Targeting Bromodomain and Extra-Terminal (BET) Family ... · Jonathan Welti1, Adam Sharp1,2,Wei...

Translational Cancer Mechanisms and Therapy

Targeting Bromodomain and Extra-Terminal(BET) Family Proteins in Castration-ResistantProstate Cancer (CRPC)Jonathan Welti1, Adam Sharp1,2,Wei Yuan1, David Dolling1, Daniel Nava Rodrigues1,Ines Figueiredo1, Veronica Gil1, Antje Neeb1, Matthew Clarke1, George Seed1,Mateus Crespo1, Semini Sumanasuriya1,2, Jian Ning1, Eleanor Knight1, Jeffrey C. Francis1,Ashley Hughes3,Wendy S. Halsey3, Alec Paschalis1,2, Ram S. Mani4, Ganesh V. Raj4,Stephen R. Plymate5, Suzanne Carreira1, Gunther Boysen1, International SU2C/PCFProstate Cancer Dream Team, Arul M. Chinnaiyan6, Amanda Swain1, andJohann S. de Bono1,2

Abstract

Purpose: Persistent androgen receptor (AR) signaling drivescastration-resistant prostate cancer (CRPC) and confers resistanceto AR-targeting therapies. Novel therapeutic strategies to over-come this are urgently required.We evaluatedhowbromodomainand extra-terminal (BET) protein inhibitors (BETi) abrogate aber-rant AR signaling in CRPC.

Experimental Design: We determined associations betweenBET expression, AR-driven transcription, and patient outcome;and the effect and mechanism by which chemical BETi (JQ1 andGSK1210151A; I-BET151) and BET family protein knockdownregulates AR-V7 expression and AR signaling in prostate cancermodels.

Results: Nuclear BRD4 protein expression increases signifi-cantly (P � 0.01) with castration resistance in same patienttreatment-na€�ve (median H-score; interquartile range: 100;100–170) and CRPC (150; 110–200) biopsies, with higherexpression at diagnosis associating with worse outcome

(HR, 3.25; 95% CI, 1.50–7.01; P � 0.001). BRD2, BRD3, andBRD4 RNA expression in CRPC biopsies correlates withAR-driven transcription (all P � 0.001). Chemical BETi, andcombined BET family protein knockdown, reduce AR-V7expression and AR signaling. This was not recapitulated byC-MYC knockdown. In addition, we show that BETi regulatesRNA processing thereby reducing alternative splicing andAR-V7 expression. Furthermore, BETi reduce growth of prostatecancer cells and patient-derived organoids with known ARmutations, AR amplification and AR-V7 expression. Finally,BETi, unlike enzalutamide, decreases persistent AR signalingand growth (P � 0.001) of a patient-derived xenograft model ofCRPC with AR amplification and AR-V7 expression.

Conclusions: BETi merit clinical evaluation as inhibitorsof AR splicing and function, with trials demonstrating theirblockade in proof-of-mechanism pharmacodynamic studies.Clin Cancer Res; 24(13); 3149–62. �2018 AACR.

IntroductionProstate cancer is the most common cancer and second

leading cause of cancer-related death in men in the Westernworld (1). The androgen receptor (AR) remains the main

oncogenic driver for prostate cancer development and progres-sion (2–5). Despite initial robust responses to androgen dep-rivation therapy (ADT), nearly all patients with advanceddisease progress to fatal castration-resistant prostate cancer(CRPC). There is increasing evidence of persistent AR signalingas patients progress to CRPC with rising prostate-specific anti-gen (PSA), increasing steroidogenesis, overexpression of ARcoregulators, and the development of AR aberrations (6–8).The discovery of second-generation antiandrogen therapies,such as abiraterone and enzalutamide, that effectively targetAR signaling in patients with hormone-sensitive disease andCRPC has improved patient outcome (9–13). However, pri-mary and secondary resistance to both therapies is commonand may, in part, be due to the expression of constitutivelyactive AR splice variants of which AR variant 7 (AR-V7) isconsidered the most significant and best studied (14–27).Consistent with this, recent studies have demonstrated thatAR-V7 expression increases as patients progress to CRPC, andassociates with resistance to current AR-directed therapies(14, 15, 22–26). AR-V7 lacks the AR ligand–binding domain(LBD), but contains a cryptic exon 3 (derived from an intron)after AR exon 3 (27). Despite lacking a LBD, AR-V7 continues to

1The Institute for Cancer Research, London, United Kingdom. 2The RoyalMarsden NHS Foundation Trust, London, United Kingdom. 3GlaxoSmithKline,Collegeville, Pennsylvania. 4The University of Texas Southwestern MedicalCenter, Dallas, Texas. 5University of Washington/GRECC/VAPSHCS, Seattle,Washington. 6University of Michigan, Ann Arbor, Michigan.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

J. Welti and A. Sharp are the co-first authors of this article.

A. Swain and J.S. de Bono are the co-senior authors of this article.

Corresponding Author: Johann S. de Bono, The Institute of Cancer Researchand The Royal Marsden NHS Foundation Trust, London SM2 5NG, UnitedKingdom. Phone: 4420-8722-4028; Fax: 4420-8642-7979; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-3571

�2018 American Association for Cancer Research.

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signal through its transcriptionally active N-terminal domainfacilitating ligand independent AR signaling in CRPC andtreatment resistance (17–19, 27). Currently, all licensed ther-apies that modulate AR signaling do so through the AR LBD andhave little or no activity against AR-V7. The development oftherapies that overcome aberrant AR signaling in CRPC is anunmet medical need for treating lethal prostate cancer.

A promising therapeutic approach, currently undergoingclinical evaluation in CRPC patients, is to target the bromo-domain and extra-terminal (BET) family of chromatin readersthat include the bromodomain containing protein (BRD) 2,BRD3, and BRD4 (28). BRD4 is a critical coregulator of AR(29, 30). Furthermore, increased BRD4 protein stability insubstrate-binding adaptor speckle-type POZ protein (SPOP)mutant prostate cancers is associated with AR signaling (31,32). BET inhibitors (BETi) and proteolysis targeting chimera(PROTAC) induced BET protein degraders have antitumoractivity in CRPC models (29, 30, 33–36), attenuating ARsignaling and potently reducing C-MYC expression (29, 30,33, 34, 36). More recently, BETi have been shown to decreaseAR-V7 expression by regulating splicing factors required for itsgeneration (30). Taken together, these preclinical data nowneed to be validated in prospective clinical trials that evaluateBETi as a therapeutic strategy to overcome persistent AR sig-naling in CRPC.

