Abstract 5840: Radiosensitization with the c-MET inhibitor crizotinib in KRAS mutant colorectal cancer
Authors: Cuneo Kyle C. , Kurapati Himabindu , Mehta Ranjit , Lawrence Theodore S. , Nyati Mukesh K. 
Published: July 2017

Abstract
Background: KRAS mutant colorectal cancers (CRC) are resistant to many targeted therapies including cetuximab. Novel molecular targets for KRAS mutant tumors may enhance the efficacy of chemoradiation. C-MET plays important roles in treatment resistance, tumor invasion, and metastasis. In this study, we used a small molecule inhibitor of c-MET, crizotinib in cetuximab resistant mutant KRAS driven CRC cell lines and assessed radiosensitization.
Methods: To test the effect of c-MET inhibition on CRC cells we used the KRAS mutant cell lines HCT116, DLD1, and LoVo. Colony formation assays were performed to assess the effects of crizotinib and cetuximab. Immunoblot analysis was used to determine the effect of crizotinib on c-MET and downstream pathways including ERK and AKT phosphorylation. We then selected doses of crizotinib below the IC50 and assessed clonogenic survival with radiation. To study potential mechanisms of radiosensitization cell cycle analysis was performed using flow cytometry. The effect of c-MET inhibition on cellular function was assessed using cell migration assays.
Results: We first performed a series of clonogenic survival assays with crizotinib and cetuximab to determine the IC50 for each cell line. As expected, the KRAS mutant cell lines LoVo, HCT116, and DLD1 were resistant to cetuximab (surviving fraction greater than 95% at 100 μg/ml). These cell lines were sensitive to the c-MET inhibitor crizotinib with IC50 values of 14.4 nM for LoVo, 316 nM for HCT166, and 217 nM for DLD1. Immunoblot analysis showed that crizotinib at concentrations near the IC50 for each cell line attenuated c-MET phosphorylation and downstream signaling pathways including ERK and AKT. Crizotinib further blocked radiation induced c-MET phosphorylation and attenuated activation of downstream signaling pathways. We then performed a series of clonogenic survival assays to study the radiosensitizing potential of crizotinib. Pretreatment with crizotinib for 24 hours radiosensitized LoVo and DLD1 cell lines with enhancement ratios of 1.66 and 1.25, respectively. Interestingly, treatment with crizotinib for 1 hour prior to radiation had only modest dose enhancement, suggesting that prolonged exposure to crizotinib is required for optimal radiosensitization. Cell cycle analysis revealed minimal G1 arrest with crizotinib. Additionally, crizotinib completely blocked HGF and EGF induced cell migration.
Conclusions: Inhibition of c-MET with crizotinib effectively sensitizes cetuximab resistant KRAS mutant CRC cell lines to radiation. Given the important role c-MET plays in treatment resistance, crizotinib has the potential to improve the outcome of patients with locally advanced rectal cancer undergoing radiation therapy.
Citation Format: Kyle C. Cuneo, Himabindu Kurapati, Ranjit Mehta, Theodore S. Lawrence, Mukesh K. Nyati. Radiosensitization with the c-MET inhibitor crizotinib in KRAS mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5840. doi:10.1158/1538-7445.AM2017-5840