PB2062 ESTABLISHMENT OF A KNOCKDOWN MODEL FOR UNRAVELLING THE PATHOGENESIS OF MANTLE CELL LYMPHOMA

Abstract

Background:Mantle cell lymphoma (MCL) is an aggressive subtype of B cell non‐Hodgkin's lymphoma. It is a very heterogeneous disease with respect to clinical presentation and prognosis and relapses often occur with disseminated lymphoma, which are even more difficult to treat. Aberrations that drive transformation of the cell towards malignancy involve genomic changes and aberrantly expressed genes affecting the gene expression profile. All of these molecular aberrations have potential prognostic and therapeutic value.Recently, we conducted a study where we sequenced the transcriptome of four MCL patient both at diagnosis and relapse (Hansen et al., Molecular characterization of sorted malignant B cells from patients clinically identified with mantle cell lymphoma, Under review in Experimental Hematology). This study identified several genes aberrantly expressed in MCL. The aberrant expression of selected genes was validated in a larger cohort of MCL patients by qPCR. From these data two genes, Protein tyrosine phosphatase type J (PTPRJ) and leukocyte immunoglobulin like receptor A4 (LILRA4), which are, so far, uncharacterized in MCL were selected for further analyses.Aims:The aim of this study was to establish a knockdown (KD) model in lymphoma cells that can be used to investigate the function of genes, which we have identified to be aberrantly expressed in MCL patients such as PTPRJ and LILRA4.MethodsThe MCL cell lines Mino, JEKO‐1 and Granta‐519 were transfected with siRNAs and KD efficiency was evaluated by qPCR. 3 different transfection platforms were tested: Accel siRNA delivery medium (Dharmacon), Lipofectamine2000 (Invitrogen) and Electroporation with Amaxa Nucleofector 2b and Nucleofector solution V (Lonza). FOr the Accell platform a pool of 4 siRNAs targeting LILRA4 was used. For the others pre‐designed Silencer Select siRNAs were used, with 2 siRNAs targeting both PTPRJ and LILRA4. For all platforms a positive control GAPDH siRNA was included and different transfection conditions tested.Results:All cell lines were successfully modified using the Accell platform, with the highest KD efficiency in Mino for both GAPDH and LILRA4, which were downregulated by 99% and 69%, respectively. Transfection of Mino using electroporation resulted in downregulation of GAPDH by 74% and PTPRJ by 67%, while the downregulation of LILRA4 was less definite. When Lipofectamine2000 was used, expression was only marginally downregulated (24%). Consequently, an experiment was made in a cell line derived from human mesenchymal stem cells (Tert4), which has been thoroughly used for for KDs using Lipofectamine2000. In these cells, GAPDH and PTPRJ expression was downregulated by 90‐99%, validating the efficiency of the siRNAs. This also indicates that Lipofectamine2000 is not suitable for transfection of the tested MCL cell lines.Summary/Conclusion:Mino was the most successfully modified cell line tested using the Accell and Nucleofector transfection platforms, while less convincing results were obtained in Granta‐519 and JEKO‐1 cells. Future studies are planned using the MCL knockdown model for functional assays, both in cell lines and primary patient cells, investigating cancer‐related phenotypes such as proliferation and survival.Establishment of a knockdown model in MCL derived cell lines will enable experiments that can add valuable information about the altered signaling pathways involved in the pathogenesis of MCL, and may ultimately lead to identification of novel anti‐cancer targets or biomarkers.