Here, we confirm that nuclear BRD4 protein expressionincreases with castration resistance and associates with patientoutcome. Moreover, BRD2, BRD3, and BRD4 RNA expressionassociates with AR-driven transcription in CRPC clinical tumorbiopsies. We show that BETi reduce AR-V7 protein expression(as does BET family protein siRNA knockdown) by regulatinggenes critical for RNA processing and alternative splicing.Finally, we demonstrate that BETi, unlike enzalutamide, regu-late AR aberrant (driven by AR mutations, AR amplification,and AR-V7 expression) signaling and inhibit the growth ofpatient-derived models of CRPC. These data support furtherinvestigation of BETi in patients suffering from CRPC withpersistent AR oncogenic signaling following next-generationAR antagonists.

Materials and MethodsCell lines and drugs

All cell lines used in this study were grown in recommendedmedia at 37�C in 5% CO2 and are detailed in SupplementaryTable S1. All cell lines were tested for mycoplasma using theVenorGem One Step PCR Kit (Cambio) and STR-profiled usingtheCell authentication service by EurofinsMedigenomix. JQ1wasobtained from Stratech Scientific and GSK1210151A (I-BET151)was a kind gift from GlaxoSmithKline (GSK).

siRNACells were transiently transfected with siRNA as indicated. All

siRNA were ON-TARGETplus pools (Dharmacon; GE Health-care), listed in Supplementary Table S2. The siRNA was usedalong with 0.4% RNAiMax transfection reagent (Thermo FisherScientific) as per manufacturer's instructions and incubated withcells as indicated.

Quantitative reverse transcription PCRCell line RNA was extracted using the RNeasy Plus Mini Kit

(Qiagen) as per manufacturer's instructions. Patient-derivedxenograft (PDX) RNA was obtained by mechanical homogeniza-tion, reconstituted with RNeasy RLT buffer, passed through aQiashredder tube (Qiagen) and further processed with RNeasyPlusMini Kit as above. cDNAwas synthesized using the RevertaidFirst Strand cDNA Synthesis kit (Thermo Fisher Scientific). Quan-titative reverse transcription PCR (qRT-PCR) was carried out onViiA 7 System Real-Time PCR System (Life Technologies) usingthe TaqMan Universal PCR Master Mix (Applied Biosystems).TaqMan probes (Thermo Fisher Scientific) used are listed inSupplementary Table S3. Fold change in mRNA expression levelswas calculated by the comparative Ct method, using the formula2�(�(DDCt).

Western blotCells were lysed with RIPA buffer (Pierce) supplemented with

protease inhibitor cocktail (Roche). PDX lysate was obtained bymechanical homogenization, reconstituted in RIPA buffer. Pro-tein extracts (20 mg) were separated on 4% to 12% NuPAGEBis-Tris gel (Invitrogen) by electrophoresis and subsequentlytransferred onto Immobilon-P PVDF membranes of 0.45-mmpore size (Millipore). Details of primary antibodies used areprovided in Supplementary Table S4. Chemiluminescence wasdetected on the Chemidoc Touch imaging system (Bio-Rad).

Growth assaysGrowth of cells was determined using a modified sulforhoda-

mine B assay (SRB) described in Supplementary Methods (37).

RNA-sequencing, pathway analysis, and alternativesplicing events

Cell RNA was extracted from cells using the RNeasy PlusMini Kit (Qiagen) as per manufacturer's instructions. RNAquality was analyzed using Agilent Bioanalyzer RNA Nano Kit.500 ng of total RNA from each sample was used for Illumina'sTruSeq Stranded mRNA Library Prep Kit. Library quality wasconfirmed using the Agilent Bioanalyzer High SensitivityDNA Assay. The libraries were quantified and normalized byqPCR using KAPA Library Quantification Kit (Roche). Libraryclustering was performed on a cBot with Illumina HiSeq PECluster Kit v3. The libraries were sequenced as paired-end 101

Translational Relevance

Advanced prostate cancer invariably progresses to lethalcastration-resistant prostate cancer (CRPC). Resistance to cur-rent androgen receptor (AR) targeting therapies is associatedwith the development of AR aberrations including the consti-tutively active AR splice variant 7 (AR-V7). Currently, noclinically available therapies effectively inhibit aberrant ARsignaling. BRD4, a bromodomain and extra-terminal (BET)family protein, is a critical AR coregulator. We show that BRD4expression associates with patient outcome and AR-driventranscription in lethal prostate cancer. Moreover, BET inhibi-tors (BETi) reduce AR splicing and AR-V7 expression byregulating alternative splicing, abrogating AR signaling andinhibiting growth of CRPC patient-derived models. Clinicalstudies with BETi in CRPC should pursue pharmacodynamicsstudies evaluating abrogation of AR splicing and persistent ARsignaling to optimize the development of these drugs for thetreatment of CRPC.

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Clin Cancer Res; 24(13) July 1, 2018 Clinical Cancer Research3150




base pair reads on an Illumina HiSeq 2500 with an IlluminaHiSeq SBS Kit v3. Base calling and quality scoring were per-formed using Real-Time Analysis (version 1.18.64) and FASTQfile generation and de-multiplexing using CASAVA. The pairedend raw reads in FASTQ format were aligned to the referencehuman genome (hg19) using RNA sequencing spliced readmapper TopHat (v2.0.7), with default settings (38). The libraryand mapping quality were estimated using Picard tools (http://broadinstitute.github.io/picard). The alternative splicing events(skipped exons, alternative 50 splice sites, alternative 30 splice sites,mutually exclusive exons and retained introns), basedonEnsemblv61 annotation, were accessed using MATS v3.0.8 (39).

Gene expression and AR activity scoreData from 122 CRPC transcriptomes generated by the Inter-

national Stand Up To Cancer/Prostate Cancer Foundation(SU2C/PCF) Prostate Cancer Dream Teamwere downloaded andreanalyzed (3). Paired-end transcriptome sequencing reads werealigned to the human reference genome (GRCh37/hg19) usingTophat2 (v2.0.7). Gene expression, Fragments Per Kilobase oftranscript perMillionmapped reads (FPKM),was calculated usingCufflinks (40). AR activity score is an accumulationmeasurementof AR pathway activity based on 43 genes (Supplementary TableS5) regulated by AR in prostate cancer cell lines and metastaticprostate cancer (5, 41).

