The ROS1 tyrosine kinase inhibitor (TKI) crizotinib has shown dramatic effects 
in patients with non-small cell lung cancer (NSCLC) harboring ROS1 fusion genes. 
However, patients inevitably develop resistance to this agent. Therefore, a new 
treatment strategy is required for lung tumors with ROS1 fusion genes. In the 
present study, lung cancer cell lines, HCC78 harboring SLC34A2-ROS1 and ABC-20 
harboring CD74-ROS1, were used as cell line-based resistance models. 
Crizotinib-resistant HCC78R cells were established from HCC78. We 
comprehensively screened the resistant cells using a phosphor-receptor tyrosine 
kinase array and RNA sequence analysis by next-generation sequencing. HCC78R 
cells showed upregulation of HB-EGF and activation of epidermal growth factor 
receptor (EGFR) phosphorylation and the EGFR signaling pathway. Recombinant 
HB-EGF or EGF rendered HCC78 cells or ABC-20 cells resistant to crizotinib. RNA 
sequence analysis by next-generation sequencing revealed the upregulation of AXL 
in HCC78R cells. HCC78R cells showed marked sensitivity to EGFR-TKI or anti-EGFR 
antibody treatment in vitro. Combinations of an AXL inhibitor, cabozantinib or 
gilteritinib, and an EGFR-TKI were more effective against HCC78R cells than 
monotherapy with an EGFR-TKI or AXL inhibitor. The combination of cabozantinib 
and gefitinib effectively inhibited the growth of HCC78R tumors in an in vivo 
xenograft model of NOG mice. The results of this study indicated that 
HB-EGF/EGFR and AXL play roles in crizotinib resistance in lung cancers 
harboring ROS1 fusions. The combination of cabozantinib and EGFR-TKI may 
represent a useful alternative treatment strategy for patients with advanced 
NSCLC harboring ROS1 fusion genes.