HIF-1 inhibition depletes CML stem cells in combination with TKI treatment. (A) Experimental strategy. BM cells (2 × 10⁶) from BCR-ABL transgenic mice (CD45.1/2) in which leukemia had been induced via tetracycline withdrawal were transplanted into CD45.1 recipient mice. Once mice had developed leukemia 6 or 8 weeks after transplantation, they were treated with vehicle, nilotinib (TKI, 50 mg/kg per day by oral gavage), HIFi (echinomycin 10 mg/kg, intraperitoneal for 5 consecutive days, with 2 days off times, 2 cycles), or the combination for 14 days. Mice were euthanized, and PB, the BM, and the spleen were harvested for analysis (n = 6-10). (B) Total neutrophils (based on differential count) in the PB are shown. (C) Total number of LTHSCs in the spleen are shown. (D) Total number of LTHSCs per 2 femurs and 2 tibiae (4 bones) are shown. (E) BM cells from CML mice treated with vehicle, nilotinib, HIFi, or combination for 2 weeks were transplanted in limiting-dilution transplantation (500 000 cells per mouse, n = 8; 1 000 000 cells per mouse, n = 6; and 2 000 000 cells per mouse, n = 6 each) into CD45.1 recipient mice together with competing normal CD45.2 cells (100 000 cells per mouse). Donor cell engraftment in the PB was evaluated after 16 weeks. (F) Kaplan-Meier plots of CML mice treated with vehicle, TKI, HIFi, or combination for 2 weeks and then followed-up for survival (n = 10 each). Log-rank test indicated significantly increased survival for combination- vs TKI-treated mice (P = .012). (G) Irradiated NRGS mice (400 cGy) received transplantation with human CML CD34⁺ BM cells (0.5×10⁶ cells per mouse). After successful human cell engraftment was confirmed after 6 to 8 weeks, mice were treated with vehicle, nilotinib, HIFi, or combination for 14 days. Mice were euthanized and the BM was harvested for analysis (n = 6-8). (H-K) The absolute number of human CD45⁺ cells (H), human CD34⁺CD38⁺ cells (I), and human CD34⁺CD38⁻CD90⁺ cells (J) are shown. (K) The percent of human CD34⁺CD38⁻CD90⁺ cells expressing HIF-1α after vehicle and nilotinib treatment for 2 weeks are shown. Results represent mean ± SEM of multiple replicates. Significance values: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗ P < .0001. Hu, human; WT, wild-type.