Molecular characterisation and functional assessment of FGFR3 mutations in cancer

Genetic alterations such as mutations in the Fibroblast Growth Factor Receptors (FGFRs) family play important roles in the development and progression of several cancer types. How these mutations affect protein function within the cell and drive tumour growth is still unknown. Despite the development of selective inhibitors for FGFR, the majority are still being evaluated in clinical trials where their efficacy in patients is not always achieved and disease recurrence often affects responders. This project seeks to understand the receptor mechanisms and downstream molecular rewiring in tumour cells that sustain cell survival signalling through FGFR3. I have generated NIH-3T3 cellular models expressing FGFR3 cancer-associated mutants for molecular characterisation. A set of mutants were analysed for their baseline activation levels using a multiplex assay and for their downstream signalling dependencies using a targeted small molecule inhibitor screen. I have identified that FGFR3 cancer-associated alterations display increased Src phosphorylation when compared to the WT FGFR3. Paradoxically, cell lines expressing the FGFR3 S249C mutation and the FGFR3-TACC3 fusion protein were found to be resistant to Src inhibitors such as dasatinib. Interestingly, profiling these cells indicate that effective durable therapy requires the blockade of multiple downstream effectors to overcome resistance signalling pathways in the presence of FGFR inhibitor monotherapy. These include the simultaneous blockade of Erk and Src, by respectively utilising the FGFR inhibitor BGJ398 and the Src inhibitor dasatinib. Similarly, human bladder cancer cells harbouring the endogenous FGFR3 mutations R248C (639V) and Y375C (MGHU3) along with the FGFR3-TACC3 fusion (RT112M) were also found to achieve an effective and durable response with the same combination therapy. Mechanistic work in an extended panel of extracellular domain cysteine mutants in NIH-3T3 cells demonstrated that the substitution of the cysteine residue forms spontaneous dimers that promote growth under anchorage-independent conditions, a described hallmark of cancer. Moreover, results show that FGFR cysteine mutations are likely to be a useful biomarker to select for patients who will benefit from FGFR and Src inhibitors combination therapy