BCL6 Attenuates DNA Damage Sensing in Normal and Malignant B-Cells by Directly Repressing ATR.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 157-157 ◽  
Author(s):  
Stella M. Ranuncolo ◽  
Jose M. Polo ◽  
Jamil Dierov ◽  
Rita Shaknovich ◽  
Martin Carroll ◽  
...  
Keyword(s):  
Dna Damage ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Target Genes ◽  
Double Strand Breaks ◽  
B Cell Lymphomas ◽  
H2ax Phosphorylation ◽  
Strand Breaks ◽  
Damage Sensing

Abstract The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers by normal B-cells. However, the mechanisms by which BCL6 licenses germinal center formation and lymphomagenesis are unknown. To characterize this mechanism we identified BCL6 target genes by expression arrays and high throughput chromatin immunoprecipitations. Remarkably, a number of these target genes were critical mediators of DNA damage sensing checkpoints including ATR and p53. Therefore, we hypothesized that BCL6 could attenuate DNA damage sensing by silencing these genes, which is likely a critical attribute for survival and proliferation of germinal center B-cells cells undergoing somatic hypermutation (SHM) and class switch recombination (CSR). Accordingly, we found that expression of BCL6 in normal diploid fibroblasts could block cellular sensing of DNA damage as demonstrated by loss of histone 2AX (H2AX) phosphorylation and delayed repair of double strand breaks. Repression of ATR (but not p53 or other targets) was required for this phenotype. This is a physiological effect since the same result was observed when BCL6 was expressed in purified primary human tonsilar mature B-cells. Reciprocally, shRNA knockdown of BCL6 in B-cell lymphomas rescued repression of ATR, enhanced H2AX phosphorylation and accelerated repair of double strand breaks, independent of the status of p53. shRNA knockdown of BCL6 caused a marked increase of apoptosis in lymphoma cells in response to DNA damage, due to restored DNA damage checkpoint functions. Importantly, BCL6 knockdown had an identical effect on ATR levels, H2AX phosphorylation, DNA damage, and survival in purified primary human germinal center centroblasts. These results suggest that a major role of BCL6 in germinal center formation is to attenuate cellular response to DNA damage occurring as a byproduct of CSR and SHM. The same mechanism also seems to be required for lymphomagenesis, since we observed that sustained BCL6 expression in human primary mature B-cells leads to aberrant survival properties and genomic instability. Moreover, BCL6 blockade using a specific inhibitor molecule designed by our lab induces apoptosis in lymphoma cells and synergizes with DNA damaging agents. Therefore, we have identified a critical mechanism of action of the BCL6 oncoprotein in normal and pathogenic states and show that specific targeting of BCL6 could synergize with chemotherapy drugs for the therapy of B-cell lymphomas.

A Critical BCL6-Related Feedback Loop Explains the Unusual Biological Features of Germinal Center B-Cells and Their Malignant Transformation into B-Cell Lymphomas.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 224-224 ◽  
Author(s):  
Stella M. Ranuncolo ◽  
Jose M. Polo ◽  
Jamil Dierov ◽  
Martin Carroll ◽  
Ari M. Melnick
Keyword(s):  
Dna Damage ◽  
B Cells ◽  
B Cell ◽  
Dna Damage Response ◽  
Feedback Loop ◽  
Dna Damage Checkpoint ◽  
B Cell Lymphomas ◽  
Damage Sensing ◽  
Damage Response ◽  
Repression Complex

