Constitutive Expression Of BCL6 Research Articles

Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo. We show that BCL6 knock-out in SU-DHL-4 cells in vitro results in an anti-proliferative response 4–7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/Cas9 systems for probing oncogene addiction.

AID assists DNMT1 to attenuate BCL6 expression through DNA methylation in diffuse large B-cell lymphoma cell lines

The BCL6 proto-oncogene encodes a transcriptional repressor, which is required for germinal centers (GCs) formation and lymphomagenesis. Previous studies have been reported that the constitutive expression of BCL6 leads to diffuse large B cell lymphoma (DLBCL) through activation-induced cytidine deaminase (AID) mediated chromosomal translocations and mutations. However, other DLBCLs (45%) without structural variants were characterized by abnormally high level of BCL6 expression through an unknown mechanism. Herein, we report that deficiency in AID or methyltransferase 1 (DNMT1) triggers high level of BCL6 expression. AID-DNMT1 complex binds to −0.4 kb −0 kb region of BCL6 promoter and contributes to generate BCL6 methylation which results in inhibition of BCL6 expression. The proteasome pathway inhibitor MG132 induces accumulation of AID and DNMT1, causes decreased BCL6 expression, and leads to cell apoptosis and tumor growth inhibition in DLBCL cell xenograft mice. These findings propose mechanistic insight into an alternative cofactor role of AID in assisting DNMT1 to maintain BCL6 methylation, thus suppress BCL6 transcription in DLBCL. This novel mechanism will provide a new drug selection in the therapeutic approach to DLBCL in the future.

Histone demethylase LSD1 is required for germinal center formation and BCL6-driven lymphomagenesis.

Germinal center (GC) B cells feature repression of many gene enhancers to establish their characteristic transcriptome. Here we show that conditional deletion of Lsd1 in GCs significantly impaired GC formation, associated with to failure to repress immune synapse genes linked to GC exit, which are also direct targets of the BCL6 transcriptional repressor. We found that BCL6 directly binds and recruits LSD1, primarily to intergenic and intronic enhancers. Conditional deletion of Lsd1 suppressed GC hyperplasia caused by constitutive expression of Bcl6, and significantly delayed Bcl6-driven lymphomagenesis. Administration of LSD1 catalytic inhibitors had little effect on GC formation or GC derived lymphoma cells. Using a CRISPR/Cas9 domain screen we found instead that the LSD1 Tower domain was critical for LSD1 dependency in GC derived B cells. These results indicate an essential role of LSD1 in the humoral immune response, where it modulates enhancer function by forming repression complexes with BCL6.

BCL6 modulation of acute lymphoblastic leukemia response to chemotherapy.

The bone marrow niche has a significant impact on acute lymphoblastic leukemia (ALL) cell phenotype. Of clinical relevance is the frequency with which quiescent leukemic cells, in this niche, survive treatment and contribute to relapse. This study suggests that marrow microenvironment regulation of BCL6 in ALL is one factor that may be involved in the transition between proliferative and quiescent states of ALL cells. Utilizing ALL cell lines, and primary patient tumor cells we observed that tumor cell BCL6 protein abundance is decreased in the presence of primary human bone marrow stromal cells (BMSC) and osteoblasts (HOB). Chemical inhibition, or shRNA knockdown, of BCL6 in ALL cells resulted in diminished ALL proliferation. As many chemotherapy regimens require tumor cell proliferation for optimal efficacy, we investigated the consequences of constitutive BCL6 expression in leukemic cells during co-culture with BMSC or HOB. Forced chronic expression of BCL6 during co-culture with BMSC or HOB sensitized the tumor to chemotherapy induced cell death. Combination treatment of caffeine, which increases BCL6 expression in ALL cells, with chemotherapy extended the event free survival of mice. These data suggest that BCL6 is one factor, modulated by microenvironment derived cues that may contribute to regulation of ALL therapeutic response.

A Chromatin Reader That Acts As a Key to Lock in and Coordinate Recruitment of Transcription Factors and a Novel Polycomb Complex to Bivalent Chromatin Thus Driving Formation of Germinal Centers and B-Cell Lymphomas

