Abstract B020: Understanding the role of dNTP pool on double-strand break and resistance to therapy in quiescent hypoxic glioblastoma

Abstract

Abstract Glioblastoma (GBM) is the deadliest type of primary brain tumor. After surgical resection, chemotherapy, and radiotherapy (RT) are used as treatment modalities to induce double-strand breaks (DSBs), the most cytotoxic and lethal form of DNA damage. However, an inadequate oxygen supply and the high metabolic demand in rapidly growing solid tumors like GBM result in severe hypoxic regions contributing to altered cellular metabolism and resistance to these cancer therapies. These subpopulations of resistant and refractory hypoxic cancer cells are clinically significant as they lead to poor prognosis and adverse clinical outcomes in patients. Ionizing radiation (IR) and chemotherapeutics such as temozolomide induce DNA damage, such as base deletions, single-strand breaks (SSBs), and DSBs. Rapidly replicating cells are more sensitive to these therapeutic agents partly due to replication-induced conversion of SSB and other manageable damages to highly lethal DSB. A balanced deoxyribonucleoside triphosphate (dNTP) is crucial for DNA replication, repair, and maintenance of genomic stability. Cellular dNTP is mainly regulated by ribonucleotide reductase (RNR) and the SAM- and HD-domain-containing protein (SAMHD1). RNR boosts the cellular dNTP pool through the de novo synthesis while SAMHD1 depletes the pool by hydrolyzing dNTPs. Previous studies have shown that hypoxic cancer cells have low dNTP levels due to oxygen dependency of its rate limiting RNR enzyme, which could result in reduced DNA replication and cell cycle arrest. Here, we show the direct relationships between dNTP levels, DNA replication, and DSB induction in hypoxic GBM cancer cells, which is currently unknown. To bridge this knowledge gap, we exposed GBM cells to severe hypoxia and utilized cellular dNTP analysis to demonstrate that their dNTP pool can be elevated. Our result shows that elevating cellular dNTP pool relieved hypoxia-induced cell cycle arrest as determined by flow cytometry and increased their DNA replication. A single cell microscopic analysis also revealed that increasing the dNTP pool significantly enhanced the induction of DSBs following exposure to IR and bleomycin. Furthermore, we detected an increased efficacy of DSB-inducing agents in hypoxic GBM as demonstrated by apoptosis pathway activation. Our result suggest that increasing the intracellular dNTP pool in hypoxic GBM will stimulate DNA replication, increase genotoxin-induced DSB, and improve DSB-inducing therapeutics' efficacy. This study provides a preclinical justification for using cellular dNTP modulation as a novel therapeutic strategy and an adjunct to classical DSB-inducing agents in hypoxic GBM. Citation Format: Edidiong R. Usoro, Dominique Monroe, Kristen Carver, Maya Jenkins, Emily Steinbeck, Arilyn Williams, Shelby Aycox, Waaqo Daddacha. Understanding the role of dNTP pool on double-strand break and resistance to therapy in quiescent hypoxic glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B020.