Abstract 1918 Elevated dNTP Pools Synergize with Ionizing Radiation to Impair DNA Damage Repair and Promote Apoptosis in Therapy Resistant Cancers

Glioblastoma (GBM) is a deadly primary tumor of the brain, accounting for almost half of all diagnosed malignant gliomas. Ionizing radiation (IR) and some chemotherapeutics strategically target cancer cells by causing DNA double-strand breaks (DSBs). However, cancers like GBM often develop resistance to DSB-inducing therapeutics due to dysregulated DNA repair. Homologous Recombination (HR) is an error-free DSB-repair process consisting of proteins that are often dysregulated in GBM, causing overactive DNA-repair, therapy resistance, and poor patient outcomes. Proteins involved in the HR-repair process have been extensively investigated; however, therapeutic resistance remains common. Here, we provide an alternative approach to target DSB-resistant GBM by modulating the dNTP pool instead of solely targeting DNA-repair proteins. Our goal is to understand the interplay between dNTP pools and DSB- repair. dNTPs are a necessary constituent to repair DSBs; however, the role of the dNTP pool in DNA damage repair is not fully understood. Previous research shows that the dNTP pool remains unchanged after inducing DSBs, highlighting the importance of a tightly regulated dNTP pool. A neutral comet assay allowed for the quantification of accumulated double-stranded breaks in different treatment groups. These same groups were analyzed using immunofluorescent microscopy to examine distinct proteins involved in HR-mediated repair. RNA sequencing analysis was used to determine differential gene expression of treatment groups Flow staining of these group allowed for quantification of cell viability and apoptotic cell populations. Immunoblotting and immunoprecipitation aided in the determination of protein interactions. We found that increasing the dNTP pool prior to IR treatment resulted in impaired DNA DSB repair as determined by the neutral comet assay in GBM cells. When analyzing key proteins involved in homologous recombination via immunofluorescence, we observed a significant decrease in RPA70 localization to the DNA damage site, indicating a disruption in end resection, a necessary phase of HR. Our RNA sequencing results show that increasing the dNTP pool in LN229 GBM cells prior to IR treatment caused significant changes in gene expression when compared to IR treatment alone. Pathways most affected include apoptosis, p53 cell cycle arrest, and DNA repair. The pathway analysis has identified gene of interest in the synergistic dNTP + IR treatment model. Significant increases in apoptosis and decreases in viability were observed 48 hours after inducing DSBs in LN229 cells with elevated dNTP pools. Not only do these data suggest that HR efficiency is reduced due to end resection impairment, they also show that as a result, cell viability is destabilized due to prolonged DNA damage, resulting in apoptosis. The identification of this impairment may be essential in finding novel therapeutic approaches to sensitize therapy resistant GBM. This research is supported by Augusta University – Biochemistry & Molecular Biology Department Faculty Start-Up and NIH/NCI K01CA226346 to WD