Author Manuscript Published OnlineFirst on March 28, 2016; DOI: 10.1158/0008-5472.CAN-15-2357 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Title: AZD9496: An oral estrogen receptor inhibitor that blocks the growth of ER- 2 positive and ESR1 mutant breast tumours in preclinical models. 3 4 Running title: Pharmacology of SERD inhibitor in breast cancer models 5 6 Hazel M. Weir,a Robert H. Bradbury,a Mandy Lawson,a Alfred A. Rabow,a David Buttar,a 7 Rowena J. Callis,b Jon O. Curwen,a Camila de Almeida,a Peter Ballard, a Michael Hulse,a 8 Craig S. Donald,a Lyman J. L. Feron,a Galith Karoutchi,a Philip MacFaul,a Thomas Moss,a 9 Richard A. Norman,b Stuart E. Pearson,a Michael Tonge,b Gareth Davies,a Graeme E. 10 Walker,b Zena Wilson,a Rachel Rowlinson,a Steve Powell,a Claire Sadler,a Graham 11 Richmond,a Brendon Ladd, d Ermira Pazolli,c Anne Marie Mazzola,d Celina D’Cruz,d Chris 12 De Savi.d 13 14 Authors’ Affiliations: 15 aOncology iMed, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK; 16 bDiscovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG UK; c 17 H3 Biomedicine, Technology Sq, Cambridge, MA 02139,dOncology iMed, AstraZeneca R&D 18 Boston, Gatehouse Drive, Waltham, MA 02451, U.S. 19 20 Key words: SERD, estrogen receptor, metastatic breast cancer, ER mutation, ESR1 mutation. 21 combination therapy 22 23 Corresponding author: [email protected] 24 25 Disclosure of Potential Conflicts of Interest: All authors are present or past employees of 26 AstraZeneca Pharmaceuticals 27 28 Tables: 1 29 Figures: 6 30 Supplementary Tables:4 31 Supplementary Figures: 7 32 33 Precis: This article discloses the structure and pharmacology of an oral non-steroidal small 34 molecule which is potent against wild-type and mutant forms of the estrogen receptor, 35 producing anti-tumour efficacy either as a monotherapy or in combination with PI3K or cell 36 cycle inhibitors in vivo. 37 1 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 28, 2016; DOI: 10.1158/0008-5472.CAN-15-2357 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 38 39 40 Abstract 41 Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by 42 monthly intramuscular injection. Given its present limitations of dosing and route of 43 administration, a more flexible orally available compound has been sought to pursue the 44 potential benefits of this drug in patients with advanced metastatic disease. Here we report 45 the identification and characterization of AZD9496, a non-steroidal small molecule inhibitor 46 of ERα which is a potent and selective antagonist and down-regulator of ERα in vitro and in 47 vivo in ER-positive models of breast cancer. Significant tumour growth inhibition was 48 observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where 49 this effect was accompanied by a dose-dependent decrease in PR protein levels demonstrating 50 potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors 51 led to further growth inhibitory effects compared to monotherapy alone. Tumour regressions 52 were also seen in a long-term estrogen-deprived breast model, where significant down- 53 regulation of ERα protein was observed. AZD9496 bound and down-regulated clinically 54 relevant ESR1 mutants in vitro and inhibited tumour growth in an ESR1-mutant patient- 55 derived xenograft model that included a D538G mutation. Collectively, the pharmacological 56 evidence showed that AZD9496 is an oral, non-steroidal, selective estrogen receptor 57 antagonist and down-regulator in ER-positive breast cells that could provide meaningful 58 benefit to ER-positive breast cancer patients. AZD9496 is currently being evaluated in a 59 Phase 1 clinical trial. 60 2 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 28, 2016; DOI: 10.1158/0008-5472.CAN-15-2357 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 61 62 Introduction 63 With over 70% of breast cancers expressing the estrogen receptor alpha (ERα), 64 treatment with anti-hormonal therapies that directly antagonise ER function (e.g. tamoxifen) 65 or therapies that block the production of the ligand, estrogen (e.g. aromatase inhibitors) are 66 key to management of the disease both in adjuvant and metastatic settings (1, 2). Despite 67 initial efficacy seen with endocrine therapies, the development of acquired resistance 68 ultimately limits the use of these agents although the majority of tumours continue to require 69 ERα for growth. Through the use of pre-clinical models and cell lines, changes in growth 70 factor receptors have been implicated in driving resistance in cells e.g. receptor tyrosine 71 kinases (HER2), epidermal growth factor receptor (EGFR) and their downstream signalling 72 pathways, Ras/Raf/MAPK and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) 73 signalling (3, 4, 5). In some instances signalling from different growth factor receptor kinases 74 leads to phosphorylation of various proteins in the ER pathway, including ER itself, leading 75 to ligand–independent activation of the ER pathway (6). 