scholarly journals The oncoprotein LMO2 is expressed in normal germinal-center B cells and in human B-cell lymphomas

Blood ◽  
2006 ◽  
Vol 109 (4) ◽  
pp. 1636-1642 ◽  
Author(s):  
Yasodha Natkunam ◽  
Shuchun Zhao ◽  
David Y. Mason ◽  
Jun Chen ◽  
Behnaz Taidi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
B Cell Lymphoma ◽  
Hierarchical Cluster ◽  
Tissue Expression ◽  
B Cell Lymphomas ◽  
Neoplastic Tissue ◽  
Associated Proteins

Abstract We previously developed a multivariate model based on the RNA expression of 6 genes (LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2) that predicts survival in diffuse large B-cell lymphoma (DLBCL) patients. Since LMO2 emerged as the strongest predictor of superior outcome, we generated a monoclonal anti-LMO2 antibody in order to study its tissue expression pattern. Immunohistologic analysis of over 1200 normal and neoplastic tissue and cell lines showed that LMO2 protein is expressed as a nuclear marker in normal germinal-center (GC) B cells and GC-derived B-cell lines and in a subset of GC-derived B-cell lymphomas. LMO2 was also expressed in erythroid and myeloid precursors and in megakaryocytes and also in lymphoblastic and acute myeloid leukemias. It was rarely expressed in mature T, natural killer (NK), and plasma cell neoplasms and was absent from nonhematolymphoid tissues except for endothelial cells. Hierarchical cluster analysis of immunohistologic data in DLBCL demonstrated that the expression profile of the LMO2 protein was similar to that of other GC-associated proteins (HGAL, BCL6, and CD10) but different from that of non-GC proteins (MUM1/IRF4 and BCL2). Our results warrant inclusion of LMO2 in multivariate analyses to construct a clinically applicable immunohistologic algorithm for predicting survival in patients with DLBCL.

CD40L Modulates TRAIL-Induced Apoptosis in Germinal Center Derived B Cell Lymphomas.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4630-4630
Author(s):  
Marion Travert ◽  
Patricia Ame-Thomas ◽  
Thierry Fest ◽  
Céline Pangault ◽  
Gilbert Semana ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Lymphoma Cells ◽  
Over Expression ◽  
Induced Apoptosis ◽  
Transformed Cell Lines

Abstract Follicular lymphoma are characterized by the rearrangement of the bcl-2 gene, present in more than 90% of patients. Over-expression of the bcl-2 protein resulting from this translocation is associated with the inability to eradicate the lymphoma, by inhibiting apoptosis. Despite the median survival ranges from 8 to 15 years, leading to the designation of indolent lymphoma, patients with advanced-stage follicular lymphoma are not cured with current therapeutic options. Numerous reports have shown that Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in a wide variety of transformed cell lines of diverse lineage, but does not appear to kill normal cells, even though TRAIL mRNA is expressed at significant levels in most normal tissues. As cell death induced by TRAIL occurs almost exclusively in tumor cells, it suggests that this drug is safe to use as an antitumor therapy. We therefore investigated the efficiency of this cytokine to induce apoptosis in germinal center derived B cell lymphoma, despite bcl-2 over-expression. Our study was also designed to evaluate the role of CD40L, one of the main differentiation signal involved in B cell maturation during the germinal center reaction, on the regulation of TRAIL-induced apoptosis. This study was performed on three germinal center derived tumor cell lines (BL2, VAL and RL), and on normal and tumor primary cells obtained from human tonsils and lymph nodes. Our data show that normal B lymphocytes obtained from tonsil biopsies are resistant to TRAIL-mediated apoptosis, when B lymphoma cells issued from lymph node of numerous patients are significantly sensitive to the cytokine. When we treat these lymphoma cells with trimeric huCD40L, we partly rescue these cells from spontaneous apoptosis which naturally occurs after few days of culture, and reverse by 50% TRAIL-mediated apoptosis when cells were co-treated with huCD40L for 16 hours. Similar results were reproduced on some germinal center derived cell lines. BL2 was indeed found highly sensitive to TRAIL-induced apoptosis following a 24 hour exposure. On the opposite, VAL and RL were almost insensitive. We have demonstrate that apoptosis is exclusively mediated by TRAIL-R1 in BL2. Analysis of signalling pathways revealed that the protection to TRAIL-induced apoptosis by CD40L is due to some specific anti-apoptotic molecules that will be described. Genes encoding these molecules are targets of the NFκB signalling pathway activated by CD40L. Our results suggest that activation of NFκB and induction of anti-apoptotic molecules by CD40L play an important role in the protection of germinal center derived B cell lymphomas against apoptosis. Then, NFκB inhibitors may be wise to use in clinical trials in conjunction with TRAIL against follicular lymphomas.

