azd9496: an oral estrogen receptor inhibitor that blocks...

Therapeutics, Targets, and Chemical Biology

AZD9496: An Oral Estrogen Receptor InhibitorThat Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical ModelsHazel 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,RichardA.Norman2,StuartE.Pearson1,MichaelTonge2,GarethDavies1,GraemeE.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-istered to breast cancer patients by monthly intramuscularinjection. Given its present limitations of dosing and routeof administration, a more flexible orally available compoundhas been sought to pursue the potential benefits of this drugin patients with advanced metastatic disease. Here we reportthe identification and characterization of AZD9496, a nonste-roidal small-molecule inhibitor of ERa, which is a potentand selective antagonist and downregulator of ERa in vitro andin vivo in ER-positive models of breast cancer. Significanttumor growth inhibition was observed as low as 0.5 mg/kgdose in the estrogen-dependent MCF-7 xenograft model, wherethis effect was accompanied by a dose-dependent decrease inPR protein levels, demonstrating potent antagonist activity.

Combining AZD9496 with PI3K pathway and CDK4/6 inhi-bitors led to further growth-inhibitory effects compared withmonotherapy alone. Tumor regressions were also seen in along-term estrogen-deprived breast model, where significantdownregulation of ERa protein was observed. AZD9496bound and downregulated clinically relevant ESR1 mutantsin vitro and inhibited tumor growth in an ESR1-mutantpatient-derived xenograft model that included a D538Gmutation. Collectively, the pharmacologic evidence showedthat AZD9496 is an oral, nonsteroidal, selective estrogenreceptor antagonist and downregulator in ERþ breast cellsthat could provide meaningful benefit to ERþ breast cancerpatients. AZD9496 is currently being evaluated in a phase Iclinical trial. Cancer Res; 76(11); 1–12. �2016 AACR.

IntroductionWith over 70% of breast cancers expressing the estrogen recep-

tor alpha (ERa), treatment with antihormonal therapies thatdirectly antagonize ER function (e.g., tamoxifen) or therapiesthat block the production of the ligand, estrogen (e.g., aromataseinhibitors) are key tomanagement of the disease both in adjuvantand metastatic settings (1, 2). Despite initial efficacy seen withendocrine therapies, the development of acquired resistanceultimately limits the use of these agents although the majorityof tumors continue to require ERa for growth. Through the use ofpreclinical models and cell lines, changes in growth factor recep-

tors have been implicated in driving resistance in cells, for exam-ple, receptor tyrosine kinases (HER2), EGFR, and their down-stream signaling pathways, Ras/Raf/MAPK and PI3K/proteinkinase B (AKT) signaling (3–5). In some instances, signaling fromdifferent growth factor receptor kinases leads to phosphorylationof various proteins in the ER pathway, including ER itself, leadingto ligand-independent activation of the ER pathway (6).

In the metastatic setting, 500 mg fulvestrant, given as 2 � 250mg intramuscular injections, has been shown to offer additionalbenefit over the 250 mg dose with improved median overallsurvival (7, 8). Recent analysis of presurgical data from a numberof trials showed a dose-dependent effect on key biomarkers suchas ER, progesterone receptor (PR), and Ki67 labeling index (9).Although fulvestrant is clearly effective in this later treatmentsetting, there are still perceived limitations in its overall clinicalbenefit due to very low bioavailability, the time taken to achieveplasma steady-state of 3–6months andultimately the level of ERareduction seen in clinical samples (10, 11). It is widely believedthat a potent, orally bioavailable SERD that could achieve highersteady-state free drug levelsmore rapidlywould have thepotentialfor increased receptor knockdown and lead to quicker clinicalresponses, reducing the possibility of early relapses (12). More-over, the recent discovery of ESR1 mutations in metastatic breastcancer patients that had received prior endocrine treatments,where the mutated receptor is active in the absence of estrogen,

1Oncology iMed, AstraZeneca, Alderley Park, Macclesfield, UnitedKingdom. 2Discovery Sciences, AstraZeneca, Alderley Park, Maccles-field, United Kingdom. 3Oncology iMed, AstraZeneca R&D Boston,Gatehouse Drive, Waltham, Massachusetts. 4H3 Biomedicine, Cam-bridge, Massachusetts.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

CorrespondingAuthor:HazelM.Weir, AstraZeneca,Alderley Park,Macclesfield,Cheshire, SK10 4TG, United Kingdom. Phone: 016-2551-3799; Fax: 016-2551-9173; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-2357

�2016 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org OF1

Research. on December 25, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 28, 2016; DOI: 10.1158/0008-5472.CAN-15-2357

http://cancerres.aacrjournals.org/

highlights an important potential resistance mechanism. Preclin-ical in vitro and in vivo studies have shown a reduction in signalingfrom ESR1 mutants by tamoxifen and fulvestrant, but at higherdoses than required to inhibit the wild-type receptor, whichimplies that more potent SERDs may be required to treat thesepatients or to use in the adjuvant setting to limit the emergence ofmutations (13–15).

Achieving oral bioavailability has remained a key challenge inthe design of ER downregulators and until now no oral estrogenreceptor downregulators, other than GDC-0810 and RAD1901,have progressed into clinical trials, despite encouraging reportsfrom others of preclinical activity in mouse xenograft models(16–21). Here, we describe a chemically novel, nonsteroidal ERaantagonist and downregulator, AZD9496, that can be adminis-tered orally and links increased tumor growth inhibition toenhanced biomarker modulation compared with fulvestrant ina preclinical in vivo model of breast cancer. Moreover, we showthat AZD9496 is a potent inhibitor of ESR1-mutant receptors andcan drive tumor growth inhibition in a patient-derived ESR1mutant in vivo model (PDX). Hence, AZD9496 is anticipated toyield improved bioavailability and clinical benefit throughenhanced ER pathway modulation.

Materials and MethodsCell lines and culture

All AZ cell lines were tested and authenticated by short-tandemrepeat (STR) analysis. MCF-7 was obtained from DSMZ and lastSTR tested in February 2013.MDA-MB-361 (STR tested inOctober2011), MDA-MB-134 (STR tested in October 2010), T47D (STRtested in June 2012), CAMA-1 (STR tested in March 2012),HCC1428 (STR tested in December 2011), HCC70 (STR testedinOctober 2013), LnCAP (STR tested in August 2011), MDA-MB-468 (STR tested inMarch 2014), BT474 (STR tested in July 2013),PC3 (STR tested in October 2012), HCT116 (STR tested in August2011) were all obtained from the ATCC.HCC1428-LTED cell line(STR tested in November 2012) was kindly provided by CarlosArteaga (Vanderbilt University, Nashville, TN; ref. 22). Cells werecultured in RPMI1640media (Corning) supplemented with 10%heat-inactivated FCS or charcoal/dextran–treated CCS to depletehormone and2mmol/L L-glutamine (Corning). StableMCF-7 celllines expressing FLAG-tagged ESR1-mutant proteins in pTRIPZlentiviral vectors (GE Healthcare) under the control of the doxy-cycline-inducible Tet promoter were generated by lentiviral infec-tion using standard techniques.

