ABSTRACT Metastatic castration-resistant prostate cancer (mCRPC) is an incurable disease that is expected to account for ~ 34,500 deaths each year in the United States. Therapeutic options are limited for mCRPC 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 mCRPC. These alterations have been correlated with particular therapeutic vulnerabilities in prostate cancer (PCa) cells. Specifically, defects in homologous recombination repair (HRR) would predict sensitivity to inhibition of Poly (ADP-ribose) polymerase (PARP). PARP inhibitors (PARPis) 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 HRR. Cancer cells lacking BRCA1/2 depend instead on PARP-regulated DNA repair and are hypersensitive to PARPis. The U.S. FDA has approved two PARP inhibitors (olaparib and rucaparib) for treatment of mCRPC patients with HRR mutations (or deleterious BRCA1/2 mutations) based on the results from recent clinical trials. One of the major barriers to effective treatment using PARPis is how to select patients who most likely benefit from PARP inhibition. BRCA1/2 mutations can predict PARPi response with 50-60% accuracy. However, the degree to which patients with non-BCRA1/2 genomic alterations respond to PARPis remains unclear. Through genome-wide CRISPR screening, we have recently discovered that loss of MMS22L in PCa cells predicts the response to PARP inhibition. MMS22L is required for HRR of replication fork-associated DNA double strand breaks. More importantly, the MMS22L gene is frequently deleted (~14%) in prostate tumors. In addition, the results from our CRISPR screening further suggest that loss of TP53 or RB1 may render PCa cells resistance to PARPis due to upregulation of HRR gene expression, which can be overcome by combining ATR inhibition. Therefore, the goal of this project is to determine (1) to what extent loss of MMS22L confers a cellular response to PARP inhibition in preclinical PCa models; (2) to what extent inactivation of TP53 or RB1 influences PARPi response; (3) to what extent ATR inhibition re-sensitizes resistant PCa cells to PARP inhibition. The successful completion of this project will set the stage for future clinical trials in mCRPC patients with MMS22L and TP53/RB1 alterations and significantly expand the pool of eligible patients for PARP inhibition.