Identification and characterization of KRAS functional partners in isoform-defined complexes

KRAS is a member of the RAS small GTPase family and one of the most frequently mutated oncogenes in human cancer. KRAS dimerization is considered the basic signaling unit of KRAS, but the impact on the downstream signaling of its complex assembly and disassembly at the cell membrane is still unknown. It has been demonstrated that wild-type KRAS can act as tumor suppressor in cells with heterozygous KRAS mutation. The molecular basis for the inhibitory effect of wild-type KRAS is complex formation with mutant KRAS, whilst dimerization-deficient wild-type KRAS is no longer able to exert growth-inhibitory effect on mutant KRAS. The different effects induced by wild-type/mutant KRAS and mutant/mutant KRAS dimers could be explained by the recruitment of specific interactors at the cellular membrane forming different complexes. Here, by using a KRAS dimerization-inducible system (KRASDIM), we induced the formation of wild-type/mutant and mutant/mutant KRAS membrane complexes with space and time control, and we performed mass spectrometry to identify specific interactors recruited by the different KRAS dimerization complexes. Based on the literature, we focused our attention on two candidates, LMO7 and RIN1, respectively recruited by wild-type/mutant and mutant/mutant KRAS dimers. To understand their biological roles, we performed loss of function assays by short hairpin RNA (shRNA)-mediated knockdown of target expression. The characterization of the KRAS functional complex at the membrane could aid in the discovery of innovative therapeutic methods for KRAS-mutant cancer by revealing new biological functions of the triggered downstream signaling.