Abstract The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers (GCs) by normal B-cells. However, the mechanisms by which BCL6 licenses GC formation and lymphomagenesis remain unknown. To characterize this mechanism we identified BCL6 target genes through several methods. Most notable among these was ATR, a master DNA damage response mediator. We showed that primary BCL6-expressing GC centroblasts purified from human tonsils do not express ATR, do not activate downstream targets of ATR (Chk1) and exhibit an overall attenuated DNA damage checkpoint response (as shown in COMET assays, H2AX phosphorylation assays, and other methods). ATR expression, the activation of ChK1, and the DNA damage phenotype were fully rescued by BCL6 loss of function (induced by shRNA or by a specific BCL6 inhibitor that we designed). BCL6 expressing DLBCL cell lines exhibited the same BCL6-dependent DNA damage response phenotype. This could be attributed almost entirely to ATR since ectopic expression of ATR could restore DNA damage sensing even in the presence of BCL6. Reciprocally, loss of ATR in BCL6 depleted B-cells could completely prevent them from restoring their damage response pathways. These effects are independent of p53, which in contrast to ATR is expressed in primary centroblasts and in many primary DLBCL cases. Gain of function experiments with BCL6 in isolated human tonsilar naïve B-cells could induce this same damage phenotype in an ATR dependent/p53 independent manner. Most remarkably, we discovered that CD40 signaling, which occurs once centroblasts mature into centrocytes in the GC light zone, rescues ATR from BCL6 mediated repression by signaling to the BCL6 repression complex through NFKB. This causes the SMRT and N-CoR corepressors to be released from BCL6 repressor complexes associated with the ATR promoter, induces ATR expression, its downstream target ChK1 activation and restores DNA damage sensing. As a consequence, B-cells that have undergone extensive damage (as a by-product of antibody affinity maturation and intense proliferation) can now undergo apoptosis, while those with lower level damage undergo cell cycle arrest, DNA repair, and further differentiation. We thus described a molecular feedback loop through which BCL6 mediates GC formation by directly repressing ATR and inducing a DNA damage checkpoint deficient phenotype, and through which subsequent CD40 signaling rescues this phenotype by disrupting the BCL6 repression complex. We showed that sustained BCL6 expression leads to DNA misrepair and genomic instability in primary B cells consistent with its role in lymphomagenesis. Reciprocally, therapeutic targeting of BCL6 synergized with chemotherapy and radiation to kill DLBCL cells (by restoring DNA damage checkpoints) and would thus be expected to improve therapeutic outcomes of DLBCL patients.

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.

scholarly journals Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein

2005 ◽  
Vol 203 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Chang Hoon Lee ◽  
Mark Melchers ◽  
Hongsheng Wang ◽  
Ted A. Torrey ◽  
Rebecca Slota ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Transcriptional Activation ◽  
Germinal Center ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Consensus Sequence ◽  
B Cell Lymphoma ◽  
Regulatory Factor

Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein–tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.

scholarly journals Immunolocalization of the ICE/Ced-3–Family Protease, CPP32 (Caspase-3), in Non-Hodgkin's Lymphomas, Chronic Lymphocytic Leukemias, and Reactive Lymph Nodes

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3817-3825 ◽  
Author(s):  
Stanislaw Krajewski ◽  
Randy D. Gascoyne ◽  
Juan M. Zapata ◽  
Maryla Krajewska ◽  
Shinichi Kitada ◽  
...  
Keyword(s):  
B Cells ◽  
Lymph Nodes ◽  
B Cell ◽  
Germinal Center ◽  
Caspase 3 ◽  
Large Cell ◽  
B Cell Lymphomas ◽  
Mantle Zone ◽  
Dynamic Regulation ◽  
Hodgkin's Lymphomas

Immunohistochemical analysis of the apoptosis-effector protease CPP32 (Caspase-3) in normal lymph nodes, tonsils, and nodes affected with reactive hyperplasia (n = 22) showed strong immunoreactivity in the apoptosis-prone germinal center B-lymphocytes of secondary follicles, but little or no reactivity in the surrounding long-lived mantle zone lymphocytes. Immunoblot analysis of fluorescence-activated cell sorted germinal center and mantle zone B cells supported the immunohistochemical results. In 22 of 27 (81%) follicular small cleaved cell non-Hodgkin's B-cell lymphomas, the CPP32-immunopositive germinal center lymphocytes were replaced by CPP32-negative tumor cells. In contrast, the large cell component of follicular mixed cells (FMs) and follicular large cell lymphomas (FLCLs) was strongly CPP32 immunopositive in 12 of 17 (71%) and in 8 of 14 (57%) cases, respectively, whereas the residual small-cleaved cells were poorly stained for CPP32 in all FLCLs and in 12 of 17 (71%) FMs, suggesting that an upregulation of CPP32 immunoreactivity occurred during progression. Similarly, cytosolic immunostaining for CPP32 was present in 10 of 12 (83%) diffuse large cell lymphomas (DLCLs) and 2 of 3 diffuse mixed B-cell lymphomas (DMs). Immunopositivity for CPP32 was also found in the majority of other types of non-Hodgkin's lymphomas studied. Plasmacytomas were CPP32 immunonegative in 4 of 12 (33%) cases, in contrast to normal plasma cells, which uniformly contained intense CPP32 immunoreactivity, implying downregulation of CPP32 in a subset of these malignancies. All 12 peripheral blood B-cell chronic lymphocyte leukemia specimens examined were CPP32 immunopositive, whereas 3 of 3 small lymphocytic lymphomas were CPP32 negative, suggesting that CPP32 expression may vary depending on the tissue compartment in which these neoplastic B cells reside. The results show dynamic regulation of CPP32 expression in normal and malignant lymphocytes.

