Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific ...events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program.
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IJS, NUK, SBMB, UL, UM, UPUK
Significance Human IgM ⁺IgD ⁺CD27 ⁺ B lymphocytes represent a large subpopulation of the human B-cell pool, but their generation is debated and their immunological functions are poorly understood. ...This work shows that these lymphocytes possess typical memory B-cell expression patterns, enabling them to differentiate rapidly into plasma cells upon restimulation. Moreover, we reveal unique features of these IgM memory B cells, their potential to reenter germinal center reactions, and their specific interaction with immunomodulatory neutrophils in early inflammatory responses. Thus, key characteristics and functions of a major human B-cell subset are elucidated.
The generation and functions of human peripheral blood (PB) IgM ⁺IgD ⁺CD27 ⁺ B lymphocytes with somatically mutated IgV genes are controversially discussed. We determined their differential gene expression to naive B cells and to IgM-only and IgG ⁺ memory B cells. This analysis revealed a high similarity of IgM ⁺(IgD ⁺)CD27 ⁺ and IgG ⁺ memory B cells but also pointed at distinct functional capacities of both subsets. In vitro analyses revealed a tendency of activated IgM ⁺IgD ⁺CD27 ⁺ B cells to migrate to B-cell follicles and undergo germinal center (GC) B-cell differentiation, whereas activated IgG ⁺ memory B cells preferentially showed a plasma cell (PC) fate. This observation was supported by reverse regulation of B-cell lymphoma 6 and PR domain containing 1 and differential BTB and CNC homology 1, basic leucine zipper transcription factor 2 expression. Moreover, IgM ⁺IgD ⁺CD27 ⁺ B lymphocytes preferentially responded to neutrophil-derived cytokines. Costimulation with catecholamines, carcinoembryonic antigen cell adhesion molecule 8 (CEACAM8), and IFN-γ caused differentiation of IgM ⁺IgD ⁺CD27 ⁺ B cells into PCs, induced class switching to IgG ₂, and was reproducible in cocultures with neutrophils. In conclusion, this study substantiates memory B-cell characteristics of human IgM ⁺IgD ⁺CD27 ⁺ B cells in that they share typical memory B-cell transcription patterns with IgG ⁺ post-GC B cells and show a faster and more vigorous restimulation potential, a hallmark of immune memory. Moreover, this work reveals a functional plasticity of human IgM memory B cells by showing their propensity to undergo secondary GC reactions upon reactivation, but also by their special role in early inflammation via interaction with immunomodulatory neutrophils.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Our knowledge about the clonal composition and intraclonal diversity of the human memory B-cell compartment and the relationship between memory B-cell subsets is still limited, although these are ...central issues for our understanding of adaptive immunity. We performed a deep sequencing analysis of rearranged immunoglobulin (Ig) heavy chain genes from biological replicates, covering more than 100,000 memory B lymphocytes from two healthy adults. We reveal a highly similar B-cell receptor repertoire among the four main human IgM⁺ and IgG⁺ memory B-cell subsets. Strikingly, in both donors, 45% of sequences could be assigned to expanded clones, demonstrating that the human memory B-cell compartment is characterized by many, often very large, B-cell clones. Twenty percent of the clones consisted of class switched and IgM⁺(IgD⁺) members, a feature that correlated significantly with clone size. Hence, we provide strong evidence that the vast majority of Ig mutated B cells—including IgM⁺IgD⁺CD27⁺ B cells—are post-germinal center (GC) memory B cells. Clone members showed high intraclonal sequence diversity and high intraclonal versatility in Ig class and IgG subclass composition, with particular patterns of memory B-cell clone generation in GC reactions. In conclusion, GC produce amazingly large, complex, and diverse memory B-cell clones, equipping the human immune system with a versatile and highly diverse compartment of IgM⁺(IgD⁺) and class-switched memory B cells.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The pathogenesis of T‐cell large granular lymphocytic leukemia (T‐LGL) is poorly understood, as STAT3 mutations are the only known frequent genetic lesions. Here, we identified non‐synonymous ...alterations in the TNFAIP3 tumor suppressor gene in 3 of 39 T‐LGL. In two cases these were somatic mutations, in one case the somatic origin was likely. A further case harbored a SNP that is a known risk allele for autoimmune diseases and B cell lymphomas. Thus, TNFAIP3 mutations represent recurrent genetic lesions in T‐LGL that affect about 8% of cases, likely contributing to deregulated NF‐κB activity in this leukemia.
