MiR-28 Silencing In Germinal Center-Derived Lymphomas

Abstract Abstract 703 In order to gain insights into the role of microRNA (miRNAs) in mature B cell function and lymphomagenesis, we performed a comprehensive miRNA expression profiling of normal mature B cells and of germinal center (GC)-derived lymphomas. miRNA expression profiles were generated using a commercial array platform designed to interrogate approximately 700 miRNAs. Normal GC B cells were isolated from tonsil tissue of 5 pediatric healthy donors and subjected to miRNA profiling in parallel with tumor specimens obtained from 10 Burkitt Lymphoma (BL), 16 Follicular Lymphoma (FL) and 20 Diffuse Large B Cell Lymphoma (DLBCL) patients. Each tumor type displayed a distinct miRNA profile and appeared to be clearly separated from the normal counterpart. Interestingly, a set of miRNAs was expressed in normal GC cells, but not in lymphoma samples, suggesting that structural and/or functional loss of miRNAs occur during lymphomagenesis. Among these, miR-28 was found to be up-regulated in GC B cells, while it was completely silenced in BL and significantly reduced in a large fraction of DLBCL and FL. Quantitative RT-PCR analysis confirmed miR-28 reduced expression in these GC-derived lymphoma subtypes including both primary biopsies and cell lines. MiR-28 is an intragenic miRNA encoded by the LPP gene locus, located on chromosome 3q28. Deletions affecting miR-28 and LPP were previously reported in FL (Schwaenen et al. Genes Chromosomes Cancer 2009; 48:39-54) and similarly we identified LPP deletions in about 9% of DLBCL investigated for copy number alterations by SNP arrays. Further FISH analyses performed in cell lines lacking miR-28 expression (12 DLBCL and 27 BL) failed to identify chromosomal aberrations in the LPP locus, suggesting that mechanisms other than genetic losses, possibly of epigenetic nature, are involved in miR-28 silencing in lymphomas. In order to investigate the effects of miR-28 in lymphoma cells, we generated stable lymphoma cell lines displaying inducible expression of miR-28. Re-expression of miR-28 in lymphoma cells led to a retarded growth due to a combination of G1-cell-cycle arrest and increased apoptosis. Furthermore, lymphoma cells expressing miR-28 lost their clonogenic properties as shown by their inability to form colonies in soft agar. Taken together these results suggest a tumor suppressor function for miR-28 in lymphoma cells. Toward the identification of the direct miR-28 target genes, we implemented computational target prediction methods with gene expression profiling data obtained from the miR-28 engineered cell lines. Approximately two thousand miR-28 direct candidate targets were computationally predicted, 88 of which displayed transcriptional down-regulation upon miR-28 induction, suggesting that miR-28 may affect the stability of the target transcript. The candidate targets included genes involved in the control of cell proliferation and apoptosis and in cell signaling, consistent with the phenotypic changes induced by miR-28 expression. In 4 out of 5 tested candidate targets, 3′-UTR reporter gene assay confirmed the direct effect of miR-28 on the target gene. Finally, we have generated a conditional, GC B cell-specific miR-28 knock-out mouse model, which will provide critical insights on the physiologic as well as the tumor suppressor role of miR-28 in vivo. Disclosures: No relevant conflicts of interest to declare.

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Integrative Genomics Reveals a Role for GNA13 in Lymphomagenesis within the Germinal Center Niche

Blood ◽

10.1182/blood.v124.21.782.782 ◽

2014 ◽

Vol 124 (21) ◽

pp. 782-782

Author(s):

Jane Healy ◽

Adrienne Greenough ◽

Rachel Rempel ◽

Moffitt Andrea ◽

Izidore S Lossos ◽

...

