Protein kinases play critical roles in regulating cellular processes, including growth, survival, differentiation, and apoptosis. Mutations in protein kinase are frequently associated with development and progression of cancer. These mutations, often referred to as cancer driver mutations can lead to the continuous activation or inhibition of kinase signalling pathways, which in turn promotes oncogenic transformations. Our study focuses on the physical and functional interaction networks of 180 established driver mutations (from the COSMIC database) in 58 cancer driver protein kinases (Figure1). We generated Flp-In™ T-REx™ 293 stable cell lines harbouring the mutations including those in the FGFR family (FGFR1/2/3/4).

Fibroblast growth factor receptors (FGFRs) are a family of essential transmembrane receptors involved in diverse cellular activities. Mutations of FGFR including amplifications, activating mutations, rearrangements and fusions lead to the imbalance of signalling pathways resulting in oncogenic transformation of healthy cells. Here we present the impact of such driver mutations in the FGFR family on cancer signalling networks using a multi-proteomics strategy.

Utilizing affinity purification mass spectrometry (APMS) and proximity labelling (BioID), we mapped the protein-protein interaction networks for each mutated kinase relative to its wild-type form. This allowed us to understand the altered interactor profiles and the signalling pathways affected by the mutations. Further, phosphoproteomics was applied to illustrate the phosphorylation landscape induced by the mutations, including specific phosphosites. To functionally validate the consequences of the mutations, kinase-dead mutants of FGFR family were also expressed and analysed.

Our findings indicate a significant upregulation of several critical cancer driver proteins, including ABL2, ARAF, PIK3CA, PIK3CB, PLCG1 and STAT5B by FGFR mutations. These changes lead to distinct signalling cascades as confirmed by pathway-specific reporter assays. In addition, confocal microscopy and MS-microscopy demonstrated notable changes in the subcellular localization of the mutated FGFRs which may account for their altered interaction profiles. Many of the identified proteins are attractive candidates for therapeutic drug targeting in FGFR-induced cancers.

Future directions of this research will focus on the validation of the functional relevance of the cancer driver proteins by gene silencing and specific drug targeting experiments as well as immunohistochemical analysis on tissue from the mutation-confirmed cancers of interest followed by whole proteome analysis from the tissue samples to provide a comprehensive understanding of the global effect of the mutation. This multi-proteomics strategy allows the characterization of the complex signalling changes induced by FGFR mutations and identifies new targets for precision cancer medicine.