Selective Androgen Receptor Modulator RAD140 Inhibits The

Published OnlineFirst October 3, 2017; DOI: 10.1158/1078-0432.CCR-17-0670

Cancer Therapy: Preclinical Clinical Cancer Research Selective Androgen Receptor Modulator RAD140 Inhibits the Growth of Androgen/Estrogen Receptor–Positive Breast Cancer Models with a Distinct Mechanism of Action Ziyang Yu, Suqin He, Dannie Wang, Hitisha K. Patel, Chris P. Miller, Jeffrey L. Brown, Gary Hattersley, and Jamal C. Saeh

Abstract

þ Purpose: Steroidal androgens suppress androgen receptor and AR/ER breast cancer patient-derived xenografts (PDX). Activa- þ estrogen receptor positive (AR/ER ) breast cancer cells and were tion of AR and suppression of ER pathway, including the ESR1 used to treat breast cancer, eliciting favorable response. The gene, were seen with RAD140 treatment. Coadministration of þ current study evaluates the activity and efficacy of the oral selective RAD140 and palbociclib showed improved efficacy in the AR/ER þ AR modulator RAD140 in in vivo and in vitro models of AR/ER PDX models. In line with efficacy, a subset of AR-repressed genes breast cancer. associated with DNA replication was suppressed with RAD140 Experimental Design: A series of in vitro assays were used to treatment, an effect apparently enhanced by concurrent admin- determine the affinity of RAD140 to 4 nuclear receptors and istration of palbociclib. evaluate its tissue-selective AR activity. The efficacy and pharma- Conclusions: RAD140 is a potent AR agonist in breast cancer codynamics of RAD140 as monotherapy or in combination with cells with a distinct mechanism of action, including the AR- þ palbociclib were evaluated in AR/ER breast cancer xenograft mediated repression of ESR1. It inhibits the growth of multiple þ models. AR/ER breast cancer PDX models as a single agent, and in Results: RAD140 bound AR with high affinity and specificity combination with palbociclib. The preclinical data presented here þ and activated AR in breast cancer but not prostate cancer cells. Oral support further clinical investigation of RAD140 in AR/ER breast administration of RAD140 substantially inhibited the growth of cancer patients. Clin Cancer Res; 23(24); 1–13. 2017 AACR.

Introduction combination therapies now represent a new generation of standard of care for this indication. Breast cancer is the second leading cause of cancer-related death Recent histopathologic studies revealed that the androgen in women, with an estimated 246,660 newly diagnosed cases and receptor (AR) is the most commonly expressed hormone receptor 40,450 deaths in the United States alone in 2016 (1). Breast cancer þ in breast cancer, with 75% to 90% of ER and approximately 30% is a heterogeneous disease categorized into several histopatho- of ER breast cancers expressing AR (6, 7). Although AR is widely logic subtypes based on the status of estrogen receptor a (ER), present in breast cancer tissue, accumulating evidence has dem- progesterone receptor (PR), and HER2 receptor. Although ER- þ onstrated that the role of AR in these tumors is subtype dependent positive (ER ) breast cancers are now treated with standard-of- (8–11). AR antagonists including bicalutamide and enzalutamide care agents targeting the ER axis, including tamoxifen, fulvestrant, have been shown to reduce the growth of AR-positive but ER , and aromatase inhibitors (AI), and HER2-positive tumors are including triple-negative, breast cancer in preclinical models treated with HER2 inhibitors, such as trastuzumab, novel thera- þ (12–14) and in patients (11). In contrast, in ER breast cancers, peutic approaches are still needed to address resistance emerging AR has been considered antiproliferative and has been associated from these established regimens (2, 3). More recently, combined with a favorable prognosis. Clinically, until the 1970s, breast administration of ER-targeted therapies with the inhibitors of cancers were often treated with nonselective steroidal androgens cyclin-dependent kinase (CDK) 4/6 (4) or mTOR (5) have yielded þ including testosterone derivatives and danazol with response improved therapeutic efﬁcacy in ER breast cancer, and these rates of 20% to 25% (9, 15–18). In line with these clinical experiences, preclinical studies have also shown treatment with classic androgens, including DHT, reduced the growth of AR and þ þ Radius Health, Inc., Waltham, Massachusetts. ER (AR/ER ) breast cancer cells in vitro (19–21) and in vivo (22). Note: Supplementary data for this article are available at Clinical Cancer Classic androgen-based therapy for breast cancer declined due to Research Online (http://clincancerres.aacrjournals.org/). its virilizing effects, potential risk of further aromatization to Corresponding Authors: Ziyang Yu, Radius Health, Inc., 950 Winter Street, estrogens, and the emergence of ER-targeted agents including Waltham, MA 02451. Phone: 617-551-4065; Fax: 617-551-4701; E-mail: tamoxifen. However, it is worth noting that more recent clinical [email protected]; and Jamal C. Saeh, [email protected] evidence showed androgen treatment also led to remission in doi: 10.1158/1078-0432.CCR-17-0670 patients who were progressing after ER-targeted therapy with 2017 American Association for Cancer Research. objective response rate around 17% to 39% (23, 24). Fulvestrant

