Ataxia-telangiectasia mutated kinase disruption enhances the efficacy of radiation therapy in spatially-directed diffuse midline gliomas

Diffuse midline gliomas (DMGs) are lethal brain tumors characterized by p53-inactivating mutations and oncohistone H3.3K27M mutations that rewire the cellular response to genotoxic stress. Using RCAS/tv-a retroviruses and Cre recombinase to inactivate p53 and induce native H3.3K27M mutations in a lineage- and spatially-directed manner, we generated primary mouse tumors that recapitulate human DMG. Disrupting ataxia-telangiectasia mutated kinase (ATM) enhanced the efficacy of radiation therapy in murine and patient-derived DMG models, increasing survival. Microscopy-based in situ sequencing was used to spatially resolve transcriptional profiles in >750,000 single cells with or without ATM disruption and radiation therapy, revealing altered immune-neoplastic and endothelial cell interactions after treatment. An allelic series of primary murine DMG models with different p53 mutations confirmed that transactivation-independent p53 activity is a key mediator of radiosensitivity after ATM disruption. Our findings contribute primary DMG mouse models with deep profiling and reveal the mechanisms of treatment response to an actionable therapeutic strategy. Overall design: Bisulfite methylation sequencing and data analysis were performed by Novogene. Briefly, K27M mutant (nPH) and matched K27M wildtype (nP) tumor-bearing mice (n = 4 biological replicates per group) were generated, and tumor-bearing brains embedded in FFPE. Tumor regions were identified in the brains on matched H+E slides, and tumor microdissection was performed . Genomic DNA was isolated, spiked with lambda bacteriophage DNA (to serve as an internal negative control), fragmented to 200-400 base pairs, and bisulfite treatment was performed to convert unmethylated cytosines into uracil via deamination. After methylation sequencing adapter ligation, double strand DNA synthesis, and library size selection, PCR amplification was performed followed by Illumina sequencing. FastQC was used for quality control on the raw reads.