AZD9496: An oral estrogen receptor inhibitor that blocks the growth

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AZD9496: An oral estrogen receptor inhibitor that blocks the growth
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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
De Savi.d
Authors’ Affiliations:
a
Oncology iMed, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK;
b
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.
combination therapy
Corresponding author: [email protected]
Disclosure of Potential Conflicts of Interest: All authors are present or past employees of
AstraZeneca Pharmaceuticals
Tables: 1
Figures: 6
Supplementary Tables:4
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.
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Abstract
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Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by
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monthly intramuscular injection. Given its present limitations of dosing and route of
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administration, a more flexible orally available compound has been sought to pursue the
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potential benefits of this drug in patients with advanced metastatic disease. Here we report
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the identification and characterization of AZD9496, a non-steroidal small molecule inhibitor
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of ERα which is a potent and selective antagonist and down-regulator of ERα in vitro and in
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vivo in ER-positive models of breast cancer. Significant tumour growth inhibition was
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observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where
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this effect was accompanied by a dose-dependent decrease in PR protein levels demonstrating
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potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors
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led to further growth inhibitory effects compared to monotherapy alone. Tumour regressions
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were also seen in a long-term estrogen-deprived breast model, where significant down-
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regulation of ERα protein was observed. AZD9496 bound and down-regulated clinically
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relevant ESR1 mutants in vitro and inhibited tumour growth in an ESR1-mutant patient-
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derived xenograft model that included a D538G mutation. Collectively, the pharmacological
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evidence showed that AZD9496 is an oral, non-steroidal, selective estrogen receptor
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antagonist and down-regulator in ER-positive breast cells that could provide meaningful
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benefit to ER-positive breast cancer patients. AZD9496 is currently being evaluated in a
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Phase 1 clinical trial.
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Introduction
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With over 70% of breast cancers expressing the estrogen receptor alpha (ERα),
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treatment with anti-hormonal therapies that directly antagonise ER function (e.g. tamoxifen)
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or therapies that block the production of the ligand, estrogen (e.g. aromatase inhibitors) are
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key to management of the disease both in adjuvant and metastatic settings (1, 2). Despite
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initial efficacy seen with endocrine therapies, the development of acquired resistance
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ultimately limits the use of these agents although the majority of tumours continue to require
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ERα for growth. Through the use of pre-clinical models and cell lines, changes in growth
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factor receptors have been implicated in driving resistance in cells e.g. receptor tyrosine
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kinases (HER2), epidermal growth factor receptor (EGFR) and their downstream signalling
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pathways, Ras/Raf/MAPK and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)
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signalling (3, 4, 5). In some instances signalling from different growth factor receptor kinases
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leads to phosphorylation of various proteins in the ER pathway, including ER itself, leading
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to ligand–independent activation of the ER pathway (6).
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In the metastatic setting, 500 mg fulvestrant, given as 2 x 250 mg intramuscular
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injections, has been shown to offer additional benefit over the 250mg dose with improved
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median overall survival (7, 8). Recent analysis of pre-surgical data from a number of trials
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showed a dose-dependent effect on key biomarkers such as ER, progesterone receptor (PR)
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and Ki67 labeling index (9). Although fulvestrant is clearly effective in this later treatment
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setting, there are still perceived limitations in its overall clinical benefit due to very low
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bioavailability, the time taken to achieve plasma steady-state of 3-6 months and ultimately
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the level of ERα reduction seen in clinical samples (10, 11). It is widely believed that a
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potent, orally bioavailable SERD that could achieve higher steady-state free drug levels more
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rapidly would have the potential for increased receptor knock-down and lead to quicker
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clinical responses, reducing the possibility of early relapses (12). Moreover, the recent
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discovery of ESR1 mutations in metastatic breast cancer patients that had received prior
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endocrine treatments, where the mutated receptor is active in the absence of estrogen,
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highlights an important potential resistance mechanism. Pre-clinical in vitro and in vivo
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studies have shown a reduction in signalling from ESR1 mutants by tamoxifen and fulvestrant
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but at higher doses than required to inhibit the wild type receptor which implies that more
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potent SERDs may be required to treat these patients or to used in the adjuvant setting to
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limit the emergence of mutations (13, 14, 15).
Achieving oral bioavailability has remained a key challenge in the design of ER
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down-regulators and until now no oral estrogen receptor down-regulators, other than GDC-
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0810 and RAD1901, have progressed into clinical trials, despite encouraging reports from
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others of pre-clinical activity in mouse xenograft models (16, 17, 18, 19, 20, 21). Here we
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describe a chemically novel, non-steroidal ERα antagonist and down-regulator, AZD9496,
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that can be administered orally and links increased tumour growth inhibition to enhanced
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biomarker modulation compared to fulvestrant in a preclinical in vivo model of breast cancer.
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Moreover we show that AZD9496 is a potent inhibitor of ESR1 mutant receptors and can
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drive tumour growth inhibition in a patient-derived ESR1 mutant in vivo model (PDX). Hence
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AZD9496 is anticipated to yield improved bioavailability and clinical benefit through
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enhanced ER pathway modulation.
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Methods
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Cell lines and culture
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All AZ cell lines were tested and authenticated by short-tandem repeat (STR) analysis. MCF-
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7 was obtained from DSMZ and last STR tested February 2013. MDA-MB-361 (STR tested
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October 2011), MDA-MB-134 (STR tested October 2010), T47D (STR tested June 2012),
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CAMA-1 (STR tested March 2012) HCC1428 (STR tested December 2011) HCC70 (STR
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tested October 2013), LnCAP (STR tested August 2011), MDA-MB-468 (STR tested March
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2014), BT474 (STR tested July 2013), PC3 (STR tested October 2012), HCT116 (STR
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tested August 2011) were all obtained from the ATCC. HCC1428-LTED cell line (STR
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tested November 2012) was kindly provided by Carlos Arteaga (22). Cells were cultured in
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RPMI-1640 media (Corning) supplemented with 10% heat-inactivated foetal calf serum
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(FCS) or charcoal/dextran-treated CCS to deplete hormone and 2mmol/L L-glutamine
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(Corning).. Stable MCF-7 cell lines expressing FLAG-tagged ESR1 mutant proteins in
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pTRIPZ lentiviral vectors (GE Healthcare) under the control of the doxycycline inducible Tet
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promoter were generated by lentiviral infection using standard techniques.
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Biochemical and in vitro cell assays
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Binding, ER agonism, antagonism, down-regulation and cell proliferation assays were carried
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out as described previously (23). BIAcore affinity measurements and immunoblotting from
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compound treated cells are described in Supplementary Methods.
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Protein production, crystallization and structure determination
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Protein expression, purification and crystallization of the ERα ligand-binding domain was
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carried out as previously described (24). X-ray diffraction data were collected at the ESRF on
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beamline ID23-1 on an ADSC detector. Data were processed using XDS (25) as implemented
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within EDNA (26) and scaled and merged using SCALA (27). The structure of ERα in
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complex with AZD9496 was solved by molecular replacement using AmoRE and an internal
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ERα structure as the search model (28). Quality checks on the protein structures were carried
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out using the validation tools in Coot (29). The final structure has been deposited in the
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Protein Databank (PDB) with the ID code given in Supplementary Table S1.
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SILAC and mass spectrometry
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SILAC assays were carried out as previously described (30) and detailed in Supplementary
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methods. Compound treated samples were analysed by mass spectrometry using relative
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peptide quantification by selected reaction monitoring (SRM). Degradation half-life was
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measured using the one-phase exponential decay equation in GraphPad PRISM (Y=Span.e-
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K.X
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decreases to Plateau with a rate constant K.
+Plateau) where X is time and Y is response which starts out as Span+Plateau and
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Rat Uterine and Xenograft Studies
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All studies involving animals in the United Kingdom were conducted in accordance with UK
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Home Office legislation, the Animal Scientific procedures Act 1986 (ASPA) and
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AstraZeneca Global Bioethics policy or with US federal, state and Institutional guidelines in
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an AAALAC-accredited facility. The rat uterine weight assay for the measurement of
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estrogenic activity were performed as described previously (31). Details of individual
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models, protein analysis of tumour fragments and pharmacokinetic analysis of plasma
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samples are included in Supplementary Methods.
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Results
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AZD9496 is a selective ERα antagonist, down-regulator and inhibitor of ER positive
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tumour cell growth
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AZD9496 ((E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-
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tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid, Fig. 1A) was identified through
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structure based design and iterative medicinal chemistry structure-activity-relationship (SAR)
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studies from a novel ER binding motif (32). A directed ER binding screen was established to
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identify novel binding motifs that could then be optimised for both cellular phenotype and
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potency while maintaining good pharmacokinetic properties (23). Unlike fulvestrant, and
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other well known historical modulators of the ER receptor, including hydroxy-tamoxifen
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(Fig. 1B), AZD9496 lacks a phenolic moiety in which the phenol mimics the A-ring phenol
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group of the endogenous ligand estradiol but gains its potency through a different series of
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novel interactions with the ER protein (Fig. 1C). The indole NH picks up an interaction by
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forming a hydrogen bond to the carbonyl of Leu-346, with additional lipophilic interactions
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achieved by both the chiral methyl substituent, adjacent to the ring nitrogen and the N-
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substituted isopropyl fluoro side-chain occupying a “lipophilic hole” in the ER protein. The
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acrylic acid side chain picks up an unusual acid-acid co-localization with Asp-351 in the
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Helix-12 region of the ER that has been proposed to be crucial for achieving a down-
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regulator-antagonist profile (33). AZD9496 showed high oral bioavailability across three
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species (F% 63, 91 and 74, rat, mouse and dog, respectively) with generally low volume and
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clearance across species, albeit a higher clearance in mouse. The percent free levels in human
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plasma of 0.15% were 5 fold higher than those measured for fulvestrant.
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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
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vitro cell assays were developed specifically to measure ERα down-regulation, agonism and
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antagonism through simultaneous measurement of ER and PR protein levels in cells after
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treatment with AZD9496 (23). PR expression is known to be regulated by ER transcription
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and was measured as a marker of ER activation in cells (34). The potency of AZD9496
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approached that observed with fulvestrant in achieving pM IC50 in ERα binding, down-
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regulation and antagonism and significant cell growth inhibition in hormone-depleted growth
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conditions. Adding 250 nM tamoxifen significantly lowered the IC50 value, as expected, if
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AZD9496 and tamoxifen are competing for binding to the LBD (Table 1A). The potent
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down-regulation of ERα was also verified by immunoblotting techniques (Supplementary
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Fig. S1).
