AZD9496: an Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1- Mutant Breast Tumors in Preclinical Models Hazel M

Home , Fulvestrant

Published OnlineFirst March 28, 2016; DOI: 10.1158/0008-5472.CAN-15-2357 Cancer Therapeutics, Targets, and Chemical Biology Research

AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1- Mutant Breast Tumors in Preclinical Models Hazel M. Weir1, Robert H. Bradbury1, Mandy Lawson1, Alfred A. Rabow1, David Buttar1, Rowena J. Callis2, Jon O. Curwen1, Camila de Almeida1, Peter Ballard1, Michael Hulse1, Craig S. Donald1, Lyman J.L. Feron1, Galith Karoutchi1, Philip MacFaul1, Thomas Moss1, Richard A. Norman2, Stuart E. Pearson1, Michael Tonge2,Gareth Davies1,Graeme E.Walker2, Zena Wilson1, Rachel Rowlinson1, Steve Powell1, Claire Sadler1, Graham Richmond1, Brendon Ladd3, Ermira Pazolli4, Anne Marie Mazzola3, Celina D'Cruz3, and Chris De Savi3

Abstract

Fulvestrant is an estrogen receptor (ER) antagonist admin- Combining AZD9496 with PI3K pathway and CDK4/6 inhi- istered to breast cancer patients by monthly intramuscular bitors led to further growth-inhibitory effects compared with injection. Given its present limitations of dosing and route monotherapy alone. Tumor regressions were also seen in a of administration, a more flexible orally available compound long-term estrogen-deprived breast model, where significant has been sought to pursue the potential benefits of this drug downregulation of ERa protein was observed. AZD9496 in patients with advanced metastatic disease. Here we report bound and downregulated clinically relevant ESR1 mutants the identification and characterization of AZD9496, a nonste- in vitro andinhibitedtumorgrowthinanESR1-mutant roidal small-molecule inhibitor of ERa,whichisapotent patient-derived xenograft model that included a D538G and selective antagonist and downregulator of ERa in vitro and mutation. Collectively, the pharmacologic evidence showed in vivo in ER-positive models of breast cancer. Significant that AZD9496 is an oral, nonsteroidal, selective estrogen þ tumor growth inhibition was observed as low as 0.5 mg/kg receptor antagonist and downregulator in ER breast cells þ dose in the estrogen-dependent MCF-7 xenograft model, where that could provide meaningful benefittoER breast cancer this effect was accompanied by a dose-dependent decrease in patients. AZD9496 is currently being evaluated in a phase I PR protein levels, demonstrating potent antagonist activity. clinical trial. Cancer Res; 76(11); 1–12. 2016 AACR.

Introduction tors have been implicated in driving resistance in cells, for example, receptor tyrosine kinases (HER2), EGFR, and their down- With over 70% of breast cancers expressing the estrogen recep- stream signaling pathways, Ras/Raf/MAPK and PI3K/protein tor alpha (ERa), treatment with antihormonal therapies that kinase B (AKT) signaling (3–5). In some instances, signaling from directly antagonize ER function (e.g., tamoxifen) or therapies different growth factor receptor kinases leads to phosphorylation that block the production of the ligand, estrogen (e.g., aromatase of various proteins in the ER pathway, including ER itself, leading inhibitors) are key to management of the disease both in adjuvant to ligand-independent activation of the ER pathway (6). and metastatic settings (1, 2). Despite initial efficacy seen with In the metastatic setting, 500 mg fulvestrant, given as 2 250 endocrine therapies, the development of acquired resistance mg intramuscular injections, has been shown to offer additional ultimately limits the use of these agents although the majority benefit over the 250 mg dose with improved median overall of tumors continue to require ERa for growth. Through the use of survival (7, 8). Recent analysis of presurgical data from a number preclinical models and cell lines, changes in growth factor recep- 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 1Oncology iMed, AstraZeneca, Alderley Park, Macclesfield, United Kingdom. 2Discovery Sciences, AstraZeneca, Alderley Park, Maccles- setting, there are still perceived limitations in its overall clinical field, United Kingdom. 3Oncology iMed, AstraZeneca R&D Boston, benefit due to very low bioavailability, the time taken to achieve 4 Gatehouse Drive, Waltham, Massachusetts. H3 Biomedicine, Cam- plasma steady-state of 3–6 months and ultimately the level of ERa bridge, Massachusetts. reduction seen in clinical samples (10, 11). It is widely believed Note: Supplementary data for this article are available at Cancer Research that a potent, orally bioavailable SERD that could achieve higher Online (http://cancerres.aacrjournals.org/). steady-state free drug levels more rapidly would have the potential Corresponding Author: Hazel M. Weir, AstraZeneca, Alderley Park, Macclesfield, for increased receptor knockdown and lead to quicker clinical Cheshire, SK10 4TG, United Kingdom. Phone: 016-2551-3799; Fax: 016-2551- responses, reducing the possibility of early relapses (12). More- 9173; E-mail: Hazel.Weir@astrazeneca.com over, the recent discovery of ESR1 mutations in metastatic breast doi: 10.1158/0008-5472.CAN-15-2357 cancer patients that had received prior endocrine treatments, 2016 American Association for Cancer Research. where the mutated receptor is active in the absence of estrogen,

