FLT3 TK inhibition reduces glucose uptake and central carbon metabolism without affecting glutamine uptake. (A-D) Time-course analysis of glucose and glutamine uptake from media by MV411 (A-B) and MOLM13 (C-D) cells treated with AC220 1 nM or vehicle control (mean ± SEM, n = 3; ****P < .0001; 2-way ANOVA with Bonferroni’s multiple comparisons). (E) Total and isotopologue abundance of selected glycolytic intermediates and products measured by LC-MS analysis in MV411 cell extracts treated with AC220 1 nM or vehicle control and grown in media containing uniformly labeled ¹³C [U-¹³C₆] glucose (GLC) (mean ± SEM, n = 5; ***P = .0004 for total 3-phosphoglycerate and P = .0007 for total lactate; 2-tailed paired t test). (F-G) Extracellular acidification rate (ECAR) of MV411 (F) and MOLM13 (G) cells treated with AC220 1nM or vehicle control (mean ± SEM, n = 3, ****P < .0001, ***P = .0003, 2-way ANOVA with Bonferroni’s multiple comparisons). (H) Total and isotopologue levels of selected TCA cycle intermediates in MV411 cells treated as in panel E (mean ± SEM, n = 5; *** P = .0009,**P = .0011; ns, not significant; 2-tailed, paired Student t test). A.U., arbitrary units. (I) Percentage of total levels of citrate and aspartate provided respectively by the ¹³C₂ and ¹³C₃ fraction following AC220 treatment as in panel E (mean ± SEM, n = 5; ****P < .0001; ns, not significant; 2-way ANOVA with Bonferroni’s multiple comparisons). (J-K) Volcano plot for gene expression changes by RNA sequencing (n = 2 for each cell line) of MV411 and MOLM13 cells treated with AC220 1 nM compared with vehicle control, highlighting reduced expression of glycolysis genes (top) and the minimal effects on TCA cycle genes (bottom). (L-M) Quantitative PCR validation in MV411 (L) and MOLM13 (M) for the reduced expression of lactate dehydrogenase (LDHA) and glucose transporter (GLUT3) after treatment with AC220 1 nM (top) (mean ± SEM, MV411 n = 4, MOLM13 n = 5; for LDHA: **P = .0053, *** P = .0005; for GLUT3, MV411: *P = .0150; MOLM13: *P = .0387; 2-tailed, paired Student t test) and for the lack of changes in expression in glutamine metabolism genes (GLS) and glutamate dehydrogenase (GLUD1) (bottom) (mean ± SEM, n = 4; ns, not significant by 2-tailed, paired Student t test).