Mutational activation of the Kras oncogene occurs in 40% of colon cancers, which affects over 150,000 people and approximately 45,000 people will die of the disease in the United States per year. Although advances in early detection and clinical management have led to improved survival, the future of colon cancer therapy lies in personalized molecular medicine, where physicians tailor an individual's treatment to the mutation(s) that have occurred in their cancer. While activating mutations at codons 12, 13, and 61 of K-Ras were originally identified in cancers, a broader assessment of RAS mutations has led to the identification of weaker, non-canonical activating mutations at codon 146 (typically an alanine to threonine substitution), which occurs in approximately 4% of colorectal cancers. Kras mutations at codons 12 or 13 are associated with poor prognosis and show resistance to both conventional and targeted therapies, while mutations at codon 146 is associated with a more favorable clinical outcome in patients with colorectal cancer. Identification of a K-RAS mutant that would prove to be a potential target for therapeutic intervention in colorectal cancer would clearly be valuable. In order to fully understand the implications of these observations, and to utilize the information for therapeutic benefit, we need to study the different mutant forms of K-Ras in an experimentally tractable system. Using both genetically engineered mouse models and human colorectal cancer cell lines we will assess the molecular and phenotypic similarities and differences between distinct forms of mutant K-Ras. Consistent with previous reports of the biochemical activities of the different mutants, we found that K-RasA146T exhibited enhanced GTP-binding relative to wild type, but significantly lower GTP-binding relative to K-RasG12D. Interestingly, A146T animals exhibited an intermediate tissue-level hyperplasia phenotype, consistent with the intermediate biochemical phenotype, but exhibit a hyper-proliferation phenotype that is equal to G12D. Unlike our observation from genetically engineered mice, we found that cell lines carrying codon 146 mutations had levels of K-Ras-GTP that were equal to cell lines carrying codon 12/13 mutations. At a functional level, we found that codon 146 mutations enhanced sodium butyrate-induced apoptosis in CRC cells, just like codon 12 mutations, and that loss of K-RasA146T suppressed proliferation at a level similar to loss of K-RasG12D. In the context of Apc-mutant colonic tumors, activation of K-RasA146T led to an increase in tumor number and a decrease in animal life span compared to control animals, similar to K-RasG12D animals. In the future we will employ computational modeling and network-level signaling pathway measurements to characterize how signaling pathways are affected by the mutation of K-RAS A146T compared to both wild type and other K-Ras mutants. We expect that these studies will provide a rational for broader K-RAS testing in the clinic beyond the common alleles at codon 12 and 13 and allow for the development of novel therapies to treat K-RAS mutant colorectal cancer patients.

Citation Format: Jessica J. Gierut, Kevin M. Haigis. Functional characterization of K-RAS A146T in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B06. doi: 10.1158/1557-3125.RASONC14-B06