Massively Parallel High Throughput Sequencing Identifies Novel Micrornas in Normal and Malignant B Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3350-3350
Author(s):  
Jenny Zhang ◽  
Dereje D. Jima ◽  
Yuan Gao ◽  
Han Wu ◽  
Jun Zhu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
High Throughput ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Plasma Cells ◽  
Expression Patterns ◽  
Microrna Expression ◽  
Massively Parallel ◽  
B Cell Malignancies

Abstract Background: MicroRNAs (miRNAs) are small non coding RNAs that have been shown to play a regulatory role in a number of different settings including development, hematopoiesis and lineage-selection. The expression patterns of miRNAs in various cellular processes and in various normal and malignant tissues are an area of active exploration. Bioinformatic analyses of the genome suggest that there might be thousands of miRNAs encoded in the genome. However, thus far only about 600 unique miRNAs have been identified in humans. The role of microRNAs in B cell malignancies is poorly understood. Mature B cells comprise naive, germinal center, memory and plasma cells. These B cell stages comprise the majority of leukemias and lymphomas. We have previously demonstrated a role for microRNAs in the regulation of key transcription factors and oncogenes including PRDM1, LMO2 and MYBL1. We hypothesized that microRNAs play a role in the pathogenesis of lymphomas and have applied high throughput sequencing to understand the pattern and function of microRNA expression in normal B cells and their malignant counterparts. Methods: CD19+ mature B cells were obtained from normal patients undergoing tonsillectomy and sorted using flow cytometry into naive, germinal center, memory and plasma cells. We also obtained cells from tumor cell lines derived from mantle cell lymphoma (Mino, JVM2), Burkitt lymphoma (Ramos, BL41) and multiple myeloma (H929, U266), as well as patient tumor samples derived from Burkitt lymphoma and diffuse large B cell lymphomas. From these cells, we purified small RNAs (18-25 nucleotides) and ligated sequencing adapters to these small RNAs and subjected them to 15 cycles of PCR amplification. The constructs were then subjected to massively parallel high throughput sequencing (Illumina) in picoliter wells to identify millions of sequences per sample. Sequences thus identified were matched to the genome and microRNAs were identified based on their characteristic stem-loop secondary structure, thermodynamic stability, and evidence of processing with the microRNA-related enzymes drosha and dicer. Results: Using massively parallel high-throughput sequencing of small RNAs isolated from these B cell subsets, we analyzed a total of 62,293,147 sequences (> 1.6 billion bases). We found that 261 known microRNAs are expressed in normal and malignant B cells, a number that is three times higher than previously recognized. Our work also identified the expression of 86 novel miRNAs in normal and malignant B cells, many of which appear to target genes important in B cell differentiation including BCL6, NLK, EBF, as well as oncogenes including MYC, LMO2, and CCND1. We found no evidence of decreased expression of microRNAs in B cell malignancies, in contrast to the described down-regulation of microRNAs in tumors from other lineages. On the other hand, there were striking differences between the microRNA expression patterns in normal and malignant B cells, although B cell malignancies still frequently express miRNAs that are characteristic of their normal B cell counterpart. Each malignancy had a characteristic pattern of microRNA expression that was distinct from other malignancies and normal B cells. Conclusion: Through high throughput sequencing, we have discovered novel microRNAs that target important oncogenes including BCL6, MYC, LMO2, and CCND1, suggesting a role for microRNAs in B cell lymphomas.

A Comprehensive Identification of the Microrna Transcriptome and Its Application in B Cell Malignancies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2403-2403
Author(s):  
Cassandra L. Jacobs ◽  
Dereje D Jima ◽  
Jenny Zhang ◽  
Cherie Dunphy ◽  
Kristy L. Richards ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
High Throughput ◽  
Germinal Center ◽  
High Throughput Sequencing ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Microrna Expression ◽  
B Cell Malignancies

