Venetoclax-azacitidine resistanceisassociated with upregulation of MCL-1 and FLT3 signaling and could be partly overcome by gilteritinib. (A) Fifty percent inhibitory concentrations (IC50s) for venetoclax, azacitidine, and gilteritinib in sensitive and venetoclax-azacitidine–resistant HL60 cells were measured by treating cells in triplicate with the drugs in 7 concentrations (1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, and 10 μM) for 48 hours and staining with MTS reagent. IC50 was calculated at grcalculator.org, and representative results of 3 independent experiments are shown. (B) MCL-1 levels in sensitive and resistant HL60 cells as estimated by western blotting. Blot is representative of 3 independent experiments. (C) Resistant HL60 cells were treated in technical triplicate with venetoclax (0, 10, 20, 50, 100, 500, and 1000 nM) and gilteritinib (0, 100, 500, and 1000 nM) for 48 hours. Viability was assessed by staining with MTS reagent. Data are presented as mean ± standard deviation from 1 of 3 independent experiments. (D) Effect of venetoclax-gilteritinib combination on colony-formation capacity of resistant HL60 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 shown. Bliss scores are given in a range of −100 to 100, with 100 as maximum Bliss score. (E) Normalized enrichment score (NES) plot for FLT3 (left) and MAPK (right) signaling in sensitive vs venetoclax-azacitidine–resistant HL60 cells. (F) Heat map of MAPK (left) and FLT3 (right) signaling–associated proteins differentially expressed in sensitive vs venetoclax-azacitidine–resistant HL60 cells. ∗∗∗∗∗P < .000005.
