Identification of FOXO-dependent gene signatures in MM cells. Independent LME-1 FOXO1 knockout clones (n = 2), MM1.S FOXO3 knockout clones (n = 2), and XG-3 FOXO3 knockout clones and their respective control clones (n = 2) were treated for 20 hours with 2.5 μM MK2206 AKT inhibitor, or left untreated, and subjected to GEP. (A) GSEA enrichment plot of upregulated FOXO3 target genes (DELPUECH_FOXO3_TARGETS_UP) in WT “cas9 only” (WT) clones treated for 20 hours with 2.5 μM MK2206 (‘WTMK’; left side of the plot) vs MK2206-treated FOXO knockout clones, untreated WT, and FOXO knockout clones (‘REST’; right side of the plot) from the LME-1, MM1.S, and XG-3 HMCLs combined. False discovery rate (FDR), enrichment score (ES), normalized enrichment score (NES), and P value are indicated in the enrichment plot. (B) Area proportional Venn diagrams depicting the number of genes that are upregulated (left panel) or downregulated (right panel) in a FOXO-dependent fashion upon AKT inhibition. Genes that overlap in all 3 HMCLs are listed alongside the Venn diagrams. Differentially expressed genes between the groups were defined based on P values, using the following cutoffs: LME-1, P < .15; MM1.S, P < .01; XG-3, P < .02 (analysis of variance corrected for multiple testing by FDR). (C) k-means clustering results (10 rounds, 2 groups, blue and red boxes) and z score heat maps based on the genes that are downregulated upon AKT inhibition in a FOXO-dependent fashion and overlapped in all 3 HMCLs (Figure 3B) in a patient GEP dataset. This set contains GEP and survival data of 542 patients with MM. Blue depicts downregulated gene expression, and red depicts upregulated gene expression. (D) Kaplan-Meier plot depicting overall survival of patients with MM from the GEP dataset, using k-means clustering–derived groups representing high and low expression of FOXO target genes.