TET2 mutations in Diffuse Large B-Cell Lymphoma: The Role of TET2 in the Regulation of Methylation Patterns at TET2 Target Genes

Abstract Abstract 1364 Introduction: Cytosine methylation (mC) is a major DNA modification in higher eukaryotic genomes, which is involved in transcriptional silencing. A large amount of data has shown that patterns of DNA methylation are perturbed in hematological cancers including diffuse large B-cell lymphoma (DLBCL). The discovery that the TET hydroxylases convert mC to hydroxymethylcytosine (hmC) is a major break through for our understanding of how DNA methylation is deregulated. Multiple reports describe TET2 (Ten-Eleven Translocation 2) loss-of-function mutations in myeloid malignancies, and a recent study shows that TET2 inactivation perturbs both myeloid and lymphoid development in the mouse, and identifies TET2 mutations in ∼2% of human B-cell lymphoma (Quivoron et al, Cancer Cell 20, 1–14, 2011). Aims: In the present study our aims are to determine the frequency and clinical impact of TET2 mutations in DLBCL, to identify TET2 target genes in CD34+ cells, normal- and malignant B-cells, and evaluate the role of TET2 mutations on the methylation pattern at TET2 targets genes in normal and malignant hematopoiesis. Methods: DNA was isolated from fresh frozen DLBCL (n=110), normal CD34+ cells and B-cells, and a TET2 mutant DLBCL-cell line. Mutation scanning was performed by denaturing gradient gel electrophoresis (DGGE) and automated sequencing. Global methylation profiling was done by Illumina Infinium microarrays, methylation at individual genes by methylation specific melting curve analysis and pyrosequencing. Global mC and hmC patterns were determined by DNA immunoprecipitation and promoter array analysis in cell lines, B-cells and CD34+ cells. TET2 target genes were identified by ChIP followed by deep sequencing. Gene expression by Nimblegen custom made arrays and RT-qPCR. Results: We identified TET2 mutations in 15% of primary diffuse DLBCL, including missense mutation in the catalytic domain (n=8, 2 of which showed allelic loss), loss-of-function mutations (n=7, one of which showed allelic loss), and missense mutation outside the catalytic domain (n=1 with allelic loss). Somatic origin of these mutations was verified in 11 of the 16 cases where matched normal tissue was available. No difference in overall survival was observed between TET2mut and TET2wt cases (P=0.17). To a large extent, the TET2 targets genes identified by ChIP seq analysis were overlapping in CD34+ cells, normal- and malignant B-cells. Gene ontology analysis showed that TET2 target genes are mainly involved in DNA metabolism and repair, metabolic processes and cell cycle homeostasis. Global methylation in TET2mut and TET2wt cases and gene expression data are being analyzed in DLBCL samples. In addition, the distribution patterns of hmC and mC at TET2 target genes and the relation to gene expression is being analyzed in a TET2 mutant DLBCL cell line, normal B-cells and CD34+ cells. Conclusion and further analyses: Here, we show that TET2 mutations are frequent in DLBCL, and identify the TET2 target genes in CD34+ cells, and in normal and malignant B-cells. The role of TET2 mutations for global methylation and for the methylation patterns at TET2 target genes will be presented at the meeting. By investigating the clinical implications of TET2 mutations we aim to identify DLBCL subsets that may benefit from hypomethylating therapy. Furthermore, the identification of hypermethylated TET2 target genes will hopefully contribute to molecular understanding of how TET2 mutations induces malignant transformation. Disclosures: Christensen: EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company. Helin:EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company.

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

Blood ◽

10.1182/blood.v122.21.3805.3805 ◽

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.

