Yulei Shen

Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.

Genome-wide miRNA profiling of mantle cell lymphoma reveals a distinct subgroup with poor prognosis

miRNA deregulation has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Using a high-throughput quantitative real-time PCR platform, we performed miRNA profiling on cyclin D1-positive MCL (n = 30) and cyclin D1-negative MCL (n = 7) and compared them with small lymphocytic leukemia/lymphoma (n = 12), aggressive B-cell lymphomas (n = 138), normal B-cell subsets, and stromal cells. We identified a 19-miRNA classifier that included 6 up-regulated miRNAs and 13 down regulated miRNA that was able to distinguish MCL from other aggressive lymphomas. Some of the up-regulated miRNAs are highly expressed in naive B cells. This miRNA classifier showed consistent results in formalin-fixed paraffin-embedded tissues and was able to distinguish cyclin D1-negative MCL from other lymphomas. A 26-miRNA classifier could distinguish MCL from small lymphocytic leukemia/lymphoma, dominated by 23 up-regulated miRNAs in MCL. Unsupervised hierarchical clustering of MCL patients demonstrated a cluster characterized by high expression of miRNAs from the polycistronic miR17-92 cluster and its paralogs, miR-106a-363 and miR-106b-25, and associated with high proliferation gene signature. The other clusters showed enrichment of stroma-associated miRNAs, and also had higher expression of stroma-associated genes. Our clinical outcome analysis in the present study suggested that miRNAs can serve as prognosticators.

MicroRNA expression profiling identifies molecular signatures associated with anaplastic large cell lymphoma

Anaplastic large-cell lymphomas (ALCLs) encompass at least 2 systemic diseases distinguished by the presence or absence of anaplastic lymphoma kinase (ALK) expression. We performed genome-wide microRNA (miRNA) profiling on 33 ALK-positive (ALK[+]) ALCLs, 25 ALK-negative (ALK[-]) ALCLs, 9 angioimmunoblastic T-cell lymphomas, 11 peripheral T-cell lymphomas not otherwise specified (PTCLNOS), and normal T cells, and demonstrated that ALCLs express many of the miRNAs that are highly expressed in normal T cells with the prominent exception of miR-146a. Unsupervised hierarchical clustering demonstrated distinct clustering of ALCL, PTCL-NOS, and the AITL subtype of PTCL. Cases of ALK(+) ALCL and ALK(-) ALCL were interspersed in unsupervised analysis, suggesting a close relationship at the molecular level. We identified an miRNA signature of 7 miRNAs (5 upregulated: miR-512-3p, miR-886-5p, miR-886-3p, miR-708, miR-135b; 2 downregulated: miR-146a, miR-155) significantly associated with ALK(+) ALCL cases. In addition, we derived an 11-miRNA signature (4 upregulated: miR-210, miR-197, miR-191, miR-512-3p; 7 downregulated: miR-451, miR-146a, miR-22, miR-455-3p, miR-455-5p, miR-143, miR-494) that differentiates ALK(-) ALCL from other PTCLs. Our in vitro studies identified a set of 32 miRNAs associated with ALK expression. Of these, the miR-17∼92 cluster and its paralogues were also highly expressed in ALK(+) ALCL and may represent important downstream effectors of the ALK oncogenic pathway.

Distinct gene expression profiles in different B-cell compartments in human peripheral lymphoid organs

BackgroundThere are three major B-cell compartments in peripheral lymphoid organs: the germinal center (GC), the mantle zone (MNZ) and the marginal zone (MGZ). Unique sets of B-cells reside in these compartments, and they have specific functional roles in humoral immune response. MNZ B cells are naïve cells in a quiescent state and may participate in GC reactions upon proper stimulation. The adult splenic MGZ contains mostly memory B cells and is also known to provide a rapid response to particulate antigens. The GC B-cells proliferate rapidly and undergo selection and affinity maturation. The B-cell maturational process is accompanied by changes in the expression of cell-surface and intracellular proteins and requires signals from the specialized microenvironments.ResultsWe performed laser microdissection of the three compartments for gene expression profiling by cDNA microarray. The transcriptional program of the GC was dominated by upregulation of genes associated with proliferation and DNA repair or recombination. The MNZ and MGZ showed increased expression of genes promoting cellular quiescence. The three compartments also revealed distinct repertoires of apoptosis-associated genes, chemokines and chemokine receptors. The MNZ and GC showed upregulation of CCL20 and CCL18 respectively. The MGZ was characterized by high expression of many chemokines genes e.g. CXCL12, CCL3, CCL14 and IFN-associated genes, consistent with its role in rapid response to infections. A stromal signature was identified including genes associated with macrophages or with synthesis of extracellular matrix and genes that influenced lymphocyte migration and survival. Differentially expressed genes that did not belong to the above categories include the well characterized BCL6 and CD10 and many others whose function is not known.ConclusionsTranscriptional profiling of B-cell compartments has identified groups of genes involved in critical molecular and cellular events that affect proliferation, survival migration, and differentiation of the cells. The gene expression study of normal B-cell compartments may additionally contribute to our understanding of the molecular abnormalities of the corresponding lymphoid tumors.