Keyword(s):

Cell Migration ◽

B Cells ◽

Tumor Suppressor ◽

B Cell ◽

Mouse Models ◽

Cell Lines ◽

Germinal Center ◽

Burkitt Lymphoma ◽

B Cell Lymphoma

Abstract Nonhodgkin Lymphoma (NHL) is among the most common cancer subtypes, with approximately >350,000 new cases diagnosed annually worldwide. The vast majority of NHLs arise from germinal center (GC) B cells. We and others have identified GNA13 as one of the most frequently mutated genes in GC-derived lymphomas, including ~30% of Burkitt Lymphoma and ~25% of Germinal Center B Cell-like (GCB) Diffuse Large B cell Lymphoma. Despite this association, the role of GNA13 in lymphomagenesis remains elusive. In human breast and prostate cancer, GNA13 behaves as an oncogene, with increased expression linked to cellular invasion and metastasis. Intriguingly, GNA13 mutations in GCB DLBCL and Burkitt Lymphoma are frequently inactivating, possessing a high number of nonsense and missense mutations in conserved domains. This suggests that GNA13 may function as a tumor suppressor in the context of lymphoma, in contrast to its role in solid tumors. The purpose of this study is to define the role of GNA13 in GC B cells and to clarify how GNA13 loss may contribute to lymphoma within the germinal center niche. We first investigated the expression pattern of GNA13 in lymphocyte populations from normal human tonsil. Our data demonstrated that GNA13 is enriched in GC B cells by quantitative PCR and immunohistochemistry. To determine the effect of GNA13 abundance on global mRNA expression patterns, we performed RNA sequencing on lymphoma derived cell lines. Using this method, we found that GNA13 knockdown and overexpression was highly correlated with GC dark and light zone gene signatures, respectively. We next devised a proteomics approach to identify potential GNA13 binding partners in GCs. Lysates from lymphoma-derived cell lines overexpressing FLAG-tagged GNA13 were subjected to immunoprecipitation with M2-antibody bound magnetic beads, followed by LC-MS/MS. Our results demonstrated an enrichment of proteins involved in focal adhesion, consistent with the known involvement of GNA13 in processes of cytoskeletal reorganization and cell migration. We next explored the role of GNA13 in vivo. Since GNA13 mutations are a unique feature of GC-derived lymphomas, we developed mouse models that would allow us study GNA13 exclusively in the germinal center context. We generated B cell and GC specific GNA13 knockout mice by crossing GNA13fl/fl mice with MB1-Cre and AID-Cre strains. After immunization with sheep red blood cells, both B cell and GC specific GNA13 deficient mice possessed normal levels of B, T and GC cells within secondary lymphoid sites including Peyer’s patches and spleen, suggesting that GNA13 is not essential for GC formation. GC B cells from both GNA13 deficient strains demonstrated enhanced cellular motility toward GC directed chemokines CXCL12, CXCL13 and S1P using in vitro transwell migration assays. Furthermore, B cells isolated from GNA13 deficient animals showed enhanced RhoA activity. These data suggested that GNA13 inhibits GC B cell migration and RhoA mediated cell motility in normal conditions. Loss of GNA13 may then deregulate normal chemokine gradient signaling, resulting in global increases in GC migration. We also demonstrated that GNA13 deficient B cells possess elevated levels of phosphorylated AKT, an effect potentiated by the addition of CXCL12 and S1P. AKT signaling is known to promote cell survival in a variety of cell types, which may further promote oncogenesis. In this study, we have synthesized the complementary approaches of next generation sequencing, proteomics and genetic mouse models to gain novel insight into the biological function of GNA13, a gene that is mutated in a high proportion of GC-derived lymphomas. As a whole, our work suggests that GNA13 serves as a tumor suppressor during the germinal center reaction. The acquisition of inactivating GNA13 mutations may promote lymphoma by allowing cells to physically escape the germinal center niche and evade apoptosis while continuing to express GC signature genes. Affected cells may be subjected to persistent somatic hypermutation, which, over time, could result in the accumulation of additional oncogenic mutations, culminating in development of GC-derived lymphoma. Disclosures No relevant conflicts of interest to declare.

