TMET-05. Tracing Nutrient Utilization and α-KG Production in H3K27M and H3-WT DIPG Using13C-Labeled Substrates

Abstract DIPG exhibits elevated levels of α-ketoglutarate (α-KG), a key co-factor for DNA and histone demethylases, which drives epigenetic reprogramming and promotes tumor growth. Glucose, glutamine, acetate, and ketone body (e.g., betahydroxybutyrate, BHB) are the major nutrient sources that contribute to α-KG synthesis via TCA cycle in the tumor cells. Here, we examine the role of the above nutrients in the synthesis of α-KG in H3.3K27M, H3.1K27M mutants and H3WT patient-derived DIPG cells using 13C- tracer based isotopomer analysis. DIPG cells were grown as neurospheres and were incubated for 24 hours with 11.0 mM [U-13C]glucose or 4.0 mM [U-13C]glutamine or [1,2-13C]acetate or [U-13C]BHB. Cells were washed with cold PBS and were harvested in 1.5 mL 50% MeOH. Cell lysates were analyzed by GC-MS for mass isotopomer distributions of various metabolites involved in the glycolysis and TCA cycle. We determined α-KG production from glucose, glutamine, acetate, and BHB in patient-derived DIPG cultures: SU-DIPG-13 (H3.3K27M), SU-DIPG-36 (H3.1K27M), and SU-DIPG-48 (H3WT). Glucose contributed comparably across lines (13.2 ± 3.5, 9.0 ± 3.0, and 13.1 ± 2.0), indicating no mutation-specific differences. Glutamine was the dominant α-KG source in all lines, with markedly elevated levels in H3.1K27M (61.8 ± 1.1) compared to H3WT (40.9 ± 5.9) and H3.3K27M (29.5 ± 1.9), suggesting genotype-dependent glutaminolysis. Acetate-derived α-KG was substantially higher in H3K27M-mutant lines (27.0 ± 0.9 and 20.0± 0.7) than in H3WT (2.0 ± 0.4), indicating a mutation-associated shift toward acetate metabolism. BHB contribution to α-KG was modest but comparable to glucose across all lines: 8.0 ± 1.1 (H3.3K27M), 5.3 ± 0.5 (H3.1K27M), and 9.4 ± 0.2 (H3WT), highlighting BHB as a consistent alternative substrate. These findings reveal that H3K27M-mutant DIPG preferentially utilizes glutamine and acetate to sustain α-KG production and epigenetic remodeling. Thus, targeting glutamine and acetate metabolism may offer therapeutic potential in treating H3K-mutant DIPG.