Large B-cell Lymphoma Cell Lines Research Articles

Vav‐1 expression correlates with NFκB activation and CD40‐mediated cell death in diffuse large B‐cell lymphoma cell lines

Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy with a variable response to therapy. We have previously shown that DLBCL cell lines differ in their susceptibility to CD40-mediated cell death, and that resistance to CD40-targeted antibodies correlated with increased expression of markers of immature B-cell and absence of Vav-1 mRNA. We used gene expression profiling to investigate the mechanism of CD40 resistance in these cell lines, and found that resistance correlated with lack of Vav-1 and inability to activate NFκB upon CD40 ligation. Analysis of tissue microarrays of 213 DLBCL cases revealed that Vav-1 expression correlated with a higher proliferative index and the presence of the post-germinal centre marker Irf-4. Our results suggest that Vav-1 expression may be associated with activated B-cell DLBCL origin and higher proliferative activity, and indicate Vav-1 as a potential marker to identify tumours likely to respond to CD40-targeted therapies.

Novel IL-21 signaling pathway up-regulates c-Myc and induces apoptosis of diffuse large B-cell lymphomas

AbstractInterleukin-21 (IL-21), a member of the IL-2 cytokine family, has diverse regulatory effects on natural killer (NK), T, and B cells. In contrast to other cytokines that are usually immunostimulatory, IL-21 can induce apoptosis of murine B cells at specific activation-differentiation stages. This effect may be used for treatment of B-cell malignancies. Herein we report that diffuse large B-cell lymphoma (DLBCL) cell lines exhibit widespread expression of the IL-21 receptor (IL-21R) and that IL-21 stimulation leads to cell-cycle arrest and caspase-dependent apoptosis. IL-21 also induces apoptosis in de novo DLBCL primary tumors but does not affect viability of human healthy B cells. Furthermore, IL-21 promotes tumor regression and prolongs survival of mice harboring xenograft DLBCL tumors. The antilymphoma effects of this cytokine are dependent on a mechanism involving IL-21–activated signal transducer and activator of transcription 3 (STAT3) up-regulating expression of c-Myc. This up-regulation promotes a decrease in expression of antiapoptotic Bcl-2 and Bcl-XL proteins triggering cell death. Our results represent one of the first examples in which the STAT3–c-Myc signaling pathway, which can promote survival and oncogenesis, can induce apoptosis in neoplastic cells. Moreover, based on IL-21's potency in vitro and in animal models, our findings indicate that this cytokine should be examined in clinical studies of DLBCL.

Histone acetyltransferase p300 is a coactivator for transcription factor REL and is C-terminally truncated in the human diffuse large B-cell lymphoma cell line RC-K8

Human c-Rel (REL) is a member of the NF-κB family of transcription factors. REL’s normal physiological role is in the regulation of B-cell proliferation and survival. The REL gene is amplified in many human B-cell lymphomas and overexpression of REL can transform chicken lymphoid cells. In this report, histone acetyltransferase p300 enhanced REL-induced transactivation and interacted with REL both in vitro and in REL-transformed chicken spleen cells and the B-lymphoma cell line RC-K8, in which REL is constitutively active and required for proliferation. However, due to a deletion in the EP300 locus, only a C-terminally truncated form of p300 is expressed in RC-K8 cells. These results suggest a role for p300 in REL-mediated oncogenic activity in B lymphoma.

Anti-CD20 Antibody GA101 in Combination with Chemotherapy or Flavopiridol in Mantle Cell Lymphoma.

