TKI-addicted ROS1-rearranged cells are destined to survival or death by the intensity of ROS1 kinase activity

Sci Rep. 2017 Jul 17;7(1):5519. doi: 10.1038/s41598-017-05736-9.

Abstract

ROS1 rearrangement is observed in 1-2% of non-small cell lung cancers (NSCLC). The ROS1 tyrosine kinase inhibitor (TKI) crizotinib has induced marked tumour shrinkage in ROS1-rearranged cancers. However, emergence of acquired resistance to TKI is inevitable within a few years. Previous findings indicate that cabozantinib overcomes secondary mutation-mediated crizotinib-resistance in ROS1-fusion-positive cells. Here we attempted to establish cabozantinib-resistant cells by N-ethyl-N-nitrosourea mutagenesis screening using CD74-ROS1-expressing Ba/F3 cells. Two resistant cell lines with CD74-ROS1 F2004V or F2075C mutations, which are homologous to ALK F1174 or F1245 mutations, survived in the presence of a low dose of ROS1-TKI. Removal of ROS1-TKI from these TKI-addicted cells induced excessive activation of ROS1 tyrosine kinase followed by apoptosis. We succeeded in recapturing the TKI-addicted phenotype using doxycycline-inducible CD74-ROS1 mutant over-expression in Ba/F3 cells, suggesting that excessive ROS1 oncogenic signaling itself induced apoptosis instead of cell growth. Phosphoproteomic analysis and high-throughput inhibitor screening revealed that excessive ROS1 signaling in the TKI-addicted cells phosphorylated or activated apoptosis-related molecules such as FAF1 or p38. Collectively, our findings partly clarify molecular mechanisms of excessive ROS1 oncogenic signaling that mediates paradoxical induction of apoptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anilides / pharmacology
  • Antigens, Differentiation, B-Lymphocyte / genetics
  • Antigens, Differentiation, B-Lymphocyte / metabolism
  • Apoptosis / drug effects*
  • Cell Line, Tumor
  • Doxycycline / pharmacology
  • Drug Resistance, Neoplasm / genetics
  • Ethylnitrosourea / pharmacology
  • Histocompatibility Antigens Class II / genetics
  • Histocompatibility Antigens Class II / metabolism
  • Humans
  • Imidazoles / pharmacology
  • Mutagenesis, Site-Directed
  • Phosphopeptides / analysis
  • Protein Kinase Inhibitors / pharmacology*
  • Protein Structure, Tertiary
  • Protein-Tyrosine Kinases / chemistry
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Pyridines / pharmacology
  • Signal Transduction / drug effects
  • Tandem Mass Spectrometry
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • p38 Mitogen-Activated Protein Kinases / metabolism

Substances

  • Anilides
  • Antigens, Differentiation, B-Lymphocyte
  • Histocompatibility Antigens Class II
  • Imidazoles
  • Phosphopeptides
  • Protein Kinase Inhibitors
  • Proto-Oncogene Proteins
  • Pyridines
  • invariant chain
  • cabozantinib
  • Protein-Tyrosine Kinases
  • ROS1 protein, human
  • p38 Mitogen-Activated Protein Kinases
  • Doxycycline
  • Ethylnitrosourea
  • 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole