Specific Micro-RNA Expression Profile in Hodgkin Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 381-381
Author(s):  
Lu Ping Tan ◽  
Geert Harms ◽  
Tjasso Blokzijl ◽  
Rikst Nynke Schakel ◽  
Johan Gibcus ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Differentially Expressed ◽  
Cell Phenotype ◽  
Qrt Pcr

Abstract Introduction Classical Hodgkin lymphoma (cHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) differs not only in the form of histology and reactive background but also in the phenotypes of the tumor cells. Although tumor cells from both HL subtypes are originated from the germinal center (GC) B cell, gene expression studies show that lymphocytic and histiocytic (L&H) cells from NLPHL resembles normal B cells while Hodgkin/Reed-Sternberg cells (H/RS) from cHL demonstrate a loss of B cell phenotype and have significant overlap with primary mediastinal B cell lymphoma (PMBL). Recently, a new class of small RNAs, namely the micro-RNAs (miRNAs), has been identified. It is now known that at the post-transcriptional level, miRNAs negatively regulate gene expression in a sequence specific manner. Unique miRNAs expression patterns have been reported in various tissue types and also during a wide range of physiological states, such as cell proliferation, development, differentiation, apoptosis and hypoxia. As miRNAs play important roles in many cellular processes, it is proposed that there is a link between aberrant miRNA expression and loss of B cell phenotype in cHL. Methods In this study, miRNA profiles from cell lines of various B cell lymphoma subtypes were examined by qRT-PCR. Also, several B cell subsets were sorted from tonsil by FACS and the miRNA profiles studied by qRT-PCR. Some of the miRNAs are analyzed by in situ hybridization (ISH) in both HL tissue and tonsil samples. Results The miRNA profiling data indicated that cHL cell lines cluster together with PMBL while DEV, an NLPHL cell line, clusters together with CB. Upon validation of differentially expressed miRNAs on a cell line panel of 33 cell lines by monoplex qRT-PCR, 5/8 miRNAs identified as differentially expressed between cHL and GC B cells, were confirmed. Four out of six miRNAs differentially expressed between cHL and PMBL, were also confirmed as being differentially expressed in a larger cell panel. A high degree of overlap was observed between the most abundantly expressed miRNAs in the four HL cell lines. Expression of these miRNAs in HRS cells was verified by ISH in HL tissue samples. miRNA profiles of naive, GC and memory B cells display unique patterns. The overall miRNA expression levels were much lower than observed in the cell lines. Results of miRNA ISH in tonsil tissue demonstrated a specific staining pattern for each miRNA. These data indicate that miRNAs are particularly important for subsets of lymphocytes. Conclusion Several miRNAs that are expressed specifically in Hodgkin lymphoma have been identified. However, the effect of the aberrant expressions of these miRNAs in HL is yet to be elucidated, as the targets of these miRNAs remain unknown.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 266-266 ◽  
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.

FOXO1 is a tumor suppressor in classical Hodgkin lymphoma

Blood ◽  
2012 ◽  
Vol 119 (15) ◽  
pp. 3503-3511 ◽  
Author(s):  
Linka Xie ◽  
Alexey Ushmorov ◽  
Frank Leithäuser ◽  
Hanfeng Guan ◽  
Christian Steidl ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Nuclear Export ◽  
Cell Lymphoma ◽  
Ectopic Expression ◽  
B Cell Lymphoma ◽  
Classical Hodgkin Lymphoma ◽  
Non Hodgkin Lymphoma

Abstract The FOXO transcription factors control proliferation and apoptosis in different cell types. Their activity is regulated by posttranslational modifications, mainly by the PI3K-PKB pathway, which controls nuclear export and degradation. We show that FOXO1 is highly expressed in normal germinal center B cells as well as in non-Hodgkin lymphomas, including follicular lymphoma, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia, and mantle cell lymphoma. In contrast, in 31 of 32 classical Hodgkin lymphoma (cHL) cases, Hodgkin and Reed-Sternberg cells were FOXO1 negative. Neoplastic cells of nodular lymphocyte-predominant Hodgkin lymphoma were negative in 14 of 20 cases. FOXO1 was down-regulated in cHL cell lines, whereas it was expressed in non-Hodgkin lymphoma cell lines at levels comparable with normal B cells. Ectopic expression of a constitutively active FOXO1 induced apoptosis in cHL cell lines and blocked proliferation, accompanied with cell-cycle arrest in the G0/G1 phase. We found that, in cHL cell lines, FOXO1 is inactivated by multiple mechanisms, including constitutive activation of AKT/PKB and MAPK/ERK kinases and up-regulation of microRNAs miR-96, miR-182, and miR-183. These results suggest that FOXO1 repression contributes to cHL lymphomagenesis.

