Dasatinib Induces Apoptosis in Chronic Lymphocytic Leukemia and Enhances the Activity of Rituximab and Fludarabine.

Abstract Chronic lymphocytic leukemia (CLL) is a disease characterized by the monoclonal accumulation of well-differentiated CD5 B cells. Previously, we reported that CLL cells that use unmutated immunoglobulin VH (IgVH) genes and/or express ZAP-70 are more responsive to signaling induced by ligation of surface IgM compared to cells that use mutated IgVH and lack expression of ZAP-70 (Chen et al. Blood 2005) Because signaling through the Ig receptor appears to play a role in the pathogenesis or progression of CLL, targeting the Ig signal transduction pathway in CLL might have therapeutic utility, particularly for those patients with high-risk disease. Dasatinib (Sprycel) is a tyrosine kinase inhibitor (TKI) with antiproliferative activity against hematological and solid tumor cell lines and it is FDA approved for the treatment of patients with CML and Ph+ ALL. Contrary to Imatinib (Gleevec), Dasatinib is a potent TKI not only of the Abl family of kinases but also of Src kinases, which regulate Ig-receptor signal transduction and govern the early events following Ig receptor ligation. Because Dasatinib TKI profile and its potential role inhibiting BCR signaling we studied its in vitro activity in CLL. Primary leukemia cells from 40 different CLL patients were evaluated. We found that Dasatinib, but not Imatinib, induced apoptosis in CLL cells at doses that were pharmacologically achievable. The IC50 for Dasatinib was in the 30–100 nM range. Interestingly, the pro-apoptotic activity of Dasatinib in CLL cells was not observed in normal B, T cells or blood mononuclear cells of healthy donors, suggesting that Dasatinib has a specific effect on CLL cells. Dasatinib induced apoptosis in CLL cells in a time and concentration dependent manner. Peak apoptosis occurred after 2 hours of in vitro exposure. Leukemia cells that expressed ZAP-70 were significantly more sensitive to Dasatinib-induced apoptosis than CLL cells lacking expression of ZAP-70. In addition, Dasatinib enhanced in vitro the pro-apoptotic activity of Rituximab and Fludarabine in CLL cells. Dasatinib treatment of CLL cells induced changes in the expression profile of apoptosis related genes as well as changes in apoptosis related proteins such as cleavage of PARP-1 and Caspases. Moreover, CLL cells treated in vitro with Dasatinib showed reduced tyrosine kinase activity measured by ELISA and also by immunoblots of CLL-cell lysates using specific phospho-TK antibodies. In addition, Ig receptor signaling following surface IgM ligation was decreased in CLL cells that were pre-treated with Dasatinib relative to that of untreated CLL cells. In conclusion, Dasatinib induces apoptosis of CLL cells at low nanomolar concentrations that do not appear to affect the viability of B cells, T cells, or blood mononuclear cells of healthy adults. CLL cells that expressed ZAP-70 were significantly more sensitive to Dasatinib than CLL cells that lacked expression of ZAP-70. This process was associated with impairment of BCR signaling, decrease TK activity and regulation of genes and proteins related to apoptosis. In addition, treatment of CLL cells with Dasatinib enhanced the in vitro activity of Rituximab and Fludarabine. These results reveal that Dasatinib is potentially active in CLL, providing a rationale for clinical trials evaluating its clinical activity in the treatment of patients with this disease.