Published OnlineFirst December 2, 2013; DOI: 10.1158/1535-7163.MCT-13-0817 Molecular Cancer Cancer Biology and Signal Transduction Therapeutics Acquired Resistance to Dasatinib in Lung Cancer Cell Lines Conferred by DDR2 Gatekeeper Mutation and NF1 Loss Ellen M. Beauchamp1, Brittany A. Woods1,7, Austin M. Dulak1, Li Tan3, Chunxiao Xu1, Nathanael S. Gray2, Adam J. Bass1,6, Kwok-kin Wong1,4, Matthew Meyerson1,5,6, and Peter S. Hammerman1,6 Abstract The treatment of non–small cell lung cancer has evolved dramatically over the past decade with the adoption of widespread use of effective targeted therapies in patients with distinct molecular alterations. In lung squamous cell carcinoma (lung SqCC), recent studies have suggested that DDR2 mutations are a biomarker for therapeutic response to dasatinib and clinical trials are underway testing this hypothesis. Although targeted therapeutics are typically quite effective as initial therapy for patients with lung cancer, nearly all patients develop resistance with long-term exposure to targeted drugs. Here, we use DDR2-dependent lung cancer cell lines to model acquired resistance to dasatinib therapy. We perform targeted exome sequencing to identify two distinct mechanisms of acquired resistance: acquisition of the T654I gatekeeper mutation in DDR2 and loss of NF1. We show that NF1 loss activates a bypass pathway, which confers ERK dependency downstream of RAS activation. These results indicate that acquired resistance to dasatinib can occur via both second-site mutations in DDR2 and by activation of bypass pathways. These data may help to anticipate mechanisms of resistance that may be identified in upcoming clinical trials of anti-DDR2 therapy in lung cancer and suggest strategies to overcome resistance. Mol Cancer Ther; 13(2); 475–82. Ó2013 AACR. Introduction disease, which is diagnosed in 40,000 to 50,000 patients per For patients with lung adenocarcinoma, the most com- year in the United States (13). The majority of patients mon subtype of non–small cell lung cancer (NSCLC), present with locally advanced or metastatic disease. genomic studies have identified several targetable molec- Recent studies of lung SqCC have identified recurrent ular alterations. Alterations in EGFR (1–3) and ALK (4, 5), alterations in tyrosine kinases including amplifications, and more recently, BRAF (6, 7), ROS1 (8, 9), and RET (10– mutations, and translocations of fibroblast growth factor 12), have been associated with marked responses to small receptors (FGFR; refs. 14–18) and mutations in the dis- coidin domain receptor 2 gene (DDR2; refs. 19, 20). molecule kinase inhibitors in clinical trials and therapies FGFR1 DDR2 targeting EGFR and ALK genomic alterations are U.S. amplification and mutation have been Food and Drug Administration (FDA) approved. These associated with response to targeted agents in preclin- targeted therapies are both more efficacious and less toxic ical models and in early-phase clinical trials (19, 21, 22). than standard chemotherapy in selected populations and These studies have led to interest in the development of have led to a paradigm shift in the management of lung targeted therapeutic approaches specifically for lung adenocarcinoma. SqCCs given that there are no targeted agents approved Lung squamous cell carcinoma (lung SqCC) is the in this disease. Several ongoing clinical trials are inves- second most common subtype of NSCLC and is a lethal tigating therapeutic biomarkers in lung SqCCs, includ- ing FGFR mutations and amplifications as well as BRAF, PIK3CA,andDDR2 mutations (www.clinicaltrials.gov). Authors' Affiliations: Departments of 1Medical Oncology and 2Biological In lung adenocarcinoma, the success of targeted ther- Chemistry and Molecular Pharmacology, Dana-Farber Cancer Institute; apies has been limited by the observation that nearly all 3Department of Biological Chemistry and Molecular Pharmacology, Har- vard Medical School; 4Belfer Institute of Applied Cancer Sciences; 5Depart- treated patients develop acquired resistance to the tar- ment of Pathology, Brigham and Womens Hospital, Boston; 6Broad Insti- geted agent over time. Studies of lung cancer cell lines tute Cancer Program, Cambridge, Massachusetts; and 7Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New with sensitizing mutations and patient-derived rebiopsy York, New York specimens have demonstrated that acquired resistance Note: Supplementary data for this article are available at Molecular Cancer can occur as a result of secondary mutations in the original Therapeutics Online (http://mct.aacrjournals.org/). target, such as EGFR T790M (23, 24) and ALK L1196M (25, Corresponding Author: Peter S. Hammerman, Dana-Farber Cancer Insti- 26), or