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To understand the binding kinetics of AZD9496 to human ERα ligand binding
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domain (LBD), binding studies were performed in the presence of increasing concentrations
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of AZD9496 and association (kass) and dissociation (kdiss) rate constants were calculated.
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AZD9496 was calculated as having an apparent dissociation rate constant (kdiss) for ERα
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LBD of 0.00043 sec-1, at 25 ºC equating to a half-life (t ½) of 27 ± 2 min and the affinity
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derived from the ratio of the rate constants gave a KD of 0.33 nM (Supplementary Table
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S2). This is in good agreement with the binding IC50 of 0.82 nM and correlates well with
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previously published data on estrogen and tamoxifen binding to ER LBD (35). AZD9496
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showed pM equipotent binding to both ERα and ERβ isoforms as expected given their high
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sequence homology and similarity in tertiary structure in their LBDs (36) and highly selective
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binding compared to progesterone (~650 fold), glucocorticoid (~11223 fold) and androgen
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(~36375 fold) receptor LBDs (Supplementary Table S3). Selectivity for ER was also
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evident by measuring cell growth inhibition in a panel of ER +ve and ER -ve cell lines
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(Supplementary Table S4). No significant growth inhibition was seen in any ER -ve cell
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lines (EC50 >10 μM) in contrast to the low nM activity seen in a range of ER +ve cell lines.
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AZD9496 directly targets ERα for down-regulation in vitro
To measure the rate at which AZD9496 down-regulated ERα, stable isotope
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labelling of amino acids (SILAC) experiments were performed in MCF-7 cells using mass
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spectroscopy analysis to detect ERα protein levels after treatment with compounds. Addition
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of AZD9496 or fulvestrant increased the rate of degradation of the isotope-labelled ERα
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compared to DMSO control. Levels of newly synthesised ERα peptide were also reduced
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following AZD9496 and fulvestrant treatment, presumably due to on-going degradation of
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newly-synthesised ERα protein by compound present, whereas ERα protein levels continued
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to increase over time in the presence of tamoxifen or DMSO (Fig 2). The t½ of ERα was
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decreased from 3 hours, in the presence of DMSO, to 0.75 hours with 100 nM AZD9496 and
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0.6 hours with 100 nM fulvestrant. (Supplementary Fig. S2). Down-regulation of ER occurs
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through the 26S proteosomal pathway as no decrease in ERα protein level in MCF-7 cells
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was seen in the presence of the proteosome inhibitor MG132 (Supplementary Fig. S3).
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Additionally, ERα down-regulation was shown to be reversible in compound wash-out
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experiments, where ERα levels increased in a time-dependent manner back to basal levels
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over a 48 hour period following compound removal. (Supplementary Fig. S4).
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Agonism of ERα in human breast and uterine cells
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In contrast to an AstraZeneca reference compound (AZ10557841) which has known ERα
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agonist activity in breast cells, AZD9496 was shown to cause no increase in PR protein levels
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but rather a slight decrease below the detected basal levels (Fig. 3A). Some endocrine
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therapies, such as tamoxifen, can act as partial ER agonists in certain tissues e.g.
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endometrium, due to conformational changes that take place at the ER and the resulting effect
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on co-factor recruitment and transcriptional gene activation from the tamoxifen-bound
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receptor (37). To test if AZD9496 could act as a partial agonist in other tissues, agonist
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effects were measured in vivo in a previously validated immature female rat model designed
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to detect agonistic properties of test compounds by measuring increases in uterine weight
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(31). AZD9496, given once daily orally at 5 and 25 mg/kg produced statistically significant
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increases in uterine weight compared to the fulvestrant control (p<0.001) but significantly
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lower than tamoxifen (p=0.001) (Fig. 3B). Histological staining of uterine tissue samples
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also showed that the lengthening of the endometrial cells in the rat uteri appeared to have
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decreased compared to tamoxifen (Fig. 3C). In further studies, ERα protein levels were
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reduced with fulvestrant and AZD9496, but not tamoxifen, compared to relevant vehicle
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controls, whereas PR levels were significantly increased with tamoxifen alone (Fig. 3D).