76 In the metastatic setting, 500 mg fulvestrant, given as 2 x 250 mg intramuscular 77 injections, has been shown to offer additional benefit over the 250mg dose with improved 78 median overall survival (7, 8). Recent analysis of pre-surgical data from a number of trials 79 showed a dose-dependent effect on key biomarkers such as ER, progesterone receptor (PR) 80 and Ki67 labeling index (9). Although fulvestrant is clearly effective in this later treatment 81 setting, there are still perceived limitations in its overall clinical benefit due to very low 82 bioavailability, the time taken to achieve plasma steady-state of 3-6 months and ultimately 83 the level of ERα reduction seen in clinical samples (10, 11). It is widely believed that a 84 potent, orally bioavailable SERD that could achieve higher steady-state free drug levels more 3 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 28, 2016; DOI: 10.1158/0008-5472.CAN-15-2357 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 85 rapidly would have the potential for increased receptor knock-down and lead to quicker 86 clinical responses, reducing the possibility of early relapses (12). Moreover, the recent 87 discovery of ESR1 mutations in metastatic breast cancer patients that had received prior 88 endocrine treatments, where the mutated receptor is active in the absence of estrogen, 89 highlights an important potential resistance mechanism. Pre-clinical in vitro and in vivo 90 studies have shown a reduction in signalling from ESR1 mutants by tamoxifen and fulvestrant 91 but at higher doses than required to inhibit the wild type receptor which implies that more 92 potent SERDs may be required to treat these patients or to used in the adjuvant setting to 93 limit the emergence of mutations (13, 14, 15). 94 Achieving oral bioavailability has remained a key challenge in the design of ER 95 down-regulators and until now no oral estrogen receptor down-regulators, other than GDC- 96 0810 and RAD1901, have progressed into clinical trials, despite encouraging reports from 97 others of pre-clinical activity in mouse xenograft models (16, 17, 18, 19, 20, 21). Here we 98 describe a chemically novel, non-steroidal ERα antagonist and down-regulator, AZD9496, 99 that can be administered orally and links increased tumour growth inhibition to enhanced 100 biomarker modulation compared to fulvestrant in a preclinical in vivo model of breast cancer. 101 Moreover we show that AZD9496 is a potent inhibitor of ESR1 mutant receptors and can 102 drive tumour growth inhibition in a patient-derived ESR1 mutant in vivo model (PDX). Hence 103 AZD9496 is anticipated to yield improved bioavailability and clinical benefit through 104 enhanced ER pathway modulation. 105 Methods 106 Cell lines and culture 107 All AZ cell lines were tested and authenticated by short-tandem repeat (STR) analysis. MCF- 108 7 was obtained from DSMZ and last STR tested February 2013. MDA-MB-361 (STR tested 4 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 28, 2016; DOI: 10.1158/0008-5472.CAN-15-2357 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 109 October 2011), MDA-MB-134 (STR tested October 2010), T47D (STR tested June 2012), 110 CAMA-1 (STR tested March 2012) HCC1428 (STR tested December 2011) HCC70 (STR 111 tested October 2013), LnCAP (STR tested August 2011), MDA-MB-468 (STR tested March 112 2014), BT474 (STR tested July 2013), PC3 (STR tested October 2012), HCT116 (STR 113 tested August 2011) were all obtained from the ATCC. HCC1428-LTED cell line (STR 114 tested November 2012) was kindly provided by Carlos Arteaga (22). Cells were cultured in 115 RPMI-1640 media (Corning) supplemented with 10% heat-inactivated foetal calf serum 116 (FCS) or charcoal/dextran-treated CCS to deplete hormone and 2mmol/L L-glutamine 117 (Corning).. Stable MCF-7 cell lines expressing FLAG-tagged ESR1 mutant proteins in 118 pTRIPZ lentiviral vectors (GE Healthcare) under the control of the doxycycline inducible Tet 119 promoter were generated by lentiviral infection using standard techniques.