Abstract
Abstract Background: Despite the excellent initial responses of ALK-rearranged lung cancer to the ALK tyrosine kinase inhibitors (TKIs), most patients develop acquired resistance through several mechanisms, including ALK amplification, mutation or activation of alternative signaling pathways. In this study, we investigated the effect of dual-targeting EGFR and ALK pathways in NSCLC cells that are resistant to ALK inhibitors. Methods: The cell proliferation, apoptosis and cell cycle analysis were measured by spectrophotometry and flow cytometry. The combination effect of EGFR TKI (afatinib or dacomitinib) and ALK TKI (crizotinib or LDK378) was evaluated using a combination index (CI). We analyzed EGFR ligand levels by ELISA and expression of ALK, EGFR and its downstream molecules were detected by Western blot assay after exposure to TKIs. Results: NCI-H3122CR1 that was resistant to crizotinib and NCI-H3122 cell lines became resistant to LDK378 through the step-wise increase of the drug, resulting in H3122CR1LR1 and H3122LR1 cells, respectively. The resistant cell lines exhibited 10-14-fold higher IC50 values for crizotinib or LDK378 compared with parental cells. The genetic alterations of ALK or EGFR genes were not found in H3122CR1, H3122LR1, and H3122CR1LR1 cells. We analyzed EGFR signaling pathways in these cells to determine their roles in mediating ALK inhibitor resistance. We observed that H3122CR1, H3122LR1 and H3122CR1LR1 cells had higher phospho-EGFR than parental cells. In these cells, mRNA and protein levels of amphiregulin and EGF were up-regulated. Combined treatment with EGFR TKI and LDK378 synergistically inhibited cell growth (CI<1) and induced apoptosis in H3122LR1 and H3122CR1LR1 cells. Similar results were observed in H3122CR1 and SNU-2535 harboring ALK G1269A mutation cells, when exposed to afatinib and crizotinib. The combination of EGFR and ALK TKIs decreased the phosphorylation of several downstream molecules in H3122LR1 and H3122CR1LR1 cells, whereas EGFR or ALK TKI alone did not reduce the phosphorylation levels. Conclusion: Our results suggest that dual inhibition of ALK and EGFR effectively abrogate ALK and its downstream signals and induce apoptosis in ALK TKIs-resistant, ALK-rearranged NSCLC cells. Therefore, dual-targeting of EGFR and ALK might be a promising strategy for ALK-rearranged NSCLC patients who showed acquired resistance to crizotinib and/or LDK378. Citation Format: Soyeon Kim, Tae Min Kim, Yong-Oon Ahn, Bhumsuk Keam, Se-Hoon Lee, Dae Seog Heo, Dong-Wan Kim. Synergistic inhibition of ALK inhibitor-resistant lung cancer cells by dual-targeting EGFR and ALK pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1842. doi:10.1158/1538-7445.AM2014-1842
Published Version
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