Identifying Cancer-Relevant Mutations in the DLC START Domain using Evolutionary and Structure-Function Analyses

Rho GTPase signaling promotes proliferation, invasion, and metastasis in a broad spectrum of cancers. Rho GTPase activity is regulated by the Deleted in Liver Cancer (DLC) family of bonafide tumor suppressors which directly inactivate Rho GTPases by stimulating GTP hydrolysis. In addition to a RhoGAP domain, DLC proteins contain a StAR-related lipid transfer (START) domain. START domains in other organisms bind hydrophobic small molecules and can regulate interacting partners or co-occurring domains through a variety of mechanisms. In the case of DLC proteins, their START domain appears to contribute to tumor suppressive activity. However, the nature of this START-directed mechanism, as well as the identities of relevant functional residues, remain virtually unknown. Using the Catalogue of Somatic Mutations in Cancer (COSMIC) dataset, and evolutionary and structure-function analyses, we identify several conserved residues likely to be required for START-directed regulation of DLC-1 and DLC-2 tumor suppressive capability. This pan-cancer analysis shows that conserved residues of both START domains are highly-overrepresented in cancer cells from a wide range tissues. Interestingly, in DLC-1 and DLC-2, three of these residues form multiple interactions at the tertiary structural level. Further, mutation of any of these residues is predicted to disrupt interactions and thus destabilize the START domain. As such, these mutations would not have emerged from traditional hotspot scans of COSMIC. We propose that evolutionary and structure-function analyses are an underutilized strategy which could be used to unmask cancer-relevant mutations within COSMIC. Our data also suggest DLC-1 and DLC-2 as high- priority candidates for development of novel therapeutics targeting their START domain. Simple Summary Deleted in Liver Cancer (DLC) proteins are tumor suppressors that contain a StAR-related lipid transfer (START) domain. Little is known about the DLC START domain including the residues that mediate its activation. Using the Catalogue of Somatic Mutations in Cancer (COSMIC) dataset, evolutionary, and structure-function analyses, we identify key functional residues of DLC START domains. Mutations in these residues are significantly overrepresented in numerous cancers in multiple tissue types. In other contexts, START domains bind hydrophobic small molecules and stimulate regulatory outputs of interacting partners or co-occurring domains. The identification of functional residues in the DLC START domain may thus have implications for targeted manipulation of DLC tumor suppressive activity. Critically, these residues would not have been identified using traditional queries of COSMIC. Thus, we propose tandem evolutionary and structure-function approaches are an underutilized strategy to unmask cancer-relevant mutations within COSMIC.