Abstract Fibroblast growth factor receptors (FGFR1-4) are essential receptor tyrosine kinases that play a crucial role in controlling cellular fate, guiding tissue development, and maintaining physiological balance. Cancer-driving mutations in FGFRs are found in many tumour types, but exactly how each mutation alters receptor behaviour, protein interactions, and signalling pathways is still not fully understood. In this study, we systematically analysed wild-type, kinase-dead, and two cancer driver mutants for each FGFR paralogue by generating stable, isogenic Flp-In™ T-REx™ 293 cell lines which were engineered under inducible expression for each FGFR variant. We performed affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID) to generate high-confidence mutation-level map of FGFR interactomes, integrating both stable and transient protein associations. To understand the functional implications of the altered interactome we performed phosphoproteomics on both global lysates and affinity-purified complexes. This enabled quantification of pathway-specific signalling changes and differentiation between the catalytic and scaffolding roles of mutant receptors. Distinct signalling rewiring patterns were observed between wild-type and mutant FGFRs such as preferred activation of MAPK versus PI3K-AKT signalling pathway and revealed mutation-specific signalling biases consistent with recent models of RTK signalling divergence. Functional assays in NIH3T3 and U2OS cells, including colony formation, immunofluorescence, cell cycle profiling, and reporter assays, validated the differential oncogenic potential and subcellular localisation of each variant. We further evaluated the effects of clinically relevant FGFR inhibitors on the viability and signalling pathway activity across all mutants, identifying both drug-sensitive and drug-resistant classes linked to specific mutations, including gatekeeper substitutions (e.g., FGFR4-V550L) and extracellular loop alterations (e.g., FGFR2-S252W). Together, our findings establish the first paralogue-wide, mutation-resolved signalling atlas of the FGFR family. This integrative approach of structural, functional and pharmacological analysis provides systematic insight into oncogenic mechanism and therapeutic sensitivity and establishes a broadly applicable framework for the functional annotation of kinase driver mutations in cancer. Citation Format: Iftekhar Mahmud Chowdhury, Xiaonan Liu, Markku Varjosalo, . Mapping mutation-specific interaction and signalling networks across FGFR1-4 to understand cancer-associated rewiring abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7658.
Chowdhury et al. (Fri,) studied this question.