Cancer is a multistep process reflecting genetic alterations that drive progressive transformation of normal cells into highly proliferative malignant cells. Deregulation of cellular signalling is one of the key traits in cancer, allowing cells to breach anticancer defence mechanisms. The most frequently mutated oncogene in cancer is KRAS, encoding a small GTPase protein involved in controlling the activity of critical signalling pathways that regulate normal cellular proliferation, such as the PI3K and ERK pathways. The prevalence of different codon substitutions in the KRAS gene varies in different tissues. There is emerging evidence supporting the notion that different codon substitutions may trigger different biological effects. We hypothesise that different codon substitutions in KRAS can trigger different feedbacks and signalling dynamics that may result in varying fitness advantages in different tissues.

I use quantitative Western blotting and Modular Response Analysis with KRAS isogenic cell lines to characterise how KRAS substitutions at codon G12 perturb the topology and dynamics of the ERK signalling network as a first step to test this hypothesis. My work has identified two mutant-specific interactions in the ERK pathway: a MEK to RAF inhibition seen strongest in G12A, G12C and G12D cells, and a RAF to ERK activation (or loss of inhibition) seen in all mutants compared to WT. Antibody array data suggests the potential role of JNK and TYK2 in mediating these interactions, respectively, and thus my work has provided preliminary, albeit testable, hypotheses for elucidating the possible mechanisms responsible for this rewiring of the ERK pathway. My work has also identified the presence of a second BRAF form present only in the mutants with the strongest MEK to RAF inhibition. It appears this KRAS-mutant specific BRAF form is most likely a splice variant that has enhanced dimerisation capabilities with CRAF. RAF dimerisation is one mechanism via which RAF inhibitors fail to be effective in treating KRAS mutant cancers. This exemplifies the importance of characterising the identity and role of this BRAF form, as it may have implications on development of mutant-specific therapies for KRAS-driven cancers. ERK pathway activation can also regulate the transcription of many downstream targets. I have attempted to characterise KRAS-dependent gene expression changes with RNA sequencing data, with the aim of understanding key differences between mutant and WT KRAS cells and to also understand how the gene expression profiles change over time during the very early steps in oncogenesis. Preliminary data suggests mutant-specific differential gene transcription that may be linked to the RAF inhibition via MEK and/or the second BRAF form. My work currently does not explain the specific mechanisms behind the interactions identified with MRA, however, it does lay down the foundation and provides hypotheses that can be tested in the lab. Gaining mechanistic insight into the pathogenicity of cancer driver mutations and their differential role in different tissues is of fundamental importance to understand how different mutations shape the evolution of cancer clones during carcinogenesis and to design optimal targeted therapeutic strategies.