Search

In response to T-cell-dependent antigens, mature B cells in the secondary lymphoid organs are stimulated to form germinal centers (GCs), which are histological structures deputed to antibody affinity maturation, a process associated with immunoglobulin gene editing by somatic hypermutation (SHM) and class switch recombination (CSR). GC B cells are heterogeneous and transition across multiple stages before being eliminated by apoptosis or committing to post-GC differentiation as memory B cells or plasma cells. In order to explore the dynamics of SHM and CSR during the GC reaction, we identified GC subpopulations by single-cell (sc) transcriptomics and analyzed the load of immunoglobulin variable (V) region mutations as well as the isotype class distribution in each subpopulation. The results showed that the large majority of GC B cells display a quantitatively similar mutational load in the V regions and analogous IGH isotype class distribution, except for the precursors of memory B cells (PreM) and plasma cells (PBL). PreM showed a bimodal pattern with about half of the cells displaying high V region germline identity and enrichment for unswitched IGH, while the rest of the cells carried a mutational load similar to the bulk of GC B cells and showed a switched isotype. PBL displayed a bias toward expression of IGHG and higher V region germline identity compared to the bulk of GC B cells. Genes implicated in SHM and CSR were significantly induced in specific GC subpopulations, consistent with the occurrence of SHM in dark zone cells and suggesting that CSR can occur within the GC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Corinaldesi, Holmes, Shen, Grunstein, Pasqualucci, Dalla-Favera and Basso.)

In response to T cell-dependent antigens, mature B cells are stimulated to form germinal centers (GCs), the sites of B cell affinity maturation and the cell of origin (COO) of most B cell lymphomas. To explore the dynamics of GC B cell development beyond the known dark zone and light zone compartments, we performed single-cell (sc) transcriptomic analysis on human GC B cells and identified multiple functionally linked subpopulations, including the distinct precursors of memory B cells and plasma cells. The gene expression signatures associated with these GC subpopulations were effective in providing a sc-COO for ∼80% of diffuse large B cell lymphomas (DLBCLs) and identified novel prognostic subgroups of DLBCL., Competing Interests: Disclosures: N. Compagno is currently employed at Novartis. No other disclosures were reported., (© 2020 Holmes et al.)

Inactivating mutations of the CREBBP and EP300 acetyltransferases are among the most common genetic alterations in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). Here, we examined the relationship between these two enzymes in germinal center (GC) B cells, the normal counterpart of FL and DLBCL, and in lymphomagenesis by using conditional GC-directed deletion mouse models targeting Crebbp or Ep300. We found that CREBBP and EP300 modulate common as well as distinct transcriptional programs implicated in separate anatomic and functional GC compartments. Consistently, deletion of Ep300 but not Crebbp impaired the fitness of GC B cells in vivo. Combined loss of Crebbp and Ep300 completely abrogated GC formation, suggesting that these proteins partially compensate for each other through common transcriptional targets. This synthetic lethal interaction was retained in CREBBP-mutant DLBCL cells and could be pharmacologically targeted with selective small molecule inhibitors of CREBBP and EP300 function. These data provide proof-of-principle for the clinical development of EP300-specific inhibitors in FL and DLBCL., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The gene encoding the MEF2B transcription factor is mutated in germinal center (GC)-derived B cell lymphomas, but its role in GC development and lymphomagenesis is unknown. We demonstrate that Mef2b deletion reduces GC formation in mice and identify MEF2B transcriptional targets in GC, with roles in cell proliferation, apoptosis, GC confinement, and differentiation. The most common lymphoma-associated MEF2B mutant (MEF2B D83V ) is hypomorphic, yet escapes binding and negative regulation by components of the HUCA complex and class IIa HDACs. Mef2b D83V expression in mice leads to GC enlargement and lymphoma development, a phenotype that becomes fully penetrant in combination with BCL2 de-regulation, an event associated with human MEF2B mutations. These results identify MEF2B as a critical GC regulator and a driver oncogene in lymphomagenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for the germinal center (GC) reaction and is implicated in lymphomagenesis. BCL6 protein stability is regulated by F-box protein 11 (FBXO11)-mediated ubiquitination and degradation, which is impaired in ∼6% of diffuse large B-cell lymphomas that carry inactivating genetic alterations targeting the FBXO11 gene. In order to investigate the role of FBXO11 in vivo, we analyzed GC-specific FBXO11 knockout mice. FBXO11 reduction or loss led to an increased number of GC B cells, to an altered ratio of GC dark zone to light zone cells, and to higher levels of BCL6 protein in GC B cells. B-cell receptor-mediated degradation of BCL6 was reduced in the absence of FBXO11, suggesting that FBXO11 contributes to the physiologic downregulation of BCL6 at the end of the GC reaction. Finally, FBXO11 inactivation was associated with the development of lymphoproliferative disorders in mice., (© 2016 by The American Society of Hematology.)