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Nuclear CD40 interacts with c-Rel and enhances proliferation in aggressive B-cell lymphoma

Blood ◽

10.1182/blood-2007-02-073080 ◽

2007 ◽

Vol 110 (6) ◽

pp. 2121-2127 ◽

Cited By ~ 39

Author(s):

Hai-Jun Zhou ◽

Lan V. Pham ◽

Archito T. Tamayo ◽

Yen-Chiu Lin-Lee ◽

Lingchen Fu ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Target Genes ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Promoter Activation ◽

Growth And Survival ◽

Survival Mechanisms ◽

Lymphoma Therapy ◽

Receptor Family

Abstract CD40 is an integral plasma membrane–associated member of the TNF receptor family that has recently been shown to also reside in the nucleus of both normal B cells and large B-cell lymphoma (LBCL) cells. However, the physiological function of CD40 in the B-cell nucleus has not been examined. In this study, we demonstrate that nuclear CD40 interacts with the NF-κB protein c-Rel, but not p65, in LBCL cells. Nuclear CD40 forms complexes with c-Rel on the promoters of NF-κB target genes, CD154, BLyS/BAFF, and Bfl-1/A1, in various LBCL cell lines. Wild-type CD40, but not NLS-mutated CD40, further enhances c-Rel–mediated Blys promoter activation as well as proliferation in LBCL cells. Studies in normal B cells and LBCL patient cells further support a nuclear transcriptional function for CD40 and c-Rel. Cooperation between nuclear CD40 and c-Rel appears to be important in regulating cell growth and survival genes involved in lymphoma cell proliferation and survival mechanisms. Modulating the nuclear function of CD40 and c-Rel could reveal new mechanisms in LBCL pathophysiology and provide potential new targets for lymphoma therapy.

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

Blood ◽

10.1182/blood.v126.23.699.699 ◽

2015 ◽

Vol 126 (23) ◽

pp. 699-699 ◽

Cited By ~ 1

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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Reproducing Transformation of Indolent B-cell Lymphoma by T-cell Immunosuppression of L.CD40 Mice

10.1101/477273 ◽

2018 ◽

Author(s):

Christelle Vincent-Fabert ◽

Alexis Saintamand ◽

Amandine David ◽

Mehdi Alizadeh ◽

François Boyer ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Mouse Model ◽

B Cell ◽

Immune Suppression ◽

Clinical Course ◽

Cell Lymphoma ◽

Immune Surveillance ◽

B Cell Lymphoma

AbstractTransformation of an indolent B-cell lymphoma is associated with a more aggressive clinical course and poor survival. The role of immune surveillance in the transformation of a B-cell indolent lymphoma towards a more aggressive form is poorly documented. To experimentally address this question, we used the L.CD40 mouse model, which is characterized by B-cell specific continuous CD40 signaling, responsible for spleen indolent clonal or oligoclonal B-cell lymphoma after one year in 60% cases. Immunosuppression was obtained either by T/NK cell depletion or by treatment with the T-cell immunosuppressive drug cyclosporin A. Immunosuppressed L.CD40 mice had larger splenomegaly with increased numbers of B-cells in both spleen and peripheral blood. High-throughput sequencing of immunoglobulin variable segments revealed that clonal expansion was increased in immunosuppressed L.CD40 mice. Tumor B cells of immunosuppressed mice were larger with an immunoblastic aspect, both on blood smears and spleen tissue sections, with increased proliferation rate and increased numbers of activated B-cells. Collectively, these features suggest that immune suppression induced a shift from indolent lymphomas into aggressive ones. Thus, as a preclinical model, immunosuppressed L.CD40 mice reproduce aggressive transformation of an indolent B-cell tumor and highlight the role of the immune surveillance in its clinical course, opening new perspective for immune restoration therapies.Summary statementHighlighting the role of immune surveillance, transformation of indolent B-cell lymphoma into an aggressive malignancy is experimentally reproduced after T-cell immune suppression in the L.CD40 preclinical mouse model.

