Identifying splicing factors critical for androgen receptor variant generation in castrate resistant prostate cancer

Abstract

Resistance to androgen receptor (AR)-targeted therapies represent a major clinical challenge in prostate cancer (PCa). A key mechanism of resistance in patients who progress to an advanced form of the disease, termed castrate-resistant PCa (CRPC), is the generation of alternatively spliced androgen receptor variants (AR-Vs). Unlike wild-type or mutant fulllength AR (FL-AR) isoforms, AR-Vs are refractory to the current repertoire of receptortargeting agents and hence drive tumour progression. Our understanding of the splicing factors that control AR-V splicing is limited. Therefore, an unbiased, global analysis of splicing factors important for AR-V generation is vital to provide new targets for advanced PCa therapy. A stable Cas9-inducible CWR22Rv1-AR-EK derivative cell line, termed CWR22Rv1-AR-EKiCas9, was developed which: (i) retains endogenous AR-V expression but lacks FL-AR; and (ii) expresses Cas9 under a doxycycline-inducible promotor. This cell line model was validated for use in an optimised bespoke splicing factor library CRISPR/Cas9 knockout screen, utilising free guide RNAs in a multi-384-well format to assay, by indirect immunofluorescence and highthroughput microscopy, AR-V abundance following gene knockout. This yielded several novel splicing factor candidate genes for further validation as regulators of AR across several CRPC cell line models. Two of the novel splicing factors candidates identified in the CRISPR/Cas9 knockout screen, MFAP1 and CWC22, were shown to be required for generation of FL-AR and/or AR-V mRNA transcripts across several CRPC cell line models. Depletion of these splicing factors resulted in reduced AR-V protein abundance and anti-proliferative effects in several CRPC cell lines. Global transcriptomic analysis revealed dysregulated cellular pathways/functions in response to MFAP1 and CWC22 depletion, including genes associated with DDR. Overall, the project has identified novel AR-V splicing regulators that control several therapeutically-tractable pathways which alongside targeting of splicing processes could provide novel cellular vulnerabilities which can be exploited in CRPC.