Huifeng Niu

BCL6 interacts with the transcription factor Miz-1 to suppress the cyclin-dependent kinase inhibitor p21 and cell cycle arrest in germinal center B cells

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.

Chromosomal translocations cause deregulated BCL6 expression by promoter substitution in B cell lymphoma.

The BCL6 gene codes for a zinc‐finger transcription factor and is involved in chromosomal rearrangements in 30–40% of diffuse large‐cell lymphoma (DLCL). These rearrangements cluster within the 5′ regulatory region of BCL6 spanning its first non‐coding exon. To determine the functional consequences of these alterations, we have analyzed the structure of the rearranged BCL6 alleles and their corresponding RNA and protein species in two DLCL biopsies and one tumor cell line which carried the t(3;14)(q27;q32) translocation involving the BCL6 and immunoglobulin heavy‐chain (IgH) loci. In all three cases, the breakpoints were mapped within the IgH switch region and the BCL6 first intron, leading to the juxtaposition of part of the IgH locus upstream and in the same transcriptional orientation to the BCL6 coding exons. An analysis of cDNA clones showed that these recombinations generate chimeric IgH‐BCL6 transcripts which initiated from IgH germline transcript promoters (I mu or I gamma 3), but retain a normal BCL6 coding domain. In the tumor cell line, the chimeric I gamma 3‐BCL6 allele, but not the germline BCL6 gene, was transcriptionally active and produced a normal BCL6 protein. These findings indicate that t(3;14) translocations alter BCL6 expression by promoter substitution and imply that the consequence of these alterations is the deregulated expression of a normal BCL6 protein.

Molecular pathogenesis of non-Hodgkin's lymphoma: the role of Bcl-6.

Non-Hodgkins 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.

BCL6 Controls the Expression of the B7-1/CD80 Costimulatory Receptor in Germinal Center B Cells

The BCL6 proto-oncogene encodes a transcriptional repressor required for the development of germinal centers (GCs) and implicated in the pathogenesis of GC-derived B cell lymphoma. Understanding the precise role of BCL6 in normal GC formation and in lymphomagenesis depends on the identification of genes that are direct targets of its transcriptional repression. Here we report that BCL6 directly controls the expression of B7–1/CD80, a costimulatory receptor involved in B–T cell interactions critical for the development of T cell–mediated antibody responses. Upon CD40 signaling, transcription of the CD80 gene is induced by the nuclear factor (NF)-κB transcription factor. Our results show that BCL6 prevents CD40-induced expression of CD80 by binding its promoter region in vivo and suppressing its transcriptional activation by NF-κB. Consistent with a physiologic role for BCL6 in suppressing CD80, the expression of these two genes is mutually exclusive in B cells, and BCL6-defective mice show increased expression of CD80 in B cells. The results suggest that BCL6 may directly control the ability of B cell to interact with T cells during normal GC development. In addition, these findings imply that T–B cell interactions may be disrupted in B cell lymphoma expressing deregulated BCL6 genes.

Molecular pathogenesis of B cell malignancy: the role of BCL-6.

Human malignancies displaying a mature B cell phenotype include non-Hodgkin lymphoma, (NHL), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM). Analogous to most cancer types, the pathogenesis of these malignancies represents a multistep process involving the progressive and clonal accumulation of multiple genetic lesions affecting proto-oncogenes and tumor suppressor genes. However, several important features distinguish the mechanism and type of genetic alterations associated with NHL, CLL, and MM (Table 1).

BCL-6 AND THE MOLECULAR PATHOGENESIS OF B-CELL LYMPHOMA

The results presented identify the first genetic lesion associated with DLCL, the most clinically relevant form of NHL. Although no proof yet exists of a role for these lesions in DLCL pathogenesis, the feature of the BCL-6 gene product, its specific pattern of expression in B cells, and the clustering of lesions disrupting its regulatory domain strongly suggest that deregulation of BCL-6 expression may contribute to DLCL development. A more precise definition of the role of BCL-6 in normal and neoplastic B-cell development is the goal of ongoing study of transgenic mice engineered either to express BCL-6 under heterologous promoters or lacking BCL-6 function due to targeted deletions. In addition to contributing to the understanding of DLCL pathogenesis, the identification of BCL-6 lesions may have relevant clinical implications. DLCL represent a heterogeneous group of neoplasms which are treated homogeneously despite the fact that only 50% of patients experience long-term disease-free survival (Schneider et al. 1990). The fact that BCL-6 rearrangements identify biologically and clinically distinct subsets of DLCL suggests that these lesions may be useful as markers in selection of differential therapeutic strategies based on different risk groups. Furthermore, the BCL-6 rearrangements can be used to identify and monitor the malignant clone with sensitive PCR-based techniques. Since clinical remission has been observed in a significant fraction of DLCL cases, these markers may serve as critical tools for sensitive monitoring of minimal residual disease and early diagnosis of relapse (Gribben et al. 1993).

