PRDM11 Is a Novel Putative Tumor Suppressor In Aggressive B Cell Lymphoma

Abstract Abstract 1002 Introduction and Aim: Deregulation of epigenetic factors contributes together with genetic alterations to the development of cancer. The PR domain proteins (PRDMs) constitute a sub-family of the SET domain family of histone methyl transferases and consist of 17 family members. Several of the PRDMs have been characterized as tumorsuppressors, including PRDM2, PRDM3, PRDM5 and PRDM16, but the mechanisms are largely unknown. In a functional screen with overexpressed MYC, we found that shRNA mediated down regulation of PRDM11 facilitates oncogenic transformation. Given the importance of MYC in lymphoma development it is essential to understand the genetic settings that facilitate MYC-induced transformation. We thus set out to investigate the function of PRDM11 in lymphomagenesis. Methods: To identify PR/SET proteins collaborating with MYC in cellular transformation a retroviral vector based library of shRNA targeting 61 SET and PR domain genes from mice and humans was generated. Primary mouse embryo fibroblasts (MEFs) were transduced with the library and with the Myc overexpression vector. A conditional Prdm11 knockout (KO) mouse strain and crosses to the Eμ-Myc tumor prone strain were generated to evaluate the tumorsuppressor potential of Prdm11 in vivo. The expression levels of PRDM11 in B cell lymphoma cell lines were evaluated by RT-qPCR and immunohistochemistry staining. PRDM11 expression level in Diffuse Large B Cell Lymphoma (DLBCL) patients was assessed by immunohistochemistry staining of DLBCL tissue microarrays (TMAs) according to the H-score. In order to investigate whether the PRDM11 expression was regulated by epigenetic mechanisms we performed H3K27me3 and H3K4me3 ChIP analysis and DNA methylation specific melting curve analysis. DGGE mutation scanning was applied to analyze 17 lymphoma cell lines and 77 DLBCL patients for point mutations. Results: We found that overexpression of Prdm11 in MEFs diminished growth and induced apoptosis in a manner independent of p53 and the intrinsic apoptotic pathway. Furthermore, Prdm11 (KO) MEFs grew faster than their wildtype (WT) littermate controls and transformed in the presence of oncogenic Myc. Prdm11 KO mice were viable and fertile with no apparent phenotype. The Eμ-Myc mice selectively express the Myc transgene in the B-cell lineage and develop malignant lymphomas with a mean latency of 100–120 days. Importantly, we found that loss of Prdm11 potently accelerated lymphomagenesis in the Eμ-Myc mouse (p<0,0006) and induced the incidence time from 111 to 75 days. To investigate the function if PRDM11 in humans, PRDM11 expression levels were evaluated in a panel of human DLBCL and Mantle Cell Lymphoma (MCL) cell lines. Compared to normal B-cells and reactive lymph nodes, PRDM11 mRNA expression levels were significantly lower or absent in 16/19 lymphoma cell lines. PRDM11 immunohistochemistry staining of DLBCL TMAs showed lower levels compared to reactive lymph nodes. PRDM11 immunohistochemistry staining in reactive lymph nodes was mainly localized to the activated B cells in the germinal centres. Interestingly, low or absent PRDM11 expression is associated with significantly worse overall survival (p = 0,011, univariate analysis of 22 patients). We are currently in the process of investigating the prognostic significance of PRDM11 expression in another 100 patients with DLBCL. These data will be presented at the meeting. We have found an inverse correlation between the expression level of PRDM11 and the presence of the repressive chromatin mark H3K27me3 at the PRDM11 promoter by ChIP analysis. H3K27me3 is less enriched at the promoter of PRDM11 in normal B cells as well as in cell lines with EZH2 missense mutation. DNA methylation was not detected in 17 lymphoma cell lines or 77 DLBCL patients and 3 PRDM11 sequence variants were also present in the germ line. In conclusion: PRDM11 is a novel putative tumor suppressor in mice and men, whose downregulation may be associated with poor prognosis in DLBCL. H3K27 trimethylation of the PRDM11 promoter may be a novel target for therapy in DLBCL. Disclosures: No relevant conflicts of interest to declare.