The EZH2 Polycomb H3K27 histone methyltransferase and the BCL6 transcriptional repressor are highly expressed in germinal center (GC) B cells and are each targeted by activating somatic mutations in DLBCL. In previous work we demonstrated that i) EZH2 is required for GC formation, ii) EZH2 Y641 somatic mutation drives GC hyperplasia and lymphomagenesis and iii) that WT and mutant EZH2 function by creating bivalent chromatin at promoters of genes that regulate cell cycle and terminal differentiation. In other work we showed that BCL6 represses gene promoters specifically by recruiting BCOR complex (a non-canonical form of PRC1 complex); and that BCL6 and EZH2 bind to many of the same promoters, although they do not physically interact. Finally we reported that the presence of both EZH2 and BCL6 was required for BCOR complex to stably associate with chromatin. This led us to hypothesize that BCL6 and EZH2 may be mutually dependent in normal and malignant B-cells.We found that conditional deletion of EZH2 from already established GC B-cells abrogates the ability of constitutive IµBCL6 expression to induce GC hyperplasia. A similar effect was observed by administering the specific EZH2 inhibitor GSK503. For the reciprocal experiments we developed two new mouse models: a conditional BCL6 knockout and a conditional EZH2Y641F knockin in the EZH2 endogenous locus. We showed that EZH2Y641F knockin mice developed GC hyperplasia, but this effect is abrogated by conditional deletion of BCL6 from established GC B cells. Given cell line data suggesting that BCOR might be important for the effect of EZH2 and BCL6 we next generated GC BCOR conditional knockout mice and observed that loss of BCOR not only disrupts GC formation but also abrogates the GC hyperplasia when crossed into the conditional EZH2Y641F knockin mice. Reversal of the EZH2Y641F knockin GC hyperplasia was also achieved by treating these mice with a novel small molecule called 1085 that potently disrupts BCL6 binding to BCOR. Notably, 1085 synergized with EZH2 inhibitors to kill DLBCL cells in vitro and eradicate DLBCL xenografts in vivo. Collectively these data show that EZH2, BCL6 and BCOR are mutually dependent both in the context of the normal GC reaction as well as in the setting of lymphoma precursor lesions driven by constitutive BCL6 expression or mutant EZH2.We next wished to determine the basic mechanism that explains the functional connection between EZH2 and BCL6-BCOR. Using ChIP-seq in primary human GC B cells we showed that there is significant overlap between BCL6-BCOR complex binding with EZH2 and bivalent chromatin. Using ChIP-re-ChIP we found that EZH2, BCOR and BCL6 are all localized to the same chromatin at bivalent gene promoters. Previously we showed that EZH2 mediated H3K27me3 is required for BCOR recruitment. However it was not clear why or how H3K27me3 is important for this effect. We postulated that a histone reader protein might bind to BCOR and recruit it to H3K27me3 marked chromatin. We therefore performed tandem affinity purification of BCOR followed by mass spectrometry to identify putative partner proteins and identified the chromodomain containing protein CBX8 as a novel BCOR partner. Notably CBX8 is the only H3K27 chromodomain protein that is upregulated in GC B cells. We confirmed endogenous BCOR-CBX8 interaction in human DLBCL cells. We showed that CBX8 is recruited to bivalent chromatin in an H3K27me3 dependent manner, and CBX8 depletion results in loss of BCOR complex assemble. Most strikingly, CBX8 knockdown was a phenocopy of EZH2 shRNA or EZH2 inhibitor. CBX8 loss resulted in derepression of EZH2 targets, and phenocopied the effect of EZH2 shRNA or inhibitors (proliferation arrest followed by plasma cell differentiation and then apoptosis). Therefore, CBX8 is shown for the first time to recruit non-canonical PRC1 and to explain how EZH2 cooperates with BCL6 to repress transcription and mediate the GC and DLBCL phenotype. PRC2 functions in a mutually interdependent manner with BCL6 to jointly enable stable association of non-canonical PRC1 complex with bivalent chromatin.In summary, our data establish a paradigm whereby EZH2 functions in a coordinate manner with transcription factors to transiently suppress genes in a cell context-specific manner relevant to its function in normal immunity as well as DLBCL. Our results also provide a rational basis for clinical translation of combined EZH2 and BCL6 inhibitors. DisclosuresLevine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Self-Enforcing Feedback Activation between BCL6 and Pre-B Cell Receptor Signaling Defines a Distinct Subtype of Acute Lymphoblastic Leukemia

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.

