Cell lines generated from a chronic lymphocytic leukemia mouse model exhibit constitutive Btk and Akt signaling
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Simar Pal Singh1,2,3, Saravanan Y. Pillai1, Marjolein J.W. de Bruijn1, Ralph Stadhouders1,4, Odilia B.J. Corneth1, Henk Jan van den Ham5, Alice Muggen2, Wilfred van IJcken6, Erik Slinger7, Annemieke Kuil8, Marcel Spaargaren8, Arnon P. Kater7, Anton W. Langerak2 and Rudi W. Hendriks1
1Department of Pulmonary Medicine, Erasmus MC, Rotterdam, The Netherlands
2Department of Immunology, Erasmus MC, Rotterdam, The Netherlands
3Post graduate school Molecular Medicine, Erasmus MC, Rotterdam, The Netherlands
4Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona Spain
5Department of Virosciences, Erasmus MC, Rotterdam, The Netherlands
6Center for Biomics, Erasmus MC, Rotterdam, The Netherlands
7Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands
8Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands
Rudi W. Hendriks, email: [email protected]
Keywords: B-cell receptor (BCR), bruton’s tyrosine kinase (Btk), chronic lymphocytic leukemia (CLL), ibrutinib, idelalisib
Received: March 02, 2017 Accepted: May 03, 2017 Published: May 26, 2017
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature CD5+ B cells in blood. Spontaneous apoptosis of CLL cells in vitro has hampered in-depth investigation of CLL pathogenesis. Here we describe the generation of three monoclonal mouse cell lines, EMC2, EMC4 and EMC6, from the IgH.TEμ CLL mouse model based on sporadic expression of SV40 large T antigen. The cell lines exhibit a stable CD5+CD43+IgM+CD19+ CLL phenotype in culture and can be adoptively transferred into Rag1–/– mice. RNA-seq analysis revealed only minor differences between the cell lines and their primary tumors and suggested that NF-κB and mTOR signaling pathways were involved in cell line outgrowth. In vitro survival and proliferation was dependent on constitutive phosphorylation of Bruton’s tyrosine kinase (Btk) at Y551/Y223, and Akt(S473). Treatment of the cell lines with small molecule inhibitors specific for Btk (ibrutinib) or PI3K (idelalisib), which is upstream of Akt, resulted in reduced viability, proliferation and fibronectin-dependent cell adhesion. Treatment of cell line-engrafted Rag1–/– mice with ibrutinib was associated with transient lymphocytosis, reduced splenomegaly and increased overall survival. Thus, by generating stable cell lines we established a novel platform for in vitro and in vivo investigation of CLL signal transduction and treatment modalities.
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