Unveiling the Role of MicroRNA155 in Diffuse Large B Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2024-2024
Author(s):  
Deepak Rai ◽  
Shailaja Karanti ◽  
Patricia Dahia ◽  
Ricardo C.T. Aguiar
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
B Cell ◽  
Cell Lines ◽  
Mhc Class Ii ◽  
B Cell Lymphoma ◽  
Tissue Cell ◽  
Transcription Analysis ◽  
Mrna Cleavage

Abstract MicroRNAs (miR) are non-coding RNAs that regulate gene expression by pairing to 3UTRs of target genes inducing translational repression or mRNA cleavage. New evidence suggests that the latter mechanism markedly contributes to miRNA effects. Hence, global gene expression analyses may help elucidate the functional role of miRNA by recognizing pathways modified by their abnormal expression and identifying direct targets. MiR155, the product of the non coding gene BIC, is overexpressed in lymphomas and its role in tumorigenesis is supported by the development of B-cell malignancies in miR155 transgenic mice. However, the functional consequences of miR155 overexpression in tumor development remain unclear. To address this issue, we developed a semi-quantitative RT-PCR assay that specifically amplifies either the nuclear unspliced BIC mRNA (target of the RNase III Drosha) or the spliced BIC mRNA. We found a marked correlation between the expression levels of these two mRNAs, which in turn agreed with the levels of mature miR155 detected in northern blots. Of the 22 DLBCL cell lines studied, only 5 (DHL6, Ly3, Ly10, Farage, RCK-8) expressed significantly high levels of BIC and miR155. To isolate the effects of miR155 in DLBCL we genetically modified its expression and performed global transcription analysis on microarray. In brief, we cloned the BIC transcript in a MSCV-GFP bicistronic retrovirus and confirmed in transduced HeLa cells that the mature miR155 was expressed when this transcript was driven by an LTR promoter. Next, we used two DLBCL cell lines with low levels of miR155 (Ly8 and Ly19) to generate unique populations expressing miR155 or MSCV alone. RNA was isolated from GFP-sorted cells, hybridized to the Affymetrix U133Plus2.0 chip and the data analyzed with dChip. Remarkably, and in agreement with the role of miRNAs, supervised analysis (fold diff >1.7) revealed a vast predominance (>90%) of downregulated genes when comparing miR155 expressing cells to MSCV only. These gene groups included predicted miR155 targets and were significantly enriched for molecules involved in the immune response (p<.001), including MHC class II, chemokine receptors, TDT, NFAT and CD24, a particularly relevant target for miR155 inhibition since its activation induces apoptosis in lymphomas. To validate and extend our findings, we queried public expression datasets of primary DLBCL. First, we used our cell lines expression data to confirm that the BIC probe in the Affymetrix chip reliably reflected the expression of miR155. Since DLBCL entails at least two groups of tumors reflecting distinct normal B-cells (GC and ABC) and the expression of miRNA and its targets are highly tissue/cell specific, we compartmentalized these analyses within the groups of ABC and GC tumors. We selected the tumors within each group with the highest and lowest levels of BIC (20% percentile) and performed unsupervised hierarchical clustering analysis (filtering parameters 0.5<SD/mean<10). In agreement with our data in the GC-type miR155-expressing cell lines, we found that in GC-, but not in ABC tumors, the expression of BIC inversely correlated with that of the genes related to the immune response. Notably, the pro-apoptotic molecule CD24 was significantly downregulated (p<0.02) in BIC overexpressing primary DLBCL, underscoring the need for further characterization of the signals relayed by this surface molecule and its potential as a rational drug target. Our data start to delineate the effects of miR155 in DLBCL and show the potential of expression arrays to identify miRNA targets modified by mRNA cleavage.

