TCA cycle disruption compromises nucleotide biosynthesis in AML. (A) Schematics of the de novo purine (upper) and pyrimidine (lower) biosynthesis pathways highlighting aspartate (Asp)-dependent steps. (B) LC-MS for measurement of the levels of selected nucleotide intermediates (shown in heat map) was performed on p53^R172H AML cells at 48 hours after knockdown with the indicated shRNAs. Data are shown as log₂ fold change relative to shControl-treated cells. P values are indicated (2-way ANOVA with Sidak multiple comparisons test; n = 3-6 per condition). (C-D) Labeling of IMP and AMP. 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 fraction (%) of IMP or AMP labeling from ¹³C₆-glucose (C) or ¹⁵N₁ glutamine (D). (E-G) The fraction (%) of UMP labeling from ¹³C₆-glucose (E), ¹⁵N₁-glutamine (F), or ¹³C₅-glutamine (G) was measured. For panels C-G, P values are indicated for total percentage of labeled metabolites in shControl (Ctl) vs shOgdh groups (Wilcoxon rank-sum test or unpaired t test as appropriate; n = 3-6 per condition). (H) Levels or ratios of selected TCA cycle and nucleotide metabolites in PDX1 cells. LC-MS for measurement of the indicated metabolites was performed at 7 days after knockdown with the indicated shRNAs. Data are shown as either peak area (a.u) or a ratio. P values are indicated (1-way ANOVA with Sidak multiple comparisons test; n = 3 per condition). CA, carbamoyl aspartate; GDP; guanosine diphosphate; GTP, guanosine-5'-triphosphate; Pi; inorganic phosphate; R5P, ribose 5-phosphate.