Purine salvage promotes treatment resistance in H3K27M-mutant diffuse midline glioma

Abstract

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are a fatal form of brain cancer. These tumors often carry a driver mutation on histone H3 converting lysine 27 to methionine (H3K27M). DMG-H3K27M are characterized by altered metabolism and resistance to standard of care radiation (RT) but how the H3K27M mediates the metabolic response to radiation and consequent treatment resistance is uncertain. We performed metabolomics on irradiated and untreated H3K27M isogenic DMG cell lines and observed an H3K27M-specific enrichment for purine synthesis pathways. We profiled the expression of purine synthesis enzymes in publicly available patient data and our models, quantified purine synthesis using stable isotope tracing, and characterized the in vitro and in vivo response to de novo and salvage purine synthesis inhibition in combination with RT. DMG-H3K27M cells activate purine metabolism in an H3K27M-specific fashion. In the absence of genotoxic treatment, H3K27M-expressing cells have higher relative activity of de novo synthesis and apparent lower activity of purine salvage demonstrated via stable isotope tracing of key metabolites in purine synthesis and by lower expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the rate-limiting enzyme of purine salvage into IMP and GMP. Inhibition of de novo guanylate synthesis radiosensitized DMG-H3K27M cells in vitro and in vivo. Irradiated H3K27M cells upregulated HGPRT expression and hypoxanthine-derived guanylate salvage but maintained high levels of guanine-derived salvage. Exogenous guanine supplementation decreased radiosensitization in cells treated with combination RT and de novo purine synthesis inhibition. Silencing HGPRT combined with RT markedly suppressed DMG-H3K27M tumor growth in vivo. Our results indicate that DMG-H3K27M cells rely on highly active purine synthesis, both from the de novo and salvage synthesis pathways. However, highly active salvage of free purine bases into mature guanylates can bypass inhibition of the de novo synthetic pathway. We conclude that inhibiting purine salvage may be a promising strategy to overcome treatment resistance in DMG-H3K27M tumors.