scholarly journals Suppression of EZH2 Accelerates MYC-Driven Lymphomagenesis By Inhibition of Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3009-3009 ◽  
Author(s):  
Iris Appelmann ◽  
Claudio Scuoppo ◽  
Vishal Thapar ◽  
Daniela Ledezma ◽  
Amaia Lujambio ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Human Cancer ◽  
B Cell Lymphoma ◽  
Metastatic Potential ◽  
Loss Of Function ◽  
Wild Type ◽  
Proliferating Cells ◽  
Gain Of Function

Abstract EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), which also contains the non-catalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). This complex methylates histone H3 at lysine 27 (H3K27) which, together with H3K4 methylation, generates a bivalent “code” that primes genes for either expression or silencing. EZH2 is highly expressed in stem cells and many proliferating cells, downregulated in differentiated cells and frequently altered in cancer in ways that point to a context dependent role for this gene. For instance, overexpression of EZH2 has been described in prostate and breast cancer, where this overexpression is associated with invasive growth metastatic potential and poor clinical outcome. More recently, both gain and loss of function mutations of EZH2 were identified in human cancer. In particular, activating mutations of EZH2 affecting a tyrosine at position 641 located within the SET domain (Y641) were observed in lymphoma. In striking contrast to wild type EZH2 which catalyzes the monomethylation of H3K27 very efficiently and shows less efficient catalytic activity in the subsequent di- and trimethylation reactions, the mutation generates a neomorphic protein with enhanced catalytic activity and efficiency for di- and tri-methylation, hinting toward a “cooperation” of both wild type and mutant EZH2 to increase total H3K27me3 levels and representing a functional equivalent of EZH2 overexpression in human lymphoma. Gain of function mutations in EZH2 are frequent in Diffuse Large B-cell Lymphoma (DLBCL; 22%) and in Follicular Lymphoma (FL; 7%) and recent data implicate EZH2 as an oncogene in DLBCL and FL lymphomas. In contrast to DLBCL and FL, EZH2 mutations have so far never been identified in MYC-driven B-cell Non-Hodgkin Lymphoma (B-NHL), and EZH2 expression is suppressed in Burkitt’s Lymphoma (BL), a lymphoma for which a MYC translocation is pathognomonic, suggesting that in this context EZH2 could have a role different from its role in DLBCL and FL. We probed the role of EZH2 in MYC-driven lymphomagenesis by mimicking the loss of function mutations by RNAi mediated suppression of EZH2 and the gain of function mutations by over-expression of the Y641 mutant. Our results show that suppression of EZH2, but not overexpression of the EZH2 Y641 mutant, accelerates MYC-driven lymphomagenesis by attenuating apoptosis. Our model recapitulates the transcriptional signature of a subset of B-NHLs driven by MYC overactivation and EZH2 suppression. Taken together, our data imply EZH2 as a tumor suppressor in the context of MYC activation and thus raise a warning for the use of EZH2-targeted therapies in some B-NHLs subtypes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Activated B-Cell DLBCL with Downstream Activation of Survival Signaling Requires PIM Kinase Activity to Maintain Oncoprotein Translation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4496-4496
Author(s):  
Tara L. Peters ◽  
Ana A Tula-Sanchez ◽  
Lingxiao Li ◽  
Praechompoo Pongtornpipat ◽  
Mengdie WANG ◽  
...  
Keyword(s):  
B Cell ◽  
Conflicts Of Interest ◽  
B Cell Lymphoma ◽  
Protein Translation ◽  
Cyclin D3 ◽  
Cell Of Origin ◽  
Loss Of Function ◽  
Western Blots ◽  
Kinase Inhibition ◽  
Highly Sensitive