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Cell culture and treatment Translational Relevance The ZR-75-1, HCC1428, and T47D breast cancer cells and The expression of the androgen receptor (AR) in estrogen LNCaP prostate cancer cells were purchased from ATCC and receptor (ER)–positive breast cancer has been well described. maintained in medium and condition as recommended by the Androgens inhibit the proliferation of AR and ER-positive vendor. All cell lines involved in the study were tested negative for þ (AR/ER ) breast cancer cells, and androgen-based treatment mycoplasma contamination (MycoAlert Detection Kit) and sub- showed clinical benefit in breast cancer patients. However, the jected to annual authentication using STR profiling (Thermo clinical utility of the classic steroidal androgen-based therapies Fisher Scientific). All the cells were under passage 15 at the time has been limited. We report here that RAD140, an oral, tissue of experiments. HCC1428 long-term estrogen-depleted (LTED) selective androgen receptor modulator, exhibited potent activ- cells were established by culturing these cells in medium with þ ity in inhibiting the growth of multiple AR/ER breast cancer 10% charcoal dextran-stripped serum (CSS) for 16 weeks, and the xenograft models. The coadministration of RAD140 with expressions of AR, ER, and PR were confirmed by Western blotting þ CDK4/6 inhibitor elicited enhanced efficacy in these AR/ER (Supplementary Fig. S1A). For proliferation assay, cells were models. Mechanistically, RAD140 potently regulates AR target seeded in medium with 10% CSS at 30,000 cells per well in genes, while suppressing the ER targets and the ESR1 mRNA. 24-well plates and subjected to treatment with RAD140, DHT, or These suggest a distinct AR-mediated mechanism of action DMSO for 14 days with treatments being renewed every 3 days. At compared with the traditional ER-targeted agents. RAD140 the end of the treatment period, trypsinized cells were stained with þ may present a novel therapeutic approach for AR/ER breast Trypan blue before being subjected to live cell counting in a cancer. The first-in-human study of RAD140 in recurrent breast Nexcelom Cellometer (Nexcelom Bioscience). The PC3-AR cell cancer has been initiated. line, described previously (29), was a generous gift from Dr. Steve Balk of Beth Israel Deaconess Medical Center (Boston, MA) and Dr. Changemeng Cai of University of Massachusetts (Boston, MA). For ZR-75-1 cell-based assays, cells were seeded and incu- at second line or later in large phase III trials elicited objective bated in medium with 5% CSS for 48 hours. Cells were then fi response rates of 2.1% to 11% (25–28). treated with 17b-estradiol at 1 nmol/L nal concentration or þ The high prevalence of AR observed in ER breast cancers, vehicle (ethanol) before being incubated with DMSO, RAD140 along with the prior clinical efficacy demonstrated with classic (Radius Health, Inc., as described previously; ref. 30), or DHT androgens, provides a strong rationale for the development of a (Sigma Aldrich) in the presence or absence of enzalutamide or new generation of oral, selective AR agonists and to further ARN-509 (Selleckchem) for 24 hours. The experiments were þ exploit their therapeutic benefits in AR/ER breast cancer. Here, performed in duplicate. The nuclear and cytoplasmic fractions we describe the tissue-selective AR agonist activity of RAD140, of the cells were prepared using the NE-PER Nuclear and Cyto- fi an oral nonsteroidal selective AR modulator (SARM), and its plasmic Extraction Kit (Thermo Fisher Scienti c) following the distinct AR-mediated mechanism of action including the sup- manufacturer's instructions. pression of ESR1. RAD140 exhibited potent antitumor activity þ AR reporter gene assays in the in vivo models of AR/ER breast cancer both as a single AR reporter gene assay was carried out using a Cignal Androgen agent and in combination with the CDK4/6 inhibitor palbo- Receptor Reporter (luc) Kit (Qiagen). ZR-75-1 breast cancer cells, ciclib. RAD140 represents a new generation of tissue-selective LNCaP, and PC3-AR prostate cancer cells were transfected with a AR agonists and potentially a novel therapeutic option for þ tandem androgen-responsive element (ARE)-driven firefly lucif- AR/ER breast cancer. erase construct and a Renilla luciferase construct in RMPI1640 media containing 5% CSS. Forty-eight hours later, transfected Materials and Methods cells were treated with DMSO, RAD140, or DHT. The luciferase Nuclear receptor binding assay activity was determined using the Dual-Luciferase Reporter Assay The PolarScreen nuclear receptor competitor assays (Thermo (Promega) as a representation of AR transcription activity and Fisher Scientific) for AR, ER, PR, and glucocorticoid receptor (GR) normalized to the activity of Renilla luciferase. Each set was carried were used to determine the binding affinity and specificity of out in triplicate. The values were presented as average fold changes RAD140. Briefly, RAD140 and fluorescence-labeled ligands SD over the vehicle control. (Fluormones) for AR, ER, PR, or GR, respectively, were added to each reaction well, and the plate was incubated for 4 hours at In vivo efficacy study room temperature in the dark. Polarization values for each well All study protocols were reviewed by Institutional Animal Care were measured and plotted against the concentration of RAD140. and Use Committees and Radius Health, Inc., and conducted in As a reference, the binding affinity of the standard ligands DHT, compliance with U.S. and European regulations of protection of 17b-estradiol, progesterone, and dexamethasone (for AR, ER, PR, laboratory animals. The HBCx-22, HBCx-21, and HBCx-3 breast and GR, respectively) was also measured. The relative binding cancer patient-derived xenograft (PDX) models were established affinity of RAD140 versus the 4 standard ligands is reflected by the and evaluated at XenTech by implanting tumor fragments sub- ratios of IC50 (standard ligand)/IC50 (RAD140). A commercially cutaneously in the flank female athymic nude mice (Foxn1nu, available spectrum screen was performed for RAD140 (MDS Envigo RMS). The ST897 breast cancer PDX model was estab- Pharma Services). The binding of RAD140 (1 mmol/L) to a panel lished in female athymic nude mice (Foxn1nu, Charles River of cellular targets was assessed (Supplementary Table S1). Appre- Laboratories) and studies conducted at South Texas Accelerated ciable binding was defined as a larger than 50% inhibition of the Research Therapeutics. These models were characterized as ER and reference radio-ligand. PR positive and negative for HER2 overexpression based on IHC

