RAF-MEK1/2-ERK1/2 signalling is a focus for targeted cancer drug development with many RAF-MEK1/2 inhibitors now in clinical trials. Detecting biomarkers of pathway blockade, particularly those that are non-invasive, is key for assessing target modulation and drug efficacy and thereby aiding identification of promising pharmaceuticals in early stage trials to prioritise for pivotal trials. Using magnetic resonance spectroscopy (MRS), we have shown a reduction in lactate levels in BRAF mutant human melanoma cells and xenografts following treatment with MEK1/2 selective inhibitors. Here we will a) identify and assess the metabolic biomarkers of response to novel classes of RAF/MEK1/2 inhibitors (BRAF-selective, MEK1/2 selective and RAF+MEK1/2 selective) in human cancer cells with varying BRAF and RAS mutation status, b) investigate the metabolic mechanisms involved in response and resistance to treatment and c) assess the translatability of any metabolic biomarkers in in vivo tumour models and patient tumour tissues, to identify the value of incorporating in vivo measurements into clinical trials. We will assess the effect of RAF/MEK1/2 signalling inhibition in human cancer cells with varying BRAF and RAS mutation status in vitro using 1H, 31P and 13C MRS combined with molecular and biochemical assays to investigate changes in metabolism and metabolic pathway fluxes and enzyme activities associated with response and resistance to treatment. These investigations will then be extended to a) tumour models in vivo where we aim to delineate the impact of any therapy-driven physiological changes (using MR imaging) on metabolism and b) RAF-MEK1/2 inhibitor-treated patient samples to assess the clinical relevance of our findings. This work will define the potential of metabolic changes as non-invasive imaging biomarkers for the action of RAF/MEK1/2-targeted agents and will be a major step in translating findings from the laboratory to the clinic.