Transcription factor CCAAT/enhancer-binding protein alpha and critical circadian clock downstream target gene PER2 are highly deregulated in diffuse large B-cell lymphoma

Abstract Genomic amplification of the 2p arm has been identified as a recurrent alteration in classical Hodgkin lymphoma, follicular lymphoma, primary mediastinal large B cell lymphoma and diffuse large B-cell lymphoma (DLBCL). We previously reported that 2p15 was gained in 25 out of 100 DLBCL patients by use of a genome-wide array-comparative genomic hybridization (array-CGH). In DLBCL with 2p amplification, genomic co-amplification of REL and BCL11A has been observed. Recent studies suggest that REL amplification is infrequently associated with nuclear REL expression and NFkB activation. In an attempt to identify the target gene at 2p15 amplification, we made BAC contig array CGH glasses for 2p15 region with 33 BAC clones covering 4.5Mb, and found that seven samples of the DLBCL with 2p amplification displayed alterations. REL and BCL11A were located within majority of the gained regions. The minimal common region of amplification was mapped to 0.5 Mb and we found that this region did not include BCL11A. To investigate the relationship between genomic gains and gene expression, we performed real-time quantitative polymerase chain reaction (RQ-PCR) analysis. The results indicated that REL, rather than BCL11A, is the target of 2p15 alterations in DLBCL.

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CEBPalpha and a Critical Circadian Clock Downstream Target Gene, PER2, Are Highly Dysregulated in Diffuse Large B-Cell Lymphoma.

Blood ◽

10.1182/blood.v114.22.3454.3454 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3454-3454

Author(s):

Nils Heinrich Thoennissen ◽

Gabriela B. Iwanski ◽

BaoNgan Doan ◽

Sigal Gery ◽

Jonathan W Said ◽

...

Keyword(s):

Circadian Clock ◽

B Cell ◽

Cell Lines ◽

Clock Gene ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Circadian Clock Gene ◽

Downstream Target ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Abstract 3454 Poster Board III-342 Non-Hodgkin's lymphoma (NHL) is a heterogeneous clinicopathologic entity characterized by distinct cells of origin and unique cytogenetic and molecular aberrations. Recently, we found by transcription profiling of CCAAT/enhancer-binding proteins (CEBPs) that they could regulate expression of the core circadian clock gene Period (PER) 2. We now report that C/EBPalpha and PER2 are highly dysregulated specifically in diffuse large B-cell lymphoma (DLBCL), one of the commonest type of high-grade NHL. Real time RT-PCR analysis of human samples of diffuse large B-cell lymphoma (DLBCL; n = 37), mantle cell lymphoma (MCL; n = 12), follicular lymphoma (FL; n = 12), and Burkitt's lymphoma (BL; n = 6) compared to normal tonsil samples (n = 10) revealed a markedly downregulated expression of both CEPBalpha and PER2 specifically in the DLBCL samples. The expression of these two genes was also significantly decreased in the 7 human DLBCL cell lines, OCI-Ly1, -Ly4, -Ly7, -LY10, and SUDHL-4, -6, -16. In contrast, mRNA levels of CEBPalpha and PER2 in samples of MCL, FL or BL showed no significant change compared to the normal tonsils, which were confirmed in cell lines of MCL (SP-49, Jeko-1, NCEB-1), FL (FLK-1) and BL (Daudi). Moreover, PER1, another important circadian rhythm gene of the Period family, showed no differences of expression in any of the NHL samples compared to control lymph nodes. The murine pro-B lymphoid cell line Ba/F3 had low levels C/EBPalpha, and forced expression of the transcription factor resulted in decreased growth and increased apoptosis as measured by trypan blue cell count and Annexin V (FACS), suggesting that this transcription factor may induce apoptosis in lymphoma. Moreover, transfection of Ba/F3 cells with a zinc-inducible CEBPalpha gene increased PER2 expression levels by 7-fold. The analysis of the PER2 promoter region showed several potential CEBPalpha binding sites, and chromatin immunoprecipitation (ChIP) as well as luciferase reporter assay experiments using Ba/F3 cells revealed that CEBPalpha can directly bind to the PER2 promoter and induce its expression. Interestingly, treatment of Ba/F3 and several DLBCL cell lines (Ly-4, SUDHL-4, -6) with the histone deacetylase-inhibitor, SAHA, significantly induced expression of both CEBPalpha and PER2, which was associated with inhibition of cell growth and apoptosis. Our further studies explored the consequences of expressing PER2 on cell proliferation. Forced expression of PER2 in Ba/F3 cells led to substantial growth reduction, G0/G1 cell cycle arrest, and altered protein expression of apoptosis-related genes. The protein level of Bcl-X(L) was downregulated, whereas levels of Bax and PARP cleavage activity were upregulated compared with control cells transfected with empty plasmid. In summary, our results show for the first time that both CEBPalpha and its key downstream target circadian clock gene, PER2, are highly dysregulated in DLBCL. Our data strongly suggest that these genes may play a role in the pathogenesis of this disorder and should be considered for therapeutic manipulation. Disclosures No relevant conflicts of interest to declare.