Profiles Of De Novo CD25-Positive Mature B-Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4308-4308
Author(s):  
Shin-ichiro Fujiwara ◽  
Raine Tatara ◽  
Kiyoshi Okazuka ◽  
Iekuni Oh ◽  
Ken Ohmine ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
High Expression ◽  
B Cell Lymphomas ◽  
Cns Involvement ◽  
Cd25 Expression

Abstract Background Interleukin 2 (IL-2) is an important cytokine that controls the proliferation and differentiation of not only T- but also B-lymphocytes. Recently, we reported that CD25 (IL-2 receptor alpha chain, IL-2R) is expressed in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and high expression of CD25 in the two types of lymphoma is correlated with a poor prognosis following chemotherapy regimens containing rituximab (ASH annual meeting, 2011 118:2666, 2012 120:1543). We evaluated the clinical significance of CD25 expression in a larger series of different mature B-cell lymphomas (BCL). Patients and Methods Four hundred and thirty-seven newly diagnosed patients who were admitted to our hospital between 2002 and 2013 were retrospectively evaluated. Lymph node or related tissue biopsy samples of BCL were analyzed using flow cytometry, as follows: 182 patients, DLBCL; 92, FL; 48, chronic lymphocytic leukemia (CLL); 21, mantle cell lymphoma (MCL); 23, marginal zone lymphoma (MZL); 8, Burkitt lymphoma (BL); 18, B-cell lymphoma unclassifiable with features intermediate between BL and DLBCL (BL/DLBCL); 5, lymphoplasmacytic lymphoma (LPL); and 39, reactive lymphadenopathy with sufficient B-cells. CD25-positivity was defined as >20% of clonal B-cells in a gated region. Results CD25 expression in patients with MCL, CLL, MZL, and DLBCL was significantly higher than that in patients with reactive lymphadenopathy (P<0.001,<0.001, =0.019, and <0.001, respectively). BL and FL, which were derived from germinal center B-cells, did not express CD25. These results indicate that pre- or post- germinal center-derived B-cells, activated by IL-2/IL-2R signaling, may give rise to CD25+ BCL such as CD25+ MCL, CLL, MZL, and DLBCL. The highest median CD25 expression (41.5%) was observed in MCL. CD25 expression was higher in MCL than CD5+ BCL (CLL and CD5+ DLBCL) (median, 41.5 vs. 16.9%, respectively; P<0.001). With a cut-off value of 60% CD25-positivity, patients with CD25-high (>60%) MCL (n=9) were not treated with aggressive chemotherapy regimens such as Hyper-CVAD due to their age and characteristics, compared with those with CD25-low (<60%) MCL (n=12) (11.1 vs. 72.7%, respectively, P=0.021). In patients with CLL, the range of CD25 expression was wide (0.4-90.7%), and 29 patients (60%) showed CD25-positivity (CD25+ CLL). CD25+ CLL showed higher soluble IL-2R (sIL-2R) levels and an inferior overall survival (OS) than CD25- CLL (median sIL-2R, 2,195 vs. 706 U/ml P=0.047; 5-year OS, 62.7 vs. 100%; P=0.037). There was a significant correlation between levels of CD25 and sIL-2R (r=0.53, P=0.0053). It is clinically important to distinguish between DLBCL and BCL involving MYC oncogene rearrangement (BL and BL/DLBCL, MYC+ BCL). The former showed higher CD25 expression than the latter (median, 10.2 vs. 2.1%, respectively, P=0.04). The progression-free survival rate (PFS) after rituximab containing chemotherapy was inferior in patients with CD25+ DLBCL (n=72) than those with CD25- DLBCL (n=110) and MYC+ BCL (5-year PFS, 49 vs. 70.4, 66.3%, respectively). In patients with DLBCL, central nerve system (CNS) involvement was observed in 15 patients (7 at diagnosis and 8 at relapse). CD25+ DLBCL showed a higher frequency of CNS involvement than CD25– DLBCL (13.8 vs. 4.5%, respectively, P=0.049). Regarding MZL, CD25 was highly expressed in nodal MZL, but it showed a low expression in splenic MZL. Regarding the sites of extranodal MZL, CD25 expression was lower in the thyroid than at other sites (median, 5.1 vs. 21.2%, respectively, P=0.37). There were some differences between CD25+ (n=9) and CD25- (n=14) MZL concerning the presence of B symptoms (33.3 vs. 0%, respectively) and advanced stage (66.6 vs. 35.7%, respectively). Conclusion CD25 expression using flow cytometry can potentially provide diagnostic and prognostic implications on BCL patient. The high expression of CD25 in MCL and CLL suggests the possibility of targeted anti-CD25 immunotherapy. These findings may shed light on the role of CD25 expression in B-cell lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.

