZeneca, Alderley Park, Macclesfield, UK. The authors
also thank Carlos Arteaga for use of the HCC1428-LTED cell line.
1. Davies C, Godwin J, Gray R, Clarke M, Cutter D, et al. Early Breast Cancer Trialists'
Collaborative Group (EBCTCG), Relevance of breast cancer hormone receptors and other
factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised
trials. Lancet 2011;378:771-84.
2. Cuzick J, Sestak I, Baum M, Buzdar A, Howell A, Dowsett M, et al. Effect of anastrozole
and tamoxifen as adjuvant treatment for early-stage breast cancer: 10-year analysis of the
ATAC trial. Lancet Oncol 2010;11:1135-41.
3. Nicholson RI, Hutcheson IR, Jones HE, Hiscox SE, Giles M, Taylor KM, et al. Growth
factor signalling in endocrine and anti-growth factor resistant breast cancer. Rev Endocr
Metab Disord 2007;8:241-53.
4. Lupien M, Meyer CA, Bailey ST, Eeckhoute J, Cook J, Westerling T, et al. Growth factor
stimulation induces a distinct ER(alpha) cistrome underlying breast cancer endocrine
resistance. Genes Dev. 2010 Oct 1;24(19):2219-27.
5. Massarweh S, Schiff R. Resistance to endocrine therapy in breast cancer: exploiting
estrogen receptor/growth factor signaling crosstalk. Endocr Relat Cancer. 2006 Dec;13
6. Ali S, Coombes RC. Endocrine-responsive breast cancer and strategies for combating
resistance. Nat Rev Cancer 2002;2:101-12.
7. Di Leo A, Jerusalem G, Petruzelka L, Torres R, Bondarenko IN, Khasanov R, et al. Final
overall survival: fulvestrant 500 mg vs 250 mg in the randomized CONFIRM trial. J Natl
Cancer Inst 2014;106:337.
8. Kuter I, Gee JM, Hegg R, Singer CF, Badwe RA, Lowe ES, et al. Dose-dependent change
in biomarkers during neoadjuvant endocrine therapy with fulvestrant: results from
NEWEST, a randomized Phase II study. Breast Cancer Res Treat 2012;133:237-46.
9. Robertson JF, Lindemann J, Garnett S, Anderson E, Nicholson RI, Kuter I, et al. A good
drug made better: the fulvestrant dose-response story. Clin Breast Cancer 2014;14:381-9.
10. Robertson JF, Harrison M. Fulvestrant: pharmacokinetics and pharmacology. Br J Cancer
2004;90 Suppl 1:S7-10.
11. Howell A, Sapunar F. Fulvestrant revisited: efficacy and safety of the 500-mg dose. Clin
Breast Cancer 2011;11:204-10.
12. Robertson JF. Fulvestrant (Faslodex) -- how to make a good drug better. Oncologist
13. Li S, Shen D, Shao J, Crowder R, Liu W, Prat A, et al. Endocrine-therapy-resistant ESR1
variants revealed by genomic characterization of breast-cancer-derived xenografts. Cell
14. Toy W, Shen Y, Won H, Green B, Sakr RA, Will M, et al. ESR1 ligand-binding domain
mutations in hormone-resistant breast cancer. Nat Genet 2013;45:1439-45.
15. Jeselsohn R, Yelensky R, Buchwalter G, Frampton G, Meric-Bernstam F, GonzalezAngulo AM, et al. Emergence of constitutively active estrogen receptor-alpha mutations in
pretreated advanced estrogen receptor-positive breast cancer. Clin Cancer Res
16. Willson TM, Henke BR, Momtahen TM, Charifson PS, Batchelor KW, Lubahn DB, et al.
3-[4-(1,2-Diphenylbut-1-enyl)phenyl]acrylic acid: a non-steroidal estrogen with functional
selectivity for bone over uterus in rats. J Med Chem 1994;37:1550-2.
17. Hoffmann J, Bohlmann R, Heinrich N, Hofmeister H, Kroll J, Kunzer H, et al.
Characterization of new estrogen receptor destabilizing compounds: effects on estrogensensitive and tamoxifen-resistant breast cancer. J Natl Cancer Inst 2004;96:210-8.
18. Tan Q, Blizzard TA, Morgan JD, Birzin ET, Chan W, Yang YT, et al. Estrogen receptor
ligands. Part 10: Chromanes: old scaffolds for new SERAMs. Bioorg Med Chem Lett
19. Suzuki N, Liu X, Laxmi YR, Okamoto K, Kim HJ, Zhang G, et al. Anti-breast cancer
potential of SS5020, a novel benzopyran antiestrogen. Int J Cancer 2011;128:974-82.
