LAUSR

Individuals with germline heterozygous mutations of breast cancer susceptibility genes BRCA1 or BRCA2 have a 50 to 80% lifetime risk of developing breast cancer, yet the mechanisms of these cancers remain largely unknown. There is growing evidence that BRCA1 and BRCA2 heterozygosity confer haploinsufficiency in normal human mammary epithelial cells for their multiple known functions. Haploinsufficiency for BRCA1 leads to defects in differentiation, replication stress response, premature senescence and genomic instability in human mammary epithelial cells. Deficiencies in error-free DNA damage repair has been observed in genetically engineered cells as well as primary human mammary epithelial cells carrying BRCA1 heterozygous mutations. There is relatively less research on understanding the role of haploinsufficiency for BRCA2 in human mammary epithelial cells. It has been reported that haploinsufficiency for BRCA2 leads to genomic instability via unscheduled R-loops. We previously showed that accumulation of sub-chromosomal copy number variations (CNVs) and replication stress-induced DNA damage, together with attenuated checkpoint and apoptotic responses, constitute a haplo-insufficient phenotype in mammary epithelial cells of BRCA2 mutation carrier patients. Common fragile sites (CFS) are found in all individuals and represent a normal component of chromosome structure. Numerous studies have shown that CFSs are prone to deletions and rearrangements in many cancers, a situation referred as CFS instability. Importantly, Fanconi Anemia/BRCA pathway has been previously reported to have a role in regulating CFS instability. In this study, we further explored a link between BRCA2 haploinsufficiency and CFS instability. We induced replication stress with DNA polymerase inhibitor aphidicolin (APH) treatment in control and BRCA2- deficient human mammary epithelial cells. We then performed DAPI banding to map and score the cytogenic lesions in metaphase spreads cells, in the absence or presence of APH treatment. Our results demonstrated that BRCA2- deficient human mammary epithelial cells showed increased frequency of CFS regions such as FRA18B. Correspondingly, BRCA2-deficient cells exhibited a failure to activate CHK1, a central coordinator of the response to replication stress and DNA damage, in response to APH treatment, despite normal levels of total CHK1 protein. ATR/CHK1 pathway is a major regulator of CFS stability. Thus, our results provide support for BRCA2 haploinsufficiency leading to CFS instability in human mammary epithelial cells. These studies will yield unanticipated opportunities for improved risk assessment and prevention strategies in high-risk patients. Citation Format: Mihriban Karaayvaz, Kavya Vipparthi, Antony Caputo. Haploinsufficiency for BRCA2 leads to common fragile site instability in human mammary epithelial cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6519.