scholarly journals Long Non Coding RNA Signatures with Diagnostic and Prognostic Significance in Aggressive B-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1759-1759 ◽  
Author(s):  
Jiayu Yu ◽  
Alyssa Bouska ◽  
Waseem Lone ◽  
Chengfeng Bi ◽  
Tayla Heavican ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Fold Change ◽  
Differentially Expressed ◽  
Non Coding Rna ◽  
Long Non Coding Rna ◽  
Aggressive B Cell Lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL) displays significant heterogeneity with regard to clinical, pathological, and genetic features. Using gene expression profiling, we have delineated 3 molecular subgroups within DLBCL: germinal center B cells (GCB-DLBCL), activated B cells (ABC-DLBCL), and primary mediastinal B-cell lymphoma (PMBL),while these defined subgroups show characteristic mutation profiles and oncogenic pathways, a small set of cases of DLBCL still remain "unclassifiable" (UC). Burkitt lymphoma (BL) is characterized by the t(8;14)(q24;q32) with distinct morphological and immunophenotypic features. The diagnostic distinction between BL and DLBCL is challenging in a subset of cases that have overlapping morphological, immunohistochemical, and even molecular features, but is crucial for effective therapy. The identification of long non-coding RNA (lncRNA) has added another critical component to cancer biology. LncRNAs are defined as a distinct set of non-protein coding transcripts longer than 200 nucleotides. The functions of a few lncRNAs have been recently elucidated of which some are thought to regulate gene-specific transcription. The goal of the current study was to identify reliable lncRNA signatures for the BL and DLBCL subgroups and evaluate their usefulness as prognostic biomarkers. We examined the expression of lncRNAs from our earlier studies using Affymetrix-HG-U133 plus 2 arrays to distinguish unique gene expression profiles between BL and DLBCL (PMID: 27292966). In the initial analysis, we compared BL (n=77) with DLBCL (n=474) and identified 275 differentially expressed lncRNAs (p=0.005, fold change>1.5). There was high expression of TCL6 and DDX-AS11 in BL. We confirmed the microarray results for TCL6 and DDX-AS11 by qRT-PCR in a subset of cases. We further tested whether expression of TCL6 and DDX-AS11 was regulated by the MYC oncogene and observed significant downregulation of these transcripts upon CRISPR/cas9 mediated deletion of the MYC promoter in the Raji cell line. We also sought to identify GCB-DLBCL and ABC-DLBCL associated lncRNAs. We observed 36 lncRNAs highly expressed in ABC-DLBCL and 40 lncRNAs highly expressed in GCB-DLBCL (P=0.005, fold change>1.5). Of the differentially expressed lncRNAs, lnc00487 and DUBR were upregulated in GCB-DLBCL, whereas lnc00944 and FUT8 were upregulated ABC-DLBCL. The observed expression differences were validated in ABC-DLBCL and GCB-DLBCL cell lines. The differentially expressed LncRNAs were also validated in other DLBCL cohorts. LncRNA00487 expression was associated with superior clinical outcome in DLBCL series treated with Rituximab (R)-CHOP (p=0.01), and gene expression and overall survival (OS) were validated in another DLBCL series treated with R-CHOP (PMID:22437443). In the present study, we found that lncRNAs are differentially expressed in aggressive B cell lymphoma and could be useful as diagnostic or prognostic markers. They may play an important role in lymphoma biology and further studies of their functions are warranted. Disclosures No relevant conflicts of interest to declare.

