Abstract 6010: BRCA2 reversion alleles confer PARPi resistance via homology-directed repair, not fork protection

Abstract

Abstract BRCA2 reversion alleles have been identified in relapsed, treatment resistant cancer patients. The reversions restore the open reading frame of the BRCA2 protein often resulting in significant sequence deletions with unknown functional consequences. No reports to date have introduced patient reversion mutations into a BRCA2 null cell model to analyze which BRCA2 functions are re-established and whether all or a subset of activities are required for PARPi resistance. Our study utilized BRCA2 reversion alleles identified through sequencing of circulating tumor DNA (ctDNA) from PARPi resistant ovarian cancer patients. We rigorously evaluated whether altered BRCA2 reversion proteins restore homology-directed repair (HDR), fork protection, or gap suppression to determine the specific activities required for PARPi resistance. We found that reverted BRCA2 proteins are functional for HDR by assessing survival response to PARPi, analyzing RAD51 foci at DSBs, DR-GFP reporter assays, RAD51 binding, and CRISPR/Cas9 gene targeting assays. Surprisingly, using the DNA fibercombing approach to assess replication fork dynamics, we identify two separation-of-function mutants, T1974 and C1654_M1890, which are deficient in fork protection, yet confer PARPi resistance. The mutations are located within the BRC 5-8 domain of BRCA2 proposed to play an important role in RAD51 nucleoprotein filament stability. Biochemical analyses of the purified proteins using single molecule FRET (smFRET) indicate reduced filament stability potentially explaining the deficit in fork protection. In conclusion, these separation-of-function BRCA2 mutations provide new mechanistic insight into the underlying biology of BRCA2 and reveal the specific functions needed to overcome PARPi-mediated cell death. Citation Format: Ryan Jensen, Gemma Moore, Sudipta Lahiri. BRCA2 reversion alleles confer PARPi resistance via homology-directed repair, not fork protection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6010.