Roger J. Daly

Different molecular classifications for gastric cancer (GC) have been proposed based on multi‐omics platforms with the long‐term goal of improved precision treatment. However, the GC (phospho)proteome remains incompletely characterized, particularly at the level of tyrosine phosphorylation. In addition, previous multiomics‐based stratification of patient cohorts has lacked identification of corresponding cell line models and comprehensive validation of broad or subgroup‐selective therapeutic targets. To address these knowledge gaps, we applied a reverse approach, undertaking the most comprehensive (phospho)proteomic analysis of GC cell lines to date and cross‐validating this using publicly available data. Mass spectrometry (MS)‐based (phospho)proteomic and tyrosine phosphorylation datasets were subjected to individual or integrated clustering to identify subgroups that were subsequently characterized in terms of enriched molecular processes and pathways. Significant congruence was detected between cell line proteomic and specific patient‐derived transcriptomic subclassifications. Many protein kinases exhibiting ‘outlier’ expression or phosphorylation in the cell line dataset exhibited genomic aberrations in patient samples and association with poor prognosis, with casein kinase I isoform delta/epsilon (CSNK1D/E) being experimentally validated as potential therapeutic targets. Src family kinases were predicted to be commonly hyperactivated in GC cell lines, consistent with broad sensitivity to the next‐generation Src inhibitor eCF506. In addition, phosphoproteomic and integrative clustering segregated the cell lines into two subtypes, with epithelial–mesenchyme transition (EMT) and proliferation‐associated processes enriched in one, designated the EMT subtype, and metabolic pathways, cell–cell junctions, and the immune response dominating the features of the other, designated the metabolism subtype. Application of kinase activity prediction algorithms and interrogation of gene dependency and drug sensitivity databases predicted that the mechanistic target of rapamycin kinase (mTOR) and dual specificity mitogen‐activated protein kinase kinase 2 (MAP2K2) represented potential therapeutic targets for the EMT and metabolism subtypes, respectively, and this was confirmed using selective inhibitors. Overall, our study provides novel, in‐depth insights into GC proteomics, kinomics, and molecular taxonomy and reveals potential therapeutic targets that could provide the basis for precision treatments.