Pharmacogenetic Approaches Identify ANTXR1 As a Potential Mediator of Response to Therapeutic NF-κB Inhibition in Diffuse Large B Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 234-234
Author(s):  
Soham D. Puvvada ◽  
Cassandra L Love ◽  
Vladimir Grubor ◽  
Jenny Zhang ◽  
Jason Smith ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Exome Sequencing ◽  
Cell Lines ◽  
Expression Profiling ◽  
B Cell Lymphoma ◽  
Lymphoid Malignancies ◽  
Whole Exome

Abstract Abstract 234 Background: NF-κB is a family of transcription factors known to play an essential role in the development & survival of lymphocytes. In recent years, it has been clear that aberrant NF-κB activation is a hallmark of various lymphoid malignancies and appears to be associated with chemotherapy resistance and adverse prognosis. The canonical NF-κB pathway is frequently engaged in lymphoid malignancies wherein activated IKK phosphorylates IκB proteins inducing IκB polyubiquination and subsequent proteasomal proteolytic degradation; this allows for release and nuclear translocation of NF-κB dimers to activate target gene transcription. Gene Expression Profiling (GEP) has identified two distinct sub groups of Diffuse Large B cell Lymphoma (DLBCL). While the Activated B cell (ABC) type shows constitutive activation of NF-κB, the role for NF-κB activation in Germinal Center B Cell (GCB) DLBCL is currently unclear. Since NF-κB inhibition has been identified as a therapeutic possibility in DLBCLs, it is important to define the role of this pathway and its modulators. In this study, we sought to investigate key regulators of the NF-κB pathway that might mediate a therapeutic response to IKKβ inhibition of NF-κB in DLBCL including the GCB subtype. Through GEP and Exome Sequencing, we demonstrate that ANTXR1 is a key mediator of response to IKKβ inhibition in DLBCL. Methods/Results: We obtained a novel selective inhibitor of IKKβ, TLX-2001 that has been found to be safe in animal models. IC50 were obtained on 61 cell lines representing various lymphomas including DLBCL (N=25) using cell viability MTT assays. The drug showed efficacy in both ABC and GCB DLBCL cell lines at physiologically achievable concentrations. These results were unsurprising in ABC DLBCLs which are known to depend on NF-κB activation, but the lethality of this selective drug in GCB DLBCLs was unexpected. To better understand the role of individual genes in the response in GCB DLBCLs, gene expression profiling was performed on 61 cell lines using Human Gene 1.0 ST Array. We found that ANTXR1 expression significantly correlated with NF-κB resistance (p = 0.035). Additionally, we sequenced the exomes of DLBCL tumors (N=95) and matched normal tissue (N=34). 95 cases of DLBCLs consisted of 73 cases of primary human DLBCLs and 22 DLBCL cell lines. Whole exome sequencing was performed using the Agilent solution-based system of exon capture to sequence all protein coding exons in the CCDS database. We identified 465 recurrently somatically mutated genes in these DLBCL cases, and found that mutation status of ANTXR1 was associated with high sensitivity to IKKβ inhibition (p =0.015) of NF-κB. Cell lines with non-synonymous mutations in ANTXR1 had over 3-fold lower IC50 (mean= 2.39 μM) compared to cell lines with no mutation in ANTXR1 (mean IC50 = 8.72μM). Discussion/Conclusion: ANTXR1 is the docking receptor for bacillus anthracis toxin, and anti-tumor responses have been observed in mice injected with recombinant engineered anthrax toxin. It is also known as TEM8 and maps to chromosome 2p13.1. Increased levels of TEM8 (tumor endothelial marker 8) have been noted in various malignancies including melanoma. Our data suggest that pharmacogenetic approaches that combine gene expression profiling and whole exome sequencing are useful tools for identifying novel genes that modulate therapeutic responses in lymphoma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Feedback inhibition of immunoglobulin gene rearrangement by membrane mu, but not by secreted mu heavy chains.

