scholarly journals Methylome-based cell-of-origin modeling (Methyl-COOM) identifies aberrant expression of immune regulatory molecules in CLL

2020 ◽  
Author(s):  
Justyna A. Wierzbinska ◽  
Reka Toth ◽  
Naveed Ishaque ◽  
Karsten Rippe ◽  
Jan-Philipp Mallm ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Biology ◽  
Disease Onset ◽  
Lymphocytic Leukemia ◽  
B Cell Differentiation ◽  
Cell Of Origin ◽  
Aberrant Expression ◽  
Aberrant Transcript ◽  
Epigenetic Patterns

ABSTRACTBackgroundIn cancer, normal epigenetic patterns are disturbed and contribute to gene expression changes, disease onset and progression. The cancer epigenome is composed of the epigenetic patterns present in the tumor-initiating cell at the time of transformation, and the tumor-specific epigenetic alterations that are acquired during tumor initiation and progression. The precise dissection of these two components of the tumor epigenome will facilitate a better understanding of the biological mechanisms underlying malignant transformation. Chronic lymphocytic leukemia (CLL) originates from differentiating B cells, which undergo extensive epigenetic programming. This poses the challenge to precisely determine the epigenomic ground-state of the cell-of-origin in order to identify CLL-specific epigenetic aberrations.MethodsWe developed a linear regression model, methylome-based cell-of-origin modeling (Methyl-COOM), to map the cell-of-origin for individual CLL patients based on the continuum of epigenomic changes during normal B cell differentiation.ResultsMethyl-COOM accurately maps the cell-of-origin of CLL and identifies CLL-specific aberrant DNA methylation events that are not confounded by physiologic epigenetic B cell programming. Furthermore, Methyl-COOM unmasks abnormal action of transcription factors, altered super-enhancer activities, and aberrant transcript expression in CLL. Among the aberrantly regulated transcripts were many genes that have previously been implicated in T cell biology. Flow cytometry analysis of these markers confirmed their aberrant expression on malignant B cells at the protein level.ConclusionsMethyl-COOM analysis of CLL identified disease-specific aberrant gene regulation. The aberrantly expressed genes identified in this study might play a role in immune-evasion in CLL and might serve as novel targets for immunotherapy approaches. In summary, we propose a novel framework for in silico modeling of reference DNA methylomes and for the identification of cancer-specific epigenetic changes, a concept that can be broadly applied to other human malignancies.

The Opposing Role of Histone Deacetylase 10 (HDAC10) and HDAC11 in Proliferation/Survival of Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1363-1363 ◽  
Author(s):  
Eva Sahakian ◽  
Bijal D. Shah ◽  
John Powers ◽  
Susan Deng ◽  
Oscar Merino ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Cell Line ◽  
Cell Biology ◽  
Lymphocytic Leukemia ◽  
B Cell Malignancies ◽  
Aberrant Expression ◽  
Over Expression