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Micro-RNA Gene Deregulation by Chromosome Translocations with Fragile Genomic Regions in B-Cell Lymphoma Cells

Blood ◽

10.1182/blood.v112.11.786.786 ◽

2008 ◽

Vol 112 (11) ◽

pp. 786-786

Author(s):

Bjoern Schneider ◽

Stefan Nagel ◽

Maren Kaufmann ◽

Hans G. Drexler ◽

Roderick A.F. MacLeod

Keyword(s):

Multiple Myeloma ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Micro Rna ◽

Lymphoma Cell ◽

Chromosome Rearrangements ◽

Causal Connection

Abstract Micro-RNA (miR) genes posttranscriptionally modulate target gene expression via imperfect 3′-UTR matching sequences and play key roles in development, homeostasis and cancer. Little is known how miR genes are themselves regulated, or deregulated in cancer. Chief paradigm for neoplastic miR deregulation concerns miR-17/92 cluster members subject to genomic amplification in B-cell lymphoma. While the repeated occurrence of oncogenic miR genes at or near chromosomal breakpoints in cancer links chromosome fragility to oncogenic miR deregulation, direct evidence of a causal connection remains tenuous. We found that t(3;7)(q27;q32) in a B-cell lymphoma cell line joins 5′-BCL6 to a noncoding region of chromosome 7 inside a common chromosomal fragile site (FRA7H). In these cells hybrid mRNA was absent, unlike canonical BCL6 translocations which involve promoter exchange yielding hybrid mRNA. Affected cells instead showed downregulation of miR-29b-1, the only gene located within FRA7H - a recurrent transcriptional feature of B-cell lymphoma subsets. In another BCL6 translocation, t(3;13)(q27;q31)t(13;12)(q31;p11), which 5′-RACE also showed to be non-fusogenic, long distance inverse (LDI)-PCR revealed junction of 5′-BCL6 to chromosome 13 sequences inside the miR-17/92 host gene MIRH1 (alias c13orf25). FISH using a sensitive tyramide amplification protocol with c13orf25 clones confirmed the presence of a cryptic BCL6-MIRH1 rearrangement. Surprisingly, reverse transcriptase quantitative (q) PCR assay revealed weak MIRH1 expression using 3′-primers. In contrast, repeating the assay using more central primers covering the miR-17/92 coding region showed massive upregulation. 3′-RACE confirmed a novel high level MIRH1 transcript truncated by 3.1 kbp. Quantitative genomic PCR and FISH excluded miR-17/92 genomic copy number alteration, while LDI-PCR analysis showed that formation of truncated MIRH1 involved multiple DNA cuts at 3q27 (x1), 12p11 (x1), and 13q31 (x5) – the last including a complex excision/inversion/insertion rearrangement. Stress induced DNA duplex destabilization (SIDD) analysis revealed that 6 of 7 breaks precisely coincided with fragility peaks. Taken together, these data suggest a novel role for BCL6 translocations in the deregulation of miR genes near sites of chromosome or DNA instability. BCL6 has been shown to suppress p53 in germinal center B-cells thus protecting B-cells from apoptosis induced by DNA damage, offering a possible explanation for chromosome rearrangements associated with genomic fragility therein. Chromosomal MIRH1 dysregulation is not limited to BCL6 expressing lymphomas, however: cytogenetic investigations performed on diverse leukemia-lymphoma cell lines, including those derived from multiple myeloma and plasma cell leukemia, showed 11/50 with cytogenetic rearrangements at or near MIRH1. In sister cell lines sequentially established at diagnosis and relapse of multiple myeloma, only the latter showed miR-17/92 chromosomal rearrangement and upregulation. Interestingly miR overexpression was limited to miR-92, while miR-17/18 were barely expressed. FISH analysis and qPCR showed that discrepant expression was associated with rearrangement upstream of MIRH1. In brief, our data show that like other cancer genes, oncogenic miRs are subject to dysregulation mediated by structural chromosome rearrangements.

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Anti-CD20 Antibody Rituximab and Anti-CD23 Antibody IDEC-152 Induce Apoptosis of Malignant B-Cells in Combination with Chemical Antagonists of XIAP.