Abstract 4781 BackgroundMantle cell lymphoma (MCL) responds only transiently to conventional chemotherapy resulting in a dismal long-term prognosis. At a molecular level it is characterised by the chromosomal translocation t(11;14)(q13;q32), which leads to constitutive over-expression of the cell cycle regulatory protein cyclin D1. GA101 is a third generation, glycoengineered type II IgG1 anti-CD20 monoclonal antibody with superior direct cell death induction by targeting a type II epitope and enhanced antibody dependent cellular cytotoxicity (ADCC). High efficacy in lymphoma cell lines has led to combination experiments with various chemotherapeutic compounds or the CDK-inhibitor Flavopiridol. MethodsUsing a MCL cell line panel (Granta-519, HBL-2, Jeko-1, Rec-1 and Z-138) and a Diffuse Large B-Cell Lymphoma cell line (Karpas-422) we determined the effect of GA101 (1 μg/ml) monotherapy as well as in combination with Fludarabine (0,25 μg/ml), Bendamustine (5 μg/ml), Mitoxantrone (0,25 and 0,5 μg/ml) and Flavopiridol (100nM) on cell proliferation and viability. Trypan-blue exclusion tests were used to analyze cell viability at 0h, 24h, 48h and 72h. The panel of MCL cell lines was treated to determine potential synergism of agent combinations. Accordingly, fractional product was calculated: synergism > 0,1; additive effect -0,1<x<0,1; antagonism < -0,1. ResultsAfter mono-exposure with GA101 (1 μg/ml), Granta-519 and Rec-1 showed the highest sensitivity (Granta: 65-75% cell reduction, Rec-1: 30-45%). Intermediate results were achieved for HBL-2 (20-30%), Z-138 and Karpas-422 (10-15%), Jeko-1 (5%). Fludarabine alone resulted in a 20-40% cell reduction. Bendamustine showed a higher efficacy in Jeko-1, Rec-1 and Z-138 (40-90%) than in Granta-519, Karpas-422 and HBL-2 (10%). Mitoxantrone treatment demonstrated a high impact on all cell lines (80-95% cell reduction). Flavopiridol induced a 65-85% cell reduction in Jeko-1, Rec-1 and Karpas-422in comparison to 30-45% in Granta-519, HBL-2 and Z-138. Additional experiments showed additive effects of all GA101 combinations resulting in 40-80% cell reduction (Fludarabine), 30-90% (Bendamustine), 85-95% (Mitoxantrone) and 60-80% (Flavopiridol). ConclusionsThese in vitro results demonstrate that the anti-CD20 monoclonal antibody GA101 alone or in combination with various chemotherapeutical compounds or the CDK-inhibitor (Flavopiridol) show a promising efficacy in MCL cell lines (additive in combination), supporting the clinical evaluation of such an innovative immuno-chemotherapy in mantle cell lymphoma. Disclosures:Klein:Roche (Glycart): Employment, Equity Ownership, Patents & Royalties. Weinkauf:Lilly Deutschland GmbH: Research Funding. Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Dreyling:Roche: Honoraria, Research Funding.

Expression and Targeted Inhibition of the Immunoregulatory Carbohydrate-Binding Lectin, Galectin 1, in EBV-Driven Post-Transplant Lymphoproliferative Disorders.

Abstract 96EBV-associated post-transplant lymphoproliferative disorders (PTLD) are potentially fatal EBV-driven B cell proliferations that develop in immunocompromised solid organ or bone marrow allograft recipients. Proliferation of EBV-infected B cells in PTLD is maintained by expression of EBV latent genes such as latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance. Management of early PTLD lesions is currently based on reduction or withdrawal of immunosuppression which increases the risk of graft rejection. For these reasons, alternative treatments that specifically boost host anti-EBV immune responses are needed.To identify novel T-cell inhibitory molecules induced by EBV and expressed in PTLDs, we compared the transcription profiles of a series of EBV-transformed lymphoblastoid B-cell lines (LCLs) and diffuse large B-cell lymphoma (DLBCL) cell lines. The LCLs overexpressed galectin-1 (Gal-1), a carbohydrate-binding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4+ Th1 and CD8+ cytotoxic T cells. Gal-1 protein expression was also uniformly high in LCLs and low or undetectable in DLBCL lines by western blotting. Immunohistochemical staining of primary tumors confirmed abundant Gal-1 expression in PTLD B-cells, whereas DLBCLs were negative.To elucidate the mechanisms responsible for Gal-1 overexpression in EBV-associated PTLD, we performed transcriptional reporter assays using cotransfected FLAG-tagged LMP1 and/or LMP2A and a luciferase reporter vector driven by Gal-1 promoter. LMP2A and LMP1 each increased Gal-1-driven luciferase expression 3- to 6-fold, respectively, and the combination of LMP2A and LMP1 was synergistic. In complementary experiments, siRNA-mediated depletion of LMP2A decreased Gal-1 protein abundance in EBV-transformed lymphoblastoid cells. The mechanism of LMP1 and LMP2A-induced Gal-1 expression in LCLs was both AP-1 and PI3K-dependent; the PI3K selective inhibitor, LY294002, markedly inhibited Gal-1 expression in these cells.Given the tolerogenic and immunosuppressive function of Gal-1 and the EBV-driven overexpression of the lectin in LCLs and primary PTLDs, we postulated that Gal-1 might be a targetable immune escape mechanism in EBV-associated malignancies. For these reasons, we assessed the neutralizing activity of a recently developed series of anti-Gal1 monoclonal antibodies. After confirming that these reagents selectively identified both recombinant and native Gal1, we asked whether certain Gal-1 monoclonal antibodies neutralized recombinant Gal-1-mediated apoptosis of EBV-specific polyclonal, largely CD8+, T-cells. Preincubation of human recombinant Gal-1 (hrGal-1) with Gal-1 monoclonal antibodies, but not with isotype-matched controls, selectively inhibited hrGal-1 induced apoptosis of EBV-specific CD8+ T-cells.Taken together, these studies indicate that EBV-transformed LCLs and primary EBV-associated PTLDs express Gal-1 in a LMP-1 and LMP2A-dependent manner. Given the acknowledged role of Gal-1 in mediating an immunopriviledged tumor microenvironment, the lectin likely inhibits the host anti-tumor response in these tumors. Antibody-mediated neutralization of secreted Gal-1 may represent a novel immunotherapeutic strategy in PTLD and other Gal1-expressing tumors. Disclosures:No relevant conflicts of interest to declare.

