Isogenic K-Ras mutant cancer cells: A novel platform for drug profiling

Ras mutations are present in approximately 49% of human colorectal cancers, with mutations in K-Ras G12 and G13 being the most common. Given their high prevalence, understanding how Ras mutations impact response to anticancer agents is crucial to the successful development of novel agents. Recent evidence suggests that not all variants of K-Ras respond in the same way to targeted agents, where some patients with G13D mutations respond better to Cetuximab compared to patients expressing G12V (De Roock, JAMA 2011). Therefore there is a need for better in vitro models to facilitate the characterisation of inhibitor selectivity and efficacy in K-Ras mutant cell systems. We created suites of isogenic X-MAN SW48 and LIM1215 colorectal cancer cells, each of which harbour one of 7 different K-Ras G12 or G13 mutations at their endogenous loci introduced via AAV-mediated homologous recombination. This system allows use of endogenous promoters and has enabled us to create a panel of human cell lines in which the only difference between the cells is the point mutation of interest. This more accurately recapitulates the genomic architecture in human tumors and provides an excellent platform for addressing the impact of K-Ras mutations on responses to therapeutic agents. We selected over 30 targeted agents and cytotoxic drugs based on their clinical relevance to colorectal cancer. We profiled the anti-proliferative activity of each of these across the suite of K-Ras variants in SW48 cells. Strikingly, we found a consistent pattern of resistance towards 5 chemically distinct MEK1/2 inhibitors across the 7 K-Ras variants compared to wild-type cells. These results were confirmed in a second suite of K-Ras mutant lines generated in LIM1215 cells, where clear resistance to MEK inhibitors was observed. To investigate the mechanism of this resistance to MEK inhibitors, we combined the anti-EGFR monoclonal antibody Cetuximab with the MEK inhibitor AZD6244, both compounds being ineffective as single agents in the K-Ras G12D and G12C mutant SW48 cells. Concentrations of Cetuximab as low as 0.01μg/ml re-sensitised previously resistant K-Ras mutant cells to the effects of AZD6244. Given that this dose of Cetuximab was ineffective alone, this result suggests that upregulation of the EGFR axis may be a potential mechanism in compensating for MEK inhibition, and these studies are on-going. In conclusion, we have created two suites of isogenic colorectal cancer cell lines each expressing a different K-Ras G12 or G13 mutation and demonstrated that K-Ras mutations impart resistance to clinical MEK inhibitors. Despite screening over 30 chemotherapeutics covering an array of targets, we did not identify any other significant differences in sensitivity between K-Ras wild-type and mutant cells, demonstrating the mechanistic relevance of this model to MEK inhibitors. Our data highlight the advantages of using this system in profiling molecularly targeted agents in order to identify potential mechanisms of resistance and in rationally identifying combinations.