Abstract Abstract 2403 Poster Board II-380 Background MicroRNAs are 18-22 nucleotide-long RNA molecules that regulate expression of genes. We and others have previously demonstrated a role for microRNAs in the pathogenesis of B cell malignancies. Computational predictions suggest that the human genome encodes several thousand microRNAs. Thus far, about 700 microRNAs have been discovered in humans, including over 200 new microRNAs in the past year alone. The ongoing discovery of microRNAs makes it difficult to comprehensively study their role in a disease group. The advent of high throughput sequencing allows the simultaneous identification of millions of transcripts, thereby providing a sensitivity that is several orders of magnitude higher than conventional methods. We hypothesized that high throughput sequencing would be an effective tool to comprehensively identify microRNAs in normal and malignant B cells. While there is an overlap between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) in morphology, immunophenotype and cytogenetics, distinguishing between BL and DLBCL is critical because there are important differences in their clinical management. We investigated whether microRNA expression could be used to reliably distinguish BL from DLBCL. Methods and Results We carefully chose 31 human samples to represent the spectrum of normal and malignant B cells including FACS-sorted naive, germinal center, memory, plasma cells, EBV transformed and activated B cells. Samples derived from B cell malignancies included B-lymphoblastic lymphoma, chronic lymphocytic leukemia (immunoglobulin gene mutated and unmutated), mantle cell lymphoma, marginal zone lymphomas, HIV-related lymphoma, BL, DLBCL (activated and germinal center type), primary mediastinal B cell lymphoma, Hodgkin lymphoma, and multiple myeloma. We applied massively parallel, high-throughput sequencing of the 18-22 nt RNAs from these cases and generated a total of 255,624,785 sequences (∼5 billion bases). Using a computational approach that we have previously validated with normal B cells, we identified the expression of 429 known microRNAs in normal and malignant B cells, a number that is over three times higher than previously recognized in any tissue type. We also identified the expression of 302 novel microRNAs in normal and malignant B cells. The vast majority of these microRNAs were highly conserved in multiple species. As a proof of principle, we generated a custom microarray that included all the known human, and viral microRNAs, as well as 302 novel microRNAs identified by sequencing, and applied it to the clinically important distinction of BL from DLBCL. Biopsy samples were collected from 104 patients (BL, N=25, DLBCL, N=79) treated at 9 institutions that comprise an international consortium. All cases were reviewed for pathology diagnosis and profiled for microRNA expression. We constructed a Bayesian predictor to distinguish BL from DLBCL based on the microRNA expression. The predictor performance was tested using leave-one-out cross-validation. We also applied gene expression profiling to the cases of DLBCL to identify the molecular subsets of DLBCL: activated B cell like and germinal center B cell like DLBCL. The microRNA profiles of these cases were equally efficacious in distinguishing the DLBCL subsets. The predictor constructed based on microRNA expression was over 90% accurate in distinguishing BL from DLBCL, using pathology diagnosis as the gold standard. Further, microRNA-based predictor was also over 90% accurate in the distinction of the molecular subsets of DLBCL, compared to the gold standard of gene expression-profiling. As additional validation, we performed in situ hybridization of selected microRNAs to directly visualize their expression using methods that are easily accessible in conventional pathology laboratories. We found excellent concordance between the expression results derived from microarrays and in situ hybridization suggesting a ready path to clinical translation. Conclusion Our study represents the first comprehensive delineation of microRNA expression in B cell malignancies using high throughput sequencing. Our data suggest that microRNAs are a promising marker for the distinction of aggressive lymphomas. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Combined Loss of Tet1 and Tet2 Promotes B-Cell, but Not Myeloid Malignancies in Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3650-3650
Author(s):  
Zhigang Zhao ◽  
Lin Li ◽  
Meelad Dawlaty ◽  
Feng Pan ◽  
Zhe Li ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Cultures ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Hematological Malignancies ◽  
Cell Populations ◽  
Double Knockout ◽  
B Cell Malignancies ◽  
Myeloid Malignancies ◽  
Genome Wide