Abstract Abstract 1363 The role of HDACs in cell biology, initially limited to their effects upon histones, encompasses now more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution and the stage of cellular differentiation. Not surprisingly, HDACs have been shown to play important roles in normal B-cell biology and, aberrant expression of these proteins has been found in some B-cell malignancies1. However, the role of specific HDACs in regulation of pro-survival and cell-cycling genes in MCL and CLL still remains poorly understood. We therefore evaluated by RT-PCR the mRNA expression of specific HDACs in MCL and CLL cell lines and in primary cells from patients with these B-cell malignancies. Our analysis revealed a unique and opposing expression of HDAC10 and HDAC11 in these malignant B-cells. While HDAC11 over-expression was frequently found in MCL and CLL cells, in particular in patients with aggressive disease, an almost complete abrogation of HDAC10 was observed in malignant B-cells as compared to normal B-cell controls. These findings led us to explore the biological consequences of manipulating HDAC11 and HDAC10 in MCL and CLL cells. First, knocking-down HDAC11 (HDAC11KD) using lentiviral shRNA resulted in downregulation of cyclin D1, Cdkn1a (p21) and bcl-2. Furthermore, HDAC11KD MCL or CLL cells displayed a slower cell proliferation relative to non-target shRNA control cells. Cell cycle analysis revealed that HDAC11KD clones are arrested in G1. Conversely, over-expression of HDAC11 in the MCL cell line Z138c or in the CLL cell line MEC1 resulted in enhanced cell survival and increased proliferative capacity. Mechanistically, we have recently found that HDAC11 over-expression is associated with increased phosphorylation of STAT3, a known survival pathway in malignant B-cells. Second, HDAC10 belongs to the class II HDAC family and its biological functions remain largely unknown. Similar to our results in aggressive MCL and CLL, a decreased HDAC10 expression has been reported in patients with aggressive solid tumors2, suggesting that loss of HDAC10 expression might confer a survival advantage to malignant cells. Indeed, over-expression of HDAC10 in Z138c and MEC1 cells resulted in a rapid induction of cell death in vitro with only 5% of cells being alive at 48 hours. Our results highlight the need for a better understanding of the expression/function of specific HDACs in MCL and CLL biology. The findings of opposing roles for HDAC11 and HDAC10 in influencing cell survival and proliferation might explain the limited efficacy of pan-HDAC inhibitors (with their indiscriminate inhibition of multiple HDACs) in these B-cell malignancies, and provide support for the development of isotype-selective inhibitors targeting HDAC11. Disclosures: Chen-Kiang: Pfizer, Inc.: Research Funding.

Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2973-2979 ◽  
Author(s):  
Anne J. Novak ◽  
Richard J. Bram ◽  
Neil E. Kay ◽  
Diane F. Jelinek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocyte ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Aberrant Expression ◽  
B Lymphocyte Stimulator

B-cell chronic lymphocytic leukemia (B-CLL) is defined by the accumulation of CD5+ B cells in the periphery and bone marrow. This disease is not characterized by highly proliferative cells but rather by the presence of leukemic cells with significant resistance to apoptosis and, therefore, prolonged survival. B-lymphocyte stimulator (BLyS) is a newly identified tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis and it shares significant homology with another TNF superfamily member, APRIL. The striking effects of BLyS on normal B-cell maintenance and survival raises the possibility that it may be involved in pathogenesis and maintenance of hematologic malignancies, including B-CLL. In this study, we investigated the status of APRIL and BLyS expression, as well as their receptors, in this disease. All B-CLL patient cells studied expressed one or more of 3 known receptors for BLyS; however, the pattern of expression was variable. In addition, we demonstrate for the first time that B-CLL cells from a subset of patients aberrantly express BLyS and APRIL mRNA, whereas these molecules were not detectable in normal B cells. Furthermore, we provide in vitro evidence that BLyS protects B-CLL cells from apoptosis and enhances cell survival. Because these molecules are key regulators of B-cell homeostasis and tumor progression, leukemic cell autocrine expression of BLyS and APRIL may be playing an important role in the pathogenesis of this disease.

The Target Genes and Prognostic Significance of Mir-150 in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3859-3859
Author(s):  
Marek Mraz ◽  
Laura Z. Rassenti ◽  
Emanuela M. Ghia ◽  
Liguang Chen ◽  
Jessie-Farah Fecteau ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Biology ◽  
Fold Change ◽  
Lymphocytic Leukemia ◽  
Inverse Association ◽  
Expression Of Genes ◽  
Bcr Signaling ◽  
Indolent Disease