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AZD9496 is a potent, oral inhibitor of breast tumour growth in vivo
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The effect of chronic, oral dosing of AZD9496 was explored in MCF7 human breast
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xenografts, as a representative ER+/PR+/HER2+ breast cancer model. Good bioavailability
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and high clearance gave a terminal t½ of 5-6 hours after oral dosing in the mouse and resulted
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in significant dose-dependent tumour growth inhibition with 96% inhibition at 50 mg/kg and
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no toxicity or weight loss relative to the vehicle control group (Fig. 4A). To confirm that
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AZD9496 was targeting the ER pathway, PR protein levels were measured in tumour samples
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taken at the end of the study and a significant reduction in PR was seen which correlated with
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tumour growth inhibition. A >90% reduction in PR was seen with both 10 and 50 mg/kg
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doses and a 75% decrease even with the 0.5 mg/kg dose, demonstrating that AZD9496 can
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clearly antagonise the ER pathway (Fig. 4B). Dosing 5 mg/kg of AZD9496, the minimal
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dose required to see significant tumour inhibition, gave greater tumour growth inhibition
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compared to 5 mg/mouse fulvestrant given 3x weekly and tamoxifen given 10 mg/kg orally,
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daily (Fig. 4C). A series of pharmacodynamic in vivo studies were conducted to measure
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time taken to reach maximal inhibition of PR levels and time taken to recover back to basal
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levels. Three days of dosing with 5 mg/kg of AZD9496 gave 98% reduction of PR protein
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and continued to suppress protein levels at 48 hours (Fig. 4D) with full recovery by 72 hours
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(data not shown) which indicates a long pharmacological half life in vivo. Fulvestrant, given
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as 3 x 5 mg/mouse doses over one week, gave a 60% reduction in PR protein over a
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prolonged period with measured plasma levels ~8-fold higher than those achieved clinically,
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at steady state, with 500 mg fulvestrant (Fig. 4E). As estrogen itself is known to down-
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regulate ER protein (38, 39), we were unable to detect further decreases in ERα protein
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compared to control animals with AZD9496 or fulvestrant in the MCF-7 model presumably
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due to the high circulating plasma levels of estrogen from implanted pellets at the time
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tumour samples were taken (Supplementary Fig. S5). A mouse specific metabolite of
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AZD9496 was detected in circulating plasma at similar levels to AZD9496 and showed a
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similar pharmacokinetic profile. Testing this metabolite in the in vitro MCF-7 assays resulted
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in ~5 fold lower ERα antagonism activity and 7 fold lower ERα down-regulation activity
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than AZD9496 (data not shown). Using a PK/PD model based on PR inhibition data, at the 5
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mg/kg dose in vivo, which gives 98 % inhibition of PR, the inhibitory activity that could be
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attributed to the parent compound alone was 85% when the activity of the metabolite was
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discounted.
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To explore mechanistic effects on ER pathway regulation further, a gene
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expression analysis study was performed using tumour samples dosed at 10 mg/kg for 3 days
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with AZD9496 or tamoxifen and fulvestrant given as a single dose 5 mg dose
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subcutaneously. Tumours were taken 24 hours after the last dose of AZD9496 and tamoxifen
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and mRNA levels measured using a Human Transcriptome Array (HTA 2.0). A number of
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known ER regulated genes were examined to see if AZD9496 could antagonise these genes
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in a similar manner to Fulvestrant and tamoxifen (40-42). The heap map shows that
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AZD9496 could indeed down-regulate the mRNA levels of estrogen responsive genes
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(Supplementary Fig. S6A). As differences in the levels of mRNA down-regulation in vivo
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could be due to different PK profiles of the molecules, the effects of compounds on the
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mRNA levels of a subset of estrogen regulated genes were also measured in MCF-7 cells that
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had been treated with or without estrogen (Supplementary Fig. 6 B-E). For two of these
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genes, TFF1 and AREG, tamoxifen was shown to increase mRNA levels in the absence of
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estrogen but not with either fulvestrant or AZD9496 (Supplementary Fig. 6 D, E). Where
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transcript levels were increased to varying levels in the presence of estrogen, fulvestrant and
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AZD9496 inhibited transcript levels in a dose dependent fashion compared to tamoxifen. No
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significant effects were seen in ESR1 mRNA levels in vitro indicating that the protein down-
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regulation described previously is due to down-regulation of the protein and not a decrease in
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transcript levels per se (Supplementary Fig. 6F).
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AZD9496 causes tumour regressions in combination with PI3K pathway and CDK4/6
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inhibitors and in an estrogen deprived ER +ve model of resistance
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Acquired resistance to aromatase inhibitors and antiestrogens is driven through
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a variety of mechanisms which include effects on the ER pathway itself i.e. down-regulation
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of ER expression and altered expression of ER co-regulators, as well as activation of
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alternative pro-survival or proliferative pathways and cross-talk between growth factor
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receptors and ER pathways. This has led to a number of clinical trials with PIK3, mTOR,
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AKT inhibitors, as well as CDK4/6 inhibitors in combination with aromatase inhibitors or
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fulvestrant and early results have showed increased progression-free survival (PFS) in many
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cases (43). Combinations of AZD9496 with AZD2014 (dual mTORC1/2), AZD8835
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(PIK3Cα/δ) and Palbociclib (CDK4/6) inhibitors were tested in the MCF-7 in vivo model and
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in all cases tumour regressions seen with the combination arms alone compared to tumour
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stasis with monotherapy (Fig. 5A-C). AZD9496 was also tested in a long-term estrogen
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deprived model (LTED), using the HCC-1428 LTED cell line that was previously adapted to
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grow in the absence of estrogen and as such represents a model of aromatase inhibitor
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resistance (22). Tumour regressions were seen with both AZD9496 and fulvestrant (Fig. 5D)
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and both compounds completed ablated ER protein levels in the end of study tumours
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(Supplementary Fig. S7).