Blood ◽

10.1182/blood.v104.11.1401.1401 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1401-1401 ◽

Cited By ~ 1

Author(s):

Massimo Mangiola ◽

Kate Welsh ◽

Shinichi Kitada ◽

Irene M. Pedersen ◽

Nuzhat Pathan ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Annexin V ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Lymphoma Cell ◽

Efficient Induction ◽

Anti Cd20

Abstract We tested the effects of Rituximab (anti-CD20) and IDEC-152 (anti-CD23) on apoptosis of B-cell malignancies, using established non-Hodgkin’s B-Cell lymphoma cell lines and freshly isolated Chronic Lymphocytic Leukemia (CLL) B-cells. We used monolayers of stably transfected CHO-cells expressing FcRγIII-A to present antibody to B-cells and promote crosslinking. Established B-cell lymphomas (n = 3) were cultured in the presence of FcRγIIIA-expressing CHO monolayer with or without MAbs and apoptosis was measured by annexin V/propidium iodide staining at various times thereafter. Both antibodies induced time-dependent apoptosis of B-cell lymphoma cell lines. After 48 hrs of treatment with either Rituximab or IDEC-152, the majority of the malignant B-cells were apoptotic (remaining viable cells = 28.7% ± 0.2137% for Rituximab and 30.87% ± 0.7332% for IDEC-152). Rituximab and IDEC-152 also induced marked increases in caspase activity in B-cell lymphoma cell lines, with fold-increases above baseline control cells of 25 ± 0.9031 and 24 ± 0.3839, respectively. In contrast, neither Rituximab nor IDEC-152 induced striking effects on primary CLL B-cells (n = 6). We therefore tested the combination of Rituximab or IDEC-152 with other agents that target anti-apoptotic proteins, exploring whether more efficient induction of apoptosis can be achieved. We cultured lymphoma cell lines and primary CLL specimens with chemical antagonists of XIAP (Schimmer, et al. Cancer Cell5: 25, 2004), an anti-apoptotic protein that inhibits effector caspases. When used at concentrations where XIAP antagonists alone were non-apoptotic (approximately 2.5 μM), a significant increase in apoptosis was achieved in cultures of lymphoma and CLL cells treated with either Rituximab or IDEC-152. These findings suggest that Rituximab or IDEC-152 may more efficiently induce apoptosis of malignant B-cells when combined with an apoptosis-sensitizing agent. (Supported by CA-81534; CA-78040; and an unrestricted grant from Genentech, Inc.).

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Biomimetic Synthetic High Density Lipoprotein Nanostructures Target the SR-B1 Receptor and Differentially Manipulate Cellular Cholesterol Flux in Lymphoma Cells: A Novel Treatment Paradigm

Blood ◽

10.1182/blood.v120.21.157.157 ◽

2012 ◽

Vol 120 (21) ◽

pp. 157-157 ◽

Cited By ~ 1

Author(s):

Shuo Yang ◽

Marina G Damiano ◽

Heng Zhang ◽

Sushant Tripathy ◽

Andrey Ugolkov ◽

...

Keyword(s):

B Cells ◽

High Density Lipoprotein ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Density Lipoprotein ◽