Aberrant Somatic Hypermutation Targets an Extensive Set of Genes in Diffuse Large B-Cell Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1528-1528 ◽  
Author(s):  
Laura Pasqualucci ◽  
Roberta Guglielmino ◽  
Sami N. Malek ◽  
Urban Novak ◽  
Mara Compagno ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Chromosomal Translocations ◽  
Total N ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Genomic instability is a driving force in tumor development that can be achieved by a variety of mechanisms, such as defective chromosome segregation or inactivation of the DNA mismatch repair pathway. Although B-cell lymphomas are associated with chromosomal translocations deregulating oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has long not been described. We have reported that the somatic hypermutation process (SHM), which normally targets the immunoglobulin variable region (IgV) and BCL6 genes in germinal center (GC) B-cells, functions aberrantly in >50% of diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma (Pasqualucci et al., Nature412:341, 2001). As a consequence, multiple somatic mutations are introduced into the 5′ region of genes that do not represent physiologic SHM targets, including known proto-oncogenes such as PIM1, PAX5, RhoH/TTF and cMYC. To further define the extent of this phenomenon, termed aberrant somatic hypermutation (ASHM), and to identify additional hypermutated loci of possible pathogenetic significance in DLBCL, we screened 113 genes for the presence of mutations affecting their 5′ sequences (≥1.3 Kb from the transcription start site, the target region for SHM) in 10 DLBCL cell lines. Fifteen genes (13.3%) were found to harbor a significant number of mutations (p<0.05), with 70% of the cell lines being mutated in 7 or more genes; among these, six B-cell specific loci -BCL7A, CIITA, IRF4, LRMP, NCOA3 and SIAT1- carried 9–53 mutational events distributed in 20 to 70% of the cases, corresponding to an overall mutation frequency of 0.032–0.15% (frequency in the mutated cases: 0.07–0.25%). The same genes were found hypermutated in a panel of 20 primary DLBCL biopsies, which displayed an overall mutation load of 7 to 45 distinct events/gene (total N=125). Mutations were of somatic origin, independent of chromosomal translocations to the Ig loci and were restricted to the first 1.5–2 Kb from the promoter. In addition, analogous to previously identified SHM and ASHM targets, the mutations exhibited characteristic features, including a bias for transitions over transversions, preferential hotspot (RGYW/WRCY motifs) targeting, and higher frequencies at G:C pairs. However, in contrast to physiologic SHM targets such as IgV and BCL6, none of the 4 newly identified hypermutated genes that have been analyzed so far (BCL7A, CIITA, SIAT1, LRMP) displayed significant levels of mutations in purified normal GC B-cells as well as in other B-cell malignancies. This finding indicates that these genes represent aberrant hypermutation targets resulting from a tumor-associated malfunction, possibly a loss of target specificity of the physiologic SHM process. Considering previous results and the present survey, 17 (13%) out of 130 genes investigated have been found involved in ASHM, suggesting that this aberrant activity may involve an extensive set of target genes in DLBCL. Since the mutations affect both regulatory and coding sequences of the targeted genes, aberrant SHM may represent a major contributor to the pathogenesis of this disease and may explain in part its phenotypic and clinical heterogeneity.

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

Blood ◽  
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.

Contribution of Deregulated miRNAs to the Phenotypic Characteristic of Hodgkin Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 265-265
Author(s):  
Lu Ping Tan ◽  
Bart-Jan Kroesen ◽  
Enrico Tiacci ◽  
Gerben Duns ◽  
Erwin Seinen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Mirna Expression ◽  
Conflicts Of Interest ◽  
System Development ◽  
Cell Phenotype ◽  
Immune System Development

