Advancing therapeutic target discovery with multi-omics: Effect of unbiased phosphoproteomic and metabolic profiling in WT and KRAS mutant colorectal cancer cells on molecular interplay between protein regulation and metabolic rewiring.

e15714 Background: KRAS mutations cause significant metabolic and protein dysregulation in cancer, leading to alterations in cellular signaling pathways, promoting uncontrolled cell growth and proliferation. The metabolic reprogramming of altered KRAS function includes increased glucose, amino acid, and lipid metabolism. The changes in the cellular proteome stem from modifications in protein expression and phosphorylation. Understanding the interplay between these metabolic and protein-level alterations is crucial for developing targeted therapeutic strategies against KRAS-driven cancers. Accordingly, we performed an integrated metabolomic and phosphoproteomic analysis of HCT116 cells with and without a KRAS mutation. Methods: HCT-116 cells containing wildtype (WT, n = 5) and mutant KRAS (MUT, n = 5) were profiled to assess metabolite, protein, and phosphoprotein levels between WT and MUT cells. Metabolomic profiling was completed with LC/MS to capture polar and lipid metabolites. Data was processed through an in-house untargeted metabolomic analysis pipeline. For phosphoteomic analysis, enriched phosphopeptides and flow-through peptides were analyzed with DIA LC/MS/MS. The resulting data was processed with DIA-NN and combined into aggregated protein and phosphosite profiles. Results: The multi-omic analysis profiled 2,025 metabolites, 6,654 proteins, and 10,034 phosphosites. When considering each data type individually, all three analyte types seperated the WT and MUT cells through PCA analysis, underscoring the magnitude of dysregulation from KRAS mutation. When considering the metabolites, proteins, and phosphosites that reached statistical significance, 85 metabolites and 145 proteins were differentially abundant. Strikingly, 1,036 phosphosites were unique to either KRAS and WT samples, with ~800 of these being unique to KRAS samples. The results of a joint pathway analysis identified > 70 pathways that reached significant enrichment (p < 0.05) and had support at both the protein, metabolite, and phosphosite level. The most enriched pathway was RAS signaling. Other enriched pathways include selenocysteine synthesis, sialic acid metabolism, ROS detoxification, and respiration. Conclusions: This study showed extensive differential phosphorylation in KRAS mutant cells, suggesting a widespread rewiring of signaling networks that impacts diverse cellular functions. The multi-omic approach taken enabled the identification of previously known and novel pathways affected by KRAS signaling, offering potential new targets for therapeutic intervention in KRAS-driven cancers.