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highlights an important potential resistance mechanism. Preclin- ERa structure as the search model (28). Quality checks on the ical in vitro and in vivo studies have shown a reduction in signaling protein structures were carried out using the validation tools in from ESR1 mutants by tamoxifen and fulvestrant, but at higher Coot (29). The final structure has been deposited in the Protein doses than required to inhibit the wild-type receptor, which Databank with the ID code given in Supplementary Table S1. implies that more potent SERDs may be required to treat these patients or to use in the adjuvant setting to limit the emergence of Stable isotope labeling of amino acids and mass spectrometry mutations (13–15). Stable isotope labeling of amino acids (SILAC) assays were Achieving oral bioavailability has remained a key challenge in carried out as described previously (30) and detailed in Supple- the design of ER downregulators and until now no oral estrogen mentary Methods. Compound-treated samples were analyzed by receptor downregulators, other than GDC-0810 and RAD1901, mass spectrometry using relative peptide quantification by select- have progressed into clinical trials, despite encouraging reports ed reaction monitoring (SRM). Degradation half-life was mea- from others of preclinical activity in mouse xenograft models sured using the one-phase exponential decay equation in Graph- (16–21). Here, we describe a chemically novel, nonsteroidal ERa Pad PRISM (Y ¼ Span.e K.X þ Plateau), where X is time and Y is antagonist and downregulator, AZD9496, that can be adminis- response, which starts out as Span þ Plateau and decreases to tered orally and links increased tumor growth inhibition to Plateau with a rate constant K. enhanced biomarker modulation compared with fulvestrant in a preclinical in vivo model of breast cancer. Moreover, we show Rat uterine and xenograft studies that AZD9496 is a potent inhibitor of ESR1-mutant receptors and All studies involving animals in the United Kingdom were can drive tumor growth inhibition in a patient-derived ESR1 conducted in accordance with UK Home Office legislation, the mutant in vivo model (PDX). Hence, AZD9496 is anticipated to Animal Scientific Procedures Act 1986, and AstraZeneca Global yield improved bioavailability and clinical benefit through Bioethics policy or with US federal, state, and institutional guide- enhanced ER pathway modulation. lines in an Association for Assessment and Accreditation of Laboratory Animal Care–accredited facility. The rat uterine weight Materials and Methods assay for the measurement of estrogenic activity was performed as Cell lines and culture described previously (31). Details of individual models, protein All AZ cell lines were tested and authenticated by short-tandem analysis of tumor fragments, and pharmacokinetic analysis of repeat (STR) analysis. MCF-7 was obtained from DSMZ and last plasma samples are included in Supplementary Methods. STR tested in February 2013. MDA-MB-361 (STR tested in October 2011), MDA-MB-134 (STR tested in October 2010), T47D (STR Results tested in June 2012), CAMA-1 (STR tested in March 2012), AZD9496 is a selective ERa antagonist, downregulator, and þ HCC1428 (STR tested in December 2011), HCC70 (STR tested inhibitor of ER tumor cell growth in October 2013), LnCAP (STR tested in August 2011), MDA-MB- AZD9496 ((E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methyl- 468 (STR tested in March 2014), BT474 (STR tested in July 2013), propyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl) PC3 (STR tested in October 2012), HCT116 (STR tested in August phenyl)acrylic acid; Fig. 1A) was identified through structure-based 2011) were all obtained from the ATCC. HCC1428-LTED cell line design and iterative medicinal chemistry structure–activity relation- (STR tested in November 2012) was kindly provided by Carlos ship (SAR) studies from a novel ER-binding motif (32). A directed Arteaga (Vanderbilt University, Nashville, TN; ref. 22). Cells were ER-binding screen was established to identify novel binding motifs cultured in RPMI1640 media (Corning) supplemented with 10% that could then be optimized for both cellular phenotype and heat-inactivated FCS or charcoal/dextran–treated CCS to deplete potency while maintaining good pharmacokinetic properties hormone and 2 mmol/L L-glutamine (Corning). Stable MCF-7 cell (23). Unlike fulvestrant, and other well-known historical modula- lines expressing FLAG-tagged ESR1-mutant proteins in pTRIPZ tors of the ER receptor, including hydroxytamoxifen (Fig. 1B), lentiviral vectors (GE Healthcare) under the control of the doxy- AZD9496 lacks a phenolic moiety in which the phenol mimics the cycline-inducible Tet promoter were generated by lentiviral infec- A-ring phenol group of the endogenous ligand estradiol but gains its tion using standard techniques. potency through a different series of novel interactions with the ER protein (Fig. 1C). The indole NH picks up an interaction by forming Biochemical and in vitro cell assays a hydrogen bond to the carbonyl of Leu-346, with additional Binding, ER agonism, antagonism, downregulation, and cell lipophilic interactions achieved by both the chiral methyl substit- proliferation assays were carried out as described previously (23). uent, adjacent to the ring nitrogen, and the N-substituted isopropyl BIAcore affinity measurements and immunoblotting from com- fluoro side-chain occupying a "lipophilic hole" in the ER protein. pound-treated cells are described in Supplementary Methods. The acrylic acid side chain picks up an unusual acid–acid colocaliza- tion with Asp-351 in the helix-12 region of the ER that has been Protein production, crystallization, and structure proposed to be crucial for achieving a downregulator–antagonist determination profile (33). AZD9496 showed high oral bioavailability across three Protein expression, purification, and crystallization of the ERa species (F% 63, 91, and 74, rat, mouse, and dog, respectively) with ligand-binding domain was carried out as described previously generally low volume and clearance across species, albeit a higher (24). X-ray diffraction data were collected at the ESRF on beamline clearance in mouse. The percent free levels in human plasma of ID23-1 on an ADSC detector. Data were processed using XDS (25) 0.15% were 5-fold higher than those measured for fulvestrant. as implemented within EDNA (26) and scaled and merged using The key characteristics of a SERD is the ability to both SCALA (27). The structure of ERa in complex with AZD9496 was antagonize and downregulate ER protein without inducing any solved by molecular replacement using AmoRE and an internal partial agonist effects in breast cells. A series of in vitro cell

