Abstract 4206: Investigation into the mechanisms and therapeutic potential of R-loop-induced DNA damage in ER+ breast cancer

A potential therapeutic vulnerability of endocrine-resistant estrogen receptor alpha (ER)-positive breast cancer is R-loop-mediated DNA damage induced by 17b-estradiol (E2). This study aims to elucidate the mechanism of DNA damage and test combination therapies to exacerbate the anti-cancer effects of E2. ER+ breast cancer cells overexpressing exogenous ER, which induces sensitivity to the growth-inhibitory effects of E2, were exposed to E2 and inhibitors of DNA damage repair proteins including poly-ADP ribose polymerases (PARP) and CHK1/2. Experimental endpoints included immunoblotting, flow cytometry, immunofluorescence, DNA fiber analysis, and growth assays using cell lines and patient-derived xenografts (PDXs). Cells overexpressing ER showed R-loop-dependent DNA damage in replicating cells. Combination therapy of E2 and the PARPi olaparib increased DNA damage in replicating cells without negatively impacting the elevated replication rates induced by E2. E2 significantly increased single-stranded DNA in ER-overexpressing cells; ssDNA is known to be susceptible to damage. These effects of E2 were exacerbated by PARPi. E2 increased the proportion of cells replicating DNA, but E2 paradoxically interfered with replication fork progression as shown by DNA fiber analysis. These findings suggest that replication interference contributes to DNA damage and eventual cell death but does not impair the ability for the cell to replicate. An alternative therapeutic approach is combination therapy with E2 and Chk1/2 inhibitor. This combination successfully inhibits growth of both parental and ER overexpressing cells indicating that E2 and Chk1/2 inhibition may have clinical potential. E2 induced DNA damage mediated by R-loops can be amplified using combination therapies targeting DNA damage repair or cell cycle inhibition. While it is clear that replication conflicts plays an important role in DNA damage, the mechanism in which the DNA damage is occurring has yet to be elucidated. Citation Format: Alyssa Roberts, Nicole Traphagen, Anneka Johnson, Eugene Demidenko, Patricia Muskus, Xiaofeng Wang, Todd W. Miller. Investigation into the mechanisms and therapeutic potential of R-loop-induced DNA damage in ER+ breast cancer [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 4206.