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and later confirmed using Western blot analysis and qPCR (Sup- KLK2, FKBP5, ZBTB16, ESR1, PGR, TFF1, GREB1, BLM, FANCI, plementary Table S2; Supplementary Fig. S1). The efficacy study in LMNB1, MCM2, MCM4, MCM7, 18S, and GAPDH were pur- T-47D breast cancer cell line–derived xenograft model, estab- chased from Thermo Fisher Scientific. The PCR reaction was set up lished in NOD/Shi-scid/IL-2Rgnull mice, was conducted at WuXi with TaqMan Fast Virus 1-Step Master Mix in a Quant Studio AppTec. These xenograft models were supplemented with exog- 6 Flex qPCR system (Thermo Fisher Scientific). Samples from a enous estradiol in drinking water (HBCx-3, HBCx-21, HBCx-22, minimum of 3 tumors from each treatment group or those from and ST897) or subcutaneous pellet (0.18 mg/90-day release, a minimum of 2 replicated cell-based assays were included for the Innovative Research America) implanted 3 days prior to tumor analysis. The expression of genes of interest were normalized to cell inoculation (T-47D). The PDX-bearing mice were random- that of GAPDH or 18S and presented as the mean fold change ized once their tumor volumes reached 60 to 200 mm3,as compared with the vehicle control group. For RNA-seq analysis, described previously (31). The cell line–derived xenografts with total RNA samples were converted into cDNA libraries using the volumes between 125 and 250 mm3 were enrolled in treatment TrueSeq Stranded mRNA Sample Preparation Kit (Illumina). groups. RAD140, DHT, or fulvestrant were administered as single Libraries are quantified, normalized, and pooled before being agent or in combination with palbociclib. RAD140, palbociclib, subjected to HiSeq 2 50 bp paired end sequencing on an and the vehicle (0.5% carboxymethyl cellulose) were given orally. Illumina sequencing platform. De-multiplexed FASTQ files were Fulvestrant (clinical formulation, AstraZeneca) was given as sub- aligned to HG19 human genome using the STAR aligner version cutaneous injection. DHT was administered as subcutaneously 2.4 and genetic features were quantified using RSEM version implanted pellets (12.5 mg/60-day release, Innovative Research 1.2.14. The data discussed in this publication have been deposited America) from the day of randomization to the end of treatment in NCBI's Gene Expression Omnibus and are accessible through (14). Tumor volume and body weight were measured twice a GEO Series accession number GSE104177. The AR and ER target week. Tumor growth inhibition (TGI %) was calculated on the genes with significant changes in expression (FDR < 0.05) were basis of the change of mean tumor volume from treatment day 1 analyzed and log2 fold changes presented in heatmap. Global to the last day of assessment and presented as a percentage of that pathway analysis was performed for genes with significant of the vehicle control group [100 (1- DTVtreatment/DTVvehicle)], as changes in expression (FDR < 0.001) using the functional anno- an indication of antitumor efficacy. Statistical analyses were tation tools (Gene Ontology) available on the National Health performed for the changes in individual tumor volume (DTV, Institute DAVID Bioinformatics Resources platform (https:// from treatment day 1 to the last day of assessment) between the david.ncifcrf.gov). groups compared. Results IHC RAD140 binds to AR with high affinity and high specificity Formalin-fixed paraffin-embedded sections of xenograft The binding affinity of RAD140 was assessed using fluorescence tumors underwent antigen retrieval and were then blocked using polarization-based competitive binding assays for AR, ER, PR, and 5% goat serum and avidin blocking solution. IHC was performed GR and compared against the standard ligands for each nuclear in a Leica Bond III auto-stainer (Leica Biosystems) according to the receptor. As positive controls, assays evaluating the binding of the manufacturer's instructions. The primary rabbit mAbs against ER standard ligands, including DHT, 17b-estradiol (E2), progester- (SP1) and PR (SP42) were obtained from Cell Marque. Rabbit one, and dexamethasone to AR, ER, PR, and GR, respectively, mAb against Ki67 (D2H10) and phospho-retinoblastoma protein were performed. In the competitive binding assay against the (phospho-Rb) were from Cell Signaling Technology. Mouse anti- AR-Fluoromone, RAD140 exhibited 1.6-fold lower AR affinity body against AR (441) was obtained from Dako. The AR IHC than DHT (Fig. 1A). This suggests binding affinity of RAD140 to staining protocol included the use of ImmunoDNA Background AR is slightly lower but comparable with that of natural androgen. Blocker (Bio SB). Sections shown in each figure were stained and RAD140 exhibited 33-fold lower affinity to PR compared with photographed under identical conditions. Staining and scoring of progesterone. No binding of RAD140 to ER or GR was detected. In the samples were performed by a pathologist blinded to the addition, a cellular target spectrum screen was performed to treatment groups. determine the binding selectivity of RAD140 against a broad panel of 165 molecular targets that included many pharmaco- Western blot analysis logically relevant molecules including ion channels, G-protein– Tumor lysates from the in vivo studies were prepared using Cell coupled receptors, enzymes, and nuclear hormone receptors Lysis Buffer with protease and phosphatase inhibitors (Cell Sig- (Supplementary Table S1). No appreciable interaction of RAD140 naling Technology) in a FastPrep Sample Preparation System (MP with any targets screened, other than AR and PR, was detected. Biomedicals). Cell lysates were prepared using Cell Lysis Buffer as These results indicate RAD140 binds to AR with high affinity described above. Total protein was quantitated and subjected to and specificity. Western blot analysis using antibodies against AR (PG-21, EMD Millipore; Clone 441, Thermo Fisher Scientific), ERa, PR, GAPDH, b-tubulin, and HDAC2 (Cell Signaling Technology). RAD140 is a potent tissue-selective AR agonist SARMs are selective AR ligands that have been shown to act as Gene expression analysis agonists or antagonists in a tissue-context–dependent manner Total RNA from xenograft samples and cells was extracted using (32). We previously demonstrated that RAD140 exhibits differ- the RNeasy Mini Kit (Qiagen), with a DNase incubation step ential activity in prostate versus in the muscle (30). To further included to ensure complete removal of genomic DNA. The assess the activity of RAD140 on AR transcription in tissue context, expression of genes was evaluated using qPCR and RNA sequenc- an AR reporter assay was performed in ZR-75-1, a human breast ing (RNA-seq). For qPCR, TaqMan primer and probe sets for AR, cancer cell line expressing endogenous AR and ER (21), and the