Self-Enforcing Feedback Activation Between BCL6 and Tonic Pre-B Cell Receptor Signaling in Acute Lymphoblastic Leukemia

Background and hypothesis: Like mature B cell lymphoma, pre-B ALL originates from B cell precursors that critically depend on survival signals emanating from a functional (pre-) B cell receptor (BCR). While recent work successfully introduced BCR signaling inhibitors into patient care for various subtypes of mature B cell lymphoma, it is not known whether pre-BCR signaling represents a therapeutic target in pre-B ALL and in which cytogenetic subsets targeting of pre-BCR signaling will be effective. In this study we demonstrated that ALL can be subdivided into two groups that fundamentally differ with respect to pre-BCR signalling. We identified a novel mechanism of self-enforcing feedback activation between the transcription factor BCL6 and tonic pre-BCR signaling in pre-BCR+ ALL and proposed a dual targeting strategy of both BCL6 and pre-BCR related tyrosine kinases for the treatment of patients with pre-BCR+ ALL.Results: Flow cytometry analysis of surface pre-BCR expression (λ5, VpreB), cytoplasmic μ heavy chain (μHC) expression and intracellular Ca2+ signal in 29 patient-derived pre-B ALL xenograft samples and cell lines showed pre-BCR expression and activity in a subset of pre-B ALL, including all TCF3-PBX1 cases studied (n=4) and two cases with deletions at 6q21. Studying 830 pre-B ALL cases from four clinical trials (MDACC, St. Jude, COG P9906 and ECOG E2993), tonic pre-BCR signaling and constitutive PI3K-AKT activation was found in 112 cases (13.5%), including 93% TCF3-PBX1 (53 of 57), del (6)(q21) (7 of 7), PBX1 (1q23) duplication (4 of 4), MLL-rearrangement (3 of 86), hyperdiploid (2 of 43) and other (43 of 406) pre-B ALL cases. In other major ALL subtypes, we found no evidence for pre-BCR expression and activity, including BCR-ABL1 (0 of 196) and ETV6-RUNX1 (0 of 31). We found frequent 1q23 (PBX1) duplication, TCF3-PBX1 or other PBX1-rearrangement, 6q21 (PRDM1) deletion in ALL cells with tonic pre-BCR signaling.Development of a genetic mouse model for inducible ablation of Bcl6. Pre-BCR-induced activation of BCL6 relieves PRDM1-mediated repression of pre-BCR signaling components and positively regulates pre-BCR signaling output at the transcriptional level. The clinical data (COG P9906, ECOG E2993) revealed that high mRNA levels of BCL6 at the time of diagnosis is predictive of poor clinical outcome specifically in patients with pre-BCR+ ALL but not ALL cells lacking pre-BCR expression. These findings suggest an important role of BCL6 as a cofactor of pre-BCR signaling in a large subset of ALL. To directly test the role of Bcl6- and pre-BCR interactions, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Deletion of Bcl6 in mouse pre-BCR+ ALL and expression of a dominate-negative form of BCL6 in human primary pre-BCR+ALL cells, both rapidly induced cell death, indicating BCL6 cooperates with the pre-BCR in leukemic transformation.Cooperation between pre-BCR and BCL6 signaling. Inhibition of BCL6 via the specific BCL6 inhibitor RI-BPI showed compromised colony formation and induced cell cycle arrest. Interestingly, constitutive BCL6 expression was sensitive to inhibition of SYK and SRC tyrosine kinases downstream of the pre-BCR. Treating 6 pre-BCR+ and 8 pre-BCR- patient-derived ALL samples with the SYK inhibitor (PRT06207), BTK inhibitor (Ibrutinib) or a broader SRC and BTK inhibitor Dasatinib, we observed remarkably decreased BCL6 expression and increased apoptosis in pre-BCR+ but not pre-BCR- ALL cells. In vivo treatments with Dasatinib prevented leukemia initiation and significantly prolonged survival of the recipient mice that were injected with primary pre-BCR+ ALL cells, compared to non-treatment or Nilotinib-treatment. These data demonstrate that both inhibition of BCL6 and pre-BCR signaling selectively killed patient-derived pre-BCR+ ALL cells.Conclusions: Our study identified two distinct subtypes of pre-B ALL that fundamentally differ with respect to pre-BCR signaling. Tonic pre-BCR signaling engages a BCL6-dependent, self-enforcing amplification loop. Based on these findings, we propose a dual targeting strategy of BCL6 and pre-BCR tyrosine kinases for the treatment of patients with pre-BCR+ALL. DisclosuresNo relevant conflicts of interest to declare.

