GLUT1 is required for AML cell growth and survival. (A) Flow cytometric analysis of GLUT1 expression in MLL::AF9 LSC-enriched (c-Kit⁺) cells and their normal bone marrow c-Kit⁺ counterparts. In panels B to F, MLL::AF9 cells were transduced with Glut1 sgRNAs (Glut1 sgRNA1 and sgRNA2) or a nontargeting control cloned into GFP-expressing lentiviral vectors. (B) Genetic editing in the Glut1 locus was quantified by deep sequencing within sorted GFP⁺ cells, 3 days after transduction. (C) Representative histogram of GLUT1 expression measured by flow cytometry within GFP⁺ leukemia cells, 4 days after transduction. (D) Quantification of GFP⁺MLL::AF9 leukemia cells in the bone marrow of mice 12 days after transplantation with c-Kit⁺ leukemia cells transduced with Glut1 sgRNAs or nontargeting control. The percentage of GFP⁺ cells at day 12 was normalized to the input percentage of GFP⁺ cells before transplantation, 2 days after transduction (T₀). (E) Ex vivo competition proliferation assay as measured by the percentage of GFP⁺ leukemia cells on day 2, 5, 8, and 10 after transduction, normalized to the input percentage at day 2 (T₀). (F) Kaplan-Meier survival analysis of mice that received transplantation with sorted GFP⁺ leukemia cells 2 days after transduction (n = 5 mice per group; log-rank test). Data are represented as mean ± standard deviation (SD) with an n = 3, unless otherwise stated. Significance was measured by 1-way analysis of variance (ANOVA) with the following thresholds: ∗∗∗P < .001 and ∗∗∗∗P < .0001. Refer to supplemental Figure 2 and supplemental Table 2.