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Figure 1. Binding affinity and specificity of RAD140 and its tissue-selective AR agonist activity. A, The binding affinity of RAD140 to AR, ER, PR, and GR was determined using PolarScreen Competitive Binding Assays for each of these nuclear receptors. DHT, 17b-estradiol, progesterone, and dexamethasone were included as positive control for the binding to AR, ER, PR, and GR, respectively. The relative affinity of RAD140, versus the standard ligands, to these

receptors was calculated on the basis of the IC50 values using Prism. Data presented are an average of at least three independent experiments. B, ZR-75-1 breast cancer cells and LNCaP prostate cancer cells were transfected with ARE-luc and Renilla luciferase constructs in medium containing 5% CSS. Twenty-four hours after the transfection, cells were treated with RAD140 or DHT at the indicated ﬁnal concentrations. ARE-driven luciferase activity was measured and normalized to that of Renilla luciferase. Data presented are an average of biological triplicates. C, Steroid-deprived ZR-75-1 cells were treated with ethanol (vehicle) or 1 nmol/L of 17b-estradiol (E2) for 2 hours, followed by treatment with RAD140 at 100 nmol/L in the presence or absence of ARN-509 (10 mmol/L) for overnight. Whole-cell lysates and nuclear fractions were prepared and subjected to Western blot analyses for AR. HDAC2 and b-tubulin were probed as loading controls for nuclear and whole-cell extracts, respectively. D, HCC1428 LTED cells were maintained in steroid-depleted condition and treated with RAD140 or DHT at the indicated concentrations for 14 days, with medium and compounds replenished every 3 days. At the end of the treatment, cells were stained with Trypan blue before subjected to live-cell counting in a Nexcelom Cellometer. Average cell number SD of biological triplicates is presented. , P < 0.05 for comparison with the DMSO-treated group.

AR-positive prostate cancer cell line LNCaP (29). Dose responses 1 or 10 nmol/L led to more than a 10-fold induction of AR by RAD140 were compared with the known AR agonist DHT. transcription activity (Fig. 1B, right). To rule out the possibility Treatment of steroid-depleted ZR-75-1 cells with 1, 10, and that the attenuated AR activity of RAD140 is due to the expression 100 nmol/L RAD140 induced AR transcription activity by 1.9- of a mutant AR T877A in LNCaP cells, we also performed AR to 2.9-fold, comparable with the induction seen with DHT at the reporter assay in PC3-AR cells, which express ectopic wild-type AR same concentrations (Fig. 1B, left). In contrast, in the low-passage, (29). Similar to the ﬁnding in LNCaP cells, RAD140 exhibited androgen-sensitive LNCaP cells treated with RAD140 at 0.1 to much attenuated activity on ectopic wild-type AR in prostate 10 nmol/L, no induction of AR activity was observed while DHT at cancer cells compared with DHT (Supplementary Fig. S2). These

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þ results indicated that RAD140 is a tissue-specific AR agonist, agent is effective in inhibiting the growth of AR/ER breast cancer with selective activity in breast cancer cells but not in prostate xenografts. cancer cells. We then examined the changes in common breast cancer Classic AR agonists are known to induce AR conformation biomarkers in the HBCx-22 xenografts treated with RAD140 or change and subsequent localization to the nuclei, while pure AR fulvestrant using IHC. As shown in Fig. 2C, HBCx-22 tumors in the antagonist, such as enzalutamide and ARN-509 act partially via control group were positive for AR, ER, and PR, consistent with blocking AR nuclear localization (33, 34). We then examined prior characterization of this model (Supplementary Fig. S1). An whether RAD140 induces the nuclear expression of AR in breast apparently increased AR expression, predominantly localized in cancer cells. Treatment of ZR-75-1 cells with RAD140 led to the nuclei, was seen in RAD140-treated tumors. This demonstrat- increased AR expression in the nuclear fraction while the estrogen ed that RAD140 possesses AR agonist properties, such as stabi- (E2) showed no apparent effect (Fig. 1C). The positive control, lizing AR and promoting its nuclear localization, which are DHT, substantially increased the nuclear expression of AR. ARN- commonly seen with classic androgens (37, 38). Interestingly, 509 substantially reduced the nuclear expression induced by DHT RAD140 treatment led to substantially decreased expression of ER and RAD140, suggesting a direct antagonism against these two AR and its downstream target PR. A profound decrease in Ki67 agonists. This provided further evidence that RAD140 is an AR expression was also seen in these RAD140-treated tumors, indi- agonist in breast cancer cells. cating a suppressive effect on the proliferation of these tumor cells. Treatment with fulvestrant also led to substantial decreases in ER, þ RAD140 monotherapy suppresses the growth of AR/ER breast PR, and Ki67 expression, to levels seemingly comparable with cancer PDX models those seen with RAD140. Furthermore, the epithelial nature of the It has been previously reported that DHT inhibits the prolif- terminal tumor mass was confirmed using IHC assays for cyto- þ eration of ER breast cancer cells driven by supplemental estradiol keratin (CK) 18 and vimentin (Supplementary Fig. S4). This (21). We evaluated the activity of RAD140 and DHT in an suggests SARM and SERD both inhibit the ER pathway and breast þ estrogen-independent AR/ER breast cancer line HCC1428 LTED, cancer cell proliferation. in which ER signaling appears to be active as judged by PR expression (Supplementary Fig. S1). RAD140 exhibited inhibito- Pharmacodynamic analysis reveals RAD140-mediated ry effect on the proliferation of these endocrine-resistant breast regulation of AR and ER target genes and ESR1 cancer cells at concentrations as low as 10 nmol/L, with an It has been proposed that androgens suppress ER signaling in þ apparent maximal effect seen at 100 nmol/L (Fig. 1D). This was AR/ER breast cancer cells, which may contribute to the anti- comparable with the inhibitory effect seen with DHT in the 0.1 to proliferative activity (19, 21). To better understand the mecha- þ 10 nmol/L range. To further assess how the AR agonist activity of nism of action of the SARM RAD140 in AR/ER breast cancer RAD140 translates into antitumor activity in vivo, RAD140 was models, we examined the pharmacodynamic changes with a focus þ evaluated as a single agent in AR/ER breast cancer PDX models. on the AR and ER pathways. In the HBCx-22 model, a profound Oral administration of RAD140 induced significant TGI of 76%, induction of AR target genes, including FKBP5, KLK2, and 59%, and 58% when compared with the vehicle control groups in ZBTB16, was seen in RAD140-treated tumors (Fig. 3A, top) while HBCx-22, HBCx-3, and HBCx-21, respectively (Fig. 2A, Supple- the expression of the AR gene was not affected. Fulvestrant, as mentary Fig. S3A and S3B). In the HBCx-22 model, fulvestrant, a expected, did not induce the expression of these AR target genes, þ selective ER downregulator (SERD) and standard of care for ER although a slight increase in AR message was seen. The ER target breast cancer dosed at 1 mg weekly, exhibited a statistically genes PGR, TFF1, and GREB1 were found to be substantially significant antitumor activity, as judged by a TGI of 59%, com- suppressed in RAD140-treated tumors to levels comparable or parable with a previous report of this model and indicative of its even lower than that seen with fulvestrant (Fig. 3A, bottom). dependency on ER (31). It has been previously determined that Interestingly, in these RAD140-treated tumors, the mRNA expres- the 1 mg weekly administration of fulvestrant achieves exposure sion of ESR1, the coding gene of ER, was profoundly suppressed. equivalent to the 500 mg monthly dose in human (35). The In contrast, the fulvestrant treatment did not lead to appreciable changes in tumor volume of the RAD140- or fulvestrant-treated change in ESR1 gene expression. At protein level, both RAD140 HBCx-22 xenografts appeared to be lower compared with those and fulvestrant exhibited a profound effect in decreasing the treated with vehicle (Fig. 2B). To further verify the concept of expression of ER and PR in HBCx-22 xenografts (Fig. 3B, left), þ inhibiting the growth of AR/ER breast cancer tumors with AR consistent with the IHC findings described above. Similar þ agonists, T-47D cell line–derived xenografts were treated with decrease in ER and PR expression was seen in HBCx-3 AR/ER DHT, RAD140, or fulvestrant (Supplementary Fig. S3C). Treat- xenografts treated with RAD140 (Fig. 3B, middle). In the T-47D ment with DHT or RAD140 inhibited the growth of these T-47D xenografts, treatment with RAD140, DHT, or fulvestrant also led tumors to a comparable extent. Fulvestrant also elicited potent to decreased ER and PR expression (Fig. 3B, right). growth inhibition. In addition, the activity of the combination of Next, the modulation of AR and ER target genes by RAD140 was fulvestrant with RAD140 was evaluated in HBCx-22 model, but further examined in vitro. As determined in ARE-luc reporter assay no appreciable improvement in tumor growth inhibition was and proliferation assay (Fig. 1B and D), RAD140 at 100 nmol/L observed, suggesting a potential overlap in the pathway inhibition exhibited maximal effects, comparable with DHT at 10 nmol/L; induced by these two agents in this model (Supplementary therefore, 100 nmol/L was selected as the RAD140 concentration Fig. S3D). This observation is consistent with previous clinical for further in vitro assays. In ZR-75-1 cells treated with RAD140, experience with anastrozole–fulvestrant combination and pre- substantial induction of FKBP5 and ZBTB16 messages was seen clinical data of fulvestrant–RAD1901 combination, both of which (Fig. 3C), which was comparable with that seen with DHT. The showed such intense ER blockade failed to yield additional benefit induction of these AR targets by RAD140 or DHT was completely (35, 36). Together, these results indicated that RAD140 as a single blocked by the antagonist enzalutamide. The expression of the AR

