A Novel SND1-BRAF Fusion Confers Resistance to c-Met Inhibitor PF-04217903 in GTL16 Cells though MAPK Activation Nathan V. Lee1, Maruja E. Lira1, Adam Pavlicek1, Jingjing Ye2¤, Dana Buckman1, Shubha Bagrodia1, Sreesha P. Srinivasa1, Yongjun Zhao3, Samuel Aparicio4,5, Paul A. Rejto1, James G. Christensen 1 , 1 Keith A. Ching * 1 Oncology Research Unit, Pfizer Global Research & Development, San Diego, California, United States of America, 2 Global Pre-Clinical Statistics, Pfizer Global Research & Development, San Diego, California, United States of America, 3 Michael Smith Genome Sciences Centre, BC Cancer Research Centre, Vancouver, British Columbia, Canada, 4 Molecular Oncology, BC Cancer Agency, Vancouver, British Columbia, Canada, 5 Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada Abstract Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling. Citation: Lee NV, Lira ME, Pavlicek A, Ye J, Buckman D, et al. (2012) A Novel SND1-BRAF Fusion Confers Resistance to c-Met Inhibitor PF-04217903 in GTL16 Cells though MAPK Activation. PLoS ONE 7(6): e39653. doi:10.1371/journal.pone.0039653 Editor: Keiran Smalley, The Moffitt Cancer Center & Research Institute, United States of America Received November 23, 2011; Accepted May 24, 2012; Published June 22, 2012 Copyright: 2012 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work described in the manuscript was funded by Pfizer Inc. (www.pfizer.com). Employees of the funder actively contributed to study design, data collection and analysis, decision to publish, and preparation of the manuscript. Competing Interests: Nathan Lee, Maruja Lira, Adam Pavlicek, Jingjing Ye, Dana Buckman, Shubha Bagrodia, Sreesha Srinivasa, Paul Rejto, James Christensen, and Keith Ching were paid employees of Pfizer. Yongjun Zhao and Samuel Aparicio were funded by Pfizer. Pfizer maintains the patents and marketing rights to the compounds PF-04217903, PF-04880594, and Xalkori used in this study. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: keith.ching@pfizer.com ¤ Current address: Division of Biostatistics, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, Maryland, United States of America Introduction Aberrant receptor tyrosine kinase (RTK) activity provides growth and survival signals crucial for the development and progression of many cancers. Treatment of patients with targeted inhibitors of key oncogenic kinase drivers such as sunitinib, erlotinib, gefitinib, and imatinib have demonstrated clinical success [1]. However, despite successful clinical outcomes in select patient populations, the development of resistance to targeted inhibitors can result in disease progression and limit therapeutic effectiveness. Notably, the emergence of secondary mutations or upregulation of compensatory pathways in response to RTK inhibition often arises after a period of initial efficacy [2,3,4,5]. The c-Met/HGFR receptor tyrosine kinase is a promising therapeutic target as mutations of c-Met (in papillary renal cell carcinoma, childhood hepatocellular carcinoma) and focal ampli- fications of the MET gene locus (in NSCLC, GBM, esophageal and gastric cancers) may indicate an oncogenic dependence on c- Met signaling [6,7,8]. For instance, cell lines and xenograft tumors bearing amplification of the MET gene locus are very responsive to c-Met inhibitors such as the highly selective small molecule PF- 04217903 (METi). Not unlike the eventual resistance that emerges against other RTK inhibitors, several studies have described development of resistance to c-Met inhibitors via c-Met amplifi- cation [9] or c-Met mutations that prevent the inhibitor from binding [10]. Additionally, the activation of EGFR/ERBB family receptors [2,11,12], KRAS, BRAF, or AKT [13] can also overcome c-Met inhibition. To anticipate potential resistance, we utilized an in vitro screen to select for METi resistant clones of GTL16, a c-Met dependent gastric carcinoma cell line that harbors a high-level focal amplification of the MET gene locus [12,14]. Here we report a novel escape mechanism of GTL16 treated with METi. Molecular characterization of resistant clones reveals a genomic rearrange- PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39653