What's New?
T‐cell large granular lymphocytic leukemia (T‐LGL) is often associated with mutations in the STAT3 gene, but associations with other genetic mutations remain unknown. Here the authors identified non‐synonymous mutations in the gene encoding TNF‐alpha‐induced protein 3 (TNFAIP3), a negative regulator of NF‐kappa B signaling, in three patients with T‐LGL. The study underscores the important role of NF‐kappa B activity in this otherwise poorly understood lymphoid malignancy.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Chemokine receptors are G-protein-coupled cell surface receptors, which dissociate upon activation by their ligands and cause downstream signaling. Several studies have revealed the crucial ...contribution of chemokine receptors and their ligands in normal B-cell differentiation and development of hematopoietic malignancies. The Richter syndrome (RS) represents the clinico-pathologic transformation of chronic lymphocytic leukaemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). Due to the lack of knowledge on the chemokine receptor, we aimed to investigate their expression profile in patients with CLL and Richter syndrome. Therefore, we investigated the mRNA expression levels of 18 known chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, XCR1, CX3CR1) by using semi-quantitative real-time PCR on seven samples of paired (CLL and transformed DLBCL) RS samples, additionally four CLL samples -all of them subsequently transformed into DLBCL-, and eight transformed DLBCL samples originating from CLL. Additionally, 30 samples of de-novo DLBCL, including 10 germinal center B-cell (GCB) lymphomas, 12 non-germinal center B-cell lymphomas (non-GCB), and 8 unclassified DLBCL were included. Four samples of naïve B-cells (CD5 neg), CD5+ naïve B-cells and CD27+ memory B-cells (n=12) served as non-neoplastic controls.
No differences in the chemokine receptor profile were detected between CD5+ and negative naïve B-cells. When comparing CD27+ memory B-cells to naïve B-cells a significant lower expression level was found for CCR7 (7-fold), CXCR4 (4-fold), and CXCR5 (1.5 fold). CCR7 (5-fold) and CXCR4 (5-fold) were also lower expressed in CD27+ memory B-cells compared to CD5+ naïve B-cells.
Five out of 18 chemokine receptors were differentially expressed comparing the distinct normal B-cell subsets with RS samples. Comparing CLL samples and RS samples to CD5+ naïve B-cells, CXCR4 (12-fold for CLLs and 10-fold for RS samples) and CXCR5 (2-fold for CLLs and 2.4-fold for RS samples) were lower expressed, whereas CXCR3 (10-fold for CLLs and 8.5-fold for the transformed samples) was higher expressed and CCR5 de-novo expressed. Compared to naïve B-cells, the same chemokine receptors were deregulated: CXCR4 (10-fold for CLLs and 8.5-fold for the RS samples) and CXCR5 (2-fold for CLLs and 2.4-fold for the transformed samples) were lower expressed, CXCR3 (45-fold for CLLs and 30-fold for the transformed samples) was higher expressed and CCR5 was de-novo expressed. Comparing CLL samples and transformed RS samples to CD27+ memory B-cells, CCR5 (5.1-fold for CLLs and 4.3-fold for the RS samples) and CCR7 (8.7-fold for CLLs and 10-fold for the transformed samples) were higher expressed in both malignancies.
Only one chemokine receptor was found to be differentially expressed in our seven paired RS samples: CCR6 showed a trend of a higher expression (1.4-fold) in CLL components.
Considering RS and GCB DLBCL, CCR1, CCR5, and CXCR6 were found to be significantly down-regulated in RS (at least 4-fold), in contrast to CCR7 and CXCR4, which showed higher expression levels in RS (6-fold). CCR1 and CCR5 were lower expressed comparing RS and non-GCB DLBCL (25-fold and 8-fold), whereas CCR7 again, together with CXCR7, was higher expressed (3- fold and 6-fold respectively).
