A novel patient-derived cell line originated at the time of crizotinib resistance displays a mesenchymal phenotype

Abstract

Lung cancers that harbor genomic ALK alterations are clinically responsive to pharmacologic ALK inhibition. Crizotinib, an orally available small-molecule inhibitor of the ALK tyrosine kinase, was approved for the treatment of ALK+ lung cancer patients. Unfortunately, as seen with other tyrosine kinases inhibitors (TKIs) in clinical use, most patients whose disease initially responds to crizotinib eventually develop progressive disease. To elucidate mechanisms of acquired resistance to ALK TKIs, we established and characterized novel ALK+ cell lines from patients with acquired resistance to ALK TKI therapy. In particular, we developed a cell line (designated STM) from a patient with an EML4-ALK (E6;A20, variant 3) fusion that developed acquired resistance to crizotinib. Fluorescence in situ hybridization confirmed that the cell line retained the ALK rearrangement. STM cells had decreased sensitivity to crizotinib and second-generation ALK inhibitors, including ceritinib, alectinib, and X-396. Morphologic changes were observed in this patient-derived cell line, the tumor cells had a spindle form, characteristic of the epithelial-mesenchymal transition (EMT). Loss of expression of the epithelial marker, E-cadherin, was accompanied by strong expression of the mesenchymal markers, vimentin and N-cadherin in STM cells. Enhanced invasion and migration capabilities were observed in STM cells consistent with a mesenchymal phenotype. Src has been shown to play a role in E-cadherin regulation and EMT. Thus, treatment of cells with dasatinib, a Src inhibitor, suppressed cell growth in STM cells, but not in ALK+/TKI sensitive cell lines. The addition of a low dose of dasatinib sensitized STM cells to the antiproliferative effects of crizotinib. STM cells were subjected to genetic analysis to identify new genetic anomalies that could be driving resistance. Top hits are being evaluated. To our knowledge, this is the first report that demonstrates EMT occurring in an ALK+ crizotinib resistant clinical sample. Collectively these data support EMT as a mechanism of resistance to crizotinib and identifies dasatinib as a potential therapeutic for treatment of crizotinib resistance associated with EMT.