Biochemical and in vitro cell assaysBinding, ER agonism, antagonism, downregulation, and cell

proliferation assays were carried out as described previously (23).BIAcore affinity measurements and immunoblotting from com-pound-treated cells are described in Supplementary Methods.

Protein production, crystallization, and structuredetermination

Protein expression, purification, and crystallization of the ERaligand-binding domain was carried out as described previously(24). X-raydiffractiondatawere collected at the ESRFonbeamlineID23-1 on anADSCdetector. Datawere processed using XDS (25)as implemented within EDNA (26) and scaled and merged usingSCALA (27). The structure of ERa in complex with AZD9496 wassolved by molecular replacement using AmoRE and an internal

ERa structure as the search model (28). Quality checks on theprotein structures were carried out using the validation tools inCoot (29). The final structure has been deposited in the ProteinDatabank with the ID code given in Supplementary Table S1.

Stable isotope labeling of amino acids and mass spectrometryStable isotope labeling of amino acids (SILAC) assays were

carried out as described previously (30) and detailed in Supple-mentary Methods. Compound-treated samples were analyzed bymass spectrometry using relative peptide quantification by select-ed reaction monitoring (SRM). Degradation half-life was mea-sured using the one-phase exponential decay equation in Graph-Pad PRISM (Y ¼ Span.e�K.X þ Plateau), where X is time and Y isresponse, which starts out as Span þ Plateau and decreases toPlateau with a rate constant K.

Rat uterine and xenograft studiesAll studies involving animals in the United Kingdom were

conducted in accordance with UK Home Office legislation, theAnimal Scientific Procedures Act 1986, and AstraZeneca GlobalBioethics policy or with US federal, state, and institutional guide-lines in an Association for Assessment and Accreditation ofLaboratory Animal Care–accredited facility. The rat uterineweightassay for themeasurement of estrogenic activity was performed asdescribed previously (31). Details of individual models, proteinanalysis of tumor fragments, and pharmacokinetic analysis ofplasma samples are included in Supplementary Methods.

ResultsAZD9496 is a selective ERa antagonist, downregulator, andinhibitor of ERþ tumor cell growth

AZD9496 ((E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methyl-propyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; Fig. 1A) was identified through structure-baseddesign and iterative medicinal chemistry structure–activity relation-ship (SAR) studies from a novel ER-binding motif (32). A directedER-binding screen was established to identify novel binding motifsthat could then be optimized for both cellular phenotype andpotency while maintaining good pharmacokinetic properties(23). Unlike fulvestrant, and other well-known historical modula-tors of the ER receptor, including hydroxytamoxifen (Fig. 1B),AZD9496 lacks a phenolic moiety in which the phenol mimics theA-ring phenol groupof the endogenous ligand estradiol but gains itspotency through a different series of novel interactions with the ERprotein (Fig. 1C). The indoleNHpicks up an interaction by forminga hydrogen bond to the carbonyl of Leu-346, with additionallipophilic interactions achieved by both the chiral methyl substit-uent, adjacent to the ring nitrogen, and the N-substituted isopropylfluoro side-chain occupying a "lipophilic hole" in the ER protein.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 beenproposed to be crucial for achieving a downregulator–antagonistprofile (33). AZD9496 showedhigh oral bioavailability across threespecies (F% 63, 91, and 74, rat, mouse, and dog, respectively) withgenerally low volume and clearance across species, albeit a higherclearance in mouse. The percent free levels in human plasma of0.15% were 5-fold higher than those measured for fulvestrant.

The key characteristics of a SERD is the ability to bothantagonize and downregulate ER protein without inducing anypartial agonist effects in breast cells. A series of in vitro cell

Weir et al.

Cancer Res; 76(11) June 1, 2016 Cancer ResearchOF2




assays were developed specifically to measure ERa downregu-lation, agonism, and antagonism through simultaneous mea-surement of ER and PR protein levels in cells after treatmentwith AZD9496 (23). PR expression is known to be regulated byER transcription and was measured as a marker of ER activationin cells (34). The potency of AZD9496 approached thatobserved with fulvestrant in achieving pmol/L IC50 in ERabinding, downregulation, and antagonism and significant cellgrowth inhibition in hormone-depleted growth conditions.Adding 250 nmol/L tamoxifen significantly lowered the IC50

value, as expected, if AZD9496 and tamoxifen are competingfor binding to the ligand-binding domain (LBD; Table 1). Thepotent downregulation of ERa was also verified by immuno-blotting techniques (Supplementary Fig. S1).

To understand the binding kinetics of AZD9496 to human ERaLBD, binding studieswere performed in the presence of increasingconcentrations of AZD9496 and association (kass) and dissocia-tion (kdiss) rate constants were calculated. AZD9496 was calcu-

lated as having an apparent dissociation rate constant (kdiss) forERa LBD of 0.00043/sec, at 25�C equating to a half-life (t1/2) of27 � 2 minutes and the affinity derived from the ratio of therate constants gave a Kd of 0.33 nmol/L (SupplementaryTable S2). This is in good agreement with the binding IC50 of0.82 nmol/L and correlates well with previously published dataon estrogen and tamoxifen binding to ER LBD (35). AZD9496showed pmol/L equipotent binding to both ERa and ERbisoforms, as expected, given their high sequence homologyand similarity in tertiary structure in their LBDs (36), and highlyselective binding compared with progesterone (�650-fold),glucocorticoid (�11,223-fold), and androgen (�36,375-fold)receptor LBDs (Supplementary Table S3). Selectivity for ER wasalso evident bymeasuring cell growth inhibition in a panel of ERþ

and ER� cell lines (Supplementary Table S4). No significantgrowth inhibition was seen in any ER� cell lines (EC50 >10 mmol/L) in contrast to the low nanomolar activity seen in arange of ERþ cell lines.

Figure 1.Crystal structure of the LBD of ERa incomplex with AZD9496. A, two-dimensional chemical representationof AZD9496. B, two-dimensionalchemical representation of 4-hydroxytamoxifen. C, three-dimensionalstructure of the complex betweenAZD9496 and the LBD of ERa. Theprotein and ligand structures arerepresented as sticks with carbon andfluorine atoms colored gray and lightblue, respectively. The van der Waalsradii of the protein atoms are shownasa continuous surface highlighting theligand-binding pocket of the ERa. Keyprotein residues are labeled and polarinteractions are shown as dotted lines.

