Burkitt Lymphoma-derived Cell Line Research Articles

Novel lectin-based chimeric antigen receptors target Gb3-positive tumour cells

The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.

Synthesis of polyenylpyrrole derivatives with selective growth inhibitory activity against T-cell acute lymphoblastic leukemia cells

T-cell acute lymphoblastic leukemia (T-ALL) is a hardly curable disease with a high relapse rate. 20 analogs were synthesized based on the structures of two kinds of fungi-derived polyenylpyrrole products (rumbrin (1) and auxarconjugatin-B (2)) to suppress the growth of T-ALL-derived cell line CCRF-CEM and tested for growth-inhibiting activity. The octatetraenylpyrrole analog gave an IC50 of 0.27 μM in CCRF-CEM cells, while it did not affect Burkitt lymphoma-derived cell line Raji and the cervical cancer cell line HeLa, or the oral cancer cell line HSC-3 (IC50 > 10 μM). This compound will be a promising compound for developing T-ALL-specific drugs.

HLL2-Cys-PBD, a New Site-Specifically Conjugated, Pyrrolobenzodiazepine (PBD) Dimer-Based Antibody Drug Conjugate (ADC) Targeting CD22-Expressing B-Cell Malignancies.

We previously described the development of hLL2-PBD, an ADC composed of the humanized monoclonal antibody epratuzumab (hLL2) against human CD22, stochastically conjugated to SG3249, a cathepsin B-cleavable valine-alanine PBD payload. SG3249, also known as tesirine, is a potent and clinically validated PBD payload currently being employed in the clinic in three ADCs: the CD25-targeted ADCT-301, the CD19-targeted ADCT-402, both for the treatment of lymphoma and leukemia, and the DDL3-targeted rovalpituzumab tesirine for the treatment of small cell lung cancer. hLL2-PBD demonstrated potent and specific in vitro and in vivo activity in the CD22-positive human Burkitt's lymphoma-derived cell lines Ramos, Daudi and WSU-DLCL2. Here, we describe the development and pre-clinical characterization of hLL2-cys-PBD, an ADC composed of an engineered version of epratuzumab (hLL2-cys), site-specifically conjugated to the PBD payload tesirine. The average drug to antibody ratio of hLL2-cys-PBD is 1.74.In vitro, hLL2-cys-PBD showed potent and specific cytoxtoxic activity against a panel of CD22-expressing human lymphoma cell lines, including Ramos, Daudi, WSU-DLCL2 and SU-DHL-4. Moreover, hLL2-cys-PBD was efficiently internalized by CD22-expressing cells and, consistent with the proposed mode of action of PBD dimer warheads, it induced DNA inter-strand cross-linking after a two hour exposure period, as measured by the modified single cell gel electrophoresis (comet) assay. In contrast, a non-CD22-targeted, site-specific, PBD-containing control ADC did not yield appreciable DNA cross-links.In vivo, hLL2-PBD showed potent, dose-dependent, and durable anti-tumor activity in subcutaneously implanted Ramos and WSU-DLCL2 xenograft models. In the Ramos model, a single dose of hLL2-cys-PBD at 1 mg/kg induced complete regression (CR) in all ten treated mice, with 9/10 tumor free survivors (TFS) at the end of the study on day 60. In the WSU-DLCL2 model, a single dose of hLL2-cys-PBD at 3 mg/kg resulted in 3/10 partial regression, 7/10 CR and 2/10 TFS at the end of the study on day 60. The toxicity of hLL2-cys-PBD was evaluated in rat (single dose study) and in cynomolgus monkey (repeat dose study). In rat, hLL2-cys-PBD was well tolerated at 2 mg/kg. In cynomolgus monkey, hLL2-cys-PBD elicited the expected rapid B-cell depletion and it was well tolerated at 0.6 mg/kg.Together, these data demonstrate that hLL2-cys-PBD has potent anti-tumor activity against CD22-expressing hematological tumors and warrants further development of this ADC into the clinic. DisclosuresZammarchi:ADC Therapeutics: Employment. Corbett:ADC Therapeutics: Research Funding. Adams:Medimmune: Employment. Mellinas-Gomez:Medimmune: Employment. Tyrer:Medimmune: Employment. Dissanayake:ADC Therapeutics: Employment. Sims:ADC Therapeutics: Employment. Havenith:ADC Therapeutics: Employment. Chivers:ADC Therapeutics: Employment. Willimas:MedImmune: Employment. Howard:MedImmune/ ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hartley:Spirogen Ltd/ADC Therapeutics: Employment, Equity Ownership, Research Funding. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Recurrent Mutations in Focal Adhesion Genes Including the RhoA Signaling Axis Are a Defining Feature of Germinal Center (GC) Derived B Cell Lymphomas and Promote Aberrant Migration and Cellular Adhesion within the GC Niche