The pathways regulating formation of the germinal center (GC) dark zone (DZ) and light zone (LZ) are unknown. In this study we show that FOXO1 transcription factor expression was restricted to the GC DZ and was required for DZ formation, since its absence in mice led to the loss of DZ gene programs and the formation of LZ-only GCs. FOXO1-negative GC B cells displayed normal somatic hypermutation but defective affinity maturation and class switch recombination. The function of FOXO1 in sustaining the DZ program involved the trans-activation of the chemokine receptor CXCR4, and cooperation with the BCL6 transcription factor in the trans-repression of genes involved in immune activation, DNA repair, and plasma cell differentiation. These results also have implications for the role of FOXO1 in lymphomagenesis because they suggest that constitutive FOXO1 activity might be required for the oncogenic activity of deregulated BCL6 expression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mutations in the gene encoding the KMT2D (or MLL2) methyltransferase are highly recurrent and occur early during tumorigenesis in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the functional consequences of these mutations and their role in lymphomagenesis are unknown. Here we show that FL- and DLBCL-associated KMT2D mutations impair KMT2D enzymatic activity, leading to diminished global H3K4 methylation in germinal-center (GC) B cells and DLBCL cells. Conditional deletion of Kmt2d early during B cell development, but not after initiation of the GC reaction, results in an increase in GC B cells and enhances B cell proliferation in mice. Moreover, genetic ablation of Kmt2d in mice overexpressing Bcl2 increases the incidence of GC-derived lymphomas resembling human tumors. These findings suggest that KMT2D acts as a tumor suppressor gene whose early loss facilitates lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cells. Eradication of KMT2D-deficient cells may thus represent a rational therapeutic approach for targeting early tumorigenic events.

Germinal centres (GCs) are involved in the selection of B cells secreting high-affinity antibodies and are also the origin of most human B cell lymphomas. Recent progress has been made in identifying the functionally relevant stages of the GC and the complex trafficking mechanisms of B cells within the GC. These studies have identified transcription factors and signalling pathways that regulate distinct phases of GC development. Notably, these factors and pathways are hijacked during tumorigenesis, as revealed by analyses of the genetic lesions associated with various types of B cell lymphomas. This Review focuses on recent insights into the mechanisms that regulate GC development and that are relevant for human B cell lymphomagenesis.

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center (GC) formation and whose de-regulation is involved in lymphomagenesis. Although substantial evidence indicates that BCL6 exerts its function by repressing the transcription of hundreds of protein-coding genes, its potential role in regulating gene expression via microRNAs (miRNAs) is not known. We have identified a core of 15 miRNAs that show binding of BCL6 in their genomic loci and are down-regulated in GC B cells. Among BCL6 validated targets, miR-155 and miR-361 directly modulate AID expression, indicating that via repression of these miRNAs, BCL6 up-regulates AID. Similarly, the expression of additional genes relevant for the GC phenotype, including SPI1, IRF8, and MYB, appears to be sustained via BCL6-mediated repression of miR-155. These findings identify a novel mechanism by which BCL6, in addition to repressing protein coding genes, promotes the expression of important GC functions by repressing specific miRNAs.

BCL6 is a transcriptional repressor required in mature B cells during the germinal center (GC) reaction. Multiple mechanisms act coordinately to timely modulate BCL6 expression at transcriptional and post-transcriptional levels. BCL6 prevents premature activation and differentiation of GC B cells and provides an environment tolerant of the DNA breaks associated with immunoglobulin gene remodeling mechanisms involved in the production of high-affinity antibodies of different isotypes. The critical functions exerted by BCL6 during normal B-cell development can be hijacked by the malignant transformation process. Indeed, BCL6 is targeted by genetic aberrations and acts as an oncogene in GC-derived lymphomas. The aberrations affecting BCL6 interfere with the multiple levels of regulation that grant a fine tuning of BCL6 expression and activity in physiologic conditions. This review summarizes the current knowledge on BCL6 function and its role in lymphomagenesis., (© 2012 John Wiley & Sons A/S.)

