Proteomic Mapping of the Interactome of KRAS Mutants Identifies New Features of RAS Signalling Networks and the Mechanism of Action of Sotorasib

SIMPLE SUMMARY: Cancer is caused by changes in DNA called mutations that alter the way proteins work. We know that RAS proteins are one of the most commonly mutated proteins in cancer cells. These proteins work like a light switch and mutant RAS remain in the ON position sending signals to the cell to keep dividing when they should not. Until recently, it was thought that all RAS mutations were equal, but differences among these mutants have been identified. Here we used a technique called proteomics to decipher the differences among RAS mutants. We find that each mutant binds a different set of proteins and can regulate different signals. We also find that a clinically approved drug that inhibits one RAS mutant regulates the interaction of RAS proteins with other proteins. Our findings extend our knowledge of how the RAS mutants work, which can potentially be used to improve cancer treatments. ABSTRACT: RAS proteins are key regulators of cell signalling and control different cell functions including cell proliferation, differentiation, and cell death. Point mutations in the genes of this family are common, particularly in KRAS. These mutations were thought to cause the constitutive activation of KRAS, but recent findings showed that some mutants can cycle between active and inactive states. This observation, together with the development of covalent KRASG12C inhibitors, has led to the arrival of KRAS inhibitors in the clinic. However, most patients develop resistance to these targeted therapies, and we lack effective treatments for other KRAS mutants. To accelerate the development of RAS targeting therapies, we need to fully characterise the molecular mechanisms governing KRAS signalling networks and determine what differentiates the signalling downstream of the KRAS mutants. Here we have used affinity purification mass-spectrometry proteomics to characterise the interactome of KRAS wild-type and three KRAS mutants. Bioinformatic analysis associated with experimental validation allows us to map the signalling network mediated by the different KRAS proteins. Using this approach, we characterised how the interactome of KRAS wild-type and mutants is regulated by the clinically approved KRASG12C inhibitor Sotorasib. In addition, we identified novel crosstalks between KRAS and its effector pathways including the AKT and JAK-STAT signalling modules.