Our data indicate a difference in the chemokine receptor profile within normal B-cell subsets. These differences are also reflected in the different expression profile of low and high aggressive component of CLL/RS compared to the distinct B cell subtypes. Hence, in future these multiple deregulated CC and CXC receptors might serve as a further hint in identifying the cell of origin of different B-cell malignancies.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Knowledge of the cell-of-origin is essential for the full understanding of the causes of a malignant disease and for the rational design of targeted therapies. The B cell compartment is ...composed of a highly complex mixture of subtypes, each with distinct phenotypes and roles within the immune system. In chronic lymphocytic leukemia (CLL), heterogeneity in the biology and clinical course of the disease is thought to be linked to divergent cellular origins. We and others have previously shown that the epigenome of CLL, as measured by the global pattering of DNA methylation, is highly clonal and remarkably stable over time and thus represents a powerful approach to trace founder subtype populations. Here we combine epigenomic and transcriptomic analysis using next-generation sequencing approaches to compare CLL cells to highly purified and specific B cell subpopulations at various stages of maturation. We find that B cell maturation involves substantial unidirectional epigenetic programming that occurs as a continuum throughout the transition between naïve to fully-mature memory B cell subpopulations. Combining 258 CLL cases using Illumina 450K analysis reveals that all CLLs arise from a discrete window within the spectrum of B cell maturation that is more similar to mature B cells, with the majority of cases clustering at two distinct points correlating with unmutated IGHV versus highly mutated (<95% homology) IGHV genes; however, a significant number (~20%) of cases arise at various points between these two clusters. Next we show that using RNA-seq, broad differences in global expression patterns mirror the degree of epigenetic programming achieved by individual CLLs. Progressively further programming is paralleled by a transition from an aggressive to indolent expression pattern, indicated by the decrease in the expression levels of genes with known roles in promoting CLL cell survival, such as ZAP70, BTK, TCL1a, MCL1, miR-155 and others. Using DNA methylation and ChIP-seq data to compare the sequence and chromatin features of genomic regions that are programmed in normal B cell maturation versus CLL, reveals that although a myriad of transcription factors and pathways connected to immune cell function are involved in normal epigenetic programming in B cells, aberrant CLL-specific alterations involve excess activity of NFAT and EGR gene families and, paradoxically, a reduction of AP-1 activity. To further investigate the role of immediate-early genes, RNA-seq analysis of in vitro-activated CLL cells revealed a strong association between the degree of epigenetic programming and the specific inducibility of EGR2 and c-FOS, supporting a functional role of these genes in aberrant DNA methylation programming. Finally, in an independent clinically well-annotated cohort of 349 CLL cases, we demonstrate that the degree of epigenetic programming is significantly associated with time to treatment and overall survival in patients. Collectively, this work demonstrates that instead of a distinct cell(s)-of-origin, CLL is rather derived from a continuum of possible programming states, and that the degree of programming achieved by a particular CLL at the time of transformation dictates its global gene expression pattern and clinical outcome. Furthermore, a parallel assessment of B cell maturation with CLL development permits a refinement of the disease-specific, early molecular events, highlighting the dysregulation of particular transcription factors and pathways in the pathogenesis of aggressive versus indolent disease.
Citation Format: Christopher C. Oakes, Marc Seifert, Assenov Yassen, Lei Gu, Martina Przekopowitz, Amy Ruppert, Andrius Serva, Sandra Koser, David Brocks, Daniel Lipka, Olga Bogatyrova, Daniel Mertens, Marc Zapatka, Peter Lichter, Hartmut Doehner, Ralf Kueppers, Thorsten Zenz, Stephan Stilgenbauer, John Byrd, Christoph Plass. Progressive epigenetic programming during B cell maturation yields a continuum of clonal disease phenotypes with distinct etiologies in chronic lymphocytic leukemia. abstract. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B25.