Novel CTCF binding at a site in exon1A of BCL6 is associated with active histone marks and a transcriptionally active locus

BCL6 is a zinc-finger transcriptional repressor, which is highly expressed in germinal centre B-cells and is essential for germinal centre formation and T-dependent antibody responses. Constitutive BCL6 expression is sufficient to produce lymphomas in mice. Deregulated expression of BCL6 due to chromosomal rearrangements, mutations of a negative autoregulatory site in the BCL6 promoter region and aberrant post-translational modifications have been detected in a number of human lymphomas. Tight lineage and temporal regulation of BCL6 is, therefore, required for normal immunity, and abnormal regulation occurs in lymphomas. CCCTC-binding factor (CTCF) is a multi-functional chromatin regulator, which has recently been shown to bind in a methylation-sensitive manner to sites within the BCL6 first intron. We demonstrate a novel CTCF-binding site in BCL6 exon1A within a potential CpG island, which is unmethylated both in cell lines and in primary lymphoma samples. CTCF binding, which was found in BCL6-expressing cell lines, correlated with the presence of histone variant H2A.Z and active histone marks, suggesting that CTCF induces chromatin modification at a transcriptionally active BCL6 locus. CTCF binding to exon1A was required to maintain BCL6 expression in germinal centre cells by avoiding BCL6-negative autoregulation. Silencing of CTCF in BCL6-expressing cells reduced BCL6 mRNA and protein expression, which is sufficient to induce B-cell terminal differentiation toward plasma cells. Moreover, lack of CTCF binding to exon1A shifts the BCL6 local chromatin from an active to a repressive state. This work demonstrates that, in contexts in which BCL6 is expressed, CTCF binding to BCL6 exon1A associates with epigenetic modifications indicative of transcriptionally open chromatin.

Bcl6 as a Novel Therapeutic Target in Multiple Myeloma (MM).

Abstract 295BCL6 (B- cell lymphoma 6) is a proto-oncogene encoding a transcriptional repressor and regulates germinal center B cell differentiation. It is deregulated by chromosomal translocations or aberrant somatic hypermutation in a subset of diffuse large B cell lymphomas (Polo, JM. et al. PNAS, 2007). Importantly, small peptide inhibitor of BCL6 mediates cytotoxicity in primary human DLBCL cells both in vitro and in vivo, without effecting normal lymphoid tissue (Cerchietti, LC. et al. Blood, 2009). In this study, we examined modulation of Bcl6 expression and its sequelae in human MM cells in the context of bone marrow (BM) microenvironment.By Western blot analysis, constitutive expression of Bcl6 was either undetectable or very weakly expressed in all MM cell lines (MM.1S, MM.1R, RPMI8226, RPMI-LR5 H929, OPM1 and OPM2) except U266 cells. Importantly, however, Bcl6 expression was markedly upregulated by co-culture of MM cells with bone marrow stromal cells (BMSCs). Since U266 has high baseline phopho-STAT3 and BMSC-induced Bcl6 upregulation in other MM cell lines was associated with phosphorylation of STAT3, we hypothesized that JAK/STAT3 signaling might mediate Bcl6 expression in MM cells in the context of BM microenvironment. Indeed, anti-IL-6 neutralizing Ab significantly blocked BMSC co-culture-induced Bcl6 expression. To assess the clinical relevance of Bcl6 expression observed in MM cell lines, we examined its expression in patient MM cells by tissue microarray immunohistochemical analysis, confirming that Bcl6 was strongly positive within the nucleus in all cases. To obtain direct evidence that IL-6 triggered Bcl6 expression, we cultured MM cells with recombinant human IL-6; as expected, IL-6 strongly triggered Bcl6 expression in MM cell lines in a dose- and time-dependent fashion whereas other cytokines (ie, IGF-1, VEGF, IL-3) did not. Importantly, Bcl6 was also induced by IL-6 in primary tumor cells from MM patients. Real time RT-PCR confirmed that both BMSC co-culture and IL-6 treatment upregulated expression of Bcl6 mRNA. Furthermore, oncostatin-M, which also induces phospho-STAT3, similarly upregulated Bcl6. Conversely, pan-JAK inhibitor AG490 and STAT3 siRNA markedly downregulated Bcl6 expression in U266 cells, associated with downregulation of phospho-STAT3. Taken together, these results indicate that gp130 family member cytokines upregulate Bcl6 expression in MM cells via JAK/STAT3 signaling. We also found that TNFα also triggered Bcl6 in MM cell lines (MM.1S, RPMI8226, OPM1 and OPM2) and primary MM tumor cells; however, TNFα-induced upregulation of Bcl6 was independent on STAT3 activation. Importantly, IKKβ inhibitor MLN120B significantly reduced Bcl6 expression in MM cells triggered by TNFα. Taken together, these results suggested that TNFα-induced Bcl6 expression is mediated via the canonical NF-κB signaling pathway. Finally, to examine the biologic significance of Bcl6 inhibition in MM cells, we downregulated Bcl6 expression using lentiviral shRNA, in the presence or absence of IL-6. Importantly, downregulation of Bcl6 significantly decreased the number of MM cells, associated with decreased S and G2/M phase on cell cycle analysis. Our results therefore suggest that Bcl-6 expression is modulated via both JAk/STAT3 and NF-κB pathways in MM cells. Therefore Bcl6 represents a novel therapeutic target in MM.since targeting these cascades could downregulate Bcl-6 expression, and inhibit growth of MM cells in the bone marrow milieu. Disclosures:Raje:Millennium: Speakers Bureau. Munshi:: . Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium: Research Funding.