Assembly of a transcriptional and post-translational molecular interaction network in B cells, the human B-cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC). Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre-replication, replication, and mitosis. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.

BCL6 is a transcriptional repressor required for mature B-cell germinal center (GC) formation and implicated in lymphomagenesis. BCL6's physiologic function is only partially known because the complete set of its targets in GC B cells has not been identified. To address this issue, we used an integrated biochemical-computational-functional approach to identify BCL6 direct targets in normal GC B cells. This approach includes (1) identification of BCL6-bound promoters by genome-wide chromatin immunoprecipitation, (2) inference of transcriptional relationships by the use of a regulatory network reverse engineering approach (ARACNe), and (3) validation of physiologic relevance of the candidate targets down-regulated in GC B cells. Our approach demonstrated that a large set of promoters (> 4000) is physically bound by BCL6 but that only a fraction of them is repressed in GC B cells. This set of 1207 targets identifies several cellular functions directly controlled by BCL6 during GC development, including activation, survival, DNA-damage response, cell cycle arrest, cytokine signaling, Toll-like receptor signaling, and differentiation. These results define a broad role of BCL6 in preventing centroblasts from responding to signals leading to exit from the GC before they complete the phase of proliferative expansion and of antibody affinity maturation.

BCL6 is a transcriptional repressor which has emerged as a critical regulator of germinal centers (GC), the sites where B cells are selected based on the production of antibodies with high affinity for the antigen. BCL6 is also a frequently activated oncogene in the pathogenesis of human B cell lymphomas, most of which derive from the GC B cells. A thorough understanding of the biological role of BCL6 in normal B cell development and lymphomagenesis depends upon the identification of the full set of genes that are targets of its transcriptional regulatory function. Recently, the identification of BCL6 targets has been implemented with the use of genome-wide chromatin immunoprecipitation and gene expression profiling approaches. A large set of promoters have been shown to be physically bound by BCL6, but only a fraction of them appears to be subjected to transcriptional repression in GC B cells. This set of BCL6 targets points to a number of cellular functions which are likely to be directly controlled by BCL6 during GC development, including activation, survival, DNA-damage response, cell cycle arrest, cytokine-, toll-like receptor-, TGFbeta-, WNT-signaling, and differentiation. Overall, BCL6 is revealing its dual role of "safe-keeper" in preventing centroblasts from responding to signals leading to a premature exit from the GC and of contributor to lymphomagenesis by allowing the instauration of conditions favorable to malignant transformation., ((c) 2010 Elsevier Inc. All rights reserved.)

The BCL6 proto-oncogene encodes a transcriptional repressor necessary for the development of germinal centers (GCs) and directly implicated in lymphomagenesis. Post-GC development of B cells requires BCL6 downregulation, while its constitutive expression caused by chromosomal translocations leads to diffuse large B cell lymphoma (DLBCL). Herein we identify a signaling pathway that downregulates BCL6 expression in normal GC B cells and is blocked in a subset of DLBCL due to alterations in the BCL6 gene. Activation of the CD40 receptor leads to NF-kappaB-mediated induction of the IRF4 transcription factor, which, in turn, represses BCL6 expression by binding to its promoter region. A subset of DLBCL displays chromosomal translocations or mutations that disrupt the IRF4-responsive region in the BCL6 promoter and block its downregulation by CD40 signaling.

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center formation and has been linked to lymphomagenesis. BCL6 functions by directly binding to specific DNA sequences and suppressing the transcription of target genes. Here we report an alternative mechanism by which BCL6 controls the transcription of genes lacking a BCL6 binding site and show that this mechanism was required for the prevention of tumor suppressor p53-independent cell cycle arrest in germinal center B cells. BCL6 interacted with the transcriptional activator Miz-1 and, via Miz-1, bound to the promoter and suppressed transcription of the cell cycle arrest gene CDKN1A. Through this mechanism, BCL6 may facilitate the proliferative expansion of germinal centers during the normal immune response and, when deregulated, the pathological expansion of B cell lymphomas.

