MEK-ERK signaling is a crucial pathway and a therapeutic target in NCAM⁺ AML. (A) Quantitative phosphoproteomics of stable isotope labeled Scr- and shNCAM_2-expressing NOMO-1 cells. Left, Volcano plot displaying differential phosphorylation sites of the phosphoproteomic data. Right, Selected differential phosphorylation sites that have a documented impact on the protein function. Data were provided from 3 independent experiments. Statistical analysis was performed with the Limma package in R. (B) List of the 5 most enriched GO biological processes (GO BP) terms for enriched phosphorylation in the indicated groups. (C) RNA-seq was performed after Dox-mediated induction of scrambled shRNA or shNCAM_2 in MOLM-14, SKM1 and NOMO-1 cells for 72 hours and gene set enrichment analysis was performed using the GSEA preranked on all the expressed transcripts. Shown are the gene signatures that most significantly decrease after NCAM downregulation. (D) Samples of the TCGA dataset were preselected for the top (NCAM1_high) and bottom (NCAM1_low) quintile of NCAM1 expression. Gene set enrichment analysis was performed using GSEA preranked on all expressed genes in NCAM1_high and -low samples and compared for overlapping enrichment with the curated MSigDB data sets. Shown are selected enrichments based on the terms shown in panels A-C. (E) Heatmap of sensitivity of leukemic cell lines upon treatment with different small-molecule inhibitors that target the MEK-ERK signaling pathway. Analysis was performed on data derived from the Genomic in cancer sensitivity⁵¹  project. (F) AML cell lines were treated with Trametinib (50 nM) for 24, 48, and 72 hours as indicated. Cellular proliferation was assessed by MTT absorbance and compared by 2-tailed Student t test. Error bars represent mean of 3 measurements ± SD of mean. (G) NOMO-1, SKM-1 and MOLM-14 cells were treated with Ara-C, Trametinib or in combination for 48 hours. Cell viability was measured by MTT, while treatment synergy was measured using the Loewe model (see “Materials and methods”). Shown are dose-response curves in the top panels and 3-dimensional diffusion plots demonstrating treatment synergism in the bottom panels.