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

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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: Hazel.Weir@astrazeneca.com 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

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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.

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

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

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.

120

121 Biochemical and in vitro cell assays

122 Binding, ER agonism, antagonism, down-regulation and cell proliferation assays were carried

123 out as described previously (23). BIAcore affinity measurements and immunoblotting from

124 compound treated cells are described in Supplementary Methods.

125

126 Protein production, crystallization and structure determination

127 Protein expression, purification and crystallization of the ERα ligand-binding domain was

128 carried out as previously described (24). X-ray diffraction data were collected at the ESRF on

129 beamline ID23-1 on an ADSC detector. Data were processed using XDS (25) as implemented

130 within EDNA (26) and scaled and merged using SCALA (27). The structure of ERα in

131 complex with AZD9496 was solved by molecular replacement using AmoRE and an internal

132 ERα structure as the search model (28). Quality checks on the protein structures were carried

133 out using the validation tools in Coot (29). The final structure has been deposited in the

134 Protein Databank (PDB) with the ID code given in Supplementary Table S1.

135

136 SILAC and mass spectrometry

137 SILAC assays were carried out as previously described (30) and detailed in Supplementary

138 methods. Compound treated samples were analysed by mass spectrometry using relative

139 peptide quantification by selected reaction monitoring (SRM). Degradation half-life was

140 measured using the one-phase exponential decay equation in GraphPad PRISM (Y=Span.e-

141 K.X+Plateau) where X is time and Y is response which starts out as Span+Plateau and

142 decreases to Plateau with a rate constant K.

143

144 Rat Uterine and Xenograft Studies

145 All studies involving animals in the United Kingdom were conducted in accordance with UK

146 Home Office legislation, the Animal Scientific procedures Act 1986 (ASPA) and

147 AstraZeneca Global Bioethics policy or with US federal, state and Institutional guidelines in

148 an AAALAC-accredited facility. The rat uterine weight assay for the measurement of

149 estrogenic activity were performed as described previously (31). Details of individual

150 models, protein analysis of tumour fragments and pharmacokinetic analysis of plasma

151 samples are included in Supplementary Methods.

152

153 Results

154 AZD9496 is a selective ERα antagonist, down-regulator and inhibitor of ER positive 155 tumour cell growth 156 157 AZD9496 ((E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-

158 tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid, Fig. 1A) was identified through

159 structure based design and iterative medicinal chemistry structure-activity-relationship (SAR)

160 studies from a novel ER binding motif (32). A directed ER binding screen was established to

161 identify novel binding motifs that could then be optimised for both cellular phenotype and

162 potency while maintaining good pharmacokinetic properties (23). Unlike fulvestrant, and

163 other well known historical modulators of the ER receptor, including hydroxy-tamoxifen

164 (Fig. 1B), AZD9496 lacks a phenolic moiety in which the phenol mimics the A-ring phenol

165 group of the endogenous ligand estradiol but gains its potency through a different series of

166 novel interactions with the ER protein (Fig. 1C). The indole NH picks up an interaction by

167 forming a hydrogen bond to the carbonyl of Leu-346, with additional lipophilic interactions

168 achieved by both the chiral methyl substituent, adjacent to the ring nitrogen and the N-

169 substituted isopropyl fluoro side-chain occupying a “lipophilic hole” in the ER protein. The

170 acrylic acid side chain picks up an unusual acid-acid co-localization with Asp-351 in the

171 Helix-12 region of the ER that has been proposed to be crucial for achieving a down-

172 regulator-antagonist profile (33). AZD9496 showed high oral bioavailability across three

173 species (F% 63, 91 and 74, rat, mouse and dog, respectively) with generally low volume and

174 clearance across species, albeit a higher clearance in mouse. The percent free levels in human

175 plasma of 0.15% were 5 fold higher than those measured for fulvestrant.

176 The key characteristics of a SERD is the ability to both antagonise and down-

177 regulate ER protein without inducing any partial agonist effects in breast cells. A series of in

178 vitro cell assays were developed specifically to measure ERα down-regulation, agonism and

179 antagonism through simultaneous measurement of ER and PR protein levels in cells after

180 treatment with AZD9496 (23). PR expression is known to be regulated by ER transcription

181 and was measured as a marker of ER activation in cells (34). The potency of AZD9496

182 approached that observed with fulvestrant in achieving pM IC50 in ERα binding, down-

183 regulation and antagonism and significant cell growth inhibition in hormone-depleted growth

184 conditions. Adding 250 nM tamoxifen significantly lowered the IC50 value, as expected, if

185 AZD9496 and tamoxifen are competing for binding to the LBD (Table 1A). The potent

186 down-regulation of ERα was also verified by immunoblotting techniques (Supplementary

187 Fig. S1).

188 To understand the binding kinetics of AZD9496 to human ERα ligand binding

189 domain (LBD), binding studies were performed in the presence of increasing concentrations

190 of AZD9496 and association (kass) and dissociation (kdiss) rate constants were calculated.