Two Newly Characterized Germinal Center B-Cell-Associated Genes, GCET1 and GCET2, Have Differential Expression in Normal and Neoplastic B Cells

A group of genes are highly expressed in normal germinal center (GC) B cells and GC B-cell-derived malignancies based on cDNA microarray analysis. Two new genes, GCET1 (germinal center B-cell expressed transcript 1) and GCET2, were cloned from selected expressed sequence tags (IMAGE clone 1334260 and 814622, respectively). GCET1 is located on chromosome 14q32 and has four splicing isoforms, of which the longest one is 1787 bp and encodes a 435-amino acid protein. GCET2 is located on 3q13.13, and the cloned fragment is 3270 bp, which encodes a protein of 178 amino acids. Blast search showed that GCET1 has a highly conserved serine proteinase inhibitor (SERPIN) domain and is located on a chromosomal locus containing seven other SERPIN family members. GCET2 is a likely homologue of the mouse gene M17, a GC-expressed transcript. Analysis of the GCET2 protein sequence indicated that it may be involved in signal transduction in the cytoplasm. Northern blot and real-time polymerase chain reaction analyses confirmed that GCET1 is highly restricted to normal GC B cells and GCB-cell-derived cell lines. Although GCET2 is also a useful marker for normal and neoplastic GC B cells, it has a wider range of expression including immature B and T cells. Real-time polymerase chain reaction assay showed that both GCET1 and GCET2 are preferentially expressed in follicular lymphoma and diffuse large B-cell lymphoma with GC B-cell differentiation, confirming previous microarray gene expression analysis, but neither one is entirely specific. Multiple markers are necessary to differentiate the GCB from the activated B-cell type of diffuse large B-cell lymphoma with a high degree of accuracy.

The miR-17-92 MicroRNA Cluster Is Regulated by Multiple Mechanisms in B-Cell Malignancies

A cluster of six microRNAs (miRNAs), miR-17-92, is processed from the transcript of C13orf25, a gene amplified in some lymphomas and solid tumors. We find that levels of the miRNAs in the cluster do not vary entirely in parallel with each other or with the primary RNA in B-cell lines or normal cells, suggesting that processing or stability of the miRNAs is differentially regulated. Using luciferase reporter assays, we identified the region required for maximum promoter activity. Additional deletions and mutations indicated that the promoter is regulated by the collaborative activity of several transcription factors, most of which individually have only a moderate effect; mutation of a cluster of putative SP1-binding sites, however, reduces promoter activity by 70%. MYC is known to regulate C13orf25; surprisingly, mutation of a putative promoter MYC-binding site enhanced promoter activity. We found that the inhibitory MYC family member MXI1 bound to this region. The chromatin structure of a >22.5-kb region encompassing the gene contains peaks of activating histone marks, suggesting the presence of enhancers, and we confirmed that at least two regions have enhancer activity. Because the miR-17-92 cluster acts as an important oncogene in several cancers and targets genes important in regulating cell proliferation and survival, further studies of its transcriptional control are warranted.

Molecular distinctions between pediatric and adult mature B-cell non-Hodgkin lymphomas identified through genomic profiling

Burkitt lymphoma (BL) predominates in pediatric patients, whereas diffuse large B-cell lymphoma (DLBCL) is uncommon. In contrast to adults, BL and DLBCL are treated similarly in children and both entities have superior outcomes in children compared with adults. Gene expression profiling (GEP) and miRNA expression profiling clearly differentiated pediatric DLBCL from BL, forming distinct clusters regardless of patient age. However, pathway analysis of GEP data identified minor differences between corresponding pediatric and adult tumors. Predominance (6:1) of the germinal center B-cell subtype to activated B-cell subtype was found among pediatric DLBCL. Two cases were molecularly classified as primary mediastinal B-cell lymphoma. We observed frequent abnormalities in 8q24 in pediatric DLBCL, including MYC rearrangement in 31% (5 of 16) and gain or amplification in 50% (6 of 12) nonrearranged cases. MYC rearrangement was present in 96% (23 of 24) BL cases. Array-based CGH analysis identified abnormalities that are shared between adult and pediatric DLBCL (+12q15, +19q13, -6q), and abnormalities unique to the pediatric cases (-4p14, -19q13.32, +16p11.2), suggesting distinct pathogenetic mechanisms relative to age. Elucidation of the underlying target genes may provide insight into factors that modulate outcome and could provide potential novel therapeutic targets with less toxicity for pediatric patients with B-cell non-Hodgkin lymphoma.

