FOXO activation impairs proliferation of MM cells. (A) GSEA enrichment plots show a significant depletion of cell cycle and proliferation-associated gene sets in WT “cas9 only” (WT) clones treated for 20 hours with 2.5 μM MK2206 (‘WTMK’; left side of the plots) vs MK2206-treated FOXO knockout clones and untreated WT and FOXO knockout clones (‘REST’; right side of the plots). For GSEA, GEP datasets from the LME-1, MM1.S, and XG-3 HMCLs were combined. False discovery rate (FDR), enrichment score (ES), normalized enrichment score (NES), and P values are indicated in the enrichment plots. (B) Bromodeoxyuridine incorporation cell cycle analysis of HMCLs (n = 8) treated for 20 hours with 2.5 μM MK2206. 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 Fisher's least significant difference posttest) was performed on the percentages of cells in S phase (*P < .05, **P < .01, ***P < .001). The mean values of 3 experiments are depicted. (C) AKT inhibition leads to a FOXO-dependent G1 phase arrest. Cell cycle analysis of LME-1, MM1.S, and XG-3 HMCLs and their respective FOXO1 or FOXO3 knockout clones were treated for overnight with 2.5 μM MK2206 (MK). 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 clones (***P < .001, ****P < .0001). The mean values of 3 experiments are depicted. (D) Immunoblot analysis of cell cycle and proliferation-associated proteins in LME-1, MM1.S, and XG-3 control clones and their respective FOXO1 or FOXO3 knockout clones treated overnight with increasing concentrations of 0/0.5/2.5 μM MK2206. β-actin was used as loading control.