PDX development CP50Ametastatic lymphnode biopsy fromapatient withCRPCwho

had received all standard of care therapies for prostate cancer wasdivided and implanted subcutaneously into two non-obese dia-betic (NOD) scid gamma (NSG) (JAX Mice) male mice (termedCP50 PDX). Tumor growth was observed 6 months after implan-tation. Passaging of tumors was performed by implanting tumorfragments of 3� 3� 3 mm subcutaneously into male NSGmice.To generate a castrate CP50 PDX line, tumors that had beenpassaged four times were castrated when they reached 300 to400 mm3 and were harvested when they reached a size of around1200 mm3. Subsequent passages were done in castrated mice.Individual CP50 PDX experiments are detailed in SupplementaryMethods. All animal studies were ethically reviewed and carriedout in accordance with Animals (Scientific Procedures) Act 1986.

Patient-derived organoid and PDX-organoid developmentPatient-derived organoid (PDO) and CP50 PDX-organoid

(PDX-O; from CP50 PDX) were generated and maintained aspreviously described (42). PDOswere successfully generated from22 of 36 (61%) metastatic CRPC biopsies between October 2015and April 2016; nine of which were available for growth experi-ments. Briefly, freshmetastatic CRPCbiopsies or CP50 PDX tissuewere washed in phosphate-buffered saline (PBS) supplementedwith 0.5% BSA, 10 mmol/L ROCK inhibitor (AbMole) and sub-sequently minced into small pieces followed by collagenase IIdigestion (1:250 dilution; Thermo Fisher Scientific) for 30 to 45minutes at 37�C. Digested biopsies were then washed in PBSsupplemented as above and crude cell suspension was furtherseparated into single cells using a 20-mm cell strainer (BD Bios-ciences). The resulting single cells were collected by centrifugationand suspended in organoid medium before mixing with growthfactor–depleted Matrigel (Corning). For PDO and CP50 PDX-Ogrowth experiments, the mixture was plated in 5-mL drops in 96-well optical plates. Drug treatment was started 24 hours after

seeding and growth was determined using CellTiter-Glo(Promega) after 7 days of treatment. For CP50 PDX-O RNAanalysis, the mixture was plated in 30-mL drops in 24-well tissueculture plates. Drug treatment was started 24 hours after seedingand organoids were harvested in cold PBS after 48 hours oftreatment. RNA extraction was performed using the Quick-RNAMini Prep Kit (Zymo Research) according to the manufacturer'sprotocol and RNA analysis performed as described above.

Patients and tissue samplesPatients were identified from a population of men with CRPC

treated at the Royal Marsden NHS Foundation Trust. All patientshad given written informed consent and were enrolled in insti-tutional protocols approved by the Royal Marsden NHS Foun-dation Trust Hospital (London, United Kingdom) ethics reviewcommittee (reference No. 04/Q0801/60). Human biologicalsamples were sourced ethically and their research use was inaccord with the terms of the informed consent provided. Patientsand tissue samples identified can be found in SupplementaryMethods.

Tissue analysisIHC for BRD4, AR-V7, and full-length AR (AR-FL) was per-

formedonpatient samples andCP50PDXas described previously(Supplementary Methods; refs. 25, 26, 43).

Statistical analysisAll statistical analyses were performed using Stata v13.1 or

GraphPad Prism v6 and are indicatedwithin all figures and tables.Detailed methods for clinical statistical analysis can be found inSupplementary Methods.

ResultsBRD4 protein expression at diagnosis associates with prostatecancer patient overall survival and increases further as patientsdevelop castration-resistant disease

To investigate the clinical significance of nuclear BRD4 expres-sion in prostate cancer we evaluated tumor samples from 53patients, 15 with only diagnostic (archival) hormone-sensitiveprostate cancer (HSPC) biopsies and 38 patients with matchedHSPC and CRPC biopsies (Supplementary Fig. S1). BRD4 anti-body specificity for IHC was confirmed using BRD4 knockdownand overexpression in LNCaP95 cells (Supplementary Fig. S2).H-scores (HS) were determined by IHC for nuclear BRD4 expressionin all patient biopsies (Fig. 1A); median nuclear BRD4 expressionat diagnosis in 53 HSPC biopsies was 100 [interquartile range(IQR): 100–150] with BRD4 expression (continuous variable; per100 HS) being significantly associated with time to castrationresistance (TTCRPC;HR, 2.16; 95%CI, 1.25–3.73; P¼ 0.006) andoverall survival (OS; HR, 2.23; 95% CI, 1.23–4.02; P ¼ 0.008).Furthermore, patients with high nuclear BRD4 expression (HS �100; n ¼ 41) had a significantly shorter median TTCRPC[1.8 years vs. 9.1 years; HR, 4.75; 95% confidence interval (CI),2.11–10.68; P � 0.001) and OS (5.5 years vs. 12.6 years; HR,3.25; 95% CI, 1.50–7.01; P � 0.001) compared to patients withlow nuclear BRD4 expression (HS < 100; n ¼ 12; Fig. 1B and C).Patients with low BRD4 expression at diagnosis had lower inci-dence of lymph node involvement (P ¼ 0.006) and fewer pre-sented with distant metastasis, although not statistically signifi-cant (P¼ 0.12); andweremore likely to receive curative treatment

Targeting BET Family Proteins in CRPC

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(P ¼ 0.002; Supplementary Table S6). Thirty-eight patients withmatched HSPC and CRPC biopsies were used to investigate theclinical significance of nuclear BRD4 expression as patients devel-opedCRPC.Nuclear BRD4 expression increased significantly (P¼0.008) as patients progressed from HSPC (median; IQR: HS 100;100–170) to CRPC (HS 150; 110–200; Fig. 1D). Nuclear BRD4expression (continuous variable; per 100 HS) in CRPC biopsieswas not significantly associatedwithOS (HR, 0.63; 95%CI, 0.32–1.24; P ¼ 0.18). Although SPOP-mutant prostate cancer havebeen associated with increased BRD4 protein expression, wefound no clear association between SPOP mutations (4 of 39cases with SPOP status) and BRD4 protein expression (Supple-mentary Fig. S3; refs. 31, 32). Taken together, these data suggestthat higher nuclear BRD4 expression at diagnosis, but not CRPC,is associated with poorer patient outcome. In addition, nuclearBRD4 expression increases as patients progress from HSPC toCRPC, suggesting a role in disease progression and treatmentresistance.

