Abstract 4130: Signaling specificity by individual KRAS mutations modulate response to targeted agents in colorectal cancer

Abstract

Abstract Results from a number of trials in metastatic colorectal cancer (CRC) have demonstrated that oncogenic KRAS mutations confer clinical resistance to cetuximab and panitumumab, two monoclonal antibodies targeting the EGFR. As a consequence, the European Medicine Agency and the FDA have restricted drug prescription to patients carrying wild-type KRAS tumors. KRAS mutations are present in approximately 35-40% of CRC samples. Of these, over 70% consist of alterations affecting codon 12 (with G12D and G12V variants representing approximately 34% and 23%, respectively) and about 20% are found at codon 13 (with G13D changes being by far the most prevalent). Previous studies including small cohort of patients have occasionally reported that few CRC patients with KRAS mutant tumors received clinical benefit from treatment with cetuximab. We noted that these unusual clinical responses were observed in patients affected by tumors carrying specifically the KRAS G13D change. For this reason, we hypothesized that oncogenic alleles affecting KRAS codons 12 or 13 would result in different response to anti-EGFR monoclonal antibodies. To investigate this, we used a forward genetics approach and expressed – by targeted homologous recombination – the seven most frequently mutated KRAS alleles in two wild-type colorectal cancer cell lines, SW48 and LIM1215, both of which are sensitive to anti-EGFR monoclonal antibodies. At least two independent knock-in clones of each genotype were isolated, and the successful targeting of KRAS was validated by gDNA and cDNA sequencing. Biochemical analysis confirmed that all mutant cells displayed activated Ras, as shown by the levels of GTP-bound Ras measured by pull-down assay. However, we noted differences in the phosphorylation status of KRAS effectors, such as MEK, ERK and AKT, depending upon individual mutations or incubation with cetuximab. Notably, we observed that cetuximab was able to impair proliferation of G13D mutated cells, while G12V or G12D clones were not affected. In addition, cetuximab administration prominently delayed the growth of tumors formed by wild-type or KRAS G13D mutant cells grown as xenografts in immunocompromised mice. In contrast, KRAS G12V or G12D tumors were insensitive to treatment in vivo. These preclinical findings were corroborated in a retrospective analysis showing that patients with G13D-mutated tumors treated with cetuximab had a longer overall and progression-free survival than patients with other KRAS mutated tumors (De Roock W*, Jonker DJ*, Di Nicolantonio F*, et al. JAMA, 2010;304(16):1812-20). Overall, these results indicate that the KRAS G12V, G12D and G13D mutations differently affect response to cetuximab in preclinical models. Further work is ongoing to characterize in detail less frequent KRAS codon 12 mutants (G12A, G12C G12R, G12S) in our panel of isogenic colorectal cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4130. doi:10.1158/1538-7445.AM2011-4130