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

Hayato Ogura,Yuka Nagatake-Kobayashi,Naoya Fujita,Ryohei Katayama,Jun Adachi,Takeshi Tomonaga

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.

Highlights

ROS1 was initially identified as the homolog of the proto-oncogene v-ros, the transforming gene of the avian sarcoma virus UR21
The acquisition of tyrosine kinase inhibitor (TKI) resistance other than by secondary mutations has been reported in oncogene-driven lung cancers, such as crizotinib resistance mediated by amplification of the Anaplastic lymphoma kinase (ALK) fusion gene in NSCLC16, gefitinib resistance mediated by activation of a bypass pathway through MET amplification or activation in EGFR-positive NSCLC17, 18, or ceritinib resistance mediated by the over-expression of ABCB1 in ALK-rearranged NSCLC19
Since the cabozantinib-addicted mutant Ba/F3 cells died when TKI was removed, for F2004V- or F2075C-mutated cells, IC50 values were calculated by comparing the cell viability with 10 nM of cabozantinib; for wild-type CD74-ROS1 cells, the IC50 value was calculated without cabozantinib

Summary

Introduction

ROS1 was initially identified as the homolog of the proto-oncogene v-ros, the transforming gene of the avian sarcoma virus UR21. To elucidate mechanisms underlying acquired resistance to crizotinib in cancer cells harbouring the ROS1 fusion gene, we previously performed N-ethyl-N-nitrosourea (ENU) mutagenesis screening of Ba/F3 cells expressing CD74-ROS1 and identified multiple crizotinib resistant mutations including G2032R solvent front mutation and demonstrated that cabozantinib effectively inhibited the G2032R ROS1 mutation[20]. These clones were intermediately resistant to cabozantinib but, surprisingly, could not survive in the total absence of cabozantinib because of their own excessive ROS1 signaling They could grow only in the presence of low doses of ROS1-TKIs which controlled their ROS1 kinase activity to an appropriate level. By ENU mutagenesis screening, we identified cells that harbour CD74-ROS1 which were resistant to and addicted to ROS1-TKIs. We found that ROS1 signaling was excessively activated in these cells by removal of the ROS1-TKI, inducing apoptosis mainly in a caspase-8-dependent manner. Our findings may lead to elucidation of some as yet undefined aspects of drug-resistant cancer cells

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Results

Conclusion