Nonhodgkin lymphomas (NHLs) are among the most common cancer subtypes, with approximately >350,000 new cases diagnosed annually worldwide. The majority of NHLs arise from germinal center (GC)-derived B cells. Through network analysis we demonstrated that mutations in focal adhesion pathway genes, including RHOA and GNA13, are a defining characteristic of GC-derived B cell lymphoma, occurring in ~75% of the samples analyzed, and less than 10% of ABC DLBCLs and mantle cell lymphomas. We and others have previously described RHOA mutations in GC-derived B cell lymphomas including DLBCL and Burkitt lymphoma, but the functional consequences of these mutations remain unknown.We examined the patterns of RHOA mutation in DLBCL through targeted gene sequencing in 335 tumor-normal pairs. Cell of origin analysis revealed that there were 128 GCB DLBCLs in this group, with RHOA mutations occurring in 15 (11.7%) of GCB cases and none of the 207 ABC cases. RHOA mutations also occurred in 10% of Burkitt lymphoma cases (total N=66) and zero mantle cell lymphoma cases (total N=54). All the RHOA mutations were missense, and were predicted computationally to be of high impact. A significant proportion of the identified RHOA mutations affect evolutionarily conserved residues within the protein's GTP binding domain, further suggesting a functional importance. Interestingly, inactivating G17V mutations, found in 15% of peripheral T cell and 35-70% of angioimmunoblastic T cell lymphomas, never occurred in DLBCLs. RHOA is thought to signal downstream of GNA13 in a variety of cell types to regulate cellular adhesion and migration. In our study, human mutations in GNA13 and RHOA were mutually exclusive, and GC B cell specific GNA13 deletion in the AID-Cre Gna13 mouse model resulted in reduced levels of active RHOA, suggesting a pathway effect.To determine if the altered migration patterns noted in GNA13 deficient mice were due to loss of RHOA signaling, we crossed RhoA conditional knockout mice with mice expressing Cre under the AID promoter to generate deletion of RhoA exclusively in GC B cells. We then compared these mice with AID-Cre Gna13 knockout mice in parallel experiments. We found that GNA13 deficient mice demonstrate altered GC B migration dynamics, evidence of GC zonal disorganization, and reduced levels of filamentous actin. We also found that GNA13 is required for focal adhesion formation in the human Burkitt lymphoma derived cell line Raji. Similar to the GNA13 deficient state, we found that RHOA deficient GC B cells have altered dark zone and light zone dynamics, with an increased proportion of GC B cells expressing GC light zone markers, and reduced levels of filamentous actin.These data suggest that focal adhesion genes, including RHOA and GNA13, may promote lymphoma in GC B cells via aberrant migration and cellular adhesion, processes normally essential to intrazonal cycling, affinity selection, and GC B cell maturation. To our knowledge, this represents the in vivo characterization of RHOA in GC B cells and RHO mutations in B cell lymphomas and points to an important oncogenic role for RHOA in the germinal center niche. DisclosuresNo relevant conflicts of interest to declare.

Abstract 637: Preclinical activity of hLL2-PBD, a novel anti-CD22 antibody-pyrrolobenzodiazepine (PBD) conjugate in models of non-Hodgkin lymphoma

Abstract CD22 is a type I transmembrane sialoglycoprotein, which expression is restricted to the B-cell lineage. CD22 is also found highly expressed on most malignant mature B cells, including follicular non-Hodgkin lymphoma (NHL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), small lymphocytic lymphoma (SLL) and chronic lymphocytic leukemia (CLL). The differential and favorable expression profile of CD22 in normal and tumor tissues, together with its rapid internalization upon binding with a ligand or antibody, make CD22 an attractive target for an antibody drug conjugate (ADC) mediated treatment of B-cell malignancies. hLL2-PBD is an ADC composed of the humanized monoclonal antibody epratuzumab (hLL2) against human CD22 stochastically conjugated via a valine-alanine cleavable, maleimide linker to a PBD dimer. PBD dimers represent a novel class of payloads, which bind in the DNA minor groove and form highly cytotoxic DNA interstrand cross-links. The average drug to antibody ratio (DAR) of hLL2-PBD is 2. In vitro, hLL2-PBD showed potent and specific cytotoxicity, as assessed by the MTS assay, in the CD22-positive human Burkitt's lymphoma-derived cell lines Ramos and Daudi and the human diffuse large B-cell lymphoma-derived cell line WSU-DLCL2. In vivo, hLL2-PBD demonstrated potent anti-tumor efficacy in subcutaneously (s.c.) implanted Daudi and Ramos xenograft models. In the Daudi model, hLL2-PBD, administered as a single dose at either 0.1 or 0.3 mg/kg, achieved dose-dependent anti-tumor activity, resulting in significant increase in survival. At 0.3 mg/kg, hLL2-PBD induced complete tumor regression in all treated animals. In the Ramos xenograft model, a single dose of 1 mg/kg of hLL2-PBD achieved strong tumor regression. Moreover, hLL2-PBD achieved a superior anti-tumor activity compared to Hu10F4-vcMMAE, a CD22-targeting ADC with the auristatin payload vcMMAE and a DAR of 4 (Genentech Inc.), when tested at 1 mg/kg, single dose. Pharmacokinetic (PK) analysis of hLL2-PBD in CB.17 SCID mice showed a favorable PK profile, with a half-life of approximately 12 days. In conclusion, these data demonstrate the potent in vitro and in vivo anti-tumor activity of hLL2-PBD against CD22-positive hematological tumors and warrant further development of this ADC into the clinic. Citation Format: Francesca Zammarchi, David Williams, Karin Havenith, Francois D'Hooge, Philip W. Howard, John A. Hartley, Patrick van Berkel. Preclinical activity of hLL2-PBD, a novel anti-CD22 antibody-pyrrolobenzodiazepine (PBD) conjugate in models of non-Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 637. doi:10.1158/1538-7445.AM2015-637

