Abstract 1473: POLQ facilitates annealing mediated long deletions at chromosomal break in homologous recombination deficient cancers

Tumors deficient in homologous recombination (HRD) predominantly rely on alternative DNA repair pathways for maintaining genome stability, especially once end resection has been initiated after a double strand break (DSB). These back up repair mechanisms, especially microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA), once considered minor contributors to genome stability, have garnered increased attention as recent evidence supports their significant roles in tumorigenesis and their potential as therapeutic targets. Consequently DNA polymerase theta (POLQ), a key regulator of MMEJ, emerges as an attractive target for the selective treatment of HRD cancers, and recent efforts in the development of POLQ inhibitors have demonstrated their potential to enhance the efficacy of existing therapies and overcome resistance mechanisms. In our study, we investigated the genetic drivers of the under-investigated SSA and its connection to the related MMEJ pathway in human cells. A previously unidentified role for POLQ in regulating the frequency of SSA was observed in addition to its role in MMEJ. Beyond its established role as a gap-filling polymerase, we show that the helicase activity of POLQ is critical for homology-mediated annealing in both MMEJ and SSA. POLQ is shown to be a key facilitator of SSA through its ability to process DNA ends and promote annealing of homologous sequences with at least 80% sequence identity (homeology), resulting in a range of deletion sizes. We also provide evidence for these homeology-dependent SSA-like repair scars in tumor genomes and show that they are associated with HRD and elevated POLQ expression, suggesting the utilization of these mechanisms as a critical repair pathway in these tumors. These findings suggest POLQ is the ideal target for therapies of HRD cancer. Citation Format: Manisha Jalan, Erika S. Buechelmaier, Jonathan Rafailov, Yingjie Zhu, Xin Pei, Sebastian Brylka, Joan Gomez-Aguilar, Niamh McDermott, Nadeem Riaz, Marcin Imielinski, Simon N. Powell. POLQ facilitates annealing mediated long deletions at chromosomal break in homologous recombination deficient cancers [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 1473.