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Figure 2. The effect of oral administration of RAD140 on the growth of AR/ERþ breast cancer PDX. A, Mean tumor volumes SEM of HBCx-22 breast cancer xenografts treated with vehicle, RAD140 (100 mg/kg once daily), or fulvestrant (1 mg once weekly) for the indicated period of time (n ¼ 8/group). B, Changes in tumor volume of individual HBCx-22 xenografts from the beginning to the end of the treatment. C, IHC analysis of HBCx-22 xenografts collected at the end of the study was performed using antibodies against AR, ER, PR, and Ki67. Representative images of three individual tumors from each treatment group are shown.

gene did not seem to be affected by RAD140 or DHT treatment. pathway and ER pathway genes appear to be comparable, suggest- The ER target genes PGR and TFF1 were induced by 1 nmol/L of ing an AR-speciﬁc effect. Furthermore, competitive blockade of AR E2. Treatment with either RAD140 or DHT was found to repress activation by enzalutamide effectively reversed the AR and ER the E2-induced expression of these genes. Similar to the effects pathway modulation by RAD140 or DHT. The incomplete rever- seen in xenograft models, treatment with RAD140 or DHT sal of ESR1 suppression by enzalutamide may be due to the decreased ESR1 mRNA. The effects of RAD140 and DHT on AR relatively lower afﬁnity of this compound compared with AR

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Figure 3. Expression of AR and ER pathway genes and ESR1 in RAD140-treated AR/ERþ breast cancer cells and xenografts. A, Tumor samples from the HBCx-22 collected 6 hours after last treatment were subjected to qPCR analysis for the expression of AR, ER, and the indicated downstream target genes. B, Samples from HBCx-22, HBCx-3, and T-47D xenograft models were collected 6 hours after last treatment and subjected to Western blot analysis for the expressionof AR, ER, and PR. C, Steroid-deprived ZR-75-1 cells were pretreated with 1 nmol/L of E2 for 2 hours and then treated with RAD140 (100 nmol/L) or DHT (10 nmol/L) in the presence or absence of enzalutamide (10 mmol/L) for 24 hours. RNA of the treated cells was isolated and subjected to qPCR analysis for the expression of AR, ESR1, and the indicated downstream targets.

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Figure 4. Global pathway modulation by RAD140 in the HBCx-22 PDX model. A, Heatmaps of mRNA expression fold change of the known AR and ER pathway genes in RAD140- versus vehicle-treated tumors (n ¼ 3/group), with downregulation represented by shades of blue and upregulation by shades of red. Gene Ontology analyses of the signiﬁcantly upregulated (B) or downregulated (C) genes in RAD140-treated tumors.