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The Role of DNA Repair in the Pathogenesis of Activation Induced Cytidine Deaminase Dependent B Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.397.397 ◽

2011 ◽

Vol 118 (21) ◽

pp. 397-397

Author(s):

Xiwen Gu ◽

Carmen J. Booth ◽

David G. Schatz ◽

Matthew P. Strout

Keyword(s):

Dna Repair ◽

B Cells ◽

B Cell ◽

Median Survival ◽

Cell Lymphoma ◽

Cytidine Deaminase ◽

B Cell Lymphoma ◽

Enzyme Activation ◽

Activation Induced Cytidine Deaminase

Abstract Abstract 397 Upon antigenic stimulation of B cells, germinal centers (GCs) are formed where somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes serve to diversify the immune response. SHM and CSR are initiated by the enzyme activation induced cytidine deaminase (AID) through the conversion of C/G base pairs to U-G mismatches. These mismatches are processed by UNG-dependent base excision repair (BER) and MSH2-dependent mismatch repair (MMR) pathways to yield mutations (for SHM) and DNA strand lesions (for CSR). Despite this essential role in immune diversification, the intrinsic activity of AID as a DNA mutator poses a threat to genomic integrity. Indeed, aberrant targeting of AID activity is associated with translocations and point mutations of proto-oncogenes associated with B cell malignancies. A specific dependence on AID in the pathogenesis of lymphomas of GC B cell origin is exemplified in Iμ-Bcl6 knock-in mice. These mice develop a diffuse large B cell lymphoma (DLBL) that resembles the human disease but are protected from development of this lymphoma when crossed onto an Aid-deficient background. To investigate the role of Aid-associated DNA repair in the pathogenesis of this disease, we crossed Iμ-Bcl6 mice onto a background deficient in BER (Ung−/−) and MMR (Msh2−/−). Young healthy Iμ-Bcl6 and Iμ-Bcl6 Ung−/−Msh2−/− mice displayed a normal number and distribution of B cells and normal architecture of lymphoid organs. Five of 28 Iμ-Bcl6 mice (17.9%) became sick starting at ∼12 months of age. Historically, median survival in these mice has not been reached and ∼80% survive to 15 months. In contrast, 21 of 28 Iμ-Bcl6 Ung−/−Msh2−/−mice (75%) developed disease with an onset of ∼3 months and had a median survival of 6.2 months (p<0.0001). All 5 of the Iμ-Bcl6 mice and the majority of Iμ-Bcl6 Ung−/−Msh2−/−mice developed B cell lymphoma with splenic involvement and variable nodal involvement. Five of the Iμ-Bcl6 Ung−/−Msh2−/−mice developed other cancers (3 T cell lymphomas, 1 pre-B cell lymphoma and 1 colon adenocarcinoma). Tumors from both genotypes expressed a mature B cell phenotype (B220+ IgM+ Igκ+ CD138-) and morphology revealed loss of normal lymphoid architecture with infiltration by lymphoid blasts. Additional staining demonstrated expression of at least one GC marker (Fas, GL7 and/or PNA). Similar to Iμ-Bcl6 mice, while many of the Iμ-Bcl6 Ung−/−Msh2−/−tumors had clonal mutated Ig heavy chain gene variable regions, two of the tumors were identified as oligoclonal, suggesting a preceding lymphoproliferative stage. In the absence of Ung and Msh2, Aid-generated U-G mismatches are not recognized and are simply replicated, causing only C/G to T/A transition mutations and no strand lesions. Thus, as expected, all Ig mutations in Iμ-Bcl6 Ung−/−Msh2−/−mice were C/G to T/A transitions. Lymphomas from Iμ-Bcl6 mice have been found to harbor numerous chromosome translocations and aneuploidies. Although additional analyses are underway, spectral karyotyping of 3 Iμ-Bcl6 Ung−/−Msh2−/−tumors revealed 2 with normal cytogenetics and 1 with a 40–41,XX,t(2;17),+15,+19. Surprisingly, sequence analysis of several known Aid target genes (cMyc, Pim1, RhoH, Pax5, Cd79a, Fas, H2ax and OcaB) in tumors from 3 Iμ-Bcl6 Ung−/−Msh2−/− mice did not identify any clonal mutations. However, non-clonal C/T to T/A transition mutations in cMyc were present at a frequency of 1.2 × 10−4, suggestive of ongoing Aid activity. The presence of Aid activity but absence of off-target Aid-mediated clonal SHM suggests that either other genes are targeted by Aid or that Aid has a secondary role in lymphomagenesis such as epigenetic reprogramming, as has been shown in iPS cells. Nonetheless, the incidence of Aid-dependent lymphomagenesis in the absence of Aid-associated DNA repair is significantly increased and the latency is greatly shortened. Altogether, this data suggests that Aid-associated BER and MMR pathways afford a protective effect against the development of Aid-dependent GC B cell lymphomas such as DLBL. To investigate the role of the individual Aid-associated DNA repair pathways, we have also generated Iμ-Bcl6 Ung−/− and Iμ-Bcl6 Msh2−/− single knockout mice. These studies are ongoing but preliminary results suggest that while the effect of Ung and Msh2 deficiency on lymphomagenesis may be synergistic, Msh2 might play a more critical role in preventing Aid-mediated genomic instability. Disclosures: No relevant conflicts of interest to declare.

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NFAT2 Is a Critical Regulator Of Anergy Induction In CLL

Blood ◽

10.1182/blood.v122.21.869.869 ◽

2013 ◽

Vol 122 (21) ◽

pp. 869-869

Author(s):

Melanie Märklin ◽

Jonas S. Heitmann ◽

B. Sc. ◽

David Worbs ◽

B. Sc. ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

Transgenic Mice ◽

B Cell ◽

Gene Expression Analysis ◽

Target Genes ◽

Flow Cytometric Analysis ◽

Knock Out ◽

Flow Cytometric

Abstract NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. Expression of CD38 and ZAP70 define a subgroup of patients with enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggessive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype refering to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL and in anergy induction in CLL cells. For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eµ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eµ enhancer. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 wks to which the animals eventually succumb at an average age of 10 months. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10). To identify novel NFAT2 target genes in CLL cells, we performed a comparative gene expression analysis on CLL cells with intact NFAT2 expression and on CLL cells with NFAT2 deletion using affymetrix microarrays. Mice with NFAT2 knock out exhibited a significantly more aggressive disease course with accelerated accumulation of CD5+CD19+ CLL cells and a significantly reduced life expectancy (200 vs. 325 days) as compared to control animals. Flow cytometric analysis at distinct time points showed a pronounced infiltration by CD5+ B cells of the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5+ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5+ B cells with concomitant expression of ZAP70 and CD38. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5+CD19+ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko exhibited significantly higher rates of proliferation than control animals. To identify NFAT2 target genes resonsible for the observed alterations in the disease phenotype, we subsequently peformed a gene expression analysis with CD5+CD19+ CLL cells from TCL1+NFAT2 ko mice with CLL cells from TCL1+ mice serving as controls. Here, we detected a significantly altered expression of 22 genes associated with B cell anergy in the TCL1+NFAT2 ko cohort. The vast majority of these genes was expressed significantly less in the absence of NFAT2 with Lck, Pacsin1, Hspa14 and CD166 constituting the strongest hits with up to 10fold reduced gene expression. Downregulation of the identified target genes was subsequently confirmed using RT-PCR and Western Blotting. In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells. In addition, we could show that NFAT2 controls the expression of several anergy-associated genes and that its absence prevents the acquisition of an anergic phenotype by the CLL cells correlating with a significantly more aggressive course of the disease. Taken together, our data demonstrate that NFAT2 plays an essential role in the pathogenesis of CLL and implicate this transcription factor as a potential target in its treatment. Disclosures: No relevant conflicts of interest to declare.

