Distinct Role of Src Family Kinase Inhibitors in Burkitt Lymphoma Cells Vs. Diffuse Large B-Cell Lymphoma Cells

Abstract Introduction: The Non-Hodgkin lymphomas (NHLs) are a heterogeneous group of malignancies, with approximately 85% of NHL belonging to the B-cell lineage. Src family kinases (SFKs) are non-receptor intracellular tyrosine kinases which are important in the regulation of multiple signaling pathways including cell proliferation, tumor invasiveness, angiogenesis and apoptosis. Syk is another predominant tyrosine kinase expressed in B-cell lines in addition to SFKs. We attempted to correlate SFK and Syk inhibitor efficacy with the presence of phospho-SFK or phospho-Syk in lymphoma cell lines and tissues. Methods: Cell proliferation was measured with WST-1 reagent. Apoptotic assay was performed with Annexin-V and 7-AAD by flow cytometry (FC, FACSCalibur, BD Bioscience). Phospho-Src (Y416) antibody (cell signaling Technology, CSL) was used for immunoblotting and immunohistochemistry. (IHC, Discovery, Ventana Medical Systems). Phospho-Syk (Y525/526) antibody (CSL) was used for FC and immunoblotting. Results: In a screening for the effects of different kinases' inhibitors on B-cell lymphoma lines, we observed that SFK inhibitors, PP2 and dasatinib (Sprycel, Bristol Myers Squibb), inhibited proliferation and caused dose-dependent apoptosis induction at 24 h (PP2: 31% at 10 mM; dasatinib: 39% at 100 nM) in Burkitt's lymphoma cell line Raji. The apoptotic induction was associated with cleavage of caspase-3 and caspase-8. The ability of SFK inhibitors to induce apoptosis in Raji cells paralleled high level expression of constitutive phospho- SFK (Y416). In contrast to this Burkitt's line, diffuse large B-cell lymphoma (DLBCL) lines (Sud-HL4, Sud-HL-6 and OCI-LY3, OCI-LY10) were less-sensitive to these SFK inhibitors but showed apoptosis induction upon exposure to the Syk inhibitor (piceatannol & syk inhibitor IV). Interestingly, the DLBCL lines that were resistant to SFK inhibitors had undectable or low levels of phospho-SFK (Y416); while their susceptibility to the Syk inhibitor-induced apoptosis paralleled detectable constitutive phospho-Syk (Y525/526). Immunohistochemical staining of burkitt's lymphoma tissues and a tissue microarray panel of NHL indicated 13/20 (65%) of Burkitt's lymphoma, 3/5 of small lymphocytic lymphoma, 2/5 of mantle cell lymphoma, 3/10 of follicular lymphoma, 2/5 of DLBCL, 2/5 of marginal zone lymphoma, 1/5 of lymphoblastic lymphoma are positive for phospho-Src (Y416). Staining of normal tonsil tissue showed germinal center cells are strong positive for phospho-Src (Y416), while marginal zone cells are weak positive and plasma cells are negative. We are currently testing the correlation of phospho-Src (Y416) expression in fresh NHL tissues and their sensitivity to Src family kinase inhibitors. Conclusion: These data suggest that rational application of molecularly targeted therapy for aggressive NHL is possible by directly examining key signaling nodes promoting survival and proliferation. For instance, the clinical SFK inhibitor dasatinib is currently being examined in a clinical trial for NHL (NCT00550615). Our results suggest that profiling patients' lymphoma cells for phospho-SFK could optimize therapeutic efficacy and minimize unnecessary treatment-related side effects.