Direct Transcriptional Repression of BCL2 by BCL6 in Germinal Centre B Cells and Its Disruption in B Cell Lymphomas with BCL2 Locus Alterations

The human proto-oncogene BCL6 encodes a BTB/POZ-zinc finger transcriptional repressor that is required for germinal centre (GC) development and is expressed in the majority of normal GC B cells and in the majority of B cell lymphoma (B-NHL), including follicular lymphoma (FL) and a subset of diffuse large B cell lymphomas (DLBCLs). Deregulation of BCL6, by chromosomal translocation or somatic hypermutation, is implicated in the pathogenesis of B-NHL. The precise function of BCL6 in GC development and lymphomagenesis is still unclear also due to the very few direct BCL6 target genes that have been identified. In order to identify physiologically relevant BCL6 direct target genes, we used a novel approach combining high-throughput biochemical (ChIP-on-chip), bioinformatics (ARACNe) (Basso et al., Nature Genetics, 2005) and gene expression profile analysis (Klein et al., Proc. Natl. Acad. Sci., 2004) of normal GC B cells. The results (see abstract by Saito et al.) have identified a core of bona fide target genes whose promoter is bound by BCL6, whose transcription is dynamically linked to BCL6, and whose expression is down-regulated in GC B cells. Among the genes meeting these stringent criteria, we found BCL2, encoding the anti-apoptotic molecule whose expression is deregulated by chromosomal translocations or gene amplification in the majority of FL and in a subset of DLBCL. Further investigations showed that BCL6 represses BCL2 transcription by binding specific DNA sites within the BCL2 promoter region. Suppression of BCL6 expression via specific siRNAs leads to increased levels of BCL2, and, conversely, constitutive BCL6 expression prevents B cell receptor (BCR)-induced upregulation of BCL2 in B cells. Consistent with a physiological role for BCL6- mediated BCL2 suppression, immunohistochemical analysis shows that BCL2 expression is absent in GC B cells where BCL6 is highly expressed. Notably, the comparative analysis of BCL6 and BCL2 expression in B-NHL cell lines showed a conserved inverse correlation between BCL6 and BCL2 levels in cell lines carrying a normal BCL2 locus. However, cell lines carrying chromosomal translocations or amplifications affecting the BCL2 gene displayed the pathologic co-expression of both proteins, suggesting that the alterations affecting the BCL2 locus prevent BCL6-mediated suppression. These results indicate that one critical role of BCL6 is the modulation of antiapoptotic function in GC B cells via BCL2 transcriptional repression, suggesting a mechanism by which B cells may die in the GC if not rescued by BCR and CD40 engagement, both of which downregulate BCL6 while inducing BCL2 expression. Alterations of the BCL2 locus may contribute to lymphomagenesis by making the gene resistant to suppression by BCL6, and therefore allowing B cells to avoid apoptosis in the absence of antigen- and T-cell-mediated rescue.

BCL6 represses CHEK1 and suppresses DNA damage pathways in normal and malignant B-cells

BCL6 is a transcriptional repressor protein that is expressed in a developmentally regulated fashion during B-cell maturation. Specifically, BCL6 is required for formation of germinal centers in response to T-cell dependent antigen activation. Germinal center B-cells feature the ability to tolerate rapid proliferation and simultaneous genetic recombination. Genetic lesions that cause constitutive expression of BCL6 are commonly associated with diffuse large B-cell lymphomas (DLBCL). Recent studies show that BCL6 contributes to the germinal center phenotype by directly repressing genes involved in sensing or responding to DNA damage including ATR, TP53 and CDKN1A. The CHEK1 protein is activated through phosphorylation by the ATR kinase domain in response to DNA damage. Activated CHEK1 can phosphorylate and modulate the activity a number of proteins including p53, providing a link between ATR sensing of DNA damage and p53 checkpoint activity. Herein we show that BCL6 can directly bind to a DNA consensus element in the CHEK1 promoter and repress its expression in normal and malignant B-cells. DLBCL cells can be killed by a specific BCL6 peptide inhibitor (BPI) that interferes with corepressor binding to the BCL6 BTB domain. BPI could reactivate CHEK1 in DLBCL cells, suggesting that its induction might contribute to BPI anti-lymphoma effects. Therefore, BCL6 can suppress multiple genes involved in a common pathway sensing, transducing and responding to genotoxic stress.

