Abstract 231: Polymerase iota drives aberrant mitotic DNA synthesis and replication stress in BRCA2 deficient human mammary epithelial cells.

Abstract Germline mutations in BRCA2 greatly increase breast cancer risk, yet the early molecular events preceding tumor initiation remain incompletely defined. Prior work shows that mammary epithelial cells with compromised BRCA2 function, including cells from mutation carriers, exhibit replication-associated genome instability. Here, we investigated the role of polymerase iota (Pol ι), an error-prone translesion polymerase upregulated in breast cancer and linked to replication stress.Using primary human mammary epithelial cells (HMECs) from BRCA2 mutation carriers and BRCA2-silenced MCF10A cells, we found that loss of BRCA2 augments Pol ι expression at both mRNA and protein levels. We demonstrate that Pol ι is required for mitotic DNA synthesis (MiDAS), a compensatory pathway engaged when DNA under-replication persists into mitosis. Pol ι silencing significantly reduced MiDAS in BRCA2-silenced cells, but not in wild-type cells, revealing a context-specific dependency on Pol ι for completing mitotic replication.Unexpectedly, silencing Pol ι in BRCA2-silenced cells reduced chromosomal breaks, 53BP1 bodies, and micronuclei, lesions typically associated with impaired MiDAS. Moreover, Pol ι loss markedly decreased replication stress markers, including phosphorylated γH2AX and RPA. These findings indicate that Pol ι -mediated MiDAS in the setting of BRCA2 deficiency is not protective but instead amplifies replication stress and genome instability.Our results uncover a previously unrecognized regulatory interaction in which BRCA2 restrains Pol ι, and loss of this restraint permits aberrant Pol ι -driven MiDAS that exacerbates genome instability. This work identifies Pol ι as a potential early intervention target for reducing replication stress in individuals carrying BRCA2 mutations. Citation Format: Kavya Vipparthi, Ariana Bellare, Resul Ozbilgic, Mihriban Karaayvaz. Polymerase iota drives aberrant mitotic DNA synthesis and replication stress in BRCA2 deficient human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 231.