Abstract Abstract 265 In Hodgkin Lymphoma (HL), the Hodgkin Reed-Sternberg (HRS) cells are a minority of large mono- or multi-nucleated B cells characterized by a loss of B cell phenotype, constitutive NF-kB activation, a disturbed cell cycle and anti-apoptotic features. In this study we investigated the role of deregulated miRNA expression in the pathogenesis of HL. MiRNA in situ hybridization (ISH) in HL tissue was performed to determine expression of miRNAs previously reported to be highly abundant in HL cell lines, in HRS cells. Next we identified the miRNA-targetome of two HL cell lines by immunoprecipitation of RISC in untransfected and transfected cell lines. miRNA ISH confirmed expression of miR-17-5p, miR-24, miR-106a, miR-146a, miR-150, miR-155, miR-181b and miR-210 in HRS cells. Ago2-immunoprecipitation followed by microarray analysis of the co-immunoprecipitated mRNA revealed that the miRNA-targetome of HL comprises of about 2,500 genes. Inhibition of the anti-miR-17 seed family revealed that about 500 of these genes are regulated by miRNAs of the miR-17 seed family. Gene ontology (GO) analysis for the total miRNA-targetome of HL showed a significant enrichment of genes involved in the regulation of cell cycle, apoptosis, immune system development and NF-kB cascade. The miRNA-targetome of HL contained several genes known to be mutated in HRS cells, including A20, FAS, NFKB1A, NFKB1E, PERP and SOCS1. Also, using previously reported gene expression data, we defined a set of genes downregulated in HL cell lines (L428 and L1236) compared to germinal center B cells (GCB) and compared them to the miRNA-targetome of the same cell lines. This resulted in the identification of 149 genes in L428 and 183 genes in L1236 that were subjected to miRNA mediated repression. Unexpectedly, only a few of all the reported inactivated genes in HRS cells that might contribute to loss of B cell phenotype (MYBL1 and CXCR4) were found to be regulated by miRNAs in HL. In conclusion, we confirmed the expression of miRNAs in the HRS cells of HL tissue and identified miRNA repressed genes in HL. Our data indicated that aberrant miRNA expression contributes to the deregulation of apoptosis, cell cycle, and NF-kB pathways but not loss of B cell phenotype in HL. Disclosures: No relevant conflicts of interest to declare.

PIM as a Rational Target for B-Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3946-3946
Author(s):  
Cristina Gomez-Abad ◽  
Helena Pisonero ◽  
Juan F Leal ◽  
Giovanna Roncador ◽  
Jose A. Martinez-Climent ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Specific Inhibitor ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Micromolar Range ◽  
Large B Cell ◽  
Stat Pathway

Abstract Abstract 3946 Poster Board III-882 INTRODUCTION The Pim kinases are a family of serine/threonine kinases composed by three members: Pim1, Pim2 and Pim3, involved in the phosphorylation and regulation of several proteins that are essential for cell cycle progression, metabolism or apoptosis (BAD, p21, p27KIP, AKT, Mdm2 and cMyc, among them). Overexpression, translocation or amplification of Pim family have been described in many human cancers, including B-cell Non Hodgkin's Lymphoma, Multiple Myeloma, Prostate cancer and Pancreatic cancer. In addition, 50% of patients diagnosed with diffuse large B-cell Lymphoma (DLBCL) present somatic mutations in Pim1. Despite of its important role in cancer progression, very few chemical inhibitors have been described in the literature, being effective all of them in the high micromolar range. PURPOSE Validating PIM as a rational therapeutic target in B-cell lymphoma, developing tools for patient stratification and pharmacodynamic studies on PIM inhibition. MATERIAL AND METHODS Gene expression profiling and Copy Number data were obtained from a series of 94 B-cell Non-Hodgkin Lymphoma patients (DLBCL, FL, MALT, MCL and NMZL). The effect of Pim inhibition was checked on cell lines by using a novel specific inhibitor for the Pim family (ETP-39010). Newly produced antibodies and RT-PCR primers and protocols were standarized. RESULTS Gene expression data revealed high Pim isoforms expression in a subset of patients with Mantle cell lymphoma (MCL), and Diffuse Large B-cell lymphoma (DLBLC)-ABC type. CGH analysis focused on chromosomal regions containing Pim family and its main regulatory upstream pathway (JAK/STAT) was performed. Heterozygous gains of Pim1 (6p21.2) and Pim3 (22q13.33) were identified in 13.6% of DLBCL patients and in 4.2% of MCL. Alterations in JAK/STAT pathway were also detected in 59.1% of DLBCL patients, and 37.5% of MCL patients presented any alteration in JAK/STAT pathway, being frequent losses of JAK2 chromosomal region. Analysis of additional pathways involved in the up-stream regulation of Pim family disclosed heterozygous gains of PIK3C3 in 40.9% of DLBCL patients, and gains of PIK3CA in 45.9% of MCL patients. Lymphoma cell lines (15) derived from both MCL (9) and ABC-DLBLC (6) subtype, have been analyzed by qRT-PCR and Western-blot, showing variable expression levels of Pim1, Pim2 and Pim3. IC50 obtained for the ETP-39010 compound is in the low micromolar range for the MCL (0.7-8.7 micromolar) and DLBCL-ABC (0.8-10.3 micromolar) cell lines. Since Pim kinase family phosphorilate multiple sites of Bad and AKT, we have checked the inhibition of its phosphorilation as molecular biomarkers for the ETP-39010 effect. Our data show an inhibition of at least 20% of pBad (S112) and almost a complete inhibition of pAKT (S473) 4h after treatment. In addition, cell cycle arrest at G1 and induction of apoptosis were observed 24h after the treatment. CONCLUSION Pim family genes are a rational therapeutic target in MCL and DLBCL-ABC lymphoma subtypes. Stratification and pharmacodynamic markers have been developed for PIM inhibition using a novel specific inhibitor compound -ETP-39010-. Disclosures: No relevant conflicts of interest to declare.