B Cell Lymphoma ◽

High Density ◽

Lymphoma Cell ◽

Lymphoma Cells

Abstract Abstract 157 We report a nanoparticle-enabled therapeutic approach to B cell lymphoma using synthetic, high-density lipoprotein nanoparticles (HDL-NP). Like natural HDLs, biomimetic HDL-NPs target scavenger receptor type B-1 (SR-B1), a high-affinity HDL receptor expressed by lymphoma cells. Functionally, and unlike natural HDL, a gold nanoparticle template used to control HDL-NP synthesis enables differential manipulation of cellular cholesterol flux through SR-B1. Recent evidence in lymphoblasts and myeloblasts from patients with acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) demonstrates enhanced uptake of cholesterol through high-density lipoprotein (HDL) carriers, which may result in increased cell proliferation. We therefore hypothesized that by targeting SR-B1, we could manipulate cholesterol flux in lymphoma cells thereby targeting cellular signaling pathways that would lead to cell death and offer an innovative approach to the treatment of lymphoma and other cancers. Methods: To accomplish this, we developed a biomimetic spherical nanoparticle (HDL-NP) with surface chemical properties similar to natural HDL, including the ability to sequester cholesterol. Biomimetic HDL-NPs are synthesized using a 5 nm diameter gold (Au) nanoparticle (NP) as a size- and shape-restrictive template on which to assemble the surface chemical components of natural HDLs, including phospholipids and the HDL-defining apolipoprotein A1 (Apo A1). Importantly, the core AuNP template occupies the real estate in natural cholesterol-rich HDLs reserved for esterified cholesterol, which inherently limits the ability of HDL-NPs to deliver cholesterol. We incubated the HDL-NPs with various lymphoma cell lines, and similarly tested the HDL-NPs in a xenograft model. Results: We first examined gene expression profiles of diffuse large B-cell lymphoma (DLBCL), Burkitt Lymphoma (BL) and normal B cells from patient samples in a database generated using Affymetrix U133plus 2.0 arrays in order to establish the prevalence of SR-B1 expression. We compared the expression of SR-B1 in BL cases (n=20), and DLBCL cases (n=40) that were further subdivided as activated B-cell (ABC)-like DLBCL (n=20), and germinal center (GC)-like DLBCL (n=20) to normal naive (n=3) and memory (n=3) B cells obtained from healthy donors. We found that SR-B1 was expressed at two to four-fold higher levels in the lymphomas (ABC and GC) compared with normal B cells. Next, we determined the expression of SR-B1 in lymphoma cell lines and normal peripheral lymphocytes by immunoblotting, and we found that SR-B1 is expressed in multiple B cell lymphoma cell lines, but not in Jurkat, a T-cell line, and is not expressed by normal human lymphocytes. Incubation of HDL-NP with Ramos, LY-3 and SUDHL-4 resulted in a dose-dependent decrease in cell viability and apoptosis (Figure 1) of the Ramos and SUDHL-4 cells, less so in LY-3 cells, and not in the Jurkat line. This required the nanoparticle construct and could not be duplicated by individual components of that construct, and was reversible with addition of acetylated low-density lipoprotein, indicating that the SR-B1 receptor was targeted. Xenograft experiments with SCID beige mice (C.B-Igh-1b/GbmsTac-Prkdcscid-Lystbg N7) bearing Ramos and Jurkat flank tumor xenografts confirmed the activity of the HDL-NP (Figure 2). Conclusion: We report a template-directed and bio-functional therapeutic nanostructure that could shift the paradigm for treating lymphoma and other cancers. A combination of SR-B1 binding and manipulation of cholesterol flux is responsible for selective induction of apoptosis in B cell lymphoma. Disclosures: Thaxton: Aurasense: Employment, Equity Ownership.

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Development of a New Mouse Model for Mature B Cell Lymphoma and Resistance to Fas Restoration-Triggered Apoptosis

Blood ◽

10.1182/blood.v128.22.1757.1757 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1757-1757

Author(s):

Eiji Sugihara ◽

Norisato Hashimoto ◽

Satoru Osuka ◽

Ueno Sayaka ◽

Shinichiro Okamoto ◽

...

Keyword(s):

B Cells ◽

Mouse Model ◽

B Cell ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Lymphoma Cells ◽

Hodgkin's Lymphomas

Abstract Non-Hodgkin's lymphomas (NHLs) include mature B cell lymphomas such as Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL), which are derived from germinal center (GC) B cells. The pro-apoptotic receptor Fas (CD95) is normally expressed in GC B cells and has been considered to be implicated in the pathogenesis of lymphomas. However, little is known about how Fas is regulated during lymphomagenesis. In this study, we developed a new ex vivo-based simple mouse model for mature B cell lymphoma by the transplantation of Ink4a/Arf (Cdkn2a)-deficient GC B cells that were retrovirally transduced with c-Myc. We found that Fas expression was downregulated at protein and mRNA levels in all formed lymphomas. To determine the role of Fas downregulation in lymphomagenesis and established lymphoma cells, we performed shRNA-mediated knockdown of Fas in c-Myc-GC B cells and retroviral transduction of Fas in lymphoma cells. As a result of transplantations, Fas downregulation was critical for both lymphomagenesis and maintenance of lymphoma cell survival, suggesting that GC-derived lymphomas require sustained Fas downregulation, probably to escape immune surveillance. We further found that CD40 signal activation in mouse lymphoma cells restored Fas expression thorough multiple signaling pathways including NFkB, PI3K and MAPKs (SAPK, MEK and p38). Restored Fas expression significantly induced apoptosis after FasL treatment, suggesting that Fas restoration is a potential therapeutic strategy for lymphomas. Similarly, human BL and DLBCL cell lines mostly demonstrated Fas downregulation, which was restored by CD40L stimulation. While half of the lymphoma cell lines exhibited sensitivity to FasL treatment upon Fas restoration, the other cell lines were resistant to it. We identified that Livin, a member of IAP family, is highly expressed in these resistant cell lines and is a poor prognostic factor for BL and DLBCL patients. Knockdown of Livin by shRNA and an inhibitor targeting Livin sensitized the resistant cells to Fas restoration-triggered cell death. Thus, the resistant lymphoma cells may acquire Livin during lymphoma development. Correctively, these findings suggest that Fas can be restored in lymphoma cells and thereby induce apoptosis with FasL treatment, and that Livin is a promising therapeutic target for NHLs resistant to Fas restoration-triggered apoptosis. Disclosures Okamoto: Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Astellas Pharma Inc.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Teijin Pharma Limited: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding. Saya:Daiichi Sankyo Co., Ltd.: Research Funding; Aqua Therapeutics Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Nihon Nohyaku Co., Ltd.: Research Funding.

