SYK Inhibition Targets CD38−Mediated Survival and Proliferation Signals in Chronic Lymphocytic Leukemia Cells

Abstract 800

Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of mature monoclonal B cells in the blood, secondary lymphoid tissue, and marrow. The highly variable prognosis of the disease may be predicted using a number of biomarkers, including the expression level of CD38. Human CD38 is a surface glycoprotein with enzymatic activity and the ability to mediate cell-cell interactions by binding the non-substrate ligand CD31. CD31/CD38 interactions drive CLL proliferation and chemotaxis, and increase CXCL-12-mediated signals and homing of CLL cells towards lymphoid organs. We and others have previously identified SYK, a key component of the BCR signalling pathway, as a candidate for targeted therapy in CLL due to its enhanced expression and activity and the apoptotic effects of pharmacological SYK inhibition even in the context of the microenvironment. In this study, we demonstrate direct involvement of SYK in the CD38 signaling pathway and that SYK inhibition may prevent CD38−mediated CLL cell proliferation and migration.

CD38 stimulation of primary CLL cells by its ligand CD31 resulted in SYK activation as indicated by phospho-protein analysis by flow cytometry (n=18, p=0.0002) and immunoblotting. Analysis of CD38 expression on CLL cells by flow cytometry with SYK inhibition using the pharmacological SYK inhibitor R406 (Sellek) revealed a significant decrease of CD38 by 31% (n=25, p<0.0001). Particularly, CLL cells with high CD38 expression showed a substantial downregulation following SYK inhibition. Since CD38 expression is associated with poor clinical outcome, the proliferative signals delivered by its ligation might be critical for this association. Therefore, we tested the influence of SYK inhibition on proliferative signals as assessed by the expression of the proliferation marker Ki-67. 12 hours of stimulation with CD31 resulted in up-regulation of Ki-67 in CLL cells, an effect completely abrogated by concomitant treatment with the SYK inhibitor R406 (n=8). Since the expression of CD38 is regulated by different pathways including CD40 ligation and SYK involvement has been implicated in CD40 signaling, we evaluated whether R406 is capable to inhibit CD38 upregulation induced by CD40L. Indeed, in vitro stimulation with 3 μg/ml recombinant CD40L for 30 hours resulted in a significant increase of CD38 expression (p=0.0059) and was reversed by concomitant SYK inhibition (p=0.0142, n=9). As positive control, we treated CLL cells with ATRA that upregulated CD38 in myeloid cells. ATRA is directly translocated to the nucleus without requirement of signal transduction. Hence, SYK inhibition did not influence ATRA-mediated CD38 upregulation. In order to functionally characterize the role of SYK in the CD38/CD31 interaction in CLL cells, we analyzed the expression of the anti-apoptotic molecule MCL-1 in the presence and absence of SYK inhibition with and without CD38 stimulation. CD31 ligation enhanced MCL-1 expression, an effect reversed when CLL cells were coincubated with R406 (n=8).

Taken together, our data demonstrate direct involvement of SYK in the CD38 signaling pathway. Beyond the known effects of SYK inhibition on chemokine- and integrin mediated CLL-stroma interactions, these data establish interference with the CD31/CD38 axis as novel therapeutic mode of action of SYK inhibition in CLL. Since CD38 expression identifies CLL cells with high proliferative and migratory function and clinically associates with poor responsiveness to chemotherapy SYK inhibition may be a particularly promising therapeutic option in CLL patients expressing CD38.