Baf A1 preferentially targets functionally defined LSCs and leads to the accumulation of damaged mitochondria. (A) Primary cells from patients with AML were grown in methylcellulose with the indicated drug (7.5 µM PTL, 2.5 µM Ara-C, 40 µM CQ, 50 nM Baf A1, and 10 mM 3-MA). Data are shown as the number of CFUs, compared with those in untreated cells. After the primary plating was quantified, the cells were collected for a secondary replating (technical replicates). Error bars represent ± standard error of the mean (SEM). Statistical analysis compared each treatment mean with that of Baf A1. (B) NSG mice were inoculated with samples from 2 patients with AML (AML 1 and AML 2) and treated with DMSO or Baf A1 (1 mg/kg) intraperitoneally 2 times per week for 4 weeks (5 mice per group). The BM was then harvested from the mouse recipients of the primary transplant, measured for AML burden by flow cytometry for human and mouse CD45, and injected via the tail vein into the second set of NSG mice. The AML burden of the secondary transplant was measured after 6 to 8 weeks. (C) Electron micrographs of healthy donor CB or AML18 samples after overnight treatment with Baf A1 (25 nM). AML cells demonstrated an increase in the number of mitochondria. Original magnification ×15 000. (D) CD34⁺-enriched samples from patients with AML were treated with 25 nM Baf A1 for 48 hours, then stained for mitochondria using MitoID (2 technical replicates). Data are shown as mean fluorescence intensity (MFI) of MitoID. (E-G) AML samples or MOLM-13 cells were treated with Baf A1 or CQ for 48 hours. Viability of all samples was >85%, by trypan blue exclusion. (E) Representative MitoStress Test profile determined with a Seahorse XF analyzer. (F) MitoID MFI (≥3 biological replicates; 3 technical replicates). (G) Average maximal respiration (≥3 biological replicates; 3 technical replicates). Error bars represent ± standard deviation. *P < .05; ** P < .01; *** P < .001. ns, not significant.