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AZD9496 antagonises and down-regulates mutant ER in vitro and in vivo
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The recent identification of ESR1 mutations in patients that had received one or more
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endocrine therapy treatments has led to the discovery that these mutations can drive cell
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proliferation in the absence of estrogen and may constitute a resistance mechanism to some
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endocrine agents. In vitro binding studies were performed using wt, D538G and Y537S
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LBDs and both AZD9496 and fulvestrant were able to bind to mutant LBD with nM potency
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although 2-3 fold reduced compared to wt (Table 1B). The different IC50 values obtained for
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binding of AZD9496 and fulvestrant to wt ERα compared to the value in Table 1A is likely
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due to different sources of protein used in the Lanthascreen kit assays versus the laboratory
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derived wt and ESR1 mutant proteins. Down-regulation of ER mutant proteins was measured
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using MCF-7 doxycycline inducible stable cell lines in vitro. ER protein levels increased on
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induction with 1 μg/ml doxycyline and led to increased levels of PR in the absence of
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estrogen. AZD9496 and fulvestrant were able to down-regulate all mutant ER proteins and
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decreased PR levels whereas tamoxifen appeared to stabilise ER protein levels and reduced
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PR levels to a lesser extent (Fig. 6A). To explore activity against an ESR1 mutant in vivo,
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tumour growth inhibition studies were performed in a patient-derived xenograft (PDX)
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model, CTC-174, which harbours a D538G mutation in the LBD and constituted 31% of the
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transcripts variants identified by RNA-seq analysis. Tumours were grown from implanted
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tumour fragments either with or without estrogen and in both cases viable tumours grew.
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Given published data that higher doses of SERM/SERDs may be needed to inhibit ESR1
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mutants (14, 15), 25mg/kg of AZD9496 was used alongside 5mg/mouse of fulvestrant which
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was known to achieve higher PK levels in mouse than the current clinical dose. AZD9496
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and fulvestrant inhibited tumour growth by 66% and 59% respectively, compared to
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tamoxifen which gave 28% inhibition. Efficacy correlated with antagonism of the pathway
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with AZD9496 giving a 94% decrease in PR levels compared to 63% with fulvestrant (Fig.
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6B and C). In the absence of estrogen, and given the significant PR changes seen with
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25mg/kg, the dose of AZD9496 was lowered but a 5 mg/kg dose still achieved growth
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inhibition of ~70% and led to a significant decrease of 73% in ERα protein levels at the end
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of study (Fig. 6D and E). Given the similarities in growth inhibition either with or without
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estrogen, residual tumour growth is likely to be due to either additional mutations (e.g.
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PIKCA N345K) and/or growth factor signalling pathways involved in driving tumour growth
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in addition to the ER pathway.
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Discussion
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Despite the enhanced clinical benefits of directly antagonising and down-regulating ER and
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thereby targeting any ligand-independent ER driven pathways, the pharmacokinetic and IHC
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biomarker data for ER, PR and Ki67 from clinical trials with fulvestrant would suggest that a
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plateau of effect has not been reached, and there is scope to further improve on the 50%
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reduction in ER levels seen after 6 months of treatment (8, 12). As one of the potential draw-
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backs associated with fulvestrant is the very low bioavailability which is seen both in patients
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and in pre-clinical animal models, a potent, orally bioavailable SERD could have the
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potential to significantly antagonise ER activity and increase ERα receptor knockdown
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versus fulvestrant due to higher exposures. Our approach focused on identifying novel ER
14
352
motifs with established drug-like properties that could then be optimized for cellular
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phenotype and potency and led to the identification of AZD9496 which showed pM potency
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in vitro in ER+ve cell lines only. Previous structural analysis of a number of SERMs with
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ERα LBD identified key interactions with Asp 351 which was shown to be critical for the
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antagonist activity of anti-estrogens such as raloxifene, lasofoxifene and GW5638 through
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shielding or neutralisation of the negative charge. Unlike tamoxifen however, GW5638
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repositioned residues Helix-12 through its acrylate side chain, increasing the exposed
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hydrophobic surface of ERα resulting in destabilisation and ultimately 26S proteosomal
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degradation of the receptor. The acrylic acid moiety of AZD9496 is co-localized with the Asp 351
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residue in the helix-12 region of ER, in an unusual acid-acid interaction and it is therefore proposed
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that AZD9496 also repositions residues associated with the helix-12 via specific contacts with the N-
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terminus region resulting in a mixed antagonist and down-regulator profile (33, 44). Evidence that
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AZD9496 was directly binding and down-regulating the receptor at the same site as other
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anti-estrogens was seen by the reduced potency observed on prior addition of tamoxifen to
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the down-regulation assay (Table 1A). Mechanistically we have shown that AZD9496 has
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very similar effects on antagonism, ERα down-regulation and agonism to fulvestrant. Even
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where ERα was over-expressed in MCF-7 cells, unlike tamoxifen, AZD9496 was able to
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down-regulate and antagonise the over-expressed receptor (Fig 6A). Our transcriptional
370
studies in both MCF-7 cells and the in vivo model also demonstrated antagonism of the
371
transcriptional activity of the receptor as seen by decreased mRNA levels of ER-activated
372
genes in a dose dependent manner (Supplementary Fig. 6).
373
Unlike fulvestrant, AZD9496 did show some degree of agonism in endometrial
374
tissue (Fig. 3). This effect was dose independent and would indicate that there are subtle
375
differences in how AZD9496 interacts with ERα compared to tamoxifen and fulvestrant and
376
therefore the extent to which tissue specific co-activators can interact with the receptor.
15
377
Given the widespread use of tamoxifen in the adjuvant setting for 5-10 years, the low level of
378
agonism seen with AZD9496 is not viewed as a deterrent for clinical development. A number
379
of studies have looked at the incidence of endometrial cancer in patients taking tamoxifen
380
over a number of years and despite the small increase in the incidence of endometrial cancer,
381
the benefits of tamoxifen greatly outweigh any risks of developing uterine malignancy in
382
post-menopausal patients (45, 46).