A “Survival of the Fittest” Mechanism for Weeding Out Potentially Lymphomagenic B-Cells during Germinal Center B-Cell Differentiation.

During normal T-cell dependent immune responses, activated B-cells differentiate into germinal center (GC) centroblasts, which tolerate simultaneous genomic recombination and rapid clonal expansion in order to produce high affinity antibodies. Upregulation of the BCL6 transcriptional repressor is required for centroblasts to acquire this phenotype, since it can repress DNA damage sensing and checkpoint genes. Genetic lesions that cause constitutive expression of BCL6 are common oncogenic events in human diffuse large B-cell lymphomas (DLBCL) and presumably contribute to malignant transformation by sustaining the centroblast phase and facilitating accumulation of genetic errors. We hypothesized that centroblasts must have evolved a mechanism to rapidly terminate the genomic instability phenotype in order to limit the likelihood of malignant transformation. A recent report (Allen et. al. PMID: 17185562) showed that centroblasts and GC T-cells make direct physical contact for ∼30 minutes during affinity maturation. We wondered whether CD40 signaling from T-cells could disrupt the function of BCL6 within this timeframe. Accordingly, we found in ChIP assays that CD40 signaling in B-cells can disrupt the ability of BCL6 to recruit the SMRT and N-CoR corepressors within minutes, at which time RNA polymerase II moves from the promoter to the exons of BCL6 target genes, histones became acetylated, and mRNA levels increase. We showed that signaling from CD40 to N-CoR was dependent on NFkB, while signaling to SMRT appeared to be related to MAP kinase phosphorylation cascades. Two BCL6 targets regulated in this manner are ATR and p53. Accordingly, although CD40 can promote survival of intact GC B-cells, CD40 induced cell death of centroblasts in the presence of higher levels of DNA damage led to cell death, rather than survival. Washout of CD40 after 60 minutes to emulate transient T-cell contact permitted BCL6 target gene mRNA levels to return to their repressed levels, demonstrating that this is a reversible process, which could allow centroblasts that pass quality control to either continue proliferation or undergo terminal differentiation. In order to identify direct targets of BCL6 subject to this regulatory mechanism, we performed ChIP-on-chip of BCL6 in primary human tonsilar centroblasts on a 24,000 promoter array. 915 genes were identified with a cut-off of 99.9th percentile (FDR<0.026), while 1880 genes were identified with a cutoff at the 99th percentile (FDR<0.12). These natural BCL6 targets are functionally related to DNA damage response, transcriptional repression, protein translation, NFkB signaling and others, re-expression of which may play a critical role in facilitating quality control of centroblasts during affinity maturation. Taken together, these data suggest that transient CD40 signaling in the GC might allow T-cells to “weed out” heavily damaged centroblasts while at the same time promoting survival of only the “fittest” B-cells, which could undergo differentiation or additional rounds of proliferation. Others have shown that sustained CD40 signaling can downregulate BCL6 at longer timepoints, possibly reflecting longer exposure of B-cells to this signaling pathway that might occur towards the end of their cycle through the germinal center. Therefore, CD40 can inhibit BCL6 through two different mechanisms, each with potentially different functions during B-cell maturation. Loss of either mechanism is likely to contribute to lymphomagenesis.

The BCL6 Oncogene Drives an Epigenomic Program Linking Stem Cells to the Pathogenesis of Human Diffuse Large B Cell Lymphoma in Mice.