The malignant phenotype combines characteristics that are acquired and inherited from the normal cell of origin. Hematological malignancies and related disease subtypes are thought to arise from ...diverse cell types that may reflect various developmental stages within the hematopoetic lineage. The contribution of different normal cell states and processes to the biological and clinical features of malignancy is not well understood. In chronic lymphocytic leukemia (CLL), two or three subtypes have been identified by variation in the degree of somatic IGHV mutations and recently uncovered epigenetic differences, respectively, suggesting that these subtypes derive from distinct normal B cell subsets at different stages of maturity. However, in CLL, as well as in most malignancies, the full possible extent of maturity states and the relative contribution of normal versus malignant developmental programs to the malignant phenotype have not been defined in a high-resolution manner.
It is widely accepted that epigenetic patterns are important to establish and stabilize cellular phenotypes. Using whole genome bisulfite sequencing and sequence-specific methods, we assessed the dynamic DNA methylation events that occur during the maturation of B cells using six highly purified B cell subsets representing various stages of maturation. We confirmed previous reports that broad epigenetic programming affects about 25% of the genome from naïve to memory B cells, and further revealed that B cell subpopulations of intermediate maturity retained increasing degrees of the maturation program resulting in a singular developmental trajectory. Maturation was driven in part by the activity of a specific set of transcription factors (e.g. AP-1, EBF1, RUNX3, OCT2, IRF4 and NFkB). Using the developmental epigenetic signature defined by transcription factor binding site (TFBS) programming in normal cells to compare to tumor cells of 268 CLL revealed that tumors have the potential to derive from a continuum of possible maturation states that are reflected in the maturation stages of normal cells. Using RNA sequencing to measure gene expression, we found the degree of maturation achieved in tumors closely associates with the acquisition of a more indolent pattern of gene expression, evidenced by progressive downregulation of CLL oncogenes, such as ZAP70, TCL1 and BTK. Further assessment of the level of DNA methylation maturity in an independent sample cohort of 348 CLL cases revealed a quantitative, continuous relationship with increasingly favorable clinical outcomes.
Although the majority of methylation differences found between tumor subtypes are naturally present in normal B cells, by identifying changes that are only present in CLL we further uncovered a previously unappreciated pathogenic role of transcription factor dysregulation. Specifically, a blockade in the epigenetic maturation of EBF and AP-1 TFBSs was found to define low-programmed (less mature, poor outcome) CLL cases and was associated with transcriptional and genetic loss of EBF1 and FOS transcription factors in tumor cells. Aberrantly acquired DNA methylation events in CLL were linked to excess activity of specific transcription factor families, namely EGR and NFAT. Intriguingly, we show that recurrent somatic mutations within the DNA binding domain of EGR2 selectively influence the methylation status of its cognate binding sites in mutant cases, establishing a role for this transcription factor in epigenetic dysregulation in CLL.
Collectively, this work reveals that a unique epigenetic maturation signature, directed by normal developmental processes, defines individual CLL cases resulting in a spectrum of maturity across tumors. The majority of DNA methylation differences observed between individual CLLs reflects the state of maturity of the founder cell and profoundly influences the disease phenotype. We further propose that in CLL the disease-specific state results, in part, by dysregulation of key transcription factors that imbalance the normal B cell epigenetic program.
Kipps:Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; AbbVie: Consultancy, Research Funding. Stilgenbauer:AbbVie: Consultancy, Other: travel grants, Research Funding; Amgen: Consultancy, Other: travel grants, Research Funding; Boehringer-Ingelheim: Consultancy, Other: travel grants, Research Funding; Celgene: Consultancy, Other: travel grants, Research Funding; Hoffman-LaRoche: Consultancy, Honoraria, Other: travel grants, Research Funding; Genentech: Consultancy, Other: travel grants, Research Funding; Genzyme: Consultancy, Other: travel grants, Research Funding; Gilead: Consultancy, Other: travel grants, Research Funding; GlaxoSmithKline: Consultancy, Other: travel grants, Research Funding; Janssen: Consultancy, Other: travel grants, Research Funding; Mundipharma: Consultancy, Other: travel grants, Research Funding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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