Differential Patterns of Gene Expression and CD20 Localisation by the Type II Anti-CD20 Antibody Obinutuzumab (GA101) Compared with the Type I Antibody Rituximab Suggests Binding to Functionally Distinct CD20 Subpopulations on B-Cell Lymphoma Cell Lines,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3721-3721
Author(s):  
Gerhard Niederfellner ◽  
Olaf Mundigl ◽  
Alexander Lifke ◽  
Andreas Franke ◽  
Ute Baer ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mechanisms Of Action ◽  
Type I ◽  
Type Ii ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract Abstract 3721 The anti-CD20 antibody rituximab has become central to the treatment of B-cell malignancies over the last decade. Recently, it has been shown that anti-CD20 antibodies can be divided into two types based on their mechanisms of action on B cells. Rituximab is a type I antibody that redistributes CD20 into lipid rafts and promotes complement-dependent cytotoxicity (CDC), while the type II, glycoengineered antibody GA101 has lower CDC activity but higher antibody-dependent cellular cytotoxicity and direct cell death activity. In preclinical studies GA101 was superior to rituximab in B-cell killing in vitro, depletion of B cells from whole blood, and inhibition of tumour cell growth in lymphoma xenograft models. GA101 is currently being evaluated in Phase II/III trials, including comparative studies with rituximab. To investigate the differences in direct effects of GA101 and rituximab on B-cell lymphoma signaling, we have analysed the effects of antibody binding on gene expression in different B-cell lines using a GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Rituximab and GA101 rapidly induced gene expression changes in SUDHL4 and Z138 cells, including regulation of genes associated with B-cell-receptor activation such as EGR2, BCL2A1, RGS1 and NAB2. The effects on gene expression differed markedly between different cell lines and between the two antibodies. SUDHL4 cells showed pronounced changes in the gene expression pattern to rituximab treatment, while Z138 cells, which represent a different B-cell stage, showed less pronounced changes in gene expression. The reverse was true for GA101, suggesting not only that the signaling mediated by CD20 differs in different cell lines, but also that in a given cell line the two types of antibodies bind CD20 molecules with different signaling capacity. For each cell line, gene expression induced by other type I antibodies (LT20, 2H7, MEM97) was more like rituximab and that induced by other type II antibodies (H299/B1, BH20) was more like GA101 in terms of the number of genes regulated and the magnitude of changes in expression. Unbiased hierarchical clustering analysis of gene expression in SUDHL4 could discriminate type I from type II antibodies, confirming that the two classes of antibody recognised CD20 complexes with inherently different signalling capacities. By confocal and time-lapse microscopy using different fluorophores, rituximab and GA101 localised to different compartments on the membrane of lymphoma cells. GA101/CD20 complexes were relatively static and predominantly associated with sites of cell–cell contact, while rituximab/CD20 complexes were highly dynamic and predominantly outside areas of contact. These findings suggest that type II antibodies such as GA101 bind distinct subpopulations of CD20 compared with type I antibodies such as rituximab, accounting for the differences in mechanisms of action and anti-tumour activity between these antibodies. Disclosures: Niederfellner: Roche: Employment. Mundigl:Roche: Employment. Lifke:Roche: Employment. Franke:Roche: Employment. Baer:Roche: Employment. Burtscher:Roche: Employment. Maisel:Roche: Employment. Belousov:Roche: Employment. Weidner:Roche: Employment. Umana:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Defining The Role Of Microrna-146a In B Cell Lymphomagenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3805-3805
Author(s):  
Jorge Contreras ◽  
Jayanth Kumar Palanichamy ◽  
Tiffany Tran ◽  
Dinesh S. Rao
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Expression Patterns ◽  
Significant Proportion ◽  
B Cell Lymphoma ◽  
Gene Expression Patterns

