Inhibition of GSK3 partially relieves AKT dependency of MM cells. (A) GSK3 inhibition partially rescued AKT inhibitor–induced cell death in HMCLs (n = 5). Cells were cotreated with 3.2 μM MK2206 and 1 μM GSK3 inhibitor (GSK3 inh.) CHIR99021 for 3 days. Mean ± SEM of 3 independent experiments are shown (**P < .01, ****P < .0001; unpaired Student t test with Welch’s correction). (B) Partial rescue of AKT inhibitor–induced cell death in primary MM patient plasma cells (n = 4). Cells were treated for 3 days with 2.5 μM MK2206 and 1 μM CHIR99021. Specific cell death was calculated based on 7-AAD viability dye staining and flow cytometry. Means of 3 technical replicates are displayed (*P < .05; Wilcoxon signed-rank test). (C) Immunoblot analysis of AKT, FOXO1, FOXO3, phospho-AKT-S473, and phospho-Thr24 FOXO1/phospho-Thr32 FOXO3 in LME-1 cells and MM1.S cells treated overnight with 2.5 μM MK2206, with or without 1 μM CHIR99021. β-actin was used as loading control. (D) Bromodeoxyuridine incorporation cell cycle analysis of HMCLs (n = 5) treated overnight with 2.5 μM MK2206 and 1 μM CHIR99021. Bromodeoxyuridine incorporation and DNA content were assessed by using flow cytometry. Sub-G1 phase (dead) cells were excluded from the analysis. Percentages of cells in the G1, S, and G2 phases of the cell cycle are depicted. Statistical analysis (1-way analysis of variance with Bonferroni’s multiple comparison test) was performed on the percentages of cells in S phase compared with untreated control cultures. Cultures were performed in triplicate (*P < .05, **P < .01).