FOXR1 Activation in B-Cell Lymphoma

Abstract Several recurrent chromosomal aberrations targeting 11q23 have been described in diffuse large B-cell lymphoma (DLBCL). Here, we describe a novel fusion between RPS25 and FOXR1, two neighboring genes on 11q23 in the DLBCL cell line U-2932. RNAseq identified the fusion mRNA and genomic cloning localized the breakpoint to intron 2/3 of RPS25 and to the promoter region of FOXR1 (bp -3532). The cell line consists of two genetically distinct clones that represent subclones of the patients tumor.1 We confirmed RPS25/FOXR1 fusion in the patients DNA and in one of the two cell line subclones suggesting that the fusion had occurred at some later stages of tumor development. Physiological FOXR1 expression is restricted to the early stages of embryogenesis. Ectopic expression as result of 11q23 intrachromosomal deletion-fusion had so far only been described in neuroblastoma.2 In-frame fusions with the 5´ genes MLL and PAFAH1B2 led to the overexpression of FOXR1.2 In accordance with the notion that also in DLBCL a constitutively expressed 5´ gene (RPS25) might be responsible for the ectopic expression of FOXR1, FOXR1 levels were 1000x higher in the fusion-positive than in the fusion-negative U-2932 subclone. Expression array analyses showed that the RPS25/FOXR1 positive U-2932 subclone had the highest FOXR1 expression level of 55 B-lymphoma cell lines tested, three log-scales higher than the average expression level. Santo et al.2 reported that FOXR1 acts as negative regulator of fork-head box factor-mediated transcription and suggested a possible role in tumorigenesis. We describe for the first time that FOXR1 fusions also occur in lymphoma. In-silico analyses show that the aberrant expression of FOXR1 is rare, but recurrent in various forms of lymphoma. Given the potential oncogenic role of FOXR1, cell line U-2932 with one FOXR1-positive and one FOXR1-negative subclone appear to be a promising model for functional analysis of FOXR1-mediated cellular events. 1 Quentmeier H, Amini RM, Berglund M, et al. U-2932: two clones in one cell line, a tool for the study of clonal evolution. Leukemia. 2013;27(5):1155-1164. 2 Santo EE, Ebus ME, Koster J, et al. Oncogenic activation of FOXR1 by 11q23 intrachromosomal deletion-fusions in neuroblastoma. Oncogene 2012;31(12):1571-1581. Disclosures No relevant conflicts of interest to declare.

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Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽

10.1182/blood.v120.21.2417.2417 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2417-2417

Author(s):

Olga Ritz ◽

Jochen K Lennerz ◽

Karolin Rommel ◽

Karola Dorsch ◽

Elena Kelsch ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Ectopic Expression ◽

B Cell Lymphoma ◽

Proximal Promoter ◽

Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

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Effect of Knockdown of CD44 Expression by shRNA On Biologicalfeatures of Activated B-Cell-Like Diffuse Large B Cell Lymphoma Cell Line

Blood ◽

10.1182/blood.v120.21.5080.5080 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5080-5080

Author(s):

Ru Feng ◽

Xiaolei Wei ◽

Wenbing Duan ◽

Fen Huang ◽

J. Jessica Yu ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Late Phase ◽

B Cell Lymphoma ◽

Cd44 Expression ◽

Knock Down ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Abstract 5080 Objective To study the effect of CD44 down-regulation with lentivirus-mediated small hairpin RNA (shRNA) interference on the biological features of activated B-cell-like diffuse large B-cell lymphoma (DLBCL) cell line SUDHL-2. Methods Lentiviral vector for RNAi of CD44, which contained green fluorescent protein(GFP) was constructed. Package cells phoenix293 was used to produce virus stocks. Then SUDHL-2 cells were infected with the recombinant lentivirus and the cells with CD44 knock-down were selected by FACS for GFP expression. CD44 expression in the cells was determined by RT-PCR and FACS. The proliferation, apoptosis and invasion were evaluated by MTT methods, FACS and transwell migration assay, respectively. Results DNA sequencing demonstrated that the lentivirus RNAi vector was constructed successfully. Clones of SUDHL-2 cells infected with the recombinant lentivirus were selected and exhibited substantial knock-down of CD44 mRNA and protein expression compared with the control cells. The proliferation ability of cloned SUDHL-2 cells were inhibited. The apoptosis rates at early and late phase were 6. 26%, 36. 40% respectively in cloned SUDHL-2 cells and in control cells were 2. 9%, 2. 56% at early phase and 6. 1%, 6. 58% at late phase. The migrating number of cloned SUDHL-2 cells(34. 53±8. 05)% was also significantly decreased compared with the control cells, 78. 67±2. 64% and 78. 00±6. 13% (P=0. 290). Conclusion The lentivirus-mediated shRNA of CD44 is efficient in down-regulating CD44 expression and inducing apoptosis, inhibiting proliferation and invasiveness of SUDHL-2 cells, suggesting that CD44 might have an oncogene role in the tumorigenesis and progression of ABC-DLBCL. Disclosures: No relevant conflicts of interest to declare.