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Constitutive NF-κB and NFAT activation leads to stimulation of the BLyS survival pathway in aggressive B-cell lymphomas

Blood ◽

10.1182/blood-2005-10-4042 ◽

2006 ◽

Vol 107 (11) ◽

pp. 4540-4548 ◽

Cited By ~ 122

Author(s):

Lingchen Fu ◽

Yen-Chiu Lin-Lee ◽

Lan V. Pham ◽

Archito Tamayo ◽

Linda Yoshimura ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Cell Survival ◽

B Lymphocyte ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Non Hodgkin Lymphoma ◽

Survival Pathway ◽

Differentiation And Proliferation

AbstractB-lymphocyte stimulator (BLyS), a relatively recently recognized member of the tumor necrosis factor ligand family (TNF), is a potent cell-survival factor expressed in many hematopoietic cells. BLyS binds to 3 TNF-R receptors, TACI, BCMA, BAFF-R, to regulate B-cell survival, differentiation, and proliferation. The mechanisms involved in BLYS gene expression and regulation are still incompletely understood. In this study, we examined BLYS gene expression, function, and regulation in B-cell non-Hodgkin lymphoma (NHL-B) cells. Our studies indicate that BLyS is constitutively expressed in aggressive NHL-B cells, including large B-cell lymphoma (LBCL) and mantle cell lymphoma (MCL), playing an important role in the survival and proliferation of malignant B cells. We found that 2 important transcription factors, NF-κB and NFAT, are involved in regulating BLyS expression through at least one NF-κB and 2 NFAT binding sites in the BLYS promoter. We also provide evidence suggesting that the constitutive activation of NF-κB and BLyS in NHL-B cells forms a positive feedback loop associated with lymphoma cell survival and proliferation. Our findings indicate that constitutive NF-κB and NFAT activations are crucial transcriptional regulators of the BLyS survival pathway in malignant B cells that could be therapeutic targets in aggressive NHL-B.

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Targeting MYC-Driven B-Cell Lymphoma By Inhibition of the Histone Methyltransferase DOT1L

Blood ◽

10.1182/blood-2018-99-115475 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2839-2839 ◽

Cited By ~ 2

Author(s):

Anagha Deshpande ◽

Benson Chen ◽

Parham Ramezani-Rad ◽

Alessandro Pastore ◽

Luyi Zhao ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Cell Lines ◽

Target Genes ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Advisory Committees ◽

Genome Wide

Abstract Aberrant activation of the MYC proto-oncogene is a recurrent feature in human B-cell lymphomas of diverse sub-types, correlating with adverse prognosis and therapy resistance. Direct pharmacological MYC-targeting has proved difficult, but recent studies have shown that targeting chromatin regulators critical for MYC-driven oncogenesis may provide alternative avenues for therapeutic intervention. Recently, it has been demonstrated that MYC-driven oncogenesis in certain solid tumors is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L. We hypothesized that B-cell lymphomas with hyperactive MYC-signaling might be responsive to DOT1L inhibition. In order to test this hypothesis, we tested the effect of the DOT1L inhibitor Pinometostat (EPZ-5676) on a panel of human B-cell lymphoma cell lines featuring elevated MYC. Pinometostat treatment reduced global H3K79 methylation levels, accompanied by a time and dose-dependent decrease in proliferation of several Burkitt's lymphoma cell lines including P493-6, Daudi and Raji. We observed that key MYC-target genes including CDK4, PPAT and NPM1 were significantly downregulated upon Pinometostat treatment, suggesting that DOT1L is required for the transcriptional activation of MYC-target genes in these cells. Pinometostat-treated B-lymphoma cells showed a significant decrease of cells in S-phase compared to controls as assessed by BrdU-labeling assays. Similar results were also obtained in a panel of B-cell lymphoma cell lines with MYC-rearrangements including mantle cell lymphoma (MCL) cell lines Jeko-1, JVM2, Mino-1 and Maver-1 and the diffused large B-cell lymphoma (DLBCL) cell line Karpas 422. Next, we sought to investigate whether the DOT1L-dependence of MYC-driven B-cell lymphoma could be reproduced in a well-defined model of MYC-driven B-cell lymphoma. Towards this end, we utilized a mouse model in which expression of the Cre recombinase from a B cell specific promoter leads to ectopic expression of a transgenic human MYC allele and concomitant deletion of the tumor suppressor Pten in B cells. Similar to our in vitro studies, Pinometostat treatment led to a significant reduction in proliferation of B-cell lymphoma cells from these mice with an IC50 of 0.5 µM. Furthermore, we sought to ascertain whether these findings reflected on-target effects related to DOT1L inhibition. Therefore, we deleted DOT1L using CRISPR/Cas9 in B-cell lymphoma cell lines and assessed the effect on proliferation using competitive-proliferation assays. We observed that DOT1L-deletion progressively diminished the relative growth of anti-DOT1L sgRNA-expressing P493-6 and Jeko1 cells compared to non-targeted cells invitro. In order to test the requirement for DOT1L in lymphoma propagation in vivo, we performed intravenous injections of equal number of Jeko-1 cells with either anti-DOT1L or anti-Renilla control sgRNAs into sub-lethally irradiated non-obese diabetic/severe combined immunodeficiency mice (NOD/SCID) mice. Mice injected with control anti-Renilla sgRNAs succumbed to disease with a median latency of 34 days while the latency of disease in the anti-DOT1L sgRNA cohort was 45 days. In summary, DOT1L depletion significantly delayed disease latency in this invivo disseminated model of B-cell lymphoma (P=0.02). We then performed transcriptomic analyses of Pinometostat-treated B-cell lymphoma cell lines compared to DMSO-treated counterparts using RNA-seq. Gene-set enrichment analysis (GSEA) of RNA-seq data from three different B-cell lymphoma cell lines demonstrated that Pinometostat treatment significantly decreased the expression of MYC-target genes. In order to investigate the intriguing role of DOT1L in regulating MYC-target gene expression, we used ChIP-seq to assess the genome-wide occupancy of MYC following DOT1L inhibitor treatment. Strikingly, our studies demonstrated that DOT1L inhibition significantly reduced the chromatin occupancy of MYC. Taken together, our experiments demonstrate the role of DOT1L in MYC-driven B-cell lymphoma pathogenesis invitro and invivo. Furthermore, our genome-wide studies demonstrate the importance of DOT1L for genomic MYC occupancy. Based on these findings, we propose that therapeutic DOT1L targeting may be a viable strategy in MYC-driven B-cell lymphoma. Disclosures Weigert: Roche: Research Funding; Novartis: Research Funding. Rickert:Pfizer: Employment. Ren:Elli Lilly: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arima Genomics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deshpande:Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.

