Obtaining an integrated understanding of oncogenic RAS signalling

The RAS family of small GTPases act as signalling hubs regulating cell proliferation and differentiation. The physiological importance of RAS signalling is evident as about 25% of all human cancers harbour mutations in ras genes, where kras is most frequently mutated (about 18%) (COSMIC, v94). However, there is no anti-Ras inhibitor available except the one that targets G12C oncogenic mutation through the thiol group of the cysteine 12. As G12C mutation contributes to just about 10% of kras oncogenic mutations, it is vital to develop effective inhibitors against other oncogenic Ras variants. Towards this goal, we need to understand the mode of action of oncogenic RAS molecules.

The way Ras activates downstream effectors is through direct protein-protein interactions. 56 human proteins are found to have a domain termed either Ras Binding Domain (RBD) or Ras Association domain (RA), that features a ubiquitin-like ββαββαβ fold. Some of them are experimentally proven to act as Ras effectors. Representative examples include RAF kinases that prime ERK pathway activation, PI3 Kinase that leads to Akt activation and RalGEFs that act as a GDP-GTP exchange factor (GEF) for small GTPases, RalA and RalB.

Although the essentiality of RAS-effector interactions in the oncogenic RAS signalling is well-recognised, the dynamic nature of these interactions has been elusive.

• Does one molecule of RAS simultaneously interact with multiple effectors?
• Does the RAS molecule jump between different effectors?
• Does interaction with one of the effectors influence the next interaction?
• Do all effectors contribute equally to cause the oncogenic-Ras phenotype? Or, some of them play a more important role than others?

We will address these questions in the PhD project by combining various techniques, including single-molecule analysis using optical microscopy, structural biology (X-ray crystallography, NMR and cryo-electron microscopy), biochemistry (protein purification, biolayer interferometry and surface plasmon resonance) and live-cell imaging of human culture cells where genes encoding relevant signalling molecules are to be edited by CRISPR-Cas9 technology. Successful delivery of the project will bring a novel concept of RAS signalling and help design inhibitors targeting RAS signalling.