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HDAC6 Inhibition Increases CD20 Level and Improves The Efficacy Of Anti-CD20 Monoclonal Antibodies

Blood ◽

10.1182/blood.v122.21.4406.4406 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4406-4406

Author(s):

Malgorzata Bobrowicz ◽

Michal Dwojak ◽

Kamil Bojarczuk ◽

Magdalena Winiarska ◽

Jakub Golab

Keyword(s):

Monoclonal Antibodies ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Cd20 Antigen ◽

Anti Cd20 ◽

Hodgkin's Lymphomas

Abstract CD20, an integrate membrane protein expressed on the surface of normal and malignant B-cells is widely used as a molecular target for monoclonal antibodies (mAbs) in the therapy of non-Hodgkin’s lymphomas and chronic lymphocytic leukemia (CLL). Accumulating evidence indicates that CD20 can be modulated at several levels, both transcriptional and posttranscriptional and its up-regulation would result in increased efficacy of anti-CD20 mAbs. CD20 antigen has been reported to be regulated epigenetically e.g. by histone deacetylases (HDACs). The results of our preliminary experiments show that use of non-selective HDAC inhibitors as well as blocking the activity of a single HDAC isoform - HDAC6 leads to up-regulation of CD20 protein in B-cell lymphoma cell lines and increase of the efficacy of therapy with anti-CD20 mAbs. Since HDAC6 is engaged mainly in the acetylation of non-histone substrates and the observed up-regulation of CD20 molecule does not seem to rely on transcriptional mechanisms we postulate that HDAC6 is engaged in processes of CD20 trafficking or/and degradation. CD20 being a membrane bound protein is most probably undergoing endocytosis. However, this process and the role of HDAC6 in its regulation has not been explored so far. The aim of this study was to understand how the inhibition of HDAC6 activity influences CD20 level in normal and malignant B-cells. We wanted to determine the mechanisms underlying this phenomena. This study required use of B-cell lymphoma cell lines as well as lymphocytes infected with Epstein-Barr virus and normal B- lymphocytes. Several HDAC pan-inhibitors and HDAC6 inhibitors were tested. To assess the membrane level of CD20 antigen, FITC-anti-CD20 staining was performed followed by cytometric analysis. The influence of HDACi on total level of CD20 protein was assessed in Western blotting. The complement-dependent cytotoxicity (CDC) assay was performed using rituximab and ofatumumab as well as human serum as a source of complement. Cell cytotoxicity was assessed by propidium iodide staining followed by cytometric analysis. The influence of HDAC inhibition on the transcription of CD20 was examined by qRT-PCR using SyBR Green and hydrolysis probes. The activity of CD20 promoter after inhibition of HDAC was assessed in Dual Luciferase Assay. The colocalisation of CD20 with other proteins that may influence its trafficking/degradation was assessed using immunocytochemistry with specific antibodies and observed under confocal microscope. The results of our study strongly suggest that combining HDACi with anti-CD20 antibodies can be an effective therapeutic modality for patients suffering from B-cell malignancies. Extensive experiments aiming at determining what factors are engaged in the regulation of CD20 by HDAC6 are planned. Disclosures: No relevant conflicts of interest to declare.

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