383
A PK-PD-efficacy model was established, integrating diverse endpoints such as
384
drug exposure, PR modulation and inhibition of tumour growth (data not shown). The model
385
reflected relative contributions of AZD9496 and its active mouse metabolite to the overall
386
biomarker modulation and efficacy. Given the half life of the parent drug and metabolite are
387
around 5 hours, to explain the prolonged PD effect observed with AZD9496 at 48 hours, an
388
indirect response model for PR was developed which estimated a degradation rate constant of
389
around 23 hours for PR. This modulation in PR was linked directly to tumour growth
390
inhibition by a proportional decrease of the tumour growth constant, and correlated well with
391
the wide range of measured efficacies.
392
We consistently saw enhanced tumour growth inhibition of AZD9496
393
compared to fulvestrant in the MCF-7 in vivo model (Fig. 4). This model contains high
394
circulating levels of plasma estradiol over the dosing period of the study as a result of the
395
estrogen pellets used to drive growth of the MCF-7 cells as a xenograft. With plasma levels
396
between 750 and 1000 pg/ml over day 21-35, this model is more representative of the pre-
397
menopausal setting where estrogen levels can vary between 20 – 400 pg/ml (47). As the
398
receptor binding data does not suggest a significant difference in affinity for the receptor
399
between AZD9496 and fulvestrant, increased efficacy is likely due to the higher free drug
400
levels of AZD9496 versus fulvestrant in this model. It would therefore be interesting to test
16
401
the activity of potent oral SERDs, such as AZD9496, in the pre-menopausal setting where
402
there is no enhanced risk of endometrial cancer observed for tamoxifen.
403
We were unable to detect a reduction in ER protein levels in the MCF-7 in vivo
404
model with AZD9496 at any dose tested (data not shown). Having shown in vitro that
405
estradiol can efficiently down-regulate ERα protein, we postulate that down-regulation of
406
ERα by estradiol is already taking place in vivo given the high levels of circulating estrogen
407
and that any additional changes by addition of a SERD are difficult to detect using the
408
current methods. This was supported by measuring ERα down-regulation in in vivo models,
409
such as CTC-174 and HCC1428LTED, not requiring estrogen supplements, and showing
410
potent down-regulation of ERα by AZD9496 (Fig. 6E, Supplementary Fig. S7). The level of
411
ERα reduction seen in the HCC1428 model is greater than seen clinically, with PK levels 5
412
fold higher than steady state plasma levels of 500 mg fulvestrant, and supports the concept
413
that if increased plasma levels of an active SERD agent could be achieved clinically, greater
414
ERα knock-down may be possible. Pre-clinically, this appears to translate to enhanced
415
tumour growth inhibition.
416
Achieving increased serum levels of a biologically active SERD could be
417
particularly beneficial in the recently identified ESR1 mutant patient setting. Pre-clinical in
418
vivo studies using PDX models derived from ESR1 mutant patients grown in the absence of
419
estrogen supplementation have shown that high doses of fulvestrant (>500 mg/kg) can lead to
420
tumour regression and significant decreases in ER protein levels but at significantly higher
421
doses than could be achieved clinically (48). Our preclinical data would indicate that both
422
AZD9496 and fulvestrant can potently bind and down-regulate D538G and Y537C/N/S ERα
423
proteins in vitro and significant tumour growth inhibition rather than regression seen in vivo.
424
This is in contrast to published work showing relatively little down-regulation of Y537N
425
mutant protein in vitro with increasing doses of fulvestrant (15) but could be attributed to
17
426
different expression systems and cell lines used for the analysis. It is notable that the Dox-
427
inducible mutant receptors do not appear to be constitutively down-regulated in a similar
428
manner to wt ER exposed to estradiol (Supplementary Fig. S1). This may due to the level of
429
over-expression of the receptor in each stable cell line and it will be interesting to compare
430
these findings from engineered expression systems to those from genetically altered mutant
431
cell lines where expression is under control of the native promoter. Although very high doses
432
of both compounds were not tested in the PDX CTC-174 ESR1 mutant model, the lack of any
433
significant dose response might indicate the need to inhibit other growth factor activated
434
pathways in addition to the ER pathway to achieve significant anti-tumour growth. As trials
435
commence to explore treatment of ESR1 mutant patients with new oral SERDs, data will
436
hopefully emerge that will indicate whether lack of response is due to inability to fully inhibit
437
mutated ER receptors or whether in these advanced metastatic patients, other mutations and
438
pathways are or become dominant in driving tumour growth.
439
In metastatic breast cancer the median survival time is still around two years as
440
a result of acquired endocrine resistance, highlighting the need for additional therapies (43).
441
This has led to a number of clinical trials using PI3K-Akt-mTOR pathway inhibitors in
442
combination with AIs as well as cyclin-dependent kinase inhibitors such as palbociclib which
443
was recently approved for combination treatment with letrozole in the metastatic breast
444
cancer. Results from Phase III BOLERO-2 trial also demonstrated the PFS benefits of a
445
steroidal AI plus everolimus in patients that had progressed on a non-steroidal AI (49). More
446
recently further trials have been initiated using fulvestrant as the combination endocrine
447
partner which will allow analysis and understanding of any additional benefit from
448
combination with a SERD versus AIs in this setting. Our data clearly demonstrated enhanced
449
combination effects on tumour growth of an oral SERD agent with either dual mTORC1/2,
450
PIKCα/δ or CDK4/6 inhibitors and furthermore opens up the possibility of using a combined
18
451
endocrine and targeted inhibitor approach with longer term dosing in the adjuvant setting,
452
assuming emerging data from on-going metastatic trials supports the testing of combinations
453
in early breast disease. The identification of a potent, orally bioavailable compound such as
454
AZD9496 is a step forward in the next generation of SERD agents to undergo clinical
455
evaluation as a future monotherapy or combination partner of choice.