191 AZD9496 was calculated as having an apparent dissociation rate constant (kdiss) for ERα

192 LBD of 0.00043 sec-1, at 25 ºC equating to a half-life (t ½) of 27 ± 2 min and the affinity

193 derived from the ratio of the rate constants gave a KD of 0.33 nM (Supplementary Table

194 S2). This is in good agreement with the binding IC50 of 0.82 nM and correlates well with

195 previously published data on estrogen and tamoxifen binding to ER LBD (35). AZD9496

196 showed pM equipotent binding to both ERα and ERβ isoforms as expected given their high

197 sequence homology and similarity in tertiary structure in their LBDs (36) and highly selective

198 binding compared to progesterone (~650 fold), glucocorticoid (~11223 fold) and androgen

199 (~36375 fold) receptor LBDs (Supplementary Table S3). Selectivity for ER was also

200 evident by measuring cell growth inhibition in a panel of ER +ve and ER -ve cell lines

201 (Supplementary Table S4). No significant growth inhibition was seen in any ER -ve cell

202 lines (EC50 >10 μM) in contrast to the low nM activity seen in a range of ER +ve cell lines.

203

204 AZD9496 directly targets ERα for down-regulation in vitro

205 To measure the rate at which AZD9496 down-regulated ERα, stable isotope

206 labelling of amino acids (SILAC) experiments were performed in MCF-7 cells using mass

207 spectroscopy analysis to detect ERα protein levels after treatment with compounds. Addition

208 of AZD9496 or fulvestrant increased the rate of degradation of the isotope-labelled ERα

209 compared to DMSO control. Levels of newly synthesised ERα peptide were also reduced

210 following AZD9496 and fulvestrant treatment, presumably due to on-going degradation of

211 newly-synthesised ERα protein by compound present, whereas ERα protein levels continued

212 to increase over time in the presence of tamoxifen or DMSO (Fig 2). The t½ of ERα was

213 decreased from 3 hours, in the presence of DMSO, to 0.75 hours with 100 nM AZD9496 and

214 0.6 hours with 100 nM fulvestrant. (Supplementary Fig. S2). Down-regulation of ER occurs

215 through the 26S proteosomal pathway as no decrease in ERα protein level in MCF-7 cells

216 was seen in the presence of the proteosome inhibitor MG132 (Supplementary Fig. S3).

217 Additionally, ERα down-regulation was shown to be reversible in compound wash-out

218 experiments, where ERα levels increased in a time-dependent manner back to basal levels

219 over a 48 hour period following compound removal. (Supplementary Fig. S4).

220 Agonism of ERα in human breast and uterine cells

221 In contrast to an AstraZeneca reference compound (AZ10557841) which has known ERα

222 agonist activity in breast cells, AZD9496 was shown to cause no increase in PR protein levels

223 but rather a slight decrease below the detected basal levels (Fig. 3A). Some endocrine

224 therapies, such as tamoxifen, can act as partial ER agonists in certain tissues e.g.

225 endometrium, due to conformational changes that take place at the ER and the resulting effect

226 on co-factor recruitment and transcriptional gene activation from the tamoxifen-bound

227 receptor (37). To test if AZD9496 could act as a partial agonist in other tissues, agonist

228 effects were measured in vivo in a previously validated immature female rat model designed

229 to detect agonistic properties of test compounds by measuring increases in uterine weight

230 (31). AZD9496, given once daily orally at 5 and 25 mg/kg produced statistically significant

231 increases in uterine weight compared to the fulvestrant control (p<0.001) but significantly

232 lower than tamoxifen (p=0.001) (Fig. 3B). Histological staining of uterine tissue samples

233 also showed that the lengthening of the endometrial cells in the rat uteri appeared to have

234 decreased compared to tamoxifen (Fig. 3C). In further studies, ERα protein levels were

235 reduced with fulvestrant and AZD9496, but not tamoxifen, compared to relevant vehicle

236 controls, whereas PR levels were significantly increased with tamoxifen alone (Fig. 3D).