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Establishment and Characterization of a Novel Human Cell Line of Triple Hit Lymphoma, AMU-ML1, Carrying t(2;18)(p11.2;q21) and t(3;8;14)(q27;q24;q32) That Involve BCL2, BCL6 and MYC

Blood ◽

10.1182/blood.v118.21.5210.5210 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5210-5210

Author(s):

Norikazu Tsunekawa ◽

Ichiro Hanamura ◽

Hidesuke Yamamoto ◽

Hisao Nagoshi ◽

Motohiro Wakabayashi ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Human Cell Line ◽

Culture Cell ◽

World Health ◽

Primary Tumors ◽

Health Organization

Abstract Abstract 5210 Mature B-cell lymphomas with both BCL2 and MYC translocations to IG loci are rarely identified and most of them are classified as B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (IL) in the new World Health Organization classification. In this study, we established a novel human lymphoma cell line, AMU-ML1, from pericardial effusion (PE) of a patient with IL before the initiation of chemotherapy and analyzed its characters. A 60-year-old male was admitted to our hospital because of PE and diagnosed as having IL from the atypical lymphocytes in PE. He was treated with rituximab plus hyper-CVAD and other regimens but died of lymphoma approximately 10 months after diagnosis without reaching complete remission. The cells from patient's PE at diagnosis were cultured in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS). After 5 months of culture, cell proliferation became continuous with 10% FBS and the cell line was designated as AMU-ML1 (Aichi Medical University, malignant lymphoma, no. 1) after confirmation that the cells began growing again after the conventional freeze-thaw procedure. AMU-ML1 cells were positive for CD10, CD19, CD20, CD79a, HLA-DR and cytoplasmic lambda chain and negative for CD3, CD4, CD5, CD8, CD13, CD23, CD33 and CD56 by flow cytometry analysis and showed a complex karyotypes including t(2;18)(p11.2;q21) and t(3;8;14)(q27;q24;q32) by G-banding analysis. This profile is consistent with the profile of the patient's cells. Spectral karyotyping and fluorescent in situ hybridization analysis of AMU-ML1 cells revealed that t(2;18)(p11.2;q21) was IGL/BCL2 and t(3;8;14)(q27;q24;q32) was BCL6/MYC, MYC/IGH and IGH/BCL6. Subcutaneous transplantation of AMU-ML1 cells into NOD/scid mice treated with anti-asialo GM1 antibody resulted in formation of primary tumors. Thus, the AMU-ML1 cell line is useful for studying the biological consequences of IL with triple hit of BCL2, BCL6 and MYC, and possibly invasion to PE of lymphoma. Disclosures: No relevant conflicts of interest to declare.

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Mir-155 Enhances B-Cell Lymphoma Growth By Targeting TBRG1

Blood ◽

10.1182/blood.v126.23.4820.4820 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4820-4820 ◽

Cited By ~ 1

Author(s):

Joost Kluiver ◽

Izabella Slezak-Prochazka ◽

Melanie Winkle ◽

Lydia Visser ◽

Arjan Diepstra ◽

...

Keyword(s):

B Cell ◽

Molecular Mechanisms ◽

Target Genes ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Ectopic Expression ◽

B Cell Lymphoma ◽

Argonaute 2 ◽

Rna Immunoprecipitation

Abstract Several studies have indicated an important role for miR-155 in the pathogenesis of B-cell lymphoma. Highly elevated levels of miR-155 were indeed observed in most B-cell lymphomas with the exception of Burkitt lymphoma (BL). However, the molecular mechanisms that underlie the oncogenic role of miR-155 in B-cell lymphoma are not well understood. To identify the miR-155 targets relevant for B-cell lymphoma, we performed RNA immunoprecipitation of Argonaute 2 in Hodgkin lymphoma (HL) cells upon miR-155 inhibition and in BL cells upon ectopic expression of miR-155. We identified 54 miR-155-specific target genes in BL cells and confirmed miR-155 targeting of DET1, TBRG1 (NIAM1), TRIM32, HOMEZ, PSIP1 and JARID2. Five of these targets are also regulated by endogenous miR-155 in HL cells. Both overexpression of miR-155 and inhibition of expression of the miR-155 target gene TBRG1 increased proliferation of BL cells. In primary B-cell lymphoma TBRG1-positive cases have significant lower levels of miR-155 as compared to TBRG1-negative cases, suggesting that TBRG1 is also regulated by miR-155 in primary B-cell lymphoma. Thus, our data indicate that miR-155 plays an oncogenic role in B-cell lymphoma by targeting the tumor suppressor TBRG1. Disclosures No relevant conflicts of interest to declare.