1988 ◽  
Vol 168 (4) ◽  
pp. 1363-1381 ◽  
Author(s):  
J Manz ◽  
K Denis ◽  
O Witte ◽  
R Brinster ◽  
U Storb
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
B Cell ◽  
Cell Lines ◽  
Gene Rearrangement ◽  
Feedback Inhibition ◽  
Allelic Exclusion ◽  
Immunoglobulin Gene ◽  
Heavy Chains

Previous work (6-10) has shown that allelic exclusion of Ig gene expression is controlled by functionally rearranged mu and kappa genes. This report deals with the comparison of membrane mu (micron) and secreted mu (microsecond) in promoting such feedback inhibition. Splenic B cell hybridomas were analyzed from transgenic mice harboring a rearranged kappa gene alone or in combination with either an intact rearranged mu gene or a truncated version of the mu gene. The intact mu gene is capable of producing both membrane and secreted forms of the protein, while the truncated version can only encode the secreted form. The role of the microsecond was also tested in pre-B cell lines. Analysis of the extent of endogenous Ig gene rearrangement revealed that (a) the production of micron together with kappa can terminate Ig gene rearrangement; (b) microsecond with kappa does not have this feedback effect; (c) microsecond may interfere with the effect of micron and kappa; and (d) the feedback shown here probably represents a complete shutoff of the specific recombinase by micron + kappa; the data do not address the question of mu alone affecting the accessibility of H genes for rearrangement.

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.

Gene Expression Signature of Host Immune Response Is Predictive of Follicular Lymphoma Patient Survival in Independent Cohorts, and Correlates with Transformation to Diffuse Large B-Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2951-2951
Author(s):  
Ash A Alizadeh ◽  
Andrew J Gentles ◽  
Sylvia K Plevritis ◽  
Ronald Levy
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Immune Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell ◽  
Independent Cohort

Abstract Abstract 2951 Poster Board II-927 Background: Expression signatures of infiltrating immune cells [1] have been shown to predict survival in follicular lymphoma (FL), but have not been cross-validated in independent patient cohorts [2,3]. These signatures may relate biologically to the frequency of infiltrating including T-cells and macrophages, or to specific transcription programs within tumor cells and/or the tumor microenvironment. We sought to evaluate the validity of this model in an independent cohort of patients with FL, assessing its relationship to outcomes including histological transformation and death. Methods: The immune response (IR) predictor score proposed by Dave et al. [1] was applied to gene expression data from an independent cohort of 88 FL patients [4] with known survival outcomes and history of transformation to diffuse large B-cell lymphoma (DLBCL). Genes (n=66) corresponding to IR1 and IR2 signatures were mapped from Affymetrix microarrays [1] to a custom cDNA array [4] via Entrez Gene ID, and the composite IR score was calculated per the scheme proposed by Dave et al. Results: The IR score was predictive of patient outcome in the 88 patient test set as a continuous variable (p=0.001, HR=2.01, 95% CI 0.50-1.30). Partitioning of patients into high and low risk groups based on the median IR score across the cohort robustly separated survival curves (Figure A). The IR score was significantly higher in FL patients known to undergo transformation to DLBCL (Figure B: mean IR score of -0.6 in non-transforming FL vs. -0.2 in transforming FL; p∼10-11, t-test). Conclusions: The IR score of Dave et al. was highly significant as a predictor of survival in the independent patient cohort [4]. Moreover, the score was significantly associated with propensity of FL to transform to DLBCL. To our knowledge, immune cell infiltration has not previously been implicated in transformation. 1. Dave SS et al. (2004) Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. N Engl J Med 351(21): 2159-2169. 2. Tibshirani R (2005) Immune signatures in follicular lymphoma. N Engl J Med 352: 1496-1497. 3. Chu G Hong WJ, Warnke R, Chu G (2005). Immune Signatures in Follicular Lymphoma (Corres). N Engl J Med. 352: 1496-1497. 4. Glas AM et al. (2005) Gene expression profiling in follicular lymphoma to assess clinical aggressiveness and to guide the choice of treatment. Blood 105(1): 301-307. Disclosures: No relevant conflicts of interest to declare.

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.

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.

