Abstract 2820: Defining the role of purine metabolism in radiation resistance in H3K27M–mutant diffuse midline glioma

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). Using liquid chromatography/mass spectrometry “snapshot” metabolomics, we determined that human H3K27M-expressing cells (referred to as K27M cells going forward) possess altered metabolic profiles distinct from their H3-wildtype (H3WT) counterparts characterized by enrichment in metabolites associated with purine metabolism. RT-treated K27M isogenic-paired cells displayed K27M-specific increases in the abundance of both hypoxanthine and guanine and decreased abundance of guanosine, which can be degraded into guanine from mature guanylate species (GMP/GDP/GTP). Hypoxanthine and guanine are the substrates for HGPRT (encoded by HPRT1), the rate-limiting enzyme in purine salvage. Using publicly available data, pediatric high-grade gliomas (pHGG) expressing K27M were found to have decreased HPRT1 transcript compared to H3WT tumors, and we found decreased HGPRT protein expression in K27M cell lines compared to their isogenic H3WT counterparts. The decreased abundance of guanosine, increased abundance of both purine salvage metabolites following RT, and decreased expression of HGPRT suggest impaired guanylate purine salvage in K27M cells that is exacerbated by RT. We hypothesized that K27M cells are deficient in purine salvage and may rely on de novo purine synthesis (DNPS) for purine production following RT. Consistent with this hypothesis, we found that a clinically available, blood-brain penetrant inhibitor of DNPS increased RT-mediated killing of K27M neurospheres in vitro. Combination RT+DNPS inhibition (DNPSi) 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 interrogated metabolic pathway utilization with stable isotope tracing. Using 2,8-deuterium-hypoxanthine to interrogate purine salvage and 15N-glutamine to interrogate de novo purine synthesis, we found an RT-mediated increase in purine salvage that could mediate resistance to DNPSi inhibition. Future experiments will determine whether inhibition of purine salvage has efficacy for those tumors that recur or progress following treatment with RT and DNPSi inhibition. Citation Format: Erik R. Peterson, Peter Sajjakulnukit, Andrew Scott, Caleb Heaslip, Costas Lyssiotis, Maria G. Castro, Daniel R. Wahl. Defining the role of purine metabolism in radiation resistance in H3K27M–mutant diffuse midline glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2820.