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CD40L Modulates TRAIL-Induced Apoptosis in Germinal Center Derived B Cell Lymphomas.

Blood ◽

10.1182/blood.v108.11.4630.4630 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4630-4630

Author(s):

Marion Travert ◽

Patricia Ame-Thomas ◽

Thierry Fest ◽

Céline Pangault ◽

Gilbert Semana ◽

...

Keyword(s):

Follicular Lymphoma ◽

B Cell ◽

Cell Lines ◽

Germinal Center ◽

B Cell Lymphoma ◽

B Cell Lymphomas ◽

Lymphoma Cells ◽

Over Expression ◽

Induced Apoptosis ◽

Transformed Cell Lines

Abstract Follicular lymphoma are characterized by the rearrangement of the bcl-2 gene, present in more than 90% of patients. Over-expression of the bcl-2 protein resulting from this translocation is associated with the inability to eradicate the lymphoma, by inhibiting apoptosis. Despite the median survival ranges from 8 to 15 years, leading to the designation of indolent lymphoma, patients with advanced-stage follicular lymphoma are not cured with current therapeutic options. Numerous reports have shown that Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in a wide variety of transformed cell lines of diverse lineage, but does not appear to kill normal cells, even though TRAIL mRNA is expressed at significant levels in most normal tissues. As cell death induced by TRAIL occurs almost exclusively in tumor cells, it suggests that this drug is safe to use as an antitumor therapy. We therefore investigated the efficiency of this cytokine to induce apoptosis in germinal center derived B cell lymphoma, despite bcl-2 over-expression. Our study was also designed to evaluate the role of CD40L, one of the main differentiation signal involved in B cell maturation during the germinal center reaction, on the regulation of TRAIL-induced apoptosis. This study was performed on three germinal center derived tumor cell lines (BL2, VAL and RL), and on normal and tumor primary cells obtained from human tonsils and lymph nodes. Our data show that normal B lymphocytes obtained from tonsil biopsies are resistant to TRAIL-mediated apoptosis, when B lymphoma cells issued from lymph node of numerous patients are significantly sensitive to the cytokine. When we treat these lymphoma cells with trimeric huCD40L, we partly rescue these cells from spontaneous apoptosis which naturally occurs after few days of culture, and reverse by 50% TRAIL-mediated apoptosis when cells were co-treated with huCD40L for 16 hours. Similar results were reproduced on some germinal center derived cell lines. BL2 was indeed found highly sensitive to TRAIL-induced apoptosis following a 24 hour exposure. On the opposite, VAL and RL were almost insensitive. We have demonstrate that apoptosis is exclusively mediated by TRAIL-R1 in BL2. Analysis of signalling pathways revealed that the protection to TRAIL-induced apoptosis by CD40L is due to some specific anti-apoptotic molecules that will be described. Genes encoding these molecules are targets of the NFκB signalling pathway activated by CD40L. Our results suggest that activation of NFκB and induction of anti-apoptotic molecules by CD40L play an important role in the protection of germinal center derived B cell lymphomas against apoptosis. Then, NFκB inhibitors may be wise to use in clinical trials in conjunction with TRAIL against follicular lymphomas.

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New Pathogenetic Insights Into Classical Hodgkin Lymphoma Revealed by Gene Expression Profiling of Microdissected Hodgkin/Reed-Sternberg Cells.

