Src family kinases (SFKs) were described to be overexpressed in chronic lymphocytic leukemia (CLL). We wished to examine the effects of the Src and Abl kinase inhibitor dasatinib on the intracellular signaling and survival of CLL cells. Dasatinib showed a dose-and time-dependent reduction of global tyrosine phosphorylation and of activating phosphotyrosine levels of SFKs. Treatment with 100 nM dasatinib led to decreased levels of the activated, phosphorylated forms of Akt, Erk1/2, and p38, and induced PARP cleavage through caspase activity. In Mec1 and JVM-3 cell lines, dasatinib increased p53 protein levels and inhibited proliferation.

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Resistance towards apoptotic stimuli mediated by overexpression of antiapoptotic factors or extracellular survival signals like B-cell receptor stimulation (BCR) are considered to be responsible for accumulation of malignant B cells in CLL . TOSO, also known as Fas-inhibitory molecule 3 (FAIM3), was identified as overexpressed candidate gene in CLL based on re-evaluation of publicly available microarray data sets. Based on primary CLL samples from 106 patients, TOSO expression was compared to healthy donor B cells using quantitative real-time PCR, western-blot, flow cytometry and immunohistochemistry. To reveal underlying mechanisms of TOSO overexpression, B-cell receptor (BCR) and CD40Ligand stimulation as well as bone marrow stroma cell co-incubation was performed. Apoptotic resistance was assessed by annexin V/7-AAD flow cytometry in context of CH11-Fas-agonistic antibody. TOSO was identified to exhibit elevated relative expression of 6.8 compared to healthy donor B cells using quantitative real-time PCR (p=0.004). High levels of TOSO expression in CLL correlated with high leukocyte count, advanced Binet stage, previous need for chemotherapy and unmutated IgVH status. CD38+ CLL subsets harboring proliferative activity showed significantly enhanced TOSO expression. Immunohistochemistry revealed upregulation of TOSO in lymph nodes of CLL patients. In lymph nodes derived from healthy donors TOSO was detected in single plasmocytoid cells within the germinal center and in the marginal zone. No specific staining was seen in follicular lymphomas. CLL-specific upregulation of TOSO was confirmed by RT-PCR, samples of follicular lymphomas, DLBCL, marginal zone lymphoma and Hodgkin cell lines did not reveal TOSO up-regulation. We evaluated functional mechanisms of aberrant TOSO expression in CLL cells and identified TOSO expression significantly being induced by BCR-stimulation compared to control cells (relative expression (RE) 8.25 vs. 4.86, p=0.013). In contrast, CD40L signaling significantly reduced TOSO expression (RE 2.60; p=0.007). Spontaneous apoptosis of CLL cells was significantly reduced by BCR-stimulus, in CD40Ligandstimulated CLL samples a slight sensitization towards Fas-mediated apoptosis was seen. In summary, we show that the anti-apoptotic factor TOSO is associated with progressive disease and enhanced in the proliferative CD38+ cell subset. Both association with unmutated IgVH and the specific induction of TOSO via the BCR suggest autoreactive BCR signaling as a key mediator of apoptosis resistance in CLL. Down-regulation of TOSO by CD40Ligand in the context of CD40Ligand-mediated Fas-sensitization of CLL reveal TOSO as a new anti-apoptotic factor in CLL. CLL-specific over-expression of the transmembrane protein might further offer new therapeutic strategies in CLL treatment.