IntroductionDiffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous disease comprising at least 3 major subtypes with distinct molecular, biologic, and clinical properties: activated B cell-like DLBCL (ABC-DLBCL), germinal center B cell-like DLBCL (GCB-DLBCL), and primary mediastinal B-cell lymphoma. 1,2 Although the overall cure rate for DLBCL reaches more than 50% with the current therapies such as R-CHOP (rituximab plus cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone/prednisolone), less than 40% of ABC-DLBCL patients are cured. [2][3][4] Therefore, new therapy approaches efficient for this and other DLBCL subtypes are highly desirable.The transcription factor NF-B controls the expression of a wide range of genes involved in cell proliferation, survival, stress response, angiogenesis, and inflammation. 5,6 NF-B activity is tightly regulated by multiple signaling pathways, and abnormal NF-B activation has been linked to cancer development and progression. [7][8][9] Constitutive NF-B activation has been observed in high frequency in all of the main DLBCL subtypes, especially in ABC-DLBCL, with more than 90% of the tumors showing nuclear NF-B, the hallmark of its activation. 9-14 A recent genomic study revealed that more than 60% of ABC-DLBCLs and approximately 30% of GCB-DLBCLs harbor somatic mutations in multiple components of NF-B signaling pathways, such as the BCR, CD40, and TLR pathways. 15 Significantly, it has been demonstrated that constitutive NF-B signaling is required for the proliferation and survival of ABC-DLBCL cell lines. 11,13,16,17 All of these observations suggest a primary role for constitutive NF-B signaling in the pathogenesis of DLBCL and, therefore, the NF-B signaling pathway may represent a rational therapeutic target in DLBCL. 9,11,18 Ubiquitination, the covalent attachment of the ubiquitin (Ub) molecule to target proteins, regulates diverse cellular processes. 19 Ubiquitination proceeds through a stepwise enzymatic cascade involving 3 classes of enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The E1 enzyme activates Ub in an ATP-dependent manner and transfers the activated Ub to an E2 enzyme through the formation of a thioester bond between the carboxy terminus of Ub and the active site cysteine of the E2, generating an E2 and Ub thioester conjugate (referred to as E2ϳUb). The E2 then cooperates with an E3 to attach the Ub to a lysine residue of a substrate. Ub itself can serve as a substrate and the process can undergo multiple rounds, resulting in the formation of polyubiquitin chains. 19,20 Because Ub has 7 lysine residues and any one of them can be conjugated to another Ub, polyubiquitin chains of different linkages with distinct functional properties are formed in cells. For example, lysine 48 (K48)-linked polyubiquitin chains typically target substrates for proteasomal degradation, whereas K63-linked polyubiquitin chains function as scaffolds to assemble protein complexes in NF-B signaling and DNA repair. [...

Previous studies in sea urchin embryos have demonstrated that nuclearization of beta-catenin is essential for initial steps in the specification of endoderm and mesenchyme, which are derived from vegetal blastomeres. This process begins at the 4th and extends through the 9th cleavage stage, an interval in which the SpSoxB1 transcription regulator is downregulated by beta-catenin-dependent gene products that include the transcription repressor SpKrl. These observations raise the possibility that SpSoxB1 removal is required to allow vegetal development to proceed. Here we show that elevated and ectopic expression of this factor suppresses differentiation of all vegetal cell types, a phenotype that is very similar to that caused by the suppression of beta-catenin nuclear function by cadherin overexpression. Suppression of vegetal fates involves interference at the protein-protein level because a mutation of SpSoxB1 that prevents its binding to DNA does not significantly reduce this activity. Reduction in SpSoxB1 level results in elevated TCF/Lef-beta-catenin-dependent expression of a luciferase reporter gene in vivo, indicating that in the normal embryo this protein suppresses the primary vegetal signaling mechanism that is required for specification of mesenchyme and endoderm. Surprisingly, normal expression of SpSoxB1 is required for gastrulation and endoderm differentiation, as shown by both morpholino-mediated translational interference and expression of a dominant negative protein. Similar gain-of-function and loss-of-function assays of a closely related factor, SpSoxB2, demonstrate that it, too, is required for gastrulation and that its overexpression can suppress vegetal development. However, significant phenotypic differences are apparent in the two perturbations, indicating that SpSoxB1 and SpSoxB2 have at least some distinct developmental functions. The results of all these studies support a model in which the concentration of SpSoxB factors must be tightly regulated along the animal-vegetal axis of the early sea urchin embryo to allow beta-catenin-dependent specification of endoderm and mesenchyme cell fates as well as to activate target genes required for gastrulation.