Blood ◽

10.1182/blood.v114.22.266.266 ◽

2009 ◽

Vol 114 (22) ◽

pp. 266-266 ◽

Cited By ~ 1

Author(s):

Enrico Tiacci ◽

Verena Brune ◽

Susan Eckerle ◽

Wolfram Klapper ◽

Ines Pfeil ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Gene Expression Profiling ◽

Hodgkin Lymphoma ◽

Cell Lines ◽

Expression Profiling ◽

Expression Profiles ◽

B Cell Lymphoma ◽

Cell Phenotype

Abstract Abstract 266 Background. Previous gene expression profiling studies on cHL have been performed on whole tissue sections (mainly reflecting the prominent reactive background in which the few HRS cells are embedded), or on cHL cell lines. However, cultured HRS cells do not likely reflect primary HRS cells in all aspects, being derived from end-stage patients and from sites (e.g. pleural effusions or bone marrow) which are not typically involved by cHL and where HRS cells lost their dependence on the inflammatory microenvironment of the lymph node. Methods. ∼1000–2000 neoplastic cells were laser-microdissected from hematoxylin/eosin-stained frozen sections of lymph nodes taken at disease onset from patients with cHL (n=16) or with various B-cell lymphomas (n=35), including primary mediastinal B-cell lymphoma (PMBL) and nodular lymphocyte-predominant Hodgkin lymphoma (nLPHL). After two rounds of in vitro linear amplification, mRNA was hybridized to Affymetrix HG-U133 Plus 2.0 chips. Expression profiles were likewise generated from sorted cHL cell lines and several normal mature B-cell populations. Results. Primary and cultured HRS cells, although sharing hallmark cHL signatures such as high NF-kB transcriptional activity and lost B-cell identity, showed considerable transcriptional divergence in chemokine/chemokine receptor activity, extracellular matrix remodeling and cell adhesion (all enriched in primary HRS cells), as well as in proliferation (enriched in cultured HRS cells). Unsupervised and supervised analyses indicated that microdissected HRS cells of cHL represent a transcriptionally unique lymphoma entity, overall closer to nLPHL than to PMBL but with differential behavior of the cHL histological subtypes, being HRS cells of the lymphocyte-rich and mixed-cellularity subtypes close to nLPHL cells while HRS cells of NS and LD exhibited greater similarity to PMBL cells. HRS cells downregulated a large number of genes involved in cell cycle checkpoints and in the maintenance of genomic integrity and chromosomal stability, while upregulating gene and gene signatures involved in various oncogenic signaling pathways and in cell phenotype reprogramming. Comparisons with normal B cells highlighted the lack of consistent transcriptional similarity of HRS cells to bulk germinal center (GC) B cells or plasma cells and, interestingly, a more pronounced resemblance to CD30+ GC B cells and CD30+ extrafollicular B cells, two previously uncharacterized subsets that are transcriptionally distinct from the other mature B-cell types. Conclusions. Gene expression profiling of primary HRS cells provided several new insights into the biology and pathogenesis of cHL, its relatedness to other lymphomas and normal B cells, and its enigmatic phenotype. Disclosures: No relevant conflicts of interest to declare.

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The Transcriptional Network Governed by Interferon Regulatory Factor (IRF8) In Germinal Center B Cell Lymphomas.

Blood ◽

10.1182/blood.v116.21.3638.3638 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3638-3638

Author(s):

Dong-Mi Shin ◽

Chang-Hoon Lee ◽

Herbert Morse

Keyword(s):