Sodium arsenite induces apoptosis and Epstein-Barr virus reactivation in lymphoblastoid cells.

Epstein-Barr virus (EBV) is associated with several malignancies, including carcinomas, such as nasopharyngeal carcinoma, and lymphomas, such as Burkitt's lymphoma and Hodgkin's lymphoma. The Latent Membrane Protein 1 (LMP1) is the major oncogene protein of EBV as its expression is responsible for the induction of cell transformation, immortalization and proliferation. Arsenic trioxide was shown to induce a cytotoxic effect on nasopharyngeal cancer cells associated with LMP1 down-regulation. However, the effect of arsenic on EBV-associated lymphoproliferative malignancies has been less studied. We investigated the effect of two different arsenical compounds, arsenic trioxide (As2O3) and sodium arsenite (NaAsO2) on the induction of cell death in P3HR1 cells, an Epstein-Barr virus-positive Burkitt lymphoma derived cell line. Both compounds inhibited cell growth and induced cell death. By flow-cytometry and Western blot analysis, we provide evidence that NaAsO2 induced caspase-dependent apoptosis whereas As2O3 triggered autophagic cell death. Furthermore, we show that NaAsO2 treatment led to a dramatic decrease of the expression level of LMP1 and the cellular protein PML. Importantly, this down-regulation was associated with a reactivation of EBV lytic cycle through the induction of immediate-early proteins Zta and Rta. These results are in agreement with a model in which LMP1 maintains EBV in a latent state by stabilizing PML expression. Altogether, our results suggest that NaAsO2 would represent a better therapeutic candidate than As2O3 in EBV-induced B lymphoma for its capacity to promote viral reactivation.

The radial nuclear positioning of genes correlates with features of megabase-sized chromatin domains

A nonrandom radial nuclear organization of genes has been well documented. This study provides further evidence that radial positioning depends on features of corresponding ∼1Mbp chromatin domains (CDs), which represent the basic units of higher-order chromatin organization. We performed a quantitative three-dimensional analysis of the radial nuclear organization of three genes located on chromosome 1 in a DG75 Burkitt lymphoma-derived cell line. Quantitative real-time polymerase chain reaction revealed similar transcription levels for the three selected genes, whereas the total expression strength (TES) calculated as the sum of transcription of all genes annotated within a surrounding window of about 1Mbp DNA differed for each region. Radial nuclear position of the studied CDs correlated with TES, i.e., the domain with the highest TES occupied the most interior position. Positions of CDs with stable TES values were stably maintained even under experimental conditions, resulting in genome-wide changes of the expression levels of many other genes. Our results strongly support the hypothesis that knowledge of the local chromatin environment is essential to predict the radial nuclear position of a gene.

Antitumor Activity of Amphibian Ribonucleases, Onconase and R-Amhinase, on Tumor Cells From B-Cell Lymphoproliferative Disorders

