CX-5461 reduces the L-GMP and LIC population in MLL-driven AML. M/E p53WT or p53null AML cells were engrafted into recipient C57Bl/6 mice and treated with CX-5461 for 48 or 72 hours (p53WT 40 mg/kg, p53null 35 mg/kg). (A) The L-GMP population (% of the GFP⁺ tumor cells that are GMP⁺) was determined by flow cytometry at 48 and 72 hours (p53WT: *P = .0224, **P = .0011, n = 5; p53null: **P = .0017; n = 4). (B) Serial dilution transplant experiment. M/E tumor cells from the BM of either a CX-5461 or vehicle-treated mouse were re-injected into recipient C57Bl/6 mice (500 000 cells: n = 5 per group; 10 to 100 000 cells: n = 9 per group) (500 000 cells vehicle vs CX-5461, P = .0027; 100 000 cells vehicle vs CX-5461, P < .0001; 10 000 cells vehicle vs CX-5461, P = .0078; and 1000 cells vehicle vs CX-5461, P < .0001). (C) Single-dose CX-5461 treatment reduces LIC frequency. (D) Drug treatment reduces the clonogenic capacity in methylcellulose (****P < .0001; n = 5). (E) CX-5461 (500 nM) reduces colony forming potential in primary human MLL and non-MLL rearranged AML. Representative image from 1 patient sample. (F) Schematic overview of the patient-derived AML (relapsed/refractory, NOS, FAB M1, and non-MLL) xenograft experiments. (G) CX-5461 (40 mg/kg) therapy (total of 6 doses) reduced the percentage of hCD45⁺ tumor cells in the BM of mice with established human disease (**P = .0075; n = 6). (H) Colony formation of sorted hCD45⁺ tumor cells is significantly reduced post–CX-5461 therapy ex vivo (**P = .0048; n = 4). (I) Delayed disease latency in secondary recipient transplanted with sorted CX-5461–treated hCD45⁺ tumor cells assessed by analysis of disease burden in the BM of secondary recipient (****P < .0001; n = 6). Unpaired 2-tailed Student t test. Graphs represent mean ± SEM. CI, confidence interval; FAB, French-American-British; FACS, fluorescence-activated cell sorting; NOS, not otherwise specified; n.s., not significant; pb, peripheral blood.