Pharmacological inhibition of GLUT1 by BAY-876 selectively targets MLL::AF9 leukemia cells. (A) Dose-dependent inhibitory effect of 1-10 000 nM BAY-876 on c-Kit⁺ normal and c-Kit⁺MLL::AF9 leukemia cells assessed by total ATP levels after 24-hour treatment. The percentage of inhibition in viability is normalized to DMSO-treated controls, and IC₅₀ values are indicated. (B) Flow cytometry quantification of cell numbers of normal and c-Kit⁺MLL::AF9 leukemia cells upon 72-hour treatment with 0.01-10 000 nM BAY-876. (C-E) Dose-dependent effects on (C) glucose uptake, (D) autophagic vesicle load, and (E) early-/late-stage apoptosis status in MLL::AF9 leukemia cells after 24- to 72-hour treatment with BAY-876. (F) Flow cytometric quantification of viable cells after 72-hour treatment with BAY-876 alone or 1 μM CQ. Data is normalized to DMSO-treated control, and synergistic effects are marked with the letter “S.” (G) Schematic experimental outline for assessment of the inhibitory effect of BAY-876 on leukemia cells in vivo. For the experiment with a defined end point, mice received transplantation with 1 × 10⁵MLL::AF9 c-Kit⁺ cells, and 3 days after transplantation, were randomized into groups receiving daily dosing of 4 mg/kg BAY-876 or vehicle (n = 4-6 mice per group). After 10 days of treatment, animals were euthanized (end point) and leukemia burden was assessed. For the survival experiment, mice that received transplantation were treated with 4 mg/kg BAY-876 or vehicle for 21 days and monitored for survival (n = 5-6 mice per group). (H) Longitudinal comparison of body weight, (I) visual comparison of spleen size, and (J) percentage of leukemic occupancy in the BM, spleen, and peripheral blood of mice treated with BAY-876 or vehicle treatment. Significance was measured by unpaired 2-tailed Student t test. (K) Kaplan-Meier survival analysis of mice inoculated with c-Kit⁺MLL::AF9 leukemia cells and treated with vehicle or BAY-876 for 21 days (n = 5-6 per group; log-rank 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; and ∗∗∗∗P < .0001. Illustration in panel G created using BioRender. Refer to supplemental Figure 5. ATP, adenosine triphosphate; BM, bone marrow; IC₅₀, half-maximal inhibitory concentration; RLU, relative luminescence units.