FOXO represses the expression of metabolic genes in MM cells. (A) GSEA in MMPCs from newly diagnosed patients with MM (n = 75) vs bone marrow plasma cells obtained from healthy donors (n = 15) showing that the shared FOXO–repressed gene set is enriched in MMPCs, indicating lower FOXO activity in these cells. FDR, ES, NES, and P-value are shown in the plots. (B) GSEA in Cas9-CTRL HMCL clones treated overnight with 2.5 μM MK2206 (‘CTRL+MK’) vs the combination of untreated CTRL clones and FOXO KO clones, either treated overnight with 2.5 μM MK2206 or left untreated (‘REST’). Datasets from the HMCLs LME-1, MM1.S, and XG-3 were combined for GSEA. Enrichment plots for Reactome glycolysis, KEGG TCA cycle, and GSEA Hallmark_oxidative_phosphorylation gene sets are shown. FDR, ES, NES, and P-values are shown in the plots. (C) Heatmaps showing (z-score transformed) expression of Reactome glycolysis genes (upper panels) and KEGG_TCA cycle genes (lower panels) in LME-1 CTRL clones and LME-1 FOXO1-knockout (FOXO1-/-) clones (left panels), MM1.S CTRL clones and FOXO3-KO (FOXO3-/-) (middle panels), and XG-3 CTRL clones and FOXO3-KO clones (right panels) that were treated overnight with 2.5 μM MK2206 or left untreated. Z-score values are depicted, ranging from −3 (blue) indicating low expression to 3 (red) indicating high expression. (D) Schematic representation of the glycolysis metabolic pathway and the TCA cycle. Enzymes (purple boxes), metabolites, and conversions (arrows) are indicated. For the glycolysis pathway, red, blue, and yellow symbols indicate >1.5-fold (FOXO-dependent) decrease in gene expression in LME-1, MM1.S, and XG-3, respectively. For the TCA cycle a >1.2-fold (FOXO-dependent) decrease in gene expression is indicated. (E) Schematic overview of k-means clustering approach (10 rounds) to define 2 groups of patients with MM (n = 542) based on the expression of the experimentally defined shared FOXO–repressed gene set identified in the LME-1, MM1.S, and XG-3 HMCLs, as previously described by us.¹⁹ Groups of patients with MM defined by k-means clustering were labeled as ‘FOXO high’ (n = 387) and ’FOXO low’ (n = 155). GSEA enrichment plots are shown, interrogating Reactome for glycolysis, KEGG for TCA cycle, and GSEA for Hallmark_oxidative_phosphorylation gene sets. CTRL, control; ES, enrichment score; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes; KO, knockout; NES, normalized enrichment score.