Abstract Abstract 3859 Chronic lymphocytic leukemia (CLL) is the first disease in which miRNAs (hsa-miR-15a-16–1) were directly linked to cancer pathogenesis (Calin et al. PNAS, 2002). We and others have also shown that expression of certain miRNAs associates with disease activity in patients with CLL (Calin et al. NEJM, 2005; Mraz et al. Blood, 2012; Mraz et al. Leukemia, 2009). Moreover, patients with more aggressive disease have CLL cells that generally express unmutated IGHV and/or ZAP-70 and have a miRNA expression profile that differs from that of CLL cells from patients with indolent disease (Calin et al. NEJM, 2005). However, we still have very limited understanding of how miRNAs affect CLL cell-biology and expression of genes that play a critical role in either promoting or arresting the disease. We used pooled samples from 10 CLL patients to screen (TaqMan miRNA Cards-ABI, 750 miRNAs) for abundantly expressed miRNAs that could hypothetically influence CLL B cell biology. We identified miR-150 as the most abundant miRNA in CLL cells and also as being strongly expressed when compared to CD19+ blood lymphocytes of normal adults (N=5, P=0.008). This miRNA already has been reported to influence the differentiation and gene expression of normal B cells (Xiao et al. Cell, 2007) suggesting its possible relevance for CLL B cell biology. We examined additional CLL cell samples (N=168) and confirmed high miR-150 levels and also noted heterogeneity in its expression between CLL cells of patients with aggressive versus indolent disease. In our cohort, CLL cells of patients that expressed ZAP-70 (20% cut-off, N=74) or had unmutated IGHV (N=72) expressed significantly lower median-levels of miR-150 (fold change −1.7 and −2.0 respectively, p<0.005). Moreover, the lower levels of miR-150 also directly associated with higher response to stimulation of B-cell receptor (BCR) on CLL cells with anti-IgM (P<0.05, N=36, quantified by flow cytometric measurement of calcium mobilization). To understand the gene network regulated by miR-150 in CLL we performed array-based transcriptome analyses (HG-U133 Plus 2.0, Affymetrix) of 110 patient samples, which identified differential expression of 215 genes between CLL cells expressing low versus high levels of miR-150 (SAM analysis of upper and lower terciles). Thirty-eight of these 215 genes (17%) are predicted targets of miR-150 (determined by TargetScan, www.targetscan.org). Two well annotated genes (GAB1 and FOXP1) have evolutionary conserved binding sides for miR-150 in their 3‘UTRs, suggesting the possible importance of miR-150 in their regulation. GAB1 is an adaptor molecule and plays a key role in variety of cell signaling pathways (PLCγ, Ras/Erk, PI3K/Akt, CrkL). Interestingly, GAB1 modulates PI3K/Akt-pathway through binding domain identical to Bruton’s tyrosine kinase (Rameh et al. JBC, 1997) and is a key molecule involved in regulating BCR-signaling (Ingham et al. JBC, 1998, 2001), a process that factors prominently in the pathogenesis and progression of CLL. FOXP1 is an essential participant in the transcriptional regulatory network of B lymphopoiesis and has been identified as playing a role in disease progression of other B-cell lymphomas (Hu et al. Nat Immunol, 2006). The immunoblot analysis of GAB1 and FOXP1 in CLL cells confirmed their higher protein levels in cases with low miR-150 expression (P<0.005, fold change >10.0). Importantly, cells with higher expression of GAB1 or FOXP1 were more responsive to BCR stimulation in vitro (P<0.01, N=36) and higher expression of each associates with shorter overall survival (OS) (13.9 vs. 22.7 years, 13.9 vs. 21.1 years; N=168; P<0.05). Most notably, a reverse trend was observed for miR-150, where higher levels (>median) were associated with significantly longer OS (not-reached vs. 13.9 years, N=168, P=0.006). Additionally, the expression level of miR-150 was an independent predictor of OS and time to first treatment (TTFT) in multivariate analyses, which included IGHV status, ZAP-70, CD38, Rai stage, gender, and age (OS HR: 3.4 [CI 1.4–8.6], P=0.009; TTFT HR: 2.3 [CI 1.3–4.2], P=0.004). We conclude that there is an inverse association between high-risk disease and expression of miR-150, which may reflect its capacity to regulate the expression of genes encoding proteins that may contribute to BCR-signaling and/or survival of CLL B cells. Disclosures: No relevant conflicts of interest to declare.

Notch2 is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3742-3747 ◽  
Author(s):  
Rainer Hubmann ◽  
Josef D. Schwarzmeier ◽  
Medhat Shehata ◽  
Martin Hilgarth ◽  
Markus Duechler ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Core Promoter ◽  
Lymphocytic Leukemia ◽  
Mobility Shift ◽  
Aberrant Expression ◽  
Barr Virus ◽  
Epstein Barr ◽  
Cell Chronic Lymphocytic Leukemia

Members of the Notch family encode transmembrane receptors that modulate differentiation, proliferation, and apoptotic programs of many precursor cells, including hematopoietic progenitors. Stimulation of Notch causes cleavage followed by translocation of the intracellular domain (NotchIC) to the nucleus, where it activates transcription of CBF1 responsive genes. The aim of this study was to elucidate the mechanisms leading to the overexpression of CD23, a striking feature of B-cell chronic lymphocytic leukemia (B-CLL) cells. By electrophoretic mobility shift assays, we identified a transcription factor complex (C1) that binds sequence specific to one known and 4 newly identified putative CBF1 recognition sites in the CD23a core promoter region. With the use of Epstein-Barr virus (EBV)–infected B cells as a model for CBF1 mediated CD23a expression, C1 was found to be EBV inducible. Supershift assays revealed that the nuclear form of Notch2 is a component of C1 in B-CLL cells, supporting a model in which NotchIC activates transcription by binding to CBF1 tethered to DNA. Transient transfection of REH pre–B cells with an activated form of Notch2 induced endogenous CD23a, confirming thatCD23a is a target gene of Notch2 signaling. Finally, reverse transcription-polymerase chain reaction and kinetic analysis demonstrated that the Notch2 oncogene is not only overexpressed in B-CLL cells but might also be related to the failure of apoptosis characteristic for this disease. In conclusion, these data suggest that deregulation of Notch2 signaling is involved in the aberrant expression of CD23 in B-CLL.

scholarly journals VLA-4 Expression and Activation in B Cell Malignancies: Functional and Clinical Aspects

2020 ◽  
Vol 21 (6) ◽  
pp. 2206 ◽  
Author(s):  
Andrea Härzschel ◽  
Antonella Zucchetto ◽  
Valter Gattei ◽  
Tanja Nicole Hartmann
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Types ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Clinical Aspects ◽  
Neoplastic Disease ◽  
B Cell Differentiation ◽  
B Cell Malignancies ◽  
Bcr Signaling

Lineage commitment and differentiation of hematopoietic cells takes place in well-defined microenvironmental surroundings. Communication with other cell types is a vital prerequisite for the normal functions of the immune system, while disturbances in this communication support the development and progression of neoplastic disease. Integrins such as the integrin very late antigen-4 (VLA-4; CD49d/CD29) control the localization of healthy as well as malignant B cells within the tissue, and thus determine the patterns of organ infiltration. Malignant B cells retain some key characteristics of their normal counterparts, with B cell receptor (BCR) signaling and integrin-mediated adhesion being essential mediators of tumor cell homing, survival and proliferation. It is thus not surprising that targeting the BCR pathway using small molecule inhibitors has proved highly effective in the treatment of B cell malignancies. Attenuation of BCR-dependent lymphoma–microenvironment interactions was, in this regard, described as a main mechanism critically contributing to the efficacy of these agents. Here, we review the contribution of VLA-4 to normal B cell differentiation on the one hand, and to the pathophysiology of B cell malignancies on the other hand. We describe its impact as a prognostic marker, its interplay with BCR signaling and its predictive role for novel BCR-targeting therapies, in chronic lymphocytic leukemia and beyond.

scholarly journals Patterns of microRNA expression characterize stages of human B-cell differentiation

Blood ◽  
2009 ◽  
Vol 113 (19) ◽  
pp. 4586-4594 ◽  
Author(s):  
Jenny Zhang ◽  
Dereje D. Jima ◽  
Cassandra Jacobs ◽  
Randy Fischer ◽  
Eva Gottwein ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Microrna Expression ◽  
Lymphocytic Leukemia ◽  
Cell Phenotype ◽  
Tumor Type ◽  
B Cell Differentiation ◽  
B Cell Malignancies

Abstract Mature B-cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells constitute the majority of leukemias and lymphomas. These malignancies often maintain the characteristics of the normal B cells that they are derived from, a feature that is frequently used in their diagnosis. The role of microRNAs in mature B cells is largely unknown. Through concomitant microRNA and mRNA profiling, we demonstrate a potential regulatory role for microRNAs at every stage of the mature B-cell differentiation process. In addition, we have experimentally identified a direct role for the microRNA regulation of key transcription factors in B-cell differentiation: LMO2 and PRDM1 (Blimp1). We also profiled the microRNA of B-cell tumors derived from diffuse large B-cell lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia. We found that, in contrast to many other malignancies, common B-cell malignancies do not down-regulate microRNA expression. Although these tumors could be distinguished from each other with use of microRNA expression, each tumor type maintained the expression of the lineage-specific microRNAs. Expression of these lineage-specific microRNAs could correctly predict the lineage of B-cell malignancies in more than 95% of the cases. Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B cells.

scholarly journals Regulation of expression of a human intercellular adhesion molecule (ICAM-1) during lymphohematopoietic differentiation

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1896-1903
Author(s):  
AW Boyd ◽  
SM Dunn ◽  
JV Fecondo ◽  
JG Culvenor ◽  
U Duhrsen ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Adhesion Molecule ◽  
Intercellular Adhesion ◽  
Lymphocytic Leukemia ◽  
Intercellular Adhesion Molecule ◽  
Cell Contact ◽  
Small Subset ◽  
B Cell Differentiation ◽  
Immune Functions

The intercellular adhesion molecule (ICAM-1) is a cell-surface molecule which binds to leukocyte function antigen-1 (LFA-1) and regulates both leukocyte adhesion to endothelial cells and immune functions requiring cell-cell contact. Membrane expression of ICAM-1 is highly regulated on all hematopoietic lineages. Cell membrane antigen is significantly expressed on a small subset of bone marrow (BM) progenitors but is weak or absent on all cell lineages once they enter the circulation. However, strong expression on tissue macrophages and germinal center B cells suggested that activated cells may show upregulated expression. When B cells, T cells, macrophages, or granulocytes were activated in vitro by suitable mitogens, ICAM-1 expression was induced in all cases. Parallel studies of hematopoietic tumors demonstrated a heterogeneity of expression which correlated with expression on their normal cellular counterparts. In particular, a striking correlation between expression on B-cell tumors and corresponding stages of B-cell differentiation was noted. The widely varying expression of ICAM-1 contrasts with LFA-1 which, while variable, is nevertheless significantly positive at all stages of differentiation. This suggests that the major regulation of homotypic adhesion mediated by the LFA-1/ICAM-1 linkage occurs through control of ICAM-1 expression. In keeping with this notion, ICAM-1 expression was also correlated with the “adhesiveness” of B-lymphoid tumors. Large solitary lymphoma masses showed intense expression of ICAM-1. Conversely, chronic lymphocytic leukemia (CLL) cells and lymphoma cells from tumors exhibiting diffuse, widespread lymph node disease showed weak expression. These observations are discussed in relation to the role of ICAM-1 in regulation of lymphoid recirculation and the biology of lymphoid tumors.

Regulation and Function of NOTCH2 in B-CLL Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1138-1138
Author(s):  
Rainer Hubmann ◽  
Martin Hilgarth ◽  
Susanne Schnabl ◽  
Dita Demirtas ◽  
Josef D. Schwarzmeier ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Target Gene ◽  
Active Form ◽  
Lymphocytic Leukemia ◽  
B Cell Differentiation ◽  
Cell Fate Decisions ◽  
Mutational Status ◽  
And Function