237

238 AZD9496 is a potent, oral inhibitor of breast tumour growth in vivo

239 The effect of chronic, oral dosing of AZD9496 was explored in MCF7 human breast

240 xenografts, as a representative ER+/PR+/HER2+ breast cancer model. Good bioavailability

241 and high clearance gave a terminal t½ of 5-6 hours after oral dosing in the mouse and resulted

242 in significant dose-dependent tumour growth inhibition with 96% inhibition at 50 mg/kg and

243 no toxicity or weight loss relative to the vehicle control group (Fig. 4A). To confirm that

244 AZD9496 was targeting the ER pathway, PR protein levels were measured in tumour samples

245 taken at the end of the study and a significant reduction in PR was seen which correlated with

246 tumour growth inhibition. A >90% reduction in PR was seen with both 10 and 50 mg/kg

247 doses and a 75% decrease even with the 0.5 mg/kg dose, demonstrating that AZD9496 can

248 clearly antagonise the ER pathway (Fig. 4B). Dosing 5 mg/kg of AZD9496, the minimal

249 dose required to see significant tumour inhibition, gave greater tumour growth inhibition

250 compared to 5 mg/mouse fulvestrant given 3x weekly and tamoxifen given 10 mg/kg orally,

251 daily (Fig. 4C). A series of pharmacodynamic in vivo studies were conducted to measure

252 time taken to reach maximal inhibition of PR levels and time taken to recover back to basal

253 levels. Three days of dosing with 5 mg/kg of AZD9496 gave 98% reduction of PR protein

254 and continued to suppress protein levels at 48 hours (Fig. 4D) with full recovery by 72 hours

255 (data not shown) which indicates a long pharmacological half life in vivo. Fulvestrant, given

256 as 3 x 5 mg/mouse doses over one week, gave a 60% reduction in PR protein over a

257 prolonged period with measured plasma levels ~8-fold higher than those achieved clinically,

258 at steady state, with 500 mg fulvestrant (Fig. 4E). As estrogen itself is known to down-

259 regulate ER protein (38, 39), we were unable to detect further decreases in ERα protein

260 compared to control animals with AZD9496 or fulvestrant in the MCF-7 model presumably

261 due to the high circulating plasma levels of estrogen from implanted pellets at the time

262 tumour samples were taken (Supplementary Fig. S5). A mouse specific metabolite of

263 AZD9496 was detected in circulating plasma at similar levels to AZD9496 and showed a

264 similar pharmacokinetic profile. Testing this metabolite in the in vitro MCF-7 assays resulted

265 in ~5 fold lower ERα antagonism activity and 7 fold lower ERα down-regulation activity

266 than AZD9496 (data not shown). Using a PK/PD model based on PR inhibition data, at the 5

267 mg/kg dose in vivo, which gives 98 % inhibition of PR, the inhibitory activity that could be

268 attributed to the parent compound alone was 85% when the activity of the metabolite was

269 discounted.

270 To explore mechanistic effects on ER pathway regulation further, a gene

271 expression analysis study was performed using tumour samples dosed at 10 mg/kg for 3 days

272 with AZD9496 or tamoxifen and fulvestrant given as a single dose 5 mg dose

273 subcutaneously. Tumours were taken 24 hours after the last dose of AZD9496 and tamoxifen

274 and mRNA levels measured using a Human Transcriptome Array (HTA 2.0). A number of

275 known ER regulated genes were examined to see if AZD9496 could antagonise these genes

276 in a similar manner to Fulvestrant and tamoxifen (40-42). The heap map shows that

277 AZD9496 could indeed down-regulate the mRNA levels of estrogen responsive genes

278 (Supplementary Fig. S6A). As differences in the levels of mRNA down-regulation in vivo

279 could be due to different PK profiles of the molecules, the effects of compounds on the

280 mRNA levels of a subset of estrogen regulated genes were also measured in MCF-7 cells that

281 had been treated with or without estrogen (Supplementary Fig. 6 B-E). For two of these

282 genes, TFF1 and AREG, tamoxifen was shown to increase mRNA levels in the absence of

283 estrogen but not with either fulvestrant or AZD9496 (Supplementary Fig. 6 D, E). Where

284 transcript levels were increased to varying levels in the presence of estrogen, fulvestrant and

285 AZD9496 inhibited transcript levels in a dose dependent fashion compared to tamoxifen. No

286 significant effects were seen in ESR1 mRNA levels in vitro indicating that the protein down-

287 regulation described previously is due to down-regulation of the protein and not a decrease in

288 transcript levels per se (Supplementary Fig. 6F).

