Poster Session II: Lymphoma biology & translational research
Time- and tissue-specific expression of transgenes is essential for the accurate representation of human disease in in vivo models. The translocation t(2;5) is the hallmark of anaplastic large cell lymphoma (ALCL). It involves the anaplastic lymphoma kinase gene (ALK) and the nucleophosmin gene (NPM), which leads to the expression of the fusion neo-oncogene NPM-ALK (NA), acting as a constitutively active tyrosine kinase. Current approaches for the generation of mouse models of ALK+ ALCL are based on retroviral or transgenic overexpression of the cDNA of NA resulting in high NA expression levels and a quick polyclonal disease development, thus not accurately modeling a clonal disease. Current generation of conditional transgenic mouse models is relatively cumbersome and not amenable to high throughput analysis since they required de novo generation and characterization of genetically modified mice.
To address these limitations, we tried to develop a novel approach taking advantage of a translational stop cassette flanked by loxP recombination sites, which can be incorporated into viral expression constructs and enables controlled transgene activation in suitable Cre-transgenic mouse lines.
We developed a conditional retroviral expression approach, by which we incorporated the translational stop cassette flanked by loxP recombination sites into hematopoietic stem cells from transgenic mice expressing Cre recombinase from lineage-specific promoters. We applied this recombination-based conditional expression system to hematopoietic stem cells from Lck-Cre transgenic mice restricting NPM-ALK oncogene activity to the T-cell compartment. Diseased mice were characterized by FACS, IHC and microarray analyses.
By applying this approach to target expression of the NPM-ALK oncogene to T-cells in mice, we were able to generate a new model of ALK+ ALCL lymphoma. Flow cytometric analyses, IHC stainings and microarray data uniformly confirmed CD30 (Ki-1) expression and upregulation of markers of cytotoxic T-cell activation of our primary cells from NPM-ALK mice compared to other T-cell lymphoma cell lines.
The conditional retroviral expression approach described in this study enables rapid time- and tissue-controlled in vivo gene expression, refining and facilitating GEMM development. Using this sophisticated approach we restricted NA oncogene expression to T-cells through a floxed translational stop cassette, enabling the generation of a new murine model of a ALK+ ALCL lymphoma, which faithfully recapitulates multiple features of the human disease, including expression of the CD30 (Ki-1) marker.