B Cells ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Germinal Center ◽

Expression Profiling ◽

Transcriptional Network ◽

Human And Mouse ◽

Chip Chip ◽

Mouse Lines

Abstract Abstract 3638 The transcription factor IRF8, is highly expressed in germinal center (GC) centroblastic B cells and diffuse large B cell lymphomas (DLBCL). IRF8 is known to play roles in GC formation as well as in early B cell fate commitment, functioning both as a transcriptional activator and repressor; however, the transcriptional network regulated by IRF8 has not been elucidated yet. To identify direct targets of IRF8 on a genome-wide scale, we used ChIP-chip and expression profiling to study human and mouse lymphoma cell lines of GC origin – human Ly1, Odh1 and Val and mouse NFS201, NFS202, and NFS205. The IRF8-negative human multiple myeloma cell line, MMS1, and mouse plasmacytoma cell line, MPC11, were used as negative controls. For the human lines, 271 IRF8 target genes with common binding sites in all three cell lines, but not in negative cell line, were highly enriched for genes involved in innate immunity and adaptive immunity. Two well-established IRF8 target motifs- GAAANNGAAA and GGAANNGAAA - were significantly enriched in promoters of these genes compared to sequences 1kb upstream. Studies of the mouse lines identified 871 IRF8 targets with the lowest number found in NFS205. NFS205 expressed little PU.1, a common partner of IRF8 in DNA binding. PU.1 ChIP-chip analyses of the mouse lines allowed us to classify IRF8 targets into those that are PU.1-associated and PU.1-independent. Analyses of expression profiling of siIRF8-treated cells were used to identify transcriptionally active IRF8 targets. Gene Set Enrichment Analysis (GSEA) revealed significantly high enrichment of IRF8 ChIP targets as well as significant enrichment of genes involved in hematopoietic development. Finally 52 IRF8 targets common to human and mouse GC neoplasms were identified and characterized as contributing to antigen presentation, BCR and CD40 signaling, and interferon signaling, which can contribute differentially to the normal GC reaction and B cells transformation. These results define the contribution of IRF8 to the transcriptional network in GC B cells and provide new insights into the workings of the GC reaction in health and disease. This work was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. Disclosures: No relevant conflicts of interest to declare.

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Cohesin Core Complex Gene Dosage Contributes to Germinal Center Derived Lymphoma Phenotypes and Outcomes

Frontiers in Immunology ◽

10.3389/fimmu.2021.688493 ◽

2021 ◽

Vol 12 ◽

Author(s):

Martin A. Rivas ◽

Ceyda Durmaz ◽

Andreas Kloetgen ◽

Cristopher R. Chin ◽

Zhengming Chen ◽

...

Keyword(s):

B Cells ◽

Tumor Suppressor ◽

B Cell ◽

Cell Fate ◽

Plasma Cell ◽

Germinal Center ◽

Gene Dosage ◽

B Cell Lymphoma ◽

Dlbcl Patient ◽

Atpase Subunit

The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that Smc3 functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.

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Inactivation of the PRDM1/BLIMP1 Gene in Non-GC-Type Diffuse Large B-Cell Lymphoma.

Blood ◽

10.1182/blood.v106.11.2614.2614 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2614-2614

Author(s):

Laura Pasqualucci ◽

Mara Compagno ◽

Jane Houldsworth ◽

Stefano Monti ◽

Adina Grunn ◽

...

Keyword(s):

