Alexandra Schrader

Down-regulation of BLIMP1α by the EBV oncogene, LMP-1, disrupts the plasma cell differentiation program and prevents viral replication in B cells: implications for the pathogenesis of EBV-associated B-cell lymphomas

An important pathogenic event in Epstein-Barr virus (EBV)-associated lymphomas is the suppression of virus replication, which would otherwise lead to cell death. Because virus replication in B cells is intimately linked to their differentiation toward plasma cells, we asked whether the physiologic signals that drive normal B-cell differentiation are absent in EBV-transformed cells. We focused on BLIMP1α, a transcription factor that is required for plasma cell differentiation and that is inactivated in diffuse large B-cell lymphomas. We show that BLIMP1α expression is down-regulated after EBV infection of primary germinal center B cells and that the EBV oncogene, latent membrane protein-1 (LMP-1), is alone capable of inducing this down-regulation in these cells. Furthermore, the down-regulation of BLIMP1α by LMP-1 was accompanied by a partial disruption of the BLIMP1α transcriptional program, including the aberrant induction of MYC, the repression of which is required for terminal differentiation. Finally, we show that the ectopic expression of BLIMP1α in EBV-transformed cells can induce the viral lytic cycle. Our results suggest that LMP-1 expression in progenitor germinal center B cells could contribute to the pathogenesis of EBV-associated lymphomas by down-regulating BLIMP1α, in turn preventing plasma cell differentiation and induction of the viral lytic cycle.

High myc activity is an independent negative prognostic factor for diffuse large B cell lymphomas

Gene expression profiling has recently enabled the reclassification of aggressive non‐Hodgkin lymphomas (aNHL) into distinct subgroups. In Burkitt lymphoma (BL) aberrant c‐Myc activity results from IG‐MYC translocations. However, MYC aberrations are not limited to BLs and then have a negative prognostic impact. In this study, we investigated to which extent aberrant c‐Myc activity plays a functional role in other aNHL and whether it is independent from MYC translocations. Based on a combined microarray analysis of human germinal center (GC) B cells transfected with c‐Myc and 220 aNHLs cases, we developed a “c‐Myc index.” This index measures the extent to which lymphomas express c‐Myc responsive genes. It comprises genes that are affected in a variety of tumors compared to normal tissue. This supports the view that aberrant c‐Myc expression in GC B cells triggers a tumor‐like expression pattern. As expected, the “c‐Myc index” is very high in molecular Burkitt lymphoma (mBL), but more importantly also high within other aNHL. It constitutes a negative prognostic marker independent of established risk factors and of the presence of a MYC translocation. Our data provide new insights into the role of c‐Myc activity in different lymphomas and raises the question of treatment changes for those patients under risk.

Suppression of the LMP2A target gene, EGR‐1, protects Hodgkin's lymphoma cells from entry to the EBV lytic cycle

Hodgkins lymphoma is unusual among B cell lymphomas, in so far as the malignant Hodgkin/Reed–Sternberg (HRS) cells lack a functional B cell receptor (BCR), as well as many of the required downstream signalling components. In Epstein–Barr virus (EBV)‐positive cases of Hodgkins lymphoma, HRS cells express the viral latent membrane proteins (LMP)‐1 and ‐2A. LMP2A is thought to contribute to the pathogenesis of Hodgkins lymphoma by providing a surrogate BCR‐like survival signal. However, LMP2A has also been shown to induce the virus‐replicative cycle in B cells, an event presumably incompatible with lymphomagenesis. In an attempt to resolve this apparent paradox, we compared the transcriptional changes observed in primary HRS cells with those induced by LMP2A and by BCR activation in primary human germinal centre (GC) B cells, the presumed progenitors of HRS cells. We found a subset of genes that were up‐regulated by both LMP2A expression and BCR activation but which were down‐regulated in primary HRS cells. These genes included EGR1, an immediate‐early gene that is required for BCR‐induced entry to the virus‐replicative cycle. We present data supporting a model for the pathogenesis of EBV‐positive Hodgkins lymphoma in which LMP2A‐expressing HRS cells lacking BCR signalling functions cannot induce EGR1 and are consequently protected from entry to the virus lytic cycle. The primary microarray data are available from GEO (http://www.ncbi.nlm.nih.gov/geo/) under series Accession No 46143. Copyright