BET proteins associate with AR activity in castration-resistantprostate cancer

In light of BRD4 protein levels increasing as patients developCRPC, and its association with AR signaling, we next determinedwhether BRD4 and other BET familymembers associated with ARactivity in CRPC patient samples (29, 30, 34). AR activity scorewas determined using two previously described gene expressionsignatures (Supplementary Table S5; refs. 5, 41). Analysis ofRNA-sequencing (RNA-seq) data obtained from 122 CRPCbiopsies demonstrated BRD2 to be most highly expressed inCRPC compared to BRD3, BRD4, and C-MYC, a downstreamtarget of BET proteins (Fig. 1E; ref. 44). BRD2, BRD3, BRD4,and, to a lesser extent, C-MYC RNA expression all significantlycorrelated with AR activity score in CRPC biopsies (all P �0.001; Fig. 1F–I). B2M, a frequently used housekeeping controlgene, did not associate with AR activity score (P ¼ 0.6; Fig. 1J).Taken together these results indicate that BRD2, BRD3, andBRD4 are expressed and associate with AR activity in patientswith CRPC.

I-BET151 downregulates AR-V7 and inhibits AR signaling inprostate cancer cell lines

BETis have been reported to regulate full length AR (AR-FL)signaling, and expression of AR splice variants (including AR-V7)implicated in CRPC development and resistance to current ther-apies (14, 15, 22–26, 29, 30, 34). To investigate this further, wedetermined the effect of I-BET151, a potent inhibitor of BRD2,BRD3, and BRD4, on AR-FL and AR-V7 expression, and ARsignaling in BRD4 and AR-V7 expressing prostate cancer cell lines(Supplementary Fig. S4; ref. 45). LNCaP95 (androgen indepen-dent and enzalutamide resistant), 22Rv1 (androgen indepen-dent), and VCaP (androgen dependent) cells were treated

with increasing concentrations of I-BET151 for 48 hours priorto analysis by quantitative real-time PCR (qRT-PCR) andWesternblot. I-BET151 significantly reduced AR-V7 and C-MYC expres-sion with only higher concentrations of I-BET151 having limitedimpact on AR-FL levels in LNCaP95 and VCaP cells (Fig. 2A andC). Furthermore, I-BET151 treatment significantly reduced expres-sion of AR-regulated transcripts (PSA and TMPRSS2; Fig. 2B andD). Interestingly, I-BET151 did not significantly impact AR-V7 orAR-FL expression in 22Rv1 cells but still reduced C-MYC expres-sion and inhibited AR signaling (Fig. 2E and F). This may, at leastin part, be due to the association of a genomic rearrangementwithabrogated AR generation in 22Rv1 cells and suggests that I-BET151–mediated AR-V7 regulation may not be through tran-scriptional regulation alone (46). A second BETi, JQ1, was used toconfirm these data (Supplementary Fig. S5). Furthermore, weinvestigated whether I-BET151 treatment was associated with achange in drug target expression. BRD2, BRD3, and BRD4 (VCaPcells), and BRD2 (LNCaP95 cells), RNA levels increased inresponse to I-BET151 treatment (Supplementary Fig. S6A andS6B). In contrast, I-BET151 exposure led to decreased BRD3 andBRD4 RNA expression in LNCaP95, and no significant change inBRD2, BRD3, and BRD4 RNA expression in 22Rv1 (Supplemen-tary Fig. S6A and S6C). In summary, these data suggest, as withJQ1 and other BETis, that I-BET151 regulates AR signaling inandrogen-dependent VCaP cells but also in androgen-indepen-dent LNCaP95 and 22Rv1 cells. However, the ability of I-BET151to regulateAR-V7 expression seems specific to LNCaP95andVCaPcells, which have not been described to have a genomic rearrange-ment of AR.

BRD2, BRD3, and BRD4 knockdown recapitulatesAR-V7–mediated regulation of I-BET151 in aC-MYC–independent manner

I-BET151 is a potent inhibitor of BRD2, BRD3, and BRD4activity, and is known to have downstream inhibitory effects onC-MYC expression (44, 45). Having shown that BRD2, BRD3,BRD4, and C-MYC are expressed in CRPC biopsies, and correlatewith AR transcriptional activity, we proceeded to investigatewhich BET family members were critical for the AR-V7 mediatedeffects of I-BET151 in prostate cancer cells. We determined theeffect of individual (BRD2 or BRD3 or BRD4) and combined BETprotein knockdown by siRNA for 72 hours on AR-FL and AR-V7expression, C-MYC expression, and AR signaling in these cell lines(Fig. 3A–F; Supplementary Fig. S7). In both LNCaP95 (Fig. 3A–C)and VCaP (Fig. 3D–F) cells, knockdown of BRD2, BRD3, andBRD4 individually, or in combination, did not reduce AR-FLexpression significantly. Combined knockdown of BRD2, BRD3,and BRD4, to a greater extent than BRD4 knockdown alone,reduced AR-V7 expression in both cell line models (Fig. 3A–F).Consistent with this, treatment of LNCaP95 andVCaP cells with I-BET151 reduced AR-V7 expression (Fig. 3A, C, D, and F).

Figure 1.BET family protein expression, association with AR signaling, and patient outcome in prostate cancer. A, Representative micrographs of BRD4 detection by IHCin matched hormone sensitive prostate cancer (HSPC) and CRPC biopsies (magnification 200�; scale bar 50 mm). B and C, Kaplan–Meier curves of time toCRPC (TTCRPC) (B) and OS (C) from diagnosis for nuclear BRD4 expression at diagnosis (HSPC) by H-score < 100 (green; n¼ 12) or�100 (orange, n¼ 41). Hazardratio (HR) with 95% CIs and P values for univariate Cox survival model are shown. D, Expression (H-score) of nuclear BRD4 expression in 38 matched patientsamples at HSPC (gray) and CRPC (red). Median H-score and interquartile range is shown. P-value (� , P � 0.05; �� , P � 0.01; �� , P � 0.001) was calculatedusingWilcoxon matched-pair signed-rank test. E, BRD2, BRD3, BRD4, and C-MYC RNA expression in fragments per kilobase of transcript per million mapped reads(FPKM) for 122 CRPC transcriptomes are shown. Median FPKM and interquartile range is shown. F–J,Association between BRD2 (F), BRD3 (G), BRD4 (H), C-MYC (I),and B2M (J) expression levels and androgen receptor AR signaling score (derived from 43 AR regulated genes; see Supplementary Table S6) from 122 CRPCtranscriptomes are shown. P values were calculated using linear regression analysis.