Neurotrophin signaling through tropomyosin receptor kinases contributes to survival and proliferation of non-Hodgkin lymphoma

Neurotrophin receptor signaling has been increasingly recognized as an important factor in the development and progression of a variety of malignancies. In order to analyze the potential contribution of neurotrophin signaling to lymphoma cell survival, we investigated the role of a neurotrophin axis in promoting survival and proliferation of non-Hodgkin lymphoma (NHL) cells. The role of neurotrophins in the survival and proliferation of NHL cells was determined by exposing cells to the Trk-specific inhibitor, K252a, and then performing (3)H-thymidine incorporation and Annexin-V/propidium iodide staining. The involvement of nuclear factor-kappaB (NF-kappaB) in this process was studied using Western blot, electrophoretic mobility shift assay, and immunofluorescence assays. Here we demonstrate that both primary NHL cells and diffuse large B-cell lymphoma cell lines express Trk receptors and their neurotrophin ligands. Furthermore, these cells are sensitive to the Trk-specific inhibitor, K252a, as evidenced by the inhibition of proliferation and/or induction of apoptosis. Analysis of the mechanism into the effects of K252a revealed that, in the OCI-LY3 cell line, K252a induced a subnuclear distribution of NF-kappaB resulting in the sequestration of RelA in the nucleolus, thereby inhibiting NF-kappaB-dependent gene transcription. This results in the loss of interleukin-6 production; a known survival-promoting signal for OCI-LY3, as well as many primary diffuse large B-cell lymphomas. Thus, Trk receptors represent a novel therapeutic target for the treatment of NHL.

Phosphorylation of the activation loop tyrosines is required for sustained Syk signaling and growth factor-independent B-cell proliferation

The Syk kinase is regarded as a promising target for the treatment of antigen-driven B-cell malignancies, considering its essential role in propagating antigenic stimuli through the B-cell receptor (BCR). In certain common B-cell malignancies Syk is activated even in the absence of BCR engagement, suggesting a wider role for this kinase in lymphomagenesis. In this paper, we have profiled molecular differences between BCR-induced and constitutive Syk activation in terms of phosphorylation of regulatory tyrosine residues, downstream signaling properties and capacity to sustain B-cell proliferation. Analysis of primary chronic lymphocytic leukemia B-cells and diffuse large B-cell lymphoma cell lines revealed that constitutive and BCR-induced Syk activation differ with respect to the phosphorylation status of the regulatory tyrosines at positions 352 and 525/526, with only the first site being phosphorylated in the case of constitutive and both sites in the case of BCR-induced Syk activation. Syk phosphorylated only on Y352 is capable of downstream signaling, as evidenced by experiments with a phosphomimetic mutant in which the activation loop tyrosines (YY525/526) were replaced with phenylalanines. However, phosphorylation at YY525/526 was shown to significantly increase the enzymatic activity of Syk and to be required for sustained PLCγ2, Akt and ERK signaling as well as B-cell transformation. These data demonstrate that constitutively active Syk and Syk activated by BCR crosslinking represent separate stages of Syk activation with distinct signaling properties and transforming capacities.

