scholarly journals Overexpression of B‐cell lymphoma 6 alters gene expression profile in a myeloma cell line and is associated with decreased DNA damage response

2017 ◽  
Vol 108 (8) ◽  
pp. 1556-1564 ◽  
Author(s):  
Kenichi Tahara ◽  
Makiko Takizawa ◽  
Arito Yamane ◽  
Yohei Osaki ◽  
Takuma Ishizaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
B Cell ◽  
Cell Line ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Damage Response

scholarly journals tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma

2013 ◽  
Vol 110 (4) ◽  
pp. 1404-1409 ◽  
Author(s):  
Roy L. Maute ◽  
Christof Schneider ◽  
Pavel Sumazin ◽  
Antony Holmes ◽  
Andrea Califano ◽  
...  
Keyword(s):  
Dna Damage ◽  
B Cell ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Damage Response

Pathogenesis of Mantle-Cell Lymphoma: All Oncogenic Roads Lead to Dysregulation of Cell Cycle and DNA Damage Response Pathways

2005 ◽  
Vol 23 (26) ◽  
pp. 6364-6369 ◽  
Author(s):  
Veronica Fernàndez ◽  
Elena Hartmann ◽  
German Ott ◽  
Elias Campo ◽  
Andreas Rosenwald
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Mantle Cell ◽  
Damage Response ◽  
Cell Cycle Machinery

Mantle-cell lymphoma (MCL) is a well-defined subtype of B-cell non-Hodgkin's lymphomas (B-NHL), accounts for approximately 6% of all lymphoid neoplasms, and has a median survival of 3 to 4 years. The genetic hallmark of MCL is the chromosomal translocation t(11;14)(q13;q32) that leads to deregulation and upregulation of Cyclin D1, an important regulator of the G1 phase of the cell cycle. This genetic event is present in virtually all cases of MCL, whereas additional genetic alterations that occur in subsets of MCL have been described. Most of these alterations appear to disturb the cell cycle machinery/interfere with the cellular response to DNA damage, thus making MCL a paradigm for cell cycle and DNA damage response dysregulation in cancer in general. In particular, Cyclin D1 upregulation, genomic amplification of the cyclin-dependent kinase (CDK) -4, deletions of the CDK inhibitor p16INK4a and overexpression of BMI-1, a transcriptional repressor of the p16INK4a locus, are associated with dysregulation of the cell cycle machinery in MCL. The DNA damage response pathway is affected by frequent alterations of the ataxia-telangiectasia mutated (ATM) kinase as well as occasional inactivation of checkpoint kinase (CHK)-1 and CHK2 that are kinases that act downstream of ATM in response to detection of DNA damage. Moreover, p53 is frequently targeted by alterations in MCL. A recent gene expression profiling study defined the proliferation signature, a quantitative measure of gene expression of proliferation-associated genes as the strongest survival predictor available to date allowing the definition of prognostic MCL subgroups that differ in median survival by more than 5 years.

Genomic Instability and Activation Of The DNA Damage Response Pathway Is An Independent Predictor Of Poor Prognosis, Is Associated With MYC Expression and Is a Promising Target For Therapy In Diffuse Large B Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3015-3015
Author(s):  
Enrico Derenzini ◽  
Claudio Agostinelli ◽  
Ilaria Iacobucci ◽  
Enrica Imbrogno ◽  
Beatrice Casadei ◽  
...  
Keyword(s):  
Dna Damage ◽  
B Cell ◽  
Genomic Instability ◽  
Cell Lines ◽  
Dna Damage Response ◽  
Poor Prognosis ◽  
B Cell Lymphoma ◽  
H2ax Phosphorylation ◽  
Damage Response ◽  
Large B Cell