Disruption Of Super Enhancer-Driven Cancer Dependencies In Diffuse Large B-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3021-3021 ◽  
Author(s):  
Bjoern Chapuy ◽  
McKeown Michael ◽  
Charles Y. Lin ◽  
Stefano Monti ◽  
Margaretha GM Roemer ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Dana Farber Cancer Institute ◽  
Super Enhancer ◽  
Equity Ownership

Abstract Diffuse large B-cell lymphoma (DLBCL) exhibits significant biological and transcriptional heterogeneity which is conferred, in part, by pathologic modulation of lineage-specific and growth-associated master regulatory transcription factors (TF). Chromatin associated with TF binding sites is markedly enriched in histone proteins that are post-translationally modified by lysine side-chain acetylation. This mark facilitates the opening of chromatin and recruits a class of co-activators which recognize ε-acetyl lysine through a bromodomain. The sub-family of bromodomain and extra-terminal domain (BET) co-activators (BRD2, BRD3 and BRD4) are appealing, in part, because transgenic expression of BRD2 caused a DLBCL-like neoplasm in mice. We recently developed the first BET inhibitor, JQ1, and now explore the role of BET bromodomains in oncogenic transcription and assess BET family members as therapeutic targets in DLBCL. Nanomolar doses of JQ1 and 3 structurally dissimilar BET bromodomain inhibitors decreased the cellular proliferation of a broad panel of DLBCL cell lines of all transcriptionally defined types whereas the inactive enantiomer, JQ1R, had no effect. BRD2 and BRD4 depletion similarly decreased the proliferation of multiple DLBCL cell lines. We next explored the therapeutic potential of BET inhibition in two independent DLBCL xenotransplantation models, Ly1 and Toledo. In the first xenograft model, JQ1-treated mice had a prolongation of overall survival (p = 0.003). In the second model, JQ1-treated animals had significantly delayed tumor progression and decreased lymphomatous infiltration of spleen and bone marrow. To define the transcriptional pathways regulated by BET bromodomain proteins, we performed transcriptional profiling of multiple vehicle and JQ1-treated DLBCL cell lines. Following JQ1 treatment, we observed downregulation of multiple MYD88/TLR and BCR signaling pathway components and functionally validated MYC and E2F target gene sets. BET inhibition decreased MYC transcripts and protein in the DLBCL cell line panel suggesting that BET bromodomains directly modulate MYC transcription. In contrast, JQ1 treatment did not measurably alter E2F1 transcript or protein abundance suggesting a co-activator role of the BET bromodomains for E2F1. To explore the role of BET bromodomains in oncogenic E2F1 transcriptional signaling, we performed ChIPSeq experiments in Ly1 cells, using a chemical genetic approach. Rank-ordering of all transcriptionally active promoters based on H3K4me3 enrichment and RNA Pol II occupancy identifies pervasive binding and spatial colocalization of BRD4 and E2F1 to active promoter elements. We identified a JQ1-mediated transcriptional elongation defect across E2F1-bound promoters, responsible for the downregulation of E2F1 targets. As oncogenic TFs may signal to RNA Pol II through distal enhancer elements, we also characterized the genome-wide localization of BRD4 to enhancers in the Ly1 DLBCL cell line. Rank-ordering of enhancer regions by H3K27ac enrichment reveals that BRD4 binds to the vast majority of active enhancers in the Ly1 genome. Strikingly, the BRD4 load is asymmetrically distributed throughout the genome at enhancer sites with only a small subset of BRD-loaded “super enhancers (SE)”, 285/18330 (1.6%), accounting for 32% of all BRD4 enhancer binding in the cell. The POU2AF1 locus emerged as the most BRD4-overloaded enhancer in Ly1. BET inhibition reduced RNA Pol II elongation of POU2AF1, with a concomitant increase in promoter-paused RNA Pol II near the transcriptional start site. Accordingly, JQ1 treatment decreased POU2AF1 transcript abundance and protein expression and reduced the expression of a POU2AF1 target gene set. POU2AF1 depletion with independent shRNAs significantly decreased the proliferation of Ly1 and enforced POU2AF1 expression decreased the sensitivity of Ly1 cells to JQ1 treatment. Additional super enhancer-driven genes that were sensitive to JQ1 treatment include ones which promote and maintain the B-cell gene expression program and limit plasma cell differentiation. Our data suggest that BET inhibition limits the growth of DLBCLs by at least two complementary activities: a specific effect on genes that define a given cell type by high BRD4 loading at enhancers and the selective suppression of transcription at E2F- and MYC- driven target genes. + Contributed equally Disclosures: Qi: Patent for JQ1: holds patent for JQ1, holds patent for JQ1 Patents & Royalties. Young:Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Enzon Pharmaceuticals: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Bradner:Tensha Therapeutics: Equity Ownership, Scientific founder of Tensha which is translating drug-like derivatives of the JQ1 chemical probe of BET bromodomains used in this study, as cancer therpeutics. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity. Other; Syros Pharmaceuticals: Equity Ownership, Scientific founder of Syros which is discovering Super Enhancers as a new class of gene control elements. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity., Scientific founder of Syros which is discovering Super Enhancers as a new class of gene control elements. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity. Other.

