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Abstract Diffuse midline gliomas (DMG) are an aggressive and treatment-resistant form of brain cancer in urgent need of new and effective treatment strategies. Radiation therapy (RT) is the standard of care for DMGs, but these tumors often recur locally within the high-dose radiation field. Therefore, it is imperative to discover methods of overcoming RT resistance. DMGs are often characterized by the presence of driving mutation in the tail domain of histone H3 that converts the 27th residue from a lysine to a methionine (termed H3K27M). How the H2K27M mutation controls metabolism and RT resistance is not known. Using liquid chromatography/mass spectrometry metabolomics of irradiated and non-irradiated isogenic pairs, we found that patient-derived H3K27M-expressing cells (referred to as K27M cells going forward) possess altered metabolic profiles distinct from their K27M-KO counterparts, characterized by enrichment in metabolites associated with purine metabolism following RT treatment. Stable isotope tracing with precursors for de novo purine synthesis (15N-amide glutamine) and purine salvage (2H 2, 8-hypoxanthine) showed that cells harboring the H3K27M mutation upregulate the de novo synthesis of guanylates but downregulate guanylate salvage. Consistent with these findings, pediatric high-grade glioma tumors and cell lines expressing K27M express lower levels of HPRT1/HGPRT, the rate-limiting enzyme in purine salvage. These findings suggest that K27M cells are preferentially reliant on de novo purine synthesis for guanylate production and that inhibition of this pathway might overcome RT resistance. Consistent with this hypothesis, we found that mycophenolic acid (MPA), a clinically available, blood-brain penetrant inhibitor of de novo purine synthesis, radiosensitized K27M neurospheres in vitro. Combination RT+MPA increased survival (38d post-implantation) over RT alone (31. 5d post-implantation) in Rag1-KO mice bearing orthotopically implanted K27M tumors but did not cure tumors. To understand potential mechanisms of treatment resistance, we performed stable isotope tracing in irradiated K27M isogenic-paired cell lines. We found an H3K27M-specific, adaptive RT-mediated increase in guanylate salvage that was accompanied by increased HGPRT protein expression following RT. Additionally, we discovered that K27M xenograft tumors had high levels of the purine salvage substrate guanine. These findings suggest that K27M cells upregulate purine salvage to evade the effects of RT, which can confer resistance to inhibitors of de novo purine synthesis. Consistent with this hypothesis, H3K27M tumors in which HPRT1 was knocked down grew without difficulty but were profoundly sensitive to RT, which cured many tumors. These findings suggest that targeting the guanylate salvage pathway in DMG-K27M tumors may be a promising strategy to overcome treatment resistance. Citation Format: Erik R. Peterson, Peter Sajjakulnukit, Andrew Scott, Caleb Heaslip, Anthony Andren, Kari Wilder-Romans, Angelica Lin, Alexandra Obrien, Zitong Zhao, Li Zhang, Sriram Venneti, Carl Koschmann, Nada Jabado, Maria G. Castro, Daniel R. Wahl. Adaptive purine metabolism drives radiation therapy resistance in H3K27M-diffuse midline glioma 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 Suppl₁): Abstract nr B045.
Peterson et al. (Mon,) studied this question.