Background: The multi-tyrosine kinase inhibitor (TKI) crizotinib elicits a dramatic response in patients with non-small cell lung cancer (NSCLC) harboring the c-ros oncogene 1 (ROS1) fusion gene (Shaw et al. NEJM 2014); however, these patients inevitably develop resistance to crizotinib within a year, which limits the efficacy of crizotinib. Although reported molecular changes include ROS1 tyrosine kinase mutations, epidermal growth factor receptor (EGFR) activation, and epithelial-to mesenchymal transition, the detailed mechanism of crizotinib resistance has not been elucidated.

Methods: To explore the molecular mechanisms of acquired crizotinib resistance in detail, we used a cell line model. HCC78 cells harboring the SLC34A2-ROS1 fusion gene, which are sensitive to crizotinib, were exposed continuously to increasing concentrations of crizotinib in a step-wise manner. A crizotinib-resistant cell line designated HCC78R was established and assessed by MTT assay, Western blotting, a receptor tyrosine kinase (RTK) array, quantitative PCR (RT-PCR), ELISA, and an RNA kinome targeted kinome panel (612 genes) with next-generation sequencing.

Results: Crizotinib-resistant HCC78R (50% inhibitory concentration [IC50] 4085.9 nmol/L) was 47-fold more resistant to crizotinib than was parental HCC78 (IC50 85.8 nmol/L) (p = 0.0145) according to MTT assay. The RTK array revealed that EGFR phosphorylation was upregulated in HCC78R cells. Western blotting confirmed the activation of EGFR and its downstream signaling pathways. RT-PCR screening of EGFR ligand family member expression showed increased heparin-binding EGF-like growth factor (HB-EGF) in HCC78R compared with HCC78 cells. Consistently, addition of the HB-EGF or conditioned medium from HCC78R cells rendered the HCC78 parental cells moderately resistant to crizotinib. EGFR overexpression or activating mutations were not detected. Furthermore, RNA kinome sequencing revealed 16-fold higher AXL mRNA expression and 1.68-fold lower ROS1 fusion gene expression in HCC78R compared with HCC78. Overexpression of the AXL protein and downregulation of the ROS1 protein were confirmed by Western blotting. Monotherapy with gefitinib (EGFR-TKI) or R428 (AXL inhibitor) or cabozantinib (AXL inhibitor) moderately inhibited the growth of HCC78R cells, while combined therapy suppressed proliferation more significantly compared with each monotherapy.

Conclusions: AXL and EGFR signaling mediate resistance to crizotinib, and combination treatment of an EGFR-TKI and AXL inhibitor may be an alternative strategy in ROS1 fusion gene-driven lung cancer cells.

Citation Format: Yuka Kato, Kadoaki Ohashi, Eiki Ichihara, Shuuta Tomida, Hiroe Kayatani, Kenichiro Kudo, Daisuke Minami, Takashi Ninomiya, Toshio Kubo, Toshiyuki Kozuki, Katsuyuki Hotta, Nagio Takigawa, Mitsune Tanimoto, Katsuyuki Kiura. AXL and EGFR signaling mediate resistance to Crizotinib in non-small cell lung cancer cells harboring the ROS1 fusion gene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1889.