agonists RAD140 and DHT. These in vivo and in vitro observations addition to the targets examined by qPCR, RAD140-treated further demonstrated the AR agonist activity of RAD140 in tumors had higher levels of AR-activated genes in addition to þ AR/ER breast cancer models. More importantly, RAD140 and those shown in qPCR. A subset of the ER target genes was found to DHT both inhibited ER downstream targets, as well as the ESR1 be suppressed in RAD140-treated tumors, consistent with the gene, suggesting a novel mechanism of action of AR agonists in qPCR findings. Using a stringent filtering criteria (FDR < 0.001), inhibiting the ER pathway in breast cancer cells. we found that globally 86 genes were significantly upregulated and 77 were downregulated by at least 2-fold in RAD140-treated þ Global modulation of signaling pathways by SARM in AR/ER HBCx-22 xenografts (Fig. 4B; Supplementary Tables S3 and S4). In breast cancer xenografts the Gene Ontology analysis, these upregulated genes were found To further understand the global effect of RAD140 on signaling to be most significantly enriched in the metabolic process. This þ cascades in AR/ER breast cancer cells, RNA-seq analysis of HBCx- was consistent with the previous report describing the role of AR as 22 xenografts was performed. We examined a broader range of AR a master regulator of central metabolism and biosynthesis in and ER target genes with significantly altered expression in prostate cancer cells (39). The downregulated genes in RAD140-treated tumors (Fig. 4A). The results indicated that in RAD140-treated tumors were found to be enriched in categories

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associated with cell division, DNA replication, and cell-cycle enhanced in tumors that received both agents. These results progression. This negative regulation of gene transcription was further confirmed the inhibitory effect of RAD140 on ER signaling in line with previous reports on the transcription suppressor role cascade. Gene expression analysis of the HBCx-3 xenografts of AR (40, 41). These data further confirmed the regulation of AR treated with RAD140, palbociclib, or their combination showed and ER pathways by RAD140 and suggested a unique mechanism that the AR target genes FKBP5 and ZBTB16 were robustly induced of action of RAD140 via the AR-mediated transcription repres- in RAD140-treated tumors, while no appreciable effect on the AR sion. These data offer evidence for further uncovering the under- gene was seen (Fig. 5C, top). The ER target genes PGR and TFF1 þ lying mechanism of RAD140 as a single agent in AR/ER breast were substantially suppressed in the tumors of the RAD140- cancer models. treated groups (Fig. 5C, middle). Notably, a profound suppression of the ESR1 mRNA level was also observed in these RAD140- Enhanced antitumor activity of combined administrations of treated HBCx-3 xenografts. Palbociclib alone, in contrast, did not RAD140 with CDK4/6 inhibitor have an apparent effect on AR target genes but did seem to repress Given the observed effect of RAD140 on cell cycle and DNA PGR and induce TFF1 and ESR1 to a relatively modest degree replication–related genes, we hypothesized that combined compared with the changes induced by RAD140 treatment. administration of this SARM and a CDK4/6 inhibitor, which We next explored the underlying mechanism for the improved inhibits the phosphorylation and subsequent degradation of Rb, efficacy and enhanced inhibition of cell proliferation seen with thus blocking cell-cycle entry (42–44), may produce improved the RAD140–palbociclib combination. It has been recently þ antitumor activity in AR/ER breast cancer models. Palbociclib, a reported that in castration-resistant prostate cancer cells, activated CDK4/6 inhibitor recently approved for breast cancer treatment in AR recruits hypophosphorylated Rb to the loci of genes implicated combination with ER-targeted agents in as early as first line of in DNA replication and suppress their transcription (41). We þ therapy (45–47), was selected for this study. Two AR/ER PDX hypothesized that AR activated by RAD140 inhibits DNA repli- models, HBCx-3 (Fig. 5A) and ST897 (Fig. 5B), were treated with cation–related genes in breast cancer cells, and this effect is RAD140, palbociclib, or a combination of RAD140 with palbo- enhanced by coadministration of palbociclib. Indeed, the ciclib. Compared with the vehicle-treated group, RAD140 as HBCx-3 tumors treated with RAD140 alone had decreased expres- monotherapy produced statistically significant inhibition of sion of the DNA replication–related genes including Bloom tumor growth in these models compared with vehicle control. syndrome recQ like helicase (BLM), Fanconi anemia complemen- Palbociclib as a single agent also inhibited the growth of these tation group gene (FANCI), laminin B1 (LMNB1), and minichro- xenografts to a comparable level. Importantly, RAD140 and mosome maintenance genes 2, 4, and 7 (MCM2, MCM4, and palbociclib when administered together elicited improved effica- MCM7; Fig. 5C, bottom). These decreases were comparable with cy over palbociclib or RAD140 monotherapies. Similarly, those seen with palbociclib alone. In tumors treated with both RAD140 and palbociclib combination produced improved effi- RAD140 and palbociclib, the expression of these genes appeared þ cacy compared with palbociclib alone in an additional AR/ER to be further suppressed. Together, the enhanced effect of PDX model, HBCx-22 (Supplementary Fig. S5A). An additional RAD140–palbociclib combination on the expression of DNA TGI analysis performed for HBCx-3 xenografts treated with replication–related genes may have contributed to the enhanced RAD140 and palbociclib with lower starting volume (108 mm3) inhibition on tumor growth. versus those with higher starting volume (>108 mm3) did not reveal appreciable difference between the two subgroups, suggest- Discussion ing the combination treatment had similar effect in tumors of different starting size. As the combination of RAD140 with This study demonstrates for the first time that RAD140, an palbociclib induced apparent regression in ST897 tumors, we orally available SARM, is an AR agonist in breast cancer cells and þ examined the expression of apoptotic marker cleaved caspase-3 in suppresses the growth and proliferation of multiple AR/ER ST897 and HBCx-3 tumors treated with RAD140, palbociclib, or breast cancer cell line and xenograft models. The AR pathway combination but did not observe clear signs of apoptosis (Sup- was found to be activated in RAD140-treated breast cancer cells plementary Fig. S5B), suggesting a cytostatic but not cytotoxic and xenografts, while genes within the ER pathway, including effect. Together, these results indicate that the combination of ESR1, were suppressed. In addition, RAD140 treatment was found RAD140 with palbociclib led to improved antitumor activity to decrease the expression of DNA replication–related genes in compared with each of these agents given as single agent. breast cancer cells, consistent with previous report that these genes The expressions of AR, ER, PR, Ki67, and phospho-Rb in the were suppressed in androgen-treated prostate cancer cells. Com- HBCx-3 PDX were assessed using IHC (Supplementary Fig. S6). bined administration of RAD140 with the CDK4/6 inhibitor Similar to that seen in HBCx-22 (Fig. 2C), RAD140 treatment led palbociclib was more efficacious compared with either of the to apparent enrichment of AR in the nuclei, suggesting AR acti- agents used alone. These findings suggest a distinct mechanism of vation. ER and PR expression was profoundly decreased in tumors action of RAD140, which includes the AR-mediated suppression þ treated with a RAD140-containing regimen. Palbociclib alone of ESR1 in inhibiting AR/ER breast cancer growth. also appeared to decrease the expression of ER and PR, a finding Accumulating evidence in the recent years has further defined similar to the decrease of ER in breast cancer cells treated with a the role of AR in breast cancer and has led to renewed interests in pan-CDK inhibitor roscovitine (48). The phosphorylation of Rb evaluating AR-targeted agents for breast cancer. The high preva- þ was found to be substantially suppressed in tumors treated with lence of AR in the predominant ER subtype of breast cancers (6), palbociclib as a single agent and in combination with RAD140, clinical benefit rates as high as 39% seen with androgen therapy in while RAD140 alone did not seem to affect phospho-Rb level. breast cancer patients progressing on ER-targeted treatments Ki67 expression was suppressed in tumors treated with RAD140 (23, 24), along with prior experience with steroidal androgens or palbociclib, and this suppression appeared to be further in breast cancer together lend support to the development of a

