Combination of romidepsin (Romi) with AC220 or JQ1 exert synergistic antileukemic effects on FLT3-ITD⁺ and MLL-AF9⁺ AML, respectively. (A-B) Molm-13, THP-1, and MV4-11 cells were incubated with Romi (3 nM), AC220 (10 nM), JQ1 (100 nM), or combination as indicated for 48 hours, and apoptosis was determined by flow cytometric analysis of Annexin V/7-AAD. Percentage of annexin V⁺ cells (A) and the representative fluorescence-activated cell sorting (FACS) plots of Molm-13 cells (B). (C) Representative IF micrographs (left) from 3 independent experiments showing γH2AX foci in Molm-13 cells treated with Romi (3 nM), AC220 (10 nM), JQ1(100 nM), or combination as indicated for 48 hours, and quantification (right) of γH2AX foci (red) in nuclei (blue) per cell. Scale bar, 5 μm. (D) Western blot analysis of γH2AX levels in Molm-13 cells treated with Romi (3 nM), AC220 (10 nM), JQ1 (100 nM), or combination as indicated for 48 hours. (E) The AML cell lines with or without GADD45g knockdown were incubated with Romi (3 nM), AC220 (10 nM), JQ1 (100 nM), or combination as indicated for 48 hours; the percentage of apoptosis cells was determined by flow cytometric analysis of Annexin V and 7AAD staining. (F-G) Molm13-luc2 cells were injected intravenously into sublethally irradiated NOD/SCID mice (8 × 10⁵ cells per mouse). Five days later, mice were treated with vehicle or Romi (1.5 mg/kg), AC220 (10 mg/kg), JQ1 (50 mg/kg), or combination as indicated for 2 weeks. (F) Bioluminescence imaging of representative mice from each group taken at day 25 posttransplantation. (G) Survival curve of each group mice. P values were determined by using the log-rank test (n = 5). Data are presented as the mean ± SD of ≥3 independent experiments. Comparisons were evaluated by using the 2-tailed Student t test, and multiple groups were analyzed with the 1-way analysis of variance. *P < .05, **P < .01, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, not significant.