Figure 2.

I-BET151 treatment downregulates AR-V7 andAR signaling in androgen-dependent and -independent prostate cancer cell lines. LNCaP95 (A andB), VCaP (C andD),and 22Rv1 (E and F) were treatedwith vehicle (DMSO0.1%) or various concentrations of I-BET151 (0.1, 0.5, 1.0, 2.5, and 5.0 mmol/L) for 48 hours. The effect of I-BET151treatment on AR full-length (AR-FL), AR variant 7 (AR-V7), and C-MYC protein expression (A, C, E) and AR-FL, AR-V7, C-MYC, PSA, and TMPRSS2 RNAexpression was determined (B, D, F). Single representative Western blot shown from three separate experiments. Mean RNA expression (normalized to B2Mand vehicle; defined as 1.0) with standard deviation from three individual experiments is shown (unless otherwise stated). P values (� , P � 0.05; �� , P � 0.01;�� , P � 0.001) were calculated for each I-BET151 dose compared to vehicle (DMSO 0.1%) using unpaired Student t test.

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Interestingly, I-BET151 treatment consistently reduced PSAexpression in both VCaP and LNCaP95 cells, whereas BET proteinknockdown had no significant effect (Fig. 3C and F). In contrast,TMPRSS2 expression was not consistently reduced by I-BET151(as shown previously; Fig. 2B andD). To determine whether theseI-BET151 effects were through a C-MYC dependent mechanism,we analyzedC-MYC knockdown in LNCaP95 and VCaP cells (Fig.3A, B, D, and E). As expected, I-BET151 treatment, and combinedBRD2, BRD3, and BRD4 protein knockdown, decreased C-MYCexpression. Interestingly, however, knockdown of C-MYC alonehad no significant effect on AR and AR-V7 but significantlyincreased PSA and TMPRSS2 expression (Fig. 3A–F). Takentogether, these data demonstrate that although BRD4 knockdownalone is able to regulate AR-V7 expression and AR signaling,independent of C-MYC activity, the results are more pronounced

when BRD2, BRD3, and BRD4 are knocked down together,consistent with I-BET151 targeting these BET proteins and func-tional redundancy between family members.

I-BET151 inhibits alternative splicing in prostate cancer cellsTo further understand the mechanism of I-BET151–mediated

AR-V7 regulation in CRPC cells, we determined its effects on geneexpression and associated cellular pathways. LNCaP95 cells weretreated with I-BET151 (0.5 and 2.0 mmol/L) for 48 hours andRNA-seq analysis was performed. Prior to analyzing these data,I-BET151 regulation of AR-FL, AR-V7, C-MYC, andPSA expressionwas confirmed (as an experimental control; SupplementaryFig. S8). Consistent with previous reports, I-BET151 regulatedgenes involved in chromatin remodeling and AR signaling (Sup-plementary Table S7). In addition, I-BET151 regulated genes

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BET family protein knockdown recapitulates I-BET151 effects on AR-V7 expression and AR signaling independent of C-MYC expression. LNCaP95 (A–C) and VCaP(D–F) were transfected with 50 nmol/L control, BRD2, BRD3, BRD4, or C-MYC siRNA (LNCaP95 A and B; VCaP D and E); or 150 nmol/L control or 150 nmol/L(50 nmol/L each) BRD2, BRD3, and BRD4 siRNA with I-BET51 (0.5 mmol/L) or without (vehicle; DMSO 0.1%) for 72 hours (LNCaP95 A and C; VCaP D and F).The effect of each condition on AR-FL, AR-V7, C-MYC, and PSA protein expression (LNCaP95 A; VCaP D) and AR-FL, AR-V7, C-MYC, PSA, and TMPRSS2RNA expression was determined (LNCaP95 B and C; VCaP E and F). Single representative Western blot shown from three separate experiments. MeanRNA expression (normalized to B2M and control siRNA at equivalent concentration; defined as 1.0) with standard deviation from three individual experimentsis shown. P values (� , P � 0.05; �� , P � 0.01; �� , P � 0.001) were calculated for each condition compared to control siRNA (at equivalent concentration) usingunpaired Student t test.






Figure 4.

I-BET151 decreases AR-V7 expression by decreasing alternative splicing events. LNCaP95 cells were treatedwith vehicle (DMSO0.1%) or I-BET151 (0.5 or 2.0 mmol/L)for 48 hours prior to RNA-seq analysis being performed (single experiment in duplicate). A, Unsupervised cluster and heatmap of 137 significantly (determined bynormalized reads count difference >2.0 or <2.0 and false discovery rate <0.05) changed alternative splicing events induced by I-BET151 treatment. B, Sashimiplot represents reducedAR-V7 cryptic exon expression after I-BET151 treatment. Arcs representing splice junctions connect exons. The bridge number between exon3 and cryptic exon in intron 3 is the AR-V7 expression level. C, Alternative splicing events are schematically represented and histograms of alternative splicingmean differences between vehicle (blue dotted line; defined as 0.0) and I-BET151 (0.5 or 2.0 mmol/L) in five categories of alternative splicing events areshown (left shift; decrease in splicing events).

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whose proteins are implicated in the spliceosome and critical toRNA processing, suggesting that it regulates AR-V7 expressionthroughmodulation of alternative splicing. To further support thishypothesis, we demonstrated that treatment with I-BET151 led to adose-dependent change in alternative splicing events (Fig. 4A).Treatment with 2.0 and 0.5 mmol/L I-BET151 led to significantchanges (determined by normalized reads count difference >2.0 or<2.0 and false discovery rate <0.05) in 137 alternative splicingevents (involving 119 genes) and 47 alternative splicing events

(involving 41 genes), respectively (Supplementary Table S8). Inboth cases, the majority of the significant changes in alternativesplicing events (70% and 64%; 2.0 and 0.5mmol/L) decreasedwithI-BET151 treatment (Supplementary Table S8). Furthermore, RNA-seq analysis confirmed that I-BET151 reduced AR-V7 cryptic exonexpression (Fig. 4B) and reduced alternative splicing events inLNCaP95 cells (Fig. 4C). These data suggest that I-BET151-medi-ated regulation of AR-V7 expression is through inhibition ofalternative splicing events in prostate cancer.