B Cells ◽

Tumor Suppressor ◽

B Cell ◽

Germinal Center ◽

B Cell Lymphoma ◽

Genetic Alterations ◽

Splice Acceptor Site ◽

Splice Donor Site ◽

Nonsense Mutations ◽

Hodgkin's Lymphomas

Abstract PRDI-BF1/Blimp1 is a zinc finger transcriptional repressor expressed in post-germinal center (GC) B cells and required for terminal B cell differentiation. The PRDM1/BLIMP 1 locus lies on chromosomal band 6q21–q22.1, a region frequently deleted in B-cell non-Hodgkin’s lymphomas suggesting the existence of one or more tumor suppressor loci (Gaidano et al, Blood80:1781, 1992). To test whether genetic alterations affecting the BLIMP1 gene may be involved in DLBCL pathogenesis, we performed PCR amplification and direct sequencing of the BLIMP1 coding sequences in 136 cases, including 20 cell lines and 116 primary biopsies. Gene expression profiling analysis using the Affymetrix Gene Chip system was used in 93 cases to classify them as germinal center B-cell like (GCB, N=38), activated B-cell like (ABC, N=35) and type III (N=20). Nonsense mutations were found in 7 of 136 cases. Four of these mutations generated premature stop codons, predicting severely truncated proteins of 61 to 244 aminoacids; in three cases, a single bp substitution affecting the exon 2 splice donor site led to insertion of 101 nucleotides from intron 3 and a premature stop codon. One primary tumor case carried a mutation within the intron 3 splice acceptor site, and one cell line displayed a gene rearrangement in one allele with deletion of the second allele. Strikingly, 6 of the 7 nonsense mutations identified segregated with the ABC phenotype (the remaining case was not profiled), suggesting that these alterations may be common and specific in this subtype of DLBCL (6/35, 17%). In addition, missense mutations generating aminoacid substitutions were found in 8 additional cases, and complete lack of Blimp-1 protein expression was found in most ABC-type DLBCL cases. These observations suggest that inactivation of the BLIMP1 gene may occur also by other mechanisms, including the generation of dominant negative mutants, chromosomal deletion or epigenetic silencing, in a large fraction of DLBCL. Functional studies are currently being performed to corroborate these data. Overall, these results suggest that BLIMP1 may act as a tumor suppressor gene, whose loss or inactivation may contribute to lymphomagenesis by blocking post-GC differentiation of B cells toward plasma cells.

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B Cell Lymphomas Are Resistant towards Bone Morphogenetic Protein (BMP) Induced Growth Inhibition.

Blood ◽

10.1182/blood.v108.11.2381.2381 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2381-2381

Author(s):

Kanutte Huse ◽

Marianne B. Eide ◽

Christian Kersten ◽

Erlend B. Smeland ◽

June H. Myklebust

Keyword(s):

Growth Inhibition ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Lymphoma Cells ◽

Bmp Receptors ◽

Induced Apoptosis

Abstract Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily, and mediate their effects mainly through the Smad signalling pathway. Whereas TGF-β is well established as one of the most potent negative regulators in hematopoietic cells, the role of BMPs remains more elusive. We have previously shown that BMP-6 inhibits the growth of naïve and memory human B cells. As high BMP-6 mRNA expression is associated with poor outcome in diffuse large B cell lymphoma (DLBCL; Rosenwald et al, N Engl J Med 2002), we hypothesized that resistance towards BMP-induced growth inhibition is a possible mechanism for lymphomagenesis. In the current study, 7 B cell lymphoma cell lines (representing Burkitt lymphoma (BL) and DLBCL) and tumour material from lymphoma patients were investigated to unravel the role of BMPs in lymphomas. We analyzed the expression of BMP receptors by FACS analysis, and found variable expression of the BMP receptor type I (Alk2, Alk3 and Alk6) and type II (BMP RII, Activin RIIA and RIIB) among the cell lines and in primary lymphoma cells, suggesting variable binding of BMPs. We next investigated the effect of BMP-2, BMP-4, BMP-6 and BMP-7 on proliferation and survival of B lymphoma cell lines, and found 2 of 7 cell lines to be resistant towards BMP-2 and BMP-4 induced growth inhibition. In contrast, 4 of 7 and 7 of 7 cell lines were resistant to BMP-6 and BMP-7 induced growth inhibition, respectively. In Sudhl6 cells that were highly sensitive to BMP-2 and BMP-6 induced apoptosis and inhibition of proliferation, we demonstrated that the cytokines IL-10, CD40 Ligand and BLyS were able to counteract the negative effects induced by BMPs, while IL-2 and IL-4 were not. On the contrary, both BMP-2 and BMP-6 greatly increased anti-IgM activation induced apoptosis. In resistant lymphoma cells, the BMPs were not able to induce detectable levels or induced low levels of phosphorylated SMAD1/5/8 compared to sensitive cell lines. Low or no increase in phosphorylation of SMAD1/5/8 induced by BMPs could only partly be explained by low/ undetectable expression of BMP receptors. Hence, upregulation of inhibitory Smads (Smad6, Smad7) or mutations in receptors or Smads represent other possible mechanisms for resistance to BMPs in lymphomas, and this is currently under investigation. We also investigated if the lymphoma cells produced BMPs themselves and found that 5 of 7 cell lines and 3 of 5 primary lymphomas produced significant amounts of BMP-7. Some lymphoma cells also had detectable levels of BMP-4 and BMP-6. Our findings that lymphoma cells are resistant towards BMP-7 and to some degree BMP-6 induced growth inhibition, whereas they produce these cytokines, suggest that resistance towards BMP induced signalling in B cell lymphomas can contribute to increased tumour growth.

