Abstract 336: S49076, a kinase inhibitor of AXL, MET and FGFR with strong, selective preclinical activity against tumor cells with acquired resistance to EGFR inhibitors not carrying the T790M mutation
Authors: Bertran-Alamillo Jordi , Molina Miguel A. , Valérie Cattan , Burbridge Mike , Teixido Cristina , Codony-Servat Jordi , Codony-Servat Carles , Rosell Rafael 
Published: July 2016

Abstract
Background: Aberrant activity of MET, FGFR1 and AXL has been associated with development of resistance to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC) patients. S49076 is a potent ATP-competitive tyrosine kinase inhibitor (TKI) of MET, AXL/MER, FGFR1/2/3 currently in phase I clinical trials.
Methods: We obtained six resistant lines by treating EGFR-mutated, TKI-sensitive PC9 cells with increasing concentrations of gefitinib (GR1-5) or erlotinib (ER). The six resistant cell lines conserved the exon 19 deletion but the T790M resistance mutation only emerged in two (GR1, GR4), which remained sensitive to AZD9291, a third generation EGFR TKI. Six new AZD9291-resistant cell lines were derived from GR1 and GR4 by exposure to increasing concentrations of the inhibitor. Three of the AZD9291 resistant cell lines lost the exon 19 and T790M mutations, one conserved only the sensitizing mutation and two kept both mutations but with the T790M dropping to very low allelic fractions (1% and 0.03%). All resistant cell lines were characterized for AXL, MET, MER, FGFR1 and FGFR2 expression by Q-RT-PCR, immunohistochemistry and Western blotting. The effects of S49076 on the parental and resistant cell lines were analyzed by MTT, colony formation, basal membrane invasion and migration assays. Western blotting of key signal transduction proteins was used to gain insight in the drug's mechanism of action.
Results: AXL overexpression was the most common event related to gefitinib/erlotinib resistance in the panel of cell lines, with T790M mutation, FGFR1 or BCL-2 overexpression and MET activation being less frequent events. Regarding AZD9291 resistance, AXL upregulation was again widespread, with loss of EGFR mutations as the second most frequent event. In proliferation assays, S49076 showed strong antitumor activity against all PC9-derived cell lines with acquired resistance to EGFR TKIs (erlotinib, gefitinib or AZD9291) and not carrying the T790M mutation, with IC50 of 0.2-1.2 μM and less than 10% surviving cells at 1 μM of drug in most cases. In contrast, S49076 was less active against the parental PC9 cells and the T790M positive resistant lines, with more than 50% of surviving cells at 50 μM of drug. S49076 also inhibited the anchorage-independent growth and migration of the resistant cell lines. A correlation was found between mRNA and protein levels of AXL and sensitivity to S49076. Also, PARP cleavage and moderate but reproducible inhibition of AKT phosphorylation by S49076 were observed exclusively in the non-T790M resistant cell lines.
Conclusions: S49076 shows strong activity in preclinical assays against EGFR mutated cell lines resistant to EGFR TKIs not harboring EGFR T790M mutation and overexpressing AXL. A clinical trial of S49076 in non-T790M patients progressing to EGFR TKI and overexpressing AXL or MET has been initiated.
Citation Format: Jordi Bertran-Alamillo, Miguel A. Molina, Cattan Valérie, Mike Burbridge, Cristina Teixido, Jordi Codony-Servat, Carles Codony-Servat, Rafael Rosell. S49076, a kinase inhibitor of AXL, MET and FGFR with strong, selective preclinical activity against tumor cells with acquired resistance to EGFR inhibitors not carrying the T790M mutation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 336.