Preclinical Development Activation of IL-6R/JAK1/STAT3 Signaling Induces De Novo Resistance to Irreversible EGFR Inhibitors in Non–Small Cell Lung Cancer with T790M Resistance Mutation Sun Mi Kim 1,6 , Oh-Joon Kwon 4 , Yun Kyoung Hong 1,6 , Joo Hang Kim 1,2 , Flavio Solca 7 , Sang-Jun Ha 5 , Ross A. Soo 8 , James G. Christensen 9 , Ji Hyun Lee 3 , and Byoung Chul Cho 1,2 Abstract The secondary T790M mutation in epidermal growth factor receptor (EGFR) is the major mechanism of acquired resistance to EGFR tyrosine kinase inhibitors (TKI) in non–small cell lung cancer (NSCLC). Although irreversible EGFR TKIs, such as afatinib or dacomitinib, have been introduced to overcome the acquired resistance, they showed a limited efficacy in NSCLC with T790M. Herein, we identified the novel de novo resistance mechanism to irreversible EGFR TKIs in H1975 and PC9-GR cells, which are NSCLC cells with EGFR T790M. Afatinib activated interleukin-6 receptor (IL-6R)/JAK1/STAT3 signaling via autocrine IL-6 secretion in both cells. Inhibition of IL-6R/JAK1/STAT3 signaling pathway increased the sensitivity to afatinib. Cancer cells showed stronger STAT3 activation and enhanced resistance to afatinib in the presence of MRC5 lung fibroblasts. Blockade of IL-6R/JAK1 significantly increased the sensitivity to afatinib through inhibition of afatinib-induced STAT3 activation augmented by the interaction with fibroblasts, suggesting a critical role of paracrine IL-6R/JAK1/STAT3 loop between fibroblasts and cancer cells in the development of drug resistance. The enhancement of afatinib sensitivity by inhibition of IL-6R/JAK1/STAT3 signaling was confirmed in in vivo PC9-GR xenograft model. Similar to afatinib, de novo resistance to dacomitinib in H1975 and PC9-GR cells was also mediated by dacomitinib-induced JAK1/STAT3 activation. Taken together, these findings suggest that IL- 6R/JAK1/STAT3 signaling can be a potential therapeutic target to enhance the efficacy of irreversible EGFR TKIs in patients with EGFR T790M. Mol Cancer Ther; 11(10); 2254–64. Ó2012 AACR. Introduction The epidermal growth factor receptor (EGFR) is a proto- oncogene regulating cell proliferation, angiogenesis, and metastasis (1). EGFR abnormalities including amplifica- tion, mutation, and overexpression are frequently iden- tified in a variety of tumor types and known to exert a strong oncogenic potential (2). Gefitinib and erlotinib are first-generation EGFR tyro- sine kinase inhibitors (TKI) that block EGFR signaling pathway through reversible binding to EGFR (3). Despite a dramatic initial response to reversible EGFR TKIs in patients with EGFR activating mutations such as exon 19 in-frame deletions or exon 21 L858R point mutation, almost all patients acquire resistance to these agents because of diverse mechanisms. In 50% of these patients, resistance is derived by the occurrence of a secondary T790M mutation in exon 20 of EGFR (4, 5). Therefore, to develop an effective therapy for patients harboring EGFR T790M is important to overcome the acquired resistance to the first-generation EGFR TKIs. Recently, second-generation EGFR TKIs have been clinically developed. These agents effectively inhibit EGFR activity by irreversible binding to EGFR. Afatinib (BIBW2992) and dacomitinib (PF299804) are representa- tive second-generation irreversible EGFR TKIs currently undergoing clinical trial and have shown promising anti- tumor activity in NSCLC (6, 7). Because these agents have been reported to exhibit antitumor activity in preclinical Authors' Affiliations: 1 Institute for Cancer Research, Yonsei Cancer Center; 2 Departments of Internal Medicine, and 3 Pharmacology, Pharma- cogenomic Research Center for Membrane Transporters and Research Center for Human Natural Defense System, Yonsei University College of Medicine; 4 National Core Research Center for Nanomedical Technology; 5 Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul; 6 JE UK Institute for Cancer Research, JE UK Chemical Company, Gumi-City, Kyungbuk, Republic of Korea; 7 Depart- ment of Pharmacology, Boehringer Ingelheim Austria, Vienna, Austria; 8 Department of Haematology-Oncology, National University Cancer Insti- tute, National University Health System, Cancer Science Institute of Sin- gapore, National University of Singapore, Singapore; 9 Department of Cancer Research, Pfizer Global Research and Development, La Jolla Laboratories, La Jolla, California Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Authors: Byoung Chul Cho, Yonsei Cancer Center, Divi- sion of Medical Oncology, Department of Internal Medicine, Yonsei Uni- versity College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul, Republic of Korea. Phone: 82-2-2228-8126; Fax: 82-2-393-3652; E-mail: cbc1971@yuhs.ac and Ji Hyun Lee, Department of Pharmacology, Phar- macogenomic Research Center for Membrane Transporters and Research Center for Human Natural Defense System, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul, Republic of Korea. Phone: 82-2-2228-1743; Fax: 82-2-313-1894; E-mail: jihyni@yuhs.ac doi: 10.1158/1535-7163.MCT-12-0311 Ó2012 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 11(10) October 2012 2254 on March 31, 2017. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst August 13, 2012; DOI: 10.1158/1535-7163.MCT-12-0311