Abstract Diffuse large B cell lymphoma (DLBCL) is one of the most common Non-Hodgkin lymphomas among adults. It is a heterogeneous disease characterized by multiple mutations and translocations. Gene expression profiling studies have revealed several characteristic gene expression patterns, with two main patterns emerging, namely Germinal Center(GC) type, and Activated B Cell (ABC) type. ABC-type DLBCL shows gene expression patterns that resemble activated B-cells, with increased expression of anti-apoptotic, and pro-proliferative genes. Critically, upregulation of the NF-κB the pathway is a hallmark of ABC-type DLBCL and has been shown to be necessary for survival, and is caused by several different mutations at different levels within the pathway. Recent work has revealed the critical importance of a new class of small RNA molecules, namely microRNAs, in gene regulation. Of these, microRNA-146a (miR-146a) was discovered as an NF-κB induced microRNA that plays a role as a negative feedback regulator of this pathway by targeting adaptor proteins. To further characterize miR-146a, mice deficient for this miRNA were created, and were found to develop lymphadenopathy, splenomegaly, and myeloid proliferation. As expected, immune cells in these mice have an upregulated NF-κB pathway and many of the phenotypes can be ameliorated by inhibition of the NF-κB pathway. Importantly, a significant proportion of the animals develop B-cell lymphoma at older ages. In this study, we examined the role of miR-146a in the development of malignancy in B-cells. To accelerate the role of miR-146a in tumor formation we overlaid the miR-146a deficient allele onto the Eμ-Myc like mouse model. Eμ-Myc mice develop tumors on average by 14weeks of age. The transgenic status of animals was verified by genotyping, RNA and protein expression analyses. miR-146a sufficient and deficient animals on the Eμ-Myc background were followed for tumor latency by peripheral blood analysis and careful physical examination. Based on approved humane criteria for animal discomfort, animals were sacrificed and hematopoietic tissue was harvested for analysis. Mice deficient for miR-146a had a statistically reduced survival in comparison with miR-146a sufficient animals with a p-value of .0098 (Kaplan Meir survival analysis). Complete Blood Count of animals at time of death revealed an increase leukemia presentation in the miR-146a deficient background. FACS analysis of tumor tissue from both groups revealed an increase in the number of IgM positive tumors in the miR-146a-deficient background indicating skewing towards more mature B cell neoplasms when miR-146a is lacking. Lineage analysis of tumors verified them to be of B cell origin although a subset of miR-146a sufficient tumors had higher numbers of infiltrating myeloid cells compared to deficient animals. Furthermore, histologic analysis of hematopoietic organs showed that while infiltration remained similar in kidneys and liver, more spleens in the miR-146a deficient background tended to be less involved. Our extensive histopathologic and immunophenotypic analyses indicate that miR-146a deficiency drives a more aggressive malignant phenotype in the B-cell lineage. In keeping with this, our profiling studies of human DLBCL suggest that a subset of DLBCL show decreased expression of miR-146a. We are currently examining the status of NF-κB in the murine tumors and using high throughput sequencing approaches to delineate gene expression differences between miR-146a sufficient and deficient tumors. We anticipate the discovery of novel gene targets of miR-146a and expect that these studies will lead to improved diagnostic and therapeutic options for patients of B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

SNS01-T Exhibits Significant Anti-Tumoral Activity In Models Of Multiple Myeloma and Non-Hodgkins B Cell Lymphoma and Induces Cell Death In Malignant But Not Normal B Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 880-880
Author(s):  
Catherine A Taylor ◽  
Terence Tang ◽  
Sarah Francis ◽  
Zhongda Liu ◽  
Qifa Zheng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Tumor Volume ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
Control Group ◽  
Rpmi 8226