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BET family protein knockdown and I-BET151 inhibits the growth of androgen-dependent and -independent prostate cancer cell lines. LNCaP95 (A and B) and VCaP(D and E) were transfected with 50 nmol/L control, BRD2, BRD3, BRD4, or C-MYC siRNA; or 150 nmol/L control or 150 nmol/L (50 nmol/L each) BRD2, BRD3 andBRD4 siRNA with I-BET151 (0.5 mmol/L) or without (vehicle; DMSO 0.1%) I-BET151 and growth determined after 7 days using sulforhodamine B (SRB) colorimetricassay. LNCaP95 (C) and VCaP (F) were treated with vehicle (DMSO 0.1%) or various concentrations of I-BET151 (0.1, 0.5, 1.0, 2.5, and 5.0 mmol/L) and growthdetermined after 7 days by SRB colorimetric assay. For all experiments (A–F), mean growth (normalized to control siRNA at same concentration or vehicle)with standard deviation from four individual experiments is shown. P values (� , P� 0.05; �� , P� 0.01; �� , P� 0.001) were calculated for each condition compared tocontrol siRNA (at same concentration) or vehicle using unpaired Student t-test.






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Regulation of BET protein activity and C-MYC expression by I-BET151 is critical for prostate cancer growth inhibition

BETis have been shown to inhibit the growth of AR-depen-dent and -independent prostate cancer models (29, 30, 33–35).Next, we determined the specific target of I-BET151 critical forprostate cancer cell growth inhibition. I-BET151 inhibited thegrowth of LNCaP95 and VCaP cells in a dose-dependentmanner (Fig. 5C and F). BRD4 knockdown alone, and BRD2,BRD3, and BRD4 knockdown in combination inhibited thegrowth of LNCaP95 and VCaP cells (Fig. 5A, B, D, and E;Supplementary Fig. S9). Moreover, I-BET151 treatment syner-gized (LNCaP95, P ¼ 0.01; VCaP, P ¼ 0.002) with combinedBRD2, BRD3, and BRD4 knockdown to further decrease pros-tate cancer cell growth (Fig. 5B and E). Having confirmed that I-BET151 inhibition of AR signaling and downregulation of AR-V7 expression was independent of C-MYC, we next identifiedthat C-MYC knockdown was sufficient to inhibit LNCaP95 andVCaP cell growth (Fig. 5A and C). As previously described,these data are consistent with BETi regulation of C-MYCexpression being important for prostate cancer cell growthinhibition (33).

I-BET151 regulates AR-V7 and C-MYC expression, and inhibitsAR signaling and proliferation in CRPCpatient-derivedmodels

We and others have shown that BETi regulate the growth ofprostate cancer cell line models in vitro (Fig. 5C and F; refs. 29, 30,33–35).We sought to determine if I-BET151 inhibited the growthof nine PDOs derived frommetastatic biopsies (five lymph nodesand four bone marrow trephines) from patients with advancedCRPC who had confirmed AR aberrations (2.4 treatments: meanstandard treatments for CRPC; Supplementary Fig. S10; Supple-mentary Tables S9 and S10). Growth of three out of nine (33.3%)PDOs were inhibited by >50% with I-BET151 (Fig. 6A). Therewere no significant differences between clinical and molecularcharacteristics (including SPOP status and BRD4 protein expres-sion) of patients that these PDOs were derived from (Supple-mentary Fig. S10; Supplementary Tables S9 and S10). To allowfurther investigation of I-BET151 in patient-derived models, wedeveloped a PDX (CP50) that provided material for PDX-O(Supplementary Fig. S11A). CP50 was derived from the lymphnode biopsy of a patient with CRPC that had progressed throughall standard-of-care treatments (Supplementary Fig. S11B).Whole-exome sequencing of a lymph node biopsy (performed

6months prior to the PDXparental biopsy) from the samepatientandCP50PDX confirmed similar copy number changes includinggain in chromosome 8 (MYC locus) and AR amplification but noAR or SPOP mutations (Supplementary Fig. S11C). Treatment ofCP50 PDX-O with I-BET151 downregulated AR-V7 and C-MYCexpression, and inhibited PDX-O growth (Fig. 6B and C). Incontrast, enzalutamide did not reduce PDX-O growth, consistentwith its parental biopsy being from a patient with CRPCwho haddeveloped resistance to enzalutamide (Fig. 6B and C). AR-V7expression increases as prostate cancer patients progress to CRPCanddevelop treatment resistance (14, 15, 22–26).Consistentwiththis, castration of CP50 PDX for 14 days significantly increasedAR-V7 expression, with a less pronounced impact on AR-FL, C-MYC expression, and AR signaling (Fig. 6D and E; SupplementaryFigs. S12A, S13A, and S13B). Having shown I-BET151 reducedAR-V7 expression and alternative splicing events in prostatecancer cell lines, we explored whether I-BET151 could repressAR-V7 generation in this model. I-BET151 significantly reducedAR-V7 mRNA expression and reduced AR-V7 protein expression(Fig. 6D and E; Supplementary Figs. S12A and S13A and S13B). I-BET151 also reduced C-MYC, PSA, and (to a lesser degree)TMPRSS2. Finally, we determined the effect of I-BET151 andenzalutamide in CP50 PDX grown in castrated mice for 40 days(Supplementary Fig. S12B). I-BET151, but not enzalutamide,significantly reduced tumor growth when compared to vehicle(Fig. 6F), and significantly prolonged survival (Fig. 6G). Thesedata indicate that I-BET151 reduces the expression of AR-V7 andC-MYC, thereby inhibiting AR signaling and tumor growth inadvanced, treatment resistance, CRPC.

