DNA-PKcs inhibition sensitizes glioblastoma to radiotherapy through reprogramming of tumor cell states and immune microenvironment cell types.

Glioblastoma is the most common malignant brain tumor in adults, and radiotherapy (RT) is the most effective postoperative treatment for patients with glioblastoma. Nevertheless, glioblastoma recurrence after treatment is nearly universal, and cell state plasticity and intratumor heterogeneity underlie glioblastoma evolution and resistance to treatment. Here we integrate in vitro genome-wide CRISPR interference screens and in vivo perturb-seq in preclinical models with single-nucleus and spatial transcriptomic sequencing of human tumors to identify therapeutic vulnerabilities that overcome glioblastoma resistance to RT. Gene regulatory network modeling identifies DNA-PKcs as a RT-sensitizing target in glioblastoma cells in vitro and in vivo. Small molecule inhibition of DNA-PKcs plus RT improves survival and reprograms tumor cell states and immune microenvironment composition compared to DNA-PKcs inhibition or RT monotherapy. Bioinformatic and imaging analyses of patient-matched glioblastoma samples before and after DNA-PKcs inhibition and RT show that combination therapy drives inflammatory gene expression programs in tumor cells that recruits pro-inflammatory myeloid cells to the tumor microenvironment. Using this framework to inform rational sequential therapy in preclinical models, we show that immunomodulation in response to genomic stress after DNA-PKcs inhibition and RT primes glioblastoma for response to cGAS/STING activation. These data show that DNA-PKcs modulates tumor cell states and immune microenvironment cell types to drive resistance to RT in glioblastoma, and that targeting DNA-PKcs sensitizes glioblastomas to RT and cGAS/STING activation.