456
457
Acknowledgements
458
The authors would like to thank Elaine Hurt, Haifeng Bao, Sanjoo Jalla for access to the
459
CTC-174 in vivo model developed at MedImmune, Gaithersburg MD 20878.The authors
460
thank Paul Hemsley and Emma Linney for providing ER mutant binding assay data, Helen
461
Gingell for supporting the crystallography work and Zara Ghazoui and Henry Brown for the
462
transcript data and analysis at AstraZeneca, Alderley Park, Macclesfield, UK. The authors
463
also thank Carlos Arteaga for use of the HCC1428-LTED cell line.
464
465
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604
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23
606
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611
612
Tables
Table 1 A
In vitro ERα binding, ERα down-regulation, ERα antagonism and
MCF-7 cell growth inhibition data for AZD9496 vs. fulvestrant
AZD9496
fulvestrant
ERα binding IC50 nM
0.82
0.8
ERα down-regulation IC50 nM
0.14
0.06
ERα down-regulation IC50 nM
+ 250 nM tamoxifen
11.16
1.91
ERα antagonism IC50 nM
0.28
0.21
MCF-7 cell growth inhibition EC50 nM
0.04
0.1
LogD
2.9
Not measureable
Solubility μM
110 (crystalline)
<0.9
Human % free
0.15
0.03
Table 1 B
In vitro binding to ERα mutant LBDs
ERα LBD binding IC50 nM
AZD9496 nM
fulvestrant nM
wt
0.2
1.6
D538G
0.5
3.3
Y537S
0.6
3.8
613
614
615
Tables 1A and B. Summary of in vitro cellular potency and phenotype of AZD9496. A -
616
A series of in vitro assays were performed in MCF-7 cell in hormone-depleted medium to
617
show AZD9496 effects on ER binding, down-regulation, antagonism and inhibition of cell
618
proliferation as described in (20) using a fluorescence end-point for measured of ER and PR
619
protein levels; data shown are representative of the mean IC50 or EC50 from at least four
620
independent experiments. B - IC50s for binding of AZD9496 and fulvestrant to ERα wt and
621
mutant LBDs; data shown are representative of the mean IC50 from three independent
622
experiments.
623
24
624
625
626
627
Figure legends
628
AZD9496 (A) Two-dimensional chemical representation of AZD9496. (B) Two-dimensional
629
chemical representation of 4-hydroxy tamoxifen. (C) Three-dimensional structure of the
630
complex between AZD9496 and the ligand-binding domain of ERα. The protein and ligand
631
structures are represented as sticks with carbon and fluorine atoms coloured grey and light
632
blue respectively. The van der Waals radii of the protein atoms are shown as a continuous
633
surface highlighting the ligand-binding pocket of the ERα. Key protein residues are labelled
634
and polar interactions are shown as dotted lines.
Figure 1. Crystal structure of the ligand-binding domain of ERα in complex with
635
636
Figure 2. Effect of AZD9496, fulvestrant and tamoxifen on ERα peptide turnover in
637
MCF-7 cells
638
Cells were grown in steroid-free conditions in SILAC media containing 13C615N4 L-arginine
639
to label ERα peptide as “heavy” (blue line) and then switched to grow in media containing
640
unlabelled L-arginine to label newly synthesised protein as “normal” (red line) with (A) 0.1%
641
DMSO, (B) 300 nM tamoxifen, (C) 100 nM AZD9496 or (D) 100 nM fulvestrant for the time
642
indicated. Data shown is representative of two independent experiments.
643
644
Figure 3. ERα mediated agonism in MCF-7 cells and immature female rat endometrial
645
tissue. (A) MCF-7 cells were treated with AZD9496 or a known agonist compound
646
AZ10557841 and PR levels (response) measured using in-cell immunofluorescence
647
quantification using an Acumen Ex3 platform. Data was normalised for cell number by
648
analysing a ratio of fluorescence resulting from stimulation of two emission wavelengths and
649
used to perform curve fitting analysis. A concentration response was plotted using non-
650
linear regression analysis. (B, C) Immature female rats were dosed orally with AZD9496 or
25
651
tamoxifen or subcutaneously with fulvestrant for 3 days. PEG/Captisol, polysorbate and
652
peanut oil were used as vehicle controls for AZD9496, tamoxifen and fulvestrant
653
respectively. Uterine tissue was removed 24 hrs after the final dose and weighed before
654
tissues were formalin-fixed and paraffin embedded to allow histological staining of the
655
endometrial tissue. Significant differences in mean uterine weight are shown: ***p<0.001
656
and ** p<0.01 (D). Tissue was subsequently analysed by Western blot for levels of ERα, PR
657
and vinculin. Protein levels were measured by chemiluminescent and quantified using
658
Syngene software. ER and PR protein levels were normalised to vinculin as a loading control
659
and plotted as shown, Statistically significant differences in PR levels are shown:
660
***p<0.001, ** p<0.01 and * p<0.05.
661
662
663
664
Figure 4. In vivo efficacy of AZD9496 in MCF-7 xenograft model
665
(vehicle) or AZD9496 at doses shown (A). Tumour growth was measured by caliper at
666
regular intervals and mean tumour volumes plotted for each dosed group. Tumours from
667
treated mice collected at the end of study were analysed by Western blot for levels of PR and
668
vinculin. Protein levels were measured by chemiluminescent and quantified using Syngene
669
software. PR protein levels were normalised to vinculin as a loading control and plotted as
670
shown with standard error bars * p<0.05, *** p<0.001(B). MCF-7 xenografts were also
671
dosed daily with 5 mg/kg AZD9496, 10 mg/kg tamoxifen or 5 mg/mouse 3x weekly with
672
fulvestrant subcutaneously (C). For acute dosing studies, MCF-7 xenografts were dosed for 3
673
days with doses shown of AZD9496 and tumours collected at 24 or 48 hrs after the last dose
674
(D) or dosed with 3 doses of 5 mg/mouse of fulvestrant over 5 days and tumours collected at
675
24, 48 and 96 hours after the last dose (E). PR and vinculin protein levels in the tumours were
676
analysed and plotted as described above.
MCF-7 xenografts, grown in male SCID mice, were dosed daily with either PEG/captisol
26
677
678
679
680
681
682
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.
683
with or without AZD2014 dosed at 20 mg/kg twice daily (b.i.d) 2 days out of 7, (A) with or
684
without AZD8835 dosed at 50 mg/kg b.i.d. on day1 and 4 (B) with or without Palbociclib
685
dosed daily at 50 mg/kg (C) HCC1428LTED engrafts were grown in ovariectomised NSG
686
mice and were dosed daily with either AZD9496 or once weekly with fulvestrant at doses
687
shown (D). Tumour growth was measured by caliper at regular intervals and mean tumour
688
volumes plotted for each dosed group.
MCF-7 xenografts, grown in male SCID mice, were dosed daily with AZD9496 at 5mg/kg
689
690
Figure 6: AZD9496 is efficacious against ESR1 mutants in vitro and in vivo.
691
Stable MCF-7 cell lines containing either pTRIPZ vector alone or vector containing ESR1
692
mutants shown were induced to express mutant protein with 1μg/ml doxycycline for 24hrs
693
before treating with either 0.1% DMSO (vehicle) or 100nM of fulvestrant, AZD9496 or
694
tamoxifen for 24hrs. Immunoblotting was performed to detect expression of ERα, PR and
695
GAPDH levels (A). CTC-174 tumour fragments were grown in female NSG mice implanted
696
with estrogen pellets and dosed daily with either PEG/captisol (vehicle) or AZD9496,
697
tamoxifen and Fulvestrant at doses shown. Tumour growth was measured by caliper at
698
regular intervals and mean tumour volumes plotted for each dosed group (B). Tumours from
699
treated mice collected at the end of study were analysed by Western blot for levels of PR and
700
vinculin. Protein levels were measured by chemiluminescent and quantified using Syngene
701
software. PR protein levels were normalised to vinculin as a loading control and plotted as
702
shown ***p<0.001(C). CTC-174 tumour fragments were grown in female NSG mice without
703
estrogen pellets to enable measurement of ER levels and dosed daily with either PEG/captisol
704
(vehicle) or AZD9496 at doses shown (D). Tumours from treated mice collected at the end of
27
705
study were analysed by Western blot for levels of ERα and vinculin and measured as
706
described above * p<0.05 (E)
707
28
A
B
C
Figure 1. Crystal structure of the ligand-binding domain of ERa in complex with AZD9496
A
B
C
D
Figure 2. Effect of AZD9496, fulvestrant and tamoxifen on ERa peptide turnover in MCF-7
cells
A
B
0 .1 5
AZD9496
AZ10557841
0 .1 0
R esponse
AZD9496
AZ10557841
0 .0 5
0 .0 0
-1 0
-8
-6
-4
C o n c e n t r a t io n lo g 1 0 ( M )
- 0 .0 5
C
x 10
Immature rat: control
x 20
Fulvestrant
x 20
AZD9496
x 20
Tamoxifen
D
Figure 3 ERα mediated agonism in MCF-7 cells and immature female rat
endometrial tissue.
A
B
C
D
E
Figure 4. In vivo efficacy of AZD9496 in MCF-7 xenograft model.
A
C
B
D
Figure 5. Tumour regressions with AZD9496 in combination with mTOR, PIKC and CDK4/6
inhibitors in MCF-7 xenograft model and HCC1428 LTED model
B
D
AZD9496
AZD9496
Tamoxifen
AZD9496
Y537N
Fulvestrant
Vehicle
Tamoxifen
AZD9496
Y537C
Fulvestrant
Vehicle
Tamoxifen
D538G
Fulvestrant
Vehicle
Tamoxifen
WT
Fulvestrant
Vehicle
Tamoxifen
AZD9496
Empty Vector
Fulvestrant
Vehicle
Tamoxifen
AZD9496
Fulvestrant
Vehicle
A
Y537S
ERa
PR
GAPDH
C
E
Figure 6: AZD9496 is efficacious against ESR1 mutants in vitro and in vivo.