Abstract Objective: Tet1/2/3 are methylcytosine dioxygenases regulating cytosine methylation in the genome. Tet1 and Tet2 are abundantly expressed in HSC/HPCs and implicated in hematological malignancies. Tet2 -deletion in mice causes myeloid malignancies, while Tet1 -null mice are overtly normal early in life. Here, we investigated the overlapping and non-redundant functions of Tet1/Tet2 in HSC maintenance and hematological malignancies using Tet1/2 double knockout (DKO) mice. Methods: 1) Kinetic analysis of the hematologicalparameters on WT, Tet1-/-, Tet2-/- and DKO mice; 2) Analysis of HSC, myeloid and lymphoid progenitors and various maturation stages of B-cell populations; 3) Competitive bone marrow reconstitution assay; 4) RAN-Seq on LK cells and B220+ cells from young/undiseased and diseased DKO mice respectively; 5) Chemical labeling and affinity purification method coupled with high-throughput sequencing (hMe-Seal) to profile the genome-wide distribution of 5hmC, and methylated DNA immunoprecipitation coupled with high-throughput sequencing (MeDIP-seq) to profile 5mC in BM LK cells from young WT, Tet2-/- and DKO mice; 6) q-PCR analysis of the mRNA expression levels of Tet1 and Tet2 on BM CD19+ cells from B-ALL patients and compared to that of CD19+ B-cells from healthy controls. Results: We found that T et 1 and T et 2 are often concomitantly down-regulatedin patients with B-ALL. Therefore, it is important to investigate the effects of combined loss of Tet1 and Tet2 on the hematopoietic phenotype and development of hematological malignancies in vivo. The LSK and CMP/GMP/MEP cell populations are comparable in yound WT, Tet1-/- and DKO mice, while were significantly increasedin Tet2-/- mice. When a replating assay was performed using LSK cells, Tet2-/- LSK cell cultures had a significant higher colony formation in each round of replating, while Tet1-/- and DKO LSK cell cultures only exhibited a moderate increase in the number of colonies at P2, but not P3 and P4. Furthermore, young DKO mice had an increased percentage of CLP, BLP and Pro-/Pre-/Immature-B cell populations in their BM as compared to WT, Tet1-/- and Tet2-/- mice. Consistent to the B-lineage phenotypic analysis, DKO BM cells contained higher pre-B cell colony forming cells than the three genotypes of control mice. Interestingly, DKO mice exhibited a strikingly decreased incidence and delayed onset of myeloid malignancies compared to Tet2-/- mice and in contrast developed lethal B-cell malignancies, most closely resembling B-ALL. The loss of Tet2 or DKO leads to genome-wide alterations of both 5mC and 5hmC. Significant overlaps between the differential hydroxymethylated regions (DhMRs) or differential methylated regions (DMRs) of two genotypes of LK cells were observed. However, intriguingly, the overlaps between DhMRs and DMRs within each genotype of LK cells were minimal, indicating that DhMRs and DMRs might represent distinct loci with altered epigenetic modifications under these conditions. When the expression of a pool of 654 genes that are known to be involved in regulating hematopoietic cell development and/or promoting leukemogenesis were overlap with DhMRs and DMRs identified above, we observed significant numbers of these genes with altered either 5hmC or 5mC modifications which however did not alter their gene expression. Furthermore, RNA-Seq on B-ALL DKO B220+ cells showed alteration of a set of genes involved in B-cell development and B-cell lymphoma/leukemogenesis. Conclusion: Using Tet1/2 double knockout mice, we found that Tet1 is required for Tet2 -deletion mediated HSC dysregulation, myeloid skewing and myeloid malignancy, indicating distinct roles of the two enzymes. Tet1 loss modulates the Tet2 -deletion mediated disease phenotype, not only decreasing the incidence and delaying the onset of myeloid malignancies, but also promoting the pathogenesis of B-cell malignancies. Furthermore, our observations highlight the roles of distinct cytosine modifications, particularly 5hmC, could play in marking the specific genes and enabling cells to fate decision change upon stimulation signals. These findings provide a pathological framework for further elucidating the molecular mechanisms and critical cross talks between Tet1 and Tet2 in the pathogenesis of hematological malignancies. Disclosures No relevant conflicts of interest to declare.