CIITA or RFX coding region loss of function mutations occur rarely in diffuse large B-cell lymphoma cases and cell lines with low levels of major histocompatibility complex class II expression

Loss of major histocompatibility complex class II (MHCII) gene expression was associated with poor outcome in diffuse large B-cell lymphoma (DLBCL) in several studies.[1][1]–[3][2] The mechanism for lost expression is unknown. MHCII gene expression is controlled by several transcription factors,

Networking Modules of Canonical and Alternative NFkB Signaling in Growth and Survival Regulations of Large B Cell Lymphomas

Constitutive NF-kB activation is considered a “hallmark” in B-cell malignancies, especially in large B-cell lymphoma (LBCL), a common but heterogenous Non-Hodgkin's lymphoma type. However, the mechanism(s) of activation and interactions of NF-kB components in the nuclear compartment of B-cell lymphomas are still poorly defined. Our findings demonstrate that both the canonical and the alternative NF-kB pathways are constitutively activated in both LBCL cell lines and primary lymphoma cells. NF-kB DNA binding ELISA analysis showed that the canonical NF-kB components p65 and c-rel as well as the non-canonical NF-kB components p52 and relB are constitutively activated in 14 LBCL cell lines and in 14 primary lymphoma patients' cells (both ABC-and GCB-like subtypes). Based on the ELISA data, we found that the p65/c-rel ratio is relatively higher in ABC-like LBCL subtype when compared to GCB-like LBCL subtype, but the alternative NF-kB members' p52 and rel-B are differentially activated in both GCB- and ABC-like LBCL subtypes. Micro-tissue array (MTA) studies confirmed that both NF-kB pathways are constitutively active in LBCL biopsy-cores. The components of both NF-kB pathways were also found to merge and form functional “hybrid” NF-kB complexes in the nuclear compartment. Besides the common hetero-dimeric complexes occur between p50, p65, and c-rel of the canonical NF-kB pathway and between p52 and relB of the alternative NF-kB pathway, components of both pathways also merged to form “hybrid” dimeric complexes, such as p52-relB, p52-c-rel, relB-p65, and relB-c-rel in LBCL cells. Interestingly, p65 and c-rel binds to relB and p52, respectively, with higher affinity in GCB- than in ABC-like LBCL subtype. Our data also showed that activation of both NF-kB pathways and their cross-interaction are not restricted to only neoplastic B cells but that they are also activated and utilized in highly proliferative activated normal B-lymphocyte. Specific siRNA that target individual NF-kB members p65, c-rel, and rel-B have inhibitory activity in lymphoma cell growth, but similar p52 siRNAs do not have inhibitory activity. These studies indicated that in both LBCL cell lines and patient samples (GCB-and ABC-like subtypes), both the canonical and the alternative NF-kB pathways are not only constitutively activated but also interact to form multimeric NF-kB complexes in the nuclear compartment, giving rise to an NF-kB signaling module that not only controls lymphoma cell growth and survival but likely contributes to the heterogeneity of the disease processes. These findings reveal additional aspects of molecular heterogeneity and complexity in NF-kB signaling mechanisms and interacting transcriptional modules, contributing to the pathophysiology of LBCL. These results also highlight the critical importance of multiple constitutively active NF-kB signaling pathways in LBCL and may also contribute to therapeutic refractoriness, particularly in relapsed/refractory LBCLs.

Recruitment of the SWI/SNF Chromatin Remodeling Complex by NFATc1 in the Transcriptional Regulation of the C-MYC Oncogene in Aggressive B-Cell Lymphomas

The NFAT (nuclear factor of activated T-cells) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for cell growth and survival in hematopoietic cells. Recent experimental evidence has implicated the calcineurin/NFAT signaling pathway for involvement in the pathogenesis of various malignancies, including large B-cell lymphoma (LBCL), a non-Hodgkin's lymphoma subgroup that is generally responsive to conventional cancer therapies (R-CHOP), but relapse is common that subsequently leads to therapeutic resistance. Although we have shown previously that NFAT family member NFATc1 is constitutively activated and has the ability to maintain cell growth and survival in LBCL cell lines and primary cells, the molecular mechanism(s) underlying how NFATc1 regulates cell growth and survival in LBCL is still unclear. In this study, we demonstrate that the well-known oncogene c-myc is transcriptionally regulated by the transcription factor NFATc1 in LBCL, through a chromatin remodeling mechanism that involves the recruitment of the SWI/SNF chromatin-remodeling complex. In aggressive B-cell lymphoma cell lines, c-myc oncogene protein expression was shown to correlate with NFATc1 protein expression. We further showed that NFATc1 binds to a specific DNA binding element within the proximal c-myc promoter and up-regulates c-myc transcription. The SWI/SNF proteins Brg-1 and Brm, chromatin-remodeling proteins that utilize ATP hydrolysis for energy to modify chromatin structure in order to regulate gene expression, also were shown to bind to the NFAT binding site on the c-myc promoter. Confocal microscopic analysis showed that NFATc1 colocalizes with Brg-1, and co-immunoprecipitation assays showed that Brg-1 interacts with NFATc1. Both proteins interact with the c-myc promoter within the NFAT binding site, as demonstrated by chromatin-immunoprecipitation (ChIP) analysis. Induction of a constitutively active mutant of NFATc1 (caNFATc1) in an NFATc1 negative lymphoma cell line induces c-myc protein expression. Constitutively active NFATc1 also enhances Brg-1 binding to the c-myc promoter when analyzed by ChIP-qPCR assays, suggesting that NFATc1 recruits Brg-1 to the NFAT binding site in the c-myc promoter. Down-regulation of NFATc1 by chemical inhibitors (FK-506) or by validated shRNA of NFATc1, inhibited c-myc protein expression and in-vitro lymphoma cell growth. Our data indicates a novel control mechanism for the transcriptional regulation of c-myc in the pathophysiology of aggressive lymphoma B cells and suggests that targeting NFATc1 could have therapeutic value.

