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

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.

Detection of differential bait proteoforms through immunoprecipitation-mass spectrometry data analysis

“… 20 have characterised the differential interactome of KRAS G12C mutated protein, aiming to understand whether the mechanism of action of the AMG-510 inhibitor, is mediated by a KRAS interactome change. To supplement this study with KRAS differential proteoform information upon the inhibitor, we investigated the differential peptidoforms of exogenously expressed KRAS WT and KRAS G12C upon AMG-510 treatment (PXD043536) 20 . We checked the quality of the IP-MS data by verifying that KRAS was highly enriched in the samples (Fig.…”

“…Raw files were downloaded from the PRIDE proteomics repository 31 , for the HRAS (PXD019469) 17 , KRAS (PXD043536) 20 and BRD4 (PXD012715) 26 respectively. These data were analysed using MSFragger (FragPipe 19.2-build 11, MSFragger 3.8, philosopher 5.0.0) open search default settings 6 with additional information about the mass shifts derived from PTM-Sheppard 23 .…”

“…IP-MS raw files available at PRIDE proteomics repository 31 , for the HRAS (PXD019469) 17 , KRAS (PXD043536) 20 and BRD4 (PXD012715) 26 respectively. iPTMs analysis output available at https://github.com/SdelciLab/iPTMs in the v3.0 releases.…”

Peptidoform analysis of IP-MS data allows detection of differentially present bait proteoforms

“…However, emerging resistance to AMG-510 10 has already been already reported 11 and has become a new area of cancer research. Therefore, we investigated the differential peptidoforms of exogenously expressed KRAS WT and G12C upon AMG-510 treatment (PXD043536) 12 . We checked the quality of the IP-MS data by verifying that KRAS was highly enriched in the samples (Fig 2A) and a that high protein coverage was achieved (Fig S2A-B).…”

K-RAS Is…Complicated

“…The recent manuscript from Nolan et al [11] uses a proteomics approach to make several important contributions to the understanding of how K-RAS may function and how it responds to treatment with Sotorasib. The work confirms that different RAS mutants exhibit different protein-binding profiles, in addition the fact that the host cell type plays a major role in determining the proteins that RAS interacts with.…”