Substantial evidence indicates that signaling through the CD40 receptor (CD40) is required for germinal center (GC) and memory B-cell formation. However, it is not fully understood at which stages of B-cell development the CD40 pathway is activated in vivo. To address this question, we induced CD40 signaling in human transformed GC B cells in vitro and identified a CD40 gene expression signature by DNA microarray analysis. This signature was then investigated in the gene expression profiles of normal B cells and found in pre- and post-GC B cells (naive and memory) but, surprisingly, not in GC B cells. This finding was validated in lymphoid tissues by showing that the nuclear factor-kappaB (NF-kappaB) transcription factors, which translocate to the nucleus upon CD40 stimulation, are retained in the cytoplasm in most GC B cells, indicating the absence of CD40 signaling. Nevertheless, a subset of centrocytes and B cells in the subepithelium showed nuclear staining of multiple NF-kappaB subunits, suggesting that a fraction of naive and memory B cells may be subject to CD40 signaling or to other signals that activate NF-kappaB. Together, these results show that GC expansion occurs in the absence of CD40 signaling, which may act only in the initial and final stages of the GC reaction.

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center (GC) formation and whose deregulation by genomic lesions is implicated in the pathogenesis of GC-derived diffuse large B cell lymphoma (DLBCL) and, less frequently, follicular lymphoma (FL). The biological function of BCL6 is only partially understood because no more than a few genes have been functionally characterized as direct targets of BCL6 transrepression activity. Here we report that the anti-apoptotic proto-oncogene BCL2 is a direct target of BCL6 in GC B cells. BCL6 binds to the BCL2 promoter region by interacting with the transcriptional activator Miz1 and suppresses Miz1-induced activation of BCL2 expression. BCL6-mediated suppression of BCL2 is lost in FL and DLBCL, where the 2 proteins are pathologically coexpressed, because of BCL2 chromosomal trans- locations and other mechanisms, including Miz1 deregulation and somatic mutations in the BCL2 promoter region. These results identify an important function for BCL6 in facilitating apoptosis of GC B cells via suppression of BCL2, and suggest that blocking this pathway is critical for lymphomagenesis. [ABSTRACT FROM AUTHOR]

Non-Hodgkin's lymphomas (NHL) form a heterogeneous group of diseases, with diffuse large B-cell lymphoma (DLBCL) comprising the largest subgroup. The commonest chromosomal translocations found in DLBCL are those affecting band 3q27. In 35% of DLBCL cases, as well as in a small fraction of follicular lymphomas, the normal transcriptional regulation of Bcl-6 is disrupted by these chromosomal translocations. In addition, about three-quarters of cases of DLBCL display multiple somatic mutations in the 5' non-coding region of Bcl-6, which occur independently of chromosomal translocations and appear to be due to the IgV-associated somatic hypermutation process. Bcl-6 is a 95-kD nuclear phosphoprotein belonging to the BTB/POZ (bric-a-brac, tramtrack, broad complex/ Pox virus zinc finger) zinc finger family of transcription factors. It has been suggested that Bcl-6 is important in the repression of genes involved in the control of lymphocyte activation, differentiation, and apoptosis within the germinal center, and that its down-regulation is necessary for normal B-cells to exit the germinal center. Bcl-6 remains constitutively expressed in a substantial proportion of B-cell lymphomas. Recently, acetylation has been identified as a mode for down-regulating Bcl-6 activity by inhibition of the ability of Bcl-6 to recruit complexes containing histone deacetylases (HDAC). The pharmacologic inhibition of two recently identified deacetylation pathways, HDAC- and silent information regulator (SIR)-2-dependent deacetylation, results in the accumulation of inactive acetylated Bcl-6 and thus in cell cycle arrest and apoptosis in B-cell lymphoma cells. These results reveal a new method of regulating Bcl-6, with the potential for therapeutic exploitation. These studies also indicate a novel mechanism by which acetylation promotes transcription, not only by modifying histones and activating transcriptional activators, but also by inhibiting transcriptional repressors. [ABSTRACT FROM AUTHOR]