Constitutively Activated STAT3 Promotes Cell Proliferation and Survival in the Activated B Cell Subtype of Diffuse Large B Cell Lymphomas.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1621-1621
Author(s):  
Bihui Hilda Ye ◽  
Beibei Belinda Ding ◽  
Jian Jessica Yu ◽  
Raymond Y.-L. Yu ◽  
Lourdes M. Mendez ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
B Cell Lymphoma ◽  
Cell Development ◽  
B Cell Development ◽  
Reciprocal Relationship ◽  
B Cell Lymphomas ◽  
Large B Cell

Abstract During B cell development, cell proliferation and survival are regulated by stage-specific transcription factors. Accordingly, distinct oncogenic pathways are employed by B cell lymphomas representing different stages of B cell development. Diffuse large B cell lymphoma (DLBCL) contains at least two main phenotypic subtypes, i.e. the germinal center B cell-like (GCB-DLBCL) and the activated B cell-like (ABC-DLBCL) groups. It has been shown that GCB-DLBCL responds favorably to chemotherapy and expresses high levels of BCL6, a transcription repressor known to play a causative role in lymphomagenesis. In comparison, ABC-DLBCL has lower levels of BCL6, constitutively activated NF-kappaB and tends to be refractory to chemotherapy. In this study, we investigated the relationship between BCL6 and STAT3 expression/activation in DLBCL and normal GC B cells. Our results demonstrate that BCL6 directly inhibits transcription of the STAT3 gene by binding to two BCL6 sites in its 5′ regulatory region. As a result, high level STAT3 expression and activation are preferentially detected in ABC-DLBCL and BCL6-negative normal germinal center B cells. Specifically, in tonsillar GCs, STAT3 expression and activation is restricted to a previously uncharacterized subset of BCL6−Blimp-1− B cells in the apical light zone. The location and phenotype of these cells suggest that they are in the process of exiting the BCL6-directed GC program and transitioning to a plasma cell differentiation process governed by Blimp-1. The reciprocal relationship between BCL6 and STAT3 is also conserved in DLBCL such that STAT3 expression and activation is preferentially associated with the BCL6-low, ABC subtype. Most importantly, inactivating STAT3 by either AG490 or small interference RNA in ABC-DLBCL cells inhibits cell proliferation and triggers apoptosis. These phenotypes are accompanied by decreased expression of several known STAT3 target genes, including c-Myc, JunB and Mcl-1, and increased expression of the cell cycle inhibitor p27. In addition to identifying STAT3 as a novel BCL6 target gene, our results define STAT3 activation as a second oncogenic pathway operating in ABC-DLBCL and suggest that blocking STAT3 may be potentially therapeutic in treatment of these aggressive lymphomas.

