Structures of full-length FGFR cancer fusions and disease mutants

Cells receive signals from growth factors when they need to divide and replicate (e.g., during embryonic development, growth, or wound healing). These signals are transmitted from the outside of the cell, where the growth factor binds, to the inside, by receptor tyrosine kinases (RTKs) - proteins that sit within and span across the cell membrane. Although the structures for parts of RTKs are known, high resolution structures of whole (full-length) RTKs have yet to be determined, so our understanding of how the different domains interact to control signalling is incomplete.

In this project, we will determine structures of full-length fibroblast growth factor receptors (FGFRs), using cutting-edge atomic-resolution methods such as cryo-electron microscopy and nuclear magnetic resonance spectroscopy. FGFRs, and altered forms of FGRFs that are responsible for several types of human cancers as well as for some developmental disorders, are archetypal RTKs that control processes such as embryonic development, wound healing, and growth of new blood vessels (angiogenesis). FGFRs are normally activated by binding of fibroblast growth factors (FGFs) to their extracellular (outside of the cell) regions, but in some cancers and developmental diseases they can become altered so that they are permanently active even without FGF binding.
Understanding the structural connections between the different domains of FGFRs (e.g. extracellular and intracellular parts) is essential if we are to understand how FGFRs and other RTKs function normally - for example, how they are auto-inhibited in the resting state but then become activated. This is particularly the case for a cancer-associated variant of FGFRs whereby part of the FGFR3 gene becomes fused with part of the gene from another protein, TACC3, to generate a hybrid protein that is hyperactivated and also localises to different parts of the cell. These so-called FGFR3-TACC3 fusions are responsible for certain types of glioblastomas (aggressive brain tumours) and some bladder cancers. Understanding at a structural level how the hyperactivation occurs will improve our ability to selectively target these fusion proteins to better treat those cancers for which they are responsible.
We also anticipate that these disease-associated variants of FGFRs with aberrant activity are likely to form novel intracellular complexes with a variety of different protein partners. To fully understand how mutations may affect function we must also identify binding partners that can facilitate and regulate signal transduction. To this end another aspect of our research will be to use cross-linking mass spectrometry (XL-MS) in lab-grown cancer cells to identify these new partners and find out how those interactions contribute to the disease process.

By improving our knowledge of (normal and disease-altered) FGFR structures and cellular interactions, we aim to understand better why FGFR-targeted drug molecules are effective in some disease settings and less so in others, and how we can then develop more efficacious drug molecules. In summary, our project aims to address the following questions:

- Through solving the structures of complete FGFRs and their cancer-associated altered forms (e.g. FGFR-TACC fusions), can we better understand how extracellular signals (such as growth factor binding) are translated into the different intracellular responses generated from activated FGFRs?

- What functional complexes do FGFRs form in normal and cancer cells, and what do they tell us about potential new therapeutic drug targeting strategies?

This 3-year project grant will combine and integrate insights from an array of advanced experimental techniques, including cryo-EM, cross-linking-MS, and methyl-resolved NMR, to effect a step change in our structural understanding and knowledge of functional interactions of FGFRs, and of RTKs more broadly. Specifically, building on prior work and recent progress in our labs, we are well placed to make a major advance towards high-resolution structures of full-length RTKs by exploiting our combined expertise in FGFR signalling biology, functional protein reagent generation and analysis, and cutting-edge structural biology tools and infrastructure. Levering these assets, we are poised to understand better how FGFRs and their most common and important disease-altered forms (including gene fusions with TACC proteins) are activated and transduce signals. We will address the following previously intractable research questions:

- What conformations do RTK intracellular domains assume in intact (full-length) membrane-embedded RTKs and how do these change in response to activation status (e.g. phosphoforms) and to extracellular domain conformation and ligand-bound status?

- How do the conformational changes induced by C-terminal oncogenic gene fusions including FGFR3-TACC3 lead to hyperactivation and interactions with altered cellular networks, and do these changes in turn present new opportunities for fusion-selective targeting?