Abstract The activated B-cell cell-of-origin subtype of diffuse large B-cell lymphoma (ABC-DLBCL) requires NF-kB pathway activation to maintain the malignant phenotype. NF-kB activation is downstream of B-cell receptor (BCR) stimulation and can become constitutively turned on in ABC-DLBCL through mutations in multiple different BCR signaling intermediates. Drugs targeting upstream signaling proteins such as Bruton’s Tyrosine Kinase (BTK, ibrutinib) or protein kinase C (PKC, AEB071) have shown promising results in other lymphomas driven by BCR activation and are under evaluation in ABC-DLBCL. Many ABC-DBCL cases, however, have mutations in mediators that are downstream from these targets, particularly coiled-coil domain mutations in CARD11 that stabilize NF-kB activation and A20 loss-of-function alterations that reduce protein turnover of oncogenic NF-kB intermediates. In this study we explore a potential role for PIM kinase inhibition in ABC-DLBCL and find the clinical pan-PIM inhibitor LGH447 has promising activity in particular against cells carrying these downstream mutations. We find some ABC cells were highly sensitive to LGH447 with IC50 < 0.4 µM (OCI-Ly3 and OCI-Ly10), while some others were completely insensitive with IC50 > 10.0 µM (TMD8 and HBL-1). Strikingly, all ABC lines sensitive to LGH447 carry mutations in either CARD11, TNFAIP3 (encoding A20), or both, while insensitive typically lines lack such lesions. Insensitive lines including TMD8 and HBL-1 instead have upstream mutations in CD79B and are highly sensitive to the upstream inhibitors ibrutinib and AEB071. The PIM1-3 kinases inhibited by LGH447 have multiple targets mediating cell growth and survival, including several that activate cap-dependent protein translation activation. We find LGH447 is toxic to sensitive cells due to lost translational activation. Western blots show reduced phosphorylation of ribosomal protein S6 and 4EBP1, indicating loss of mTORC1 activity. In addition, LGH447, in a manner similar to the potent direct cap-dependent translation inhibitor silvestrol, causes knockdown of key translationally regulated oncoproteins, including c-MYC, MCL1, and Cyclin D3. We also directly monitored protein synthesis through O-Propargyl-puromycin (OP-PURO) incorporation and found a direct effect of LGH447 that was similar to silvestrol, although requiring higher concentrations. PIM’s effects on activation of protein translation therefore are required in LGH447-sensitive ABC-DLBCL cells but dispensable in insensitive cells. In conclusion, pan-PIM kinase inhibition provides a strong potential therapeutic opportunity in a subset of ABC-DLBCL. Cases that bypass upstream signaling to turn on NF-kB activation more directly also bypass pathways with redundant activation of cap-dependent translation, making them dependent on the therapeutically targetable PIM kinases to carry out this critical process. Disclosures No relevant conflicts of interest to declare.

scholarly journals KLHL14 Is a Tumor Suppressor in Diffuse Large B-Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2766-2766
Author(s):  
Jaewoo Choi ◽  
James D Phelan ◽  
Ryan M. Young ◽  
Thomas Oellerich ◽  
Da Wei Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Somatic Mutations ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Loss Of Function ◽  
Wild Type ◽  
Large B Cell Lymphoma ◽  
Inactivating Mutations ◽  
Large B Cell