scholarly journals Restriction of memory B cell differentiation at the germinal center B cell positive selection stage

2020 ◽  
Vol 217 (7) ◽  
Author(s):  
Amparo Toboso-Navasa ◽  
Arief Gunawan ◽  
Giulia Morlino ◽  
Rinako Nakagawa ◽  
Andrea Taddei ◽  
...  
Keyword(s):  
B Cells ◽  
Positive Selection ◽  
B Cell ◽  
Cell Fate ◽  
Germinal Center ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
B Cell Differentiation ◽  
Repressor Complex ◽  
Selection Stage

Memory B cells (MBCs) are key for protection from reinfection. However, it is mechanistically unclear how germinal center (GC) B cells differentiate into MBCs. MYC is transiently induced in cells fated for GC expansion and plasma cell (PC) formation, so-called positively selected GC B cells. We found that these cells coexpressed MYC and MIZ1 (MYC-interacting zinc-finger protein 1 [ZBTB17]). MYC and MIZ1 are transcriptional activators; however, they form a transcriptional repressor complex that represses MIZ1 target genes. Mice lacking MYC–MIZ1 complexes displayed impaired cell cycle entry of positively selected GC B cells and reduced GC B cell expansion and PC formation. Notably, absence of MYC–MIZ1 complexes in positively selected GC B cells led to a gene expression profile alike that of MBCs and increased MBC differentiation. Thus, at the GC positive selection stage, MYC–MIZ1 complexes are required for effective GC expansion and PC formation and to restrict MBC differentiation. We propose that MYC and MIZ1 form a module that regulates GC B cell fate.

scholarly journals Immunolocalization of the ICE/Ced-3–Family Protease, CPP32 (Caspase-3), in Non-Hodgkin's Lymphomas, Chronic Lymphocytic Leukemias, and Reactive Lymph Nodes

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3817-3825 ◽  
Author(s):  
Stanislaw Krajewski ◽  
Randy D. Gascoyne ◽  
Juan M. Zapata ◽  
Maryla Krajewska ◽  
Shinichi Kitada ◽  
...  
Keyword(s):  
B Cells ◽  
Lymph Nodes ◽  
B Cell ◽  
Germinal Center ◽  
Caspase 3 ◽  
Large Cell ◽  
B Cell Lymphomas ◽  
Mantle Zone ◽  
Dynamic Regulation ◽  
Hodgkin's Lymphomas

Abstract Immunohistochemical analysis of the apoptosis-effector protease CPP32 (Caspase-3) in normal lymph nodes, tonsils, and nodes affected with reactive hyperplasia (n = 22) showed strong immunoreactivity in the apoptosis-prone germinal center B-lymphocytes of secondary follicles, but little or no reactivity in the surrounding long-lived mantle zone lymphocytes. Immunoblot analysis of fluorescence-activated cell sorted germinal center and mantle zone B cells supported the immunohistochemical results. In 22 of 27 (81%) follicular small cleaved cell non-Hodgkin's B-cell lymphomas, the CPP32-immunopositive germinal center lymphocytes were replaced by CPP32-negative tumor cells. In contrast, the large cell component of follicular mixed cells (FMs) and follicular large cell lymphomas (FLCLs) was strongly CPP32 immunopositive in 12 of 17 (71%) and in 8 of 14 (57%) cases, respectively, whereas the residual small-cleaved cells were poorly stained for CPP32 in all FLCLs and in 12 of 17 (71%) FMs, suggesting that an upregulation of CPP32 immunoreactivity occurred during progression. Similarly, cytosolic immunostaining for CPP32 was present in 10 of 12 (83%) diffuse large cell lymphomas (DLCLs) and 2 of 3 diffuse mixed B-cell lymphomas (DMs). Immunopositivity for CPP32 was also found in the majority of other types of non-Hodgkin's lymphomas studied. Plasmacytomas were CPP32 immunonegative in 4 of 12 (33%) cases, in contrast to normal plasma cells, which uniformly contained intense CPP32 immunoreactivity, implying downregulation of CPP32 in a subset of these malignancies. All 12 peripheral blood B-cell chronic lymphocyte leukemia specimens examined were CPP32 immunopositive, whereas 3 of 3 small lymphocytic lymphomas were CPP32 negative, suggesting that CPP32 expression may vary depending on the tissue compartment in which these neoplastic B cells reside. The results show dynamic regulation of CPP32 expression in normal and malignant lymphocytes.