Abstract B-cell chronic lymphocytic leukemia (B-CLL) represents a clonal expansion of self-reactive CD5+ B-lymphocytes which seems to be resistant to apoptosis in vivo. One of the characteristics of B-CLL lymphocytes is the high expression of the B-cell differentiation/activation marker CD23 which we recently identified as a target gene for NOTCH2 signaling. NOTCH2 is implicated in the development/homeostasis of murine CD5+ (B-1a) B-cells, suggesting a function for human NOTCH2 in B-CLL leukemogenesis. Here we show that peripheral B-CLL lymphocytes overexpress a transcriptionally active form of NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 expression, and cytogenetics). Although the majority of unstimulated B-CLL samples downregulate their N2IC activity in vitro, DNA-bound N2IC complexes could be maintained by the protein kinase C (PKC) activator TPA (12-O-tetradecanoylphorbol 13-acetate) accompanied by an upregulation of the NOTCH2 target gene CD23 and increased B-CLL cell viability. These effects are sensitive to the PKC-δ selective inhibitor Rottlerin. In 80% of B-CLL cases, NOTCH2 signaling was found to be resistant to the γ-secretase inhibitors (GSI’s) Dapt and compound E, indicative for the expression of truncated forms of NOTCH2 which do not require γ-secretase for processing and function. Inhibition of NOTCH2 either by Dapt in GSI sensitive B-CLL cases or, more specifically, by RNA interference downregulates CD23 expression on the mRNA and protein level and sensitizes B-CLL cells for apoptosis. Since self-reactive B-cells are normally eliminated either by chronic (anergy) or apoptotic (negative selection) B-cell receptor (BCR) signaling, we asked whether NOTCH2 modulates B-cell fate decisions triggered by the BCR. For this reason, we stably transduced the human B-cell line BL41 with constitutive active forms of NOTCH2 and found that NOTCH2 inhibits BCR mediated apoptosis induced by surface-IgM cross-linking. In summary, the data demonstrate that NOTCH2 signaling is deregulated in B-CLL cells and might be critically involved in the PKC-dependent maintenance of their malignant phenotype.

Differentiation-Stage-Specific Expression of MicroRNAs in B-Lymphocytes and Diffuse Large B-Cell Lymphomas (DLBCL)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 805-805 ◽  
Author(s):  
Raquel Malumbres ◽  
Robert Tibshirani ◽  
Elena Cubedo ◽  
Kristopher A Sarosiek ◽  
Xiaoyu Jiang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Memory B Cells ◽  
Microrna Expression ◽  
B Cell Differentiation ◽  
Cell Of Origin ◽  
Specific Expression ◽  
Differentiation Stage

Abstract B-cell development and differentiation are complex processes controlled by distinct programs of transcriptional control. A large set of transcriptional factors together or in succession control this process and their deregulation may result in block of differentiation or malignant transformation. MicroRNAs are small RNAs that orchestrate cellular functions by modulating the level of their targeted proteins by either translational arrest or transcript degradation, and play a key role in cell differentiation, apoptosis, proliferation and cancer development. An increasing number of transcription factors are being found targeted by microRNAs. Emerging evidence suggests that differentiation stage-specific expression of microRNAs occurs in the hematopoietic system and during T cell differentiation. Only limited information exists on microRNA expression in normal B cell differentiation and its malignant counterparts. Herein we analyzed microRNA expression profiles in distinct peripheral B cell differentiation stages-naïve, germinal center (GC) centroblasts and memory cells as well as tonsilar T cells. Furthermore, microRNA profiling was performed in germinal center-like (GCB-like) and activated B-cell-like (ABC-like) DLBCL cell lines originating from distinct B-cell differentiation stages. RNA, extracted with mirVana kit (AMBION) from B cell subsets and T cells enriched from normal tonsils was hybridized on LC Sciences (Houston, TX) microarrays harboring 470 human microRNAs probes (Sanger miRBase Release 9.1). Expression of selected microRNAs was confirmed by ABI RT-PCR methodology. Unsupervised clustering of microRNAs with values present in at least 50% of the samples (122 probes) resulted in perfect differentiation-stage clustering of samples. Application of Statistical Analysis of Microarrays (SAM) and Prediction Analysis of Microarrays (PAM) methods (FDR= 10%) identified a 47 microRNA cell of origin classifier for B-cells differentiation stage; 27 of these microRNAs were upregulated and 20 downregulated in centroblasts compared to memory B-cells. MicroRNAs belonging to paralog microRNA clusters (e.g. miR17-92-1, miR363-106a and miR25-106b) demonstrated similar patterns of expression in specific differentiation stages. To identify specific microRNA targets, miRanda, TargetScan and PicTar programs were used. To experimentally confirm the targets, we assessed the effects of specific microRNAs on the expression levels of targeted proteins and on the luciferase reporter under the control of the wild type and mutated 3′ UTR regions of putative target genes. Using this experimental approach we identified lymphocyte-stage-specific microRNAs which expression inversely correlated and might regulate the expression of LMO2, BLIMP1 and IRF4 proteins distinctively expressed at different differentiation stages of B lymphocytes. For example, miR223, which expression is low in GC cells but is high in naïve and memory B cells, downregulates the expression of LMO2. We next analyzed microRNA expression in DLBCL cell lines. Clustering analysis, using the 47 microRNA cell of origin classifier perfectly classified GCB-like and ABC-like cell lines. Interestingly, the expression of microRNAs in both GCB-like and ABC-like DLBCL cell lines was more similar to normal centroblasts than to memory B cells, suggesting that both may originate from distinct subpopulations of GC lymphocytes. The similarity of microRNA expression in cell lines to centroblasts was striking, with only 16 microRNAs (1 upregulated and 15 downregulated in cell lines) showing noticeable differences in levels of expression compared to normal cells. These microRNAs might be involved in the process of lymphoma transformation. SAM analysis aimed to differentiate GCB-like and ABC-like cell lines identified 11 microRNAs, only 3 of which were present in the cell of origin classifier. This observation suggests that there is also a difference in expression of microRNAs not directly related to the distinct cell of origin between the DLBCL subtypes. In summary, our results demonstrate that the microRNA profile changes during the GC reaction as well as during malignant transformation. Specific microRNAs can regulate key transcription factors controlling the processes of lymphocyte differentiation and transformation.

