Characterisation of [superscript L597V]BRAF in cancer

BRAF is a component of the RAF/MEK/ERK signalling cascade which controls fundamental cellular activities, including proliferation, cell survival, differentiation and motility. Deregulation of this pathway is common in cancer and ~7% of cancers have a mutation in BRAF. Leu597 is the fifth most commonly mutated residue in BRAF-mutated human cancers, and a substitution to a valine is one of only seven BRAF mutations that are found in both cancer and a group of developmental syndromes known as RASopathies. A major question is how the mutation can be associated with both diseases. In this study, using an autochthonous model and HEK 293[superscript T] cells, [superscript L597V]BRAF was shown to have weak kinase and MEK/ERK-inducing activity. It did not induce morphological transformation or foci formation, nor confer a growth advantage or induce early immortalisation. Therefore, the mutation was found not to be a driver oncogene. [superscript L597V]BRAF mutations are found to co-exist with other oncogenic mutations in human cancer. Using cells derived from a conditional knock-in mouse model, we showed that [superscript L597V]BRAF synergises with [superscript G12D]Kras to induce cell changes more reminiscent of the high activity mutant [superscript V600E]Braf. Double mutant [superscript L597V]BRAF and [superscript G12D]Kras cells have higher Braf and Craf kinase activity than single mutants, which translates to higher Mek/Erk activity to a similar level to [superscript V600E]Braf. These cells were more morphologically transformed than Braf[superscript +/L597V] and Kras[superscript +/G12D] cells alone. RAF inhibitors induced paradoxical activation of Erk in [superscript L597V]Braf-expressing cells, and this was shown to be through heterodimerisation and activation of Craf. These results caution against the use of RAF inhibitors in treatment of RASopathy and cancer patients with the [superscript L597V]BRAF mutation. Aged Braf[superscript +/L597V] mice developed some predisposition to tumour formation. The tumours were benign, and one out of eight tumours was heterozygous for [superscript Q61L]Hras. This supports the idea that [superscript L597V]BRAF is insufficient to induce cancer, but epistatically modifies other oncogenes to promote cancer progression by hyperactivation of the Erk pathway.