AKT prevents FOXO1- or FOXO3-induced cell death of MM cells. (A) Immunoblot analysis of CRISPR/Cas9–generated FOXO1 and FOXO3 knockout clones of the LME-1 (n = 2), MM1.S (n = 4), and XG-3 (n = 4) HMCLs. β-actin was used as loading control. (B) AKT inhibitor–induced cell death is dependent on the presence of FOXO1 in LME-1 and on FOXO3 in MM1.S and XG-3. Cloned knockout and control HMCLs were treated for 3 days with various concentrations of the MK2206 AKT inhibitor. Two to 4 independently established clones were analyzed per condition. Red bars depict FOXO1 knockout clones; blue bars depict FOXO3 knockout clones. Mean ± standard error of the mean (SEM) of 3 independent experiments are shown (**P < .01, ***P < .001, ****P < .0001; 1-way analysis of variance with Dunnett’s multiple comparison test). (C) AKT inhibitor–induced cell death in HMCLs can be rescued by FOXO1 inhibition (n = 5). HMCLs were treated for 3 days with 3.2 μM MK2206 AKT inhibitor, with (orange bars) or without (purple bars) 100 nM of the FOXO1 inhibitor AS1842856. Mean ± SEM of 3 independent experiments are shown (*P < .05, **P < .01, ***P < .001, ****P < .0001; unpaired Student t test with Welch’s correction). (D) Cell death of primary MM patient plasma cells induced by AKT inhibitor MK2206 (2.5 μM) can be overcome by the FOXO1 inhibitor AS1842856 (n = 5). Cells were treated for 3 days with 3.2 μM MK2206 AKT inhibitor, with (orange bars) or without (purple bars) 100 nM of the FOXO1 inhibitor AS1842856. Mean ± SEM of 3 technical replicates are shown (***P < .001, ****P < .0001; unpaired Student t test with Welch’s correction). Specific cell death in these experiments was determined by 7-AAD viability dye staining and flow cytometry. ns, not significant.