Pharmacology of SERD Inhibitor in Breast Cancer Models

www.aacrjournals.org Cancer Res; 76(11) June 1, 2016 OF3




AZD9496 directly targets ERa for downregulation in vitroTo measure the rate at which AZD9496 downregulated ERa,

SILAC experiments were performed in MCF-7 cells using massspectroscopy analysis to detect ERa protein levels after treat-ment with compounds. Addition of AZD9496 or fulvestrantincreased the rate of degradation of the isotope-labeled ERacompared with DMSO control. Levels of newly synthesized ERapeptide were also reduced following AZD9496 and fulvestranttreatment, presumably due to ongoing degradation of newlysynthesized ERa protein by compound present, whereas ERaprotein levels continued to increase over time in the presence oftamoxifen or DMSO (Fig. 2). The t1/2 of ERa was decreasedfrom 3 hours, in the presence of DMSO, to 0.75 hours with 100nmol/L AZD9496 and 0.6 hours with 100 nmol/L fulvestrant(Supplementary Fig. S2). Downregulation of ER occurs throughthe 26S proteosomal pathway as no decrease in ERa proteinlevel in MCF-7 cells was seen in the presence of the proteosomeinhibitor MG132 (Supplementary Fig. S3). In addition, ERadownregulation was shown to be reversible in compoundwash-out experiments, where ERa levels increased in a time-dependent manner back to basal levels over a 48-hour periodfollowing compound removal (Supplementary Fig. S4).

Agonism of ERa in human breast and uterine cellsIn contrast to an AstraZeneca reference compound

(AZ10557841), which has known ERa agonist activity in breastcells, AZD9496was shown to causeno increase inPRprotein levelsbut rather a slight decrease below the detected basal levels(Fig. 3A). Some endocrine therapies, such as tamoxifen, can actas partial ER agonists in certain tissues, for example, endometrium,due to conformational changes that take place at the ER and theresulting effect on cofactor recruitment and transcriptional geneactivation from the tamoxifen-bound receptor (37). To testwheth-er AZD9496 could act as a partial agonist in other tissues, agonisteffects were measured in vivo in a previously validated immaturefemale rat model designed to detect agonistic properties of testcompounds by measuring increases in uterine weight (31).AZD9496, given once daily orally at 5 and 25 mg/kg producedstatistically significant increases in uterine weight compared withthe fulvestrant control (P < 0.001) but significantly lower thantamoxifen (P¼0.001; Fig. 3B).Histologic staining ofuterine tissuesamples also showed that the lengthening of the endometrial cellsin the rat uteri appeared to have decreased compared with tamox-

ifen (Fig. 3C). In further studies, ERa protein levels were reducedwith fulvestrant and AZD9496, but not tamoxifen, comparedwithrelevant vehicle controls, whereas PR levels were significantlyincreased with tamoxifen alone (Fig. 3D).

AZD9496 is a potent, oral inhibitor of breast tumor growthin vivo

The effect of chronic, oral dosing of AZD9496 was explored inMCF-7 human breast xenografts, as a representative ERþ/PRþ/HER2þ breast cancer model. Good bioavailability and high clear-ance gave a terminal t1/2 of 5–6hours after oral dosing in themouseand resulted in significant dose-dependent tumor growth inhibi-tionwith 96% inhibition at 50mg/kg andno toxicityorweight lossrelative to the vehicle control group (Fig. 4A). To confirm thatAZD9496 was targeting the ER pathway, PR protein levels weremeasured in tumor samples taken at the end of the study and asignificant reduction in PR was seen, which correlated with tumorgrowth inhibition. A >90% reduction in PR was seen with both 10and 50 mg/kg doses and a 75% decrease even with the 0.5 mg/kgdose, demonstrating that AZD9496 can clearly antagonize the ERpathway (Fig. 4B). Dosing 5mg/kg of AZD9496, theminimal doserequired to see significant tumor inhibition, gave greater tumorgrowth inhibition compared with 5 mg/mouse fulvestrant given 3times weekly and tamoxifen given 10mg/kg orally, daily (Fig. 4C).A series of pharmacodynamic in vivo studies were conducted tomeasure time taken to reach maximal inhibition of PR levels andtime taken to recover back to basal levels. Three days of dosingwith5 mg/kg of AZD9496 gave 98% reduction of PR protein andcontinued to suppress protein levels at 48 hours (Fig. 4D), withfull recovery by 72 hours (data not shown), which indicates a longpharmacologic half-life in vivo. Fulvestrant, given as 3 � 5 mg/mouse doses over one week, gave a 60% reduction in PR proteinover a prolonged period with measured plasma levels approxi-mately 8-fold higher than those achieved clinically, at steadystate, with 500 mg fulvestrant (Fig. 4E). As estrogen itself isknown to downregulate ER protein (38, 39), we were unable todetect further decreases in ERa protein compared with controlanimals with AZD9496 or fulvestrant in the MCF-7 modelpresumably due to the high circulating plasma levels of estro-gen from implanted pellets at the time tumor samples weretaken (Supplementary Fig. S5). A mouse-specific metabolite ofAZD9496 was detected in circulating plasma at similar levels toAZD9496 and showed a similar pharmacokinetic profile. Test-ing this metabolite in the in vitro MCF-7 assays resulted inapproximately 5-fold lower ERa antagonism activity and 7-foldlower ERa downregulation activity than AZD9496 (data notshown). Using a pharmacokinetic/pharmacodynamic modelbased on PR inhibition data, at the 5 mg/kg dose in vivo, whichgives 98% inhibition of PR, the inhibitory activity that could beattributed to the parent compound alone was 85% when theactivity of the metabolite was discounted.

To explore mechanistic effects on ER pathway regulation fur-ther, a gene expression analysis study was performed using tumorsamples dosed at 10mg/kg for 3 dayswith AZD9496or tamoxifenand fulvestrant given as a single dose 5 mg dose subcutaneously.Tumors were taken 24 hours after the last dose of AZD9496 andtamoxifen and mRNA levels measured using a Human Transcrip-tome Array (HTA 2.0). A number of known ER-regulated geneswere examined to see whether AZD9496 could antagonize thesegenes in a similar manner to fulvestrant and tamoxifen (40–42).The heatmap shows that AZD9496 could indeed downregulate

Table 1. In vitroERabinding, ERa downregulation, ERa antagonism, andMCF-7cell growth inhibition data for AZD9496 versus fulvestrant

AZD9496 Fulvestrant

ERa binding IC50 nmol/L 0.82 0.8ERa downregulation IC50 nmol/L 0.14 0.06ERa downregulation IC50 nmol/Lþ 250 nmol/L tamoxifen

11.16 1.91

ERa antagonism IC50 nmol/L 0.28 0.21MCF-7 cell growth inhibitionEC50 nmol/L

0.04 0.1

LogD 2.9 Not measureableSolubility mmol/L 110 (crystalline) <0.9Human % free 0.15 0.03

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,antagonism, and inhibition of cell proliferation as described in ref. 20 using afluorescence endpoint to measure of ER and PR protein levels; data shown arerepresentative of the mean IC50 or EC50 from at least four independentexperiments.