Direct Growth Inhibition of Both Mouse and Human B Cell Lymphomas by CpG Oligodeoxynucleotides

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2854-2854
Author(s):  
Reiko E Yamada ◽  
David J Betting ◽  
Michael Ahdoot ◽  
Kristopher K Steward ◽  
John M Timmerman
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Cpg Oligodeoxynucleotides ◽  
Mantle Cell ◽  
Class B ◽  
Human B Cell

Abstract Abstract 2854 Immunostimulatory CpG oligodeoxynucleotides (ODN) are potent activators of T cell immunity and antibody-dependent cellular cytotoxicity (ADCC), and under study as immunotherapeutic agents for a variety of cancers, including B cell lymphomas. Recently, anti-CD20 antibody-CpG conjugates have been shown to eradicate rituximab-resistant B cell lymphoma in a syngeneic murine lymphoma model (D. Betting et al, ASH 2009). CpG is known to strongly stimulate the proliferation of normal B cells. Paradoxically, CpG has been reported to markedly inhibit the in vitro growth of the murine B cell lymphoma A20 (J. Li et al, J. Immunol. 2007), thereby prompting us to investigate the direct effects of CpGs on the growth of human B cell lymphomas. We first demonstrated that CpGs, especially those of the B class, potently inhibited proliferation of the A20 mouse B cell line in vitro by up to 81.5% (class A 58.7% and class C 52.7%). Moreover, in non-tumor bearing mice intratumoral injections of CpG activated normal B cells, while mice bearing subcutaneous A20 tumors showed suppressed tumor growth after CpG injections. Similarly, in humans, CpGs strongly stimulated the proliferation of normal peripheral blood B cells (stimulation index for class B 27.5 at 5 μg/ml). A panel of 12 human lymphoma cell lines (DLBCL, Burkitt's, mantle cell) were cultured in the presence or absence of varying concentrations of CpGs of A, B, or C classes (50, 10, or 2 μg/ml) or control ODN. Proliferation was measured by [3H]-thymidine incorporation in quadruplicate 72 hour cultures, and apoptosis measured by Annexin-V and PI flow cytometry. In contrast to the stimulation observed with normal human B cells, the proliferation of all 12 lymphoma lines were inhibited by CpGs. The strongest inhibitory effects were seen with CpG 7909, a class B CpG under clinical development for cancer therapy (Pfizer, PF-3512676). Raji cells were inhibited by 77.9%, 40.7%, and 8.8% at CpG concentrations of 50, 10, and 2 μg/ml, respectively (p≤0.01 for all comparisons vs. media alone). Among the 12 tested cell lines, the percentage growth inhibition using 50 μg/ml CpG 7909 was 61.2–80.4% for germinal center-type DLBCL (SUDHL-4, SUDHL-6, OCI-Ly19), 50–59.5% for activated B cell-type DLBCL (SUDHL-2, OCI-Ly3, OCI-Ly10), 56.4–79.3% for Burkitt's lymphomas (Raji, Ramos, Daudi, BJAB), and 69.6–69.9% for mantle cell lymphomas (Jeko-1, Granta-519). Interestingly, although all of the human cell lines expressed TLR9 by semi-quantitative RT-PCR, inhibition in the proliferation levels did not correlate with TLR9 expression levels. CpG 7909 also induced significant levels of apoptosis in Raji and Jeko-1 cells, 10.1% and 27.6% respectively at 50 μg/ml. In conclusion, we have demonstrated that CpGs have divergent effects on normal versus malignant B cells in both mouse and human systems. Delivery of CpG to mouse lymphoma cells inhibited their growth in vivo, while normal mouse B cells were activated. Furthermore, CpGs directly inhibit the proliferation of a large panel of human B cell lymphomas representing the majority of aggressive histologies. These results provide a novel mechanism of action for CpGs as therapeutic agents for B cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

Integrative Genomics Reveals a Role for GNA13 in Lymphomagenesis within the Germinal Center Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 782-782
Author(s):  
Jane Healy ◽  
Adrienne Greenough ◽  
Rachel Rempel ◽  
Moffitt Andrea ◽  
Izidore S Lossos ◽  
...  
Keyword(s):  
Cell Migration ◽  
B Cells ◽  
Tumor Suppressor ◽  
B Cell ◽  
Mouse Models ◽  
Cell Lines ◽  
Germinal Center ◽  
Burkitt Lymphoma ◽  
B Cell Lymphoma