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The BCL6 Proto-Oncogene Suppresses p53 Expression in Germinal-Center B Cells.

Blood ◽

10.1182/blood.v104.11.212.212 ◽

2004 ◽

Vol 104 (11) ◽

pp. 212-212 ◽

Cited By ~ 1

Author(s):

Ryan T. Phan ◽

Huifeng Niu ◽

Masumichi Saito ◽

Katia Basso ◽

Giorgio Cattoretti ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Promoter Region ◽

Target Genes ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

P53 Expression ◽

Cycle Arrest ◽

Bcl6 Expression

Abstract The proto-oncogene BCL6 encodes a BTB/POZ-zinc finger transcriptional repressor that is necessary for germinal center (GC) formation and is implicated in the pathogenesis of B-cell lymphoma. In ~50% diffuse large cell lymphoma and 10% follicular lymphoma, BCL6 gene expression is deregulated by chromosomal translocations or mutations that affect its 5′ regulatory region. The precise function of BCL6 in GC development and lymphomagenesis is unclear since very few BCL6 direct target genes have been identified. We report that BCL6 suppresses p53-dependent and p53-indepenent growth arrest and apoptosis responses in GC B cells. BCL6 directly suppresses the transcription of the p53 gene, as demonstrated by (1) chromatin immunoprecipitation (ChIP) assays showing that BCL6 binds the p53 promoter region in vivo; and (2) transient transfection/reporter assays identifying within the p53 promoter region two BCL6-binding sites that mediate BCL6-mediated suppression of p53 transcription. Accordingly, suppression of BCL6 expression via specific siRNA leads to increased expression of p53 both under basal condition and in response to DNA damage. Consistent with a physiological role for BCL6-mediated p53 suppression, immunohistochemical analysis shows that p53 expression is absent in GC B cells where BCL6 is highly expressed. In addition, our data reveal that BCL6 inhibits the p53-independent activation of the p21/WAF1 cell cycle arrest gene by binding to Miz-1, a transcription factor involved in p21 activation. Consistent with a role of BCL6 in inhibiting p53-related cell cycle arrest and apoptotic responses, constitutive expression of BCL6 suppresses p53 expression and p53-target genes (P21 and PUMA) and protects B cell lines from apoptosis induced by DNA damage. These results indicate that one function of BCL6 is to allow GC B cells (centroblasts) to constitutively proliferate and to sustain the physiologic DNA breaks required for immunoglobulin switch recombination and somatic hypermutation without inducing p53-related responses. These findings also imply that B cell lymphoma with deregulated BCL6 expression are functionally p53-negative and impaired in apoptotic responses.