Diffuse large-B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. The BCL6 oncogene, a transcriptional repressor, is frequently involved in DLBCL pathogenesis. Constitutive expression of BCL6 in GC cells leads to larger GCs and development of DLBCL in mice, although constitutive expression of BCL6 earlier in the B-cell lineage has the opposite effect and abrogates B-cell development. Limitations in our knowledge of how BCL6 mediates transcriptional programming to impose the malignant phenotype presents a barrier towards improved understanding and therapeutic targeting of B-cell lymphomas. Recent advances in stem cell biology suggest that many tumors are sustained by a pool of biological distinct cancer stem cells (CSCs), which may be uniquely susceptible to epigenomic programming by oncogenes and require specific therapeutic targeting. Although CSCs have not yet been identified in DLBCL, we hypothesized that such cells may exist in these tumors and their characterization could be critical for studies of lymphomagenesis and therapeutics. In order to test this hypothesis our group developed a mouse model where BCL6 is expressed transiently in the stem cell compartment at levels comparable to normal GC cells. These animals undergo completely normal hematopoiesis, except that 100% develop lymphomas within 20 to 28 weeks. Extensive histopathologic, molecular and expression array phenotyping demonstrate that these tumors are most similar to human DLBCLs. However, these tumor cells do not express BCL6, and so must be transcriptionally programmed at the stem cell level to behave in a malignant fashion. Moreover, transplantation of DLBCL cells does not propagate the tumor whereas transplantation of the CSCs rapidly re-establishes the DLBCL in recipient mice. In-depth microarray and QPCR transcriptional profiling demonstrate that BCL6 imposes a B cell lymphoma program in the stem cell compartment. This transcriptional programming subsequently becomes latent during hematopoiesis and reappears only in the mature B-cell fraction. We reasoned that this effect must be dependent on epigenetic programming imposed by BCL6 in CSCs. To determine whether this is the case we compared and contrasted the epigenetic signature of CSCs, and DLCBL tumors in the transgenic animals vs. those of normal stem cells and mature B-cells in control animals. This was performed using the HELP (HpaII tiny fragment enrichment by ligation mediated PCR) method with a custom designed high-density oligonucleotide microarray that allows quantitative detection of DNA methylation at 24,000 murine gene promoters. The data show that BCL6 imposes a unique DNA hypermethylation signature in CSCs that persists in the DLBCL cells. Taken together, our results suggest that BCL6 mediates transcriptional and epigenomic programming in stem cells that can latently persist during differentiation (thus allowing early B-cell development to proceed) and impose the DLBCL phenotype in their mature progeny. Since human lymphomas exhibit similar transcriptional programming, therapeutic targeting of the BCL6 epigenomic program might enhance the power of anti-lymphoma regimens to eradicate the disease.

BCL6 programs lymphoma cells for survival and differentiation through distinct biochemical mechanisms

AbstractThe BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCLs). Constitutive expression of BCL6 mediates lymphomagenesis through aberrant proliferation, survival, and differentiation blockade. Binding of BCL6 to the SMRT/N-CoR corepressors mediates the BCL6 survival effect in DLBCL. Although the basis for differentiation blockade is unknown in DLBCL, recent data suggest that BCL6 binding to the MTA3 corepressor might be involved. We report that BCL6 and MTA3 are coexpressed in normal germinal center B cells and DLBCL. Depletion of MTA3 in DLBCL cells induced a differentiation-related BCL6 target gene (PRDM1), but not target genes involved in survival. Accordingly, MTA3 and PRDM1 expression are mutually exclusive in germinal center B cells. We performed chromatin immunoprecipitation (ChIP)–on-chip mapping of the PRDM1 locus, identifying a novel BCL6 binding site on intron 3 of the PRDM1 gene, and show that BCL6 recruits MTA3 to this site. In DLBCL cells, MTA3 depletion induced plasmacytic differentiation but did not decrease viability of DLBCL cells. However, MTA3 depletion synergized with a specific BCL6 inhibitor that blocks SMRT/N-CoR binding to decrease DLBCL viability. Taken together, these results show that BCL6 regulates distinct transcriptional programs through the SMRT/N-CoR and MTA3 corepressors, respectively, and provides a basis for combinatorial therapeutic targeting of BCL6.

BCL6 Programs Lymphoma Cells for Survival and Differentiation through Distinct Biochemical Mechanisms, Both of Which Can Be Therapeutically Targeted.

