Abstract 1818: Enhancing double-strand break induction in hypoxic glioblastoma cells through dNTP modulation and its potential therapeutic implications

Abstract Glioblastoma (GBM), the most lethal primary brain tumor, remains resistant to current treatment modalities that include surgical resection, chemotherapy, and radiotherapy (RT). A critical challenge in GBM therapy is the presence of severe hypoxic tumor regions caused by the rapid growth of solid tumors and poor vascularization. Hypoxia drives therapeutic resistance by impairing DNA damage induction and repair mechanisms, particularly double-strand breaks (DSBs), which are the most cytotoxic lesions induced by RT and chemotherapeutic agents.Cellular deoxyribonucleoside triphosphate (dNTP) pools, tightly regulated by ribonucleotide reductase (RNR) and SAM- and HD-domain-containing protein (SAMHD1), play a pivotal role in DNA replication and repair. Hypoxia disrupts this balance by reducing dNTP synthesis due to the oxygen dependency of RNR, a rate-limiting enzyme, leading to diminished replication and cell cycle arrest. These metabolic adaptations hinder the efficacy of DSB-inducing agents in hypoxic GBM cells. Notably, rapidly replicating cells are more sensitive to cancer therapeutic agents, partly due to replication-induced conversion of single-strand breaks and other manageable damages into highly lethal DSBs.To address this resistance mechanism, we investigated the role of dNTP modulation in enhancing DNA damage and therapeutic outcomes in hypoxic GBM cells. Our study reveals that pharmacologically increasing dNTP levels in hypoxic GBM cells restored cell cycle progression and DNA replication, thereby sensitizing cells to DSB-inducing therapies. Specifically, we found that: 1. Elevated dNTP pools reversed hypoxia-induced cell cycle arrest: Flow cytometry analysis demonstrated a marked increase in S-phase entry upon dNTP modulation under hypoxic conditions. 2. Increased DSB formation following therapy: Single-cell microscopic analysis revealed enhanced DSB induction in hypoxic GBM cells treated with ionizing radiation (IR) or bleomycin after dNTP elevation. 3. Enhanced apoptotic response: Pharmacological dNTP modulation amplified apoptosis activation following DSB induction, indicating improved therapeutic efficacy. This study establishes the critical link between dNTP availability, DNA replication, and hypoxia-induced therapeutic resistance in GBM. By leveraging dNTP modulation, we provide a novel preclinical strategy to overcome hypoxia-mediated resistance to DSB-inducing therapies. Our findings support further exploration of this approach as an adjunctive treatment to improve outcomes in patients with hypoxic GBM. Citation Format: Edidiong R. Usoro, Dominique Monroe, Kristen Carver, Emily Steinbeck, Arilyn Williams, Waaqo Daddacha. Enhancing double-strand break induction in hypoxic glioblastoma cells through dNTP modulation and its potential therapeutic implications [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 1818.