Nuclear Tumor Necrosis Factor Receptor-associated Factor 6 in Lymphoid Cells Negatively Regulates c-Myb-mediated Transactivation through Small Ubiquitin-related Modifier-1 Modification

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor/scaffold protein that mediates several important signaling pathways, including the tumor necrosis factor-R:NF-kappaB pathway, involved in immune surveillance, inflammation, etc. Because most studies of TRAF6 function have focused primarily on its role as an adaptor molecule in signaling pathways in the cytoplasm, the potential functions of TRAF6 in other cellular compartments has not been previously investigated. Here, we demonstrate that TRAF6 resides not only in the cellular cytoplasm but is also found in the nuclei of both normal and malignant B lymphocytes. TRAF6 does not possess a nuclear localization signal but enters the nucleus through the nuclear pore complex containing RanGap1. Chromatin immunoprecipitation cloning experiments demonstrated that nuclear TRAF6 associates with c-Myb within the 5'-end of the c-Myb promoter. Further analysis showed that nuclear TRAF6 is modified by small ubiquitin-related modifier-1, interacts with histone deacetylase 1, and represses c-Myb-mediated transactivation. Thus, TRAF6 negatively regulates c-Myb through a novel repressor function in the nuclei of both normal and malignant B-lymphocytes that could represent a novel control mechanism that maintains cell homeostasis and immune surveillance.

Potentially oncogenic B-cell activation–induced smaller isoforms of FOXP1 are highly expressed in the activated B cell–like subtype of DLBCL

The FOXP1 forkhead transcription factor is targeted by recurrent chromosome translocations in several subtypes of B-cell non-Hodgkin lymphomas, where high-level FOXP1 protein expression has been linked to a poor prognosis. Western blotting studies of diffuse large B-cell lymphoma (DLBCL) cell lines unexpectedly identified the atypical high-level expression of 2 smaller, 60 to 65 kDa, FOXP1 isoforms in all 5 of those with the activated B cell (ABC)-like DLBCL subtype and in a subgroup of primary DLBCL. The anti-FOXP1 (JC12) monoclonal antibody cannot distinguish FOXP1 isoforms by immunohistochemistry, a finding that may be clinically relevant as high-level expression of the full-length FOXP1 protein was observed in some germinal center-derived DLBCLs. ABC-like DLBCL-derived cell lines were observed to express 2 novel, alternatively spliced FOXP1 mRNA isoforms, encoding N-terminally truncated proteins. These transcripts and the smaller protein isoforms were induced as a consequence of normal B-cell activation, which thus represents an additional mechanism for up-regulating FOXP1 expression in lymphomas. The expression of potentially oncogenic smaller FOXP1 isoforms may resolve the previously contradictory findings that FOXP1 represents a favorable prognostic marker in breast cancer and an adverse risk factor in B-cell lymphomas.

Nuclear CD40 interacts with c-Rel and enhances proliferation in aggressive B-cell lymphoma

AbstractCD40 is an integral plasma membrane–associated member of the TNF receptor family that has recently been shown to also reside in the nucleus of both normal B cells and large B-cell lymphoma (LBCL) cells. However, the physiological function of CD40 in the B-cell nucleus has not been examined. In this study, we demonstrate that nuclear CD40 interacts with the NF-κB protein c-Rel, but not p65, in LBCL cells. Nuclear CD40 forms complexes with c-Rel on the promoters of NF-κB target genes, CD154, BLyS/BAFF, and Bfl-1/A1, in various LBCL cell lines. Wild-type CD40, but not NLS-mutated CD40, further enhances c-Rel–mediated Blys promoter activation as well as proliferation in LBCL cells. Studies in normal B cells and LBCL patient cells further support a nuclear transcriptional function for CD40 and c-Rel. Cooperation between nuclear CD40 and c-Rel appears to be important in regulating cell growth and survival genes involved in lymphoma cell proliferation and survival mechanisms. Modulating the nuclear function of CD40 and c-Rel could reveal new mechanisms in LBCL pathophysiology and provide potential new targets for lymphoma therapy.