The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCL). Constitutive expression of BCL6 has been proposed to mediate lymphomagenesis through several mechanisms, including evasion of cell death, proliferation and differentiation blockade. We show here that BCL6 mediates these effects through distinct mechanisms. First, we show that blocking the association of the SMRT corepressor with BCL6 using our specific peptide inhibitor (BPI) abrogates only the survival effects of BCL6 but has no effect on differentiation. Accordingly, BPI upregulates survival genes such as ATR and p53, but not genes associated with differentiation such as Blimp1, XBP and Syndecan. In contrast, BCL6 shRNA upregulates both survival and differentiation genes and induces both cell death and differentiation. We and others have shown that BCL6 can also directly bind to the MTA3 corepressor, which is implicated in differentiation of Burkitt lymphoma cells. We found that BCL6 and MTA3 are co-expressed in DLBCL cells and primary human centroblasts (the precursor cell for most DLBCLs). The endogenous BCL6 and MTA3 proteins interacted in DLBCLs cells in co-immunoprecipitation experiments. In contrast to SMRT blockade with BPI, siRNA depletion of MTA3 induced expression of the Blimp1, XBP and Syndecan but not p53 and ATR. MTA3 depletion induced plasmacytic differentiation within 72 hours as shown in functional assays and by surface markers. We performed ChIP on chip using custom arrays densely tiling with oligonucleotides covering the entire genomic loci of 20 BCL6 target genes. Interestingly, BCL6 formed different types of repression complexes at differentiation genes (Complex with MTA3/NuRD) vs. survival genes (complex with SMRT and N-CoR). BCL6-mediated repression of genes involved in survival and differentiation thus depend on distinct biochemical mechanisms. The relevance of these findings for human disease was underscored by the fact that we found a statistically significant positive correlation between MTA3 and BCL6 gene expression in a database of 176 human DLBCLs (p<0.00001). Likewise, protein expression of BCL6 and MTA3 was also highly correlated (p<0.00001) (74 cases examined) and staining for Blimp1 revealed mutually exclusive expression from MTA3. Taken together these results illustrate the basic mechanisms through which BCL6 mediates DLBCL lymphomagenesis and provide the basis for powerful targeted therapy regimens that could be translated to the clinical setting.

The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells

The human proto-oncogene BCL6 encodes a BTB/POZ-zinc-finger transcriptional repressor that is necessary for germinal-centre formation and is implicated in the pathogenesis of B-cell lymphoma. The precise function of BCL6 in germinal-centre development and lymphomagenesis is unclear because very few direct BCL6 target genes have been identified. Here we report that BCL6 suppresses the expression of the p53 (also known as tp53) tumour suppressor gene and modulates DNA damage-induced apoptotic responses in germinal-centre B cells. BCL6 represses p53 transcription by binding two specific DNA sites within the p53 promoter region and, accordingly, p53 expression is absent in germinal-centre B cells where BCL6 is highly expressed. Suppression of BCL6 expression via specific short interfering RNA leads to increased levels of p53 messenger RNA and protein both under basal conditions and in response to DNA damage. Most notably, constitutive expression of BCL6 protects B cell lines from apoptosis induced by DNA damage. These results suggest that an important function of BCL6 is to allow germinal-centre B cells to tolerate the physiological DNA breaks required for immunoglobulin class switch recombination and somatic hypermutation without inducing a p53-dependent apoptotic response. These findings also imply that deregulated BCL6 expression contributes to lymphomagenesis in part by functional inactivation of p53.

The BCL6 Proto-Oncogene Suppresses p53 Expression in Germinal-Center B Cells.

The proto-oncogene BCL6 encodes a BTB/POZ-zinc finger transcriptional repressor that is necessary for germinal center (GC) formation and is implicated in the pathogenesis of B-cell lymphoma. In ~50% diffuse large cell lymphoma and 10% follicular lymphoma, BCL6 gene expression is deregulated by chromosomal translocations or mutations that affect its 5′ regulatory region. The precise function of BCL6 in GC development and lymphomagenesis is unclear since very few BCL6 direct target genes have been identified. We report that BCL6 suppresses p53-dependent and p53-indepenent growth arrest and apoptosis responses in GC B cells. BCL6 directly suppresses the transcription of the p53 gene, as demonstrated by (1) chromatin immunoprecipitation (ChIP) assays showing that BCL6 binds the p53 promoter region in vivo; and (2) transient transfection/reporter assays identifying within the p53 promoter region two BCL6-binding sites that mediate BCL6-mediated suppression of p53 transcription.Accordingly, suppression of BCL6 expression via specific siRNA leads to increased expression of p53 both under basal condition and in response to DNA damage. Consistent with a physiological role for BCL6-mediated p53 suppression, immunohistochemical analysis shows that p53 expression is absent in GC B cells where BCL6 is highly expressed. In addition, our data reveal that BCL6 inhibits the p53-independent activation of the p21/WAF1 cell cycle arrest gene by binding to Miz-1, a transcription factor involved in p21 activation. Consistent with a role of BCL6 in inhibiting p53-related cell cycle arrest and apoptotic responses, constitutive expression of BCL6 suppresses p53 expression and p53-target genes (P21 and PUMA) and protects B cell lines from apoptosis induced by DNA damage. These results indicate that one function of BCL6 is to allow GC B cells (centroblasts) to constitutively proliferate and to sustain the physiologic DNA breaks required for immunoglobulin switch recombination and somatic hypermutation without inducing p53-related responses. These findings also imply that B cell lymphoma with deregulated BCL6 expression are functionally p53-negative and impaired in apoptotic responses.