Weir et al.





the mRNA levels of estrogen-responsive genes (SupplementaryFig. S6A). As differences in the levels of mRNA downregulation invivo could be due to different pharmacokinetic profiles of themolecules, the effects of compounds on the mRNA levels of asubset of estrogen-regulated genes were also measured in MCF-7cells that had been treated with or without estrogen (Supplemen-tary Fig. S6B–S6E). For two of these genes, TFF1 and AREG,tamoxifen was shown to increase mRNA levels in the absence ofestrogen but not with either fulvestrant or AZD9496 (Supple-mentary Fig. S6D and S6E).Where transcript levels were increasedto varying levels in the presence of estrogen, fulvestrant andAZD9496 inhibited transcript levels in a dose-dependent fashioncompared with tamoxifen. No significant effects were seen inESR1 mRNA levels in vitro, indicating that the protein down-regulation described previously is due to downregulation of theprotein and not a decrease in transcript levels per se (Supplemen-tary Fig. S6F).

AZD9496 causes tumor regressions in combination with PI3Kpathway and CDK4/6 inhibitors and in an estrogen-deprivedERþ model of resistance

Acquired resistance to aromatase inhibitors and antiestrogensis driven through a variety of mechanisms, which include effectson the ER pathway itself, i.e., downregulation of ER expressionand altered expression of ER coregulators, as well as activation ofalternative prosurvival or proliferative pathways and cross-talkbetween growth factor receptors and ER pathways. This has led toa number of clinical trials with PIK3, mTOR, AKT inhibitors, aswell as CDK4/6 inhibitors in combination with aromatase inhi-bitors or fulvestrant and early results have shown increasedprogression-free survival (PFS) inmany cases (43). Combinationsof AZD9496 with AZD2014 (dual mTORC1/2), AZD8835(PIK3Ca/d) and palbociclib (CDK4/6) inhibitors were tested in

the MCF-7 in vivo model and in all cases tumor regressions seenwith the combination arms alone compared with tumor stasiswith monotherapy (Fig. 5A–C). AZD9496 was also tested in along-term estrogen-deprivedmodel (LTED), using theHCC-1428LTED cell line that was previously adapted to grow in the absenceof estrogen and as such represents amodel of aromatase inhibitorresistance (22). Tumor regressions were seen with both AZD9496and fulvestrant (Fig. 5D) and both compounds completed ablat-ed ER protein levels in the end-of-study tumors (SupplementaryFig. S7).

AZD9496 antagonizes and downregulates mutant ER in vitroand in vivo

The recent identification of ESR1mutations in patients that hadreceived one or more endocrine therapy treatments has led to thediscovery that these mutations can drive cell proliferation in theabsence of estrogen andmay constitute a resistancemechanism tosome endocrine agents. In vitro binding studies were performedusing wt, D538G, and Y537S LBDs and both AZD9496 andfulvestrant were able to bind tomutant LBDwith nmol/L potencyalthough 2- to 3-fold reduced compared with wt (Table 2). Thedifferent IC50 values obtained for binding of AZD9496 andfulvestrant to wt ERa compared with the value in Table 1 is

Figure 2.Effect of AZD9496, fulvestrant, andtamoxifen on ERa peptide turnover inMCF-7 cells. Cells were grown insteroid-free conditions in SILACmedia containing 13C6

15N4 L-arginineto label ERa peptide as "heavy" (blueline) and then switched to grow inmedia containing unlabeled L-arginineto label newly synthesized protein as"normal" (red line) with 0.1% DMSO(A), 300 nmol/L tamoxifen (B), 100nmol/L AZD9496 (C), or 100 nmol/Lfulvestrant (D) for the time indicated.Data shown is representative of twoindependent experiments.

Table 2. In vitro binding to ERa mutant LBDs

ERa LBD binding IC50 nmol/LAZD9496 nmol/L Fulvestrant nmol/L

wt 0.2 1.6D538G 0.5 3.3Y537S 0.6 3.8

NOTE: IC50s for binding ofAZD9496and fulvestrant toERawtandmutant LBDs;data shown are representative of the mean IC50 from three independentexperiments.






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 compoundAZ10557841 and PR levels (response) measured using in-cell immunofluorescence quantification using an Acumen Ex3 platform. Data was normalized forcell number by analyzing a ratio of fluorescence resulting from stimulation of two emission wavelengths and used to perform curve fitting analysis. Aconcentration response was plotted using nonlinear regression analysis. B and C, immature female rats were dosed orally with AZD9496 or tamoxifen orsubcutaneously 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 allowhistologic staining of the endometrial tissue. Significant differences in mean uterine weight are shown: �� , P < 0.001; �� , P < 0.01. D, tissue was subsequentlyanalyzed byWestern blot analysis for levels of ERa, PR, and vinculin. Protein levels weremeasured by chemiluminescent and quantified using Syngene software. ERand 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.

Weir et al.





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) orAZD9496 at doses shown. Tumor growth was measured by caliper at regular intervals and mean tumor volumes plotted for each dosed group. B, tumors fromtreated mice collected at the end of study were analyzed byWestern blot analysis for levels of PR and vinculin. Protein levels were measured by chemiluminescentand 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 xenograftswere also dosed dailywith 5mg/kgAZD9496, 10mg/kg tamoxifen, or 5mg/mouse three timesweeklywith fulvestrantsubcutaneously. 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 or48hours after the last dose (D) or dosedwith three doses of 5mg/mouseof fulvestrant over 5daysand tumors collected at 24, 48, and96hours 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.






