Stavroula Ntoufa

Democritus University of Thrace, Komotina, East Macedonia and Thrace, Greece

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Publications (11)37.32 Total impact

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    ABSTRACT: We recently reported that chronic lymphocytic leukemia (CLL) subgroups with distinct clonotypic BCRs present discrete patterns of TLR expression, function, and/or tolerance. In this study, to explore whether specific types of BCR/TLR collaboration exist in CLL, we studied the effect of single versus concomitant BCR and/or TLR stimulation on CLL cells from mutated (M-CLL) and unmutated CLL (U-CLL) cases. We stimulated negatively isolated CLL cells by using anti-IgM, imiquimod, and CpG oligodeoxynucleotide for BCR, TLR7, and TLR9, respectively, alone or in combination for different time points. After in vitro culture in the absence of stimulation, differences in p-ERK were identified at any time point, with higher p-ERK levels in U-CLL versus M-CLL. Pronounced p-ERK induction was seen by single stimulation in U-CLL, whereas BCR/TLR synergism was required in M-CLL, in which the effect was overall limited in scale. An opposite pattern was observed regarding induction of apoptosis, as studied by Western blotting for the cleaved fragment of poly(ADP-ribose) polymerase, and the active isoform of caspase-8, with M-CLL responding even to single stimulation, contrasting with U-CLL that showed minimal response. Our findings suggest that concomitant engagement of BCR and TLR leads to differential responses in CLL depending on the mutational status of the BCR. Differential intensity and duration of responses in M-CLL versus U-CLL indicates that the differences in signal transduction between the two subgroups may be primarily quantitative rather than qualitative.
    The Journal of Immunology 04/2014; · 5.52 Impact Factor
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    ABSTRACT: The chromatin modifier EZH2 is overexpressed and associated with inferior outcome in mantle cell lymphoma (MCL). Recently, we demonstrated preferential DNA methylation of HOX genes in MCL compared with chronic lymphocytic leukemia (CLL), despite these genes not being expressed in either entity. Since EZH2 has been shown to regulate HOX gene expression, to gain further insight into its possible role in differential silencing of HOX genes in MCL vs. CLL, we performed detailed epigenetic characterization using representative cell lines and primary samples. We observed significant overexpression of EZH2 in MCL vs. CLL. Chromatin immune precipitation (ChIP) assays revealed that EZH2 catalyzed repressive H3 lysine 27 trimethylation (H3K27me3), which was sufficient to silence HOX genes in CLL, whereas in MCL H3K27me3 is accompanied by DNA methylation for a more stable repression. More importantly, hypermethylation of the HOX genes in MCL resulted from EZH2 overexpression and subsequent recruitment of the DNA methylation machinery onto HOX gene promoters. The importance of EZH2 upregulation in this process was further underscored by siRNA transfection and EZH2 inhibitor experiments. Altogether, these observations implicate EZH2 in the long-term silencing of HOX genes in MCL, and allude to its potential as a therapeutic target with clinical impact.
    Epigenetics: official journal of the DNA Methylation Society 10/2013; 8(12). · 4.58 Impact Factor
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    ABSTRACT: Critical processes of B-cell physiology, including immune signaling through the B-cell receptor (BcR) and/or Toll-like receptors (TLRs), are targeted by microRNAs. With this in mind and also given the important role of BcR and TLR signaling and microRNAs in chronic lympocytic leukemia (CLL), we investigated whether microRNAs could be implicated in shaping the behavior of CLL clones with distinct BcR and TLR molecular and functional profiles. To this end, we examined 79 CLL cases for the expression of 33 microRNAs, selected on the following criteria: (i) deregulated in CLL versus normal B-cells; (ii) differentially expressed in CLL subgroups with distinct clinicobiological features; and, (iii) if meeting (i)+(ii), having predicted targets in the immune signaling pathways. Significant up-regulation of miR-150, miR-29c, miR-143 and miR-223 and down-regulation of miR-15a was found in mutated versus unmutated CLL, with miR-15a showing the highest fold difference. Comparison of two major subsets with distinct stereotyped BcRs and signaling signatures, namely subset #1 (IGHV1/5/7-IGKV1(D)-39, unmutated, bad prognosis) versus subset #4 (IGHV4-34/IGKV2-30, mutated, good prognosis) revealed differences in the expression of miR-150, miR-29b, miR-29c and miR-101, all down-regulated in subset #1. We were also able to link these distinct microRNA profiles with cellular phenotypes, importantly showing that in subset #1 miR-101 down-regulation is associated with overexpression of the EZH2 protein, which has been associated with clinical aggressiveness in other B-cell lymphomas. In conclusion, specific miRNAs differentially expressed among CLL subgroups with distinct BcR and/or TLR signaling may modulate the biological and clinical behavior of the CLL clones.
