DNAR-13. Uncovering novel mediators of radiation sensitivity in glioblastoma using functional genomic screening

Abstract Radiation therapy is a crucial component of the standard of care for newly diagnosed glioblastoma patients. However, the majority of tumors inevitably recur, most within the prior radiation field. Despite its broad clinical application, the molecular mechanisms that mediate response to radiation therapy in glioblastoma remain poorly understood. To address this knowledge gap, we systematically profiled the radiation sensitivity of four glioblastoma cell lines (T98G, SF295, LN18, DBTRG) and four patient-derived neurospheres (BT112, BT145, BT359, CPDM0095). The neurospheres were characterized at the genomic, transcriptomic and clinical levels. These models exhibited a spectrum of responses to radiation, with LD50s ranging from 1.5 to 8 Gy. To identify molecular factors that sensitize glioblastoma cells to radiation, we conducted a genome-wide CRISPR inactivation screen in T98G cells treated with or without a sub-lethal dose of radiation. Cells were transduced with the Inzolia multiplex CRISPR/Cas12a knockout library, targeting 19,687 single genes, 4,435 paralog pairs, 376 paralog triples, and 100 paralog quads. Cells were then divided into a control condition and an irradiated condition (3 Gy). After 18 days in culture, genomic DNA was harvested, and the relative abundance of each guide RNA array was compared between the two conditions. Targets preferentially depleted in the irradiated condition included known radiation sensitizers such as ATM and PRKDC, validating the screen’s performance. Gene set enrichment analysis (KEGG) of the top 200 hits revealed enrichment of DNA repair pathways, including homologous recombination and non-homologous end joining, as well as pathways related to the spliceosome, cell cycle, endocytosis, ubiquitin-mediated proteolysis, and pyrimidine metabolism. Notably, three components of the translesion synthesis pathway (REV1, REV3L, MAD2L2), not previously implicated in radiation response, also emerged as top candidates. A mini-pool library of the top 300 hits was subsequently generated and is being used for validation in additional glioblastoma cell lines and neurospheres. These findings provide a foundation for uncovering novel mechanisms of radiation response and nominate new targets for potential radiosensitization strategies.