AbstractAbstract 2842 Background:Several B-cell-derived malignancies still remains incurable. A promising approach that involves targeting RNA either by the use of specific antisense oligonucleotides or cytostatic/cytotoxic ribonucleases (RNases) is being promoted. Two amphibian RNases, onconase (ONC; ranpirnase) and, more recently, R-Amphinase (r-Amph), have already been developed, but studied so far mainly in solid tumors. In this report we demonstrate ex vivo and in vivo antitumor activity of ONC and, for the first time, R-Amph against tumor cells from different B-cell lymphoid malignancies. Material and methods:Pro-apoptotic effects of ONC or r-Amph alone and in combinations with doxorubicin (DOX) or purine analoques, fludarabine (FA) and cladribine (2-chlodeoxyadenozine; 2-CDA) were assessed ex vivo in peripheral blood samples of untreated patients with acute lymphoblastic leukemia; ALL, N=15) and chronic lymphocytic leukemia (CLL, N=45). Lymphocytes obtained from 31 healthy volunteers was also treated. Additionally, we tested in vitro activity of both agents alone or in combination with doxorubicin (DOX) on cell lines derived from Burkitt lymphoma (Raji) and with DOX, vincristin (VCR), dexamethasone (DEX), mafosfamide (MAF) or anti-CD20 antibody, rituximab (RIT) on diffuse large B-cell lymphoma (DLBCL)-derived cells (Toledo). Moreover, multiple myeloma (MM) cells were treated in vitro (RPMI 8226) and ex vivo (bone marrow cells). Drug-induced apoptosis was assessed after 48–72 hr incubation by the flow cytometry Annexin-V assay. Additionally, a drop of mitochondrial potential, activation of caspases -8, -9 and -3, and expression of several apoptosis-regulating proteins were investigated. Compensated apoptotic index (CAI) was calculated as a difference in % of apoptotic cells between the drug-treated sample and the parallel untreated culture. Results:Based on preliminary studies on different ex vivo and in vitro experimental models, concentrations 10–20 mg/ml of ONC and 20–60 mg/ml of r-Amph were chosen for final studies as the lowest doses inducing significant pro-apoptotic effect. In similar manner concentrations 50 ng/ml of 2-CdA, 1mg/ml of FA, 10–20 mg/ml of DOX, 20 mg/ml of RIT, 1ng/ml of VCR, 20 mg/ml of DEX and 1 mg/ml of MAF were selected.In CLL cells, significant effect of both ONC and r-Amph was evident after 72 hrs of treatment (median CAI 17.1% and 13.9%; vs untreated control cells p=0.009 and 0.012, respectively). Importantly, the same doses did not affect survival of healthy lymphocytes. Combination of ONC, but not r-Amph, with 2-CdA or FA exerted significantly stronger effect than single drugs (p<0.005).In case of ALL cells only ONC+DOX combination showed significantly higher pro-apoptotic effect compared to the single agents (p<0.005).In MM cells, only r-Amph alone induced significant cytotoxity vs. control (p<0.05 both in vitro and ex vivo).Both ONC and r-Amph showed significant anti-tumor activity in Burkitt lymphoma-derived Raji cell line (vs. control - p=0.007 and p=0,001, respectively). Moreover, combinations of ONC or r-Amph with DOX induced higher pro-apoptotic effect than single agents (p<0.001).Interestingly, both ONC and r-Amph were highly active against DLBCL-derived cell line and even at the lowest concentrations induced significant toxicity. We mimicked in vitro DLBCL standard therapeutic model using RIT, MAF, DOX, VCR and DEX (R-CHOP regimen). We found, that addition of either ONC or r-Amph to this scheme significantly amplified pro-apoptotic effect against DLBCL cell (p<0.001 vs. “R-CHOP”).The mechanism of both ONC and r-Amph cytotoxicity in all examined models was induction of apoptosis, evidently dependent on both, mitochondrial and external caspase-activation pathways (caspase -9, -8, -3; decline of mitochondrial potential). Both ONC and r-Amph induced over-expression of Bax and Bak proteins and down-regulation of Bcl-2 expression. Conclusions:Our study revealed strong pro-apoptotic activity of ONC and r-Amph in both CLL and aggressive B-cell lymphomas, with less impact for surviving of ALL or MM cells. Amplification of anti-tumor effect of R-CHOP regimen against DLBCL cells by studied RNases suggests that application of such combination of drugs may be considered to increase the cure rate in this aggressive lymphoma. Certainly, this strategy requires further studies, including an in vivo preclinical models and, eventual clinical trials. Disclosures:Ardelt:TAmiR Biotechnology, Inc: Employment, Membership on an entity's Board of Directors or advisory committees. Ardelt:Alfacell: Employment. Saxena:TAmiR Biotechnology, Inc: Employment. Muniz:TAmiR Biotechnology, Inc: Employment, Membership on an entity's Board of Directors or advisory committees.