20. Lai A, Kahraman M, Govek S, Nagasawa J, Bonnefous C, Julien J, et al. Identification of
GDC-0810 (ARN-810), an orally bioavailable Selective Estrogen Receptor Degrader
(SERD) that demonstrates robust activity in tamoxifen-resistant breast cancer xenografts. J
Med Chem 2015[Epub ahead of print].
21 Garner F, Shomali M, Paquin D, Lyttle R, Hattersley G. RAD1901: a novel, orally
bioavailable selective estrogen receptor degrader that demonstrates antitumour activity in
breast cancer xenograft models. Anti-Cancer Drugs 2015;26:948-956.
22. Miller TW, Balko JM, Ghazoui Z, Dunbier A, Anderson H, Dowsett M, et al. A gene
expression signature from human breast cancer cells with acquired hormone independence
identifies MYC as a mediator of antiestrogen resistance. Clin Cancer Res 2011;17:202434.
23. Callis R, Rabow A, Tonge M, Bradbury R, Challinor M, Roberts K, et al. A Screening
Assay Cascade to Identify and Characterize Novel Selective Estrogen Receptor
Downregulators (SERDs). J Biomol Screen 2015 [Epub ahead of print].
24. Degorce SL, Bailey A, Callis R, De Savi C, Ducray R, Lamont G, et al. Investigation of
(E)-3-[4-(2-Oxo-3-aryl-chromen-4-yl)oxyphenyl]acrylic Acids as Oral Selective Estrogen
Receptor Down-Regulators. J Med Chem 2015;58:3522-33.
25. Kabsch W. Xds. Acta Crystallogr D Biol Crystallogr 2010;66:125-32.
26. Incardona MF, Bourenkov GP, Levik K, Pieritz RA, Popov AN, Svensson O. EDNA: a
framework for plugin-based applications applied to X-ray experiment online data analysis.
J Synchrotron Radiat 2009;16:872-9.
27. Evans P. Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr
28. Navaza J. Implementation of molecular replacement in AMoRe. Acta Crystallogr D Biol
29. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta
Crystallogr D Biol Crystallogr. 2010;66:486-501.
30. Ong S, Mann M. A practical recipe for stable isotope labeling by amino acids in cell
culture (SILAC). Nat Protocols. 2007;1:2650-2660.
31. Wakeling AE, O'Connor KM, Newboult E. Comparison of the biological effects of
tamoxifen and a new antioestrogen (LY 117018) on the immature rat uterus. J Endocrinol
32. Bradbury R, De Savi C, Rabow A, Norman R, de Almeida C, Andrews D. Optimization of a
novel binding motif to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (AZD9496), a
potent and orally bioavailable selective estrogen receptor downregulator and antagonist. J
Med. Chem 2015 manuscript submitted.
33. Wu YL, Yang X, Ren Z, McDonnell DP, Norris JD, Willson TM, et al. Structural basis
for an unexpected mode of SERM-mediated ER antagonism. Mol Cell 2005;18:413-24.
34. Horwitz KB, McGuire WL. Estrogen control of progesterone receptor in human breast
cancer. Correlation with nuclear processing of estrogen receptor. J Biol Chem
35. Rich RL, Hoth LR, Geoghegan KF, Brown TA, LeMotte PK, Simons SP, et al. Kinetic
analysis of estrogen receptor/ligand interactions. Proc Natl Acad Sci USA 2002;99:856267.
36. Pike AC, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstrom O, et al.
Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a
partial agonist and a full antagonist. EMBO J 1999;18:4608-18.
37. Grese TA, Sluka JP, Bryant HU, Cullinan GJ, Glasebrook AL, Jones CD, et al. Molecular
determinants of tissue selectivity in estrogen receptor modulators. Proc Natl Acad Sci U S
38. Lonard DM, Nawaz Z, Smith CL, O'Malley BW. The 26S proteasome is required for
estrogen receptor-alpha and coactivator turnover and for efficient estrogen receptor-alpha
transactivation. Mol Cell. 2000; 5:939-48.
39. Reid G, Hübner MR., Métivier R, Brand H et al. Cyclic, proteosome-mediated turnover
of unliganded and liganded ERα on responsive promoters is an integral feature of estrogen
signaling. Mol Cell 2003; 11:695-707.
40. Ghosh MG, Thompson DA, Weigel RJ. PDZK1 and GREB1 are estrogen-regulated genes
expressed in hormone-responsive breast cancer. Cancer Res 2000;60:6367-75.
41. Sauve K, Lepage J, Sanchez M, Heveker N, Tremblay A. Positive feedback activation of
estrogen receptors by the CXCL12-CXCR4 pathway. Cancer Res 2009;69:5793-800.