Global gene expression changes of in vitro stimulated human transformed germinal centre B cells as surrogate for oncogenic pathway activation in individual aggressive B cell lymphomas

BackgroundAggressive Non-Hodgkin lymphomas (NHL) are a group of lymphomas derived from germinal centre B cells which display a heterogeneous pattern of oncogenic pathway activation. We postulate that specific immune response associated signalling, affecting gene transcription networks, may be associated with the activation of different oncogenic pathways in aggressive Non-Hodgkin lymphomas (NHL).MethodologyThe B cell receptor (BCR), CD40, B-cell activating factor (BAFF)-receptors and Interleukin (IL) 21 receptor and Toll like receptor 4 (TLR4) were stimulated in human transformed germinal centre B cells by treatment with anti IgM F(ab)2-fragments, CD40L, BAFF, IL21 and LPS respectively. The changes in gene expression following the activation of Jak/STAT, NF-кB, MAPK, Ca2+ and PI3K signalling triggered by these stimuli was assessed using microarray analysis. The expression of top 100 genes which had a change in gene expression following stimulation was investigated in gene expression profiles of patients with Aggressive non-Hodgkin Lymphoma (NHL).Resultsα IgM stimulation led to the largest number of changes in gene expression, affecting overall 6596 genes. While CD40L stimulation changed the expression of 1194 genes and IL21 stimulation affected 902 genes, only 283 and 129 genes were modulated by lipopolysaccharide or BAFF receptor stimulation, respectively. Interestingly, genes associated with a Burkitt-like phenotype, such as MYC, BCL6 or LEF1, were affected by αIgM. Unique and shared gene expression was delineated. NHL-patients were sorted according to their similarity in the expression of TOP100 affected genes to stimulated transformed germinal centre B cells The αIgM gene module discriminated individual DLBCL in a similar manner to CD40L or IL21 gene modules. DLBCLs with low module activation often carry chromosomal MYC aberrations. DLBCLs with high module activation show strong expression of genes involved in cell-cell communication, immune responses or negative feedback loops. Using chemical inhibitors for selected kinases we show that mitogen activated protein kinase- and phosphoinositide 3 kinase-signalling are dominantly involved in regulating genes included in the αIgM gene module.ConclusionWe provide an in vitro model system to investigate pathway activation in lymphomas. We defined the extent to which different immune response associated pathways are responsible for differences in gene expression which distinguish individual DLBCL cases. Our results support the view that tonic or constitutively active MAPK/ERK pathways are an important part of oncogenic signalling in NHL. The experimental model can now be applied to study the therapeutic potential of deregulated oncogenic pathways and to develop individual treatment strategies for lymphoma patients.

Genomic data integration using guided clustering

MOTIVATION In biomedical research transcriptomic, proteomic or metabolomic profiles of patient samples are often combined with genomic profiles from experiments in cell lines or animal models. Integrating experimental data with patient data is still a challenging task due to the lack of tailored statistical tools. RESULTS Here we introduce guided clustering, a new data integration strategy that combines experimental and clinical high-throughput data. Guided clustering identifies sets of genes that stand out in experimental data while at the same time display coherent expression in clinical data. We report on two potential applications: The integration of clinical microarray data with (i) genome-wide chromatin immunoprecipitation assays and (ii) with cell perturbation assays. Unlike other analysis strategies, guided clustering does not analyze the two datasets sequentially but instead in a single joint analysis. In a simulation study and in several biological applications, guided clustering performs favorably when compared with sequential analysis approaches. AVAILABILITY Guided clustering is available as a R-package from http://compdiag.uni-regensburg.de/software/guidedClustering.shtml. Documented R code of all our analysis is included in the Supplementary Materials. All newly generated data are available at the GEO database (GSE29700). CONTACT rainer.spang@klinik.uni-regensburg.de SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Dissecting the molecular pathogenisis of Burkitt lymphoma