likely due to different sources of protein used in the Lanthascreenkit assays versus the laboratory-derived wt and ESR1-mutantproteins. Downregulation of ER-mutant proteins was measuredusing MCF-7 doxycycline-inducible stable cell lines in vitro. ERprotein levels increased on induction with 1 mg/mL doxycyclineand led to increased levels of PR in the absence of estrogen.AZD9496 and fulvestrant were able to downregulate all mutantER proteins and decreased PR levels, whereas tamoxifen appearedto stabilize ER protein levels and reduced PR levels to a lesserextent (Fig. 6A). To explore activity against an ESR1mutant in vivo,tumor growth inhibition studies were performed in a PDXmodel, CTC-174, which harbors a D538G mutation in the LBDand constituted 31%of the transcripts variants identified by RNA-seq analysis. Tumors were grown from implanted tumor frag-ments either with or without estrogen and in both cases viabletumors grew. Given published data that higher doses of SERM/SERDsmay be needed to inhibit ESR1mutants (14, 15), 25mg/kg

of AZD9496 was used alongside 5 mg/mouse of fulvestrant,which was known to achieve higher pharmacokinetic levels inmouse than the current clinical dose. AZD9496 and fulvestrantinhibited tumor growth by 66% and 59% respectively, comparedwith tamoxifen, which gave 28% inhibition. Efficacy correlatedwith antagonism of the pathway with AZD9496 giving a 94%decrease in PR levels comparedwith 63%with fulvestrant (Fig. 6Band C). In the absence of estrogen, and given the significant PRchanges seen with 25 mg/kg, the dose of AZD9496 was loweredbut a 5 mg/kg dose still achieved growth inhibition of approx-imately 70% and led to a significant decrease of 73% in ERaprotein levels at the end of the study (Fig. 6D and E). Given thesimilarities in growth inhibition either with or without estrogen,residual tumor growth is likely to be due to either additionalmutations (e.g., PIKCA N345K) and/or growth factor signalingpathways involved in driving tumor growth in addition to the ERpathway.

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-7xenografts, 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 daysout of 7 (A),with orwithoutAZD8835 dosed at 50mg/kg twice daily on day 1 and4 (B), andwith orwithout palbociclib dosed daily at 50mg/kg (C). D, HCC1428LTEDengrafts were grown in ovariectomized NSG mice and were dosed daily with either AZD9496 or once weekly with fulvestrant at doses shown. Tumor growthwas measured by caliper at regular intervals and mean tumor volumes plotted for each dosed group. p.o., orally; q.d., once daily.

Weir et al.





Figure 6.AZD9496 is efficacious against ESR1mutants in vitro and in vivo. A, stable MCF-7 cell lines containing either pTRIPZ vector alone or vector containing ESR1mutantswere 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 offulvestrant, AZD9496, or tamoxifen for 24 hours. Immunoblotting was performed to detect expression of ERa, PR, and GAPDH levels. B, CTC-174 tumor fragmentswere grown in female NSG mice implanted with estrogen pellets and dosed daily with either PEG/captisol (vehicle) or AZD9496, tamoxifen, and fulvestrantat doses shown. Tumor growth was measured by caliper at regular intervals and mean tumor volumes plotted for each dosed group. C, tumors from treated micecollected at the end of study were analyzed by Western blot analysis for levels of PR and vinculin. Protein levels were measured by chemiluminescent andquantified using Syngene software. PR protein levels were normalized to vinculin as a loading control and plotted as shown. �� , P < 0.001. D, CTC-174tumor 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 treatedmice collected at the endof studywere analyzed byWestern blot analysis for levels of ERa and vinculinand measured as described above. � , P < 0.05. p.o., orally; q.d., once daily.






DiscussionDespite the enhanced clinical benefits of directly antagonizing

and downregulating ER and thereby targeting any ligand-inde-pendent ER-driven pathways, the pharmacokinetic and IHC bio-marker data for ER, PR, and Ki67 from clinical trials with fulves-trant would suggest that a plateau of effect has not been reached,and there is scope to further improve on the 50% reduction in ERlevels seen after 6 months of treatment (8, 12). As one of thepotential drawbacks associated with fulvestrant is the very lowbioavailability, which is seen both in patients and in preclinicalanimalmodels, a potent, orally bioavailable SERD could have thepotential to significantly antagonize ER activity and increase ERareceptor knockdown versus fulvestrant due to higher exposures.Our approach focused on identifying novel ER motifs withestablished drug-like properties that could then be optimized forcellular phenotype and potency and led to the identification ofAZD9496, which showed picomolar potency in vitro in ERþ celllines only. Previous structural analysis of a number of SERMswithERa LBD identified key interactions with Asp 351, which wasshown to be critical for the antagonist activity of antiestrogenssuch as raloxifene, lasofoxifene, and GW5638 through shieldingor neutralization of the negative charge. Unlike tamoxifen, how-ever, GW5638 repositioned residues helix-12 through its acrylateside chain, increasing the exposed hydrophobic surface of ERa,resulting in destabilization and ultimately 26S proteosomal deg-radation of the receptor. The acrylic acid moiety of AZD9496 iscolocalized with the Asp 351 residue in the helix-12 region of ERin an unusual acid–acid interaction, and it is therefore proposedthat AZD9496 also repositions residues associated with the helix-12 via specific contacts with the N-terminus region, resulting in amixed antagonist and downregulator profile (33, 44). Evidencethat AZD9496 was directly binding and downregulating thereceptor at the same site as other antiestrogens was seen by thereduced potency observed on prior addition of tamoxifen to thedownregulation assay (Table 1). Mechanistically, we have shownthat AZD9496 has very similar effects on antagonism, ERa down-regulation, and agonism to fulvestrant. Even where ERa wasoverexpressed in MCF-7 cells, unlike tamoxifen, AZD9496 wasable to downregulate and antagonize the overexpressed receptor(Fig. 6A). Our transcriptional studies in both MCF-7 cells and thein vivomodel also demonstrated antagonism of the transcription-al activity of the receptor as seen by decreased mRNA levels of ER-activated genes in a dose-dependentmanner (Supplementary Fig.S6).

Unlike fulvestrant, AZD9496did show somedegree of agonismin endometrial tissue (Fig. 3). This effect was dose independentand would indicate that there are subtle differences in howAZD9496 interacts with ERa compared with tamoxifen andfulvestrant and therefore the extent to which tissue specific coac-tivators can interactwith the receptor. Given thewidespread use oftamoxifen in the adjuvant setting for 5–10 years, the low level ofagonism seen with AZD9496 is not viewed as a deterrent forclinical development. A number of studies have looked at theincidence of endometrial cancer in patients taking tamoxifen overa number of years and despite the small increase in the incidenceof endometrial cancer, the benefits of tamoxifen greatly outweighany risks of developing uterine malignancy in postmenopausalpatients (45, 46).

A pharmacokinetic/pharmacodynamic efficacy model wasestablished, integrating diverse endpoints such as drug exposure,

PR modulation, and inhibition of tumor growth (data notshown). The model reflected relative contributions of AZD9496and its active mouse metabolite to the overall biomarker mod-ulation and efficacy. Given the half-life of the parent drug andmetabolite are around 5 hours, to explain the prolonged phar-macodynamic effect observed with AZD9496 at 48 hours, anindirect responsemodel for PR was developed, which estimated adegradation rate constant of around 23 hours for PR. This mod-ulation in PR was linked directly to tumor growth inhibition by aproportional decrease of the tumor growth constant, and corre-lated well with the wide range of measured efficacies.