DiscussionAdvanced prostate cancer responds to ADT, but invariably

progresses to lethal CRPC with incontrovertible evidence ofongoing AR signaling. This has led to advances in antiandrogentherapy, with the successful development of abiraterone andenzalutamide (9–13). Despite these advances, primary resistanceis common and secondary resistance on treatment inevitable inCRPC, in part due to expression of constitutively active AR splicevariants that evade current antiandrogen treatment strategies(14, 15, 21–26). One promising strategy undergoing clinicalevaluation in CRPC is targeting BET family proteins (Clinical-Trials.gov identifier: NCT02711956, NCT03150056). Several

Figure 6.I-BET151 decreases AR-V7 expression and inhibits growth in prostate cancer patient-derived models. A, Nine PDO were developed from metastatic biopsies andtreated with vehicle (DMSO 0.1%) or I-BET151 (0.5 or 5.0 mmol/L) and growth determined after 7 days using CellTiter-Glo assay. Mean growth (normalized tovehicle; defined as 1.0) with standard deviation from one experiment performed in duplicate is shown (unless otherwise stated). � , Low organoid number. B, PDX-Ofrom PDX CP50 was treated with vehicle (DMSO 0.1%) or 1.0 mmol/L I-BET151 or 10 mmol/L enzalutamide for 7 days. Mean growth (normalized to vehicle;defined as 1.0) with standard deviations from one experiment performed in triplicate is shown. P values (� , P� 0.05; �� , P� 0.01; �� , P� 0.001) were calculated foreach condition compared to vehicle (DMSO0.1%) using unpaired Student t-test.C,AR-FL, AR-V7, andC-MYCRNAexpressionwas determined.MeanRNAexpression(normalized to B2M and vehicle; defined as 1.0) is shown from a single experiment.D and E, PDX CP50micewere either left intact (n¼ 2; unshaded box) or castrated(n¼8; shadedbox) for 7 daysprior to being treatedwith either vehicle (two intactmice and four castratedmice)or 15mg/kg I-BET151 (four castratedmice) for 11 days.The effect of each condition on AR-FL and AR-V7 protein expression (D) and AR-FL, AR-V7, C-MYC, PSA, and TMPRSS2 RNA expression wasdetermined (E). Mean RNAexpression (normalized to B2M and vehicle-castrate; defined as 1.0)with standard deviation from the number ofmice described is shown.P values (� , P� 0.05; ��, P� 0.01; �� , P� 0.001) were calculated for each condition compared to vehicle-castrate using unpaired Student t test. F andG, PDX CP50were castrated and once tumor volume reached 300mm3 treatedwith either vehicle (n¼6) or 15mg/kg I-BET151 (n¼ 7) or 10mg/kg enzalutamide (n¼ 6) daily untilreaching 300% of starting volume or 40 days treatment. Mean growth (normalized to start; defined as 100%) with standard deviation was determined foreach tumor (F). P values (ns, nonsignificant; � , P� 0.05; ��, P� 0.01; �� , P� 0.001) were calculated using a longitudinal mixed effect model with per mouse randomintercept and slope. Time to reach 300% growth was used as a surrogate endpoint for survival (G). One mouse in both the I-BET151 and enzalutamidetreatment group was culled before 300% growth (^^, 294% and ^, 266%; as reached legal limit due to increased baseline volume) and censored for analysis. MedianOS, HR with 95% CIs, and P values for univariate Cox survival model are shown. Median OS for I-BET151 was not reached (NR) at 40 days treatment.






BETi, and PROTAC induced BET protein degraders have shownpromising efficacy in preclinical models of CRPC, regulating ARsignaling and AR-V7 expression (29, 30, 33–36). Although themechanismbywhich BET inhibition regulates AR signaling iswelldescribed, the ability to regulate expression of AR-V7 is not yetunderstood (29, 30, 34–36).

BET family proteins are expressed in prostate cancer (35). Herewe show that BET family proteins associate with AR activity inclinical samples and that BRD4 protein expression increases ascastration-resistant disease develops. In addition, we show thatlower BRD4 protein expression is associated with improvedpatient outcome at diagnosis but not CRPC. This is likely toreflect the demonstration that patients with lower BRD4 expres-sion are more likely to present with local disease (lymph nodenegative, no distant disease) and receive radical treatment; anobservation that warrants further investigation. SPOP mutantcancers have been identified as a subgroup of prostate cancerwith increased BRD4 expression (31, 32). Although we wereunable to confirm these findings, this is likely explained by thesmall numbers (four cases) of SPOP-mutant cases and differentpatient population. These patient-deriveddata support preclinicalstudies demonstrating BRD4 to be a critical coregulator of ARactivity and identifying its potential role in treatment resistance(29, 30, 35, 36). BETi and PROTAC-induced BET protein degra-dation have been reported to regulate AR signaling and morerecently AR-V7 expression (29, 30, 35, 36). Consistent with this,we demonstrate that the BETis, I-BET151 and JQ1, inhibit ARsignaling and reduce AR-V7 expression in VCaP and LNCaP95cells. Interestingly, both inhibitors had no effect on AR-V7 expres-sion in 22Rv1 cells, probably due to its generation in this model,in part, resulting fromAR genomic rearrangements, as opposed toalternative splicing (15, 46, 47). These data suggest that I-BET151–mediated AR-V7 regulation may be through the inhibi-tion of alternative splicing, and indicate that patient selection fortreatment with these agents should consider AR-V7 positive butAR genomic rearrangement negative tumors (30).

BETi abrogation of AR signaling is well described (29). How-ever, themechanismbywhich BETi regulate AR-V7 expression hasnot been fully explored (30, 36). Importantly, our study demon-strates that simultaneous knockdown of BRD2, BRD3, and BRD4is more effective at downregulating AR-V7 expression than indi-vidual BET family protein knockdown, suggesting a degree offunctional redundancy between BET family proteins (45). Asexpected, combined BET family protein knockdown regulatesC-MYC expression. However, I-BET151-mediated C-MYC regula-tion was not needed for abrogation of AR signaling and reducedAR-V7 expression; indeed,C-MYCknockdown alonehadno effecton AR-V7 expression and increased AR signaling. These dataindicate that in BETi early clinical trials the pharmacodynamicconfirmation of downregulated AR signaling, C-MYC and AR-V7in CRPC serial biopsies should be demonstrated to support dose/schedule selection.

AR-V7 is transcribed from alternatively spliced transcripts of ARmRNA (48). Although not fully understood, BETi have beenshown to regulate expression of splicing factors possibly key toAR-V7 generation (30, 49). Critically, we show that I-BET151regulates the expression of previously undescribed genes associ-ated with the spliceosome complex and genes important for RNAprocessing. Furthermore, we present for the first time thatI-BET151 regulates AR-V7 transcript expression and alternativesplicing events in prostate cancer cells. Taken together, these data

provide further evidence that BETis inhibit AR-V7 generationthrough regulationofRNAprocessing and inparticular alternativesplicing (30, 49). Aberrant splicing has been implicated in thepathogenesis of other (beyond prostate cancer) malignant andnonmalignant disease processes (50). In light of our findings,further studies are now warranted to determine whether BETisprovide a novel therapeutic approach to inhibit RNA processingand abrogate development/progression of these diseases.