289

290 AZD9496 causes tumour regressions in combination with PI3K pathway and CDK4/6

291 inhibitors and in an estrogen deprived ER +ve model of resistance

292 Acquired resistance to aromatase inhibitors and antiestrogens is driven through

293 a variety of mechanisms which include effects on the ER pathway itself i.e. down-regulation

294 of ER expression and altered expression of ER co-regulators, as well as activation of

295 alternative pro-survival or proliferative pathways and cross-talk between growth factor

296 receptors and ER pathways. This has led to a number of clinical trials with PIK3, mTOR,

297 AKT inhibitors, as well as CDK4/6 inhibitors in combination with aromatase inhibitors or

298 fulvestrant and early results have showed increased progression-free survival (PFS) in many

299 cases (43). Combinations of AZD9496 with AZD2014 (dual mTORC1/2), AZD8835

300 (PIK3Cα/δ) and Palbociclib (CDK4/6) inhibitors were tested in the MCF-7 in vivo model and

301 in all cases tumour regressions seen with the combination arms alone compared to tumour

302 stasis with monotherapy (Fig. 5A-C). AZD9496 was also tested in a long-term estrogen

303 deprived model (LTED), using the HCC-1428 LTED cell line that was previously adapted to

304 grow in the absence of estrogen and as such represents a model of aromatase inhibitor

305 resistance (22). Tumour regressions were seen with both AZD9496 and fulvestrant (Fig. 5D)

306 and both compounds completed ablated ER protein levels in the end of study tumours

307 (Supplementary Fig. S7).

308

309 AZD9496 antagonises and down-regulates mutant ER in vitro and in vivo

310 The recent identification of ESR1 mutations in patients that had received one or more

311 endocrine therapy treatments has led to the discovery that these mutations can drive cell

312 proliferation in the absence of estrogen and may constitute a resistance mechanism to some

313 endocrine agents. In vitro binding studies were performed using wt, D538G and Y537S

314 LBDs and both AZD9496 and fulvestrant were able to bind to mutant LBD with nM potency

315 although 2-3 fold reduced compared to wt (Table 1B). The different IC50 values obtained for

316 binding of AZD9496 and fulvestrant to wt ERα compared to the value in Table 1A is likely

317 due to different sources of protein used in the Lanthascreen kit assays versus the laboratory

318 derived wt and ESR1 mutant proteins. Down-regulation of ER mutant proteins was measured

319 using MCF-7 doxycycline inducible stable cell lines in vitro. ER protein levels increased on

320 induction with 1 μg/ml doxycyline and led to increased levels of PR in the absence of

321 estrogen. AZD9496 and fulvestrant were able to down-regulate all mutant ER proteins and

322 decreased PR levels whereas tamoxifen appeared to stabilise ER protein levels and reduced

323 PR levels to a lesser extent (Fig. 6A). To explore activity against an ESR1 mutant in vivo,

324 tumour growth inhibition studies were performed in a patient-derived xenograft (PDX)

325 model, CTC-174, which harbours a D538G mutation in the LBD and constituted 31% of the

326 transcripts variants identified by RNA-seq analysis. Tumours were grown from implanted

327 tumour fragments either with or without estrogen and in both cases viable tumours grew.

328 Given published data that higher doses of SERM/SERDs may be needed to inhibit ESR1

329 mutants (14, 15), 25mg/kg of AZD9496 was used alongside 5mg/mouse of fulvestrant which

330 was known to achieve higher PK levels in mouse than the current clinical dose. AZD9496

331 and fulvestrant inhibited tumour growth by 66% and 59% respectively, compared to

332 tamoxifen which gave 28% inhibition. Efficacy correlated with antagonism of the pathway

333 with AZD9496 giving a 94% decrease in PR levels compared to 63% with fulvestrant (Fig.

334 6B and C). In the absence of estrogen, and given the significant PR changes seen with

335 25mg/kg, the dose of AZD9496 was lowered but a 5 mg/kg dose still achieved growth

336 inhibition of ~70% and led to a significant decrease of 73% in ERα protein levels at the end

337 of study (Fig. 6D and E). Given the similarities in growth inhibition either with or without

338 estrogen, residual tumour growth is likely to be due to either additional mutations (e.g.

339 PIKCA N345K) and/or growth factor signalling pathways involved in driving tumour growth

340 in addition to the ER pathway.

