HDAC8 inhibition enhances AC220-mediated elimination of FLT3-ITD⁺ AML. (A) MV4-11 cells and MOLM-13 cells were transduced with lentiviral vectors expressing doxycycline-inducible control shRNA (shCtrl) or HDAC8 shRNA (shHDAC8-1 and shHDAC8-2). Doxycycline was added to induce shRNA expression for 48 hours. Then cells were treated with vehicle (dimethyl sulfoxide) or AC220 (5 nM) for another 24 hours. The apoptosis of cells was analyzed by annexin V/propidium iodide (PI) labeling. (B-C) MV4-11 and MOLM-13 cells were treated with the indicated concentrations of AC220 and/or 22d (10 μM). (B) The survival of cells was analyzed by annexin V/PI labeling. (C) Cell growth was evaluated using a Cell Counting Kit-8 (CCK-8) assay. ****P < .0001 vs vehicle control;  ^P < .05,  ^^P < .01,  ^^^P < .001,  ^^^^P < .0001 vs AC220 treatment alone. (D) Primary FLT3-ITD⁺ AML blasts were treated as indicated (AC220, 20 nM; 22d, 10 μM), and apoptosis was analyzed by annexin V/PI labeling. (E) Primary FLT3-ITD⁺ AML (n = 6) blasts were treated with AC220 (20 nM), 22d (10 μM), or the combination for 48 hours. Cell survival was analyzed by annexin V/PI labeling. (F) FLT3-ITD⁺ AML (n = 6) CD34⁺ cells, treated as indicated, were plated in methylcellulose culture, and erythroid or myeloid colonies were enumerated after 14 days. (G) Primary FLT3-ITD⁺ AML (n = 4) blasts, transduced with shCtrl or shHDAC8 vectors, were cultured or not with AC220 (20 nM) for 72 hours. Cell survival was analyzed by annexin V/PI labeling. (H) FLT3-ITD⁺ AML (n = 4) CD34⁺ cells, transduced with shCtrl or shHDAC8 vectors, were cultured or not with AC220 (20 nM) for 72 hours. Colony-formation assays were performed, and erythroid or myeloid colonies were enumerated after 14 days. (I) NOD-SCID mice were transplanted with MV4-11 cells (2 × 10⁶ per mouse) and treated as indicated (AC220, 10 mg/kg per day; 22d, 100 mg/kg per day). The survival of diseased mice was monitored. (J) MV4-11 cells transduced with shCtrl or shHDAC8 vectors were transplanted into NOD-SCID mice, which were then treated as indicated. The survival of diseased mice was monitored. Data are mean ± standard error of the mean. In (A) and (E-J), *P < .05, **P < .01, ***P < .001, ****P < .0001.