scholarly journals Mouse model of Epstein–Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease

2017 ◽  
Vol 114 (18) ◽  
pp. 4751-4756 ◽  
Author(s):  
Takeharu Minamitani ◽  
Yijie Ma ◽  
Hufeng Zhou ◽  
Hiroshi Kida ◽  
Chao-Yuan Tsai ◽  
...  
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Germinal Center ◽  
Epstein Barr Virus ◽  
Target Genes ◽  
Nk Cell ◽  
Barr Virus ◽  
Epstein Barr

Epstein–Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

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.

The Alternative Breakpoint Region (ABR) of BCL6 Is An Active Transcribed Region in Germinal Center B Cells and May Serve as An Enhancer

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4465-4465
Author(s):  
Yulei Shen ◽  
Himabindu Ramachandrareddy ◽  
Wing C. Chan ◽  
Timothy McKeithan
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Regulatory Elements ◽  
Breakpoint Region ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
B Cell Differentiation ◽  
B Cell Lymphomas ◽  
Bcl6 Expression

Abstract Chromosomal translocations at 3q27 involving BCL6 occur in two different regions. Rearrangements with breaks within the major breakpoint region (MBR), comprising the first exon and part of the first intron of BCL6, are among the most common genetic abnormalities in of B-cell non-Hodgkin lymphoma, whereas breaks within an alternative breakpoint region (ABR), located between 245 and 285 kb 5′ to BCL6, have also been reported in follicular lymphoma grade and a small group (6.4%) of diffuse large B-cell lymphomas (DLBCLs). As a result of the MBR translocation, BCL6 expression is deregulated by promoter substitution with either immunoglobulin (Ig) genes or non-Ig genes as partners. A role for deregulated BCL6 expression in the pathogenesis of DLBCL has previously been confirmed in a mouse model. However, the biological role of the more distant ABR region is still not known. Using real-time PCR, we identified in the ABR region an evolutionarily conserved DNase I hypersensitive site (named Far5) which contains a conserved composite binding site for transcription factors PU.1 and IRF4, both of which play important roles in B-cell differentation. Further studies demonstrated that chromatin in the Far5 region, 190kb upstream of BCL6 promoter, has an open configuration in DHL6, Granta 519 and U266 cell lines. Far5 DNA showed enhancer activity by a luciferase reporter assay. PU.1 binds to Far5 in vivo (DHL16 cell line) by a chromatin immunoprecipitation (ChIP) assay, and PU.1 binds in vitro to the conserved PU.1/IRF composite site in Far5 synergistically with either IRF4 or IRF8. ChIP-on-chip assays showed Far5 histone H3K4 monomethylation, a chromatin modification associated with gene enhancers and other regulatory elements. In addition, we identified a transcript upstream of the Far5 region that is specifically expressed in germinal center (GC) B cells, but not at other stages of B-cell differentation. These results indicated that Far5 may play a role in selective expression of intergenic transcripts in GC B-cells. In other genes, intergenic transcription plays a role in looping between distal regulatory regions and the promoter. Our data showed that ABR region is constitutively active in GC B-cells and may play an important role in increasing BCL6 transcription when naïve B cells differentiate into GC B-cells. Further investigation of interactions between the ABR and the BCL6 promoter will uncover the regulatory function of the BCL6 ABR in B-cell differentiation and B-cell lymphomas.

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