Updated project metadata.
Determining the balance between DNA double strand break repair (DSBR) pathways is essential for understanding treatment response in cancer. We report a novel method for simultaneously measuring non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). This approach revealed enhanced MMEJ and HR in glioblastoma (GBM) xenograft models with acquired temozolomide (TMZ) resistance. Knockdown of proteins in either pathway enhanced killing by TMZ, and a targeted screen identified pharmacological-grade dual HR/MMEJ inhibitors, including AZD1390, an ATM kinase inhibitor. AZD1390 suppressed DSB end resection, blocked phosphorylation of end protection proteins in response to DNA damage, and potentiated TMZ in treatment-naïve and treatment-resistant models. TP53-mutant GBMs were most susceptible to AZD1390 in combination with DNA-damaging agents due to elevated ATM-dependent HR/MMEJ, preferential activation of these pathways in response to DNA damage, and a defective G2/M checkpoint, which caused these GBMs to enter mitosis despite unrepaired DNA damage, leading to cell death via apoptosis. This report establishes ATM-dependent HR and MMEJ as targetable resistance mechanisms in TP53-mutant GBM and establishes an approach for simultaneously measuring multiple DSBR pathways in treatment selection and oncology research.