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Epigenetic suppression of SLFN11 in germinal center B-cells during B-cell development

PLoS ONE ◽

10.1371/journal.pone.0237554 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0237554

Author(s):

Fumiya Moribe ◽

Momoko Nishikori ◽

Tsuyoshi Takashima ◽

Daiki Taniyama ◽

Nobuyuki Onishi ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lines ◽

Germinal Center ◽

Cytosine Arabinoside ◽

Immunohistochemical Staining ◽

Cell Development ◽

B Cell Development ◽

Lymphoma Cells ◽

Epigenetic Modifiers

Background SLFN11 has recently been reported to execute cancer cells harboring replicative stress induced by DNA damaging agents. However, the roles of SLFN11 under physiological conditions remain poorly understood. Germinal center B-cells (GCBs) undergo somatic hypermutations and class-switch recombination, which can cause physiological genotoxic stress. Hence, we tested whether SLFN11 expression needs to be suppressed in GCBs during B-cell development. Objective To clarify the expression profile of SLFN11 in different developmental stages of B-cells and B-cell-derived cancers. Methods We analyzed the expression of SLFN11 by mining cell line databases for different stages of normal B-cells and various types of B-cell-derived cancer cell lines. We performed dual immunohistochemical staining for SLFN11 and B-cell specific markers in normal human lymphatic tissues. We tested the effects of two epigenetic modifiers, an EZH2 inhibitor, tazemetostat (EPZ6438) and a histone deacetylase inhibitor, panobinostat (LBH589) on SLFN11 expression in GCB-derived lymphoma cell lines. We also examined the therapeutic efficacy of these drugs in combination with cytosine arabinoside and the effects of SLFN11 on the efficacy of cytosine arabinoside in SLFN11-overexpressing cells. Results SLFN11 mRNA level was found low in both normal GCBs and GCB-DLBCL (GCB like-diffuse large B-cell lymphoma). Immunohistochemical staining showed low SLFN11 expression in GCBs and high SLFN11 expression in plasmablasts and plasmacytes. The EZH2 and HDAC epigenetic modifiers upregulated SLFN11 expression in GCB-derived lymphoma cells and made them more susceptible to cytosine arabinoside. SLFN11 overexpression further sensitized GCB-derived lymphoma cells to cytosine arabinoside. Conclusions The expression of SLFN11 is epigenetically suppressed in normal GCBs and GCB-derived lymphomas. GCB-derived lymphomas with low SLFN11 expression can be treated by the combination of epigenetic modifiers and cytosine arabinoside.

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Deletion of Murine Rhoh induces More Aggressive Diffuse Large B Cell Lymphoma (DLBCL) Via Interaction with Kaiso and Regulation of BCL-6 Expression

Blood ◽

10.1182/blood-2018-99-115238 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1574-1574

Author(s):

Hiroto Horiguchi ◽

Marioara Felicia Ciuculescu ◽

Anja Troeger ◽

Haiming Xu ◽

Christian Brendel ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Nuclear Localization ◽

Germinal Center ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Germinal Center Formation

