[Abstract unavailable

Purpose: We investigated the cytotoxicity and mechanisms of cell death of the broadspectrum histone deacetylase (HDAC) inhibitor PCI-24781, alone and combined with bortezomib in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines and primary lymphoproliferative (CLL/SLL) cells. Experimental Design: Apoptosis, mitochondrial membrane potential, cell cycle analysis, and reactive oxygen species (ROS) were measured by flow cytometry, whereas caspase activation was determined by Western blot. Nuclear factor κB (NF-κB)-related mRNAs were quantified by reverse transcription-PCR, NF-κB-related proteins by Western blotting, and NF-κB DNA-binding activity by electromobility shift assay. Finally, gene expression profiling was analyzed. Results: PCI-24781 induced concentration-dependent apoptosis that was associated with prominent G 0 /G 1 arrest, decreased S-phase, increased p21 protein, and increased ROS in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. Dose-dependent apoptosis with PCI-24781 was also seen among primary CLL/SLL cells. PCI-24781-induced apoptosis was shown to be ROS-and caspase-dependent. Combined PCI-24781/bortezomib treatment resulted in strong synergistic apoptosis in all non-Hodgkin lymphoma lines (combination indices, 0.19-0.6) and was additive in Hodgkin lymphoma and primary CLL/SLL cells. Further, PCI-24781/bortezomib resulted in increased caspase cleavage, mitochondrial depolarization, and histone acetylation compared with either agent alone. Gene expression profiling showed that PCI-24781 alone significantly down-regulated several antioxidant genes, proteasome components, and NF-κB pathway genes, effects that were enhanced further with bortezomib. Reverse transcription-PCR confirmed down-regulation of NF-κB1 (p105), c-Myc, and IκB-kinase subunits, where NF-κB DNA binding activity was decreased. Conclusion: We show that PCI-24781 results in increased ROS and NF-κB inhibition, leading to caspase-dependent apoptosis. We also show that bortezomib is synergistic with PCI-24781. This combination or PCI-24781 alone has potential therapeutic value in lymphoma.

Purpose: Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, antiviral, and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in leukemia and solid tumor cell lines.Experimental Design: We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma (MCL) line Granta, the follicular lymphoma (FL) cell line HF-1, and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL, DLBCL, and MCL.Results: We found that andrographolide resulted in dose-and time-dependent cell death as measured by MTT. Andrographolide significantly increased reactive oxygen species (ROS) production in all cell lines. To determine mechanism of cell death, we measured apoptosis by Annexin V/propidium iodide in the presence and absence of the antioxidant N-acetyl-L-cysteine (NAC), the glutathione (GSH)-depleting agent buthionine sulfoxamine (BSO), or caspase inhibitors. We found that apoptosis was greatly enhanced by BSO, blocked by NAC, and accompanied by poly(ADP-ribose) polymerase cleavage and activation of caspase-3, caspase-8, and caspase-9. We measured BAX conformational change and mitochondrial membrane potential, and using mouse embryonic fibroblast (MEF) Bax/Bak double knockouts (MEF

The E2A gene encodes two E protein/class I basic helix-loop-helix transcription factors, E12 and E47, that are essential for B lymphopoiesis. In addition to the DNA-binding and protein dimerization domain, the E proteins share two highly conserved transcription activation domains. In this study, we show that both activation domains are required for optimal E2A-dependent transcription. Surprisingly, however, neither activation domain is required for E2A to rescue B lymphopoiesis from E2A−/− hemopoietic progenitors, although the N terminus of E2A, which harbors some transcription capacity, is required. Therefore, the E protein activation domains function redundantly in promoting B cell development. In contrast, the N-terminal activation domain, AD1, is required for a newly described ability of E2A to suppress macrophage development in vitro. Our findings demonstrate distinct functionalities for the E protein activation domains in B lymphocytes and macrophages.

IntroductionDiffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults accounting for nearly 35% of all non-Hodgkin lymphomas (NHL). Significant advances have been in the treatment of DLBCL, particularly with immunochemotherapy, however approximately 30%-40% of patients still die from this malignancy. In addition, short-and long-term toxicities of chemotherapy, including secondary malignancies and leukemias, continue to adversely impact the long-term prognosis of patients. Continued investigations of novel targeted therapeutic agents in DLBCL are warranted.The RAS/RAF/mitogen-activated protein kinase 1/2 (MEK1/2)/ extracellular signal-regulated kinase 1/2 (ERK1/2) plays a prominent role in cancer biology, including hematologic malignancies, in part through the regulation of cell growth and proliferation. [1][2][3][4][5] Activating mutations in RAS and RAF lead to aberrant activation of their downstream target, MEK1/2. Directly downstream of MEK1/2, ERK1 and ERK2 are intimately involved in transducing signals from growth factor receptors and cytokine receptors after ligand binding. 2 Furthermore, ERK is the only known catalytic substrate of MEK. 6 We and others have shown that the MEK/ERK signaling pathway is constitutively active in a large number of cancers, including hematologic malignancies. 3,4,[7][8][9] Moreover, the MEK/ERK signal transduction cascade has been shown to be susceptible to pharmacologic intervention. Thus, MEK has emerged as an attractive therapeutic target in cancer.The majority of preclinical, and especially clinical trial data studying MEK inhibitors to date have emerged largely from solid tumor studies. [10][11][12][13][14] We recently showed in preclinical studies that inhibition of ERK1/2 phosphorylation by 1st generation MEK and ERK inhibitors correlated with significant cell death in a lymphoma tumor model, 15 while others showed that sublethal concentrations of a 1st generation MEK antagonist potentiated the effect of sorafemib in lymphoma cells. 16 Furthermore, we recently demonstrated that MCT-1, an oncogene directly downstream of MEK/ ERK, is over-expressed in the majority of primary DLBCLs. 15 MCT-1 is known to colocalize with ERK1/2, while phosphorylation of MCT-1 protein by ERK is critical for stabilization of MCT-1 protein and for its functional ability to promote cell proliferation. 15,17 ARRY-142886 (AZD6244, selumetinib; Astra Zeneca) is a selective nonATP-competitive 2nd generation oral MEK inhibitor studied primarily in solid tumor studies with reported nanomolar activity against purified MEK1 enzyme. [18][19][20][21][22][23] Furthermore, phase 1 and phase 2 solid tumor clinical trials have shown this agent to be well-tolerated and have encouraging clinical efficacy. [24][25][26][27] To our knowledge, minimal data are available on newer generation MEK inhibitors in lymphoma and moreover, this anti-MEK agent has never been examined in lymphoma. We sought to examine the mechanisms of action and cytotoxic effect of the novel 2nd generation MEK sma...