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

Blood ◽  
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.

Expression of Circadian Core Clock Gene Per2 Is Markedly Down-Regulated in Diffuse Large B-Cell Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4470-4470
Author(s):  
Nils Heinrich Thoennissen ◽  
Qi Cao ◽  
BaoNgan Doan ◽  
Sam Abbassi ◽  
Daniel Nowak ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Clock Gene ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Burkitt’S Lymphoma ◽  
Burkitt's Lymphoma ◽  
Large B Cell Lymphoma ◽  
Human Lymphoma ◽  
Large B Cell

Abstract Non-Hodgkin’s lymphoma (NHL) is a heterogeneous clinicopathologic entity characterized by distinct cells of origin, cytogenetic and molecular aberrations. In vivo studies recently showed that mice deficient in Period (PER) 2 developed lymphomas. Furthermore, we and others have recently suggested that the core circadian clock genes Period (Per)1 and Per2 have been linked to DNA damage response pathways. We showed that both genes are involved in tumor suppression by regulating cell cycle- and apoptosis-related genes. In mammals and other vertebrates, heterodimers of the basic helix-loop-helix-PAS transcription factors, CLOCK and BMAL1, activate transcription of the Period and Cryptochrome (Cry1 and Cry2) genes via binding to E-box sequences in their promoters. Subsequently, the PER and CRY repressor proteins accumulate in the nucleus and inhibit CLOCK:BMAL1 complexes, thereby inhibiting their own gene expression. This forms the major negative circadian feedback loop. In the present study, we focused on Per1 and Per2 and their role in different types of NHL. Real-time reverse transcriptase-polymerase chain reaction (real time RT-PCR) was performed to determine the mRNA expression levels of Per1 and Per2 in patients with diffuse large B-cell lymphoma (DLBCL; n = 37), mantle cell lymphoma (MCL; n = 12), follicular lymphoma (FL; n = 12), and Burkitt’s Lymphoma (n = 6) compared to human normal tonsil samples (n = 10). We further tested their expression in 14 different human lymphoma cell lines. The relative expression of Per2 was markedly down-regulated in all DLBCL samples compared to normal tonsils (mean fold change: − 20.5 ± 28.7; p < 0.001). On the other hand, levels of expression of Per2 in samples of either MCL (2.5 ± 2.6; p = 0.1), FL (1.1 ± 10; p = 0.9), or Burkitt’s Lymphoma (− 1.1 ± 2; p = 0.07) showed no significant change compared to the normal tonsils. Moreover, we found no significant regulation of the mRNA levels of Per1 in any of the four NHL subtypes. We confirmed our results by showing concordant results in human lymphoma cell lines. Again, Per2 expression levels were persistently down-regulated in the 7 DLBCL cell lines (OCI-Ly1, -Ly4, -Ly7, -LY10, and SUDHL-4, -6, -16; mean fold change: − 8.6 ± 5.7; p < 0.001) compared to normal tonsils, but not in MCL (SP-49, Jeko-1, NCEB-1), FL (FLK-1), Burkitt’s lymphoma (Daudi), and B-precursor acute lymphoblastic leukemia (Nalm-6, BALL-1) cell lines (p > 0.05). Western blot protein analyses showed easily detectable levels of PER2 in tonsil samples, but the protein was either not expressed or significantly reduced in expression in human DLBCL cell lines. In summary, our results strongly suggest for the first time that the disruption of the key circadian clock gene Per2 may play a role in the development of DLBCL.

scholarly journals MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Danxia Zhu ◽  
Cheng Fang ◽  
Wenting He ◽  
Chen Wu ◽  
Xiaodong Li ◽  
...  
Keyword(s):  
B Cell ◽  
Binding Site ◽  
Cell Lines ◽  
Expression Vector ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Protein Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.

scholarly journals Repurposing dasatinib for diffuse large B cell lymphoma

2019 ◽  
Vol 116 (34) ◽  
pp. 16981-16986 ◽  
Author(s):  
Claudio Scuoppo ◽  
Jiguang Wang ◽  
Mirjana Persaud ◽  
Sandeep K. Mittan ◽  
Katia Basso ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Multikinase Inhibitor ◽  
Large B Cell Lymphoma ◽  
Large B Cell

To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.

scholarly journals Genome-wide characterization of copy number variations in diffuse large B-cell lymphoma with implications in targeted therapy

