Abstract 803: Identifying a RAD18/UBC13-dependent mechanism of replication fork recovery to modulate chemoresponse in BRCA1-deficient cancers

Abstract

Abstract Mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 are associated with an increased lifetime risk of breast and ovarian cancers. While the BRCA proteins play a well-established role in double-stranded DNA break repair, recent studies have revealed an emerging role of BRCA1/2 in replication stress response. While replication forks are extensively degraded by nucleases in BRCA-deficient cancer cells, activation of specialized fork recovery mechanisms enables resumption of DNA synthesis and promotes cell survival. My project aims to determine this fork recovery mechanism in BRCA1-deficient cells and to identify potential recovery factors that can be targeted to improve chemotherapeutic response in BRCA1-mutated breast and ovarian cancers. To monitor perturbations in replication fork dynamics on a genome-wide scale, we utilize a DNA fiber assay technique measuring rates of fork recovery and replication fork degradation. In parallel, electron microscopy analysis allows direct visualization of replication fork intermediates. Cell survival assays are employed to test how loss of fork recovery factors impacts cell proliferation and chemotherapeutic response in BRCA1-deficient cells. Our results reveal that RAD18 and UBC13, which catalyze ubiquitination of Proliferating Cellular Nuclear Antigen (PCNA), promote fork recovery in BRCA1-deficient, but not BRCA2-deficient, cancer cells. Previous work has also shown that PCNA polyubiquitination by UBC13 is important for reversed fork formation in BRCA-proficient cells. However, our findings show that extensive degradation of reversed fork substrates still occurs in BRCA1-deficient cells lacking RAD18 or UBC13, indicating that PCNA polyubiquitination is not essential for fork reversal in this genetic background. In addition, loss of RAD18 in BRCA1-deficient cells significantly slows cell proliferation, and UBC13 inhibition further sensitizes cells lacking BRCA1 to the replication stress inducer Hydroxyurea (HU). Based on our findings, we hypothesize that RAD18, UBC13, and PCNA ubiquitination may represent novel targets to improve chemoresponse in BRCA1-deficient cancers that rely on fork recovery mechanisms for survival. Citation Format: Emily Cybulla, Jessica Jackson, Stephanie Tirman, Annabel Quinet, Delphine Lemacon, Alessandro Vindigni. Identifying a RAD18/UBC13-dependent mechanism of replication fork recovery to modulate chemoresponse in BRCA1-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 803.