Combination of venetoclax-gilteritinib reduces viability, increases apoptosis, and diminishes colony-formation capacity in FLT3 wild-type samples. (A) Dose-response assay for HL60 (left) and OCI-AML2 (right) cells treated with indicated concentrations of venetoclax and gilteritinib for 48 hours. Viability was assessed by staining with MTS reagent and was normalized to untreated controls. Data are presented as mean ± standard deviation (SD) of 3 technical replicates of 1 representative independent experiment (n = 3). (B) Percentage of apoptotic cells was measured by flow cytometry in HL60 and OCI-AML2 cells 24 hours after treatment with venetoclax (0, 10, and 100 nM) and gilteritinib (0 and 500 nM) upon staining with annexin V antibody and PI. Data are presented as mean ± SD of 2 independent experiments comprising 2 technical replicates each. Statistical significance was assessed using a 2-tailed Student unpaired t test. Bliss scores are given in a range of −100 to 100, with 100 as maximum Bliss score. (C) Representative images of fluorescence-activated cell sorting analysis of OCI-AML2 after 24 hours of treatment with gilteritinib (0 and 500 nM) and venetoclax (10 and 100 nM). (D) Effect of venetoclax and gilteritinib on colony-formation capacity of HL60, OCI-AML2, and OCI-AML3 cells was assessed by seeding cells in methylcellulose supplemented with the respective drugs for 10 days. Data of 3 independent experiments with 3 technical replicates each are presented. Bliss scores are given in a range of −100 to 100, with 100 as maximum Bliss score. (E) Representative microscopic images of colony-formation assays using HL60 cells treated with indicated concentrations of venetoclax and gilteritinib for 10 days. ∗P < .05, ∗∗P < .005, ∗∗∗P < .0005, ∗∗∗∗P < .00005.