Downregulation of Bfl-1 protein expression sensitizes malignant B cells to apoptosis

Elevated expression of the antiapoptotic protein Bfl-1 (A1) was previously reported in several cancer cell lines. Recently, molecular profiling of large B-cell lymphoma identified Bfl-1 as a gene signature in 'OxPhos' diffuse large B-cell lymphoma subtype and in primary mediastinal large B-cell lymphoma, suggesting that in addition to Bcl-2, Bcl-xL and Mcl-1, Bfl-1 may be a relevant target in the design of new strategies for cancer therapy. Using short hairpin RNA strategy, we show here that Bfl-1 silencing in one lymphoblastoid B-cell line and in two diffuse large B-cell lymphoma cell lines potently induces their apoptosis and sensitizes those cell lines to anti-CD20 (Rituximab)-mediated cell death as well as to apoptosis induced by chemotherapeutic molecules such as doxorubicin, vincristine, cisplatin and fludarabine. These results demonstrate for the first time that Bfl-1 is an essential protein for survival of malignant B cells and suggest Bfl-1 may represent a potential target for future drug development against B cell lymphoma.

A Cell-Based Assay for IκBα Stabilization Using A Two-Color Dual Luciferase-Based Sensor

A cell-sensor assay for stabilization of IkappaBalpha was developed in the activated B cell-like diffuse large B-cell lymphoma cell line OCI-Ly3. This cell line expresses known nuclear factor kappaB (NFkappaB) target genes due to high constitutive activity of IkappaB kinase (IKK), which phosphorylates the protein IkappaBalpha leading to proteasomal degradation of IkappaBalpha and activation of NFkappaB. The cell-sensor assay uses green and red light-emitting beetle luciferases, with the green luciferase fused to IkappaBalpha (IkappaBalpha-CBG68) and the red luciferase (CBR) present in its native state. The IkappaBalpha-CBG68 reporter functions as a sensor of IKK and proteasome activity, while CBR serves to normalize for cell number and nonspecific effects. Both reporter constructs were stably integrated and placed under the control of an inducible promoter system, which increased fold responsiveness to inhibitors when assay incubations were performed simultaneous to reporter induction by doxycycline. The assay was miniaturized to a 1,536-well plate format and showed a Z' of 0.6; it was then used to panel 2,677 bioactive compounds by a concentration-response-based screening strategy. The concentration-effect curves for the IkappaBalpha-CBG68 and CBR signals were then used to identify specific stabilizers of IkappaBalpha, such as IKK inhibitors or proteasome inhibitors, which increased the doxycycline-induced rise in IkappaBalpha-CBG68 without affecting the rise in CBR. Known and unexpected inhibitors of NFkappaB signaling were identified from the bioactive collection. We describe here the development and performance of this assay, and discuss the merits of its specific features.

The rGel/BLyS fusion toxin specifically targets malignant B cells expressing the BLyS receptors BAFF-R, TACI, and BCMA

B lymphocyte stimulator (BLyS) is crucial for B-cell survival, and the biological effects of BLyS are mediated by three cell surface receptors designated B cell-activating factor receptor (BAFF-R), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B-cell maturation antibody (BCMA). Increased expression of BLyS and its receptors has been identified in numerous B-cell malignancies. We generated a fusion toxin designated rGel/BLyS for receptor-mediated delivery of the recombinant gelonin (rGel) toxin to neoplastic B cells, and we characterized its activity against various B-cell tumor lines. Three mantle cell lymphoma (MCL) cell lines (JeKo-1, Mino, and SP53) and two diffuse large B-cell lymphoma (DLBCL) cell lines (SUDHL-6 and OCI-Ly3) expressing all three distinct BLyS receptors were found to be the most sensitive to the fusion toxin (IC(50) = 2-5 pmol/L and 0.001-5 nmol/L for MCL and DLBCL, respectively). The rGel/BLyS fusion toxin showed specific binding to cells expressing BLyS receptors and rapid internalization of the rGel component into target cells. The cytotoxic effects of rGel/BLyS were inhibited by pretreatment with free BLyS or with soluble BAFF-R, TACI, and BCMA decoy receptors. This suggests that the cytotoxic effects of the fusion toxin are mediated through BLyS receptors. The rGel/BLyS fusion toxin inhibited MCL cell growth through induction of apoptosis associated with caspase-3 activation and poly (ADP-ribose) polymerase cleavage. Our results suggest that BLyS has the potential to serve as an excellent targeting ligand for the specific delivery of cytotoxic molecules to neoplastic B cells expressing the BLyS receptors, and that the rGel/BLyS fusion toxin may be an excellent candidate for the treatment of B-cell malignancies especially MCL and DLBCL.