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer of aberrant B-lymphocytes. Although a portion of DLBCL is curable with standard immunochemotherapy, patients who fail this treatment have a poor prognosis. Recently, cancer genomics has paved the way for better understanding of the genetic basis of lymphoma pathogenesis. Characterization of point mutations and structural alterations has uncovered novel molecular targets for lymphoma therapy and provided a comprehensive view of lymphoma development. By performing multiplatform genomic analysis of DLBCL biopsy samples, we have identified KLHL14 as a recurrent target of somatic mutations in activated B-cell-like (ABC) DLBCL biopsies (10.8% of patients). KLHL14 contains a BTB (broad complex, tramtrack, and bric a brac) domain that can potentially mediate dimerization and binding to Cullin3 (CUL3)-a essential scaffold component of the Cullin-RING-based E3 ubiquitin ligase complexes. KLHL14 also contains kelch repeats that can form a B-propeller tertiary structure that can serve as a substrate-binding domain. KLHL14 is highly expressed in B-cells but is found at low levels in non-immune tissues. Deficiency of KLHL14 in mice leads to embryonic lethality while KLHL14 heterozygous mice show reduction of B-1a cells, suggesting a role for KLHL14 in B-cell homeostasis. Importantly, KLHL14 mutations are highly enriched in tumors belonging to the recently defined MCD (MYD88L265P/CD79B mutation) genetic subtype of DLBCL, the subset of ABC DLBCLs. Somatic mutations primarily localize to the N-terminus of the protein in the BTB domain and BACK (BTB and C-terminal Kelch) domain. However, the impact of these mutations as well as the molecular function of KLHL14 is largely unknown. To investigate the biological effect of KLHL14 loss of function, we used an inducible CRISPR/Cas9 system to delete KLHL14 in ABC DLBCL cell lines and monitored cell growth. Ablation of KLHL14 resulted in an increase in cell proliferation and survival, supporting a role for KLHL14 as a tumor suppressor. Next, we performed a multiplatform -omic analysis (proteomics, phosphoproteomics, ubiquitinomics, high-throughput sequencing) to explore the signaling networks and interactome of KLHL14. Whereas ectopic expression of wild-type KLHL14 altered the dynamics of tyrosine phosphorylation and ubiquitylation events in ABC DLBCL lines, KLHL14 lymphoma-associated mutant alleles had little if any effect, suggesting that they are loss-of-function variants. Gene expression profiling by RNA-sequencing revealed that KLHL14-inactivated cells have a higher NF-kB target gene expression than wild-type cells. Thus, tumor-associated inactivating mutations of KLHL14 depend on a subset of essential NF-kB-related oncoproteins for their survival and this might contribute to the proliferative advantage of DLBCL. In summary, we have uncovered a tumor suppressive function of KLHL14 and found that KLHL14 mutants promote ABC DLBCL survival by increasing NF-kB activity. These findings suggest that tumors with KLHL14 inactivating mutations may serve as a marker of resistance to anti-NF-kB treatment and provide the basis for treating MCD subtype patients with downstream NF-kB pathway inhibitors in the clinical settings. Disclosures Staudt: Nanostring: Patents & Royalties.

scholarly journals MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Danxia Zhu ◽  
Cheng Fang ◽  
Wenting He ◽  
Chen Wu ◽  
Xiaodong Li ◽  
...  
Keyword(s):  
B Cell ◽  
Binding Site ◽  
Cell Lines ◽  
Expression Vector ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Protein Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.

scholarly journals Combination Treatment with GSK126 and Pomalidomide Induces B-Cell Differentiation in EZH2 Gain-of-Function Mutant Diffuse Large B-Cell Lymphoma

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2541
Author(s):  
Sungryul Park ◽  
Seung-Hyun Jo ◽  
Jong-Hwan Kim ◽  
Seon-Young Kim ◽  
Jae Du Ha ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Zinc Finger ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
B Cell Differentiation ◽  
Gain Of Function ◽  
Polycomb Repressive Complex 2 ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates genes involved in cell lineage and differentiation through methylating lysine 27 on histone H3 (H3K27me3). Recurrent gain-of-function mutations of EZH2 have been identified in various cancer types, in particular, diffuse large B-cell lymphoma (DLBCL), through large-scale genome-wide association studies and EZH2 depletion or pharmacological inhibition has been shown to exert an antiproliferative effect on cancer cells, both in vitro and in vivo. In the current study, a combination of pomalidomide and GSK126 synergistically inhibited the growth of EZH2 gain-of-function mutant Diffuse large B-cell lymphoma (DLBCL) cells. Furthermore, this synergistic effect appeared to be dependent on cereblon (CRBN), a cellular receptor of pomalidomide, but not degradation of IKAROS family zinc finger 1 (IKZF1) or IKAROS family zinc finger 3 (IKZF3). RNA sequencing analyses revealed that co-treatment with GSK126 and pomalidomide induced specific gene sets involved in B-cell differentiation and apoptosis. Synergistic growth inhibition and B-cell differentiation were further validated in xenograft mouse models. Our collective results provide a molecular basis for the mechanisms underlying the combined therapeutic effects of PRC2 inhibitors and pomalidomide on EZH2-mutated DLBCL.