In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes.

IntroductionDiffuse large B-cell lymphoma (DLBCL) is an aggressive and the most common subtype of non-Hodgkin lymphoma, accounting for 30% to 40% of lymphoid malignancy. Although DLBCL represents one of the most therapy-responsive malignancies, only approximately 40% of the patients can be cured by the current treatments with multiple therapeutic agents. 1,2 As a result, nearly 10 000 patients die of DLBCL each year in the United States, 2 underscoring the importance of better molecular understanding of DLBCL and identifying proper therapeutic targets. On the basis of gene expression profiles, DLBCL can be divided into at least 3 subgroups: activated B-cell-like (ABC) DLBCL, germinal center B cell-like (GCB) DLBCL, and primary mediastinal B-cell lymphoma (PMBL), with the ABC and PMBL subgroups exhibiting higher levels of expression of NF-B target genes than the GCB subgroup. [3][4][5][6] It was shown that constitutive NF-B signaling is required for survival of ABC and PMBL DLBCL cells 7,8 and that small molecules that inhibit IB kinases (IKKs) are selectively toxic for these 2 DLBCL subgroup cells. 9 These studies highlight the NF-B pathway as a promising therapeutic target in B-cell lymphomas that depend on NF-B activity for proliferation and survival.The NF-B family consists of 5 members in mammals: p65 (RelA), RelB, c-Rel, NF-B1(p105/p50), and NF-B2(p100/p52). Members of NF-B proteins form homodimers or heterodimers and are retained in the cytoplasm before activation by the associated IB proteins or the precursor proteins p100 or p105, which contain the ankyrin repeats present also in the IB proteins and thus can also function as IB proteins. Extracellular signals induce NF-B activation through 2 major pathways: the classical (also called the canonical pathway) and the alternative (the noncanonical) pathways. Activation of these pathways leads to activation of IKK and degradation or processing of IB proteins, resulting in release of sequestered NF-B proteins, their subsequent translocation into the nucleus and target gene activation. While activation of the canonical NF-B pathway predominantly results in release of active p50/p65 and p50/C-Rel dimers, the alternative pathway leads to nuclear accumulation of the p52/RelB complex. [10][11][12] Abnormal activation of NF-B contributes to tumor development and progression, as well as to the resistance of cancer cells to chemotherapeutic agents and radiation therapy. 13,14 Thus, inhibition of NF-B activation represents a promising approach for cancer therapy. [13][14][15] However, as NF-B is expressed ubiquitously and is involved in a wide variety of normal cellular functions, a nonselective inhibition of NF-B activity would likely cause serious side effects. 13,16,17 To achieve the specificity required for effective therapeutic intervention, it may be necessary to target cancer cell-or signal-specific regulators of NF-B activation pathways.The B cell-activating factor belonging to the tumor necrosis factor family (BAFF, also known as BlyS, TALL-1, THANK, zTNF-4, C...

CD28 plays a critical role in regulating immune responses both by enhancing effector T cell activation and differentiation and controlling the development and function of regulatory T cells. CD28 is expressed at the cell surface as a disulfide linked homodimer that is thought to bind ligand monovalently. How ligand binding triggers CD28 to induce intracellular signaling as well as the proximal signaling pathways that are induced are not well-understood. In addition, recent data suggest inside-out signaling initiated by the T cell antigen receptor can enhance CD28 ligand binding, possibly by inducing a rearrangement of the CD28 dimer interface to allow for bivalent binding. To understand how possible conformational changes during ligand-induced receptor triggering and inside-out signaling are mediated, we examined the CD28 transmembrane domain. We identified an evolutionarily conserved YxxxxT motif that is shared with CTLA-4 and resembles the transmembrane dimerization motif within CD3ζ. We show that the CD28 transmembrane domain can drive protein dimerization in a bacterial expression system at levels equivalent to the well-known glycophorin A transmembrane dimerization motif. In addition, ectopic expression of the CD28 transmembrane domain into monomeric human CD25 can drive dimerization in murine T cells as detected by an increase in FRET by flow cytometry. Mutation of the polar YxxxxT motif to hydrophobic leucine residues (Y145L/T150L) attenuated CD28 transmembrane mediated dimerization in both the bacterial and mammalian assays. Introduction of the Y145L/T150L mutation of the CD28 transmembrane dimerization motif into the endogenous CD28 locus by CRISPR resulted in a dramatic loss in CD28 cell surface expression. These data suggest that under physiological conditions the YxxxxT dimerization motif within the CD28 transmembrane domain plays a critical role in the assembly and/or expression of stable CD28 dimers at the cell surface.