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.

Recurrent Mutations of the Exportin 1 Gene (XPO1) in Primary Mediastinal B-Cell Lymphoma: A Lysa Study

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 129-129 ◽  
Author(s):  
Fabrice Jardin ◽  
Anais Pujals ◽  
Laura Pelletier ◽  
Elodie Bohers ◽  
Vincent Camus ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Nuclear Export ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
U2os Cells ◽  
Aggressive B Cell Lymphoma

Abstract Background and aim of the study Primary mediastinal B-cell lymphoma (PMBL) is an entity of aggressive B-cell lymphoma that is clinically and biologically distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We recently detected by Whole exome sequencing a recurrent point mutation in the XPO1 (exportin 1) gene (also referred to as chromosome region maintenance 1; CRM1), which resulted in the Glu571Lys (p.E571K) missense substitution in 2 refractory/relapsed PMBL (Dubois et al., ICML 2015; Mareschal et al. AACR 2015). XPO1 is a member of the Karyopherin-b superfamily of nuclear transport proteins. XPO1 mediates the nuclear export of numerous RNAs and cellular regulatory proteins, including tumor suppressor proteins. This mutation is in the hydrophobic groove of XPO1 that binds to the leucine-rich nuclear export signal (NES) of cargo proteins. In this study, we investigated the prevalence, specificity, and biological / clinical relevance of XPO1 mutations in PMBL. Patients and methods High-throughput targeted or Sanger sequencing of 117 PMBL patients and 3 PMBL cell lines were performed. PMBL cases were defined either molecularly by gene expression profile (mPMBL cohort) or by standard histological method (hPMBL cohort) and enrolled in various LYSA (LYmphoma Study Association) clinical trials. To assess the frequency and specificity of XPO1 mutations, cases of classical Hodgkin lymphoma (cHL) and primary mediastinal grey zone lymphoma (MGZL) were analysed. Cell experiments were performed to assess the impact of the E571 mutation on the activity of selective inhibitor of nuclear export (SINE) molecules. Results XPO1 mutations were present in 28/117 (24%) PMBL cases but were rare in cHL cases (1/19, 5%) and absent from MGZL cases (0/20). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in PMBL cases defined by gene expression profiling (n = 32), as compared to hPMBL cases (n = 85, 13%). No difference in age, International Prognostic Index (IPI) or bulky mass was observed between the PMBL patients harboring mutant and wild-type XPO1 in the overall cohort whereas a female predominance was noticed in the mPMBL cohort. Based on a median follow-up duration of 42 months, XPO1 mutant patients exhibited significantly decreased PFS (3y PFS = 74% [CI95% 55-100]) compared to wild-type patients (3y PFS = 94% [CI95% 83-100], p=0.049) in the mPMBL cohort. In 4/4 tested cases, the E571K variant was also detected in cell-free circulating plasmatic DNA, suggesting that the mutation can be used as a biomarker at the time of diagnosis and during follow-up. Importantly, the E571K variant was detected as a heterozygous mutation in MedB-1, a PMBL-derived cell line, whereas the two other PMBL cell lines tested, Karpas1106 and U-2940, did not display any variants in XPO1 exon 15. KPT-185, the SINE compound that blocks XPO1-dependent nuclear export, induced a dose-dependent decrease in cell proliferation and increased cell death in the PMBL cell lines harbouring wild type or mutated alleles. To test directly if XPO1 mutation from E571 to E571K alters XPO1 inhibition by SINE compounds, the mutated protein was tested in vitro. The E571XPO1 mutated allele was transiently transfected into osteosarcoma U2OS cells which stably express the fluorescently labelled XPO1 cargo REV. Cells were treated with the clinical SINE compound selinexor, which is currently in phase I/II clinical trials and nuclear localization of REV-GFP was analysed in red transfected cells. The results showed that the nuclear export of the mutated XPO1 protein was inhibited by selinexor similarly to the wild-type XPO1 protein (Figure 1). Conclusion Although the oncogenic properties of XPO1 mutations remain to be determined, their recurrent selection in PMBL strongly supports their involvement in the pathogenesis of this curable aggressive B-cell lymphoma. XPO1 mutations were primarily observed in young female patients who displayed a typical PMBL molecular signature. The E571K XPO1 mutation represents a novel hallmark of PMBL but does not seem to interfere with SINE activity. Rev-GFP (green fluorescent) expressing U2OS cells were transfected with wild type XPO1-RFP (red fluorescent protein), XPO1-C528S-RFP, XPO1-E571K-mCherry, and XPO1-E571G-mCherry. The cells were then treated with 1µM KPT-330 for 8 hours. Figure 1. Rev-GFP expressing U2OS cells transfected with XPO1 variants. Figure 1. Rev-GFP expressing U2OS cells transfected with XPO1 variants. Disclosures Landesman: Karyopharm Therapeutics: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Argueta:Karyopharm Therapeutics: Employment. Milpied:Celgene: Honoraria, Research Funding.