2019 ◽  
Vol 2 (4) ◽  
pp. 246-258
Author(s):  
Prashanthi Dharanipragada ◽  
Nita Parekh
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Copy Number ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Copy Number Variations ◽  
Model Systems ◽  
Significant Heterogeneity ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL) is the aggressive form of haematological malignancies with relapse/refractory in ~ 40% of cases. It mostly develops due to accumulation of various genetic and epigenetic variations that contribute to its aggressiveness. Though large-scale structural alterations have been reported in DLBCL, their functional role in pathogenesis and as potential targets for therapy is not yet well understood. In this study we performed detection and analysis of copy number variations (CNVs) in 11 human DLBCL cell lines (4 activated B-cell–like [ABC] and 7 germinal-centre B-cell–like [GCB]), that serve as model systems for DLBCL cancer cell biology. Significant heterogeneity observed in CNV profiles of these cell lines and poor prognosis associated with ABC subtype indicates the importance of individualized screening for diagnostic and prognostic targets. Functional analysis of key cancer genes exhibiting copy alterations across the cell lines revealed activation/disruption of ten potentially targetable immuno-oncogenic pathways. Genome guided in silico therapy that putatively target these pathways is elucidated. Based on our analysis, five CNV-genes associated with worst survival prognosis are proposed as potential prognostic markers of DLBCL.

Characterization of the mutational profile of 11 diffuse large B-cell lymphoma cell lines

2017 ◽  
Vol 59 (7) ◽  
pp. 1710-1716 ◽  
Author(s):  
Darius Juskevicius ◽  
Anne Müller ◽  
Hind Hashwah ◽  
Pontus Lundberg ◽  
Alexandar Tzankov ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Aberrant Somatic Hypermutation Targets an Extensive Set of Genes in Diffuse Large B-Cell Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1528-1528 ◽  
Author(s):  
Laura Pasqualucci ◽  
Roberta Guglielmino ◽  
Sami N. Malek ◽  
Urban Novak ◽  
Mara Compagno ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Chromosomal Translocations ◽  
Total N ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Genomic instability is a driving force in tumor development that can be achieved by a variety of mechanisms, such as defective chromosome segregation or inactivation of the DNA mismatch repair pathway. Although B-cell lymphomas are associated with chromosomal translocations deregulating oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has long not been described. We have reported that the somatic hypermutation process (SHM), which normally targets the immunoglobulin variable region (IgV) and BCL6 genes in germinal center (GC) B-cells, functions aberrantly in >50% of diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma (Pasqualucci et al., Nature412:341, 2001). As a consequence, multiple somatic mutations are introduced into the 5′ region of genes that do not represent physiologic SHM targets, including known proto-oncogenes such as PIM1, PAX5, RhoH/TTF and cMYC. To further define the extent of this phenomenon, termed aberrant somatic hypermutation (ASHM), and to identify additional hypermutated loci of possible pathogenetic significance in DLBCL, we screened 113 genes for the presence of mutations affecting their 5′ sequences (≥1.3 Kb from the transcription start site, the target region for SHM) in 10 DLBCL cell lines. Fifteen genes (13.3%) were found to harbor a significant number of mutations (p<0.05), with 70% of the cell lines being mutated in 7 or more genes; among these, six B-cell specific loci -BCL7A, CIITA, IRF4, LRMP, NCOA3 and SIAT1- carried 9–53 mutational events distributed in 20 to 70% of the cases, corresponding to an overall mutation frequency of 0.032–0.15% (frequency in the mutated cases: 0.07–0.25%). The same genes were found hypermutated in a panel of 20 primary DLBCL biopsies, which displayed an overall mutation load of 7 to 45 distinct events/gene (total N=125). Mutations were of somatic origin, independent of chromosomal translocations to the Ig loci and were restricted to the first 1.5–2 Kb from the promoter. In addition, analogous to previously identified SHM and ASHM targets, the mutations exhibited characteristic features, including a bias for transitions over transversions, preferential hotspot (RGYW/WRCY motifs) targeting, and higher frequencies at G:C pairs. However, in contrast to physiologic SHM targets such as IgV and BCL6, none of the 4 newly identified hypermutated genes that have been analyzed so far (BCL7A, CIITA, SIAT1, LRMP) displayed significant levels of mutations in purified normal GC B-cells as well as in other B-cell malignancies. This finding indicates that these genes represent aberrant hypermutation targets resulting from a tumor-associated malfunction, possibly a loss of target specificity of the physiologic SHM process. Considering previous results and the present survey, 17 (13%) out of 130 genes investigated have been found involved in ASHM, suggesting that this aberrant activity may involve an extensive set of target genes in DLBCL. Since the mutations affect both regulatory and coding sequences of the targeted genes, aberrant SHM may represent a major contributor to the pathogenesis of this disease and may explain in part its phenotypic and clinical heterogeneity.