Burkitt lymphoma (BL) is a high grade B cell malignancy (Non-Hodgkin Lymphoma (NHL)) derived from germinal center B cells (GCB) that harbours a chromosomal translocation juxtaposing the protooncogene MYC next to the regulatory elements of one of the immunoglobulin loci. However, the precise contribution of Myc to the pathogenesis of this tumour is poorly understood. Based on the definition of a distinguishing gene expression signature for the molecular BL (mBL) with Myc as one hallmarking signature gene we are interested in getting answers to the questions (i) what are the target genes of Myc in primary human GCB cells and (ii) what is the functional significance of signature genes identified? We describe a non-viral vector based approach (Vockerodt et al. 2008) to express Myc in primary human GCB cells. Comparative gene expression profiling was performed accompanied by qRT-PCR. In addition elucidation of the function of selected signature genes in BL is accomplished. In a representative cell line with a mBL signature RNAi directed inhibition of elements of the CD40 signaling cascade was conducted. After activating this particular signaling cascade in a BL cell line we analysed respective gene expression profiles of IKKa, IKKb, TRAF2, TRAF6, Jak3, BCL-3 and p38 deficient cells. Based on these different RNAi-mediated GE-profiles we reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (Markowetz et al. 2005).

AKT-pathway inhibition in chronic lymphocytic leukemia reveals response relationships defined by TCL1.

Cell survival in chronic lymphocytic leukemia (CLL) largely depends on B-cell receptor-induced AKT activation. Gain-of-function genomic lesions of PI3K-AKT-mTOR pathway components are usually absent in CLL. We previously established that a BCR-mediated growth response in CLL is determined by the oncogene T-cell leukemia 1 (TCL1) through a sensitizer effect on AKT phospho-activation. Despite high clinical response rates following AKT-cascade inhibition in CLL, resistances in a substantial proportion of patients call for reliable pre- and post-exposure strata to better predict compound responses. Using a panel of inhibitors with differential vertical affinities in the PI3K-AKT-mTOR axis, we describe distinct patterns and determinants of sensitivities in 75 CLL samples. The compounds specifically impacted the BCR-induced physical TCL1-AKT interaction. In general, there was an efficient and tumor-selective abrogation of cell survival in suspension or protective stromal-cell cultures. However, biochemical and survival responses were heterogeneous across CLL and showed only incomplete overlap across inhibitors. Sensitivity clusters could be defined by differential responses to selective pan-PI3K inhibition vs. compounds acting more down-stream. An elevated PI3K/AKT/mTOR activation state conferred sensitivity or resistance, depending on the applied inhibitor. In fact, down-stream interception by mTOR or dual mTOR/PI3K inhibition appears more efficient in cases expressing the BCR-response and poor-risk determinants of ZAP70 or TCL1. Finally, exploiting the TCL1-AKT interaction, peptide-based TCL1-interphase mimics were potent in steric AKT antagonization and in reducing CLL cell survival. Overall, this study provides informative response relationships in AKT-pathway interception that can help refining predictive models in BCR-pathway inhibition in CLL.

Abstract 370: T-PLL cells resemble memory-type T-cells with aberrant effector functions implicating a leukemogenic cooperation of TCL1A and TCR signaling