Abstract RHOH encodes a GTPase-deficient, hematopoietic-specific small GTPase first identified as a hypermutable gene in DLBCL (Pasqualucci et al. 2001). RhoH is critical for T cell receptor signaling and Rhoh-deficient (RhohKO) mice have T cell lymphopenia (Gu et al., 2006) and loss of function mutations of RHOH are associated with Epidermodysplasia Verruciformis (Crequer et al., 2012). However, the role of RhoH in the biology of DLBCL is still unknown and its role in B lymphoid development is incompletely studied. We investigated the role of RhoH in normal germinal center formation and in a murine model of DLBCL by crossing RhohKO mice with Iµ-HABcl-6 transgenic (Bcl-6Tg) mice (Cattoretti G, et al., 2005). In young RhohKOmice, deficient development of CXCR5+ follicular T helper (Tfh) cells results in defective germinal center (GC) formation and impaired immunoglobulin switching in vivo. In spite of this defect in GC formation, RhohKO; Bcl-6Tg (KOTg) mouse demonstrated accelerated lymphoma progression associated with larger spleens and significantly earlier death (Log-rank test p<0.01, Figure 1). Immunohistochemistry data suggested increased expression of IRF-4 and enhanced expression of BCL-6 in KOTg mice, findings confirmed by immunoblot and consistent with an activated B-cell (ABC)-DLBCL phenotype. To analyze the mechanism underlying these results, B cell lymphoma cell lines from KOTg lymphoma mice were established. Multiple attempts to establish RhohWT lymphoma cell lines failed, although we also successfully established a lymphoma cell line from RhohKO; Bcl-6(ntg) (KONtg) mice. Re-expression of RhoH in these lines via retrovirus mediated gene transfer led to significantly decreased proliferation (5.9x106±9.6x105 cells vs 8.6x106±9.6x105 cells after 5-days culture; KOTg vs KOTg-RhoH, mean±SEM, p<0.05) that was associated with clear reduction in BCL-6 expression. These data suggest that BCL-6 is a direct or an indirect transcriptional target of RhoH. Our laboratory previously reported that KAISO, a dual-specific, Broad complex, Trantrak, Bric-a-brac/Pox virus, Zinc finger (POZ-ZF) transcription factor interacts and colocalizes with RhoH in the nucleus, whereas knockdown of RhoH inhibits the nuclear localization of KAISO in Jurkat cells (Mino A, et al., 2016). In addition, Kaiso has been shown to be a key regulator of spleen germinal center formation by repressing Bcl-6 expression in splenocytes (Koh D, et al., 2013). We hypothesized that the deletion of Rhoh may lead to the decreased nuclear localization of KAISO and result in increased the expression of Bcl-6. We first confirmed that RhoH bound KAISO in RhoH-transduced KO lymphoma cells by co-immunoprecipitation. Further immunoblot analysis and quantitative PCR (qPCR) demonstrated decreased BCL-6 expression in lymphoma cells in which RhoH was re-expressed (KOTg-RhoH and KONtg-RhoH) compared with empty vector-transduced lymphoma cell lines. Interestingly, p53 a BCL-6 target was increased in RhoH-transduced lymphoma cell lines. These data indicate that RhoH affects BCL-6 expression in B cell lymphoma cell lines and suggest that RhoH may be involved in DLBCL development by co-regulating BCL-6 expression affecting downstream targets via interaction with KAISO. Figure. Figure. Disclosures Williams: Bluebird Bio: Research Funding.

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OBF1 and Oct factors control the germinal center transcriptional program

Blood ◽

10.1182/blood.2020010175 ◽

2021 ◽

Author(s):

Shuang Song ◽

Chun Cao ◽

Mohamed-Amin Choukrallah ◽

Fengyuan Tang ◽

Gerhard Christofori ◽

...

Keyword(s):

Transcription Factors ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Mechanism Of Action ◽

Germinal Center ◽

Ectopic Expression ◽

Transcriptional Program ◽

Lymphoma Cells ◽

Ets Family

OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by ChIP-seq that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2 and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. shRNA knockdown experiments demonstrated that OCT1, OCT2 and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance -such as BCL6- are downregulated, while genes related to exit from the GC program -such as IRF4- are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.

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