    Molecular Medicine 04/2013; · 4.47 Impact Factor
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    ABSTRACT: Nowadays, chronic lymphocytic leukemia (CLL) is considered as a prototypic antigen-driven lymphoma, with antigenic stimuli from the microenvironment promoting tumor outgrowth. Antigen recognition is a function of both the clonotypic B cell receptor immunoglobulin (BcR IG) and various other immune sensors, e.g., the Toll-like receptors. The critical role of BcR IG-mediated signaling in CLL development and evolution is underscored by the following: the disease-biased IG gene repertoire; the subdivision of CLL based on the somatic hypermutation load of the BcR IG into two broad categories with vastly different prognosis and eventual outcome; the existence of subsets of cases with distinct, quasi-identical (stereotyped) BcR IGs; and the clinical efficacy of novel therapeutics inhibiting BcR signaling. Here, we trace the immunogenetic evidence for antigen selection in CLL and also consider the types of implicated antigens as well as the immune signaling pathways relevant for CLL ontogeny and clonal progression.
    Advances in experimental medicine and biology 01/2013; 792:1-24. · 1.83 Impact Factor
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    ABSTRACT: Gene pathway identification is an open and active research area that has attracted the interest not only of biomedical scientists but also of a large number of researchers from disciplines related to knowledge discovery from biological data. In this paper, we used Structural Equation Modeling (SEM) in order to statistically investigate the Toll-Like Receptor (TLR) signaling pathway in Chronic Lymphocytic Leukemia (CLL). Specifically, we used Path Analysis, a special case of SEM which is a statistical technique for testing and confirming causal relations based on data and qualitative assumptions. The dataset consists of Real Time PCR data for 84 genes relevant to the TLR signaling pathway, that were obtained from 192 patients with CLL that have been classified based on the mutational status of their clonotypic antigen receptors as mutated CLL (M-CLL) or unmutated CLL (U-CLL). The causal effects among genes were estimated through regression weights. In each case, the initially assumed model was based on the KEGG pathway database which provides reference pathways. The initial models were tested with respect to the M-CLL and U-CLL datasets. Modifications were made according to the statistical results (statistically significant regression weights, modification indices), concluding to models with good fit. Models were consistent to the reference pathway mostly for M-CLL and much less for U-CLL. These results go along with the well-described differences in immune signaling between the two subgroups, and may indicate that signaling in U-CLL is more impaired and/or modulated by complex regulatory networks that remain to be elucidated.
    Database and Expert Systems Applications (DEXA), 2013 24th International Workshop on; 01/2013
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    ABSTRACT: Chronic lymphocytic leukemia (CLL) can be divided into prognostic subgroups based on the IGHV gene mutational status, and is further characterized by multiple subsets of cases with quasi-identical or stereotyped B cell receptors that also share clinical and biological features. We recently reported differential DNA methylation profiles in IGHV-mutated and IGHV-unmutated CLL subgroups. For the first time, we here explore the global methylation profiles of stereotyped subsets with different prognosis, by applying high-resolution methylation arrays on CLL samples from three major stereotyped subsets: the poor-prognostic subsets #1 (n = 15) and #2 (n = 9) and the favorable-prognostic subset #4 (n = 15). Overall, the three subsets exhibited significantly different methylation profiles, which only partially overlapped with those observed in our previous study according to IGHV gene mutational status. Specifically, gene ontology analysis of the differentially methylated genes revealed a clear enrichment of genes involved in immune response, such as B cell activation (e.g., CD80, CD86 and IL10), with higher methylation levels in subset #1 than subsets #2 and #4. Accordingly, higher expression of the co-stimulatory molecules CD80 and CD86 was demonstrated in subset #4 vs. subset #1, pointing to a key role for these molecules in the crosstalk of CLL subset #4 cells with the microenvironment. In summary, investigation of three prototypic, stereotyped CLL subsets revealed distinct DNA methylation profiles for each subset, which suggests subset-biased patterns of transcriptional control and highlights a key role for epigenetics during leukemogenesis.