Abstract Nonhodgkin Lymphoma (NHL) is among the most common cancer subtypes, with approximately >350,000 new cases diagnosed annually worldwide. The vast majority of NHLs arise from germinal center (GC) B cells. We and others have identified GNA13 as one of the most frequently mutated genes in GC-derived lymphomas, including ~30% of Burkitt Lymphoma and ~25% of Germinal Center B Cell-like (GCB) Diffuse Large B cell Lymphoma. Despite this association, the role of GNA13 in lymphomagenesis remains elusive. In human breast and prostate cancer, GNA13 behaves as an oncogene, with increased expression linked to cellular invasion and metastasis. Intriguingly, GNA13 mutations in GCB DLBCL and Burkitt Lymphoma are frequently inactivating, possessing a high number of nonsense and missense mutations in conserved domains. This suggests that GNA13 may function as a tumor suppressor in the context of lymphoma, in contrast to its role in solid tumors. The purpose of this study is to define the role of GNA13 in GC B cells and to clarify how GNA13 loss may contribute to lymphoma within the germinal center niche. We first investigated the expression pattern of GNA13 in lymphocyte populations from normal human tonsil. Our data demonstrated that GNA13 is enriched in GC B cells by quantitative PCR and immunohistochemistry. To determine the effect of GNA13 abundance on global mRNA expression patterns, we performed RNA sequencing on lymphoma derived cell lines. Using this method, we found that GNA13 knockdown and overexpression was highly correlated with GC dark and light zone gene signatures, respectively. We next devised a proteomics approach to identify potential GNA13 binding partners in GCs. Lysates from lymphoma-derived cell lines overexpressing FLAG-tagged GNA13 were subjected to immunoprecipitation with M2-antibody bound magnetic beads, followed by LC-MS/MS. Our results demonstrated an enrichment of proteins involved in focal adhesion, consistent with the known involvement of GNA13 in processes of cytoskeletal reorganization and cell migration. We next explored the role of GNA13 in vivo. Since GNA13 mutations are a unique feature of GC-derived lymphomas, we developed mouse models that would allow us study GNA13 exclusively in the germinal center context. We generated B cell and GC specific GNA13 knockout mice by crossing GNA13fl/fl mice with MB1-Cre and AID-Cre strains. After immunization with sheep red blood cells, both B cell and GC specific GNA13 deficient mice possessed normal levels of B, T and GC cells within secondary lymphoid sites including Peyer’s patches and spleen, suggesting that GNA13 is not essential for GC formation. GC B cells from both GNA13 deficient strains demonstrated enhanced cellular motility toward GC directed chemokines CXCL12, CXCL13 and S1P using in vitro transwell migration assays. Furthermore, B cells isolated from GNA13 deficient animals showed enhanced RhoA activity. These data suggested that GNA13 inhibits GC B cell migration and RhoA mediated cell motility in normal conditions. Loss of GNA13 may then deregulate normal chemokine gradient signaling, resulting in global increases in GC migration. We also demonstrated that GNA13 deficient B cells possess elevated levels of phosphorylated AKT, an effect potentiated by the addition of CXCL12 and S1P. AKT signaling is known to promote cell survival in a variety of cell types, which may further promote oncogenesis. In this study, we have synthesized the complementary approaches of next generation sequencing, proteomics and genetic mouse models to gain novel insight into the biological function of GNA13, a gene that is mutated in a high proportion of GC-derived lymphomas. As a whole, our work suggests that GNA13 serves as a tumor suppressor during the germinal center reaction. The acquisition of inactivating GNA13 mutations may promote lymphoma by allowing cells to physically escape the germinal center niche and evade apoptosis while continuing to express GC signature genes. Affected cells may be subjected to persistent somatic hypermutation, which, over time, could result in the accumulation of additional oncogenic mutations, culminating in development of GC-derived lymphoma. Disclosures No relevant conflicts of interest to declare.

