CRISPR/Cas9 screenings and in silico investigations nominate low-frequency alterations in DNA repair genes as biomarkers for castration-resistant prostate cancers.

PARP inhibitors (PARPi) have received regulatory approval for the treatment of multiple tumor types, including prostate cancer (PCa), and are associated with therapeutic response in metastatic castration-resistant prostate cancer (mCRPC) patients characterized by defects in homologous recombination repair genes. Clinical trials results suggest that further improvements are possible, for instance in terms of patients’ enrolment criteria. Only 50% of BRCA1/2-deficient patients (TRITON2 trial) respond to therapy, variable antitumor activity is observed for the covered non-BRCA DNA repair gene (DRG) aberrations, and the duration of the treatment response is highly heterogeneous among responders. In this scenario, in the present study we aimed at the identification and characterization of novel low-frequency DRG alterations that could represent potential targets or biomarkers for mCRPC therapy. We exploited an interdisciplinary approach based on in vitro studies including CRISPR/Cas9 genotoxic screenings and on in silico analyses focused on mCRPC patients’ genomic and clinical data. The screenings were performed on PCa cell lines and included the use of a custom sgRNA pooled library targeting 356 DRGs and the administration of several drugs, most of which are currently used in the clinic. To characterize the full spectrum of DRG aberrations, the in silico approach included the joint allele-specific level analysis of germline variants and somatic alterations of 302 DRGs involved in the principal repair pathways while querying the SU2C-PCF dataset of 429 mCRPC. Association of identified DRG defects with patient outcome and response to androgen receptor-signalling inhibitors (ARSI) were performed. Multiple genes were validated as predictive biomarkers of PARPi or carboplatin response, while 14 DRG variants linked with worse prognosis or response to therapy were nominated by the in silico analyses. Altogether, this project allowed for the identification of novel DRG biomarkers and potential targets that could eventually improve the therapeutic options for mCRPC patients.