Abstract SNS01-T is a novel nanoparticle that is designed to selectively initiate apoptosis in B-cell cancers such as multiple myeloma and non-Hodgkins B-cell lymphomas. SNS01-T comprises a plasmid DNA (pExp5A) encoding a pro-apoptotic form of the eukaryotic translation initiation factor 5A (eIF5A) containing a single-point mutation that prevents hypusination, an eIF5A siRNA that inhibits expression of the pro-survival hypusine-eIF5A protein, and a polymer that serves to assemble the nucleic acids into a nanoparticle. SNS01-T is currently being investigated in a multi-site, open-label Phase1b/2a dose escalation study in subjects with relapsed or refractory multiple myeloma (MM), mantle cell lymphoma (MCL), or diffuse large B cell lymphoma (DLBCL). SNS01-T has demonstrated activity in MM xenograft models as well as in B cell lymphoma models of MCL and DLBCL, when administered twice weekly at doses ≥ 0.18 mg(nucleic acid)/kg. In this study we compared the ability of SNS01-T to transfect, regulate eIF5A expression, and kill MM, DLBCL, and MCL cell lines. Furthermore, the activity of SNS01-T in normal B cells was investigated. A previous study using a KAS-6/1 MM xenograft model demonstrated that the eIF5A siRNA and plasmid pExp5A both have anti-tumoral activity in MM but had a greater impact on tumour growth when combined together as SNS01-T. This finding was confirmed in this study in a second MM model (RPMI 8226) as well as in a DLBCL xenograft model. To determine the efficiency of SNS01-T transfection into malignant or normal B cells, the pExp5A plasmid and eIF5A siRNA were labeled with FITC and DY547, respectively, packaged into nanoparticles using polyethylenimine polymer, and used to transfect cultured cells. FACS analysis was used to determine the percent of the cell population transfected with plasmid, siRNA, or both. RT-qPCR was used to assess biological activity of SNS01-T by quantifying the expression of eIF5AK50R mRNA transgene and endogenous eIF5A mRNA in a variety of B cell lines. The IC50 of SNS01-T in a panel of MM, MCL, and DLBCL cell lines was determined by XTT assay. SCID mice bearing either RPMI 8226 MM tumours or SuDHL6 GCB DLBCL tumours were treated with pExp5A plasmid (formulated with PEI and control siRNA), eIF5A siRNA (formulated with PEI and a control plasmid), or SNS01-T at 0.375 mg/kg twice per week by intravenous injection. SNS01-T was able to transfect MM, MCL, and DLBCL cell lines, although the proportion of cells transfected with both plasmid and siRNA was higher in MM cells. Transfection of SNS01-T resulted in expression of the transgene as well as a statistically significant reduction in expression of eIF5A mRNA compared to untreated controls for all three cell types. In contrast, normal B cells were found to take up fluorescently-labeled SNS01-T with reduced efficiency compared to RPMI 8226 MM cells. Futhermore, SNS01-T was observed to induce cell death in RPMI 8226 MM cells but not in normal B cells. In the RPMI 8226 xenograft model, treatment with either the pExp5A plasmid alone or eIF5A siRNA alone resulted in a 66 % reduction (p < 0.0001) or 44 % reduction (p < 0.05) in tumor volume compared to the control group at day 24 of the study. In contrast, treatment with SNS01-T, which contains both the pExp5A plasmid and the eIF5A siRNA, resulted in an 86 % (p < 0.0001) reduction in tumor volume. A similar result was observed in the SuDHL6 model with a 14 % reduction or 27 % reduction (p < 0.05) in tumor volume compared to the control group at day 20 of the study following treatment with pExp5A plasmid or eIF5A siRNA, respectively. In contrast, treatment with SNS01-T resulted in a 79 % (p < 0.0001) reduction in tumor volume. Collectively, these preclinical studies indicate that SNS01-T therapy has significant potential against MM, MCL, and DLBCL. Disclosures: Taylor: Senesco Technologies: stock options Other. Dondero:Senesco Technologies: Employment. Thompson:Senesco Technologies: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

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