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Figure 1. Crystal structure of the LBD of ERa in complex with 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 LBD of ERa. The protein and ligand structures are represented as sticks with carbon and ﬂuorine atoms colored gray 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 ERa.Key protein residues are labeled and polar interactions are shown as dotted lines.

assays were developed specifically to measure ERa downregulated as having an apparent dissociation rate constant (kdiss)for lation, agonism, and antagonism through simultaneous mea- ERa LBD of 0.00043/sec, at 25 C equating to a half-life (t1/2)of surement of ER and PR protein levels in cells after treatment 27 2minutesandtheaffinity derived from the ratio of the with AZD9496 (23). PR expression is known to be regulated by rate constants gave a Kd of 0.33 nmol/L (Supplementary ER transcription and was measured as a marker of ER activation Table S2). This is in good agreement with the binding IC50 of in cells (34). The potency of AZD9496 approached that 0.82 nmol/L and correlates well with previously published data observed with fulvestrant in achieving pmol/L IC50 in ERa on estrogen and tamoxifen binding to ER LBD (35). AZD9496 binding, downregulation, and antagonism and significant cell showed pmol/L equipotent binding to both ERa and ERb growth inhibition in hormone-depleted growth conditions. isoforms, as expected, given their high sequence homology Adding 250 nmol/L tamoxifen significantly lowered the IC50 and similarity in tertiary structure in their LBDs (36), and highly value, as expected, if AZD9496 and tamoxifen are competing selective binding compared with progesterone (650-fold), for binding to the ligand-binding domain (LBD; Table 1). The glucocorticoid (11,223-fold), and androgen (36,375-fold) potent downregulation of ERa was also verified by immuno- receptor LBDs (Supplementary Table S3). Selectivity for ER was þ blotting techniques (Supplementary Fig. S1). also evident by measuring cell growth inhibition in a panel of ER To understand the binding kinetics of AZD9496 to human ERa and ER cell lines (Supplementary Table S4). No significant LBD, binding studies were performed in the presence of increasing growth inhibition was seen in any ER cell lines (EC50 > concentrations of AZD9496 and association (kass) and dissocia- 10 mmol/L) in contrast to the low nanomolar activity seen in a þ tion (kdiss) rate constants were calculated. AZD9496 was calcu- range of ER cell lines.

