Elevated dNTP Pool Levels Impair Homologous Recombination and Enhance Glioblastoma Sensitivity to Irradiation and Temozolomide

Glioblastoma (GBM) standard of care includes surgical resection followed by ionizing radiation (IR) and Temozolomide, which induce DNA double-strand breaks. Homologous recombination (HR), a critical DNA double-strand break repair pathway, is augmented in GBM, contributing to resistance and poor patient outcomes. Here, we demonstrate that increasing deoxyribonucleoside triphosphate (dNTP) levels impairs HR-mediated double-strand break repair, rendering GBM cells sensitive to IR and Temozolomide. Interestingly, combining an elevated dNTP pool level with IR and/or Temozolomide promotes the recruitment of DNA polymerase-α/primase, which is typically involved in Okazaki fragment synthesis during DNA replication, to the DNA double-strand break site, thereby interfering with DNA end resection. Specifically, higher dNTP pool levels disrupted the recruitment of HR-associated proteins such as RPA70 and RAD51, an effect reversed by Aphidicolin, a DNA polymerase-α/primase inhibitor. Impaired HR delayed IR- and/or Temozolomide-induced DNA double-strand break repair, leading to growth arrest and apoptosis. Furthermore, higher dNTP pool levels led to downregulation of DNA replication and HR-associated genes, while upregulating several pro-apoptotic genes. Increased sensitivity to IR and Temozolomide was also observed in engineered IR-resistant GBM cell lines and in naturally recurrent patient-derived GBM cells that emerge post-therapy. These findings emphasize how dNTP pool levels regulate HR and uncover a promising vulnerability that could be exploited to overcome resistance to DNA-damaging treatments in GBM and beyond.