Molecular Changes Associated with Acquired Resistance to Crizotinib in ROS1-Rearranged Non–Small Cell Lung Cancer

Abstract

Purpose: Although ROS1-rearranged non-small cell lung cancer (NSCLC) is sensitive to crizotinib, development of resistance is inevitable. Here, we identified molecular alterations in crizotinibresistant tumors from two NSCLC patients with the CD74-ROS1 rearrangement, and in HCC78 cells harboring SLC34A2-ROS1 that showed resistance to crizotinib (HCC78CR cells).Experimental Design: ROS1 kinase domain mutations were examined in fresh tumor tissues from two NSCLC patients and HCC78CR1-3 cells by direct sequencing. Ba/F3 cells expressing ROS1 secondary mutations were constructed to evaluate resistance to crizotinib. An upregulated pathway was identified using phospho-receptor tyrosine kinase array, EGFR signaling antibody array, and RNA sequencing (RNA-seq). Cell proliferation and ROS1 downstream signaling pathways were compared between HCC78 and HCC78CR1-3 cells.Results: The ROS1 G2032R mutation was identified in crizotinib-resistant tumors from one patient. Furthermore, HCC78CR1and CR2 cells harbored a novel ROS1 L2155S mutation (73.3% and 76.2%, respectively). ROS1 G2032R and L2155S mutations conferred resistance to crizotinib in Ba/F3 cells. Evidence of epithelial-to-mesenchymal transition with downregulated E-cadherin and upregulated vimentin was observed in HCC78CR1-2 cells and in the other patient. RNA-seq and EGFR signaling antibody array revealed that the EGFR pathway was significantly upregulated in HCC78CR3 versus HCC78 cells. Cells with the ROS1 mutation and upregulated EGFR were sensitive to foretinib, an inhibitor of c-MET, VEGFR2, and ROS1 and irreversible EGFR tyrosine kinase inhibitors plus crizotinib, respectively.Conclusions: Molecular changes associated with acquired crizotinib resistance in ROS1-rearranged NSCLC are heterogeneous, including ROS1 tyrosine kinase mutations, EGFR activation, and epithelial-to-mesenchymal transition.

PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations

“…Recently, the solvent front ROS1 G2032R mutant has been identified in multiple lung cancer cases following crizotinib treatment (24,41,42). ROS1…”

“…A recent phase I/II clinical trial designed to evaluate the safety and efficacy of the ALK/mesenchymal-epithelial transition factor (MET)/ROS1 inhibitor crizotinib in patients with ROS1 fusion-positive lung cancer demonstrated promising results (21,22). However, consistent with the clinical experience across a number of molecularly targeted kinase inhibitors (23), a subset of patients with ROS1 fusion kinase-positive cancer treated with crizotinib acquired mutations within the ROS1 kinase domain that confer drug resistance (21,24). Therefore, there is a clear need for the development of new agents to overcome crizotinib resistance.…”

Molecular Pathology of Lung Cancer

“…ROS1 rearranjmanı EGFR, KRAS ve BRAF diğer onkojenik değişikliklerle bir arada görülebilir (24). ROS1 rearranjmanı gösteren hasta grubunda da krizotinibe direnç gelişebilir (25,26). Gelişen direncin altında yatan tüm mekanizmalar henüz ortaya konamamış olsa da, kazanılmış ikincil ROS1 mutasyonlarının bir kısım krizotinib direncinden sorumlu olduğu gösterilmiştir (26).…”

Emerging uses of biomarkers in lung cancer management: molecular mechanisms of resistance

“…There is limited data available on the mechanisms of acquired resistance to crizotinib in ROS1 positive NSCLC, however several secondary ROS1 mutations that can cause resistance have been identified, for example the G2026M mutation (gatekeeper) or the L2155S solvent-front (D2033N) mutation which can still respond to the multi-targeted TKI, cabozantinib which has ROS inhibitory activity as well (78)(79)(80).…”

“…Other mechanisms of resistance might include gain of function mutations of KIT (81), activation of the RAS pathway due to either KRAS/NRAS mutations or to KRAS amplification (82), rat sarcoma viral oncogene homolog, EGFR activation (83), and epithelial-to-mesenchymal transition (78,84).…”