Abstract 3735: Dasatinib induces dephosphorylation of tyrosine proteins and apoptosis in both B-cell lymphoma lines and primary B-cell Non-Hodgkin Lymphoma cells in vitro for a subgroup of patients with phospho-Src (Y416) expression

Abstract

Abstract The Non-Hodgkin Lymphomas (NHLs) are a heterogeneous group of disease with differing clinical behavior and responses to treatment. Approximately 85% of NHLs belonging to the B-cell lineage. Src family kinases (SFKs) are the predominant tyrosine kinase expressed in B-cell lines and are important in regulating multiple signaling pathways. We attempted to correlate the efficacy of SFK inhibitors with the expression of p-SFKs in lymphoma cells, to examine p-Src (Y416) expression in NHL tissues, and study dasatinib-induced apoptosis in primary lymphoma cells. In screening for the effects of different kinases inhibitors on B-cell lymphoma lines, we observed that SFK inhibitors, PP2 and dasatinib, inhibited proliferation and induced apoptosis at 24 hrs (PP2: 31% at 10 μM; dasatinib: 39% at 100 nM) in Burkitt's lymphoma (BL) line Raji. Apoptosis was associated with cleavage of caspases-3 & 8. SFK inhibitor-induced apoptosis in Raji cells correlated with high level expression of p-Src. A similar effect was observed in a follicular lymphoma (FL) line DOHH2 despite expression of BCL2 in both. Raji and DOHH2 cells. In contrast, dasatinib showed no apoptotic induction in B-cell lymphoma lines Ramos (BL line) & Sud-HL4 [GC type diffuse large B-cell lymphoma (DLBCL) line] which have no detectable expression of p-Src. Dasatinib caused complete de-phosphorylation of tyrosine proteins (around 160 kDa, 110 kDa and 60 kDa) in Raji cells as early as 15 mins. We are currently trying to identify these proteins. IHC staining of B-cell NHLs by tissue microarray indicated 26/55 (47.3%) of NHL tissues are positive for p-Src, which including DLBCL, FL, BL, small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone lymphoma (MZL) and lymphoblastic lymphoma. Fresh lymphoma cells from 12 cases [DLBCL (3), FL (3), SLL (2), MZL (1), NK/T cell lymphoma (1) and two reactive cases] were studied for in vitro effects of dasatinib. 6 cases (3 FL, 2 DLBCL and one reactive case) had clear expression of p-Src and underwent dasatinib-induced apoptosis (100 nM, 24h) of B-cells gated by CD45+/CD19+ co-expression. De-phosphorylation of p-Src and cleavage of caspase-3 were observed in the two DLBCL cases. The other 6 cases had undetectable p-Src and showed no apoptotic induction upon expose to dasatinib. Purified B-cells from 3 healthy donors have no detectable p-Src and no dasatinib induced apoptosis. These data suggest that 1) rational application of molecularly targeted therapy for aggressive NHL may be possible by directly examining key signaling nodes promoting survival and proliferation. 2) dasatinib is a potential therapeutic candidate for a subgroup of aggressive NHL which express p-Src (Y416). 3) profiling patients’ lymphoma cells for phospho-SFK might be a strategy selection of such therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3735.