We consistently saw enhanced tumor growth inhibition ofAZD9496 compared with fulvestrant in the MCF-7 in vivo model(Fig. 4). This model contains high circulating levels of plasmaestradiol over the dosing period of the study as a result of theestrogen pellets used to drive growth of the MCF-7 cells as axenograft. With plasma levels between 750 and 1,000 pg/mL overday 21–35, this model is more representative of the premeno-pausal setting where estrogen levels can vary between 20 and 400pg/mL (47). As the receptor binding data does not suggest asignificant difference in affinity for the receptor betweenAZD9496 and fulvestrant, increased efficacy is likely due to thehigher free drug levels of AZD9496 versus fulvestrant in thismodel. It would therefore be interesting to test the activity ofpotent oral SERDs, such as AZD9496, in the premenopausalsetting where there is no enhanced risk of endometrial cancerobserved for tamoxifen.

We were unable to detect a reduction in ER protein levels in theMCF-7 in vivo model with AZD9496 at any dose tested (data notshown). Having shown in vitro that estradiol can efficiently down-regulate ERa protein, we postulate that downregulation of ERabyestradiol is already taking place in vivo given the high levels ofcirculating estrogen and that any additional changes by additionof a SERD are difficult to detect using the current methods. Thiswas supported by measuring ERa downregulation in in vivomodels, such as CTC-174 and HCC1428LTED, not requiringestrogen supplements, and showing potent downregulation ofERa by AZD9496 (Fig. 6E, Supplementary Fig. S7). The level ofERa reduction seen in the HCC1428 model is greater than seenclinically, with pharmacokinetic levels 5-fold higher than steady-state plasma levels of 500 mg fulvestrant, and supports theconcept that if increased plasma levels of an active SERD agentcould be achieved clinically, greater ERa knockdown may bepossible. Preclinically, this appears to translate to enhancedtumor growth inhibition.

Achieving increased serum levels of a biologically active SERDcould be particularly beneficial in the recently identified ESR1-mutant patient setting. Preclinical in vivo studies using PDXmodels derived from ESR1-mutant patients grown in the absenceof estrogen supplementation have shown that high doses offulvestrant (>500 mg/kg) can lead to tumor regression andsignificant decreases in ERprotein levels but at significantly higherdoses than could be achieved clinically (48). Our preclinical datawould indicate that both AZD9496 and fulvestrant can potentlybind and downregulate D538G and Y537C/N/S ERa proteins invitro and significant tumor growth inhibition rather than regres-sion seen in vivo. This is in contrast to published work showingrelatively little downregulation of Y537N mutant protein in vitrowith increasing doses of fulvestrant (15)but could be attributed todifferent expression systems and cell lines used for the analysis. Itis notable that the doxycycline-inducible mutant receptors do not

Weir et al.





appear to be constitutively downregulated in a similar manner towt ER exposed to estradiol (Supplementary Fig. S1). This may bedue to the level of overexpression of the receptor in each stable cellline and it will be interesting to compare these findings fromengineered expression systems to those from genetically alteredmutant cell lines where expression is under control of the nativepromoter. Although very high doses of both compoundswere nottested in the PDX CTC-174 ESR1-mutant model, the lack of anysignificant dose response might indicate the need to inhibit othergrowth factor–activated pathways in addition to the ER pathwayto achieve significant antitumor growth. As trials commence toexplore treatment of ESR1-mutant patients with new oral SERDs,data will hopefully emerge that will indicate whether lack ofresponse is due to inability to fully inhibit mutated ER receptorsorwhether, in these advancedmetastatic patients, othermutationsand pathways are or become dominant in driving tumor growth.

In metastatic breast cancer, the median survival time is stillaround two years as a result of acquired endocrine resistance,highlighting the need for additional therapies (43). This has led toa number of clinical trials using PI3K–Akt–mTOR pathway inhi-bitors in combination with AIs as well as cyclin-dependent kinaseinhibitors such as palbociclib, which was recently approved forcombination treatment with letrozole in the metastatic breastcancer. Results from the phase III BOLERO-2 trial also demon-strated the PFS benefits of a steroidal AI plus everolimus inpatients that had progressed on a nonsteroidal AI (49). Morerecently, further trials have been initiated using fulvestrant as thecombination endocrine partner, which will allow analysis andunderstanding of any additional benefit from combinationwith aSERD versus AIs in this setting. Our data clearly demonstratedenhanced combination effects on tumor growth of an oral SERDagentwith either dualmTORC1/2, PIKCa/d orCDK4/6 inhibitorsand furthermore opens up the possibility of using a combinedendocrine and targeted inhibitor approach with longer termdosing in the adjuvant setting, assuming emerging data fromongoing metastatic trials supports the testing of combinations inearly breast disease. The identification of a potent, orally bio-available compound suchasAZD9496 is a step forward in thenextgeneration of SERD agents to undergo clinical evaluation as afuture monotherapy or combination partner of choice.

Disclosure of Potential Conflicts of InterestJ.O. Curwen is an associate principal scientist at AstraZeneca. G. Davies has

ownership interest (including patents) in AstraZeneca as a shareholder.S. Powell is a team leader at Astrazeneca. G. Richmond has ownershipinterest (including patents) in AstraZeneca. All authors are current or formerAstraZeneca employees and shareholders.

Authors' ContributionsConception and design: H.M. Weir, R.H. Bradbury, M. Lawson, A.A. Rabow,J.O. Curwen, R.A. Norman, G.E. Walker, C. Sadler, G. Richmond, C. De SaviDevelopment of methodology: H.M. Weir, R.H. Bradbury, M. Lawson,C.S. Donald, L.J.L Feron, T. Moss, S.E. Pearson, G. Davies, G.E. Walker,S. Powell, C. Sadler, B. LaddAcquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): H.M. Weir, M. Lawson, R.J. Callis, M. Hulse, C.S. Donald,P.MacFaul, T.Moss, R.A.Norman, S.E. Pearson,M. Tonge, G.Davies, G.E.Walker,Z. Wilson, R. Rowlinson, B. Ladd, E. Pazolli, A.M. MazzolaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.M. Weir, M. Lawson, A.A. Rabow, D. Buttar,R.J. Callis, J.O. Curwen, C. de Almeida, P. Ballard, M. Hulse, P. MacFaul,R.A. Norman, M. Tonge, G. Davies, G.E. Walker, Z. Wilson, R. Rowlinson,S. Powell, G. Richmond, A.M. Mazzola, C.M. D'CruzWriting, review, and/or revision of the manuscript: H.M. Weir, M. Lawson,D. Buttar, P. Ballard, C.S. Donald, R.A. Norman, S.E. Pearson, M. Tonge,G. Davies, G.E. Walker, C. Sadler, G. Richmond, B. Ladd, A.M. Mazzola,C.M. D'Cruz, C. De SaviAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):H.M. Weir, M. Lawson, P. Ballard, S.E. Pearson,S. Powell, A.M. MazzolaStudy supervision: H.M. Weir, J.O. Curwen, R.A. Norman, G.E. Walker,C.M. D'CruzOther (chemistry route development): G. Karoutchi