MicroRNAs Regulate Mature B Cell Differentiation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 705-705
Author(s):  
Jenny Zhang ◽  
Dereje D. Jima ◽  
Cassandra L. Jacobs ◽  
Eva Gottwein ◽  
Grace Huang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Target Genes ◽  
Expression Patterns ◽  
Microrna Expression ◽  
Seed Sequence ◽  
B Cell Differentiation ◽  
Cell Stage

Abstract Background: Mature B cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells are common and constitute the majority of leukemias and lymphomas. MicroRNAs are known to play a role in oncogenesis, lineage-selection, and immune cell function, including early B cell differentiation. However, the full extent and function of microRNA expression during mature B cell differentiation and in B cell malignancies are not known. Methods: From normal young patients undergoing tonsillectomies, we sorted the mature B cell subsets (naive, germinal center, memory and plasma) using FACS, based on their expression of CD19, CD38, IgD and CD27. These sorted B cells were profiled for microRNA expression using a highly sensitive multiplexed real-time PCR assay, as well as for gene expression at the whole genome level using Affymetrix U133plus microarrays. miRNA targets can be predicted based on seed sequence matching of their 2–8 nt to the 3′UTR of gene transcripts. For each B cell stage, we experimentally validated microRNA regulation of predicted target genes of interest, LMO2, MYBL1 and PRDM1, by microRNA over-expression experiments and luciferase assays. Results: We found that microRNAs have a characteristic expression pattern that defines each mature B cell stage. Examination of both microRNA and mRNA expression showed that in each B cell population, the target genes predicted based on seed matching were expressed at lower levels, results that were highly significant (P<1E-10). We found that differential microRNA expression is important at every B cell stage transition, and differentially expressed microRNAs frequently target differentially expressed transcription factors. In the naive to germinal center B cell and germinal center B cell to memory cell transitions, we found that miR-223 had an inverse relationship with its predicted target genes LMO2 and MYBL1. To test this relationship predicted based on seed pairing, in Germinal Center-derived BJAB cells, we over-expressed miR-223 by introducing its precursor, and saw a subsequent knockdown of LMO2 and MYBL1 at both the mRNA and protein level. We confirmed seed sequence specificity by comparing miR-223 knockdown of luciferase reporter activity on the LMO2 3′UTR compared to its seed sequence mutant. We further found that miR-9 and miR-30 family members directly regulate PRDM1 (blimp1), a master regulator of the GC to PC transition. In U266 cells (PC-derived), introduction of miR-9 and miR-30 family precursor resulted in decreased PRDM1 protein expression, although transcript levels were not changed, consistent with previous evidence that miRNA can regulate at the post-transcriptional steps. We further profiled over 50 tumors derived from various B cell malignancies (small lymphocytic lymphoma, Burkitt lymphoma, and the molecular subsets of diffuse large B cell lymphoma) and found that these malignancies maintain the expression patterns of their respective lineage; microRNA expression profiles of normal B cells could correctly classify the lineage of these tumors in over 80% of the cases. In contrast to other malignancies, common lymphomas do not down-regulate microRNAs, but rather maintain the microRNA-expression patterns of their normal B-cell counterparts. Conclusion: Through concomitant microRNA and mRNA-profiling, we demonstrate a regulatory role for microRNAs at every stage in mature B-cell differentiation. Further, we have experimentally identified a direct role for the microRNA-regulation of key transcription factors in B-cell differentiation: LMO2, MYBL1 and PRDM1 (Blimp1). Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B-cells.

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.

Regulated immunoglobulin (Ig) RNA processing does not require specific cis-acting sequences: non-Ig RNA can be alternatively processed in B cells and plasma cells

1994 ◽  
Vol 14 (12) ◽  
pp. 7891-7898
Author(s):  
M L Peterson
Keyword(s):  
B Cells ◽  
B Cell ◽  
Rna Processing ◽  
Plasma Cells ◽  
Expression Patterns ◽  
Cell Types ◽  
Cis Acting ◽  
B Cell Maturation ◽  
Processing Pathways ◽  
Immunoglobulin Mu

