BRCA2 reversion mutation-independent resistance to PARP inhibition in prostate cancer through loss of function perturbations in the DNA pre-replication complex

Recent approvals of PARP inhibitors (PARPi) for BRCA-mutant metastatic castration resistant prostate cancer (mCRPC) necessitate an understanding of the factors that shape sensitivity and resistance. Reversion mutations that restore homologous recombination (HR) repair are detected in [~]50-80% of BRCA-mutant patients who respond but subsequently relapse, but there is currently little insight into why only [~]50% of BRCA-mutant patients display upfront resistance. To address this question, we performed a genome-wide CRISPR screen to identify genomic determinants of PARPi resistance in murine Brca2{Delta}/{Delta} prostate organoids genetically engineered in a manner that precludes the development of reversion mutations. Remarkably, we recovered multiple independent sgRNAs targeting three different members (Cdt1, Cdc6, Dbf4) of the DNA pre-replication complex (pre-RC), each of which independently conferred resistance to olaparib and the next generation PARP-1 selective inhibitor AZD5305. Moreover, sensitivity to PARP inhibition was restored in Brca2{Delta}/{Delta}, Cdc6-depleted prostate cells by knockdown of geminin, a negative regulator of Cdt1, further implicating the critical role of a functional pre-RC complex in PARPi sensitivity. Furthermore, [~]50% of CRPC tumors have copy number loss of pre-RC complex genes, particularly CDT1. Mechanistically, prostate cells with impaired pre-RC activity displayed rapid resolution of olaparib-induced DNA damage as well as protection from replication fork degradation caused by Brca2 loss, providing insight into how Brca2-mutant cancer cells can escape cell death from replication stress induced by PARP inhibition in the absence of HR repair. Of note, a pharmacologic inhibitor that targets the CDT1/geminin complex (AF615) restored sensitivity to AZD5305, providing a potential translational avenue to enhance sensitivity to PARP inhibition in BRCA-mutant cancers. SignificanceHere, we address a major limitation in the effectiveness of PARP inhibitors in BRCA-mutant prostate cancer treatment: only [~]50% of patients respond despite clear genomic evidence of defective homologous recombination. Prior efforts to study PARP inhibitor resistance in prostate cancer have been plagued by the lack of suitable cell lines. We overcame this challenge using primary prostate organoids coupled with genome-wide CRISPR screening. The key finding is that loss of function mutations in the DNA pre-replication complex confer PARP inhibitor resistance. These genes map to chromosomal regions frequently lost in prostate cancer and could therefore serve as potential biomarkers of treatment response. Pharmacologic inhibition of geminin, a negative regulator of the pre-replication complex, can restore PARP inhibitor sensitivity.