Tmet-14. probing tca cycle activity in h3wt and h3k27m-altered diffuse midline gliomas

Abstract DMG has high levels of α-ketoglutarate (α-KG), causing histone hypomethylation and promoting tumor growth. Therefore, targeting α-KG production in DMG tumors may be of therapeutic value. α-KG is synthesized in the tricarboxylic acid cycle (TCA) cycle via oxidation of multiple nutrients including glucose and glutamine. Hence, probing mitochondrial metabolism in DMG with the goal of altering α-KG levels will lead to new therapies to treat DMG. Here, we examine glucose and glutamine metabolism in H3WT and H3K27M-altered patient-derived DMG cells (Dr. Monje’s lab) using GC-MS based metabolic flux analysis. Both cells were grown as neurospheres in tumor stem medium. Neurospheres were treated with 11.0 mM [U-13C]glucose or 4.0 mM [U-13C]glutamine. The 13C-isotopomer analysis revealed that H3K27M-altered cells produced higher levels of (M+3) pyruvate (14.05 ± 5.37% vs. 7.15 ± 2.98%), and (M+3) lactate (9.46 ± 6.48 % vs.3.87 ± 1.80 %) compared to the H3WT. Whereas, the glucose derived (M+2) citrate in the TCA cycle was similar in both cases (15.81 ± 2.91% vs. 16.76 ± 4.15%). This data shows that both types of DMG possess similar TCA cycle activity leading to the synthesis of α-KG, which we measured through glutamate 13C labeling. Next, we probed [U-13C]glutamine metabolism in these two types of DMG cells as glutamine is another major source of α-KG synthesis . Both cell lines showed higher levels of glutamine-derived M+5 α-KG (34.62 ± 2.12 % vs. 42.17 ± 5.05%). Interestingly, H3K27M-altered DMG showed higher flux via reductive carboxylation generating M+5 citrate when compared to M+4 citrate produced via canonical glutamine metabolism in the TCA cycle (citrate M+5: 7.13 ± 2.14% vs. 4.33 ± 1.68%; citrate M+4: 4.04 ± 1.06% vs. 7.85 ± 1.52%). Therefore, inhibiting synthesis of α-KG in H3 K27M-altered DMGs would be of therapeutic interest in the treatment of both DMG subtypes.