Assessment of fibroblast growth factor receptor cancer-associated mutations and characterisation of endocrine signalling complexes

Receptor tyrosine kinase (RTK) signalling is frequently deregulated in cancers, developmental syndromes and metabolic diseases. The fibroblast growth factor (FGF) receptor (FGFR) family of RTKs have pleiotropic roles in development, metabolism and tissue homeostasis. A decade of deep sequencing studies has led to the discovery of somatic cancer-associated alterations in FGFRs including point mutations, gene fusions and amplifications. Missense substitutions appear most frequently in FGFR3 and FGFR2, some of which have been implicated as oncogenic drivers. This has led to rapid progress in anti-FGFR therapies. This work compares and analyses twenty five reported FGFR kinase domain (KD) variants using FGFR3 as a model. Kinase activity assays were developed to screen missense substitutions in vitro using purified FGFR3 KD proteins. It was found that hyperactive mutations did not necessarily occur frequently and conversely, frequently occurring mutation 'hotspots' were not necessarily kinase hyperactive. Cellular models of FGFR mutations suggested that hyperactive mutants activated signalling but did not always transform cells. Investigations into the ligand binding characteristics of a subset of activating mutants revealed mutation specific signatures of binding properties. However, there appeared to be no link between KD activity and stability. These results reinforce the importance of screening clinical tumours prior to treatment to provide better data-driven therapies for patients. Endocrine FGFR signalling was also explored in this work. Klotho family proteins bind to specific FGFs and FGFRs to signal through endocrine signalling complexes regulating diverse metabolic activities. This work developed and optimised methods to express and purify components of these binary and ternary signalling complexes. Preliminary biophysical experiments were then performed to begin to understand the protein-protein interactions involved in complex formation. This will inform the rational design of therapies already in development for targeting deregulated endocrine FGFR signalling in various metabolic disorders and cancer.