Combined effects of TKIs and CB839 in BCR-ABL–positive leukemia. (A) Glycolytic rate and capacity of BCR-ABL mutated K562 cells treated with vehicle control or imatinib 2 µM (mean ± SEM, n = 3, P < .0001 for both glycolytic rate and capacity, respectively; 2-way ANOVA with Bonferroni’s multiple comparison). (B) Volcano plot for gene expression changes by RNA sequencing of K562 cells treated with imatinib 2 µM or vehicle control, highlighting reduced expression of genes involved in glycolysis in treated cells (n = 2). (C) Quantitative PCR validation in K562 cells for the reduction in expression levels of GLUT3 (mean ± SEM, n = 3; **P = .0081; 2-tailed paired t test) and LDHA (mean ± SEM, n = 3; *P = .0132; 2-tailed paired t test) after treatment as in panel B. (D) Apoptosis in K562 cells treated with vehicle control, imatinib 2 µM, CB839 100 nM, imatinib and CB839 combination, and imatinib/CB839 combination + 4 mM αKG (mean ± SEM , n = 9; **P = .0019 between imatinib and imatinib + CB839; ****P < .0001 between imatinib + CB839 and imatinib + CB839 + αKG; ANOVA with Tukey’s multiple comparisons). (E) Relative cytoplasmic ROS levels in BCR-ABL mutated K562 cells treated as in panel D (mean ± SEM, n = 6; *P = .0495 between imatinib and imatinib + CB839; **P = .0089 between imatinib + CB839 and imatinib + CB839 + αKG; ANOVA with Sidak’s multiple comparisons). (F) Oxygen consumption rate in K562 cells treated as in panel D (mean ± SEM, n = 3; for basal respiration: P = .0606 between imatinib and imatinib + CB839; P < .0001 between imatinib + CB839 and imatinib + CB839 + αKG; for maximal respiration: ****P < .0001 between both imatinib vs imatinib + CB839 and imatinib + CB839 vs imatinib + CB839 + αKG; 2-way ANOVA with Bonferroni’s multiple comparisons). DMSO, dimethyl sulfoxide; ECAR, extracellular acidification rate.