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

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