Expression of Cyclin-Dependent Kinase Inhibitor p27Kip1 in AIDS-Related Diffuse Large-Cell Lymphomas Is Associated with Epstein-Barr Virus-Encoded Latent Membrane Protein 1

Knowledge of the role of cell-cycle regulators in the pathogenesis of acquired immune deficiency syndrome-related non-Hodgkins lymphomas (AIDS-NHLs) is scarce. Here we analyzed 86 systemic AIDS-NHLs and 20 AIDS-primary central nervous system lymphomas for expression of p27(Kip1), a negative regulator of cell-cycle progression belonging to the Kip family of cyclin-dependent kinase inhibitors. In parallel, we investigated the relationship between p27(Kip1), the lymphoma proliferation index, Epstein-Barr virus status, expression of cellular cyclin D3 and cyclin D1, and B-cell differentiation stage. We report that AIDS-immunoblastic lymphomas (AIDS-IBLs), either systemic or primarily localized to the central nervous system, consistently express p27(Kip1) protein (19 of 24 and 10 of 14, respectively) despite the high proliferative rate of the lymphoma clone, suggesting a failure of p27(Kip1) to inhibit the cell cycle in AIDS-IBL. Conversely, the remaining systemic AIDS-NHLs and AIDS-primary central nervous system lymphomas preferentially fail to express p27(Kip1). Expression of p27(Kip1) in Epstein-Barr virus-positive AIDS-NHLs generally associates with latent membrane protein 1 (LMP1) expression and is related to a late stage of B-cell differentiation, characterized by the BCL-6-/MUM1+/syn-1+/- phenotypic profile, whereas it seems to be unrelated to the expression of cellular cyclins. In B cells in vitro, induction of LMP-1 expression under the control of inducible promoters up-regulates expression of p27(Kip1), thus providing a putative mechanistic explanation for the association between LMP1 and p27(Kip1) observed in vivo. Overall, these data show that AIDS-IBL pathogenesis is characterized by loss of the inverse relationship between p27(Kip1) positivity and tumor growth fraction that is otherwise generally observed in normal lymphoid tissues and in most other types of NHLs.

Tracking CD40 Signaling during Normal Germinal Center Development by Gene Expression Profiling

Signaling through the CD40 receptor plays an important role in multiple events in T-cell dependent immune responses, including germinal center (GC) and memory B-cell formation and immunoglobulin isotype switching.1 CD40 signaling occurs in B cells following the interaction between the CD40 receptor on B cells and its ligand (CD40L) on T cells. The NF-κB transcription complex is the main nuclear mediator of CD40 signaling and induces the transcription of CD40 target genes. While the CD40–CD40L interaction has been extensively studied in the past years, it is not yet fully understood at which stages of B cell development signaling through CD40 occurs. In particular, while it has been shown that CD40 signaling is required for GC development, it remains to be elucidated whether this signal occurs continuously or at particular stages of the development of this structure. To address these questions, we first used gene expression profiling to identify the genes whose expression is induced or repressed upon CD40 signaling of B cells in vitro (CD40 signature). This signature was then used to track CD40 signaling in human B cells corresponding to the main developmental stages of GC—namely, naïve, centroblasts (CB), centrocytes (CC), and memory B cells. CD40 signaling was induced in a Burkitt lymphoma cell line (Ramos) by co-cultivation with fibroblasts that were stably transfected to express the CD40 ligand. After co-cultivation for 24 h, B cells were purified from fibroblasts by magnetic cell separation using anti-CD19 antibodies. The expression of the known CD40-responsive genes bfl-1, BCL-6, CD95/FAS, and CD80 was used as internal

Advances in the Understanding of the Molecular Pathogenesis of Aggressive B Cell Lymphomas

Non-Hodgkin lymphoma (NHL) include neoplasms originating from lymphoid cells and characterized by a high degree of biological and clinical heterogeneity (for review see Magrath, 1990). Most NHL derive from the B-cell lineage, in particular from mature B-cells characterized by rearranged immunoglobulin (Ig) heavy and light chain genes and by the expression of cell surface Ig and B-cell associated markers. The wide clinico-pathological heterogeneity of NHL correlates with distinct genetic lesions, particularly chromosomal translocations, associated with its pathogenesis (Table 1; Gaidano and Dalla-Favera, 1993). Among low-grade NHL, “mantle zone” lymphoma are associated in 50% of cases with the t(11;14) translocation involving the juxtaposition of the IgH locus to the BCL-1/PRAD-1/cyclin D1 gene coding for a protein involved in the control of cell cycle progression (Tsujimoto et al., 1984; Motokura et al., 1991; Raffeid et al., 1991). In follicular-type NHL(FL), the t(14;18) translocation juxtaposes the IgH locus to BCL-2 (Bakhshi et al., 1985; Tsujimoto et al., 1984; Cleary et al., 1985), a gene coding for a protein that prevents programmed cell death or apoptosis (Korsmeyer, 1992). After years of indolent course, a significant fraction of FL undergoes histologic transformation and clinical progression into Diffuse Large Cell Lymphoma (DLCL), an event which is associated with loss or mutations of the p53 tumor suppressor gene (Lo Coco et al., 1993). “De novo” DLCL are associated with rearrangements and deregulation of the BCL-6 gene, which codes for a zinc-finger transcription factor (Ye et al., 1993a, 1993b; Kerckaert et al., 1993). In Burkitt Lymphoma (BL), the t(8;14), t(8;22), and t(2;8) chromosomal translocations lead to the deregulated expression of the c-Myc proto-oncogene by juxtaposition to one of the Ig loci (Dalla-Favera et al., 1982; Taub et al., 1982; Dalla-Favera et aI., 1983; Dalla-Favera, 1991). A sizable fraction (35%) ofBL are also associated with loss or mutations of the p53 gene (Gaidano et aI., 1991).