The Role of PIM1 in the Ibrutinib-Resistant ABC Subtype of Diffuse Large B-Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 699-699 ◽  
Author(s):  
Hsu-Ping Kuo ◽  
Sidney Hsieh ◽  
Karl J. Schweighofer ◽  
Leo WK Cheung ◽  
Shiquan Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Growth ◽  
B Cell ◽  
Repeated Measures ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
Bcr Signaling

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for roughly 30% of newly diagnosed cases in the United States (US). DLBCL is a heterogeneous lymphoma, including the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, which have different gene expression profiles, oncogenic aberrations, and clinical outcomes (Alizadeh, Nature 2000; Staudt, Adv Immunol 2005). ABC-DLBCL is characterized by chronic active B-cell receptor (BCR) signaling (Davis, Nature 2010), which is required for cell survival. Thus, the BCR signaling pathway is an attractive therapeutic target in this type of B-cell malignancy. Bruton's tyrosine kinase (BTK), which plays a pivotal role in BCR signaling, is covalently bound with high affinity by ibrutinib, a first-in-class BTK inhibitor approved in the US for mantle cell lymphoma and chronic lymphocytic leukemia (CLL) patients (pts) who have received at least one prior treatment, CLL with del17p, and WaldenstršmÕs macroglobulinemia. A recent phase 2 clinical trial of single-agent ibrutinib in DLBCL pts revealed an overall response rate of 40% for ABC-DLBCL (Wilson, Nat. Med 2015); however, responses to single kinase-targeted cancer therapies are often limited by the cellÕs ability to bypass the target via alternative pathways or acquired mutations in the target or its pathway (Nardi, Curr Opin Hematol 2004; Gazdar, Oncogene 2009). The serine/threonine-protein kinase PIM1 is one of several genes exhibiting differential expression in ibrutinib-resistant ABC-DLBCL cells compared with wild-type (WT) cells. We identified and report herein the role of PIM1 in ABC-DLBCL ibrutinib-resistant cells. Methods: PIM1 gene expression was analyzed by RT-qPCR. In vitro, cell viability was assessed in the human ABC-DLBCL cell line HBL-1 after treatment with ibrutinib and/or a pan-PIM inhibitor for 3 days, and the effect on colony formation was determined 7 days post-treatment. PIM1 mutational analysis was performed with clinical tumor biopsy samples from 2 studies, PCYC-04753 (NCT00849654) and PCYC-1106-CA (NCT01325701). PIM1 protein stability was analyzed by treating cells with cycloheximide and examining protein levels at different time points up to 8 hours. Results: Gene expression profiling of ibrutinib-resistant ABC-DLBCL cells revealed an upregulation of PIM1 (15-fold increase compared with WT cells) as well as PIM2 and PIM3. We also found that, compared with single-drug treatment, in vitro cell growth