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Gene Expression Profiling Comparison between the Major Mature B-Cell Neoplasms and Their Normal Cellular and Anatomic Counterparts: Identification of Candidate Genes Potentially Involved in Lymphomagenesis.

Blood ◽

10.1182/blood.v108.11.2375.2375 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2375-2375

Author(s):

Nicolas Blin ◽

Celine Bossard ◽

Jean-Luc Harousseau ◽

Catherine Charbonnel ◽

Wilfried Gouraud ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

B Cells ◽

B Cell ◽

Gene Expression Profiling ◽

Germinal Center ◽

Expression Profiling ◽

Marginal Zone ◽

Cell Lymphoma ◽

B Cell Lymphoma

Abstract Gene expression profiling has provided new insights into the understanding of mature B cell neoplasms by relating each one to its normal counterpart, so that they can be to some extent classified according to the corresponding normal B-cell stage. Thus, diffuse large B cell (DLBCL) and follicular lymphoma (FL) have been related to a germinal center precursor whereas mantle cell lymphoma (MCL) or marginal zone lymphoma (MZL) are more likely to derive from naïve and memory B cell, respectively. However, little is still known about the physiopathology of B-cell lymphomas and particularly the deregulated pathways involved in their oncogenesis. To further investigate that point, we performed laser capture microdissection (LCM) of the three anatomic lymphoid compartments (i.e germinal center, mantle zone and marginal zone) taken from nine normal spleens and lymph nodes and magnetic cell separation of the four normal B cell subpopulations (i.e naïve B cells, centroblasts, centrocytes and memory B cells) purified from twelve normal tonsils for gene expression profiling by cDNA microarray. These molecular profiles have been compared to those of the four most frequent mature B cell neoplasms in adult (i.e DLBCL, FL, MZL and MCL), each one isolated from five previously untreated patients. Unsupervised analysis by hierarchical clustering of the normal anatomic and cellular populations could discriminate the germinal from the extra-germinal populations by genes involved in cell proliferation (e.g. E2F5, CCNB2, BUB1B and AURKB), DNA repair (e.g. PCNA and EXO1), cytokine secretion (e.g. IL8, IL10RB, IL4R and TGFBI) and apoptosis (e.g. CASP8, CASP10, BCL2 and FAS). Supervised analysis of the comparison between each B-cell lymphoma and its anatomic and cellular physiologic equivalent identified molecular deregulations concerning several genes’families characterizing the different histologic subtypes. Genes associated with cellular adhesion and ubiquitin cycle were significantly up-regulated in MCL (FCGBP, ITGAE, USP7, VCAM1) and MZL (CTGF, CDH1, ITGAE) whereas germinal center derived lymphomas (i.e. DLBCL and FL) mainly showed up-regulation of genes involved in cell proliferation (TNFRSF17, SEPT8) and immune response (FCER1G, XBP1, IL1RN). Few deregulated genes were common to the four subtypes, principally associated with cell proliferation (CYR61, GPNMB), cytosqueleton organization (EPB41L3) and carbohydrates metabolism (GNPDA1), suggesting potential similar oncogenic pathways. Those preliminary results are compatible with both subtype-specific and overall mechanisms of lympomagenesis and should be verified in a wider range of samples to confirm the oncogenic events involved in this heterogeneous disease.

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