OGDH inhibition reduces TCA cycle flux and aspartate biosynthesis. (A-B) Analysis of mitochondrial bioenergetics. Agilent Seahorse XF Cell Mito Stress Test assay was performed on Nras^G12D; MLL-AF9 AML cells (A) and p53^R172H AML cells (B) at 48 hours after OGDH depletion for the measurement of cellular oxygen consumption rate (OCR). Mitochondrial inhibitors are individually labeled and were added at the indicated time points. (C) TCA cycle and related metabolite levels in p53^R172H AML cells. LC-MS was performed at 48 hours after knockdown with the indicated shRNAs. Data are shown as a heat map of log₂ fold change relative to shControl-treated cells. Red color represents upregulated; blue color, downregulated. P values are indicated (2-way ANOVA with Sidak multiple comparisons test; n = 3-6 per condition). (D) Schematic of oxidative flux (carbons labeled in green) or reductive flux (carbons labeled in purple) through the TCA cycle by ¹³C₅-glutamine tracing. Red “X” indicates block observed in p53^R172H AML after OGDH knockdown. Labeled carbons are indicated in gray, green, or purple. (E) Fraction (%) of m+4 labeling from ¹³C₅-glutamine for the indicated metabolites in p53^R172H AML after induction of shRNAs targeting OGDH (or control) for 48 hours and labeling for 6 hours. P values indicated (1-way ANOVA with Dunnett multiple comparisons test; n = 3 per condition). (F) Fraction (%) of m+5 labeled citrate or m+3 labeled aspartate from ¹³C₅-glutamine in p53^R172H AML after induction of shRNAs targeting OGDH (or control) for 48 hours and labeling for 6 hours. P values indicated (1-way ANOVA with Dunnett multiple comparisons test or Kruskal-Wallis with Dunn multiple comparisons test as appropriate; n = 3 per condition). (G) After induction of shRNAs targeting OGDH (or control) in p53^R172H AML for 48 hours and labeling for 6 hours, LC-MS was used to determine the steady-state level and fraction of aspartate labeling from ¹³C₆-glucose (left) or ¹³C₅-glutamine (right). With ¹³C₆-glucose tracing, the m+2 and m+4 fractions represent 1 and 2 forward rotations through the TCA cycle, respectively, whereas with ¹³C₅-glutamine tracing m+2 represents 2 forward rotations and m+4 represents 1. Aspartate levels are shown as peak area in arbitrary units (a.u.) × fractional percentage of labeling with each tracer. P values are indicated for total labeled aspartate in shControl vs shOgdh groups (1-way ANOVA with Dunnett multiple comparisons test; n = 3 per condition). Ac-CoA, acetyl-coenzyme A; ctl, shControl; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; ns, not significant.