Alternative RNA processing of the heavy-chain immunoglobulin mu gene is regulated during B-cell maturation and requires competition between splice and cleavage-polyadenylation reactions that have balanced efficiencies. Studies with modified mu genes have failed to identify gene-specific sequences required for regulation. Thus, the only important feature for regulation may be the balanced competing splice and cleavage-polyadenylation reactions themselves. If this is so, then alternative RNA processing from any gene with similar competitive RNA processing pathways should also be regulated when expression is compared between B cells and plasma cells. To test this prediction, two nonimmunoglobulin genes engineered to have competing splice and cleavage-polyadenylation reactions were expressed in B cells and plasma cells. The ratios of alternative RNAs produced from both genes are different in the two cell types; like the mu gene, relatively more spliced RNA is produced in B cells than in plasma cells. Also, in a survey of mu gene expression in nine non-B-cell lines, only a T-cell line had an expression pattern similar to that of B cells; the expression patterns of all other lines resembled that of the plasma cells. Therefore, regulated mu RNA processing must be mediated by changes in general processing factors whose activity or abundance is regulated, most likely, in B cells.

scholarly journals Regulated immunoglobulin (Ig) RNA processing does not require specific cis-acting sequences: non-Ig RNA can be alternatively processed in B cells and plasma cells.

1994 ◽  
Vol 14 (12) ◽  
pp. 7891-7898 ◽  
Author(s):  
M L Peterson
Keyword(s):  
B Cells ◽  
B Cell ◽  
Rna Processing ◽  
Plasma Cells ◽  
Expression Patterns ◽  
Cell Types ◽  
Cis Acting ◽  
B Cell Maturation ◽  
Processing Pathways ◽  
Immunoglobulin Mu

Alternative RNA processing of the heavy-chain immunoglobulin mu gene is regulated during B-cell maturation and requires competition between splice and cleavage-polyadenylation reactions that have balanced efficiencies. Studies with modified mu genes have failed to identify gene-specific sequences required for regulation. Thus, the only important feature for regulation may be the balanced competing splice and cleavage-polyadenylation reactions themselves. If this is so, then alternative RNA processing from any gene with similar competitive RNA processing pathways should also be regulated when expression is compared between B cells and plasma cells. To test this prediction, two nonimmunoglobulin genes engineered to have competing splice and cleavage-polyadenylation reactions were expressed in B cells and plasma cells. The ratios of alternative RNAs produced from both genes are different in the two cell types; like the mu gene, relatively more spliced RNA is produced in B cells than in plasma cells. Also, in a survey of mu gene expression in nine non-B-cell lines, only a T-cell line had an expression pattern similar to that of B cells; the expression patterns of all other lines resembled that of the plasma cells. Therefore, regulated mu RNA processing must be mediated by changes in general processing factors whose activity or abundance is regulated, most likely, in B cells.

Germinal Center-Derived Lymphomas and Plasmacytomas in Mice with Targeted Deletion of MiR-15a/16-1 in Activated B Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 743-743
Author(s):  
Tomasz Sewastianik ◽  
Jianjun Zhao ◽  
Meng Jiang ◽  
Jianli Wang ◽  
Vinodh Pillai ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Plasma Cells ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Malignancies ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Activated B Cells