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Table 1. In vitro ERa binding, ERa downregulation, ERa antagonism, and MCF-7 ifen (Fig. 3C). In further studies, ERa protein levels were reduced cell growth inhibition data for AZD9496 versus fulvestrant with fulvestrant and AZD9496, but not tamoxifen, compared with AZD9496 Fulvestrant relevant vehicle controls, whereas PR levels were significantly a ER binding IC50 nmol/L 0.82 0.8 increased with tamoxifen alone (Fig. 3D). ERa downregulation IC50 nmol/L 0.14 0.06 ERa downregulation IC50 nmol/L 11.16 1.91 þ 250 nmol/L tamoxifen AZD9496 is a potent, oral inhibitor of breast tumor growth in vivo ERa antagonism IC50 nmol/L 0.28 0.21 MCF-7 cell growth inhibition 0.04 0.1 The effect of chronic, oral dosing of AZD9496 was explored in þ þ EC50 nmol/L MCF-7 human breast xenografts, as a representative ER /PR / þ LogD 2.9 Not measureable HER2 breast cancer model. Good bioavailability and high clear- Solubility mmol/L 110 (crystalline) <0.9 ance gave a terminal t of 5–6 hours after oral dosing in the mouse Human % free 0.15 0.03 1/2 and resulted in significant dose-dependent tumor growth inhibi- NOTE: A series of in vitro assays were performed in MCF-7 cell in hormone- depleted medium to show AZD9496 effects on ER binding, downregulation, tion with 96% inhibition at 50 mg/kg and no toxicity or weight loss antagonism, and inhibition of cell proliferation as described in ref. 20 using a relative to the vehicle control group (Fig. 4A). To confirm that fluorescence endpoint to measure of ER and PR protein levels; data shown are AZD9496 was targeting the ER pathway, PR protein levels were representative of the mean IC50 or EC50 from at least four independent measured in tumor samples taken at the end of the study and a experiments. significant reduction in PR was seen, which correlated with tumor growth inhibition. A >90% reduction in PR was seen with both 10 AZD9496 directly targets ERa for downregulation in vitro and 50 mg/kg doses and a 75% decrease even with the 0.5 mg/kg To measure the rate at which AZD9496 downregulated ERa, dose, demonstrating that AZD9496 can clearly antagonize the ER SILAC experiments were performed in MCF-7 cells using mass pathway (Fig. 4B). Dosing 5 mg/kg of AZD9496, the minimal dose spectroscopy analysis to detect ERa protein levels after treat- required to see significant tumor inhibition, gave greater tumor ment with compounds. Addition of AZD9496 or fulvestrant growth inhibition compared with 5 mg/mouse fulvestrant given 3 increased the rate of degradation of the isotope-labeled ERa times weekly and tamoxifen given 10 mg/kg orally, daily (Fig. 4C). compared with DMSO control. Levels of newly synthesized ERa A series of pharmacodynamic in vivo studies were conducted to peptide were also reduced following AZD9496 and fulvestrant measure time taken to reach maximal inhibition of PR levels and treatment, presumably due to ongoing degradation of newly time taken to recover back to basal levels. Three days of dosing with synthesized ERa protein by compound present, whereas ERa 5 mg/kg of AZD9496 gave 98% reduction of PR protein and protein levels continued to increase over time in the presence of continued to suppress protein levels at 48 hours (Fig. 4D), with tamoxifen or DMSO (Fig. 2). The t1/2 of ERa was decreased full recovery by 72 hours (data not shown), which indicates a long from 3 hours, in the presence of DMSO, to 0.75 hours with 100 pharmacologic half-life in vivo. Fulvestrant, given as 3 5mg/ nmol/L AZD9496 and 0.6 hours with 100 nmol/L fulvestrant mouse doses over one week, gave a 60% reduction in PR protein (Supplementary Fig. S2). Downregulation of ER occurs through over a prolonged period with measured plasma levels approxi- the 26S proteosomal pathway as no decrease in ERa protein mately 8-fold higher than those achieved clinically, at steady level in MCF-7 cells was seen in the presence of the proteosome state, with 500 mg fulvestrant (Fig. 4E). As estrogen itself is inhibitor MG132 (Supplementary Fig. S3). In addition, ERa known to downregulate ER protein (38, 39), we were unable to downregulation was shown to be reversible in compound detect further decreases in ERa protein compared with control wash-out experiments, where ERa levels increased in a time- animals with AZD9496 or fulvestrant in the MCF-7 model dependent manner back to basal levels over a 48-hour period presumably due to the high circulating plasma levels of estro- following compound removal (Supplementary Fig. S4). gen from implanted pellets at the time tumor samples were taken (Supplementary Fig. S5). A mouse-specific metabolite of Agonism of ERa in human breast and uterine cells AZD9496 was detected in circulating plasma at similar levels to In contrast to an AstraZeneca reference compound AZD9496 and showed a similar pharmacokinetic profile. Test- (AZ10557841), which has known ERa agonist activity in breast ing this metabolite in the in vitro MCF-7 assays resulted in cells, AZD9496 was shown to cause no increase in PR protein levels approximately 5-fold lower ERa antagonism activity and 7-fold but rather a slight decrease below the detected basal levels lower ERa downregulation activity than AZD9496 (data not (Fig. 3A). Some endocrine therapies, such as tamoxifen, can act shown). Using a pharmacokinetic/pharmacodynamic model as partial ER agonists in certain tissues, for example, endometrium, based on PR inhibition data, at the 5 mg/kg dose in vivo,which due to conformational changes that take place at the ER and the gives 98% inhibition of PR, the inhibitory activity that could be resulting effect on cofactor recruitment and transcriptional gene attributed to the parent compound alone was 85% when the activation from the tamoxifen-bound receptor (37). To test wheth- activity of the metabolite was discounted. er AZD9496 could act as a partial agonist in other tissues, agonist To explore mechanistic effects on ER pathway regulation fur- effects were measured in vivo in a previously validated immature ther, a gene expression analysis study was performed using tumor female rat model designed to detect agonistic properties of test samples dosed at 10 mg/kg for 3 days with AZD9496 or tamoxifen compounds by measuring increases in uterine weight (31). and fulvestrant given as a single dose 5 mg dose subcutaneously. AZD9496, given once daily orally at 5 and 25 mg/kg produced Tumors were taken 24 hours after the last dose of AZD9496 and statistically significant increases in uterine weight compared with tamoxifen and mRNA levels measured using a Human Transcrip- the fulvestrant control (P < 0.001) but significantly lower than tome Array (HTA 2.0). A number of known ER-regulated genes tamoxifen (P ¼ 0.001; Fig. 3B). Histologic staining of uterine tissue were examined to see whether AZD9496 could antagonize these samples also showed that the lengthening of the endometrial cells genes in a similar manner to fulvestrant and tamoxifen (40–42). in the rat uteri appeared to have decreased compared with tamox- The heatmap shows that AZD9496 could indeed downregulate

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Figure 2. Effect of AZD9496, fulvestrant, and tamoxifen on ERa peptide turnover in MCF-7 cells. Cells were grown in steroid-free conditions in SILAC 13 15 media containing C6 N4 L-arginine to label ERa peptide as "heavy" (blue line) and then switched to grow in media containing unlabeled L-arginine to label newly synthesized protein as "normal" (red line) with 0.1% DMSO (A), 300 nmol/L tamoxifen (B), 100 nmol/L AZD9496 (C), or 100 nmol/L fulvestrant (D) for the time indicated. Data shown is representative of two independent experiments.

