Abstract 1485: BRCA2 haploinsufficiency leads to mitotic DNA synthesis at common fragile sites in human mammary epithelial cells

Abstract Germline mutations in BRCA2 confer up to a 70% lifetime risk of developing breast cancer due to BRCA2’s critical roles in homologous recombination repair, replication fork stability, and cell cycle regulation. In BRCA2 mutation carriers, mammary epithelial cells are haplo-insufficient, meaning a single functional allele is inadequate to maintain genomic stability and suppress tumorigenesis. Genomic instability, a hallmark of cancer, frequently involves fragile site instability, particularly at common fragile sites (CFSs), which are hotspots for chromosomal rearrangements under replication stress. While the association between fragile site instability and cancer has been documented, the specific link between BRCA2 haploinsufficiency and CFS instability remains unexplored. To address this gap, we modeled BRCA2 haploinsufficiency in human mammary epithelial cells using small-interfering RNA (siRNA)-mediated knockdown and induced replication stress with the DNA polymerase inhibitor aphidicolin (APH). Cytogenetic analysis using DAPI banding revealed increased CFS instability, particularly at FRA18B, in BRCA2-deficient cells compared to controls. Furthermore, we observed elevated levels of mitotic DNA synthesis (MiDAS) in BRCA2-deficient cells, a process typically activated at under-replicated regions of CFSs during mitosis to preserve genome integrity. These findings demonstrate that BRCA2 haploinsufficiency promotes CFS instability and aberrant MiDAS, uncovering novel mechanisms of genomic instability in preneoplastic mammary epithelial cells. By elucidating these early molecular events in BRCA2-associated breast cancer, our study provides critical insights into the etiology of tumorigenesis and lays the groundwork for innovative strategies to stabilize the genome in high-risk individuals, potentially improving breast cancer prevention and outcomes. Citation Format: Kavya Vipparthi, Ariana Bellare, Mihriban Karaayvaz. BRCA2 haploinsufficiency leads to mitotic DNA synthesis at common fragile sites in human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1485.