Generation and characterization of sAML cells with resistance to BETi's. (A) Cultured AML cells were treated with the indicated concentrations of OTX015 for 48 hours. Apoptosis was determined by annexin V staining and flow cytometry. The LD₅₀ value for each cell line was calculated with GraphPad V7. (B) PD, CD34⁺ AML (n = 17), and sAML (n = 39) cells were treated with 1 µM OTX015 for 48 hours. The percentage of propidium iodide–positive, nonviable cells were determined by flow cytometry. (C) Schematic of the process used to generate OTX persister/resistant HEL92.1.7 (HEL) and SET-2 cells. (D) SET-2, HEL, and their OTX P/R counterparts were treated with the indicated concentrations of OTX015, JQ1, or ABBV-075 for 48 hours. Apoptosis was determined by annexin V staining and flow cytometry. (E) Relative mRNA expression in SET-2-OTX P/R and HEL-OTX P/R cells compared with parental SET-2 and HEL cells. The relative mRNA expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. *P < .05 and †P < .005 indicate values significantly greater in OTX P/R cells compared with parental cells. (F) Immunoblot analyses of HEL, HEL-OTX P/R, SET-2, and SET-2-OTX P/R cells following 10 shocks with OTX015. The expression levels of β-actin or GAPDH served as the loading control. The graph shows densitometry quantification of protein expression differences in the HEL-OTX P/R and SET-2-OTX P/R cells compared with the parental cells. Asterisk (*) indicates values significantly greater (P < .05) in OTX P/R cells compared with parental cells. (G) SET-2 and SET-2-OTX P/R cells were treated with 1 µM OTX015 for 16 hours. Following this, the cells were washed free of the drug and incubated for 2 and 8 hours. Cells were harvested and lysed, and immunoblot analyses were conducted.