I-BET151 inhibited the growth of prostate cancer cell lines.Interestingly, I-BET151 treatment modulated BRD2, BRD3, andBRD4 expression in prostate cancer cell lines, which may provideearly suggestions toward a mechanism of resistance to BETitherapy. Consistent with this; I-BET151 treatment alongside com-bined BET protein knockdown (preventing upregulation) wasmore potent than I-BET151 treatment alone. Early clinical trialswill need to quickly establish whether these data are recapitulatedin CRPC patients supporting its role as a resistancemechanism. Inaddition to prostate cancer cell lines, I-BET151 inhibited thegrowth of PDOs derived from fresh metastatic CRPC patientswho had progressed despite abiraterone and/or enzalutamidetreatment. These data suggest that BETis may provide therapeuticapproaches to overcome AR-resistant mechanisms in CRPCincluding AR amplification and AR-V7 expression that associatewith worse outcome and resistance to abiraterone and/or enza-lutamide (6, 14, 15, 22–26, 47). To further investigate this, wedeveloped a PDX-O and PDX (CP50) from a patient who hadprogressed on both abiraterone and enzalutamide with tumor ARamplification and AR-V7 protein expression. I-BET151 inhibitedthe growth of this CP50 PDX-O and downregulated AR-V7expression. Consistent with the presence of AR amplification andAR-V7 expression, enzalutamide had no growth-inhibitory effectsin this model. These data support the study of hypothesis-testingclinical trials with BETis to overcome persistent AR signaling inCRPC (29, 30, 36).

Studies have shown that AR-V7 expression increases aspatients develop resistance to endocrine therapies (15,21–26). Consistent with this apparent induction of AR-V7 inresponse to ADT, our CP50 PDX model demonstrated anincrease in AR-V7 expression upon castration that was blockedby I-BET151. These data suggest that I-BET151 can reduce AR-V7 expression, consistent with previous studies of BETis andPROTAC-induced BET protein degraders in cell-based models(30, 36). Finally, I-BET151 inhibited the growth of castrateCP50 PDX, which is enzalutamide resistant, supporting studiesassociating AR aberrations with these drug-resistant pheno-types. Although the studies were performed in a single PDX-O/PDX model, it represents a common prostate cancer patientpopulation that currently has no standard therapies with prov-en efficacy and represents an area of unmet clinical need. Thesedata provide further evidence that BETis may have clinicalutility in this patient population (15, 21–26).

In conclusion, our study supports the clinical interrogation ofBETis in patients with CRPC resistant to abiraterone and/orenzalutamide. It is likely with the repositioning of abirateroneto treating first line, advanced HSPC, that as patients developCRPC the incidence of AR-V7–positive disease will increase (9).Therefore, the development of therapies that overcome thesemechanisms of persistent AR signaling is an area of urgent unmetclinical need. BETis show promise, but ongoing clinical studieswill need to: (i) demonstrate their safety profile in light of theplethora of cellular processes they have been reported to regulate;

Welti et al.





(ii) confirm pharmacodynamic regulation of AR signaling andAR-V7 (and other splice variants) inCRPC tissue at a safe dose andschedule not causing dose-limiting thrombocytopenia; and (iii)induce durable anticancer responses. If these clinical trials providethis data, then BETis have the potential to provide a noveltherapeutic strategy to overcome AR aberrant signaling in CRPCand improve patient outcome beyond current clinically approvedtherapies.

Disclosure of Potential Conflicts of InterestG.V. Raj reports receiving speakers bureau honoraria from Janssen, Bayer,

Astellas, and Pfizer, and holds ownership interest (including patents) inGuardiumRx and EtiraRx. A.M. Chinnaiyan holds ownership interest (includingpatents) in and is a consultant/advisory board member for Oncofusion andOncopia. J.S. de Bono reports receiving speakers bureau honoraria fromAstraZeneca, and is a consultant/advisory board member for AstraZeneca;Glaxo, Smith, Kline; Genentech/Roche;MSD;Merck Serono;Menarini; Janssen;Pfizer Oncology; Daiichi Sankyo; and Sanofi Aventis. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: J. Welti, A. Sharp, A. Swain, J.S. de BonoDevelopment of methodology: J. Welti, A. Sharp, W. Yuan, I. Figueiredo,V. Gil, J. Ning, J.S. de BonoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Welti, A. Sharp, W. Yuan, I. Figueiredo,D.N. Rodrigues, V. Gil, A. Neeb, M. Crespo, J. Ning, E. Knight, J.C. Francis,A. Hughes, W.S. Halsey, S. Carreira, G. Boysen, A. Swain, J.S. de Bono

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Sharp, W. Yuan, D. Dolling, V. Gil, A. Neeb,G. Seed, J. Ning, R.S. Mani, S.R. Plymate, S. Carreira, G. Boysen, A. Swain,J.S. de BonoWriting, review, and/or revision of themanuscript: J.Welti, A. Sharp,W. Yuan,D. Dolling, I. Figueiredo, S. Sumanasuriya, A. Paschalis, R.S. Mani, G.V. Raj,S.R. Plymate, S. Carreira, G. Boysen, A.M. Chinnaiyan, A. Swain, J.S. de BonoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Welti, A. Sharp, W. Yuan, A. Neeb, M. Clarke,J. Ning, G.V. Raj, S. Carreira, J.S. de BonoStudy supervision: A.M. Chinnaiyan, J.S. de Bono

AcknowledgmentsThe authors would like to thank the Tissue Profiling Unit at the Institute of

Cancer Research for technical assistance. GSK1210151A (I-BET151) was a kindgift from GSK. Work in the de Bono laboratory was supported by funding fromtheMovember Foundation, Prostate Cancer UK,USDepartment ofDefense, theProstate Cancer Foundation, Stand Up To Cancer, Cancer Research UK, and theUK Department of Health through an Experimental Cancer Medicine Centregrant. A. Sharp is supported by the Medical Research Council, the Academy ofMedical Sciences andProstate CancerUK.Work in theMani andRaj laboratorieswas also supported by the US Department of Defense.

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 December 21, 2017; revised February 23, 2018; accepted March 14,2018; published first March 19, 2018.

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2018;24:3149-3162. Published OnlineFirst March 19, 2018.Clin Cancer Res Jonathan Welti, Adam Sharp, Wei Yuan, et al. in Castration-Resistant Prostate Cancer (CRPC)Targeting Bromodomain and Extra-Terminal (BET) Family Proteins

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