Autophagy is induced as a metabolic adaptation in AML cells after Glut1 disruption. (A-D) Mechanistic investigation of Glut1 disruption was performed in LSC-enriched (c-Kit⁺) bone marrow–derived MLL::AF9 cells, 4 days after transduction. (A) Representative histograms showing flow cytometric quantification of autophagic vesicle load (autophagosomes and autolysosomes) using the cell-permeant aliphatic Autophagy Probe Red. Mean fluorescent intensity (MFI) expression is depicted, unstained control is shown in gray. (B) Bar chart showing MFI of LC3B autophagy marker in Glut1-disrupted AML cells compared with cells transduced with nontargeting control. (C) Representative western blot and (D) its corresponding quantification of LC3B-I and LC3B-II. Data were normalized to expression in nontargeting control and to actin as a loading control (n = 2). (E) mRNA expression of Atg7 in sorted GFP⁺ (sgRNA-expressing) leukemia cells, 3 days after transduction, represented as FPKM values (n = 4). (F) Flow cytometric quantification of viable MLL::AF9 cells transduced with Glut1 sgRNAs or nontargeting control and then treated with dimethyl sulfoxide (DMSO) (vehicle) or 1 μM chloroquine (CQ) for 72 hours. Data were normalized to corresponding DMSO-treated controls, and statistics measured by unpaired 2-tailed Student t test. 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.