Spleen Tyrosine Kinase (SYK) Is Overexpressed and Represents a Potential Therapeutic Target in Chronic Lymphocytic Leukemia

B cell chronic lymphocytic leukemia (CLL), the most prevalent B cell malignancy in adults, is characterized by expansion of monoclonal mature B lymphocytes. Despite advances in treatment, the disease remains incurable warranting further efforts to identify novel molecular targets in CLL. B cell receptor (BCR) signaling contributes to apoptosis resistance in CLL limiting the efficacy of therapeutic approaches. In this study we investigated the expression of spleen tyrosine kinase (SYK), a key component of the BCR signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared to healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCγ₂, STAT3, and ERK1/2 in CLL compared to healthy B cells suggesting enhanced activation of these mediators in CLL. Separate analyses according to prognostic parameters revealed 1.8-fold higher SYK protein level in unmutated compared to mutated CLL cells determined by densitometric analysis (n=22, p=0.0031). These findings may well explain the higher BCR signaling capacity in the unmutated CLL subset. Various SYK inhibitors (piceatannol, curcumin, SYK Inhibitor II, and IV (Calbiochem)) reduced phosphorylation of the SYK downstream targets PLCγ₂, STAT3, and ERK1/2 in a time- and dose-dependent manner and induced apoptosis in the CLL cell lines Mec-1 and EHEB and primary CLL cells. SYK Inhibitor II revealed highest cytotoxic effects on primary CLL cells, but did not significantly reduce the viability of healthy B cells. Thus, apoptotic effects of this inhibitor were analyzed in a larger cohort of patient samples along with the well-established SYK inhibitor R406 (Rigel Inc.). After 48 hour treatment, relative viability of CLL cells was reduced to 76% and 44% for SYK Inhibitor II (4 mM and 10 mM) and to 66% for R406 (4 mM), respectively (n=38, p<0.0001). With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70⁺ cases. Cytotoxicity of SYK inhibitors were associated with SYK protein expression potentially predicting response to therapy (Pearson correlation coefficient: r=0.78 for SYK Inhibitor II (p<0.0001) and r=0.56 for R406 (p=0.0134)). Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared to fludarabine therapy alone. The mean viability of F-ara-A treated cells (10 mM) was reduced by 13% with SYK II and by 17% with R406 (n=10, p<0.0001), respectively. Since microenvironment enhances the viability of CLL cells and decreases their sensitivity towards chemotherapy, apoptosis rates of CLL cells incubated with SYK Inhibitor II in the presence and absence of the stromal cell line M2-10B4 were assayed. No significant change in cytotoxic effects was observed. Hence, stromal cell interactions do not impair the cytotoxicity of SYK inhibitors. Moreover, CD40L expressing T cells play an important role in the CLL microenvironment, and CD40 ligation is under discussion to induce fludarabine resistance in CLL. Therefore the effect of SYK Inhibitor II in combination with CD40 ligation was analyzed. In contrast to conventional chemotherapy, stimulation with CD40L significantly sensitized CLL cells towards SYK inhibition. In conclusion, this work establishes SYK inhibition as a rational and promising therapeutic principle in CLL. Given the preferential activity of SYK inhibitors in CLL cases with poor prognostic factors, the independence of their activity from the protective influence of the CLL microenvironment, and the synergistic and complementary action with fludarabine, we propose SYK inhibitors for clinical assessment in the therapy of CLL.