42. Vendrell JA, Magnino F , Danis E, Duchesne MJ , Pinloche S, Pons S , et al. Estrogen
regulation in human breast cancer cells of new downstream gene targets involved in
estrogen metabolism, cell proliferation and cell transformation. J Mol Endocrinology
43. Austreid E, Lonning PE, Eikesdal HP. The emergence of targeted drugs in breast cancer
to prevent resistance to endocrine treatment and chemotherapy. Expert Opin Pharmacother
44 . Liu H, Park W, Bentrem DJ, McKian KP, De Los Reyes A, Loweth JA et al. Structurefunction relationship of the raloxifene-estrogen receptor-α complex for regulating
transforming growth factor-α in breast cancer cells. J Biol Chem 2002; 277:9189-9198.
45. Davies C, Pan H, Godwin J, Gray R, Arriagada R, Raina V, et al. Long-term effects of
continuing adjuvant tamoxifen to 10 years versus stopping at 5 years after diagnosis of
oestrogen receptor-positive breast cancer: ATLAS, a randomised trial. Lancet
46. Bergman L, Beelen ML, Gallee MP, Hollema H, Benraadt J, van Leeuwen FE. Risk and
prognosis of endometrial cancer after tamoxifen for breast cancer. Lancet 2000;356:881-7.
47. Folkerd E, Dowsett M. Sex hormones and breast cancer risk and prognosis. Breast
2013;22 Suppl 2:S38-43.
48. Sen T, Li S, Shao J, Crowder R, Kitchen R, Ellis MJ. Patient-derived xenograft study
reveals the pharmacology and the role of ESR1gene aberrations in endocrine therapy
resistance of ER positive breast cancer. Can Res 2014;74:5544.
49. Piccart M, Hortobagyi GN, Campone M, Pritchard KI, Lebrun F, Ito Y, et al. Everolimus
plus exemestane for hormone-receptor-positive, human epidermal growth factor receptor2-negative advanced breast cancer: overall survival results from BOLERO-2. Ann Oncol
Table 1 A
In vitro ERα binding, ERα down-regulation, ERα antagonism and
MCF-7 cell growth inhibition data for AZD9496 vs. fulvestrant
ERα binding IC50 nM
ERα down-regulation IC50 nM
ERα down-regulation IC50 nM
+ 250 nM tamoxifen
ERα antagonism IC50 nM
MCF-7 cell growth inhibition EC50 nM
Human % free
Table 1 B
In vitro binding to ERα mutant LBDs
ERα LBD binding IC50 nM
Tables 1A and B. Summary of in vitro cellular potency and phenotype of AZD9496. A -
A series of in vitro assays were performed in MCF-7 cell in hormone-depleted medium to
show AZD9496 effects on ER binding, down-regulation, antagonism and inhibition of cell
proliferation as described in (20) using a fluorescence end-point for measured of ER and PR
protein levels; data shown are representative of the mean IC50 or EC50 from at least four
independent experiments. B - IC50s for binding of AZD9496 and fulvestrant to ERα wt and
mutant LBDs; data shown are representative of the mean IC50 from three independent
AZD9496 (A) Two-dimensional chemical representation of AZD9496. (B) Two-dimensional
chemical representation of 4-hydroxy tamoxifen. (C) Three-dimensional structure of the
complex between AZD9496 and the ligand-binding domain of ERα. The protein and ligand
structures are represented as sticks with carbon and fluorine atoms coloured grey and light
blue respectively. The van der Waals radii of the protein atoms are shown as a continuous
surface highlighting the ligand-binding pocket of the ERα. Key protein residues are labelled
and polar interactions are shown as dotted lines.
Figure 1. Crystal structure of the ligand-binding domain of ERα in complex with
Figure 2. Effect of AZD9496, fulvestrant and tamoxifen on ERα peptide turnover in
Cells were grown in steroid-free conditions in SILAC media containing 13C615N4 L-arginine
to label ERα peptide as “heavy” (blue line) and then switched to grow in media containing
unlabelled L-arginine to label newly synthesised protein as “normal” (red line) with (A) 0.1%
DMSO, (B) 300 nM tamoxifen, (C) 100 nM AZD9496 or (D) 100 nM fulvestrant for the time
indicated. Data shown is representative of two independent experiments.
Figure 3. ERα mediated agonism in MCF-7 cells and immature female rat endometrial
tissue. (A) MCF-7 cells were treated with AZD9496 or a known agonist compound
AZ10557841 and PR levels (response) measured using in-cell immunofluorescence
quantification using an Acumen Ex3 platform. Data was normalised for cell number by
analysing a ratio of fluorescence resulting from stimulation of two emission wavelengths and
used to perform curve fitting analysis. A concentration response was plotted using non-
linear regression analysis. (B, C) Immature female rats were dosed orally with AZD9496 or
tamoxifen or subcutaneously with fulvestrant for 3 days. PEG/Captisol, polysorbate and
peanut oil were used as vehicle controls for AZD9496, tamoxifen and fulvestrant
respectively. Uterine tissue was removed 24 hrs after the final dose and weighed before
tissues were formalin-fixed and paraffin embedded to allow histological staining of the
endometrial tissue. Significant differences in mean uterine weight are shown: ***p<0.001
and ** p<0.01 (D). Tissue was subsequently analysed by Western blot for levels of ERα, PR
and vinculin. Protein levels were measured by chemiluminescent and quantified using
Syngene software. ER and PR protein levels were normalised to vinculin as a loading control
and plotted as shown, Statistically significant differences in PR levels are shown:
