Identification of Selective SWI/SNF Dependencies in Enzalutamide-Resistant Prostate Cancer [CRISPR Screen]

Enzalutamide (ENZA) is a potent second-generation antiandrogen commonly used to treat hormone-sensitive and castration-resistant prostate cancer (CRPC) patients. While initially effective, the response is only temporary and the disease almost always develops resistance. Given that many ENZA-resistant tumors are not driven by specific somatic mutations, there is increasing evidence that epigenetic factors can cause ENZA resistance. To explore how resistance arises we systematically tested all the epigenetic modifiers in castration-resistant and ENZA-resistant prostate cancer models using a custom epigenetic CRISPR library. From this, we identified and validated numerous epigenetic modifiers that were selectivity essential including SMARCC2, a core component of the SWI/SNF complex (or BAF complex) that regulates gene expression by altering DNA accessibility. Additionally, our data demonstrated canonical BAF complex dependency in ENZA-resistance that was also observed following the loss of DPF2, a canonical BAF-specific component. We showed that the chromatin occupancy of SMARCC2 and BRG1 was expanded in acquired ENZA resistance to the regions that overlap with transcriptional activity and CRPC-associated transcription factors that are significantly accessible in CRPC patients. Overall, our study revealed a regulatory role for SMARCC2 in ENZA-resistant prostate cancer and demonstrated the feasibility of targeting the SWI/SNF complex in late-stage PCa. Overall design: For each cell line, Cas9-expressing cells were generated by lentiviral delivery of lentiCas9-blast (Addgene #52962) virus at multiplicity of infection (MOI) 5. Cells were selected with blasticidin for 5 days and maintained few passages before the CRISPR-Cas9 screen in selective media. For the screen, we used EPIKOL library in lentiGuide-puro (Addgene #52963) backbone that was generated in Koc University laboratories. The sgRNA library was transduced into stable Cas9-expressing PCa cell lines at low multiplicity of infection (MOI=0.3) with a median coverage of 500x cells per sgRNA. 48 hrs after transduction, cells were selected with puromycin for 3 days. At the end of selection, 4e6 cells were collected for sequencing experiments (reference point) and 13.5e6 cells were maintained further up to 15 doubling time to minimize differences in growth rates between cell populations. At 15th doubling time, 4x106 cells were collected for sequencing experiments.