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Aberrant Chromatin Modifications Mediate Ectopic Expression of Homeobox Gene NKX2-1 in B-Cell Lymphoma

Blood ◽

10.1182/blood.v120.21.3494.3494 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3494-3494

Author(s):

Stefan Nagel ◽

Corinna Meyer ◽

Maren Kaufmann ◽

Roderick AF MacLeod ◽

Hans G. Drexler

Keyword(s):

Transcription Factors ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Chromosomal Aberrations ◽

Cell Lymphoma ◽

Ectopic Expression ◽

Homeobox Gene ◽

B Cell Lymphoma ◽

Cell Leukemia

Abstract Abstract 3494 Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, and when deregulated promote cell transformation in multiple cancers including hematopoietic malignancies. In this context several members of the NKL family of homeobox genes are aberrantly expressed in acute T-cell leukemia by chromosomal aberrations. Here, analysis of 20 cell lines of T- and B-cell leukemia/lymphoma by expression arrays (Affymetrix, HGU133plus2) revealed exclusive activity of NKL homeobox gene NKX2-1 in a diffuse large B-cell lymphoma (DLBCL) cell line. NKX2-1 is physiologically expressed in embryonic lung and thyroid tissues where it regulates differentiation. RQ-PCR analysis of gene expression in primary hematopoietic samples, including bone marrow, lymph node, thymus, peripheral mononuclear blood cells, T-cells and B-cells, confirmed silencing therein highlighting ectopic expression of NKX2-1 in the cell line. Copy number analysis by genomic array data (Broad Institute), spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) excluded chromosomal rearrangements at the NKX2-1 locus in expressing cells. Comparative expression analysis of NKX2-1 negative DLBCL cell lines implicated several candidate genes involved in NKX2-1 regulation, variously encoding transcription factors (TFs), chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed TF HEY1 in ectopic NKX2-1 expression and NKX2-1 in HEY1 expression in DLBCL cells, indicating reciprocal activation of these TFs. Moreover, chromatin immunoprecipitation (ChIP) analysis demonstrated direct binding of NKX2-1 to the HEY1 promoter. HEY1 belongs to the basic helix-loop-helix family disturbing lymphoid differentiation if deregulated. Enhanced expression levels of histone H3K4 methyltransferase MLL correlated with downstream rearrangement and amplification of the MLL-locus at 11q23. SiRNA-mediated knockdown of MLL was accompanied by reduced expression of NKX2-1 but not of HEY1, showing that MLL supports expression of NKX2-1. Furthermore, ChIP analyses demonstrated presence of both activatory H3K4me3 and inhibitory H3K27me3 at the promoter region of NKX2-1, while at the HEY1 promoter only H3K27me3 was detected. Such bivalent histone marks have been described for developmental genes in progenitor cells, indicating a permissive role for aberrant chromatin structures at the NKX2-1 locus in this cell line. Chromosomal alteration del(6p22) as detected by SKY and subsequently mapped by FISH was shown to target the histone gene locus HIST1. Expression analysis at the RNA and protein levels showed elevated expression of core-histones including H2B. Additionally, mono-ubiquitinated H2B was strongly enhanced in this DLBCL cell line when analyzed by Western blot. This histone mark supports the MLL-mediated formation of active chromatin structures, suggesting cooperative action of the chromosomal aberrations targeting MLL and HIST1. Array data also indicated aberrant expression of polycomb repressor complex 2 (PRC2) members which counteract the activity of MLL. Accordingly, siRNA-mediated knockdown analyses demonstrated regulatory impacts of HOPX, E2F6 and JMJD3 in NKX2-1 expression. The potential impact of signaling pathways in NKX2-1 expression, comprising NFkB, SMADs and phosphodiesterases was confirmed by treatments with TNFa, TGFb and cAMP/cGMP, respectively. Taken together, we have identified ectopic expression of NKX2-1 in DLBCL cells, involved in an oncogenic regulative network which may compromise B-cell differentiation via activation of HEY1. Combined analyses of chromosomal alterations and comparative gene expressions identified aberrant chromatin structures underlying expression of NKX2-1, representing the central player in that network. Therefore, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancy. Disclosures: No relevant conflicts of interest to declare.