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Figure 5. Combined administration of RAD140 with CDK4/6 inhibitor inhibited AR/ERþ PDX growth. A, Mean tumor volumes SEM of HBCx-3 breast cancer xenografts treated with vehicle, RAD140 (100 mg/kg twice daily), palbociclib (75 mg/kg once daily), or a combination of RAD140 and palbociclib for the indicated period of time. n ¼ 7/group. Intergroup comparisons were carried out using Student t test based on changes in tumor volume (DTV) from the beginning to the end of the study. For the comparison between RAD140 and vehicle groups, P < 0.05. , the combination group versus palbociclib, P < 0.05; the combination group versus RAD140, P < 0.01. B, Mean tumor volumes SEM of ST897 breast cancer xenografts treated with vehicle, RAD140 (100 mg/kg twice daily), palbociclib (75 mg/kg once daily), or a combination of RAD140 and palbociclib for the indicated period of time. n ¼ 8/group. Statistical analysis was performed as in A. For the comparison between RAD140 and vehicle groups, P < 0.001. , combination group versus RAD140 group, P ¼ 0.0012; , combination group versus palbociclib group, P ¼ 0.0027; C, Tumor samples from the HBCx-3 model, as described in A, were collected 6 hours after last treatment and subjected to qPCR analysis for the expression of AR, ESR1, and their downstream targets, and the genes implicated in DNA replication including BLM, FANCI, LMNB1, MCM2, MCM4,andMCM7. Expression of these genes was normalized to that of ribosomal RNA 18S. Mean normalized expression SD from three individual tumors of each group is presented.