EBNA2 Interferes with the Germinal Center Phenotype by Downregulating BCL6 and TCL1 in Non-Hodgkin's Lymphoma Cells

Epstein-Barr virus (EBV)-negative diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma-derived cell lines infected in vitro with a recombinant EBV expressed type II/III latency. High expression of EBNA2 inversely correlated with expression of germinal center (GC)-associated genes, BCL6 and TCL1. The decreased expression of BCL6 appeared to be dose dependent, with almost complete abrogation in highly EBNA2-expressing clones. The role of EBNA2 in negative regulation of these genes was confirmed by transfection and in a hormone-inducible EBNA2 cell system. LMP1 transfection reduced expression of TCL1, but not of BCL6, in DLBCLs. The GC-associated gene repression was at the transcriptional level and CBF1 independent. A decrease in HLA-DR, surface immunoglobulin M, and class II transactivator expression and an increase in CCL3, a BCL6 repression target, was observed in EBNA2-expressing clones. Since BCL6 is indispensable for GC formation and somatic hypermutations (SHM), we suggest that the previously reported lack of SHM seen in EBNA2-expressing GC cells from infectious mononucleosis tonsils could be due to negative regulation of BCL6 by EBNA2. These findings suggest that EBNA2 interferes with the GC phenotype.

Interleukin-21-Induced Apoptosis and Cell Death of Diffuse Large B-Cell Lymphoma (DLBCL) Cell Lines and Primary Tumors Are Associated with an Induction of Bim.

IL-21, a member of the IL-2 cytokine family, is reported to have an immune-mediated anti-tumor activity against renal cell carcinoma, malignant melanoma and Non-Hodgkin's Lymphoma (NHL) cell lines in xenograph animal models. Whether IL-21 exhibits direct anti-tumor activity against NHL cell lines and primary tumors is presently unknown.We analyzed seven DLBCL and two Burkitt's lymphoma cell lines for IL-21 receptor (IL-21R) expression. IL-21R was expressed at high levels in the two Burkitt's lymphoma cell lines (RAJI and RAMOS). Out of the seven DLBCL lines, four expressed high levels of the receptor (OCI-LY-3, OCI-LY-7, OCI-LY-19, and RCK-8) while three had low receptor expression levels (OCI-LY-10, SU-DHL-4, and SU-DHL-6). IL-21 stimulation induced tyrosine phosphorylation of STAT-1, -3, and -5 as early as fifteen minutes post treatment in DLBCL lines expressing either high (OCI-LY-3 and RCK-8) or low (SU-DHL-6 and OCI-LY-10) levels of IL-21R.In six of the seven DLBCL lines tested, IL-21 dramatically inhibited or completely abolished cellular proliferation at 25 ng/mL and 100 ng/mL concentrations, respectively. In contrast, one DLBCL cell line (OCI-LY-3) exhibited a threefold increase in cellular proliferation after stimulation with 25 ng/mL IL-21, but proliferation was effectively inhibited by 100 ng/mL IL-21.Marked apoptosis and cell death were observed by flow cytometry at 72 hours post IL-21 exposure in all nine NHL cell lines tested with the exception of OCI-LY-3 which exhibited no significant change in cell viability. The IL-21-induced apoptosis was associated with an activation of caspases 3/7, 8, and 9, detected as early as 12 hours post IL-21 exposure. IL-21 also induced an increase in the levels of the pro-apoptotic protein Bim in all the cell lines exhibiting marked apoptosis. In contrast, Bim protein levels decreased in response to the IL-21 stimulation in the resistant OCI-LY-3 DLBCL cell line. IL-21 treatment led to a decrease in the protein levels of Bcl-2 in all the analyzed cell lines except OCI-LY-3. An increase in expression of Bcl-6 protein in three of the four tested DLBCL cell lines was observed in response to IL-21 exposure.In primary tumors, IL-21 induced apoptosis in two of two DLBCLs, two of three follicular lymphomas, and two of six chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B-lymphocytes or in the HeLa or 293T control cells. These results suggest that IL-21 exhibits a direct anti-tumor effect on the DLBCL cell lines and primary tumors and point to a potential applicability of IL-21 in anti-DLBCL therapy. Further work interrogating the molecular mechanism of the IL-21-induced apoptosis of DLBCL cells in vitro and in animal models is in progress.

