Abstract LB-218: Molecular changes associated with acquired resistance to crizotinib in ROS1-rearranged non-small cell lung cancer (NSCLC)
Authors: Song Ahn ah 
Published: May 2015

Abstract
Background: Although ROS1-rearranged NSCLC is sensitive to crizotinib, a dual inhibitor of c-MET and ALK, an acquired resistance is inevitable. Recently, ROS1 G2032R mutation was identified in a patient with CD74-ROS1 rearranged NSCLC who developed an acquired resistance to crizotinib. Here, we identified novel molecular changes in crizotinib-resistant tumors from a NSCLC patient with CD74-ROS1 rearrangement and in HCC78 cells harboring SLC34A2-ROS1 fusion that showed crizotinib resistance (HCC78CR1, CR2 and CR3).
Methods: HCC78CR1, -2 and -3 cells were generated by increasing dose of crizotinib to 1 μM. ROS1 kinase domain mutations were examined in fresh tumor tissues from a patient and in HCC78CR1, -2 and -3 cells by direct sequencing of individual clones. Whole-transcriptome sequencing (RNA-seq) was performed to identify up-regulated pathway in HCC78CR3 compared with HCC78. Cell proliferation, phosphorylation of RTKs and ROS1 downstream signaling pathways were compared among HCC78 and HCC78CR1, -2, -3 cell lines. BaF3 cells that constantly express ROS1 variants were constructed to evaluate a crizotinib resistance.
Results: ROS1 G2032R in crizotinib-resistant tumor from a patient and L2155S from HCC78CR1, -2 cells were identified by direct sequencing of individual clones. ROS1 G2032R mutation was identified in 100% of cDNA clones of crizotinib-resistant tumors from a patient. However, this was not found in normal tissues and crizotinib-pretreatment tumor tissues. cDNA clones of HCC78CR1 and 2 cells harbored ROS1 L2155S mutations at a frequency of 73.3% and 76.2% respectively. Each BaF3 cells expressing ROS1 G2032R or L2155S mutation exhibited 128 and 10 fold higher IC50 value to crizotinib than those expressing wild-type CD74-ROS1, respectively. Downstream signaling molecules such as ERK and SHP2 were more activated in HCC78CR1, -2 and -3 cells than in HCC78 cells. In addition, HCC78CR1 and -2 cells displayed a spindle cell-like morphology with decreased E-cadherin and increased vimentin and fibronectin expression, which was not observed in HCC78 cells. RNA-seq and qRT-PCR demonstrated that EGFR mRNA expression levels were 2.6 and 5.1-fold up-regulated in HCC78CR3 cells than in HCC78 cells, respectively. EGFR signaling antibody array also revealed that EGFR pathway is significantly up-regulated in HCC78CR3 cells than in HCC78 cells.
Conclusions: ROS1 G2032R and L2155S mutations are associated with acquired resistance to crizotinib in ROS1-rearranged NSCLC. In addition, epithelial-to-mesenchymal transition-like changes and EGFR activation may confer an acquired resistance to crizotinib.
Citation Format: Ahn ah Song. Molecular changes associated with acquired resistance to crizotinib in ROS1-rearranged non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-218. doi:10.1158/1538-7445.AM2014-LB-218