The Generation of an EML4-ALK Fusion NSCLC Isogenic Cell Line Relevant for Drug Discovery and Development
2016 CRISPR Precision Gene Editing Congress
Boston, Massachusetts, United StatesFebruary 23, 2016
Recent studies show that tumor cells derived from a subset of patients with non-small cell lung cancer (NSCLC) harbor the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene, the result of a Paracentric chromosomal inversion on the short arm of chromosome 2. The EML4-ALK oncogene, like other ALK fusion oncogenes, is a druggable target that is responsive to ALK inhibitors. However, there is a lack of EML4-ALK in vitro models for drug screening. Here we set out to generate an isogenic EML4-ALK fusion non-small cell lung cancer model in the A549 lung cancer cell line, which harbors other naturally occurring genomic aberrations inherent in non-small cell lung cancer. This model could serve as a clinically relevant drug screening cell model. In this study, we utilized the CRISPR/Cas9 genome editing platform to target endogenous loci in human cells and create the intended genomic translocation event. By employing sgRNAs-Cas9 constructs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant genomic rearrangements that resulted in the expression of EML4–ALK fusions. Breakpoint junction analysis tested after sgRNA-CRISPR/Cas9 mediated genomic DNA cleavage in A549 cells showed the successful creation of the EML4-ALK fusion found in tumor cells from a subpopulation of NSCLC patients. Furthermore, single clonal isolation and functional screening demonstrated that the EML4-ALK isogenic cell line was sensitive to ALK inhibitors relative to the parental A549 cell line. This newly developed EML4-ALK isogenic lung cancer cell line could provide a very useful tool for oncology drug discovery and development.
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