PRMT1 inhibition combined with AC220 treatment enhances elimination of FLT3-ITD⁺ AML cells. (A) Western blot analysis of the indicated total and phosphorylated proteins plus GAPDH in cells from 3 FLT3-ITD⁺ AML patients exposed to 5 µM MS023, 20 nM AC220, or a combination (left panels). Quantitative analysis of phospho-AKT (middle panel) and phospho-STAT5 (right panel) levels based on ImageJ software. (B-C) FLT3-ITD⁺ AML (n = 6) CD34⁺ cells were exposed to 5 µM MS023, 20 nM AC220, or a combination. (B) Apoptosis in the indicated groups, as analyzed by Annexin V/4′,6-diamidino-2-phenylindole (DAPI) labeling. (C) CFC analysis of the indicated cells. (D-E) FLT3-ITD⁺ AML (n = 5) CD34⁺ cells were exposed to 3 µM TC-E-5003, 20 nM AC220, or a combination. (D) Apoptosis in the indicated groups, as analyzed by Annexin V/DAPI labeling. (E) CFC analysis of the indicated cells. (F-G) FLT3-ITD⁺ AML (n = 3) CD34⁺ cells transduced with ShCtrl and ShPRMT1 vectors were cultured or not with AC220 (20 nM). (F) Apoptosis in the indicated groups, as analyzed by Annexin V/DAPI labeling. (G) CFC analysis of the indicated cells. (H) Selected CD34⁺ cells from primary human FLT3-ITD⁺ AML cells were injected into irradiated (250 cGy) NSGS mice (1 × 10⁶ cells per mouse). Following confirmation of >1% engraftment, mice were treated for 4 weeks with AC220 (10 mg/kg per day, gavage), MS023 (80 mg/kg per day, intraperitoneally, twice a day), a combination of MS023 and AC220, or vehicle (control) (n = 6 mice per group). Human cell engraftment was analyzed by flow cytometry. Secondary transplantation was also performed. (I) Representative fluorescence-activated cell sorting profile for CD45 and CD33 expression in BM of treated mice. Number of human AML CD45⁺ cells (J) and CD34⁺38⁻ cells (K) in BM of treated mice. (L) Mouse survival after treatment discontinuation. (M) Percentage of human AML cells in BM of secondary recipients at 16 weeks.*P < .05, **P < .01, ***P < .001. Results represent mean ± SD.