Abstract 3244: Role of epithelial-mesenchymal transition (EMT) in sensitivity to CNX-2006, a novel mutant-selective EGFR inhibitor which overcomes in vitro T790M-mediated resistance in NSCLC.
Authors: Galvani Elena , Giovannetti Elisa , Walter Annette O. , Tjin Robert , Dekker Henk , Petronini Pier Giorgio , Smit Egbert F. , Peters Godefridus J. 
Published: August 2014

Background: EGFR is an established target in advanced NSCLC, and the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib have been approved for the treatment of patients harbouring activating-EGFR mutations. Unfortunately, their efficacy is limited by acquired resistance, caused in ≈50% of the cases by the T790M secondary point-mutation. Several EGFR inhibitors have been developed with the aim to overcome such resistance. However, emergence of in vitro resistance due to T790M amplification has been reported for second-generation EGFR-TKIs. Therefore we evaluated the efficacy of CNX-2006, a prototype of the novel mutant-selective EGFR-TKI CO-1686, which is currently in a phase I clinical trial in previously treated mutant EGFR NSCLCs.

Methods: CNX-2006 was provided by Celgene Avilomics Research. Its antiproliferative activity was tested by sulforhodamine B assay in 12 NSCLC cell lines, previously characterized for EGFR and K-Ras mutational status and gefitinib sensitivity, including PC9GR4 and PC9DR1 (kindly provided by Dr. Jänne, Harvard University, Boston, USA). Novel CNX-2006 resistant clones have been established starting from the EGFR T790M cells H1975 and PC9GR4, and several markers have been characterized by RT-PCR, kinase array and Western blot.

Results: CNX-2006 inhibited cell proliferation independently from K-Ras mutations while it was as effective as gefitinib in activating-EGFR mutation positive cells. In the cell lines expressing wild-type EGFR CNX-2006 and gefitinib had limited anti-proliferative activity. CNX-2006 inhibited EGFR-T790M cells growth up to 1000-fold more compared to wild-type EGFR cells. EGFR inhibition was observed in cells harbouring the T790M mutation at IC50 values below 20 nM after 1 hour exposure to the drug. In contrast to gefitinib, CNX-2006 also significantly reduced the volume of tumor spheres derived from H1975 cells. Multiple CNX-2006 resistant clones were generated by exposing H1975 or PC9GR4 cells to increasing drug concentrations, leading to 30-fold resistant clones, which grow in CNX-2006 concentrations 16-20 times the initial IC50s. This resistance was retained for at least 3 months after drug removal. CNX-2006 resistant clones showed differences in expression of several biomarkers associated with EMT, such as a 3-fold reduction of E-cadherin mRNA and a 60-fold increase in MMP9 compared to the parental cells.

Conclusions: CNX-2006 is a potent, mutant-selective EGFR inhibitor with excellent in vitro activity in cells with activating EGFR mutations, as well as in cells harbouring the T790M mutation. Future studies in mechanisms underlying EMT are warranted and might be used to prevent CNX-2006 resistance.

Citation Format: Elena Galvani, Elisa Giovannetti, Annette O. Walter, Robert Tjin, Henk Dekker, Pier Giorgio Petronini, Egbert F. Smit, Godefridus J. Peters. Role of epithelial-mesenchymal transition (EMT) in sensitivity to CNX-2006, a novel mutant-selective EGFR inhibitor which overcomes in vitro T790M-mediated resistance in NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3244. doi:10.1158/1538-7445.AM2013-3244