the mRNA levels of estrogen-responsive genes (Supplementary the MCF-7 in vivo model and in all cases tumor regressions seen Fig. S6A). As differences in the levels of mRNA downregulation in with the combination arms alone compared with tumor stasis vivo could be due to different pharmacokinetic profiles of the with monotherapy (Fig. 5A–C). AZD9496 was also tested in a molecules, the effects of compounds on the mRNA levels of a long-term estrogen-deprived model (LTED), using the HCC-1428 subset of estrogen-regulated genes were also measured in MCF-7 LTED cell line that was previously adapted to grow in the absence cells that had been treated with or without estrogen (Supplemen- of estrogen and as such represents a model of aromatase inhibitor tary Fig. S6B–S6E). For two of these genes, TFF1 and AREG, resistance (22). Tumor regressions were seen with both AZD9496 tamoxifen was shown to increase mRNA levels in the absence of and fulvestrant (Fig. 5D) and both compounds completed ablat- estrogen but not with either fulvestrant or AZD9496 (Supple- ed ER protein levels in the end-of-study tumors (Supplementary mentary Fig. S6D and S6E). Where transcript levels were increased Fig. S7). to varying levels in the presence of estrogen, fulvestrant and AZD9496 inhibited transcript levels in a dose-dependent fashion AZD9496 antagonizes and downregulates mutant ER in vitro compared with tamoxifen. No significant effects were seen in and in vivo ESR1 mRNA levels in vitro, indicating that the protein down- The recent identification of ESR1 mutations in patients that had regulation described previously is due to downregulation of the received one or more endocrine therapy treatments has led to the protein and not a decrease in transcript levels per se (Supplemen- discovery that these mutations can drive cell proliferation in the tary Fig. S6F). absence of estrogen and may constitute a resistance mechanism to some endocrine agents. In vitro binding studies were performed AZD9496 causes tumor regressions in combination with PI3K using wt, D538G, and Y537S LBDs and both AZD9496 and pathway and CDK4/6 inhibitors and in an estrogen-deprived fulvestrant were able to bind to mutant LBD with nmol/L potency þ ER model of resistance although 2- to 3-fold reduced compared with wt (Table 2). The Acquired resistance to aromatase inhibitors and antiestrogens different IC50 values obtained for binding of AZD9496 and is driven through a variety of mechanisms, which include effects fulvestrant to wt ERa compared with the value in Table 1 is on the ER pathway itself, i.e., downregulation of ER expression and altered expression of ER coregulators, as well as activation of Table 2. In vitro binding to ERa mutant LBDs alternative prosurvival or proliferative pathways and cross-talk ERa LBD binding IC nmol/L between growth factor receptors and ER pathways. This has led to 50 AZD9496 nmol/L Fulvestrant nmol/L a number of clinical trials with PIK3, mTOR, AKT inhibitors, as wt 0.2 1.6 well as CDK4/6 inhibitors in combination with aromatase inhi- D538G 0.5 3.3 bitors or fulvestrant and early results have shown increased Y537S 0.6 3.8 progression-free survival (PFS) in many cases (43). Combinations NOTE: IC50s for binding of AZD9496 and fulvestrant to ERa wt and mutant LBDs; of AZD9496 with AZD2014 (dual mTORC1/2), AZD8835 data shown are representative of the mean IC50 from three independent (PIK3Ca/d) and palbociclib (CDK4/6) inhibitors were tested in experiments.

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Figure 3. ERa-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 normalized for cell number by analyzing a ratio of fluorescence resulting from stimulation of two emission wavelengths and used to perform curve fitting analysis. A concentration response was plotted using nonlinear regression analysis. B and 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 hours after the final dose and weighed before tissues were formalin-fixed and paraffin embedded to allow histologic staining of the endometrial tissue. Significant differences in mean uterine weight are shown: , P < 0.001; , P < 0.01. D, tissue was subsequently analyzed by Western blot analysis for levels of ERa, PR, and vinculin. Protein levels were measured by chemiluminescent and quantified using Syngene software. ER and PR protein levels were normalized 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.

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Figure 4. In vivo efﬁcacy of AZD9496 in MCF-7 xenograft model. A, MCF-7 xenografts, grown in male SCID mice, were dosed daily with either PEG/captisol (vehicle) or AZD9496 at doses shown. Tumor growth was measured by caliper at regular intervals and mean tumor volumes plotted for each dosed group. B, tumors from treated mice collected at the end of study were analyzed by Western blot analysis for levels of PR and vinculin. Protein levels were measured by chemiluminescent and quantiﬁed using Syngene software. PR protein levels were normalized to vinculin as a loading control and plotted as shown with standard error bars. , P < 0.05; , P < 0.001. C, MCF-7 xenografts were also dosed daily with 5 mg/kg AZD9496, 10 mg/kg tamoxifen, or 5 mg/mouse three times weekly with fulvestrant subcutaneously. D and E, for acute dosing studies, MCF-7 xenografts were dosed for 3 days with doses shown of AZD9496 and tumors collected at 24 or 48 hours after the last dose (D) or dosed with three doses of 5 mg/mouse of fulvestrant over 5 days and tumors collected at 24, 48, and 96 hours after the last dose (E). PR and vinculin protein levels in the tumors were analyzed and plotted as described above. p.o., orally; q.d., once daily.

