Glut1 disruption induces apoptosis and differentiation of MLL::AF9 leukemia cells. (A-E) Mechanistic investigation of Glut1 disruption was performed in LSC-enriched (c-Kit⁺) bone marrow–derived MLL::AF9 cells lentivirally transduced with Glut1 sgRNAs or nontargeting control, 3 or 4 days after transduction. (A) Representative contour plots showing the percentage of early- (annexin-V⁺ 4′,6-diamidino-2-phenylindole [DAPI]⁻) and late-stage (annexin-V⁺ DAPI⁺) apoptotic cells. Flow cytometric quantification of (B) cleaved PARP and (C) cleaved caspase-3 (n = 5). (D) Representative light microscopy images of May-Grünwald Giemsa–stained MLL::AF9 cells. Arrows delineate chromatin condensation in nuclei, and arrowheads indicate cells with more ample cytoplasm, reflecting monocytic differentiation (scale bar, 25 μm). (E) Representative histogram showing cell surface expression of GR-1, unstained shown in light gray. In panels F to I, RNA-seq was performed on sorted GFP⁺MLL::AF9 cells, 3 days after transduction with either Glut1 sgRNAs or nontargeting control (n = 4). Changes in messenger RNA (mRNA) expression of (F) Mpo, (G) Cebpb, (H) Hoxa9 and (I) Meis1 after Glut1 knockdown are represented as fragments per kilobase of transcript per million mapped reads (FPKM) values. Data are shown as mean ± SD (n = 3), unless otherwise stated; statistical testing was performed by 1-way ANOVA. ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. Refer to supplemental Figure 3. PARP, poly-ADP ribose polymerase; RNA-seq, RNA sequencing.