Glut1 inhibition suppresses glycolysis and reduces levels of TCA intermediates in leukemia cells. (A-B) GSEA of the transcriptional signature of Glut1 knockdown vs control obtained upon Glut1 disruption on sorted GFP⁺ (sgRNA-expressing) leukemia cells (n = 4). In panels C to G, targeted metabolomic analysis of sorted GFP⁺MLL::AF9 cells, 4 days after transduction (Glut1 sgRNA3: n = 3; Glut1 sgRNA1, Glut1 sgRNA2, and nontargeting control: n = 4). Quantification of glycolytic and PPP metabolites by LC-MS; TCA intermediates and amino acids quantified by GC-MS. (C) Graphical representation depicting alterations in the level of key metabolites after Glut1 knockdown. The direction of the change is encoded by color, noted as enrichment (red) and depletion (blue) in Glut1 sgRNA relative to control group. (D-G) Quantification of key metabolites in MLL::AF9 cells with Glut1 knockdown, with abundance normalized to cells transduced with nontargeting control. Metabolites are grouped based on their respective pathways: (D) glycolysis; (E) PPP; (F) TCA; and (G) amino acids. Significance was measured by unpaired 2-tailed Student t test. (H) Basal extracellular acidification rate (ECAR) in the presence of glucose, indicative of glycolytic rate (n = 4), normalized to cell number. (I) Representative glycolysis stress test assay, showing ECAR in basal conditions and after the addition of the indicated compounds in MLL::AF9 cells (n = 4). Glucose, oligomycin, and 2-DG were injected to final concentrations of 25 mM, 1 μM, and 50 mM, respectively. (J) HK enzymatic activity evaluated by detection of NADH (ΔA₄₅₀), and (K) secretion levels of extracellular lactate, the end product of glycolysis, was determined by colorimetry (A₅₇₀). Data are shown as mean ± SD (n = 3) and statistical testing was performed by 1-way ANOVA, unless otherwise stated. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. Refer to supplemental Figure 4 and supplemental Table 5. 2-DG, 2-deoxyglucose; FDR, false discovery rate; GC-MS, gas chromatography–mass spectrometry; HK, hexokinase; LC-MS, liquid chromatography–mass spectrometry; NADH, nicotinamide adenine dinucleotide; NES, normalized enrichment score.