Syk Kinase Inhibition by R406 Inhibits BCR Mediated B Cell Proliferation and Interferes with BCR Signal Transduction in Primary Tumor Samples.

Abstract

Background: B cells require B cell receptor (BCR) mediated survival signals during differentiation and throughout maturity. However, the CD79a/b signaling subunit of the BCR lacks independent kinase function and requires Syk kinase activity to activate the majority of downstream signaling effectors in the BCR signal transduction cascade. We have previously shown that inactivating Syk kinase with R406, a novel small molecule tyrosine kinase inhibitor, selectively kills B cells from primary human tumor samples and B cell lines (Sharman et. al ASCO 2007, Abstract # 3600). In addition, we have shown that Syk signaling is abnormally rapid, intense, and long-lived in primary human follicular lymphoma B cells (Irish et. al Blood 2006 108(9) 3135–3142). We hypothesized that R406 would prevent BCR mediated proliferation of normal B cells and interfere with BCR signaling in primary B lymphoma tumor samples.Methods: Normal peripheral blood B cells were evaluated with 3H-thymidine incorporation following BCR stimulation with anti-IgM and treatment with several concentrations of R406. Phosphospecific flow cytometry was used to evaluate signal transduction in single cell suspensions derived from primary lymphoma tumor samples and cell lines pretreated with R406.Results: Peripheral blood B cells proliferate in response to BCR stimulation. This proliferation is inhibited by R406 with IC50 of approximately 100 nM and IC100 at 1250 nM. Several concentrations of R406 were used to inhibit BCR signaling in primary tumor samples. At the lowest dose 50nM, there was little impact on downstream signaling in most samples. At the highest dose tested, 2500 nM, phosphorylation of SFK, Syk, Btk, c-Cbl, p38, Stat-3, Stat-5, Erk, and Akt was completely blocked in all patients. At this same dose, R406 did not inhibit Erk phosphorylation in response to PMA in cell lines.Conclusions: At pharmacologically relevant concentrations, R406 prevents peripheral blood B cell proliferation in response to BCR stimulation and completely blocks phosphorylation of all proteins evaluated following BCR stimulation in primary tumor samples. Signaling from PKC to Erk which classically involves Ras, Raf, and MEK is undisturbed at maximum drug concentrations. These findings are consistent with the hypothesis that Syk determines the phosphorylation status of most proteins within the BCR signaling cascade following BCR stimulation. Since B cells require BCR mediated survival signals, the anti-BCR signaling effect of R406 may be effective against B cell neoplasms. This idea is currently being tested in a phase I/II clinical trial using an orally bioavailable form of R406 in patients with relapsed and refractory B cell NHL.