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The Significance of CD44ICD in Difuse Large B- Cell Lymphoma Cell Line SUDHL2 and the Preliminary Study On the Mechanism

Blood ◽

10.1182/blood.v120.21.5087.5087 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5087-5087

Author(s):

Ru Feng ◽

Wenbing Duan ◽

Dayan Chen ◽

Xiaolei Wei ◽

Yongqiang Wei ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Annexin V ◽

B Cell Lymphoma ◽

Surface Expression ◽

Surface Markers ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Abstract 5087 Objective: Diffuse large B cell lymphoma (the diffuse large B-cell lymphoma, DLBCL), is one of the most common type of non-Hodhkin's lymphomawith high heterogeneity. γ-secretase can induce the hydrolysis of CD44, which play a key important in DLBCL, producing the CD44 intracellular domain. According to our research, the inhibition of CD44ICD can decrease the expression of NF-„KB and Stat-3 in SUDHL2 cell line from ABC-DLBCL cell line. Therefore the aim of this study was put important on the change of proliferation, apoptosis and main surface markers, ERK1/2 and phosphor-ERK1/2 in CD44 positive cell line from ABC-DLBCL after inhibiting the production of CD44ICD. Method: The surface expression of CD44 in SUDHL2 and OCI-ly3 cell lines was tested by flow cytometry, and then chose the CD44 positive cell line. Using 0. 1, 1. 0, 5. 0, 10, 25, 50, 75 and 100μM DAPT inhibited the production of CD44ICD in CD44 positive cell line respectively, and then tested the cell proliferation, apoptosis and main surface markers(CD44, CD19, CD20) by MTT analysis, Annexin V-FITC/PI and flow cytomety separately after 24h. mean while western blot was used to detected whether ERK1/2 and phosphor-ERK1/2 expressed in the chosen cell line, if they expressed in it, tested their change after blocking the release of CD44ICD for 1h. Results Conclusion The ERK1/2 signal path participated in the regulation of SUDHL2 cell line, moreover after inhibited the release of CD44ICD, both the ERK1/2 and phosphor-ERK1/2 could be regulated up, however further study must go on. Disclosures: No relevant conflicts of interest to declare.

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Long Non-Coding RNAs As Components Of The MYC Network In B Cell Lymphoma

Blood ◽

10.1182/blood.v122.21.1260.1260 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1260-1260

Author(s):

Joost Kluiver ◽

Melanie Winkle ◽

Mina Tayari ◽

Martijn Terpstra ◽

Gertrud Kortman ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Expression Patterns ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Differentially Expressed ◽

Primary Lymphoma ◽

Aberrant Expression

Abstract MYC is an important oncogenic transcription factor in B-cell lymphoma and high MYC expression is associated with aggressive behavior and poor clinical outcome. A large number of genes are regulated by MYC, several of which are shown to contribute to the MYC induced phenotype. Long non-coding (lnc)RNAs have recently emerged as a novel class of regulatory RNAs acting at the epigenetic, transcriptional or posttranscriptional level. Aberrant expression of several lncRNAs has already been implicated in various aspects of tumorigenesis. It is currently unknown to what extend MYC can regulate lncRNA expression and whether these lncRNAs contribute to the pathogenesis of B-cell lymphoma. Using an inducible MYC B cell lymphoma model and a custom microarray we investigated the expression >10,000 lncRNA loci and identified 1,820 lncRNA probes that show a MYC regulated expression pattern. Of these, 355 responded already after 4h, indicating direct MYC regulation. To identify transcripts relevant to lymphoma pathogenesis, we determined if these 355 lncRNAs were differentially expressed between primary lymphoma cases with high and low MYC expression and in addition also between MYC-high lymphoma cell lines and normal germinal center B cells. This revealed an overlap of 176 lncRNAs that were MYC regulated, aberrantly expressed in B cell lymphomas and differentially expressed between MYC-high and MYC-low lymphomas. Differential expression patterns were validated by qRT-PCR. As a first indication for lncRNA function, we isolated RNA from nuclear and cytoplasmic fractions of B cell lymphoma cell lines and determined enrichment fold in comparison to RNA isolated from the total cell lysates. Approximately 40% of all lncRNA transcripts showed specific subcellular localization, 80% nuclear and 20% cytoplasmic enriched. 31 of the 176 candidate lncRNAs were enriched in a specific cellular fraction. Furthermore, we analyzed which lncRNAs are enriched in Argonaute 2 containing complexes as an indication for lncRNA-miRNA interaction. For ∼5% of all expressed lncRNAs we found evidence for miRNA-lncRNA interactions, including 8 of the 176 differentially expressed MYC-induced lncRNAs. This study identified 176 MYC responsive lncRNAs that are deregulated in B cell lymphoma. To establish a definitive role in B cell lymphoma pathogenesis a further characterization is warranted. Disclosures: No relevant conflicts of interest to declare.

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