AcknowledgmentsThe authors thank Elaine Hurt, Haifeng Bao, Sanjoo Jalla for access to the

CTC-174 in vivomodel developed at MedImmune.The authors also thank PaulHemsley and Emma Linney for providing ER-mutant binding assay data, HelenGingell for supporting the crystallography work and Zara Ghazoui and HenryBrown for the transcript data and analysis at AstraZeneca. The authors also thankCarlos Arteaga for use of the HCC1428-LTED cell line.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 28, 2015; revised February 1, 2016; acceptedMarch 1, 2016;published OnlineFirst March 28, 2016.

References1. Early Breast Cancer Trialists' Collaborative Group (EBCTCG), Davies C,

Godwin J, Gray R, Clarke M, Cutter D, et al. Relevance of breast cancerhormone receptors and other factors to the efficacy of adjuvant tamox-ifen: patient-level meta-analysis of randomised trials. Lancet 2011;378:771–84.

2. Cuzick J, Sestak I, BaumM, Buzdar A, Howell A, Dowsett M, et al. Effect ofanastrozole and tamoxifen as adjuvant treatment for early-stage breastcancer: 10-year analysis of the ATAC trial. Lancet Oncol 2010;11:1135–41.

3. NicholsonRI, Hutcheson IR, JonesHE,Hiscox SE, GilesM, Taylor KM, et al.Growth factor signalling in endocrine and anti-growth factor resistantbreast cancer. Rev Endocr Metab Disord 2007;8:241–53.

4. Lupien M, Meyer CA, Bailey ST, Eeckhoute J, Cook J, Westerling T, et al.Growth factor stimulation induces a distinct ER(alpha) cistrome underly-ing breast cancer endocrine resistance. Genes Dev 2010;24:2219–27.

5. Massarweh S, Schiff R. Resistance to endocrine therapy in breast cancer:exploiting estrogen receptor/growth factor signaling crosstalk. Endocr RelatCancer 2006;13Suppl 1:S15–24.

6. Ali S, Coombes RC. Endocrine-responsive breast cancer and strategies forcombating resistance. Nat Rev Cancer 2002;2:101–12.

7. Di Leo A, JerusalemG, Petruzelka L, Torres R, Bondarenko IN, Khasanov R,et al. Final overall survival: fulvestrant 500mgvs 250mg in the randomizedCONFIRM trial. J Natl Cancer Inst 2014;106:337.

8. Kuter I, Gee JM, Hegg R, Singer CF, Badwe RA, Lowe ES, et al. Dose-dependent change in biomarkers during neoadjuvant endocrine therapywith fulvestrant: results fromNEWEST, a randomized Phase II study. BreastCancer Res Treat 2012;133:237–46.

9. Robertson JF, Lindemann J, Garnett S, Anderson E, Nicholson RI, Kuter I,et al. A good drug made better: the fulvestrant dose-response story. ClinBreast Cancer 2014;14:381–9.

10. Robertson JF, Harrison M. Fulvestrant: pharmacokinetics and pharmacol-ogy. Br J Cancer 2004;90Suppl 1:S7–10.

11. Howell A, Sapunar F. Fulvestrant revisited: efficacy and safety of the500-mgdose. Clin Breast Cancer 2011;11:204–10.

12. Robertson JF. Fulvestrant (Faslodex) – how to make a good drug better.Oncologist 2007;12:774–84.

13. Li S, Shen D, Shao J, Crowder R, Liu W, Prat A, et al. Endocrine-therapy-resistant ESR1 variants revealed by genomic characterization of breast-cancer-derived xenografts. Cell Rep 2013;4:1116–30.






14. ToyW, Shen Y,WonH,GreenB, Sakr RA,WillM, et al. ESR1 ligand-bindingdomain mutations in hormone-resistant breast cancer. Nat Genet 2013;45:1439–45.

15. Jeselsohn R, Yelensky R, Buchwalter G, Frampton G, Meric-Bernstam F,Gonzalez-Angulo AM, et al. Emergence of constitutively active estrogenreceptor-alpha mutations in pretreated advanced estrogen receptor-posi-tive breast cancer. Clin Cancer Res 2014;20:1757–67.

16. Willson TM, Henke BR, Momtahen TM, Charifson PS, Batchelor KW,Lubahn DB, et al. 3-[4-(1,2-Diphenylbut-1-enyl)phenyl]acrylic acid: anon-steroidal estrogen with functional selectivity for bone over uterus inrats. J Med Chem 1994;37:1550–2.

17. Hoffmann J, Bohlmann R, Heinrich N, Hofmeister H, Kroll J, Kunzer H,et al. Characterization of new estrogen receptor destabilizing compounds:effects on estrogen-sensitive and tamoxifen-resistant breast cancer. J NatlCancer Inst 2004;96:210–8.

18. Tan Q, Blizzard TA, Morgan JD, Birzin ET, ChanW, Yang YT, et al. Estrogenreceptor ligands. Part 10: Chromanes: old scaffolds for new SERAMs.Bioorg Med Chem Lett 2005;15:1675–81.

19. Suzuki N, Liu X, Laxmi YR, Okamoto K, KimHJ, Zhang G, et al. Anti-breastcancer potential of SS5020, a novel benzopyran antiestrogen. Int J Cancer2011;128:974–82.

20. Lai A, Kahraman M, Govek S, Nagasawa J, Bonnefous C, Julien J, et al.Identification of GDC-0810 (ARN-810), an orally bioavailable SelectiveEstrogen Receptor Degrader (SERD) that demonstrates robust activity intamoxifen-resistant breast cancer xenografts. J Med Chem 2015;58:4888–904.

21. Garner F, Shomali M, Paquin D, Lyttle R, Hattersley G. RAD1901: a novel,orally bioavailable selective estrogen receptor degrader that demonstratesantitumour activity in breast cancer xenograft models. Anti-Cancer Drugs2015;26:948–956.

22. Miller TW, Balko JM, Ghazoui Z, Dunbier A, Anderson H, Dowsett M, et al.A gene expression signature from human breast cancer cells with acquiredhormone independence identifies MYC as a mediator of antiestrogenresistance. Clin Cancer Res 2011;17:2024–34.

