Abstract 1112: Modulated dNTP pools and their influence on DNA repair mechanisms and apoptosis in therapy resistant cancers

Abstract

Abstract 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. Using a neutral comet assay, we quantified accumulated double-stranded breaks in different treatment groups. We then analyzed these groups using immunofluorescent microscopy to examine distinct proteins involved in HR-mediated repair. We carried out RNA sequencing analysis to determine differential gene expression. We quantified cell viability and apoptotic cell populations via flow cytometry and Alamar blue assays. We determined protein interactions using immunoblotting and immunoprecipitation assays. We found that increasing the dNTP pool prior to IR treatment led to impaired DNA DSB repair in GBM cells as determined by a neutral comet assay. When analyzing key proteins involved in HR 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. The most impacted pathways most were apoptosis, p53 cell cycle arrest, and DNA repair. The pathway analysis has identified genes 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, but 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. Citation Format: Dominique Monroe, Mercy Kehinde-Ige, Edidiong Usoro, Arilyn Williams, Emily Steinbeck, Shelby Aycox, Huidong Shi, Waaqo Daddacha. Modulated dNTP pools and their influence on DNA repair mechanisms and apoptosis in therapy resistant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1112.