Abstract 1515: Bromodomain and extra-terminal motif protein inhibitors (BETi) in metastatic castration resistant prostate cancer (mCRPC): A novel mechanism for regulating androgen receptor variant 7 (ARV7)
Authors: Welti Jonathan , Sharp Adam , Figueiredo Ines , Yuan Wei , Rodrigues Daniel Nava , Gil Veronica S. , Knight Eleanor , Ning Jian , Francis Jeff , Neeb Antje , Boysen Gunther , Swain Amanda , Bono Johann S. de 
Published: July 2017

Abstract
Persistent androgen receptor (AR) signaling is key to the development and progression of metastatic castration resistant prostate cancer (mCRPC). This is in part due to expression of constitutively active AR splice variants like AR variant 7 (ARV7), confering resistance to current anti-androgens including enzalutamide (E) and abiraterone (A). To improve the outcome for patients with mCRPC, new therapeutic strategies to overcome AR and ARV7 oncogenic signaling are urgently required. The inhibition of co-factors modulating AR signaling are currently being investigated as novel strategies to treat mCRPC. One promising candidate is BRD4, a member of the BET protein family, that binds the AR on androgen response elements and facilitates the recruitment of the transcriptional machinery. BET inhibitors (BETi) have been shown to regulate AR and ARV7 signalling, however, the exact mechanism of ARV7 regulation remains unclear. As BETi are currently being explored in clinical trials of unstratified patients with mCRPC, we investigated their potential mechanism of action in CRPC cell lines, patient derived organoids (PDOs) and a patient derived mouse xenograft (PDX). Here we demonstrate that nuclear expression of BRD4 and ARV7 increases as patients develop resistance to E and/or A and inhibition of BRD4 by BETi is sufficient to block AR and ARV7 signalling in mCRPC. Both inhibition of BRD4 by BETi and genetic knockdown of BRD4 reduced the growth of CRPC cell lines and led to down-regulation of AR and ARV7 at the mRNA and protein level. To further investigate whether BETi is sufficient to inhibit ARV7 activity in patients with mCRPC, we treated patient derived organoids (PDOs) and a mouse xenograft (PDX) grown from metastatic biopsies of patients resistant to E and/or A with BETi. In this study 5 out of 10 PDOs were sensitive to BETi. Consistent with the cell culture experiments, BETi treatment of the PDX led to down-regulation of both ARV7 mRNA and protein expression. Mechanistically, BETi might inhibit pre-mRNA splicing of AR resulting in the observed decrease of ARV7 expression. However, RNAseq analysis of the ARV7 expressing CRPC cell line LNCaP95 demonstrated an increase in total splicing events including skipped exons, retained introns, mutually exclusive exons, alternative 5’ splice site and alternative 3’ splice site after BETi treatment. Despite this, focused analysis of splicing factors and spliceosome components identified a subset of eight splicing factors being down-regulated by BET inhibition including one yet uncharacterized factor that is crucial for ARV7 expression in LNCaP95 cells. Based on our results we propose a model that BETi mediated inhibition of this novel ARV7-mRNA splicing factor may lead to decreased splicing and subsequent expression of ARV7 at both mRNA and protein level; providing a novel approach to target ARV7 in mCRPC.
Citation Format: Jonathan Welti, Adam Sharp, Ines Figueiredo, Wei Yuan, Daniel Nava Rodrigues, Veronica S. Gil, Eleanor Knight, Jian Ning, Jeff Francis, Antje Neeb, Gunther Boysen, Amanda Swain, Johann S. de Bono. Bromodomain and extra-terminal motif protein inhibitors (BETi) in metastatic castration resistant prostate cancer (mCRPC): A novel mechanism for regulating androgen receptor variant 7 (ARV7) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1515. doi:10.1158/1538-7445.AM2017-1515