High Resolution Genome-Wide Analysis of Chromosomal Alterations in Burkitt's Lymphoma

Additional chromosomal abnormalities are currently detected in Burkitt's lymphoma. They play major roles in the progression of BL and in prognosis. The genes involved remain elusive. A whole-genome oligonucleotide array CGH analysis correlated with karyotype and FISH was performed in a set of 27 Burkitt's lymphoma-derived cell lines and primary tumors. More than half of the 145 CNAs<2 Mb were mapped to Mendelian CNVs, including GSTT1, glutathione s-transferase and BIRC6, an anti-apoptotic protein, possibly predisposing to some cancers. Somatic cell line-specific CNVs localized to the IG locus were consistently observed with the 244 K aCGH platform. Among 136 CNAs >2 Mb, gains were found in 1q (12/27), 13q (7/27), 7q (6/27), 8q(4/27), 2p (3/27), 11q (2/27) and 15q (2/27). Losses were found in 3p (5/27), 4p (4/27), 4q (4/27), 9p (4/27), 13q (4/27), 6p (3/27), 17p (3/27), 6q (2/27),11pterp13 (2/27) and 14q12q21.3 (2/27). Twenty one minimal critical regions (MCR), (range 0.04–71.36 Mb), were delineated in tumors and cell lines. Three MCRs were localized to 1q. The proximal one was mapped to 1q21.1q25.2 with a 6.3 Mb amplicon (1q21.1q21.3) harboring BCA2 and PIAS3. In the other 2 MCRs, 1q32.1 and 1q44, MDM4 and AKT3 appeared as possible drivers of these gains respectively. The 13q31.3q32.1 <89.58–96.81> MCR contained an amplicon and ABCC4 might be the driver of this amplicon. The 40 Kb 2p16.1 <60.96–61> MCR was the smallest gained MCR and specifically encompassed the REL oncogene which is already implicated in B cell lymphomas. The most frequently deleted MCR was 3p14.1 <60.43–60.53> that removed the fifth exon of FHIT. Further investigations which combined gene expression and functional studies are essential to understand the lymphomagenesis mechanism and for the development of more effective, targeted therapeutic strategies.

Epigenetic Silencing of BIM Mediates Chemotherapy Resistance of Patients with Burkitt Lymphoma That Can Be Overcome by Therapeutic Reactivation of BIM in Mouse and Human Lymphoma Models

The most critical challenge in Burkitt lymphoma is to achieve complete remission (CR) with initial high-dose chemotherapy, as most of these patients will be eventually cured. However, those not achieving CR usually die early after diagnosis. Thus, to identify the refractory cases and how to overcome initial therapeutic resistance remain unsolved issues. We have previously reported that the gene encoding the pro-apoptotic BH3-only protein Bim is frequently inactivated by genetic and epigenetic mechanisms that substantially vary among the different B-cell lymphoma subgroups. Further screening of lymphoma samples revealed that silencing of BIM by promoter hyper-methylation occurred predominantly in Burkitt lymphoma, including 22 of 47 (47%) primary biopsies and 15 of 18 (83%) cell lines, while two additional Burkitt lymphoma-derived cell lines presented BIM homozygous deletion. BIM methylation was not associated with EBV tumor status or with the presence of MYC coding mutations. Quantitative ChIP revealed that most methylated lymphomas also showed deacetylation of histone 3 in BIM promoter mediated by HDAC complexes, but Bim post-transcriptional modifications were rarely observed. In contrast to lymphomas with unmethylated BIM, methylated/deacetylated tumors did not show alterations in other BCL2-family proteins, suggesting that transcriptional repression of Bim is critical and sufficient to impair apoptosis in Burkitt lymphoma. Indeed, in a series of clinically aggressive Burkitt lymphoma/leukemia, BIM hypermethylation was associated with inferior CR rate (only 24% of methylated cases achieved CR vs. 83% of unmethylated cases, p=0.0002) and shorter overall survival (p=0.007). These data suggested that Bim silencing mediated initial therapeutic resistance in Burkitt lymphoma. To test this hypothesis, several Burkitt lymphoma cell lines were genetically manipulated by ectopic expression of BIM using retroviral vectors or by RNAi, and then incubated with various chemotherapeutic agents commonly used in Burkitt lymphoma therapy. Resistance to doxorubicin and impaired apoptosis were selectively correlated with decreased Bim expression levels. Accordingly, treatment of apoptotic-resistant cell lines with demethylating and/or histone deacetilase inhibitors alone or in combination restored Bim expression and reversed chemotherapeutic resistance in vitro. Concordant results were obtained by treating Burkitt lymphoma xenografts with different Bim expression levels that were developed in RAG2−/−IL2gc−/− immunodeficient mice. Finally, B-cell lymphomas generated in Emu-MYC transgenes crossed with BIM knock-out mice were more resistant to apoptosis after doxorubicin treatment than Emu-MYC lymphomas. In summary, our data support thatchemotherapy resistance in patients with Burkitt lymphoma is frequently mediated by epigenetic alteration of pro-apoptotic Bim; andthis resistance can be overcome by reactivating Bim expression with demethylating agents such as decitabine and/or histone deacetylase inhibitors.These drugs, in combination with high-dose chemotherapy, may be used in the up-front therapy of patients with Burkitt lymphoma to prevent refractory disease.

