Generation of NPM1-ALK translocation in human T lymphocytes faithfully recapitulates the diversity of ALK+ ALCL tumor phenotype

To recapitulate the t(2;5)(p23;q35) chromosomal translocation, activated T lymphocytes from healthy donors PBMC were transfected with the RNP complex composed of the protein Cas9 with gRNA NPM1 (targeting NPM1 gene) and gRNA ALK (targeting ALK gene). Control cells were wild-type CD4+ activated lymphocytes. Cells were grown for 10-15 days in vitro prior to engraftment in vivo. Mice engrafted with NPM1-ALK+ T-cells developed T-cell lymphoma after 2-5 months. To get insights into the molecular bases of NPM1-ALK transformation, we next established the transcriptomes at an early in vitro stage and in fully transformed in vivo models. For this, we performed RNAseq analysis from 3 groups: 1-in vitro wild-type CD4+ activated lymphocytes [n=3], 2-in vitro NA-engineered CD4+ lymphocytes [n=3] and 3-in vivo derived CD4+ lymphoma cells purified by flow cytometry to distinguish CD3+ [n=3] and CD3- populations [n=3].