ABSTRACT Metastatic castration-resistant prostate cancer (CRPC) is an incurable disease that is expected to account for ~ 31,000 deaths each year in the United States. There are limited therapeutic options for metastatic CRPC patients that extend life. There is an urgent need for developing novel targeted therapies, especially personalized therapies based on genomic alterations in tumors. Recent genomic studies have revealed a variety of actionable molecular targets with underlying genomic alterations. Notably, alterations in genes involved in DNA damage response (DDR) are among the most common genetic events and enriched in metastatic CRPC. These alterations have been correlated with particular therapeutic vulnerabilities in prostate cancer (PCa) cells. Specifically, defects in homologous recombination (HR) repair would predict sensitivity to inhibition of Poly (ADP-ribose) polymerase (PARP). PARP inhibitors (PARPi) are a new type of targeted therapy, which works by preventing the enzyme PARP from repairing damaged DNA in tumor cells. BRCA1/2 encode proteins essential for HR repair. Cancer cells lacking BRCA1/2 depend instead on PARP-regulated DNA repair and are hypersensitive to PARPi. The U.S. FDA has approved two PARP inhibitors (olaparib and rucaparib) for treatment of metastatic CRPC patients with HR repair mutations (or deleterious BRCA mutations) based on the results from recent clinical trials. One of the major barriers to effective treatment using PARPi is how to select patients who most likely benefit from PARP inhibition. BRCA mutations can predict PARPi response with 50-60% accuracy. However, the degree to which patients with non-BCRA genomic alterations respond to PARPi remains unclear. Through a genome-wide CRISPR screen, we have recently discovered that loss of RNASEH2B in PCa cells may predict response to PARP inhibition. RNASEH2B is one of the three RNase H2 subunits that are thought to play a role in DNA replication. A recent study has demonstrated its function in ribonucleotide excision repair, which may contribute to PARP-trapping lesions. Importantly, analysis of TCGA datasets revealed RNASEH2B deep deletions in 17% of PCa tumors. Co-deletion of RNASEH2B and RB1 on chromosome13q14 frequently occurs. In addition, the results from our CRISPR screening further suggest that loss of TP53 may render PCa cells resistance to PARPi. Therefore, the goal of this project is to determine (1) to what extent loss of RNASEH2B confers a cellular response to PARPi in preclinical PCa models; (2) to what extent inactivation of TP53 and RB1 influences the response to PARPi. The successful implementation of this project will set the stage for future clinical trials in PCa patients with RNASEH2B/TP53/RB1 alterations and significantly expand the pool of eligible patients for PARP inhibition.