Regulation of pri-microRNA BIC transcription and processing in Burkitt lymphoma

BIC is a primary microRNA (pri-miR-155) that can be processed to mature miR-155. In this study, we show the crucial involvement of protein kinase C (PKC) and nuclear factor-kappaB (NF-kappaB) in the regulation of BIC expression upon B-cell receptor triggering. Surprisingly, Northern blot analysis did not reveal any miR-155 expression upon induction of BIC expression in the Burkitt lymphoma-derived Ramos cell line, whereas other microRNAs were clearly detectable. Ectopic expression of BIC in Ramos and HEK293 cells resulted in miR-155 expression in HEK293, but not in Ramos cells, suggesting a specific block of BIC to miR-155 processing in Ramos. In line with the results obtained with Ramos, lack of miR-155 expression after induction of BIC expression was also observed in other Burkitt lymphoma cell lines, indicating a generic and specific blockade in the processing of BIC in Burkitt lymphoma. In contrast, induction of BIC expression in normal tonsillar B cells resulted in very high levels of miR-155 expression and induction of BIC expression in Hodgkin's lymphoma cell lines. It also resulted in elevated levels of miR-155. Our data provide evidence for two levels of regulation for mature miR-155 expression: one at the transcriptional level involving PKC and NF-kappaB, and one at the processing level. Burkitt lymphoma cells not only express low levels of BIC, but also prevent processing of BIC via an, as yet, unknown mechanism.

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.

Epstein-Barr Virus Nuclear Antigen 2 trans -Activates the Cellular Antiapoptotic bfl-1 Gene by a CBF1/RBPJκ-Dependent Pathway

The human herpesvirus Epstein-Barr virus (EBV) establishes latency and promotes the long-term survival of its host B cell by targeting the molecular machinery controlling cell fate decisions. The cellular antiapoptotic bfl-1 gene confers protection from apoptosis under conditions of growth factor deprivation when expressed ectopically in an EBV-negative Burkitt's lymphoma-derived cell line (B. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000), and the EBV latent membrane protein 1 (LMP1) and its cellular functional homologue CD40 can both drive bfl-1 via an NF-kappaB-dependent enhancer element in the bfl-1 promoter (B. N. D'Souza, L. C. Edelstein, P. M. Pegman, S. M. Smith, S. T. Loughran, A. Clarke, A. Mehl, M. Rowe, C. Gélinas, and D. Walls, J. Virol. 78:1800-1816, 2004). Here we show that the EBV nuclear antigen 2 (EBNA2) also upregulates bfl-1. EBNA2 trans-activation of bfl-1 requires CBF1 (or RBP-J kappa), a nuclear component of the Notch signaling pathway, and there is an essential role for a core consensus CBF1-binding site on the bfl-1 promoter. trans-activation is dependent on the EBNA2-CBF1 interaction, is modulated by other EBV gene products known to interact with the CBF1 corepressor complex, and does not involve activation of NF-kappaB. bfl-1 expression is induced and maintained at high levels by the EBV growth program in a lymphoblastoid cell line, and withdrawal of either EBNA2 or LMP1 does not lead to a reduction in bfl-1 mRNA levels in this context, whereas the simultaneous loss of both EBV proteins results in a major decrease in bfl-1 expression. These findings are relevant to our understanding of EBV persistence, its role in malignant disease, and the B-cell developmental process.

Motexafin gadolinium modulates levels of phosphorylated Akt and synergizes with inhibitors of Akt phosphorylation

Motexafin gadolinium (MGd, Xcytrin) is a tumor-selective expanded porphyrin that targets oxidative stress-related proteins. MGd treatment of the follicular lymphoma-derived cell line HF-1 resulted in growth suppression and apoptosis whereas MGd treatment of the Burkitt's lymphoma-derived cell line Ramos resulted in growth suppression but not apoptosis. Because phosphorylation status of Akt/protein kinase B is regulated by oxidative stress, we monitored total and phosphorylated Akt (pAkt) in MGd-treated HF-1 and Ramos cells. Levels of pAkt increased within 30 minutes after MGd treatment of HF-1 but after 4 hours began to show a progressive decline to below baseline levels before cells underwent apoptosis. In MGd-treated Ramos cells, pAkt increased approximately 2-fold within 4 hours and remained persistently elevated. Because pAkt activates survival pathways, we determined if MGd-induced cell death could be enhanced by inhibiting phosphorylation of Akt. The addition of specific inhibitors of Akt phosphorylation (Akt inhibitor 1 or SH-5) reduced pAkt levels in MGd-treated HF-1 and Ramos cells and synergistically enhanced MGd-induced cell death. MGd was also evaluated in combination with celecoxib, an inhibitor of Akt phosphorylation, or docetaxel, a microtubule inhibitor that can decrease Akt phosphorylation. The combination of MGd/celecoxib or MGd/docetaxel resulted in decreased Akt phosphorylation and in synergistic cytotoxicity compared with either agent alone. These data point to a potential protective role for pAkt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by combining it with agents that inhibit Akt phosphorylation.