could be synergistically suppressed with a combination of ibrutinib and a pan-PIM inhibitor. This effect was observed in both WT (combination index (C.I.) = 0.25; synergy score = 3.18) and ibrutinib-resistant HBL-1 cells (C.I. = 0.18; synergy score = 4.98). In HBL-1 cells, this drug combination reduced colony formation and suppressed tumor growth in a xenograft model (Figure 1). In 48 DLBCL patient samples with available genomic profiling, PIM1 mutations appeared more frequently in pts diagnosed with ABC-DLBCL compared with GCB-DLBCL (5 out of 6 DLBCL pts with PIM1 mutations were ABC-subtype). 4 of these 5 pts exhibited a poor clinical response to ibrutinib, ie, 80% of ABC-DLBCL pts with PIM1 mutations had progressive disease, compared with only 13 of 26 (ie, 50%) ABC-DLBCL pts without PIM1 mutations. Subsequent characterization of the mutant PIM1 proteins (L2V, P81S, and S97N) confirmed that they were more stable than WT PIM1, suggesting increased protein levels by 2 potential mechanisms (WT PIM1 gene up-regulation or increased mutant PIM1 protein half-life). The impact of these mutations on PIM1 function and ibrutinib sensitivity is under investigation. Conclusions: Ibrutinib-resistant ABC-DLBCL cells have increased PIM1 expression, and synergistic growth suppression was observed when ibrutinib was combined with a pan-PIM inhibitor. PIM1 mutations identified in ABC-DLBCL pts with poor responses to ibrutinib contributed to increased PIM1 protein stability. A better understanding of the role of PIM1 in ibrutinib-resistant ABC-DLBCL tumors could provide a rationale for the design of combination therapies. Figure 1. Combination of ibrutinib and a pan-PIM inhibitor in the HBL-1 xenograft model. Ibrutinib and PIM inhibitor treatment suppressed tumor growth by 62% compared with the vehicle-treated group (*p < 0.01, repeated measures MANOVA adjusted univariate F-test). Figure 1. Combination of ibrutinib and a pan-PIM inhibitor in the HBL-1 xenograft model. Ibrutinib and PIM inhibitor treatment suppressed tumor growth by 62% compared with the vehicle-treated group (*p < 0.01, repeated measures MANOVA adjusted univariate F-test). Disclosures Kuo: Pharmacyclics LLC, an AbbVie Company: Employment. Hsieh:pharmacyclics LLC, an AbbVie Company: Employment. Schweighofer:Pharmacyclics LLC, an AbbVie Company: Employment. Cheung:Pharmacyclics LLC, an AbbVie Company: Employment. Wu:Pharmacyclics LLC, an AbbVie Company: Employment. Apatira:Pharmacyclics LLC, an AbbVie Company: Employment. Sirisawad:Pharmacyclics LLC, an AbbVie Company: Employment. Eckert:Pharmacyclics LLC, an AbbVie Company: Employment. Liang:Pharmacyclics LLC, an AbbVie Company: Employment. Hsu:Pharmacyclics LLC, an AbbVie Company: Employment. Chang:Pharmacyclics LLC, an AbbVie Company: Employment.

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