Constitutive Activation of the Protein Tyrosine Kinase Syk in Chronic Lymphocytic Leukemia B-Cells.

The protein tyrosine kinase Syk is a key mediator of proximal B-cell receptor (BCR) signaling. Following antigen stimulation Syk is recruited to the BCR and becomes activated by sequential phosphorylation at conserved tyrosine (Tyr) residues. The first event involves phosphorylation at Tyr352 by Lyn or other Src family kinases, followed by autophosphorylation of Tyr525/Tyr526 in the activation loop. Once activated, Syk further propagates the BCR signal by associating with adaptor proteins and phosphorylating downstream signaling molecules. Recently, translocations involving Syk have been identified in MDS and T-cell lymphoma, indicating that Syk may also function as a protooncogene. In line with this possibility, expression of a constitutively active TEL-Syk fusion protein was shown to result in growth factor-independent proliferation and transformation of mouse B-cells. These findings prompted us to investigated the activation status of Syk in primary unstimulated CLL B-cells. Western blot analysis with a phospho-specific antibody revealed substantial levels of Syk phosphorylated at Tyr352 in 29 of 54 freshly isolated CLL B-cell samples. Constitutive phosphorylation of Syk at Tyr352 was confirmed by immunofluorescence and confocal microscopy, which showed punctuate staining distributed across the plasma membrane and cytoplasm of unstimulated CLL B-cells. In contrast, control experiments with BJAB lymphoma B-cells showed phosphorylation of Syk at Tyr352 only following BCR crosslinking. To investigate which downstream signaling pathways are affected by Syk activation, we produced a constitutively active Syk mutant in which Tyr352 was substituted with aspartic acid. Transfection of Syk^Tyr352Asp in HEK293, Jurkat and BJAB cells resulted in tyrosine phosphorylation of cellular proteins and autophosphorylation of Syk at Tyr525/Tyr526, whereas no changes were observed following transfection with wild type Syk. In addition, transfection of the Syk^Tyr352Asp mutant in the human B-CLL cell line MEC1 resulted in increased phosphorylation of ERK, Akt and GSK3, indicating that these important cellular regulatory pathways are targeted by constitutively active Syk in CLL B-cells. To determine the effect of Syk activation on CLL cell survival, we cultured leukemic B-cells in the presence of R406, a recently developed and specific Syk kinase inhibitor (kindly provided by Rigel Pharmaceutics, Inc.). Assessment of CLL cell viability after 48 hours in culture showed moderate induction of apoptosis at concentrations above 600 nM in 12 of 18 investigated cases, with a maximal cytotoxic effect at 2.5 μM (20–50% apoptotic cells after normalization for spontaneous apoptosis). Interestingly, R406 at even lower concentrations (0.16 to 0.62 μM) inhibited the proliferation of CLL B-cells that was induced by stimulation with unmethylated CpG oligonucleotides, indicating that Syk kinase activity is required for leukemic cell proliferation in this setting. In summary, these data show that Syk is frequently activated in CLL B-cells, even in the absence of BCR engagement. Expression of constitutively active Syk results in activation of pathways that regulate cellular proliferation and survival, whereas inhibition of Syk kinase activity with R406 induces apoptosis and blocks CLL cell proliferation in an in vitro model. Together, these findings suggest that Syk may be a potential candidate for targeted therapy of CLL.