Characterization of effector binding preferences between oncogenic mutants KRAS4b G12D and KRAS4b G12C

Abstract

Up to 30% of cancers harbor mutations in the small GTPase RAS that maintain the protein in an active conformation and drive uncontrolled cellular proliferation, promote cell survival, and alter cellular metabolism, movement, and differentiation. The momentum of four decades of research is finally yielding promising results in targeting this previously deemed ‘undruggable’ protein. However, much remains to be understood about fundamental RAS biology and signaling. For instance, it is still unclear why specific mutations occur with non-uniform frequency across cancer types. Do RAS mutants differ in their ability to engage effector proteins and potentiate signaling pathways? Here, we optimize a cellular Bioluminescence Resonance Energy Transfer (BRET) system to help answer this question, and use BRET Saturation curves and immunoprecipitation to interrogate the ability of common mutants KRAS4b G12D and KRAS4b G12C to engage with effectors RAF1 and p110α. We were unable to identify significant differences in effector affinity with BRET saturation curves, but did find greater p110α engagement by KRAS4b G12D through GFP pull-downs in doxycycline-inducible GFP-KRAS mutant HeLa cell lines as well as transiently transfected HEK293T cells. We also observed higher AKT phosphorylation in KRAS G12D-driven HeLa and oncogenic cell lines, suggesting greater levels of PI3K pathway activation in cells carrying this mutation. We then discuss our solution-state NMR and molecular dynamics simulation studies revealing structural differences in the KRAS4b G12D protein, and how these differences may influence effector engagement and oncogenic signaling.