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Alteration of RhoH Expression Is Associated with Human Diffuse Large B-Cell Lymphoma.

Blood ◽

10.1182/blood.v104.11.4298.4298 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4298-4298

Author(s):

Yi Gu ◽

Carmelita J. Alvares ◽

Aparna C. Jasti ◽

Michael Jansen ◽

Judy Bean ◽

...

Keyword(s):

Transcription Factor ◽

B Cell ◽

Somatic Mutations ◽

Cell Lymphoma ◽

Binding Protein ◽

B Cell Lymphoma ◽

Cell Development ◽

Transcript Levels ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract An increasing number of Rho GTPase family proteins have been demonstrated to play critical roles in blood and immune cell development and function. The newly defined RhoH gene has been previously demonstrated to be mutated in lymphoma samples (Dallery et al, 1995; Pasqualucci et al, 2001). These alterations include chromosomal rearrangements and a high frequency of somatic mutations (up to 46%) in human non-Hodgkin’s lymphomas and diffuse large B-cell lymphoma. The RhoH gene encodes a novel hematopoietic-specific member of the RhoE subfamily, which is GTPase deficient, remaining in the active, GTP-bound state. Thus the activity of RhoH is likely regulated by the level of the protein expressed in the cell. The somatic mutations in the RhoH gene have been mapped to a 1.6kb hypermutable region in the intron 1, suggesting the possibility of dysregulated RhoH expression. However, levels of RhoH expression have not been directly measured in these hematopoietic tumors and so it remains unclear whether these mutations translate into aberrant RhoH expression. We utilized quantitative real-time RT-PCR to measure RhoH transcript levels in primary DLBCL patient samples. Based on morphologic and immunophenotypic analysis, 17 DLBCL positive samples and 14 normal control samples were used for our study. The levels of TATA-box binding protein (TBP) and human phosphogycerate kinase (HPGK) cDNAs were also examined simultaneously for relative expression normalization. RhoH transcript levels in a subset of the DLBCL samples were markedly reduced. In particular, 6 of 17 (~35%) tested samples showed a greater than 3-fold reduction in RhoH expression based on both RhoH/TBP and RhoH/HPGK ratios when compared with the median RhoH expression level of 14 normal samples. Overall, RhoH expression levels of the DLBCL group were significantly altered (mainly decreased) as compared with those of the normal group (p < 0.04, student T-test). To further determine correlation of the abnormal RhoH expression with somatic mutations in the hypermutable region of the RhoH gene in the DLBCL samples, we performed genomic PCR amplification and sequencing analysis of this region from the normal and DLBCL samples. In addition, we utilized a computational approach (Trafac - http://trafac.cchmc.org) to identify evolutionarily conserved putative transcription factor binding sites (TFBS) between human and other species in the hypermutable region. 13 conserved TFBS between human and mouse were identified in the hypermutable region. Mutations in the DLBCL patients are localized in 6 of these predicted TFBS, including pancreatic and duodenal homeobox 1 (PDX1), zinc-finger binding protein-89 (ZBP-89), lymphoid enhancer factor 1 (LEF-1), BRIGHT, engrailed 1 and myelin transcription factor 1 (MyT1). Interestingly, LEF-1 and BRIGHT are B cell-specific transcription activators. These results suggest that RhoH expression is frequently altered in 35–40% of DLBCL samples and mutations in the hypermutable region of the RhoH gene in several cases encompass core binding sequences of transcription factors important in B cell development.

