Functional assessment of BRAF and CRAF through Base Editor 3 mutagenesis reveals residues and regions critical for ERK1/2 signalling

Abstract

The RAS-regulated RAF-MEK-ERK1/2 signalling pathway is critical for transducing extracellular signals from receptor tyrosine kinases at the plasma membrane through to protein targets to direct cell fate. Components of this pathway are frequently mutated to drive enhanced signalling in cancer and a group of developmental diseases called RASopathies. Therapeutic targeting of class I BRAF mutants has proven successful but BRAF inhibitors have failed in cancers with wild type RAF because sub-saturating doses of inhibitors drive paradoxical activation of wild-type RAF. Base editor 3 (BE3) is a gene editing tool comprised of a nickase Cas9 fused to a cytidine deaminase and a uracil DNA glycosylase inhibitor. This can be used to generate high-efficiency C to T mutations. This thesis describes the use of a high efficiency BE3 mutation screen in an arrayed format with the high-throughput functional readout of p-ERK1/2 immunofluorescence to screen BRAF and CRAF for mutations that alter RAF kinase activity. The BE3 screen identified a number of mutations in BRAF and CRAF that maintained BRAF or CRAF protein expression, but significantly changed p-ERK1/2 content. This included several known mutations in BRAF and CRAF. For example, the two strongest hits from the screen CRAFS259F and CRAFT491I have previously been identified in cancer and RASopathies. In addition, the BRAF mutations BRAFE586K and BRAFT599I are known class II BRAF mutations, while BRAFS467L is a known class III mutation. Collectively, these results validated the screen. Mutations causing a significant change in p-ERK1/2 signalling were analysed structurally in the context of protein interactions likely altered by the mutation. This structural interpretation, combined with the effect of each mutation on p-ERK1/2, directed experiments to investigate the biological interactions driving these changes. This identified that BRAFE275K enhanced KRAS binding, identified several CRAF CR1 mutations which diminished KRAS binding, and demonstrated that mutations caused by the CRAFS259F guide led to increased ERK1/2 pathway activity, a loss in collective cell migration and a growth deficiency that was apparently ERK1/2-independent.