IL-4 Affects Proliferation, Chemosensitivity-and Rituximab Sensitivity of Germinal Center B-Cell like (GCB) and Activated B-Cell like (ABC) Diffuse Large B-Cell Lymphoma Differently.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 242-242 ◽  
Author(s):  
Hovav Nechushtan ◽  
Joseph D. Rosenblatt ◽  
Izidore S. Lossos
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Lysis ◽  
Large B Cell Lymphoma ◽  
Expression Of Genes ◽  
Large B Cell

Abstract Diffuse Large B-cell Lymphoma (DLBCL) represent a diverse group of lymphoid neoplasms with heterogeneous clinical, histological, immunophenotypic, cytogenetic and molecular genetic features. Approximately 50% of DLBCL patients are not cured by the standard combination chemotherapy regimens. DLBCL can be subclassified into GCB-like DLBCL which are characterized by expression of genes normally expressed in germinal center B cells, and having a significantly better overall survival (OS) than the ABC-like DLBCL, which are characterized by expression of genes induced during in vitro activation of normal B cells. At least two markers of the GCB-phenotype - BCL6 and HGAL - are IL-4 target genes, increased expression of which independently predicts better OS. These observations suggest that endogenous or exogenously administered IL-4 may influence behavior of DLBCL. IL-4 mRNA was detected at low levels in 5 of 7 GCB-like and in all 4 ABC-like DLBCL tumor specimens. Two of 7 GCB-like tumors showed high expression levels of IL-4 as determined by real-time RT-PCR. Examination of the effects of IL-4 on proliferation of GCB-like (SUDHL6, SUDHL4 and OCILY19) and ABC-like (OCILY10 and OCILY3) DLBCL cell lines showed that IL-4 mildly increased DNA synthesis, as assessed by thymidine incorporation, in all the GCB-like DLBCL. Conversely, IL-4 markedly decreased proliferation in the ABC-like DLBCL cell lines by inducing G1 arrest. IL-4 also differently affected the sensitivity of GCB-like and ABC-like DLBCL to doxorubicin. IL-4 reduced doxorubicin-induced cell death of ABC-like cell lines (20–50% reduction) while it markedly increased the killing of the GCB-like cells (40–80% induction). IL-4 also prevented serum starvation-induced cell death of the ABC-like DLBCL, but it increased cell death of the GCB-like DLBCL cell lines. Recently, Rituximab was shown to improve survival of DLBCL patients when added to the CHOP regimen. The precise mechanisms of its action are unknown; however present data suggest that it may affect lymphoma cells either by activation of complement lysis or by mediating ADCC. IL-4 reduced the complement mediated Rituximab cell lysis of the ABC-like cell lines, while it increased the complement mediated Rituximab cell lysis of the GCB-like DLBCL cell lines. Expression levels of surface markers that modulate complement cell lysis (CD46, CD55 and CD59) were not affected by IL-4 exposure. In contrast, IL-4 did not affect killing of GCB-like and ABC-like cells by ADCC. These observations suggest that DLBCL subtypes may respond differently to the in vivo cytokine milieu of the tumor. Different responsiveness to IL-4 may modulate tumor sensitivity to the current therapeutic modalities and can potentially be explored to augment response to chemotherapy and Rituximab.

Role of Phosphatidylinositol 3- Kinase/AKT Pathway in Diffuse Large B-Cell Lymphoma Survival.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4808-4808
Author(s):  
Shahab Uddin ◽  
Azhar R. Hussain ◽  
Prahant Bavi ◽  
Abdul K. Siraj ◽  
Khawla S. Al-Kuraya
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Pi3 Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Large B Cell Lymphoma ◽  
Pi3 Kinase Pathway ◽  
Kinase Pathway ◽  
Large B Cell

Abstract Phosphatidylinositol 3-kinase (PI3-kinase) is a key player in cell growth signaling in a number of lymphoid malignancies including myeloma and primary effusion lymphoma. However, its role in diffuse large B-cell lymphoma (DLBCL) has not been elucidated. Therefore, we have studied the PI3-kinase pathway and apoptosis in a panel of DLBCL cell lines (SUDHL4, SUDHL8, SUDHL10 and OCI-LY19). Our data show that inhibition of PI3-kinase by a specific inhibitor, LY294002, induced apoptosis as detected by Annexin V/Propidium Iodide dual staining in the majority of DLBCL cell lines. We then dissected the PI3-kinase pathway by analyzing the downstream targets of phosphorylation by Western blot. We found that AKT/PKB was constitutively phosphorylated, and thus activated, in all DLBCL cell lines. The downstream elements of AKT, ForkHead (FKHR) and GSK3 were also constitutively phosphorylated in all DLBCL cell lines. Similarly, treatment with LY294002 prevented this phenomenon in all the cell lines regardless of their final apoptotic endpoint. Inhibition of PI3-kinase activity further downstream induced cleavage of Bid in all DLBCL cells and subsequently loss of mitochondrial membrane potential and release of cytochrome c from mitochondria in all DLBCL cell lines. The release of cytochrome C led to activation of Caspases 9 and 3 and cleavage of PARP. Finally expression of the inhibitor of apoptosis, XIAP, which is also a downstream target of AKT, was compromised in the all cell lines following LY294002 treatment. Our data demonstrate that the PI3-kinase pathway plays a major role in the survival and growth of DLBCL cells. Altogether, these results suggest that blocking the PI3-kinase pathway may be a potential target for therapeutic intervention in diffuse large B-cell lymphoma.

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