Figure 5.
Overexpression of MCL-1 with S159A mutation inducesresistance to venetoclax, and cotargeting of FLT3 and AXL kinase was crucial for combination effect. (A) Protein expression of MCL-1 in parental HL60, empty vector–transduced, MCL-1–overexpressing, and MCL-1 S159A–mutated HL60 cells as analyzed by western blotting. B-actin levels are given as loading control. Data are representative of 3 independent experiments. (B) Fifty percent inhibitory concentrations (IC50s) for venetoclax, azacitidine, and gilteritinib in empty vector–transduced, MCL-1–overexpressing, and MCL-1 S159A–mutated 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. (C) Effect of 20 nM of venetoclax, 500 nM of gilteritinib, or the combination of both on viability of empty vector–transduced, MCL-1–overexpressing, and MCL-1 S159A–mutated HL60 cells. Viability was assessed by staining with MTS reagent and was normalized to untreated cells. Data are presented as mean ± standard deviation (SD) of 3 technical replicates of 1 representative independent experiment (n = 3). (D) Venetoclax-gilteritinib Bliss scores calculated from effect of the drug combination on viability of respectively transduced HL60 cells. Bliss scores are given in a range of −100 to 100, with 100 as maximum Bliss score. Bliss scores of ≥0 indicate synergy. (E) Protein expression of MCL-1, pERK, ERK, pGSK3, and GSK3 in HL60 cells treated for indicated time span with 1 μM of bemcentinib, 20 nM of venetoclax, or the combination of both as analyzed by western blotting. B-actin levels are given as loading control. Quantification was performed using ImageJ. Data are representative of 3 independent experiments. (F) Effect of 20 nM of venetoclax, 1 μM of gilteritinib, 1 μM of bemcentinib, 1 μM of quizartinib, or various combinations of the drugs on viability of HL60 cells. Viability was assessed by staining with MTS reagent and was normalized to untreated cells. Data are presented as mean ± SD of 3 technical replicates of 1 representative independent experiment (n = 3).

Overexpression of MCL-1 with S159A mutation inducesresistance to venetoclax, and cotargeting of FLT3 and AXL kinase was crucial for combination effect. (A) Protein expression of MCL-1 in parental HL60, empty vector–transduced, MCL-1–overexpressing, and MCL-1 S159A–mutated HL60 cells as analyzed by western blotting. B-actin levels are given as loading control. Data are representative of 3 independent experiments. (B) Fifty percent inhibitory concentrations (IC50s) for venetoclax, azacitidine, and gilteritinib in empty vector–transduced, MCL-1–overexpressing, and MCL-1 S159A–mutated 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. (C) Effect of 20 nM of venetoclax, 500 nM of gilteritinib, or the combination of both on viability of empty vector–transduced, MCL-1–overexpressing, and MCL-1 S159A–mutated HL60 cells. Viability was assessed by staining with MTS reagent and was normalized to untreated cells. Data are presented as mean ± standard deviation (SD) of 3 technical replicates of 1 representative independent experiment (n = 3). (D) Venetoclax-gilteritinib Bliss scores calculated from effect of the drug combination on viability of respectively transduced HL60 cells. Bliss scores are given in a range of −100 to 100, with 100 as maximum Bliss score. Bliss scores of ≥0 indicate synergy. (E) Protein expression of MCL-1, pERK, ERK, pGSK3, and GSK3 in HL60 cells treated for indicated time span with 1 μM of bemcentinib, 20 nM of venetoclax, or the combination of both as analyzed by western blotting. B-actin levels are given as loading control. Quantification was performed using ImageJ. Data are representative of 3 independent experiments. (F) Effect of 20 nM of venetoclax, 1 μM of gilteritinib, 1 μM of bemcentinib, 1 μM of quizartinib, or various combinations of the drugs on viability of HL60 cells. Viability was assessed by staining with MTS reagent and was normalized to untreated cells. Data are presented as mean ± SD of 3 technical replicates of 1 representative independent experiment (n = 3).

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