scholarly journals GUCY2D Gene Loss-of-Function Mutations Responsible for Leber Congenital Amaurosis 1

2019 ◽  
Author(s):  
Feng Xue ◽  
Tianying Wei ◽  
Junhui Sun ◽  
Yuqin Luo ◽  
Yanan Huo ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Guanylate Cyclase ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Loss Of Function ◽  
Wild Type ◽  
Leber Congenital Amaurosis ◽  
Cellular Location ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background: Leber congenital amaurosis (LCA) is a group of severe congenital neurodegenerative diseases. Variants in guanylate cyclase 2D (GUCY2D), which encoded guanylate cyclase protein (ROS-GC1) associate with LCA1, accounting for 6–21% of all LCA cases. Methods: In this study, one family with LCA1 was recruited from China. A combination of next-generation sequencing (NGS) and Sanger sequencing was used for disease-causing mutations screening. Additionally, immunohistochemistry and HPLC-coupled tandem mass-spectrometry (HPLC-MS/MS) were used to confirm the cellular location and catalytic activity of ROS-GC1 mutants, respectively. Results: We found three novel mutations (c.139_139delC, c.835G>A and c.2783G>A) in GUCY2D gene. The results showed that mutation c.139_139delC results in a truncated protein and destroys the structure of ROS-GC1 protein. Mutations c.835G>A and c.2783G>A exert no effects on cellular location, whereas significantly reduce the catalytic activity of ROS-GC1. Conclusions: Our findings highlight the clinical range of LCA. Moreover we used HPLC-MS/MS to analyze the concentration of 3', 5'-cyclic guanosine monophosphate (cGMP), suggesting that HPLC-MS/MS can be an effective alternative method to evaluate the catalytic activity of wild type (wt) and mutant ROS-GC1.

scholarly journals MicroRNA Signatures In B-Cell Lymphomas

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4155-4155
Author(s):  
Lorena Di Lisio ◽  
Margarita Sanchez-Beato ◽  
Gonzalo Gomez-Lopez ◽  
Maria E. E. Rodriguez ◽  
Santiago Montes-Moreno ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Burkitt Lymphoma ◽  
Gene Expression Regulation ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoid Tissues ◽  
Molecular Features ◽  
Lymphoma Type

Abstract Abstract 4155 Beyond the conventional criteria of lymphoma classification (integrated clinical, morphological, immunophenotypic, and molecular features) additional markers are still required for a more precise differential diagnosis and a better understanding of lymphoma pathogenesis. MicroRNAs (miRNA) are non-coding small RNAs that play an important role in gene expression regulation, contributing to cell differentiation and tumorigenesis. Specifically, miRNAs have been already described to play a relevant role in B cell differentiation, and in some cases proposed to constitute lymphoma-type specific markers and possible therapeutic targets. We explore the potential diagnostic application of miRNA expression in a large series of 147 cases including all B-cell non-Hodgkin lymphomas (NHL) major types and appropriate controls. As an example of a practical application, data were also used to identify miRNAs differentially expressed when comparing Burkitt Lymphoma (BL) and Diffuse Large B-Cell Lymphoma (DLBCL) in paraffin-embedded samples. Each lymphoma type (BL, CLL, DLBCL, FL, MCL, MZL/MALT, NMZL and SMZL) was compared to the whole series of NHL by Significant Analysis of Microarray (SAM) method. The analysis identified a set of 128 characteristic miRNAs (FDR&lt;0.01 and Fold change &gt;1.5 log2). All lymphoma types were characterized by specific miRNA signatures, reflecting cell of origin and/or discrete oncogene alterations. Of interest is also the comparison with reactive lymphoid tissues, since it revealed a specific B-cell lymphoma miRNA profile, which includes a cluster of downregulated miRNAs, such as let7 family, miR-1 and miR-200 family. Burkitt Lymphoma was also directly compared to DLBCL, and 43 miRNA selected by SAM analysis were studied in a new series of 28 BL and 43 DLBCL samples using quantitative RT-PCRIn this second step, the differential expression of a set of 19 miRNAs was confirmed between BL and DLBCL. (FDR &lt; 0.05 after t-test (limma)). These findings expand the potential diagnostic markers in lymphoma diagnosis and provide useful information on lymphoma pathogenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High frequency of inactivating tetraspanin CD37 mutations in diffuse large B-cell lymphoma at immune-privileged sites