The Normal IGHV1-69-derived B Cell Repertoire Contains “Stereotypic” Patterns Characteristic of Unmutated CLL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4370-4370
Author(s):  
Francesco Forconi ◽  
Kathleen N Potter ◽  
Isla Wheatley ◽  
Nikos Darzentas ◽  
Elisa Sozzi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Subset ◽  
Lymphocytic Leukemia ◽  
Immunoglobulin Gene ◽  
Cell Of Origin ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Germinal Center B Cell

Abstract Abstract 4370 The cell of origin of chronic lymphocytic leukemia (CLL) has long been sought and immunoglobulin gene analysis provides new clues. The immunoglobulin heavy variable gene (IGHV) status has clinical relevance in CLL, where two subsets, delineated by the absence or presence of somatic mutation, have markedly different prognoses. The unmutated subset (U-CLL), of inferior prognosis, appears to derive from a pre-germinal center B cell. In U-CLL, there is strikingly increased usage of the 51p1-related alleles of the IGHV1-69 gene, often combined with selected IGHD genes and with IGHJ6. Shared sequence “stereotypic” characteristics of the HCDR3 result, and suggest antigen selection of the leukemic clones. In this study, we have analyzed 147 51p1/IGHJ6 rearrangements from 3 healthy individuals (>51yr) and sought sequence patterns parallel to those of U-CLL. A pre-established dataset of 313 51p1/IGHJ6 rearrangements from patients with U-CLL was used as a reference. A high proportion (49/147, 33.3%) of normal sequences revealed stereotypic patterns, several (22/147, 15%) being similar to those described in U-CLL. Additional CLL-associated stereotypes, not yet reported, were detected in 7/147 sequences (4.8%). Stereotypes (13.6%) not detected in CLL were also found in 20/147 (13.6%) 51p1/IGHJ6 combinations. The HCDR2-IGHJ6 sequences were almost exclusively unmutated (143/147, 97,3% sequences had ≥98% homology to germline). Junctional amino acids in normal B cells were heterogeneous, as in the cases of CLL with stereotyped 51p1/IGHJ6 B-cell receptors. Normal B cells expressing 51p1-derived IgM (4.8% of all B-cells) had a phenotype of naïve B-cells, similar to 51p1-negative (CD27-) B cells, i.e. IgM+ IgD+ CD23+ CD38+, with a small percentage of CD5+ B cells, not found in the memory B-cell subset. This snapshot of the naïve B-cell repertoire reveals subsets of B cells closely related to those characteristic of CLL. Conserved patterns in the 51p1-encoded IgM of normal B cells suggest a restricted sequence repertoire shaped by evolution to recognize common pathogens. Proliferative pressure on these cells is the likely route to U-CLL. Disclosures: No relevant conflicts of interest to declare.

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