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A BCL6 Target Gene Signature Predicts the Biological Behavior and Classification of Diffuse Large B-Cell Lymphoma.

Blood ◽

10.1182/blood.v108.11.616.616 ◽

2006 ◽

Vol 108 (11) ◽

pp. 616-616

Author(s):

Jose M. Polo ◽

Przemyslaw Juszczynski ◽

Leandro Cerchietti ◽

Stefano Monti ◽

Kenny Ye ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Target Gene ◽

Target Genes ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Gene Signature ◽

Transcriptional Profiles ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL), the most common lymphoid malignancy, is a heterogeneous disease. These tumors are thought to arise from normal antigen-exposed B-cells that have migrated to or through the germinal center (GC). Structural abnormalities of the BCL6 locus (chromosomal translocation and aberrant somatic hypermutation) are the most common genetic abnormalities in DLBCL, occurring in over a third of these tumors. We recently developed a potent and specific BCL6 peptide inhibitor (BPI) that disrupts the interaction between BCL6 and the SMRT co-repressor complex. BPI was cytotoxic against some, but not all, BCL6 positive primary DLBCLs and DLBCL cell lines, indicating that a subset of DLBCLs was particularly dependent on BCL6 for their survival. We predicted that such cases might be identified through a specific BCL6-dependent gene signature and utilized ChIP on chip and a 24,000 promoter genomic microarray to identify BCL6 target genes. In these studies, BCL6 bound to 431 loci. Eighty percent of these candidate target genes contained a canonical BCL6 binding site and 85% of analyzed candidates were confirmed using quantitative single-locus CHIP. GO term enrichment revealed that BCL6 targets were significantly more likely to be genes associated with transcription, ubiquitylation, response to DNA damage, cell cycle and chromatin assembly/disassembly (FDR <.05). We predicted that coordinate regulation of the BCL6 targets would serve as a signature of BCL6 activity. For this reason, we asked whether the BCL6 target gene set was differentially expressed in the recently described DLBCL comprehensive clusters, “B-cell Receptor/Proliferation (BCR),” “Oxidative Phosphorylation (OxPhos),” and Host Response (HR),” using 2 large series of primary DLBCLs with available transcriptional profiles. Since HR tumors are largely defined by infiltrating host inflammatory cells, we focused on BCR and OxPhos DLBCLs. Of interest, BCR tumors more frequently exhibit BCL6 chromosomal translocations and increased BCL6 expression. Consistent with these observations, gene set enrichment analysis (GSEA) revealed highly significant differential expression of BCL6 target genes in BCR vs. OxPhos tumors (p <.0001). In contrast, GSEA of the BCL6 targets in the same DLBCLs sorted into developmental cell-of-origin groups was not significant (“GC” vs. “ABC”/”Other”, p =.25 and “GC” vs. “ABC” only, p =.082). To assess the functional significance of the BCL6 signature in the DLBCL consensus clusters, we generated a predictive algorithm based on the transcriptional profiles of a series of DLBCL cell lines to assign the lines to BCR, OxPhos or HR comprehensive clusters. Five BCR and 3 OxPhos DLBCL cell lines were selected for additional blinded functional analyses. BPI treatment upregulated BCL6 target gene expression in BCR, but not OxPhos, DLBCLs. Furthermore, BPI was significantly more effective in BCR lines than OxPhos DLBCLs (BPI IC50s of BCR vs. Ox Phos lines, 12.7±1.7 μM vs 48.9±2.6 μM, respectively, p<. 0001). Taken together, the data indicate that BCR DLBCLs are significantly more dependent on BCL6 transcriptional repression and more sensitive to targeted BCL6 inhibition. More generally, these studies suggest that DLBCL comprehensive cluster designation will help guide the targeted therapy of biologically discrete DLBCL subsets.

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