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likely due to different sources of protein used in the Lanthascreen of AZD9496 was used alongside 5 mg/mouse of fulvestrant, kit assays versus the laboratory-derived wt and ESR1-mutant which was known to achieve higher pharmacokinetic levels in proteins. Downregulation of ER-mutant proteins was measured mouse than the current clinical dose. AZD9496 and fulvestrant using MCF-7 doxycycline-inducible stable cell lines in vitro.ER inhibited tumor growth by 66% and 59% respectively, compared protein levels increased on induction with 1 mg/mL doxycycline with tamoxifen, which gave 28% inhibition. Efficacy correlated and led to increased levels of PR in the absence of estrogen. with antagonism of the pathway with AZD9496 giving a 94% AZD9496 and fulvestrant were able to downregulate all mutant decrease in PR levels compared with 63% with fulvestrant (Fig. 6B ER proteins and decreased PR levels, whereas tamoxifen appeared and C). In the absence of estrogen, and given the significant PR to stabilize ER protein levels and reduced PR levels to a lesser changes seen with 25 mg/kg, the dose of AZD9496 was lowered extent (Fig. 6A). To explore activity against an ESR1 mutant in vivo, but a 5 mg/kg dose still achieved growth inhibition of approx- tumor growth inhibition studies were performed in a PDX imately 70% and led to a significant decrease of 73% in ERa model, CTC-174, which harbors a D538G mutation in the LBD protein levels at the end of the study (Fig. 6D and E). Given the and constituted 31% of the transcripts variants identified by RNA- similarities in growth inhibition either with or without estrogen, seq analysis. Tumors were grown from implanted tumor frag- residual tumor growth is likely to be due to either additional ments either with or without estrogen and in both cases viable mutations (e.g., PIKCA N345K) and/or growth factor signaling tumors grew. Given published data that higher doses of SERM/ pathways involved in driving tumor growth in addition to the ER SERDs may be needed to inhibit ESR1 mutants (14, 15), 25 mg/kg pathway.

Figure 5. Tumor regressions with AZD9496 in combination with mTOR, PIKC, and CDK4/6 inhibitors in MCF-7 xenograft model and in HCC1428 LTED model. A–C, MCF-7 xenografts, grown in male SCID mice, were dosed daily with AZD9496 at 5 mg/kg 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 twice daily on day 1 and 4 (B), and with or without palbociclib dosed daily at 50 mg/kg (C). D, HCC1428LTED engrafts were grown in ovariectomized NSG mice and were dosed daily with either AZD9496 or once weekly with fulvestrant at doses shown. Tumor growth was measured by caliper at regular intervals and mean tumor volumes plotted for each dosed group. p.o., orally; q.d., once daily.

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Figure 6. AZD9496 is efﬁcacious against ESR1 mutants in vitro and in vivo. A, stable MCF-7 cell lines containing either pTRIPZ vector alone or vector containing ESR1 mutants were induced to express mutant protein with 1 mg/mL doxycycline for 24 hours before treating with either 0.1% DMSO (vehicle) or 100 nmol/L of fulvestrant, AZD9496, or tamoxifen for 24 hours. Immunoblotting was performed to detect expression of ERa, PR, and GAPDH levels. B, CTC-174 tumor 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. Tumor growth was measured by caliper at regular intervals and mean tumor volumes plotted for each dosed group. C, tumors from treated mice collected at the end of study were analyzed by Western blot analysis for levels of PR and vinculin. Protein levels were measured by chemiluminescent and quantiﬁed using Syngene software. PR protein levels were normalized to vinculin as a loading control and plotted as shown. , P < 0.001. D, CTC-174 tumor 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. E, tumors from treated mice collected at the end of study were analyzed by Western blot analysis for levels of ERa and vinculin and measured as described above. , P < 0.05. p.o., orally; q.d., once daily.

