Abstract
BACKGROUND/OBJECTIVES: ALK+ Anaplastic Large Cell Lymphoma (ALCL) is an aggressive T-cell lymphoma that is characterized by expression of the Anaplastic Lymphoma Kinase (ALK), which is induced by the t(2;5) chromosomal rearrangement, leading to the expression of the NPM-ALK fusion oncogene. Most previous preclinical models of ALK+ ALCL were based on overexpression of the NPM-ALK cDNA from heterologous promoters. Due to the enforced expression, this approach is prone to artifacts arising from synthetic overexpression, promoter competition and insertional variation. METHODS: To improve the existing ALCL models and more closely recapitulate the oncogenic events in ALK+ ALCL, we employed CRISPR/Cas-based chromosomal engineering to selectively introduce translocations between the Npm1 and Alk gene loci in murine cells. RESULTS: By inducing precise DNA cleavage at the syntenic loci on chromosome 11 and 17 in a murine IL-3-dependent Ba/F3 reporter cell line, we generated de novo Npm-Alk translocations in vivo, leading to IL-3-independent cell growth. To verify efficient recombination, we analyzed the expression of the NPM-ALK fusion protein in the recombined cells and could also show the t(11;17) in the IL-3 independent Ba/F3 cells. Subsequent functional testing of these cells using an Alk-inhibitor showed exquisite responsiveness towards Crizotinib, demonstrating strong dependence on the newly generated ALK fusion oncoprotein. Furthermore, a comparison of the gene expression pattern between Ba/F3 cells overexpressing the Npm-Alk cDNA with Ba/F3 cells transformed by CRISPR-mediated Npm-Alk translocation indicated that, while broadly overlapping, a set of pathways including the unfolded protein response pathway was increased in the Npm-Alk overexpression model, suggesting increased reactive changes induced by exogenous overexpression of Npm-Alk. Furthermore, we observed clustered expression changes in genes located in chromosomal regions close to the breakpoint in the new CRISPR-based model, indicating positional effects on gene expression mediated by the translocation event, which are not part of the older models. CONCLUSIONS: Thus, CRISPR-mediated recombination provides a novel and more faithful approach to model oncogenic translocations, which may lead to an improved understanding of the molecular pathogenesis of ALCL and enable more accurate therapeutic models of malignancies driven by oncogenic fusion proteins.