Cell lines generated from a chronic lymphocytic leukemia mouse model exhibit constitutive Btk and Akt signaling

Abstract

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.

Identification of Distinct Unmutated Chronic Lymphocytic Leukemia Subsets in Mice Based on Their T Cell Dependency

“…Notably, many gene sets or pathways were active in both CLL subsets, including high expression levels of MET receptor tyrosine kinase, which prolongs CLL cell survival through STAT3 and AKT phosphorylation ( 40 , 59 ). This could contribute to the enhanced constitutive activation of the p-Akt/p-S6 pathway in IgH.TE μ CLL as reported previously ( 23 , 24 ). Additionally, genes involved in KRAS signaling were highly expressed in both CLL subsets, consistent with its essential role in B cell lymphopoesis ( 60 ), particularly for B-1 cells recognizing PtC ( 61 ).…”

“…Splenic single-cell suspensions were prepared in magnetic-activated cell sorting (MACS) buffer (PBS/2mM EDTA/0.5%BSA) and naïve splenic B cells from 8–12 week-old WT C57BL/6 mice were purified by MACS, as previously described ( 24 , 29 ). Non-B cells, B-1 cells, GC B cells, and plasma cells were first labeled with biotinylated antibodies (BD Biosciences) to CD5 (53–7.3), CD11b (M1-70), CD43 (S7), CD95 (Jo2), CD138 (281-2), Gr-1 (RB6-8C5), and TER-119 (PK136) and subsequently with streptavidin-conjugated magnetic beads (Miltenyi Biotec).…”

“…Btk-mediated signaling enhanced leukemogenesis and Btk-deficiency led to a complete rescue from the disease ( 23 ). Moreover, primary CLL cells from IgH.TE μ mice or stable cell lines generated from these mice had detectable expression of p-Akt and substantial levels of p-S6, both of which function downstream of the BCR ( 23 , 24 ).…”

Role of Bruton’s tyrosine kinase in B cells and malignancies

“…In line with chronic BCR-mediated signaling, CLL cells show constitutive activation of various BCR pathway associated kinases. Hereby, BTK is essential for constitutively active pathways implicated in CLL cell survival, including AKT, ERK and NF-кB, both in patient cells and mouse models [ 133 , 141 – 143 ]. CLL cells are thought to interact with the tissue microenvironment and lymph node resident CLL cells show gene expression signatures indicative of BCR activation [ 144 , 145 ].…”

Secondary Resistant Mutations to Small Molecule Inhibitors in Cancer Cells

“…XLA patients have a deficiency in B-cell maturation resulting in very low plasma B-cell counts [299]. Constitutive BTK activation is essential for proliferation of abnormal B-cells in several types of blood malignancies including chronic lymphocytic leukemia (CLL) [300,301], mantle cell lymphoma [302,303], and lymphoplasmacytic lymphoma [304]. Remarkably, activating oncogenic BTK mutations have not been clinically identified.…”