Therapeutics , Targets , and Chemical Biology Resistance to Irreversible EGF Receptor Tyrosine Kinase Inhibitors through a Multistep Mechanism Involving the IGF 1 R Pathway

The clinical efficacy of EGF receptor (EGFR) kinase inhibitors gefitinib and erlotinib is limited by the development of drug resistance. The most common mechanism of drug resistance is the secondary EGFR T790M mutation. Strategies to overcome EGFR T790M-mediated drug resistance include the use of mutant selective EGFR inhibitors, including WZ4002, or the use of high concentrations of irreversible quinazoline EGFR inhibitors such as PF299804. In the current study, we develop drug-resistant versions of the EGFR-mutant PC9 cell line, which reproducibly develops EGFR T790M as a mechanism of drug resistance to gefitinib. Neither PF299804-resistant nor WZ4002-resistant clones of PC9 harbor EGFR T790M. Instead, they have shown activated insulin-like growth factor receptor (IGF1R) signaling as a result of loss of expression of IGFBP3 with the IGF1R inhibitor, BMS 536924, restoring EGFR inhibitor sensitivity. Intriguingly, prolonged exposure to either PF299804 or WZ4002 results in the emergence of amoredrug-resistant subclone that exhibits ERKactivation. AMEK inhibitor, CI-1040, partially restores sensitivity to theEGFR/IGF1R inhibitor combination.Moreover, an IGF1RorMEK inhibitor used in combinationwith either PF299804 or WZ4002 completely prevents the emergence of drug-resistant clones in this model system. Our studies suggest thatmore effectivemeans of inhibiting EGFRT790Mwill prevent the emergence of this commondrug resistance mechanism in EGFR-mutant non–small cell lung cancer. However, multiple drug resistance mechanisms can still emerge. Preventing the emergence of drug resistance, by targeting pathways that become activated in resistant cancers, may be a more effective clinical strategy. Cancer Res; 73(2); 834–43. 2012 AACR. Introduction EGF receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib are clinically effective therapies for patients with non–small cell lung cancer (NSCLC) with EGFR-mutant cancers (1–6). However, therapy is universally limited by the development of acquired drug resistance (7). The most common mechanism of acquired drug resistance is the secondary EGFR T790Mmutation and is detected in up to 60% of patients with EGFR-mutant NSCLC that develop acquired drug resistance to either gefitinib or erlotinib (8–10). The EGFR-mutant PC9 cell line has been used as model of drug resistance and reproducibly develops EGFR T790M following prolonged in vitro exposure to gefitinib or other reversible EGFR kinase inhibitors (11–13). Irreversible second-generation EGFR TKIs are undergoing clinical development to overcomeEGFRT790M-mediated drug resistance. The quinazoline-based irreversible TKIs, including neratinib (HKI-272), afatinib (BIBW2992), and dacomitinib (PF299804) have been shown to have potency against EGFR T790M-bearing models at high concentrations in preclinical models (14–16). However, when used at low concentrations, those achievable in patients with NSCLC being clinically treated with these agents, the resistance induced by these drugs in EGFR-mutant models, including in the PC9 cells, also occurs through the emergence of the T790Mmutation (17, 18). In accordance with these preclinical data, neratinib and afatinib has shown only modest efficacy in patients with EGFRmutant NSCLC that had developed acquired resistance to gefitinib or erlotinib (19, 20). One potential reason why neratinib or afatinib have not been effective clinically is due to the inability to achieve sufficient drug concentrations to inhibit EGFR T790M in patients with NSCLC. Dose escalation of irreversible quinazoline EGFR inhibitors is limited by on target inhibition of wild-type (WT) EGFR, which leads to dose-limiting skin rash (21, 22). These clinical observations have prompted the development of alternative strategies to inhibit EGFR T790M. One strategy is to develop mutant-selective EGFR inhibitors. In prior studies, we identified a novel class of irreversible pyrimidine inhibitors, including WZ4002, which Authors' Affiliations: Lowe Center for Thoracic Oncology, Department of Medical Oncology, Belfer Institute for Applied Cancer Science; and Departments of Cancer Biology and Biological Chemistry and Molecular Pharmacology, Dana Farber Cancer Institute, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts; and Department of Research Pharmacology, Pfizer Global Research and Development, La Jolla, California Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Pasi A. J€ anne, Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, HIM223, 450 Brookline Avenue, Boston, MA 02215. Phone: 617-632-6076; Fax: 617-582-7683; E-mail: pjanne@partners.org doi: 10.1158/0008-5472.CAN-12-2066 2012 American Association for Cancer Research. Cancer Research Cancer Res; 73(2) January 15, 2013 834 on July 22, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals. org Downloaded from Published OnlineFirst November 19, 2012; DOI: 10.1158/0008-5472.CAN-12-2066