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new generation of oral, nonsteroidal AR agonists for the treatment consistent with the previously described attenuated effect on þ of AR/ER breast cancer. prostate growth (30). It has been proposed that the tissue-selective SARMs are tissue-selective AR agonists by design and may offer AR activity of SARMs is due to the differential allosteric activation þ a novel approach to inhibit the growth of AR/ER breast cancers, of AR compared with classic androgens, which impacts the with substantially attenuated side effects commonly seen with recruitment of coactivators needed for AR-mediated gene tran- classic nontissue selective androgens (32, 49). The activity of scription (52, 53). An earlier study on the selective estrogen RAD140 are AR specific, as evidenced by the similar effects receptor modulator tamoxifen showed that its tissue selectivity observed with DHT and RAD140 on AR and its downstream may be attributed to the differential expression of ER coactivators targets, along with the reversal of these effects by AR antagonists such as SRC-1 in breast versus endometrial cancer cells (54). (Figs. 1C and 3C). In addition, due to its nonsteroidal structure, Similarly, it is conceivable that AR upon binding to RAD140 the SARM RAD140 is not subject to further conversion to estro- assumes a conformation that only allows the interaction with a gens by CYP19 aromatase, or to DHT by 5a-reductase, thus subset of coregulators that are uniquely expressed in breast þ reducing the potential risk of stimulating ER tumor growth or epithelial cells but not in the prostate. increasing virilization. As a proof-of-concept study, we report here Despite the preclinical evidence and favorable clinical outcome þ that RAD140 treatment inhibited the growth of the breast cancer seen with androgens in ER breast cancer (9, 10), improved xenograft models supplemented by exogenous estrogen. Of note, understanding of the underlying mechanism is needed to further RAD140 and its classic androgen comparator, DHT, also inhibited improve the design and development of new AR agonist-based the proliferation of an endocrine-resistant breast cancer cell line therapy. It has been proposed that activated AR suppresses ER model, HCC1428 LTED, suggesting AR agonists may inhibit the signaling by competing with ER for transcriptional coactivators or þ growth of ER breast cancer models with or without estrogen directly competing for binding sites and subsequently leads to supplementation. Furthermore, in multiple PDX models, inhibited cell proliferation (55, 56), suggesting a functional RAD140 inhibited tumor growth as a single agent, and this effect interference between these two nuclear receptors. Here, we show was enhanced by combining with palbociclib. PDX models have AR agonists, including RAD140 and DHT, activate AR target genes been demonstrated to closely recapitulate human tumors with in breast cancer cells in vitro and in vivo, while suppressing a subset regards to tumor heterogeneity and response to standard-of-care of ER target genes. Importantly, we found that treatment with agents (31, 50). Indeed, heterogeneity was observed in the PDX these AR agonists also led to substantial suppression of ER at both models used in this study, as indicated by variable levels of AR, ER, mRNA and protein levels. This suggests that AR may negatively and PR expression, suggesting a good representation of the patient regulate ER signaling by two mechanisms: while AR may compete tumors with a spectrum of expression levels of these nuclear with ER for coactivators (functional interference), it may directly receptors. Also, the growth pattern and response to RAD140 seen downregulate ER expression (direct suppression). The reduction with HBCx-3 and HBCx-22 PDX models, each evaluated in two of ER expression by AR agonists has not been extensively docu- independent studies (Figs. 2A and 5A; Supplementary Figs. S3A mented but can be seen in a study by Poulin and colleagues (57), and S5A), seemed to be consistent. This suggests good reproduc- in which DHT treatment in ZR-75-1 cells for 8 or 12 days led to ibility of these studies. Therefore, the efficacy seen with RAD140 in decreases of ER message by 50% or 65%, respectively. Another these PDX models may be suggestive of the potential therapeutic study also showed reduced ER protein level in ZR-75-1 cells benefit in breast cancer patient population. It is worth noting that treated with DHT, along with decreased expression of classic ER although RAD140 and fulvestrant led to similar tumor growth targets PGR and TFF1 (56), although the mechanism for the inhibition in HBCx-22 tumors, further studies are needed to decrease in ER expression had remained to be further elucidated. evaluate the efficacy of RAD140 relative to that of fulvestrant or Our data show both RAD140 and DHT treatment led to decreased tamoxifen in hormone-dependent models to predict its value in ESR1 expression at as early as 24 hours, which may present a novel first-line setting. It is interesting to note that although HBCx-3 mechanism by which RAD140 suppresses ER signaling and dif- (Supplementary Fig. S6) and HBCx-22 (Fig. 2) xenografts contain ferentiates from traditional ER-targeted agents that act via inhibit- comparable percentage of AR-positive cells, RAD140 induced ing ER protein function or leading to its degradation. This may higher degree of inhibition in growth and Ki-67 expression in translate into unique therapeutic benefit of SARMs in overcoming HBCx-22 than in HBCx-3. This may be due to the presence of resistance to ER-targeted therapy seen in tumors with reactivated þ higher percentage of ER cells in HBCx-22 tumors than in HBCx-3 ER signaling, either via posttranslational modification or enrich- (Supplementary Table S2). As summarized previously, AR ago- ment of ESR1 alterations. The inhibitory effect of RAD140 on the þ nists exhibit inhibitory effect on AR/ER breast cells but not on proliferation of HCC1428 LTED cells shed light on its efficacy in þ AR /ER cells (9, 51). It is conceivable that the varying degree of such E2-independent, albeit ER-active, models but further studies growth inhibition seen with SARM may be attributed to different are needed to fully evaluate the activity of SARMs in similar ER positivity of these models. models in vivo. In addition to the suppression of ER signaling, The tissue selectivity of RAD140 is evidenced by potent andro- our RNA-seq analysis suggests that RAD140 may inhibit tumor gen-like effects in bone and muscle, with much attenuated effects growth via other AR-mediated mechanisms. Of note, RAD140 in other androgen-responsive tissues, such as prostate and sem- treatment suppressed genes implicated in DNA replication and inal vesicles (30). Notably, when administered along with tes- cell-cycle progression, which were further suppressed by concur- tosterone, RAD140 was found to partially antagonize the growth rent CDK4/6 inhibition. Gao and colleagues (41) described the effect of testosterone in prostate and seminal vesicles, suggesting role of AR as a tumor suppressor in castration-resistant prostate that RAD140 acts as a competitive AR ligand with attenuated cancer (CRPC) cells by inhibiting DNA replication–related genes activity. Here, we demonstrated for the first time that the SARM via the recruitment of hypophosphorylated Rb. In addition, the RAD140 is a potent AR agonist in breast cancer cells. It exhibited clinical and preclinical efficacy seen with high-dose androgen much attenuated activity on AR in prostate cancer cells, a finding therapy in CRPC was also attributed to androgen-induced

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stabilization of AR, which prevents the relicensing of DNA for Analysis and interpretation of data (e.g., statistical analysis, biostatistics, replication and subsequent cell-cycle progression (58, 59). In the computational analysis): Z. Yu, S. He, D. Wang, J.L. Brown, G. Hattersley, current study, the AR-mediated suppression of ESR1 and its J.C. Saeh Writing, review, and/or revision of the manuscript: Z. Yu, S. He, D. Wang, downstream pathway, along with the suppression of DNA rep- C.P. Miller, J.L. Brown, G. Hattersley, J.C. Saeh lication- and cell cycle–related genes seen with RAD140 treatment Administrative, technical, or material support (i.e., reporting or organizing þ suggest a distinct mechanism of action of this SARM in AR/ER data, constructing databases): Z. Yu, J.L. Brown breast cancer cells. The potent antitumor activity and tissue- Study supervision: Z. Yu, J.L. Brown, J.C. Saeh selective AR activity, along with overall tolerability in animal models and oral availability together lend support to further Acknowledgments þ clinical investigation of RAD140 in AR/ER breast cancer patients. The authors would like to acknowledge Dr. Steven P. Balk of Beth Israel Deaconess Medical Center and Dr. Changmeng Cai of University of Massachu- setts Boston for their comments on the manuscript. The authors thank Drs. Disclosure of Potential Conﬂicts of Interest Fiona Garner and Teeru Bihani, and Margaret Ward, of Radius Health, Inc., for their comments and suggestions on the project and the manuscript. The authors C.P. Miller and G. Hattersley hold ownership interest (including patents) in Radius Health. No potential conﬂicts of interest were disclosed by the other thank Dr. Jim Humphreys and the histopathology team at Precision Pathology authors. (San Antonio, TX) for the IHC work.

The costs of publication of this article were defrayed in part by the payment of Authors' Contributions page charges. This article must therefore be hereby marked advertisement in Conception and design: Z. Yu, S. He, J.L. Brown, G. Hattersley, J.C. Saeh accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Development of methodology: Z. Yu, S. He, H.K. Patel Acquisition of data (provided animals, acquired and managed patients, Received March 7, 2017; revised July 14, 2017; accepted September 28, 2017; provided facilities, etc.): Z. Yu, S. He, H.K. Patel published OnlineFirst October 3, 2017.

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Selective Androgen Receptor Modulator RAD140 Inhibits the Growth of Androgen/Estrogen Receptor −Positive Breast Cancer Models with a Distinct Mechanism of Action

Ziyang Yu, Suqin He, Dannie Wang, et al.

Clin Cancer Res Published OnlineFirst October 3, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-0670

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2017/10/03/1078-0432.CCR-17-0670.DC1

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