The pathogenesis of the rare and aggressive T-cell prolymphocytic leukemia (T-PLL) is poorly understood, which particularly applies to a mechanistic concept around its hallmark oncogene TCL1A. Existing data implicate TCL1A as a catalytic enhancer of the oncogenic kinase AKT, a central node in a T-cell’s antigen receptor (TCR) signaling cascade, which mediates proliferation and differentiation. The levels and role of TCR activation in T-PLL’s pathogenesis are not known. To first clarify which physiological T-cell subset T-PLL cells most resemble, we performed comprehensive global gene expression profiling and immunophenotyping of primary T-PLL (n=79) in comparison to healthy-donor derived T-cell populations. Principle component analyses and gene signature alignments revealed a high similarity of T-PLL cells to (central) memory T-lymphocytes over naive T-cells. Surface markers revealed a spectrum of memory-type differentiation (n=69/79; 87%) with predominant central-memory stages (n=35/79; 44%). The usually TCR and/or CD28-coreceptor positive T-PLL cells revealed no restrictions to genetic or surface TCR-clonotypes. The abnormally high basal activation levels (surface CD25, CD38, CD69) correlated in their degree with inferior clinical outcomes (med. survival 20.8 vs 58.3 mo.; p=0.0012). In parallel, T-PLL cells lost expression of negative-regulatory TCR-co-receptors (e.g. CTLA-4, LAG3). Fittingly, TCR engagement of primary T-PLL cells revealed a trend to hyperactive intracellular responses and interleukin(IL)-2 release alongside a prominent Th1-cytokine program. T-PLL cells also showed a robust resistance to stimulation-induced cell death and agonistic CD95 ligation. TCR-derived signals (phospho-kinase induction, IL-2 release) were enhanced in vitro by the modulated presence of TCL1A with kinetics indicative of a sensitizer relationship, mainly in the CD3 axis as opposed to the CD28 branch. A mouse model with TCL1A-initiated protracted development of T-PLL (Lckpr-TCL1Atg) revealed congruent findings with the aberrant T-cell phenotype of human T-PLL. TCL1A expressing T-cells of this model, that were further equipped with monoclonal epitope-defined TCRs against ovalbumine or a chimeric-antigen-receptor (CAR) against carcinoembryonic antigen, gained a pre-leukemic growth advantage in scenarios of pulsed or continuous low-level receptor stimulation. Overall, we establish that T-PLL cells resemble antigen-experienced memory T-cells. Retention of functional effector responses to TCR stimulation and loss of restricting activation regulators underlie a highly activated phenotype and a marked resistance to death-inducing signals. TCL1A proactively enhances TCR responses and we postulate that this leukemogenic cooperation drives accumulation of memory-type cells that utilize amplified, hence permissive, low-level cognate antigen input. Note: This abstract was not presented at the meeting. Citation Format: Alexandra Schrader, Kathrin Warner, Sebastian Oberbeck, Giuliano Crispatzu, Petra Mayer, Sabine Putzer, Hans Diebner, Stephan Stilgenbauer, Georg Hopfinger, Jan Durig, Torsten Haferlach, Mark Lanasa, Ingo Roeder, Michael Hallek, Dimitrios Mougiakakos, Michael von Bergwelt-Baildon, Monika Bruggemann, Sebastian Newrzela, Hinrich Abken, Marco Herling. T-PLL cells resemble memory-type T-cells with aberrant effector functions implicating a leukemogenic cooperation of TCL1A and TCR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 370. doi:10.1158/1538-7445.AM2017-370

Identification of a new gene regulatory circuit involving B cell receptor activated signaling using a combined analysis of experimental, clinical and global gene expression data

To discover new regulatory pathways in B lymphoma cells, we performed a combined analysis of experimental, clinical and global gene expression data. We identified a specific cluster of genes that was coherently expressed in primary lymphoma samples and suppressed by activation of the B cell receptor (BCR) through αIgM treatment of lymphoma cells in vitro. This gene cluster, which we called BCR.1, includes numerous cell cycle regulators. A reduced expression of BCR.1 genes after BCR activation was observed in different cell lines and also in CD10+ germinal center B cells. We found that BCR activation led to a delayed entry to and progression of mitosis and defects in metaphase. Cytogenetic changes were detected upon long-term αIgM treatment. Furthermore, an inverse correlation of BCR.1 genes with c-Myc co-regulated genes in distinct groups of lymphoma patients was observed. Finally, we showed that the BCR.1 index discriminates activated B cell-like and germinal centre B cell-like diffuse large B cell lymphoma supporting the functional relevance of this new regulatory circuit and the power of guided clustering for biomarker discovery.