***p<0.001, ** p<0.01 and * p<0.05.
Figure 4. In vivo efficacy of AZD9496 in MCF-7 xenograft model
(vehicle) or AZD9496 at doses shown (A). Tumour growth was measured by caliper at
regular intervals and mean tumour volumes plotted for each dosed group. Tumours from
treated mice collected at the end of study were analysed by Western blot for levels of PR and
vinculin. Protein levels were measured by chemiluminescent and quantified using Syngene
software. PR protein levels were normalised to vinculin as a loading control and plotted as
shown with standard error bars * p<0.05, *** p<0.001(B). MCF-7 xenografts were also
dosed daily with 5 mg/kg AZD9496, 10 mg/kg tamoxifen or 5 mg/mouse 3x weekly with
fulvestrant subcutaneously (C). For acute dosing studies, MCF-7 xenografts were dosed for 3
days with doses shown of AZD9496 and tumours collected at 24 or 48 hrs after the last dose
(D) or dosed with 3 doses of 5 mg/mouse of fulvestrant over 5 days and tumours collected at
24, 48 and 96 hours after the last dose (E). PR and vinculin protein levels in the tumours were
analysed and plotted as described above.
MCF-7 xenografts, grown in male SCID mice, were dosed daily with either PEG/captisol
Figure 5. Tumour regressions with AZD9496 in combination with mTOR, PIKC and
CDK4/6 inhibitors in MCF-7 xenograft model and in HCC1428 LTED model.
with or without AZD2014 dosed at 20 mg/kg twice daily (b.i.d) 2 days out of 7, (A) with or
without AZD8835 dosed at 50 mg/kg b.i.d. on day1 and 4 (B) with or without Palbociclib
dosed daily at 50 mg/kg (C) HCC1428LTED engrafts were grown in ovariectomised NSG
mice and were dosed daily with either AZD9496 or once weekly with fulvestrant at doses
shown (D). Tumour growth was measured by caliper at regular intervals and mean tumour
volumes plotted for each dosed group.
MCF-7 xenografts, grown in male SCID mice, were dosed daily with AZD9496 at 5mg/kg
Figure 6: AZD9496 is efficacious against ESR1 mutants in vitro and in vivo.
Stable MCF-7 cell lines containing either pTRIPZ vector alone or vector containing ESR1
mutants shown were induced to express mutant protein with 1μg/ml doxycycline for 24hrs
before treating with either 0.1% DMSO (vehicle) or 100nM of fulvestrant, AZD9496 or
tamoxifen for 24hrs. Immunoblotting was performed to detect expression of ERα, PR and
GAPDH levels (A). CTC-174 tumour fragments were grown in female NSG mice implanted
with estrogen pellets and dosed daily with either PEG/captisol (vehicle) or AZD9496,
tamoxifen and Fulvestrant at doses shown. Tumour growth was measured by caliper at
regular intervals and mean tumour volumes plotted for each dosed group (B). Tumours from
treated mice collected at the end of study were analysed by Western blot for levels of PR and
vinculin. Protein levels were measured by chemiluminescent and quantified using Syngene
software. PR protein levels were normalised to vinculin as a loading control and plotted as
shown ***p<0.001(C). CTC-174 tumour fragments were grown in female NSG mice without
estrogen pellets to enable measurement of ER levels and dosed daily with either PEG/captisol
(vehicle) or AZD9496 at doses shown (D). Tumours from treated mice collected at the end of
study were analysed by Western blot for levels of ERα and vinculin and measured as
described above * p<0.05 (E)
Figure 1. Crystal structure of the ligand-binding domain of ERa in complex with AZD9496
Figure 2. Effect of AZD9496, fulvestrant and tamoxifen on ERa peptide turnover in MCF-7
0 .1 5
0 .1 0
0 .0 5
0 .0 0
C o n c e n t r a t io n lo g 1 0 ( M )
- 0 .0 5
Immature rat: control
Figure 3 ERα mediated agonism in MCF-7 cells and immature female rat
Figure 4. In vivo efficacy of AZD9496 in MCF-7 xenograft model.
Figure 5. Tumour regressions with AZD9496 in combination with mTOR, PIKC and CDK4/6
inhibitors in MCF-7 xenograft model and HCC1428 LTED model
Figure 6: AZD9496 is efficacious against ESR1 mutants in vitro and in vivo.