    Epigenetics: official journal of the DNA Methylation Society 11/2012; 7(12). · 4.58 Impact Factor
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    ABSTRACT: Subgroups of patients with chronic lymphocytic leukemia (CLL) have distinct expression profiles of Toll-like receptor (TLR) pathway-associated genes. To test the hypothesis that signaling through innate immunity receptors may influence the behavior of the malignant clone, we investigated the functional response triggered by the stimulaton of Toll-like receptors (TLRs) and NOD2 in 67 CLL cases assigned to different subgroups on the basis of IGHV gene usage, IGHV gene mutational status or B-cell receptor (BcR) stereotypy. Differences in the induction of co-stimulatory molecules and/or apoptosis were observed in mutated vs. unmutated CLL. Different responses were also identified in subsets with stereotyped BcRs, underscoring the idea that "subset-biased" innate immunity responses may occur independently of mutational status. Additionally, differential modulation of kinase activities was induced by TLR stimulation of different CLL subgroups, revealing a TLR7-tolerant state for cases belonging to stereotyped subset #4. The distinct patterns of TLR/NOD2 functional activity in cells from CLL subgroups defined by the molecular features of the clonotypic BcRs might prove relevant for elucidating the immune mechanisms underlying CLL natural history and for defining subgroups of patients who might benefit from treatment with specific TLR ligands.
    Molecular Medicine 03/2012; · 4.47 Impact Factor
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    ABSTRACT: Signaling through the B-cell receptor appears to be a major contributor to the pathogenesis of chronic lymphocytic leukemia. Toll-like receptors bridge the innate and adaptive immune responses by acting as co-stimulatory signals for B cells. The available data on the expression of Toll-like receptors in chronic lymphocytic leukemia are limited and derive from small series of patients. We profiled the expression of genes associated with Toll-like receptor signaling pathways in 192 cases of chronic lymphocytic leukemia and explored potential associations with molecular features of the clonotypic B-cell receptors. Chronic lymphocytic leukemia cells express all Toll-like receptors expressed by normal activated B cells, with high expression of TLR7 and CD180, intermediate expression of TLR1, TLR6, TLR10 and low expression of TLR2 and TLR9. The vast majority of adaptors, effectors and members of the NFKB, JNK/p38, NF/IL6 and IRF pathways are intermediately-to-highly expressed, while inhibitors of Toll-like receptor activity are generally low-to-undetectable, indicating that the Toll-like receptor-signaling framework is competent in chronic lymphocytic leukemia. Significant differences were identified for selected genes between cases carrying mutated or unmutated IGHV genes or assigned to different subsets with stereotyped B-cell receptors. The differentially expressed molecules include receptors, NFκB/MAPK signaling molecules and final targets of the cascade. The observed variations are suggestive of distinctive activation patterns of the Toll-like receptor signaling pathway in subgroups of cases of chronic lymphocytic leukemia defined by the molecular features of B-cell receptors. Additionally, they indicate that different or concomitant signals acting through receptors other than the B-cell receptor can affect the behavior of the malignant clone.
    Haematologica 07/2011; 96(11):1644-52. · 5.94 Impact Factor
  • Haematologica 01/2011; 96:1644-1652. · 5.94 Impact Factor
  • Antigens and Lymphomas Meeting; 01/2010
  • 19th Annual Meeting Of The Hellenic Society of Hematology; 01/2008