Spleen Tyrosine Kinase Is Involved in the CD38 Signal Transduction Pathway in Chronic Lymphocytic Leukemia

Abstract

The survival and proliferation of CLL cells depends on microenvironmental contacts in lymphoid organs. CD38 is a cell surface receptor that plays an important role in survival and proliferation signaling in CLL. In this study we demonstrate SYK's direct involvement in the CD38 signaling pathway in primary CLL samples. CD38 stimulation of CLL cells revealed SYK activation. SYK downstream target AKT was subsequently induced and MCL-1 expression was increased. Concomitant inhibition of SYK by the SYK inhibitor R406 resulted in reduced activation of AKT and prevented upregulation of MCL-1. Moreover, short-term CD38 stimulation enhanced BCR-signaling, as indicated by increased ERK phosphorylation. CXCL12-dependent migration was increased after CD38 stimulation. Treating CLL cells with R406 inhibited CD38-mediated migration. In addition, we observed marked downregulation of CD38 expression for CLL cells treated with R406 compared to vehicle control. Finally, we observed a clear correlation between CD38 expression on CLL cells and SYK-inhibitor efficacy. In conclusion, our study provides deeper mechanistic insight into the effect of SYK inhibition in CLL.

Fig 1. SYK is rapidly induced upon CD38 stimulation.

CD38 stimulation of primary CLL cells was performed using recombinant human CD31. A) Initial activation of SYK by phosphorylation of tyrosine residue 352 was detected after exposure of CLL cells to CD31 for 15 sec (n = 6, p<0.01) (left). Middle: Before and After Plot of 6 CLL patients. Right: A representative example of n = 6 independent experiments is shown. B) Trans-autophosphorylation of SYK upon CD38 activation was analyzed by western blot using a SYK Y(525/526) specific antibody. Left: densitometric analysis of pSYK western blots after CD31 ligation for 1 minute, 10, or 15 minutes (n = 8, p<0.01). Error bars indicate SEM. Right: A representative example of n = 8 independent experiments is shown.

Fig 2. SYK mediates CD38 induced AKT activation and MCL-1 expression.

A) Western blot analysis of pAKT (Ser473) induction after incubation of CLL cells with IB4 for 15 min with or without concomitant R406 treatment (n = 8, p<0.05). B) Western Blot analysis of MCL-1 expression after 24 hs of CD31 ligation with or without concomitant SYK inhibition (n = 12, p<0.05 and p<0.01). Error bars indicate SEM.

Fig 3. CD38 ligation enhances the migratory potential of CLL cells and is SYK dependent.

A) CLL cells were treated with 4 μM R406 or DMSO for 30 min and subsequently stimulated with IB4 for 30 min. Afterwards CLL cells were exposed to a CXCL12 gradient in a transwell chamber (n = 10, p <0.05 and p<0.01). B) Densitometric analysis of western blots for ERK phosphorylation in response to anti IgM stimulation: Before exposure to 5 μg/ml anti IgM for 1 min, CLL cells were treated with 4 μM R406 or vehicle control and prestimulated with CD31 for 15 min (n = 6, p<0.01 and p<0.05). A representative example is shown below.

Fig 4. CD38 surface expression is dependent on SYK.

Surface expression on CLL cells was analyzed by flow cytometry. A) Analysis of CD38 expression on CLL cells after 24 hs SYK inhibitor treatment (4μM R406 and 2 μM P505-15, respectively) (n = 8, p<0.001). B) Analysis of CD5 surface expression on SYK inhibitor treated CLL cells (n = 8, p = 0.31 and p = 0.42). C) Analysis of CD38 expression after stimulation with CD40L for 24 hs (n = 9, p<0.01 and p<0.05). Error bars indicate SEM.

Fig 5. SYK inhibition reduces CD38 surface expression by transcriptional regulation.

Primary CLL cells were treated with 4 μM R406 or 2 μM P505-15 for 24 hs. Expression of CD38, NF-kB and E2A mRNA was analyzed by Q-RT-PCR using ERCC6 as a reference. Error bars indicate SEM.

Fig 6. The effect of SYK inhibition on cell viability positively correlates with CD38 expression.

A) Primary CLL cells were tested for CD38 surface expression by flow cytometry. Cells were subsequently treated with 4 μM R406 for 24 hs. Induction of apoptosis was analyzed by Annexin V/PI staining. Correlating the rate of apoptosis with CD38 surface expression was analyzed by Spearman rank test (n = 17, p<0.01, Spearman: r = 0.73). B) Primary CLL cells were labeled with APC-labelled anti-CD38 antibody and sorted into CLL cells with CD38^high and CD38^low expression. Cells were subsequently treated with 4 μM R406 for 24 hs. Apoptosis induction was analyzed by flow cytometry using Annexin V/PI staining (n = 10, p<0.05). Error bars indicate SEM.