23. Callis R, Rabow A, Tonge M, Bradbury R, Challinor M, Roberts K, et al. Ascreening assay cascade to identify and characterize novel selective estrogenreceptor downregulators (SERDs). J Biomol Screen 2015;20:748–59.

24. Degorce SL, Bailey A, Callis R, De Savi C, Ducray R, Lamont G, et al.Investigation of (E)-3-[4-(2-Oxo-3-aryl-chromen-4-yl)oxyphenyl]acrylicacids as oral selective estrogen receptor down-regulators. J Med Chem2015;58:3522–33.

25. Kabsch W. Xds. Acta Crystallogr D Biol Crystallogr 2010;66:125–32.26. Incardona MF, Bourenkov GP, Levik K, Pieritz RA, Popov AN, Svensson O.

EDNA: a framework for plugin-based applications applied to X-ray exper-iment online data analysis. J Synchrotron Radiat 2009;16:872–9.

27. Evans P. Scaling and assessment of data quality. Acta Crystallogr D BiolCrystallogr 2006;62:72–82.

28. Navaza J. Implementation of molecular replacement in AMoRe. ActaCrystallogr D Biol Crystallogr 2001;57:1367–72.

29. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development ofCoot. Acta Crystallogr D Biol Crystallogr 2010;66:486–501.

30. Ong S, Mann M. A practical recipe for stable isotope labeling by aminoacids in cell culture (SILAC). Nat Protoc 2007;1:2650–60.

31. Wakeling AE, O'Connor KM, Newboult E. Comparison of the biologicaleffects of tamoxifen and anewantioestrogen (LY117018) on the immaturerat uterus. J Endocrinol 1983;99:447–53.

32. Bradbury R, De Savi C, Rabow A, Norman R, de Almeida C, Andrews D.Optimization of a novel binding motif to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (AZD9496), a potent and orally bioavail-

able selective estrogen receptor downregulator and antagonist. J MedChem 2015;58:8128–40.

33. Wu YL, Yang X, Ren Z, McDonnell DP, Norris JD, Willson TM, et al.Structural basis for an unexpected mode of SERM-mediated ER antago-nism. Mol Cell 2005;18:413–24.

34. Horwitz KB, McGuire WL. Estrogen control of progesterone receptor inhuman breast cancer. Correlation with nuclear processing of estrogenreceptor. J Biol Chem 1978;253:2223–8.

35. Rich RL, Hoth LR, Geoghegan KF, Brown TA, LeMotte PK, Simons SP, et al.Kinetic analysis of estrogen receptor/ligand interactions. Proc Natl Acad SciU S A 2002;99:8562–67.

36. Pike AC, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstrom O,et al. Structure of the ligand-binding domain of oestrogen receptor beta inthe presence of a partial agonist and a full antagonist. EMBO J 1999;18:4608–18.

37. Grese TA, Sluka JP, BryantHU, CullinanGJ, Glasebrook AL, Jones CD, et al.Molecular determinants of tissue selectivity in estrogen receptor modula-tors. Proc Natl Acad Sci U S A 1997;94:14105–10.

38. Lonard DM, Nawaz Z, Smith CL, O'Malley BW. The 26S proteasome isrequired for estrogen receptor-alpha and coactivator turnover and forefficient estrogen receptor-alpha transactivation. Mol Cell 2000;5:939–48.

39. Reid G, H€ubner MR, M�etivier R, Brand H, Denger S, Manu D, et al. Cyclic,proteosome-mediated turnover of unliganded and liganded ERa onresponsive promoters is an integral feature of estrogen signaling. Mol Cell2003;11:695–707.

40. Ghosh MG, Thompson DA, Weigel RJ. PDZK1 and GREB1 are estrogen-regulated genes expressed inhormone-responsive breast cancer. Cancer Res2000;60:6367–75.

41. Sauve K, Lepage J, Sanchez M, Heveker N, Tremblay A. Positive feedbackactivation of estrogen receptors by the CXCL12-CXCR4 pathway. CancerRes 2009;69:5793–800.

42. Vendrell JA, Magnino F, Danis E, Duchesne MJ, Pinloche S, Pons S, et al.Estrogen regulation in human breast cancer cells of new downstream genetargets involved in estrogen metabolism, cell proliferation and cell trans-formation. J Mol Endocrinol 2004;32:397–414.

43. Austreid E, Lonning PE, Eikesdal HP. The emergence of targeted drugs inbreast cancer to prevent resistance to endocrine treatment and chemother-apy. Expert Opin Pharmacother 2014;15:681–700.

44. Liu H, Park W, Bentrem DJ, McKian KP, De Los Reyes A, Loweth JA, et al.Structure-function relationship of the raloxifene-estrogen receptor-a com-plex for regulating transforming growth factor-a in breast cancer cells. J BiolChem 2002;277:9189–98.

45. Davies C, Pan H, Godwin J, Gray R, Arriagada R, Raina V, et al. Long-termeffects of continuing adjuvant tamoxifen to 10 years versus stopping at 5years after diagnosis of oestrogen receptor-positive breast cancer: ATLAS, arandomised trial. Lancet 2013;381:805–16.

46. Bergman L, BeelenML,GalleeMP,HollemaH, Benraadt J, van Leeuwen FE.Risk and prognosis of endometrial cancer after tamoxifen for breast cancer.Lancet 2000;356:881–7.

47. Folkerd E, Dowsett M. Sex hormones and breast cancer risk and prognosis.Breast 2013;22Suppl 2:S38–43.

48. Sen T, Li S, Shao J, Crowder R, Kitchen R, EllisMJ. Patient-derived xenograftstudy reveals the pharmacology and the role of ESR1 gene aberrations inendocrine therapy resistance of ER positive breast cancer. Can Res 2014;74:5544.

49. Piccart M, Hortobagyi GN, CamponeM, Pritchard KI, Lebrun F, Ito Y, et al.Everolimus plus exemestane for hormone-receptor-positive, human epi-dermal growth factor receptor-2-negative advanced breast cancer: overallsurvival results from BOLERO-2†. Ann Oncol 2014;25:2357–62.


Weir et al.




Published OnlineFirst March 28, 2016.Cancer Res Hazel M. Weir, Robert H. Bradbury, Mandy Lawson, et al. in Preclinical Models

-Mutant Breast TumorsESR1the Growth of ER-Positive and AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks

Updated version

10.1158/0008-5472.CAN-15-2357doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2016/03/26/0008-5472.CAN-15-2357.DC1

Access the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerres.aacrjournals.org/content/early/2016/05/20/0008-5472.CAN-15-2357To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-15-2357

http://cancerres.aacrjournals.org/content/suppl/2016/03/26/0008-5472.CAN-15-2357.DC1

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/early/2016/05/20/0008-5472.CAN-15-2357


azd9496: an oral estrogen receptor inhibitor that blocks...

Documents