Abstract MicroRNA (miR)-mediated gene regulation plays critical roles in B-cell development and dysregulated expression of miRs has been implicated in the pathogenesis of various types of B-cell malignancies. Somatic deletions of chromosome 13q14, harboring miR-15a/16-1, occurs frequently in B-cell lymphomas suggesting that members of this miR family are tumor suppressors. Consistently, mice with CD19-Cre-induced deletion of miR-15a/16-1 in early B-cells and follicular B-cells develop chronic lymphocytic leukemia (CLL). Since the 13q14 deletion is observed in a broader range of B-cell malignancies, we hypothesized that the type of B-cell malignancy resulting from miR-15a/16-1 down-regulation may depend on the stage of B-cell development at which this deletion occurs. Therefore, we generated a transgenic mouse model in which conditional deletion of miR-15a/16-1 takes place at later stages of B-cell development. To delete miR-15a/16-1 in activated B-cells, miR-15a/16-1fl/fl mice were mated with AID-Cre+/+ mice to obtain AID-Cre+/-; miR-15a/16-1fl/fl compound mice that expressed Cre recombinase from the Activation-induced Cytidine Deaminase (AID promoter) gene - a gene needed for generation of somatic hypermutations in the immunoglobulin (Ig) variable region (V) genes that is highly expressed in activated B-cells and is a well-known marker for germinal center (GC) B-cells. Expression levels of both miR-15a and miR-16-1, but not miR-15b were decreased in GC B-cells of AID-Cre+/-; miR-15a/16-1fl/fl mice as compared with control AID-Cre+/- mice when evaluated by In Situ Hybridization (ISH) analysis. Given that in humans miR-15a, b and 16 are also expressed in GC B-cells, these results demonstrate the validity of this mouse model in which the biological consequences of miR-15a/16-1 deletion can be studied. Next we assessed whether miR-15a/16-1 deletion could affect proliferation and/or survival of GC B-cells. GCs in the spleens of AID-Cre+/-; miR-15a/16-1fl/fl mice at 10 weeks of age were significantly increased in both number and size, and contained a larger number of Ki-67-positive B-cells as compared with spleens of AID-Cre+/- mice. No significant differences in the number of apoptotic cells, neither in the expression of the miR-15a/16-1 putative target BCL2 were detected, indicating that miR-15a/16-1 may play important roles in the proliferation, but not survival of GC B-cells. Apart from mild splenic enlargement and increased number and size of GCs, AID-Cre+/-, miR-15a/16-1fl/fl mice where indistinguishable from AID-Cre+/- mice between 8 and 40 weeks of age as assessed by weight and posture. However, after 48 weeks of age and at variable times thereafter, 80% (32/40) of AID-Cre+/-, miR-15a/16-1fl/fl mice but none from control cohorts (0/30) showed signs of disease. Gross pathologic examination of euthanized AID-Cre+/-; miR-15a/16-1fl/fl mice revealed enlargement of the spleen and lymph nodes. Detailed histological examination revealed in most instances an effacement of normal tissue architecture by a nodular or diffuse population of atypical lymphoid cells, or less commonly by sheets of plasma cells in interfollicular areas. Two distinct patterns of B220+BCL6+BCL2- B-cell lymphomas were identified after detailed analysis. The most common (47%) resembled human follicular lymphoma (FL) and the next in frequency (28%) resembled human diffuse large B-cell lymphoma (DLBCL). The other group of tumors (25%) resembled human plasmacytoma (PC). All three tumor subtypes were clonal, hypermutated and associated with different degrees of preservation of the dendritic meshwork in the lymph nodes. The comparison of lymphomas arising in AID-Cre+/-; miR-15a/16-1fl/fl mice and CD19-Cre+/-; miR-15a/16-1fl/fl mice corroborated that deletion of miR-15a/16-1 at different stages of B-cell development leads to distinct subtypes of B-cell malignancies. Finally, we investigated miR-15a/16-1 expression in human FL and PC and showed that miR-15a/16-1 abundance is significantly decreased in those malignancies when compared with nodal B-cells in reactive GCs and normal plasma cells in interfollicular areas respectively, suggesting that miR-15a/16-1 may play important roles in normal GC B-cell development as well as in the pathogenesis of FL and PC in humans. Disclosures Ghobrial: BMS: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria. Anderson:Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Role Of The BTB/POZ Domain Protein Miz-1 In The Development Of c-Myc Driven B-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3739-3739
Author(s):  
Julie Ross ◽  
Marissa Rashkovan ◽  
Christian Kosan ◽  
Charles Vadnais ◽  
Tarik Möröy
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tumor Cells ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Lymphoma ◽  
Chromosomal Translocation ◽  
B Cell Lymphoma ◽  
Igh Locus

