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TGF-β–activated kinase 1 (TAK1), a member of the MAPK kinase family, plays a key role in B-cell growth and development. In the present study, we examined the potential role of TAK1 as a therapeutic target for lymphoma. Here, we show that the active phosphorylated form of TAK1 is abundantly expressed in a panel of lymphoma cell lines, including mantle cell, anaplastic large cell, and Hodgkin lymphoma cell lines. Silencing TAK1 expression via the use of siRNA inhibited the activation of NF-κB and p38 and induced apoptosis in lymphoma cell lines. Moreover, submicromolar concentrations of AZ-TAK1, a novel ATP-competitive small molecule inhibitor of TAK1, dephosphorylated TAK1, p38, and IκB-α in lymphoma cell lines. These molecular events were associated with the release of cytochrome c into the cytosol, down-regulation of X-linked inhibitor of apoptosis, activation of caspase 9, and induction of apoptosis. We also demonstrate that primary lymphoma cells express TAK1 and pTAK1 and were sensitive to AZ-TAK1–mediated cell death. Collectively, our data demonstrate an essential role for TAK1 in regulating critical survival mechanisms in lymphoma and suggest that it may serve as a therapeutic target.

Abstract 1540 The PIM serine/threonine kinase family is composed of three highly homologous members; PIM1, PIM2 and PIM3, identified by the ability of the prototype member PIM1 to drive lymphomagenesis in mice. Upregulation of PIM1 and PIM2 is observed in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. PIMs are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways. PIMs can phosphorylate multiple substrates to mediate cell proliferation and survival. Here we describe the function of AZD1208, an orally available, potent and highly selective PIM inhibitor that effectively inhibits all three isoforms. AZD1208 inhibits the growth of several AML cell lines and sensitivity correlates with the level of PIM1 expression, STAT5 activation and presence of protein tyrosine kinase mutation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells in culture. This is accompanied by a dose-dependent reduction in pBAD serine 112 and other substrates, as well as an increase in cleaved caspase 3. AZD1208 suppresses the growth of MOLM-16 and KG-1a xenograft tumors in vivo in a dose proportional manner. In addition, AZD1208 leads to potent inhibition of colony growth of primary AML cells from bone marrow aspirates and downregulates phosphorylation of PIM targets. These results underscore the therapeutic potential of PIM kinase inhibition by AZD1208 for the treatment of AML. Disclosures: Keeton: AstraZeneca: Employment, Equity Ownership. Palakurthi:AstraZeneca: Employment, Equity Ownership. Alimzhanov:AstraZeneca: Employment, Equity Ownership. Grondine:AstraZeneca: Employment, Equity Ownership. Chen:AstraZeneca: Employment, Equity Ownership. Brown:AstraZeneca: Employment, Equity Ownership. McEachern:AstraZeneca: Employment, Equity Ownership. Cao:AstraZeneca: Employment, Equity Ownership. Chinnappan:AstraZeneca: Employment, Equity Ownership. Shen:AstraZeneca: Employment, Equity Ownership. Dakin:AstraZeneca: Employment, Equity Ownership. Zheng:AstraZeneca: Employment, Equity Ownership. Lamb:AstraZeneca: Employment, Equity Ownership. Wu:AstraZeneca: Employment, Equity Ownership. Chen:AstraZenenca: Employment, Equity Ownership. Lyne:AstraZeneca: Employment, Equity Ownership. Huszar:AstraZeneca: Employment, Equity Ownership.

Abstract 1692 Poster Board I-718 Transforming growth factor-b-activated kinase 1 (TAK1) is a key regulator of NF-kB activation. TAK1 can be activated by a variety of pro-inflammatory cytokines and T and B cell receptors. Recent experiments demonstrated that deletion of TAK1 results in inactivation of both JNK and NF-kB signaling resulting in massive apoptotic death of hematopoietic cells in mice. In this study, we examined the expression pattern of TAK1 and its role as a potential therapeutic 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). These lines expressed relatively low levels of the tumor suppressor protein A20. Mino and SP53 expressed high level of p-p38. 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. It also inhibits Jak2 but at a much higher concentration (IC50=0.18 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 primary mantle cell lymphoma cells were examined. 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, AZ-Tak1 decreased the level of SMAC/DIABOLO and cytochrome –C in the mitochondria, which was 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 induction of apoptosis. Furthermore, and consistant with its ability to inhibit Jak2 activity, AZ-Tak1 reduced STAT2 and STAT6 levels. AZ-Tak1 demonstrated no significant effect on bcl-2 family members. Finally, co-treatment with HDAC inhibitors demonstrated synergistic effect with low concentrations of AZ-Tak1. Collectively, our data demonstrate that targeting TAK1 by the small molecule inhibitor AZ-Tak1 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. Disclosures Palakurthi: Astra Zeneca: Employment. Byth:Astra Zeneca : Employment.