Inhibition of Tak-1 by AZ-Tak1 Impairs NF-κB Activation, Downregulates XIAP and Activates Caspase-9 Inducing Apoptosis In Mantle Cell lymphoma

Abstract 2852

TGF-b-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, plays a key role in regulating inflammation, immunity, metabolism, and cell death in a variety of cell types. It is activated in response to a variety of cytokines, including tumor necrosis factor (TNF), TGF-b, and interleukin 1 (IL-1). Upon receptor binding, TAK1 binds to adaptor proteins, and subsequently phosphorylate downstream molecules leading to activation of p38MAPK, JNK, and NF-kB. In this study, we examined the expression pattern of TAK1 and its potential therapeutic role as a target for lymphoma. First, we examined TAK1 expression in a panel of lymphoid cell lines by western blot, and found it to be highly expressed in mantle cell lymphoma cell lines (Mino, SP53, and Jeko-1). In contrast, PBL from healthy donors had no expression of TAK1 protein. TAK1 was also highly expressed in primary lymph node sections of MCL compared with benign reactive lymph nodes. Subsequently, we investigated the in vitro activity of the novel TAK1 small molecule inhibitor AZ-Tak1 in these cell lines. AZ-Tak1 is a potent and a relatively selective inhibitor of TAK1 kinase activity, with an IC50 of 0.009 mM. AZ-Tak1 treatment decreased the level of p38 and ERK in mantle cell lymphoma cells, and induced apoptosis in a dose and time dependent manner, with an IC50 of 0.1–0.5 mM. Using the annexin-V and PI staining and FACS analysis, After 48 hours of incubation, AZ-Tak1 (0.1 mM) induced apoptosis in 28%, 34% and 86% of Mino, SP53, and Jeko cells, respectively, which was increased to 32%, 42%, and 86% when 0.5 mM concentration was used. Similar activity was also observed when TAK1 expression in MCL cells was downregulated by TAK1- specific SiRNA and when primary mantle cell lymphoma specimens were examined after treatment with AZ-Tak1 for 24h (300nM). Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, AZ-Tak1 (0.5 mM) altered the level of several proteins that regulate cell growth and survival, especially members of the inhibitors of apoptosis (IAP) family. Specifically, nuclear NF-κB p65 levels were decreased, cytosolic levels of SMAC/DIABLO and cytochrome-C were increased in AZ-Tak1 treated cells, which were associated with a decrease in the level of the anti-apoptotic protein X-linked IAP (XIAP) and activation of the intrinsic apoptotic pathway as evident by activation of caspase 9, cleavage of caspase 3, and consequent cells apoptosis. Collectively, our data demonstrate that TAK1 is essential for MAPK and NF-κB activation. Inhibition of TAK1 by the small molecule inhibitor AZ-Tak1 or TAK1-SiRNA induces cell death in mantle cell lymphoma by activating the intrinsic apoptosis pathway, suggesting that targeting TAK1 may have a therapeutic value for the treatment of mantle cell lymphoma.