Hodgkin's Lymphoma Reed Sternberg Cells over Express the T-Cell Inhibitory Carbohydrate-Binding Lectin, Galectin 1: Role of AP-1 and Likely Mechanism of Tumor Immune Escape.

Primary Hodgkin lymphomas (HL) contain small numbers of neoplastic Reed-Sternberg (RS) cells within an extensive host inflammatory infiltrate, which includes few cytotoxic T-cells. The lack of an effective host anti-tumor response prompts speculation regarding the mechanisms that HL RS cells use to evade immune surveillance. To identify novel HL-specific T-cell inhibitory molecules, we compared the gene expression profiles of a series of HL and diffuse large B-cell lymphoma (DLBCL) cell lines. The HL cell lines overexpressed galectin-1 (GAL1), a carbohydrate-binding lectin that selectively induces the apoptosis of cytotoxic T cells. GAL1 mRNA abundance was 5- to 33- fold higher in HL cell lines than in DLBCL lines. GAL1 protein expression was also uniformly high in HL cell lines and low or undectable in DLBCL lines by western blotting.Immunohistochemical staining of primary tumors revealed abundant GAL1 expression in HL RS cells whereas DLBCLs were uniformly negative. To elucidate the mechanisms responsible for GAL1 over expression in HL, we analyzed the GAL1 locus and identified a candidate AP1 enhancer element 1.5 kb downstream of the GAL1 transcription start site. The predicted enhancer element increased the expression of a reporter gene (luciferase) 5-fold in a HL cell line but had no effect in either a DLBCL or fibroblast cell line. In electrophoretic mobility studies, AP1 bound to the predicted GAL1 enhancer element in a cell-type specific manner. In addition, mutation of the AP1 binding site in the GAL1 enhancer abolished its activity. Since the AP1 components, cJUN and JUN-B, are known to be overexpressed in HL, we analyzed cJUN and JUN-B copy numbers and transcript abundance in the HL cell lines using 100K high-density SNP arrays and oligonucleotide RNA microarrays. We found increased copy numbers of cJUN and/or JUN-B in all HL cell lines tested and confirmed these findings using fluorescence in situ hybridization (FISH). Increased cJUN and JUN-B copy numbers were associated with significantly higher transcript abundance in the HL cell lines. In additional functional assays, we confirmed previous reports that recombinant GAL1 induced the apoptosis of CD3+ PHA-stimulated lymphoblasts in a dose- and time-dependent manner. Taken together, these studies indicate that HL RS cells selectively over express GAL1, at least in part, via AP1-mediated induction of the GAL1 enhancer. Given the profound effects of GAL1 on cytotoxic T cells, RS-specific expression of GAL1 likely inhibits the host anti-tumor response in HL.

Comprehensive Analysis of Homeobox Genes in Hodgkin Lymphoma Cell Lines Identified Dysregulated Expression of HOXB9 Mediated by Constitutive Active ERK5 Signalling Pathway and BMI1.

Within the human genome nearly 200 homeobox genes have been identified. These are functionally involved in distinct developmental processes during embryogenesis and in the adult. Many have been implicated in cancer mostly following ectopic expression. In this study, we analyzed homeobox gene expression in Hodgkin lymphoma (HL) cell lines using degenerate primers for RT-PCR and Affymetrix U133A expression arrays followed by extended RT-PCR analysis of candidate homeobox genes in additional hematopoietic cell lines. We identified upregulated HOXB9 expression in HL, as well as in anaplastic large cell lymphoma and mediastinal B-cell lymphoma cell lines. Analysis of HOXB9 regulation in HL cells revealed activation by E2F3A, and repression by BMI1, respectively. Constitutive ERK5 pathway activation was identified in all HL cell lines analyzed together with primary HL lymph nodes biopsies. Our data show that ERK5 regulates HOXB9 expression, probably mediated via BMI1 repression. Expression analysis of microRNA gene mir-196a1 which is located upstream of HOXB9 indicated coregulation together with HOXB9. Functional analysis of HOXB9 by knockdown or overexpression assays revealed its influence in both proliferation and apoptosis in HL cells. In summary we identified upregulated expression of HOXB9 in HL, and its regulation mediated by constitutively active ERK5 signalling, thus representing novel therapeutic targets in both HL and NHL subtypes.