Abstract A067: Targeting the bromodomain and extra-terminal (BET) family proteins and beyond in metastatic castration-resistant prostate cancer (mCRPC): Overcoming aberrant androgen receptor (AR) signaling
Authors: Sharp Adam , Welti Jon , Yuan Wei , Figueiredo Ines , Gil Veronica , Rodrigues Daniel Nava , Lambros Maryou , Knight Eleanor , Ning Jian , Francis Jeff , Dolling David , Pope Lorna , Neeb Antje , Boysen Gunther , Zhu Yezi , Crespo Mateus , Paschalis Alec , Luo Jun , Plymate Stephen , Al-Lazikani Bissan , Swain Amanda , Bono Johann de 
Published: February 2019

Abstract
Despite robust responses to androgen deprivation therapy and AR targeting therapies (including abiraterone and enzalutamide), nearly all cases of advanced prostate cancer progress to lethal mCRPC and develop therapeutic resistance. This progression is associated with persistent AR signaling, in part due to expression of constitutively active AR splice variants that include AR variant 7 (AR-V7). We show that AR-V7 expression in patient biopsies (protein) and circulating tumor cells (RNA) associates with poor outcome from mCRPC. Therapies that regulate AR-V7 and induce robust anticancer responses are required to confirm the clinical importance of AR-V7 in mCRPC. One such promising approach, currently in clinical trials, is inhibitors of BET family proteins, which include BRD2, BRD3, and BRD4. Preclinical studies have shown that BRD4 binds to AR at the androgen response element and facilitates the recruitment of the transcriptional machinery. We show that BRD4 protein expression increases as patients develop mCRPC and at diagnosis associates with patient outcome and more advanced disease. In addition, through RNAseq analysis we show that expression levels of BRD2, 3, and 4 in mCRPC associated with degree of AR activity. We, and others, have shown that the use of BET inhibitors (BETi) in vitro on AR/AR-V7 expressing cell lines not only decreased AR activity but also preferentially decreased the production on AR-V7 mRNA. In light of BETi having efficacy against BRD2, 3, and 4, and all isoforms being expressed in mCRPC, we explored the effect of genetic knockdown of each isoform. We show that BETi treatment is sufficient to decrease AR-V7 mRNA and protein in CRPC cell lines. Moreover, we demonstrate that BRD4 knockdown, and to a greater extent, the combination of BRD2, 3, and 4 knockdown blocked AR and AR-V7 signaling. Furthermore, C-MYC knockdown did not recapitulate the effect of BETi and led to an increase in AR signaling. Consistent with these findings, BETi and the combination of BRD2, 3, and 4 knockdown reduced the growth of CRPC cell lines. To further investigate whether BETi is sufficient to inhibit AR-V7 production in patients with mCRPC, we treated patient-derived organoids (PDOs) and a mouse xenograft (PDX) grown from patient metastatic biopsies who had progressed on enzalutamide and/or abiraterone. In this study 4 out of 9 PDOs were sensitive to BETi. Consistent with cell culture experiments, BETi treatment of PDO and PDX led to downregulation of both AR-V7 mRNA and protein expression, and growth inhibition. In light of the pleotropic effects of BETi on cancer cell biology and potential for treatment-related toxicities, we explored whether we could identify critical factors for BETi mediated AR-V7 regulation in CRPC. The ability of BETi to regulate AR-V7 may suggest an effect of BETi on pre-mRNA splicing of AR resulting in the observed decrease of AR-V7 expression. RNAseq analysis of the AR-V7 expressing CRPC cell line LNCaP95 treated with BETi demonstrated an increase in total splicing. Despite this, focused analysis of splicing factors and spliceosome components identified a subset of eight splicing factors being downregulated by BETi treatment, including one yet-uncharacterized factor (splicing factor B; Sf-B) that is crucial for AR-V7 expression and LNCaP95 cell growth. In addition, mCRPC patients who express high levels of Sf-B had a significantly poorer outcome and the protein structure of Sf-B is druggable using the drug discovery knowledgebase canSAR. Based on our results, we propose that inhibition of Sf-B may lead to decreased splicing and expression of AR-V7; providing a novel approach to target AR-V7 in mCRPC.
Citation Format: Adam Sharp, Jon Welti, Wei Yuan, Ines Figueiredo, Veronica Gil, Daniel Nava Rodrigues, Maryou Lambros, Eleanor Knight, Jian Ning, Jeff Francis, David Dolling, Lorna Pope, Antje Neeb, Gunther Boysen, Yezi Zhu, Mateus Crespo, Alec Paschalis, Jun Luo, Stephen Plymate, Bissan Al-Lazikani, Amanda Swain, Johann de Bono. Targeting the bromodomain and extra-terminal (BET) family proteins and beyond in metastatic castration-resistant prostate cancer (mCRPC): Overcoming aberrant androgen receptor (AR) signaling [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A067.