DOT1L is required for MLL-AF9–induced acute leukemia progression in vivo. (A) Diagram of BMT procedure to examine the requirement of DOT1L in MLL-AF9 AML progression. TAM treatment began at 6 weeks after transplantation after leukemia development. (B-E) In vivo deletion of DOT1L prevents MLL-AF9 AML progression. (B) Reduced spleen size in TAM-treated 2lox/1lox MLL-AF9 recipient mice. Representative images of spleens harvested from transplanted mice at 9 weeks after transplantation (n = 3 per group). (C) TAM-induced DOT1L deletion prevents leukemic infiltration of the liver. Representative H&E staining of liver tissue sections at 9 weeks after transplantation (n = 3 per group). (D) On DOT1L depletion, the percentage of transformed donor cells in bone marrow diminishes over time. FACS analysis of bone marrow cells isolated from transplanted mice (n = 3 per group, per time point). Donor MLL-AF9 cells are Ly5.2⁺ (blue) and recipient/protector cells are Ly5.1⁺/Ly5.2⁺ (red). Percentage of donor Ly5.2 cells in the total bone marrow cell population at 9 and 12 weeks after transplantation are indicated. At 12 weeks after transplantation, cells were also stained with the stem cell/progenitor marker c-Kit. Transformed donor Ly5.2 population is gated and percentage of c-Kit⁺ cells indicated. Shown are representative images from 3 independent experiments. (E) Loss of clonogenic leukemic stem cells after in vivo DOT1L depletion. Methylcellulose colony formation assay of donor cells sorted from mice at 12 weeks after transplantation. Average CFUs are shown (n = 3). Error bars represent SD.