Figure 6.
AKT inhibition in MM cells leads to the FOXO/GSK3-mediated MCL1 down-modulation resulting in cell death. (A) Immunoblot analysis of MCL1 protein expression in WT “cas9 only” clones treated overnight with increasing concentrations of MK2206 (0, 0.5, 2.5 μM) with or without 1 μM GSK3 inhibitor (GSK3 inh.) CHIR99021, and in MK2206-treated (0, 0.5, 2.5 μM) FOXO1 or FOXO3 knockout HMCLs. (B) Immunoblot analysis of MCL1 protein expression in primary MM patient plasma cells (n = 5) treated overnight with or without 2.5 μM MK2206. (C) Immunoblot analysis of MCL1 protein stability in HMCLs (n = 5) after cycloheximide (CHX) treatment (200 µg/mL), with or without pretreatment of 2.5 µM MK2206 for 12 hours. Cells were treated with CHX as indicated by depicted time points. (D) Immunoblot analysis of MCL1 protein expression in HMCLs (n = 3) overexpressing MCL1. HMCLs expressing empty vector (EV) were used as controls. β-actin was used as loading control. (E) MCL1 overexpression rescues AKT inhibitor-induced cell death. HMCLs overexpressing MCL1 (n = 3) were cultured for 3 days with various concentrations of MK2206 (1.6, 3.2, 6.4 µM). HMCLs transduced with EV were used as controls. Specific cell death was assessed by 7-AAD viability dye staining and flow cytometry. Mean ± SEM of 3 independent experiments are shown (****P < .0001; 1-way analysis of variance with Bonferroni’s multiple comparison test). (F) Inhibition of AKT sensitizes HMCLs (n = 5) to MCL1 inhibitor–induced cell death. Cells were treated for 3 days with various concentrations of MK2206 and MCL1 inhibitor (MCL1 inh.) S63845, as indicated on the x-axis of the graphs. Percentages of specific cell death are depicted. Mean ± SEM values of 3 experiments are shown. Chou-Talalay combination index (CI) values at ED75 (effective dose causing 75% cell death) are indicated in the graphs. (G) Inhibition of AKT potentiates for MCL1 inhibitor–induced cell death in primary patient plasma cells (n = 4). Cells were treated for 3 days with a concentration of MK2206 and S63845 (2.5 µM MK2206, 100 nM S63845 for AMC_4389, 100 nM MK2206, 4 nM S63845 for AMC_1345 and AMC_6615, and 500 nM MK2206, 20 nM S63845 for AMC_0713) either as single drug or a combination. Mean ± SEM values of 3 technical replicates are shown. The ΔBliss score was calculated by subtracting the predicted cell death (Bliss) from the actual observed effect of the combined inhibitors; −1 indicates an antagonistic effect and +1 indicates a synergistic effect.

AKT inhibition in MM cells leads to the FOXO/GSK3-mediated MCL1 down-modulation resulting in cell death. (A) Immunoblot analysis of MCL1 protein expression in WT “cas9 only” clones treated overnight with increasing concentrations of MK2206 (0, 0.5, 2.5 μM) with or without 1 μM GSK3 inhibitor (GSK3 inh.) CHIR99021, and in MK2206-treated (0, 0.5, 2.5 μM) FOXO1 or FOXO3 knockout HMCLs. (B) Immunoblot analysis of MCL1 protein expression in primary MM patient plasma cells (n = 5) treated overnight with or without 2.5 μM MK2206. (C) Immunoblot analysis of MCL1 protein stability in HMCLs (n = 5) after cycloheximide (CHX) treatment (200 µg/mL), with or without pretreatment of 2.5 µM MK2206 for 12 hours. Cells were treated with CHX as indicated by depicted time points. (D) Immunoblot analysis of MCL1 protein expression in HMCLs (n = 3) overexpressing MCL1. HMCLs expressing empty vector (EV) were used as controls. β-actin was used as loading control. (E) MCL1 overexpression rescues AKT inhibitor-induced cell death. HMCLs overexpressing MCL1 (n = 3) were cultured for 3 days with various concentrations of MK2206 (1.6, 3.2, 6.4 µM). HMCLs transduced with EV were used as controls. Specific cell death was assessed by 7-AAD viability dye staining and flow cytometry. Mean ± SEM of 3 independent experiments are shown (****P < .0001; 1-way analysis of variance with Bonferroni’s multiple comparison test). (F) Inhibition of AKT sensitizes HMCLs (n = 5) to MCL1 inhibitor–induced cell death. Cells were treated for 3 days with various concentrations of MK2206 and MCL1 inhibitor (MCL1 inh.) S63845, as indicated on the x-axis of the graphs. Percentages of specific cell death are depicted. Mean ± SEM values of 3 experiments are shown. Chou-Talalay combination index (CI) values at ED75 (effective dose causing 75% cell death) are indicated in the graphs. (G) Inhibition of AKT potentiates for MCL1 inhibitor–induced cell death in primary patient plasma cells (n = 4). Cells were treated for 3 days with a concentration of MK2206 and S63845 (2.5 µM MK2206, 100 nM S63845 for AMC_4389, 100 nM MK2206, 4 nM S63845 for AMC_1345 and AMC_6615, and 500 nM MK2206, 20 nM S63845 for AMC_0713) either as single drug or a combination. Mean ± SEM values of 3 technical replicates are shown. The ΔBliss score was calculated by subtracting the predicted cell death (Bliss) from the actual observed effect of the combined inhibitors; −1 indicates an antagonistic effect and +1 indicates a synergistic effect.

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