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Discussion PR modulation, and inhibition of tumor growth (data not shown). The model reflected relative contributions of AZD9496 Despite the enhanced clinical benefits of directly antagonizing and its active mouse metabolite to the overall biomarker mod- and downregulating ER and thereby targeting any ligand-inde- ulation and efficacy. Given the half-life of the parent drug and pendent ER-driven pathways, the pharmacokinetic and IHC bio- metabolite are around 5 hours, to explain the prolonged phar- marker data for ER, PR, and Ki67 from clinical trials with fulves- macodynamic effect observed with AZD9496 at 48 hours, an trant would suggest that a plateau of effect has not been reached, indirect response model for PR was developed, which estimated a and there is scope to further improve on the 50% reduction in ER degradation rate constant of around 23 hours for PR. This mod- levels seen after 6 months of treatment (8, 12). As one of the ulation in PR was linked directly to tumor growth inhibition by a potential drawbacks associated with fulvestrant is the very low proportional decrease of the tumor growth constant, and corre- bioavailability, which is seen both in patients and in preclinical lated well with the wide range of measured efficacies. animal models, a potent, orally bioavailable SERD could have the We consistently saw enhanced tumor growth inhibition of potential to significantly antagonize ER activity and increase ERa AZD9496 compared with fulvestrant in the MCF-7 in vivo model receptor knockdown versus fulvestrant due to higher exposures. (Fig. 4). This model contains high circulating levels of plasma Our approach focused on identifying novel ER motifs with estradiol over the dosing period of the study as a result of the established drug-like properties that could then be optimized for estrogen pellets used to drive growth of the MCF-7 cells as a cellular phenotype and potency and led to the identification of þ xenograft. With plasma levels between 750 and 1,000 pg/mL over AZD9496, which showed picomolar potency in vitro in ER cell day 21–35, this model is more representative of the premeno- lines only. Previous structural analysis of a number of SERMs with pausal setting where estrogen levels can vary between 20 and 400 ERa LBD identified key interactions with Asp 351, which was pg/mL (47). As the receptor binding data does not suggest a shown to be critical for the antagonist activity of antiestrogens significant difference in affinity for the receptor between such as raloxifene, lasofoxifene, and GW5638 through shielding AZD9496 and fulvestrant, increased efficacy is likely due to the or neutralization of the negative charge. Unlike tamoxifen, how- higher free drug levels of AZD9496 versus fulvestrant in this ever, GW5638 repositioned residues helix-12 through its acrylate model. It would therefore be interesting to test the activity of side chain, increasing the exposed hydrophobic surface of ERa, potent oral SERDs, such as AZD9496, in the premenopausal resulting in destabilization and ultimately 26S proteosomal deg- setting where there is no enhanced risk of endometrial cancer radation of the receptor. The acrylic acid moiety of AZD9496 is observed for tamoxifen. colocalized with the Asp 351 residue in the helix-12 region of ER We were unable to detect a reduction in ER protein levels in the in an unusual acid–acid interaction, and it is therefore proposed MCF-7 in vivo model with AZD9496 at any dose tested (data not that AZD9496 also repositions residues associated with the helix- shown). Having shown in vitro that estradiol can efficiently down- 12 via specific contacts with the N-terminus region, resulting in a regulate ERa protein, we postulate that downregulation of ERa by mixed antagonist and downregulator profile (33, 44). Evidence estradiol is already taking place in vivo given the high levels of that AZD9496 was directly binding and downregulating the circulating estrogen and that any additional changes by addition receptor at the same site as other antiestrogens was seen by the of a SERD are difficult to detect using the current methods. This reduced potency observed on prior addition of tamoxifen to the was supported by measuring ERa downregulation in in vivo downregulation assay (Table 1). Mechanistically, we have shown models, such as CTC-174 and HCC1428LTED, not requiring that AZD9496 has very similar effects on antagonism, ERa down- estrogen supplements, and showing potent downregulation of regulation, and agonism to fulvestrant. Even where ERa was ERa by AZD9496 (Fig. 6E, Supplementary Fig. S7). The level of overexpressed in MCF-7 cells, unlike tamoxifen, AZD9496 was ERa reduction seen in the HCC1428 model is greater than seen able to downregulate and antagonize the overexpressed receptor clinically, with pharmacokinetic levels 5-fold higher than steady- (Fig. 6A). Our transcriptional studies in both MCF-7 cells and the state plasma levels of 500 mg fulvestrant, and supports the in vivo model also demonstrated antagonism of the transcription- concept that if increased plasma levels of an active SERD agent al activity of the receptor as seen by decreased mRNA levels of ER- could be achieved clinically, greater ERa knockdown may be activated genes in a dose-dependent manner (Supplementary Fig. possible. Preclinically, this appears to translate to enhanced S6). tumor growth inhibition. Unlike fulvestrant, AZD9496 did show some degree of agonism Achieving increased serum levels of a biologically active SERD in endometrial tissue (Fig. 3). This effect was dose independent could be particularly beneficial in the recently identified ESR1- and would indicate that there are subtle differences in how mutant patient setting. Preclinical in vivo studies using PDX AZD9496 interacts with ERa compared with tamoxifen and models derived from ESR1-mutant patients grown in the absence fulvestrant and therefore the extent to which tissue specific coac- of estrogen supplementation have shown that high doses of tivators can interact with the receptor. Given the widespread use of fulvestrant (>500 mg/kg) can lead to tumor regression and tamoxifen in the adjuvant setting for 5–10 years, the low level of significant decreases in ER protein levels but at significantly higher agonism seen with AZD9496 is not viewed as a deterrent for doses than could be achieved clinically (48). Our preclinical data clinical development. A number of studies have looked at the would indicate that both AZD9496 and fulvestrant can potently incidence of endometrial cancer in patients taking tamoxifen over bind and downregulate D538G and Y537C/N/S ERa proteins in a number of years and despite the small increase in the incidence vitro and significant tumor growth inhibition rather than regres- of endometrial cancer, the benefits of tamoxifen greatly outweigh sion seen in vivo. This is in contrast to published work showing any risks of developing uterine malignancy in postmenopausal relatively little downregulation of Y537N mutant protein in vitro patients (45, 46). with increasing doses of fulvestrant (15) but could be attributed to A pharmacokinetic/pharmacodynamic efficacy model was different expression systems and cell lines used for the analysis. It established, integrating diverse endpoints such as drug exposure, is notable that the doxycycline-inducible mutant receptors do not