Inactivation Of BANK1 By a Novel IGH-Associated Translocation and 5’ Hypermethylation In B-Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2497-2497 ◽  
Author(s):  
Kui Nie ◽  
Taotao Zhang ◽  
Jiong Yan ◽  
Leonardo Boiocchi ◽  
Shuhua Cheng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Methylation Status ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Intron 1 ◽  
Large B Cell

Abstract A novel IGH-associated reciprocal translocation, t(4;14)(q24;q32), was identified, along with trisomy 9, in 20 of 20 metaphases by conventional karyotyping in a case of malignant gastric post-transplant lymphoproliferative disorder (PTLD). Cloning of the translocation site by inverse PCR identified BANK1 (B-cell scaffold protein with ankyrin repeats 1), a B-cell-specific adaptor protein with putative functions in B-cell receptor and CD40 signaling, as a novel IGH translocation partner. The breakpoints were located at the Sα region of IGH and intron 1 of BANK1. The translocation juxtaposed the two genes in opposite orientations, and surprisingly, resulted in transcriptional inactivation of BANK1 as a result of dissociation of the major BANK1 promoter. While BANK1 isoforms were expressed in all tonsillar B-cells, with lower levels (∼ 5 fold) in the germinal centers (GC) compared to naïve and memory B-cells, transcription from the major promoter in the tumor was absent and transcription from the minor promoter was reduced 50% relative to GC B-cells, suggesting that the non-translocated BANK1 allele was also inactivated. The total BANK1 expression was very low (∼10% of normal GC B cells) and crytic promoter activation was not identified. Several genes (PPP3CA, MIR1255A, FLJ20021 and SLC39A8), located 180 to 440 kb away from BANK1, were analyzed for mRNA expression; there is no significant activation in any of these genes, further supporting that BANK1is indeed the target gene affected by the translocation. Interphase FISH using break-apart BANK1 probes confirmed breakpoint in the index case but did not identify translocations in additional 15 PTLDs and 68 diffuse large B-cell lymphomas (DLBCL), implying that BANK1 translocation may be a rare event. To determine if BANK1 inactivation may occur in B-cell lymphomas by other mechanisms, 23 B-cell lymphoma cell lines, including 8 Burkitt lymphoma (BL), 9 diffuse large B cell lymphoma (DLBCL), 3 primary effusion lymphoma (PEL), and 3 classical Hodgkin lymphoma (cHL) were bisulfite sequenced to assess the methylation status of 37 CpG dinucleotides in a 436 base-pair region at the 5’ end of BANK1, which extends across exon 1 into the 5’ portion of intron 1. High level of methylation (>60% methylation on average among all CpGs) was seen in all 3 cHL and 2 of 3 PEL cell lines. Regional methylation was seen in 3 of 8 BL lines and 1 of 3 PEL lines. No hypermemethylation was identified in the DLBCL lines or in normal tonsils. Hypermethylation was associated with almost complete silencing of BANK1 transcription. In the DLBCL lines and BL lines without BANK1 hypermethylation, BANK1mRNA expressions were variable, ranging from <5% to 130% of GCB cells. To confirm that BANK1 hypermethylation is present in primary lymphoma cases, methylation status of 17 of the 37 CpGs were assessed in 23 cHL cases using en bloc formalin-fixed, paraffin-embedded materials and also laser-capture micro-issected Hodgkin/Reed-Sternberg (HRS) cells. There was evidence of BANK1 hypermethylation in the tumor cells in 9 of 23 cHL. Tumor cell specificity of BANK1 hypermethylation was further confirmed in 4 cHL cases using micro-dissected HRS cells. HRS cells were negative for BANK1 in 28 of 29 cHL cases examined by immunohistochemistry, suggesting that other mechanisms other than DNA methylation may be responsible for silencing BANK1expression. To investigate whether BANK1 has biological effects on B-cells related to lymphoma development, exogenous BANK1 was re-introduced to BC3, a PEL cell line showing marked BANK1 hypermethylation with absence of BANK1 expression. We established a stable doxycycline-inducible BC3 cell line expressing BANK1. Inhibition of cell growth was observed 2 to 3 days after doxycyline induction, and the number of viable cells with transfected BANK1 was only 25% compared to BC3 cells carry vehicle alone at day 6. An analysis of 5-bromo-2’ deoxyuridine (BrdU) incorporation after 48 hours of doxycline induction revealed that the fraction of cells in S-phase was reduced by 50% in the BANK1 transfectants, suggesting that BANK1has a negative effect on cell proliferation in these B cells. In summary, we have identified a novel IGH translocation partner and provide an example of an unusual consequence (gene inactivation) of IGH-associated translocation. We provide for the first time evidence of a potential role of BANK1 down-regulation in the development of B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