Abstract Introduction and Objectives Myc-interacting zinc finger protein 1 (Miz-1; Zbtb17) is a BTB/POZ (POZ) domain transcription factor expressed in all mammalian tissues. The POZ domain is required for Miz-1 function and favors a stable association with chromatin. As a binding partner of the oncoprotein c-Myc, Miz-1 can modulate the expression of c-Myc target genes (e.g. p21Waf1, p15ink4b). c-Myc is frequently associated with a variety of leukemia and lymphoma. In particular, c-Myc takes part in the t(8;14) chromosomal translocation where it is placed under the control of transcriptional regulatory elements of the immunoglobulin heavy chain (IgH) locus. This chromosomal translocation is a hallmark of patients with Burkitt-type B-cell lymphoma (BL). Since epigenetic deregulation is often associated with tumor development, we wished to determine whether (i) Miz-1 is involved in the development of c-Myc-dependent B-cell lymphoma and whether (ii) Miz-1 influences c-Myc dependent chromatin and gene regulation. Methods We are using the Eµ-Myc mouse model to generate Burkitt type c-Myc dependent lymphomas to study the implication of Miz-1 in a c-Myc driven process of malignant transformation. Mice carrying the Eµ-Myc transgene (c-Myc gene placed under the control of Eµ enhancer of the IgH locus) overexpress c-Myc in lymphoid cells and develop a disease similar to Burkitt's lymphoma. Mice that express a conditional non-functional Miz-1 allele lacking the part coding for the POZ domain in B cells (Mb1-Cre-Miz-1DPOZ/flox mice, hereafter called DPOZ mice) were crossed with Eµ-Myc transgenic mice. Incidence and latency periods of the development of tumors in these animals were compared to Eµ-Myc animals that express a functional Miz-1 protein. Pre-cancerous mice of all genotypes were also investigated to evaluate the influence of Miz-1 during the development of the c-Myc dependant lymphomas. To address the role of Miz-1 in the deregulation of chromatin associated with c-Myc overexpression, chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) was performed on B-lymphoma and pre-tumoral B-cells and compared to normal B-cells. Binding to chromatin of Miz-1, c-Myc as well as histone marks will be assessed. RNA from each cell type will also be submitted to high throughput sequencing (RNA-seq) to correlate the binding of transcription factors with gene expression. Results Eµ-Myc mice that express the non-functional Miz-1 protein (DPOZ) in B-cells develop lymphoma later than Eµ-Myc mice that express a normal, functional Miz-1 (Figure 1). Tumors that grow in Eµ-Myc/DPOZ animals are phenotypically similar, but are smaller compared to those developing in Eµ-Myc mice. Additionally, blood analysis of sick animals reveal a lower amount of Large Unstained Cells (LUC) when Miz-1 is mutated. This suggests that Myc driven lymphoma and leukemia is impaired in the absence of a functional Miz-1 protein. Accordingly, in 40 days-old pre-tumoral Eµ-Myc/DPOZ animals compared to Eµ-Myc mice with wild type Miz-1, less pre-B and/or pro-B cells were observed in lymphoid organs and little or no LUC were present in the blood, suggesting that a c-Myc driven B-cell lymphoma develops with a longer latency period and progresses at a slower rate in the absence of a non-functional Miz-1 protein. Conclusion and ongoing work Our preliminary data indicate that Miz-1 is involved in the development of aggressive c-Myc driven B-cell lymphoma. To test the potency of Eµ-Myc/DPOZ tumor cells to initiate B-cell lymphoma, we will transplant these tumor cells into immune-deficient mice and follow the development of the transplanted cells. Also, to test whether Miz-1 would be a suitable therapeutic target for future leukemia and lymphoma treatment, we will use a conditional model (ROSA-CreER-Miz-1DPOZ/flox) where the POZ domain coding sequences of Miz-1 can be deleted after tamoxifen treatment after tumor initiation in Eµ-Myc animals. To better understand the mechanisms by which Miz-1 influences c-Myc in the process of lymphomagenesis, we are performing high throughput genomic analyses to identify genes that are differentially regulated in Eµ-Myc lymphomas when Miz-1 is functional or deficient. These analyzes should demonstrate whether a c-Myc/Miz-1 complex is required to malignantly transform B-cells or whether c-Myc and Miz-1 act independently in the development of B-cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Spontaneous Development of Plasmacytoid Tumors in Mice with Defective Fas–Fas Ligand Interactions