Blood ◽  
2019 ◽  
Vol 134 (12) ◽  
pp. 946-950 ◽  
Author(s):  
Suraya Elfrink ◽  
Charlotte M. de Winde ◽  
Michiel van den Brand ◽  
Madeleine Berendsen ◽  
Margaretha G. M. Roemer ◽  
...  
Keyword(s):  
B Cell ◽  
High Frequency ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Loss Of Function ◽  
Large B Cell Lymphoma ◽  
Key Points ◽  
Surface Localization ◽  
Cell Surface Localization ◽  
Large B Cell

Key Points Loss-of-function mutations in CD37 occur predominantly in diffuse large B-cell lymphoma at immune-privileged sites. CD37-mutated lymphoma B cells show impaired CD37 cell-surface localization, which may have implications for anti-CD37 therapies.

An Activating Mutation (Ser525Pro) within the Transactivation Domain of REL in Two Patients with Human B-Cell Lymphomas Enhances REL’s In Vitro Transforming Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2617-2617
Author(s):  
Heiko Trautmann ◽  
Daniel T. Starczynowski ◽  
Christiane Pott ◽  
Lana Harder ◽  
Norbert Arnold ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Transactivation Domain ◽  
Wild Type ◽  
Spleen Cells ◽  
B Cell Lymphomas ◽  
Chicken Spleen ◽  
Human B Cell

Abstract REL/NF-κB transcription factors are implicated in the control of apoptosis and cell growth particular in hematopoetic lineages. The REL locus at chromosomal region 2p13–16 is frequently amplified in B-cell lymphomas including diffuse-large B-cell lymphoma (DLBCL) and may play a role in lymphomagenesis. Overexpression of wild-type REL can transform chicken lymphoid cells in culture, and several experimentally-generated mutations within the REL C-terminal transactivation domain (TAD) have been previously shown to enhance REL’s transforming ability. We analysed 83 B-cell lymphomas included in the ‘Deutsche Krebshilfe’ funded network „Molecular Mechanisms in Malignant Lymphoma“ for the presence of activating mutations in the coding region of REL. We performed a systematic dHPLC screening for mutation discovery and identified an identical point mutation in two human B-cell lymphomas (a t(14;18)-positive follicular lymphoma and a mediastinal B-cell lymphoma) that changes Ser525 to Pro within the REL TAD. In the mediastinal B-cell lymphoma, the mutation in REL was proven to be of germline origin. FISH showed an amplification of the REL locus in the tumor cells of this case. Quantitative allelic discrimination of S525P indicates that the mutant REL gene was over-represented in both cases. By in vitro experiments we could show that the S525P mutation enhances the in vitro transforming ability of REL in chicken spleen cells. In addition, REL-S525P differs from wild-type REL in its ability to activate certain κB site-containing reporter plasmids in transient transfection assays. In particular, REL-S525P has a reduced ability to activate the human manganese superoxide dismutase (MnSOD) promoter in A293 cells; however, the MnSOD protein is over-expressed in REL-S525P-transformed chicken spleen cells as compared to wild-type REL-transformed cells. Ser525 of REL falls within a sequence that is similar to other known phosphorylation sites of the IκB kinase, and REL-S525P shows a reduced ability to be phosphorylated by IKKα in vitro. The S525P mutation reduces IKKα- and TNFα-stimulated transactivation by REL, as measured in GAL4 reporter assays. Furthermore, REL-S525P-transformed chicken spleen cells are more resistant to TNFα-induced cell death than cells transformed by wild-type REL. These results represent the first identification of a tumor-derived activating mutation in the REL proto-oncogene, and they suggest that the S525P mutation contributes to the development of human B-cell lymphomas by altering REL’s ability to induce target gene expression by affecting an IKKα-regulated transactivation activity.