MiR-28 Silencing In Germinal Center-Derived Lymphomas

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 703-703
Author(s):  
Christof Schneider ◽  
Roy L Maute ◽  
Pavel Sumazin ◽  
Manisha Brahmachary ◽  
Manu Setty ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Suppressor ◽  
B Cell ◽  
Cell Lines ◽  
Mirna Expression ◽  
Germinal Center ◽  
Expression Profiling ◽  
B Cell Lymphoma ◽  
Lymphoma Cells

Abstract Abstract 703 In order to gain insights into the role of microRNA (miRNAs) in mature B cell function and lymphomagenesis, we performed a comprehensive miRNA expression profiling of normal mature B cells and of germinal center (GC)-derived lymphomas. miRNA expression profiles were generated using a commercial array platform designed to interrogate approximately 700 miRNAs. Normal GC B cells were isolated from tonsil tissue of 5 pediatric healthy donors and subjected to miRNA profiling in parallel with tumor specimens obtained from 10 Burkitt Lymphoma (BL), 16 Follicular Lymphoma (FL) and 20 Diffuse Large B Cell Lymphoma (DLBCL) patients. Each tumor type displayed a distinct miRNA profile and appeared to be clearly separated from the normal counterpart. Interestingly, a set of miRNAs was expressed in normal GC cells, but not in lymphoma samples, suggesting that structural and/or functional loss of miRNAs occur during lymphomagenesis. Among these, miR-28 was found to be up-regulated in GC B cells, while it was completely silenced in BL and significantly reduced in a large fraction of DLBCL and FL. Quantitative RT-PCR analysis confirmed miR-28 reduced expression in these GC-derived lymphoma subtypes including both primary biopsies and cell lines. MiR-28 is an intragenic miRNA encoded by the LPP gene locus, located on chromosome 3q28. Deletions affecting miR-28 and LPP were previously reported in FL (Schwaenen et al. Genes Chromosomes Cancer 2009; 48:39-54) and similarly we identified LPP deletions in about 9% of DLBCL investigated for copy number alterations by SNP arrays. Further FISH analyses performed in cell lines lacking miR-28 expression (12 DLBCL and 27 BL) failed to identify chromosomal aberrations in the LPP locus, suggesting that mechanisms other than genetic losses, possibly of epigenetic nature, are involved in miR-28 silencing in lymphomas. In order to investigate the effects of miR-28 in lymphoma cells, we generated stable lymphoma cell lines displaying inducible expression of miR-28. Re-expression of miR-28 in lymphoma cells led to a retarded growth due to a combination of G1-cell-cycle arrest and increased apoptosis. Furthermore, lymphoma cells expressing miR-28 lost their clonogenic properties as shown by their inability to form colonies in soft agar. Taken together these results suggest a tumor suppressor function for miR-28 in lymphoma cells. Toward the identification of the direct miR-28 target genes, we implemented computational target prediction methods with gene expression profiling data obtained from the miR-28 engineered cell lines. Approximately two thousand miR-28 direct candidate targets were computationally predicted, 88 of which displayed transcriptional down-regulation upon miR-28 induction, suggesting that miR-28 may affect the stability of the target transcript. The candidate targets included genes involved in the control of cell proliferation and apoptosis and in cell signaling, consistent with the phenotypic changes induced by miR-28 expression. In 4 out of 5 tested candidate targets, 3′-UTR reporter gene assay confirmed the direct effect of miR-28 on the target gene. Finally, we have generated a conditional, GC B cell-specific miR-28 knock-out mouse model, which will provide critical insights on the physiologic as well as the tumor suppressor role of miR-28 in vivo. Disclosures: No relevant conflicts of interest to declare.