BCL6 promoter interacts with far upstream sequences with greatly enhanced activating histone modifications in germinal center B cells

BCL6 encodes a transcriptional repressor that is essential for the germinal center (GC) reaction and important in lymphomagenesis. Although its promoter has been well studied, little is known concerning its possible regulation by more distal elements. To gain such information, we mapped critical histone modifications associated with active transcription within BCL6 as well as far upstream sequences at nucleosomal resolution in B-cell lines and in normal naive and GC B cells. Promoter-associated and intronic CpG islands (CGIs) in BCL6 showed a reciprocal pattern of histone modifications. Gene expression correlated with a paradoxical loss from the intronic CGI of histone H3 lysine-4 trimethylation, normally associated with transcription, suggesting that the intronic CGI may interfere with transcription. In an ∼110-kb region extending 150–260 kb upstream of BCL6, highly active histone modifications were present only in normal GC B cells and a GC B-cell line; this region overlaps with an alternative breakpoint region for chromosomal translocations and contains a GC-specific noncoding RNA gene. By chromosome conformation capture, we determined that the BCL6 promoter interacts with this distant upstream region. It is likely that transcriptional enhancers in this region activate BCL6 and overcome strong autorepression in GC B cells.

Studies of a germinal centre B-cell expressed gene, GCET2, suggest its role as a membrane associated adapter protein

GCET2 (Germinal centre B‐cell expressed transcript 2; also named HGAL) is a newly cloned gene that has been shown to be a useful marker for germinal centre (GC) B cells and GC B‐cell derived malignancies, including follicular lymphomas and germinal centre B cell‐like diffuse large B‐cell lymphomas (GCB‐DLBCLs), and is a useful prognosticator for DLBCLs. We report here the biochemical and biological properties of GCET2, which may help to determine its role in the GC reaction. GCET2 is constitutively localised in the plasma membrane but is excluded from lipid rafts. GCET2 does not have a transmembrane domain, and its membrane localisation is mediated by myristoylation and palmitoylation. GCET2 has five conserved putative tyrosine phosphorylation sites, and it can be phosphorylated following pervanadate treatment in B cells. By serially mutating the five tyrosines, the third and fourth tyrosines were found to be essential for GCET2 phosphorylation. GCET2 was phosphorylated when co‐transfected into COS7 cells with protein tyrosine kinases (PTKs) LYN, LCK or SYK, and therefore it could be a substrate of these kinases in B cells. The third tyrosine site (107YENV) of GCET2 is a consensus GRB2 binding site, and GCET2 was found to associate with GRB2 through the third tyrosine following phosphorylation. Our data suggests that GCET2 may be an adaptor protein in GC B cells that transduces signals from GC B‐cell membrane to the cytosol via its association with GRB2.

Inhibition of poly(ADP-ribose) polymerase (PARP) and ataxia telangiectasia mutated (ATM) on the chemosensitivity of mantle cell lymphoma to agents that induce DNA strand breaks

There is a high incidence of genomic aberration of ataxia telangiectasia mutated (ATM) and genes encoding proteins involved in the ATM pathway in mantle cell lymphoma (MCL). It has been shown that poly(ADP‐ribose) polymerase inhibitor (PARPi) strongly enhances the cytotoxicity of agents, causing single‐strand DNA breaks in cells with impaired homologous recombination repair. Here, we show that PARPi AG14361 potentiates the cytotoxicity induced by topotecan treatment in MCL cell lines, which was not dependent on either TP53 or CHEK2 status. Inhibition and/or knockdown of ATM and BRCA2 did not potentiate the cytotoxic effect of treatment with PARPi and topotecan. With loss of function of ATM, other kinases can still mediate activation of ATM substrates as demonstrated by continued phosphorylation of CHEK2 (Thr‐68), although attenuated and delayed. These results suggest that PARPi may enhance the therapeutic efficacy of DNA damaging agents on MCL through TP53‐independent mechanisms without requiring the inhibition of either ATM or BRCA2. Copyright