Abstract 1479: Defining the interplay between DNAPKcs and shieldin during chromosomal break repair

Abstract Chromosomal DNA double strand breaks (DSBs) are the effective lesion of cancer radiotherapy and other cancer therapeutics, such as topoisomerase 2 poisons. Understanding how cancer cells repair DSBs can provide insight into cancer cell resistance to these therapeutics, as well as inform development approaches to improve cancer sensitivity. DSB repair is also significant for cancer biology because aberrant repair of DSBs can cause mutations and chromosomal rearrangements that drive cancer. Repair of DSBs occurs through several pathways, including non-homologous end joining (NHEJ). DNA-PKcs (DNA-dependent protein kinase) promotes NHEJ, inhibits HDR, and is an emerging drug target with kinase inhibitors in oncology clinical trials. DNA-PKcs phosphorylates itself and other DNA damage response proteins and promotes long-range DSB end synapsis to facilitate NHEJ. Another protein complex implicated in long-range DSB end synapsis is Shieldin, which includes the proteins 53BP1, RIF1 (Rap1-interacting factor 1), and other factors. Shieldin is critical for NHEJ to promote class switch recombination, which is a programmed DSB repair event during antibody maturation. However, the role of Shieldin in NHEJ repair outside this context remains poorly understood. Indeed, a specific gap in knowledge is the functional interplay between how DNA-PKcs and Shieldin promote synapsis of DSB ends to favor NHEJ. To study end joining outcomes, I use Cas9 reporter assays to measure end joining frequencies via flow cytometry. These reporter assays are chromosomally integrated into human HEK293 cells. Additionally, I examine end joining junctions using deep sequencing to compare parental HEK293 cell junctions between wild-type and 53BP1-knockout or RIF1-knockout cell lines. This PCR-based approach allows me to categorize junction types into four categories: No Indels, Insertions, Deletions, and Complex Indels. My studies indicate that RIF1 and 53BP1 are genetic modifiers of DNA-PKcs function during DSB repair. I will present our current efforts to address this gap in knowledge. Citation Format: Kaela Makins, Metztli Cisneros-Aguirre, Felicia Lopezcolorado, Jeremy M. Stark. Defining the interplay between DNAPKcs and shieldin during chromosomal break repair [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 1479.