B Cell Lymphomas Escape Bone Morphogenetic Protein (BMP) Induced Growth Inhibition: Downregulation of BMP Receptors and Upregulation of Inhibitory Smads.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2605-2605 ◽  
Author(s):  
Kanutte Huse ◽  
Maren Bakkebo ◽  
Morten Oksvold ◽  
Erlend B. Smeland ◽  
June H. Myklebust
Keyword(s):  
B Cells ◽  
Growth Inhibition ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Resistant Cell ◽  
Lymphoma Cell ◽  
Receptor Expression ◽  
Type I ◽  
B Cell Lymphomas

Abstract Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily and mediate their effects mainly through the Smad signaling pathway. TGF-β is one of the most potent negative regulators in hematopoietic cells, and many cancers develop reduced sensitivity towards TGF-β induced growth inhibition by several mechanisms, including functional loss of TGF-β receptors and Smad proteins. We have previously shown that BMP-6 inhibits the growth of normal peripheral blood B cells. As high BMP-6 mRNA expression is associated with poor outcome in diffuse large B cell lymphoma (DLBCL; Rosenwald et al, N Engl J Med 2002), we hypothesized that reduced sensitivity towards BMP-induced growth inhibition might contribute to lymphomagenesis. In the current study, 10 B lymphoma cell lines (representing Burkitt, DLBCL and FL) and tumor material from lymphoma patients were investigated to unravel the role of BMPs in lymphomas. We found that 5 – 7 out of 10 lymphoma cell lines were resistant towards BMP-2, -4, -6 and -7 induced growth inhibition. In comparison, only 3 of the cell lines were resistant towards TGF-β. Analysis of BMP receptor expression by FACS analysis showed that all lymphoma cell lines and the malignant B cells from primary lymphoma biopsies expressed the BMPR type I Alk-2, whereas the expression of Alk-3 and Alk-6 was variable. Interestingly, the expression of BMPRII was low or undetectable in BMP-6 resistant cell lines, whereas it was highly expressed in 3 out of 4 sensitive cell lines. Also, malignant B cells from lymphoma biopsies showed reduced levels of BMPRII, suggesting that downregulation of BMPRII is a mechanism for evading BMP induced growth inhibition. Interestingly, upregulation of Smad6 or Smad7 was seen in 3 of the BMP-6 resistant cell lines and might represent another way of escaping the inhibitory effects of BMP. The lymphoma cell lines were investigated for endogenous production of BMPs by real-time RT-PCR, and 2 out of 10 cell lines had detectable BMP-6 mRNA, whereas 7 cell lines expressed BMP-7 mRNA. Analysis of purified malignant B cells or normal tumor infiltrating T cells from patient biopsies, confirmed the expression of BMP-6 and BMP-7 in the malignant B cells. Altogether, the data suggest that escape from BMP induced growth inhibition might contribute to increased tumor growth in B cell lymphomas.

Distinct roles for PARP-1 and PARP-2 in c-Myc-driven B-cell lymphoma in mice

Blood ◽  
2021 ◽  
Author(s):  
Miguel A Galindo-Campos ◽  
Nura Lutfi ◽  
Sarah Bonnin ◽  
Carlos Martínez ◽  
Talia Velasco-Hernandez ◽  
...  
Keyword(s):  
Dna Damage ◽  
B Cells ◽  
B Cell ◽  
Immune Escape ◽  
Cell Lymphoma ◽  
Tumour Progression ◽  
Cancer Therapeutics ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Parp 1

Dysregulation of the c-Myc oncogene occurs in a wide variety of haematologic malignancies and its overexpression has been linked with aggressive tumour progression. Here, we show that Poly (ADP-ribose) polymerase (PARP)-1 and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphomas. PARP-1 and PARP-2 catalyse the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA-strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphomas, while PARP-1-deficiency accelerates lymphomagenesis in the Em-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in pre-leukemic Em-Myc B cells resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1-deficiency induces a proinflammatory response, and an increase in regulatory T cells likely contributing to immune escape of B-cell lymphomas, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centred therapeutic strategies with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumours.

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