CtBP1 Is a Novel and Essential Corepressor for BCL6 Autoregulation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3581-3581
Author(s):  
Lourdes M. Mendez ◽  
Jose Polo ◽  
Melissa Krupski ◽  
Jessica Yu ◽  
Ari M. Melnick ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Cell Lines ◽  
Cell Biology ◽  
Target Genes ◽  
Point Mutations ◽  
B Cell Lymphoma ◽  
B Cell Differentiation ◽  
Wild Type ◽  
Negative Autoregulation

Abstract BCL6 is POZ/BTB transcription repressor that is required for the germinal center (GC)- stage of B cell development and its deregulated expression underlies the development of many GC-derived B cell lymphomas such as diffuse large B cell lymphoma (DLBCL). BCL6 carries out its biological function by repressing target genes involved in various aspects of B cell biology such as DNA damage response, cell-cycle regulation and plasma cell differentiation. Recent publications indicate that BCL6 differentially utilizes its corepressor partners to silence target genes involved in different biological processes. Negative autoregulation of BCL6 is likely to play an important role in B-cell differentiation, and is frequently disrupted in DLBCL due to translocation or point mutation of the BCL6 promoter. However, from a mechanistic standpoint, it is not known how BCL6 mediates negative autoregulation. BCL6 is reported to repress its target genes through binding of the SMRT, NCoR and BCoR corepressors to its N-terminal POZ domain and through binding of the MTA3 and HDAC2 corepressors to its second repression domain. However, a BCL6 mutant unable to bind these corepressors retained near wild-type repression activity on the BCL6 promoter. The expression of endogenous BCL6 was unchanged in DLBCL cell lines treated with BCL6 Peptide Inhibitor, which selectively disrupts the association between BCL6 and its POZ domain corepressors, or with MTA3 siRNA. This led us to consider the possibility that BCL6 autoregulation proceeds through a novel corepressor. Several POZ transcription factors can interact with CtBP as their corepressor. We found BCL6 and CtBP can interact in both the ectopically expressed and endogenous settings in DLBCL cells. Moreover, our ChIP experiments demonstrate that CtBP is present in the 5′UTR of BCL6 at sites that were previously shown by us and others to mediate BCL6 negative autoregulation. Nearly half of DLBCL patients are estimated to carry translocations and “activating” point mutations in the 5′UTR of BCL6 which allow negative autoregulation to be bypassed. In DLBCL cell lines carrying BCL6 promoter mutations or translocations, CtBP was preferentially bound to the wild-type BCL6 allele. Moreover, CtBP siRNA specifically derepressed the wild-type allele sparing the translocated BCL6 allele driven by heterologous promoters. This allelic analysis of BCL6 is consistent with a model in which BCL6 recruits CtBP to carry out negative autoregulation. Tiling ChIP-on-chip of BCL6 target genes showed colocalization of CtBP in a BCL6 repression complex at only a subset of target genes, including BCL6. However, the BCL6 locus was the only target dependent exclusively on CtBP for repression. In an effort to address the corepressor requirements of BCL6 autoregulation, we have uncovered a novel BCL6 corepressor, CtBP. Our results substantiate the growing body of evidence that BCL6’s mechanism of repression is dynamic, selectively calling upon corepressors to silence different cohorts of target genes perhaps reflecting segregation of biological functions. Our study provides new insight into normal BCL6-driven biology and also informs BCL6-targeted lymphoma therapies.

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