Proteomics and phosphoproteomics of human colorectal cancer cells lacking a specific kinase activity reveal kinase-specific compensatory responses

Cell signaling regulates cell proliferation, survival, and migration, and abnormal kinase activity is often implicated in cancer. Although kinases are key targets for anticancer therapy, drug-induced compensatory signaling and pathway rewiring often drive acquired resistance. These compensatory responses enable tumor cells to maintain proliferation and survival, contributing to acquired drug resistance. In this study, we investigated adaptive responses following the knockout of four specific kinase genes, ERK2, PLK1, PIK3CA, and PAK4, using HCT-116, a human colorectal cancer cell line. Using CRISPR–Cas9, we generated individual knockout cell lines and conducted quantitative proteomic and phosphoproteomic profiling using isobaric tagging and tandem mass tag (TMTs) to evaluate alterations in the signaling landscape. Our integrated analysis quantified 7,531 proteins and 10,877 phosphopeptides, revealing kinase-specific patterns of compensatory signaling. ERK2 knockout was associated with activation of MAPK- and PI3K/AKT-related kinases, whereas PIK3CA knockout induced extensive proteomic remodeling and engagement of pro-survival phosphorylation programs, illustrating distinct modes of signaling network rewiring. Integration of kinase–substrate enrichment analysis (KSEA) with global proteomic data revealed that adaptive kinase activity was largely uncoupled from protein abundance and uncovered a synthetic lethal interaction between ERK2 loss and RPS6KB1 inhibition. Collectively, these findings elucidate how targeted kinase loss drives homeostatic signaling networks in cancer cells. By systemically characterizing cellular-level signaling changes and contextualizing them within known kinase pathways, our results provide insights into synthetic lethality and identify potential therapeutic targets to counteract adaptive resistance to kinase inhibitors.