Caspase-independent killing of Burkitt lymphoma cell lines by rituximab

Caspase-independent cell death may have a critical role to play in the therapeutic destruction of tumours. Recently it has been suggested that one of the mechanisms by which rituximab, a therapeutic anti-CD20 antibody, kills B cells is caspase-independent. In this study we show that rituximab can induce death in a variety of Burkitt lymphoma derived cell lines. Rituximab-treated cells show leakage of adenylate kinase, surface expression of phosphatidylserine, upregulation of the cellular stress protein HSP70, phosphorylation of the survival protein Akt, and depolarisation of the mitochondrial membrane but no loss of cytochrome c or apoptosis inducing factor. Caspase inhibitors do not block these events. In support of these data there is no cleavage of caspases 3, 8 and 9, poly(ADP-ribose) polymerase, BH3 interacting domain death agonist or genomic DNA. Morphologically, cells show nuclear enlargement and cytoplasmic vacuolisation. Triggering of receptor mediated death in CD95 responsive lines results in "classical" apoptosis indicating that the internal machinery necessary for apoptosis is intact in these lines. The results suggest that rituximab can kill human B cells via a caspase-independent form of programmed cell death that shares features of apoptosis and necrosis. This pathway may be relevant to the clinical efficacy of rituximab.

Motexafin Gadolinium Upregulates pAkt Levels and Synergizes with Inhibitors of Akt Phosphorylation.

Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective expanded porphyrin that targets oxidative stress related proteins. MGd treatment of the follicular lymphoma-derived cell line HF-1 resulted in growth suppression and apoptosis, whereas MGd treatment of the Burkitt's lymphoma-derived cell line Ramos resulted in growth suppression but not apoptosis. Since phosphorylation status of Akt/PKB kinase is regulated by oxidative stress, we monitored total and phosphorylated Akt (pAkt) in MGd treated HF-1 and Ramos cells. Levels of total and pAkt increased within 30 min after MGd treatment of HF-1 but within 4 hrs began to show a progressive decline to below baseline levels prior to cells undergoing apoptosis. Levels of total Akt did not increase in MGd treated Ramos cells but pAkt increased about 2 fold within 8 h and remained persistently elevated. Since pAkt activates survival pathways, we determined if MGd-induced cell death could be enhanced by inhibiting phosphorylation of Akt. The addition of specific inhibitors of Akt phosphorylation (SH-5 or Akt inhibitor 1) reduced pAkt levels in MGd treated HF-1 and Ramos cells and synergistically enhanced MGd-induced cell death. MGd was also evaluated in combination with celecoxib, an inhibitor of Akt phosphorylation, or docetaxel, a microtubule inhibitor which can decrease Akt phosphorylation. The combination of MGd/celecoxib or MGd/docetaxel resulted in decreased Akt phosphorylation and in synergistic cytotoxicity compared to either agent alone. These data point to a potential protective role for pAkt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by combining it with agents that inhibit Akt phosphorylation.

Biallelic deletion and loss of expression analysis of genes at FRA2G common fragile site in tumor-derived cell lines

Common fragile sites (CFS) are regions of chromosome instability that show gaps or breaks when cells are exposed to particular culture condition. Much evidence suggests that CFSs are causally related to cancer as breakpoints in recurrent chromosome mutations and as sites of viral integration. We investigated the FRA2G CFS (2q31) for biallelic deletions and loss of expression in a panel of 19 tumor-derived cell lines. We found that Burkitt lymphoma–derived cell line DAUDI has a biallelic deletion of eight of the nine analyzed genes. Moreover, we observed loss of expression (LOE) of the DHRS9 gene (alias RDHL), one of the deleted genes in the DAUDI cell line, in MOLT-14 and Raji cell lines derived from Burkitt lymphoma and from T-cell acute lymphoblastic leukemia, respectively. DHRS9 is involved in development and differentiation pathways.

High Expression of Micro-RNA BIC / miR155 in All Subtypes of Hodgkin Lymphoma.