341

342 Discussion

343 Despite the enhanced clinical benefits of directly antagonising and down-regulating ER and

344 thereby targeting any ligand-independent ER driven pathways, the pharmacokinetic and IHC

345 biomarker data for ER, PR and Ki67 from clinical trials with fulvestrant would suggest that a

346 plateau of effect has not been reached, and there is scope to further improve on the 50%

347 reduction in ER levels seen after 6 months of treatment (8, 12). As one of the potential draw-

348 backs associated with fulvestrant is the very low bioavailability which is seen both in patients

349 and in pre-clinical animal models, a potent, orally bioavailable SERD could have the

350 potential to significantly antagonise ER activity and increase ERα receptor knockdown

351 versus fulvestrant due to higher exposures. Our approach focused on identifying novel ER

352 motifs with established drug-like properties that could then be optimized for cellular

353 phenotype and potency and led to the identification of AZD9496 which showed pM potency

354 in vitro in ER+ve cell lines only. Previous structural analysis of a number of SERMs with

355 ERα LBD identified key interactions with Asp 351 which was shown to be critical for the

356 antagonist activity of anti-estrogens such as raloxifene, lasofoxifene and GW5638 through

357 shielding or neutralisation of the negative charge. Unlike tamoxifen however, GW5638

358 repositioned residues Helix-12 through its acrylate side chain, increasing the exposed

359 hydrophobic surface of ERα resulting in destabilisation and ultimately 26S proteosomal

360 degradation of the receptor. The acrylic acid moiety of AZD9496 is co-localized with the Asp 351

361 residue in the helix-12 region of ER, in an unusual acid-acid interaction and it is therefore proposed

362 that AZD9496 also repositions residues associated with the helix-12 via specific contacts with the N-

363 terminus region resulting in a mixed antagonist and down-regulator profile (33, 44). Evidence that

364 AZD9496 was directly binding and down-regulating the receptor at the same site as other

365 anti-estrogens was seen by the reduced potency observed on prior addition of tamoxifen to

366 the down-regulation assay (Table 1A). Mechanistically we have shown that AZD9496 has

367 very similar effects on antagonism, ERα down-regulation and agonism to fulvestrant. Even

368 where ERα was over-expressed in MCF-7 cells, unlike tamoxifen, AZD9496 was able to

369 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.

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

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

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

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.

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604 605

606 607 Tables 608 609 Table 1 A In vitro ERα binding, ERα down-regulation, ERα antagonism and 610 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 11.16 1.91 + 250 nM tamoxifen

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 0.03 611 612 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

624 Figure legends 625 626 627 Figure 1. Crystal structure of the ligand-binding domain of ERα in complex with

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.

635

636 Figure 2. Effect of AZD9496, fulvestrant and tamoxifen on ERα peptide turnover in

637 MCF-7 cells

13 15 638 Cells were grown in steroid-free conditions in SILAC media containing C6 N4 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

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 Figure 4. In vivo efficacy of AZD9496 in MCF-7 xenograft model 663 664 MCF-7 xenografts, grown in male SCID mice, were dosed daily with either PEG/captisol

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.

677 678 679 Figure 5. Tumour regressions with AZD9496 in combination with mTOR, PIKC and 680 CDK4/6 inhibitors in MCF-7 xenograft model and in HCC1428 LTED model. 681 682 MCF-7 xenografts, grown in male SCID mice, were dosed daily with AZD9496 at 5mg/kg

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.

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

705 study were analysed by Western blot for levels of ERα and vinculin and measured as

706 described above * p<0.05 (E)

707

A B

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 A Z D 9 4 9 6 AZD9496 0 .1 0 A Z 1 0 5 5 7 8 4 1

e AZ10557841

s n

o 0 .0 5

e R 0 .0 0

-1 0 -8 -6 -4 C o n c e n tra tio n lo g (M ) -0 .0 5 1 0

x 10 x 20 x 20 x 20 Immature rat: control Fulvestrant AZD9496 Tamoxifen

Figure 3 ERα mediated agonism in MCF-7 cells and immature female rat endometrial tissue.

A B

C D

Figure 4. In vivo efficacy of AZD9496 in MCF-7 xenograft model.

A B

C 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

Empty Vector WT D538G Y537C Y537N Y537S A Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Fulvestrant Fulvestrant Fulvestrant Fulvestrant Fulvestrant Fulvestrant AZD9496 AZD9496 AZD9496 AZD9496 AZD9496 AZD9496 Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen ERa

GAPDH

B C

E D

Figure 6: AZD9496 is efficacious against ESR1 mutants in vitro and in vivo.

AZD9496: An oral estrogen receptor inhibitor that blocks the growth of ER-positive and ESR1 mutant breast tumours in preclinical models.

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

Cancer Res Published OnlineFirst March 28, 2016.

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