HIF-1 inhibition enhances mitochondrial activity, ROS, and cell cycling in TKI-treated CML stem cells. scRNA sequencing analysis was performed on LSK cells from CML mice treated with vehicle, nilotinib (TKI), echinomycin (HIFi), or the combination (TKI + HIFi). (A) uMAP display of CML LSK cells treated with vehicle (n = 12 368 single cells; 4 samples), TKI (n = 19 866 single cells; 2 samples), HIFi (n = 18 686 single cells; 4 samples), or the combination (n = 19 836 single cells; 4 samples). (B) The percentage of cells within individual clusters after treatment, as indicated. (C) Flow cytometry analysis of dormant LTHSCs (CD34⁻) and cycling LTHSCs (CD34⁺) after treatment, as indicated. (D) Cell cycle analysis of LTHSCs from CML mice treated with vehicle, TKI, HIFi, or the combination (n = 6 each) using Ki67-DAPI labeling. (E) NES of hallmark gene sets (FDR < 0.05) comparing combination-, TKI-, and vehicle-treated qHSCs. (F-H) Results of SCENITH analysis showing changes in protein synthesis (metabolic activity) (F), mitochondrial dependence (G), and glycolytic capacity (H) in cycling and dormant LTHSCs from mice receiving the indicated treatments (n = 5-6). (I) t-distributed stochastic neighbor embedding plot showing TMRM fluorescence in dormant LTHSCs from mice receiving the indicated treatments (n = 6, concatenated). (J) Mitochondrial ROS levels measured using Mitosox in dormant and cycling LTHSCs from mice receiving the indicated treatments (n = 6 each). Results represent mean ± SEM of multiple replicates. Significance values: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.