We recently demonstrated that the noncoding BIC gene was highly expressed in Hodgkin Reed-Sternberg (HRS) cells of the vast majority of both classical and nodular lymphocyte predominant (NLP) Hodgkin lymphoma (HL). The BIC gene belongs to the family of noncoding mRNA-like molecules and was suggested to function through its noncoding RNA transcript. The human BIC gene shows a high degree of sequence identity to the Mouse and Gallus gallus BIC genes, which was most pronounced in a region containing a ~70 nucleotide stem-loop structure. Recently published data revealed expression of miR155 micro-RNA in murine tissue. The miR155 sequence was identical to the first 22 nucleotides of the ~70 nucleotide stem-loop structure of the BIC gene indicating that BIC is a primary micro-RNA transcript. Micro-RNAs (miRNA) are endogenous small RNAs (20–22 nucleotides) that inhibit protein translation by paring to the messages of protein-coding mRNAs. Based on recent data, it is clear that deregulation of miRNA expression, processing or stability may lead to deregulation of important cellular processes and thereby contribute to the development of cancer.In this study we analyzed the expression of miR155 in HL and NHL samples and studied the regulation of BIC/miR155 expression. Northern blot analysis revealed that HL cell lines, which all have a high level of BIC transcripts, also have high levels of the functionally mature miR155. NHL samples with low levels of BIC transcripts also have low levels of miR155. These data provide experimental support for processing of primary BIC transcripts to the functionally mature miR155 fragments and indicate that miR155 expression is specific for HL. To analyze whether BIC expression could be induced in NHL cell lines we studied several stimuli in the wild type Burkitt lymphoma derived cell line Ramos and a NF-κB deficient variant cell line. Real-time RT-PCR analysis revealed a high level of BIC and of the 70 nucleotide miR155 precursor in anti-IgM stimulated Ramos cells. Anti-IgM stimulation of the NF-κB deficient Ramos cell line revealed only low levels of BIC and pre-miR155. These data demonstrate that transactivation of NF-κB is required for continuous upregulation of BIC in Ramos cells. Stimulation of Ramos cells with PMA/calcium-ionophore, which directly activates PKC, significantly upregulated the level of BIC and pre-miR155. This suggests that activation of PKC and its downstream signaling components, including NF-κB are involved in the regulation of BIC expression. Thus, whereas HRS cells typically do not express surface immunoglobulin, upregulation of BIC in these cells may be induced by constitutively activated NF-κB.

The early transcription factor GATA‐2 is expressed in classical Hodgkin's lymphoma

Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin's lymphoma (cHL) are thought to be derived from germinal centre B-cells in almost all cases. However, expression profiling has revealed that HRS cells do not show a germinal centre B-cell-like phenotype. Although the nature of this aberrant phenotype and the underlying molecular mechanisms remain largely unknown, it has been reported that the activity of NOTCH1 plays an important role in the growth and survival of HRS cells. In some leukaemic cell lines, the effect of Notch signalling is mediated by the early transcription factor GATA-2. This and the fact that HRS cells lack expression of PU.1, which can repress Gata-2, led to an investigation of GATA-2 expression in HRS cells. GATA-2 expression was found in all the cHL-derived cell lines studied, but not in a Burkitt lymphoma-derived cell line. In addition, 50% of biopsies from patients with cHL contained GATA-2-expressing HRS cells. In contrast, neither normal germinal centre B-cells nor malignant cells of nodular lymphocyte-predominant Hodgkin's lymphoma, Burkitt lymphoma or diffuse large B-cell lymphoma expressed GATA-2. Thus, GATA-2 expression was found specifically in HRS cells of cHL, suggesting that GATA-2 is important in establishing the abnormal B-cell phenotype of HRS cells.

Nuclear factor kappa B-dependent activation of the antiapoptotic bfl-1 gene by the Epstein-Barr virus latent membrane protein 1 and activated CD40 receptor.

Suppression of the cellular apoptotic program by the oncogenic herpesvirus Epstein-Barr virus (EBV) is central to both the establishment of latent infection and the development of EBV-associated malignancies. We have previously shown that expression of the EBV latent membrane protein 1 (LMP1) in Burkitt's lymphoma cell lines leads to increased mRNA levels from the cellular antiapoptotic bfl-1 gene (also known as A1). Furthermore, ectopic expression of Bfl-1 in an EBV-positive cell line exhibiting a latency type 1 infection protects against apoptosis induced by growth factor deprivation (B. N. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000). We now report that LMP1 drives bfl-1 promoter activity through interactions with components of the tumor necrosis factor receptor (TNFR)/CD40 signaling pathway. We present evidence that this process is NF-kappa B dependent, involves the recruitment of TNFR-associated factor 2, and is mediated to a greater extent by the carboxyl-terminal activating region 2 (CTAR2) relative to the CTAR1 domain of LMP1. Activation of CD40 receptor also led to increased bfl-1 mRNA levels and an NF-kappa B-dependent increase in bfl-1 promoter activity in Burkitt's lymphoma-derived cell lines. We have delineated a 95-bp region of the promoter that functions as an LMP1-dependent transcriptional enhancer in this cellular context. This sequence contains a novel NF-kappa B-like binding motif that is essential for transactivation of bfl-1 by LMP1, CD40, and the NF-kappa B subunit protein p65. These findings highlight the role of LMP1 as a mediator of EBV-host cell interactions and may indicate an important route by which it exerts its cellular growth transforming properties.