title: Molecular Changes Associated with Acquired Resistance to Crizotinib in ROS1-Rearranged Non-Small Cell Lung Cancer.
abstract: OBJECTIVE
  Although ROS1-rearranged non-small cell lung cancer (NSCLC) is sensitive to crizotinib, development of resistance is inevitable. Here, we identified molecular alterations in crizotinib-resistant tumors from two NSCLC patients with the CD74-ROS1 rearrangement, and in HCC78 cells harboring SLC34A2-ROS1 that showed resistance to crizotinib (HCC78CR cells).
  
  
  METHODS
  ROS1 kinase domain mutations were examined in fresh tumor tissues from two NSCLC patients and HCC78CR1-3 cells by direct sequencing. Ba/F3 cells expressing ROS1 secondary mutations were constructed to evaluate resistance to crizotinib. An upregulated pathway was identified using phospho-receptor tyrosine kinase array, EGFR signaling antibody array, and RNA sequencing (RNA-seq). Cell proliferation and ROS1 downstream signaling pathways were compared between HCC78 and HCC78CR1-3 cells.
  
  
  RESULTS
  The ROS1 G2032R mutation was identified in crizotinib-resistant tumors from one patient. Furthermore, HCC78CR1 and CR2 cells harbored a novel ROS1 L2155S mutation (73.3% and 76.2%, respectively). ROS1 G2032R and L2155S mutations conferred resistance to crizotinib in Ba/F3 cells. Evidence of epithelial-to-mesenchymal transition with downregulated E-cadherin and upregulated vimentin was observed in HCC78CR1-2 cells and in the other patient. RNA-seq and EGFR signaling antibody array revealed that the EGFR pathway was significantly upregulated in HCC78CR3 versus HCC78 cells. Cells with the ROS1 mutation and upregulated EGFR were sensitive to foretinib, an inhibitor of c-MET, VEGFR2, and ROS1 and irreversible EGFR tyrosine kinase inhibitors plus crizotinib, respectively.
  
  
  CONCLUSIONS
  Molecular changes associated with acquired crizotinib resistance in ROS1-rearranged NSCLC are heterogeneous, including ROS1 tyrosine kinase mutations, EGFR activation, and epithelial-to-mesenchymal transition.
authors: Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors: Ahnah
authors: A
authors: Song
authors: Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors: Tae Min
authors: TM
authors: Kim
authors: Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea. kimdw@snu.ac.kr.
authors: Dong-Wan
authors: DW
authors: Kim
authors: Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors: Soyeon
authors: S
authors: Kim
authors: Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors: Bhumsuk
authors: B
authors: Keam
authors: Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors: Se-Hoon
authors: SH
authors: Lee
authors: Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors: Dae Seog
authors: DS
authors: Heo
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics*; D045744:Cell Line, Tumor; D000077547:Crizotinib; D004252:DNA Mutational Analysis; D019008:Drug Resistance, Neoplasm; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D056726:Genetic Association Studies; D006801:Humans; D020128:Inhibitory Concentration 50; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics*; D008875:Middle Aged; D020125:Mutation, Missense; D015514:Oncogene Proteins, Fusion; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D011720:Pyrazoles / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D014178:Translocation, Genetic
chemical_list: D000970:Antineoplastic Agents; D015514:Oncogene Proteins, Fusion; D011518:Proto-Oncogene Proteins; D011720:Pyrazoles; D011725:Pyridines; D000077547:Crizotinib; D011505:Protein-Tyrosine Kinases; C062333:ROS1 protein, human