by activation of parallel pathways, which bypass tute, 450 Brookline Avenue, Dana 810A, Boston, MA 02215. Phone: 617- the original target such as MET (27) or ERBB2 amplifica- 632-3000; Fax: 617-582-7880; E-mail: [email protected] tion (28). Preclinical models of acquired resistance have doi: 10.1158/1535-7163.MCT-13-0817 been robust in predicting mechanisms of resistance iden- Ó2013 American Association for Cancer Research. tified in the clinic (29–35) and there are substantial efforts www.aacrjournals.org 475 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst December 2, 2013; DOI: 10.1158/1535-7163.MCT-13-0817 Beauchamp et al. underway to identify strategies to overcome acquired Materials and Methods resistance to targeted agents for multiple lung cancer Cell culture genotypes. These issues are not limited to lung cancer as Lung cancer cell lines (NCI-H2286, HCC-366, and NCI- acquired resistance to targeted agents is an increasing H1703) were obtained from the ATCC and maintained in problem across many cancer cell types, and multiple RPMI 1640 (Invitrogen) plus 10% fetal calf serum (Gem- mechanisms of acquired resistance have been reported ini). Experiments were performed within 6 months of to date. receipt of cell lines from ATCC and no further authenti- Dasatinib is an FDA-approved oral tyrosine kinase cation was performed. Dasatinib was obtained from LC inhibitor, which has been studied in several trials of labs. NCI-H2286 and HCC-366 cells were grown in patients with lung cancer (36–38). Although the res- increasing concentrations of dasatinib beginning at 100 ponse rate to dasatinib as a single agent has been low in nmol/L and increasing to 1.5 mmol/L over a period of 3 general, several responders have been reported, includ- to 4 months. ing 2 individuals with DDR2 S768R mutations (19, 22) BRAF and an individual with a mutation (39), suggest- DNA sequencing ing that specific biomarkers may be predictive of Genomic DNA was prepared from HCC-366, NCI- response to dasatinib. An international clinical trial is H2286, and 2 independent clones of NCI-H2286-DR and now accruing patients with these genotypes to address HCC-366-DR, using the Qiagen DNeasy Kit. DNA was the efficacy of dasatinib in genomically selected patient quantified by Pico-Green (Invitrogen) and 200 ng used for cohorts with advanced NSCLC (NCT01514864). There solution-based hybrid capture using the Oncopanel Cap- has been no study reported to date of mechanisms of ture Kit (49). This was followed by massively parallel acquired resistance to dasatinib in the lung cancer sequencing on the Illumina Hiseq 2000. FASTQ files were population. aligned using the Picard pipeline and mutations called DDR2 is a receptor tyrosine kinase, which functions using Mutect and annotated using Oncotator using the as a cellular collagen receptor (40, 41). Activation of parental cell line as the reference sequence (20). Analysis DDR2 has been associated with a number of cellular methods are also described at www.broadinstitute.org/ phenotypes including transformation, migration, and cancer/cga. differentiation (40, 42). DDR2 mutations are present in 3% to 4% of patients with lung SqCC and have been Immunoblotting reportedinothercancertypes at comparable frequen- Cellular lysates were prepared using RIPA buffer sup- cies including lung adenocarcinoma, uterine cancer, plemented with protease and phosphatase inhibitors. stomach cancer, bladder cancer, melanoma, colorectal Immunoblotting was performed using the Nupage system cancer, and head and neck cancer (www.cbioportal. (Invitrogen) with 100 mg of lysate. Primary antibodies used org). DDR2 engages a number of downstream signaling were: NF1 (Santa Cruz Biotechnology), DDR2 (Bethyl pathways including PI3K/AKT, Src, MEK/ERK, and Laboratories), p-TYR clone 4G10 (Millipore), Erk (Cell k NF- B to drive cell survival, proliferation, migration, Signaling Technologies), p-Erk1/2 (Thr202/Tyr204; Cell and transformation (43–47). DDR2 is potently inhibited Signaling Technologies), Src (Cell Signaling Technologies), by several FDA approved tyrosine kinase inhibitors p-Src (Tyr416; Cell Signaling Technologies), Mek (Cell including dasatinib, nilotinib, imatinib, and ponatinib Signaling Technologies), p-Mek (Ser217/221; Cell Signal- (48). Although all of these kinase inhibitors are active ing Technologies), Akt (Cell Signaling Technologies), p- DDR2 against -mutated lung cancer cell lines, dasatinib Akt (Ser473; Cell Signaling Technologies), b-actin (Sigma), in vitro in vivo is the most potent and . The potency of and vinculin (Sigma). Immunoprecipitation was per- dasatinib against DDR2 has led to the design of clinical formed by incubating 2 mg of lysate with 10 mL antibody trials