KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRAS^G12D, a common mutant form, and KRAS^A146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRAS^A146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor–induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRAS^G12D and KRAS^A146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. Significance: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties. See related commentary by Hobbs and Der, p. 696. This article is highlighted in the In This Issue feature, p. 681

H-Ras, K-Ras, and N-Ras are small GTPases that are important in the control of cell proliferation, differentiation, and survival, and their mutants occur frequently in human cancers. The G-domain, which catalyzes GTP hydrolysis and mediates downstream signaling, is 95% conserved between the Ras isoforms. Because of their very high sequence identity, biochemical studies done on H-Ras have been considered representative of all three Ras proteins. We show here that this is not a valid assumption. Using enzyme kinetic assays under identical conditions, we observed clear differences between the three isoforms in intrinsic catalysis of GTP by Ras in the absence and presence of the Ras-binding domain (RBD) of the c-Raf kinase protein (Raf-RBD). Given their identical active sites, isoform G-domain differences must be allosteric in origin, due to remote isoform-specific residues that affect conformational states. We present the crystal structure of N-Ras bound to a GTP analogue and interpret the kinetic data in terms of structural features specific for H-, K-, and N-Ras.

Members of the family of RAS proto-oncogenes, discovered just over 40 years ago, were among the first cancer-initiating genes to be discovered. Of the three RAS family members, KRAS is the most frequently mutated in human cancers. Despite intensive biological and biochemical study of RAS proteins over the past four decades, we are only now starting to devise therapeutic strategies to target their oncogenic properties. Here, we highlight the distinct biochemical properties of common and rare KRAS alleles, enabling their classification into functional subtypes. We also discuss the implications of this functional classification for potential therapeutic avenues targeting mutant subtypes. Significance: Efforts in the recent past to inhibit KRAS oncogenicity have focused on kinases that function in downstream signal transduction cascades, although preclinical successes have not translated to patients with KRAS-mutant cancer. Recently, clinically effective covalent inhibitors of KRASG12C have been developed, establishing two principles that form a foundation for future efforts. First, KRAS is druggable. Second, each mutant form of KRAS is likely to have properties that make it uniquely druggable.

Ras GTPases are mutated at codons 12, 13, and 61, with different frequencies in KRas, HRas, and NRas and in a cancer-specific manner. The G13D mutant appears in 25% of KRas-driven colorectal cancers, while observed only rarely in HRas or NRas. Structures of Ras G13D in the three isoforms show an open active site, with adjustments to the D13 backbone torsion angles and with disconnected switch regions. KRas G13D has unique features that destabilize the nucleotide-binding pocket. In KRas G13D bound to GDP, A59 is placed in the Mg2+ binding site, as in the HRas-SOS complex. Structure and biochemistry are consistent with an intermediate level of KRas G13D bound to GTP, relative to wild-type and KRas G12D, observed in genetically engineered mouse models. The results explain in part the elevated frequency of the G13D mutant in KRas over the other isoforms of Ras.

RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interactin...