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Pharmacology of SERD Inhibitor in Breast Cancer Models

appear to be constitutively downregulated in a similar manner to Disclosure of Potential Conflicts of Interest wt ER exposed to estradiol (Supplementary Fig. S1). This may be J.O. Curwen is an associate principal scientist at AstraZeneca. G. Davies has due to the level of overexpression of the receptor in each stable cell ownership interest (including patents) in AstraZeneca as a shareholder. line and it will be interesting to compare these findings from S. Powell is a team leader at Astrazeneca. G. Richmond has ownership interest (including patents) in AstraZeneca. All authors are current or former engineered expression systems to those from genetically altered AstraZeneca employees and shareholders. mutant cell lines where expression is under control of the native promoter. Although very high doses of both compounds were not Authors' Contributions ESR1 tested in the PDX CTC-174 -mutant model, the lack of any Conception and design: H.M. Weir, R.H. Bradbury, M. Lawson, A.A. Rabow, significant dose response might indicate the need to inhibit other J.O. Curwen, R.A. Norman, G.E. Walker, C. Sadler, G. Richmond, C. De Savi growth factor–activated pathways in addition to the ER pathway Development of methodology: H.M. Weir, R.H. Bradbury, M. Lawson, to achieve significant antitumor growth. As trials commence to C.S. Donald, L.J.L Feron, T. Moss, S.E. Pearson, G. Davies, G.E. Walker, explore treatment of ESR1-mutant patients with new oral SERDs, S. Powell, C. Sadler, B. Ladd data will hopefully emerge that will indicate whether lack of Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H.M. Weir, M. Lawson, R.J. Callis, M. Hulse, C.S. Donald, response is due to inability to fully inhibit mutated ER receptors P. MacFaul, T. Moss, R.A. Norman, S.E. Pearson, M. Tonge, G. Davies, G.E. Walker, or whether, in these advanced metastatic patients, other mutations Z. Wilson, R. Rowlinson, B. Ladd, E. Pazolli, A.M. Mazzola and pathways are or become dominant in driving tumor growth. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, In metastatic breast cancer, the median survival time is still computational analysis): H.M. Weir, M. Lawson, A.A. Rabow, D. Buttar, around two years as a result of acquired endocrine resistance, R.J. Callis, J.O. Curwen, C. de Almeida, P. Ballard, M. Hulse, P. MacFaul, highlighting the need for additional therapies (43). This has led to R.A. Norman, M. Tonge, G. Davies, G.E. Walker, Z. Wilson, R. Rowlinson, – – S. Powell, G. Richmond, A.M. Mazzola, C.M. D'Cruz a number of clinical trials using PI3K Akt mTOR pathway inhi- Writing, review, and/or revision of the manuscript: H.M. Weir, M. Lawson, bitors in combination with AIs as well as cyclin-dependent kinase D. Buttar, P. Ballard, C.S. Donald, R.A. Norman, S.E. Pearson, M. Tonge, inhibitors such as palbociclib, which was recently approved for G. Davies, G.E. Walker, C. Sadler, G. Richmond, B. Ladd, A.M. Mazzola, combination treatment with letrozole in the metastatic breast C.M. D'Cruz, C. De Savi cancer. Results from the phase III BOLERO-2 trial also demon- Administrative, technical, or material support (i.e., reporting or organizing strated the PFS benefits of a steroidal AI plus everolimus in data, constructing databases): H.M. Weir, M. Lawson, P. Ballard, S.E. Pearson, S. Powell, A.M. Mazzola patients that had progressed on a nonsteroidal AI (49). More Study supervision: H.M. Weir, J.O. Curwen, R.A. Norman, G.E. Walker, recently, further trials have been initiated using fulvestrant as the C.M. D'Cruz combination endocrine partner, which will allow analysis and Other (chemistry route development): G. Karoutchi understanding of any additional benefit from combination with a SERD versus AIs in this setting. Our data clearly demonstrated Acknowledgments enhanced combination effects on tumor growth of an oral SERD The authors thank Elaine Hurt, Haifeng Bao, Sanjoo Jalla for access to the in vivo agent with either dual mTORC1/2, PIKCa/d or CDK4/6 inhibitors CTC-174 model developed at MedImmune.The authors also thank Paul Hemsley and Emma Linney for providing ER-mutant binding assay data, Helen and furthermore opens up the possibility of using a combined Gingell for supporting the crystallography work and Zara Ghazoui and Henry endocrine and targeted inhibitor approach with longer term Brown for the transcript data and analysis at AstraZeneca. The authors also thank dosing in the adjuvant setting, assuming emerging data from Carlos Arteaga for use of the HCC1428-LTED cell line. ongoing metastatic trials supports the testing of combinations in The costs of publication of this article were defrayed in part by the payment of early breast disease. The identification of a potent, orally bio- page charges. This article must therefore be hereby marked advertisement in available compound such as AZD9496 is a step forward in the next accordance with 18 U.S.C. Section 1734 solely to indicate this fact. generation of SERD agents to undergo clinical evaluation as a Received August 28, 2015; revised February 1, 2016; accepted March 1, 2016; future monotherapy or combination partner of choice. published OnlineFirst March 28, 2016.

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OF12 Cancer Res; 76(11) June 1, 2016 Cancer Research

AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical Models

Hazel M. Weir, Robert H. Bradbury, Mandy Lawson, et al.

Cancer Res Published OnlineFirst March 28, 2016.

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