1998 ◽  
Vol 187 (11) ◽  
pp. 1825-1838 ◽  
Author(s):  
Wendy F. Davidson ◽  
Thomas Giese ◽  
Torgny N. Fredrickson
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Significant Proportion ◽  
T Cell Subsets ◽  
Cell Populations ◽  
B Cell Malignancies

B cell malignancies arise with increased frequency in aging individuals and in patients with genetic or acquired immunodeficiency (e.g., AIDS) or autoimmune diseases. The mechanisms of lymphomagenesis in these individuals are poorly understood. In this report we investigated the possibility that mutations at the Fas (lpr) and Fasl (gld) loci, which prevent Fas-mediated apoptosis and cause an early onset benign lymphoid hyperplasia and autoimmunity, also predispose mice to malignant lymphomas later in life. Up to 6 mo of age, hyperplasia in lpr and gld mice results from the predominant accumulation of polyclonal T cell subsets and smaller numbers of polyclonal B cells and plasma cells. Here, we examined C3H-lpr, C3H-gld, and BALB-gld mice 6–15 mo of age for the emergence of clonal T and B cell populations and found that a significant proportion of aging mice exclusively developed B cell malignancies with many of the hallmarks of immunodeficiency-associated B lymphomas. By 1 yr of age, ∼60% of BALB-gld and 30% of C3H-gld mice had monoclonal B cell populations that grew and metastasized in scid recipients but in most cases were rejected by immunocompetent mice. The tumors developed in a milieu greatly enriched for plasma cells, CD23− B cells and immunodeficient memory T cells and variably depleted of B220+ DN T cells. Growth factor–independent cell lines were established from five of the tumors. The majority of the tumors were CD23− and IgH isotype switched and a high proportion was CD5+ and dull Mac-1+. Considering their Ig secretion and morphology in vivo, most tumors were classified as malignant plasmacytoid lymphomas. The delayed development of the gld tumors indicated that genetic defects in addition to the Fas/Fasl mutations were necessary for malignant transformation. Interestingly, none of the tumors showed changes in the genomic organization of c-Myc but many had one or more somatically-acquired MuLV proviral integrations that were transmitted in scid passages and cell lines. Therefore, insertional mutagenesis may be a mechanism for transformation in gld B cells. Our panel of in vivo passaged and in vitro adapted gld lymphomas will be a valuable tool for the future identification of genetic abnormalities associated with B cell transformation in aging and autoimmune mice.

scholarly journals Transcriptome and IgH Repertoire Analyses Show That CD11chi B Cells Are a Distinct Population With Similarity to B Cells Arising in Autoimmunity and Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Robert W. Maul ◽  
Michelle D. Catalina ◽  
Varsha Kumar ◽  
Prathyusha Bachali ◽  
Amrie C. Grammer ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Cell Population ◽  
Lupus Erythematosus ◽  
Chronic Infection ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Expression Patterns ◽  
Sequencing Analysis

A distinct B cell population marked by elevated CD11c expression is found in patients with systemic lupus erythematosus (SLE). Cells with a similar phenotype have been described during chronic infection, but variable gating strategies and nomenclature have led to uncertainty of their relationship to each other. We isolated CD11chi cells from peripheral blood and characterized them using transcriptome and IgH repertoire analyses. Gene expression data revealed the CD11chi IgD+ and IgD− subsets were highly similar to each other, but distinct from naive, memory, and plasma cell subsets. Although CD11chi B cells were enriched in some germinal center (GC) transcripts and expressed numerous negative regulators of B cell receptor (BCR) activation, they were distinct from GC B cells. Gene expression patterns from SLE CD11chi B cells were shared with other human diseases, but not with mouse age-associated B cells. IgH V-gene sequencing analysis showed IgD+ and IgD− CD11chi B cells had somatic hypermutation and were clonally related to each other and to conventional memory and plasma cells. However, the IgH repertoires expressed by the different subsets suggested that defects in negative selection during GC transit could contribute to autoimmunity. The results portray a pervasive B cell population that accumulates during autoimmunity and chronic infection and is refractory to BCR signaling.

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