Spleen Tyrosine Kinase Inhibition Prevents Chemokine- and Integrin-Mediated Stromal Protective Effects in Chronic Lymphocytic Leukemia.

Abstract

Abstract 2356Poster Board II-333B cell chronic lymphocytic leukemia (CLL) remains incurable despite recent therapeutic advances, and identification of novel molecular targets in CLL is highly desirable. The microenvironment provides essential growth and survival signals to CLL cells and confers resistance to cytotoxic agents, a phenomenon termed cell adhesion-mediated drug resistance (CAM-DR). The chemokine CXCL12 and the integrin ligand VCAM have been identified as important microenvironmental pathways that mediate CLL cell survival and CAM-DR. We have previously demonstrated that inhibition of spleen tyrosine kinase (SYK), a key component of the BCR signaling pathway, induces apoptosis in primary CLL cells when cultured alone or supported by stroma (Buchner et al., Cancer Res. 2009). In this study, we investigated SYK inhibition with regard to the interaction between CLL cells and the microenvironment.Stromal cell coculture resulted in SYK activation in primary CLL cells as indicated by phospho-protein analysis by flow cytometry (p<0.001) and immunoblotting. In vitro stimulation with 20 ng/ml recombinant CXCL12 or VLA-4 integrin ligation by contact to VCAM-1 precoated wells (10 μg/ml) for 15 sec had equivalent effects. SYK inhibition by the pharmacological SYK inhibitor R406 significantly reduced F-actin polymerization and Akt phosphorylation induced by CXCL12, integrin ligand, or stromal contact. In functional experiments, migration of CLL cells towards CXCL12 was reduced to 77% (p<0.001) by R406 in transwell culture. Likewise, CLL cell adhesion to VCAM was limited by SYK inhibition to 50% (p<0.01) compared to control cells in flow chamber experiments. In response to a number of physiological stimuli, including BCR ligation, integrins are activated through the modulation of their conformation to mediate high-affinity adhesion. Analysis of the expression of a high affinity epitope of CD29, the β1 chain of VLA-4, on CLL cells by flow cytometry with and without SYK inhibition revealed a significant decrease of 31% (p<0.0001), while the expression of total CD29 decreased only by 10% (p<0.01). To verify the specificity of the effects of pharmacological SYK inhibitors, we performed siRNA mediated silencing of SYK in primary CLL cells. 48 h after transfection, we observed a downregulation of SYK protein expression by flow cytometry by 27% (p<0.05), while the viability of the cells was not significantly affected for 72 h. Stimulation of control- and SYK-siRNA treated cells with VCAM significantly increased Akt phosphorylation in control but not in SYK-siRNA transfected cells. Furthermore, SYK-silenced CLL cells adhered significantly less to VCAM-coated surfaces than control-silenced CLL cells.Beyond inhibition of these defined pathways, R406 abrogated the protective effect of stromal coculture on CLL cell survival. The anti-apoptotic factor Mcl-1 was upregulated in CLL cells cultured with stroma compared to those cultured alone (mean MFI ± SEM: 144 ± 21 vs. 182 ± 23, p<0.001). SYK inhibition almost completely abolished the upregulation of Mcl-1 in stroma-cocultured CLL cells (mean MFI ± SEM: 182 ± 23 vs. 130 ± 15, p<0.001). Finally, SYK inhibition also prevented CAM-DR against fludarabine in stroma coculture.Taken together, our data demonstrate direct effects of SYK inhibition on chemokine and integrin-mediated CLL-stroma interactions and establish a novel therapeutic mechanism of action for SYK inhibitors in CLL. Indeed, a promising clinical activity in patients with relapsed/refractory CLL/SLL with an overall response rate of 54% has been reported for the oral prodrug of R406, fostamatinib disodium in CLL patients (Friedmann et al, ASH 2008). Of note, an unexplained transient but dramatic leukocyte flare during lymph node regression was reported in several of these patients. Our results offer a convincing explanation for this phenomenon and may even suggest that SYK blockade in CLL is a promising therapeutic principle not only due to inhibition of BCR signaling, but perhaps even more importantly by disrupting the contact of CLL cells with their protective microenvironment. Disclosures:No relevant conflicts of interest to declare.