Abstract Introduction Genomic instability and constitutive activation of the DNA damage response (DDR) pathway has been recently described in models of aggressive myc-driven lymphoid malignancies. The MYC oncogene has been reported to induce genomic instability by a mechanism involving replication stress. On the other hand, MYC is overexpressed in a fraction of diffuse large B-cell lymphomas (DLBCLs), and its overexpression has been reported to be associated with poor prognosis. The checkpoint kinases 1 (CHK1) and 2 (CHK2), are serine-threonine kinase involved in the DDR pathway. DDR activation triggers the phosphorylation of the histone H2AX at ser 139, a known marker of DNA damage and genomic instability. The correlation between genomic instability, MYC expression, and prognosis has not been investigated yet in DLBCL. Methods Immunohistochemistry (IHC) for phospho (γ) H2AX, pCHK1, pCHK2 was performed in tissue microarrays (TMAs) from 97 consecutive patients treated at our Institution between 2004 and 2011 with R-CHOP/CHOP-like regimens, with available paraffin embedded tissue from initial diagnosis. Moreover, to evaluate the therapeutic potential of DDR pathway inhibition in DLBCL, the DLBCL cell lines HBL-1, U2932, TMD8, SUDHL-6, BJAB, SUDHL-4 and primary DLBCL cells were incubated with the CHK inhibitor PF-0477736 (Pfizer). Results In the TMA study 57% of patients (n=55) displayed high levels of basal γH2AX (>30% of positive cells), 55% (n=53) displayed pCHK1/pCHK2 activation and of note all DLBCL cell lines showed detectable baseline activation of CHK1/CHK2 and/or H2AX phosphorylation, by western immunoblotting. γH2AX positive cases distributed equally in germinal center (GC) and in non GC DLBCLs, and were significantly associated with MYC expression (p<0.01). Five-year survival rate was 70% vs 41% for γH2AX-low and γH2AX-high patients respectively (p=0.01). Factors significantly related to the outcome in multivariate analysis were International Prognostic Index (IPI) score and γH2AX expression. Remarkably the prognostic significance of γH2AX was particularly evident in the low risk IPI group (0-2 risk factors), identifying a subgroup characterized by worse outcome (54% 5-year OS). In the in vitro study a significant growth inhibition (WST-1 assay), was evident after 48 hrs in all cell lines (IC50 10-230 nM). PF-0477736 25-500 nM induced cell death by apoptosis (annexin V- propidium iodide staining) in a time and dose dependent manner. Notably PF-0477736 demonstrated activity also in primary DLBCL cells (IC 50 of 50-500 nM, 24 hrs). We observed inhibition of phosphorylation of the downstream target CDC25c (ser 216), coupled with a marked increase in γH2AX ser 139 and CHK1 phosphorylation (ser317 and 345) following treatment. Conclusions A significant fraction of DLBCLs shows high levels of inherent genomic instability; the DDR activation marker γH2AX is a poor prognostic predictor in DLBCL and interestingly is significantly associated with MYC expression. DDR inhibition resulted to be highly effective in DLBCL cell lines and primary DLBCL cells; on treatment modifications of CHK1 and H2AX phosphorylation could be useful biomarkers of CHK inhibitors activity. These data provide strong rationale for targeting the DDR pathway and for clinical investigation of CHK inhibitors in DLBCL. Disclosures: No relevant conflicts of interest to declare.

Heightened DNA Damage Response and p53 Activation In MiR-155 Null Cells Are Context Specific and Aid Dependent

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2279-2279
Author(s):  
Hakim Bouamar ◽  
Long Wang ◽  
Manoela Ortega ◽  
An-Ping Lin ◽  
Daifeng Jiang ◽  
...  
Keyword(s):  
Dna Damage ◽  
B Cells ◽  
B Cell ◽  
Dna Damage Response ◽  
Target Genes ◽  
B Cell Lymphoma ◽  
Dna Lesions ◽  
Damage Response ◽  
P53 Target Genes

Abstract The germinal center (GC) reaction includes two critical events: somatic hypermutation (SHM) and class switch DNA recombination (CSR). These processes are tightly controlled, thus preventing excessive DNA injury which could lead to loss of normal B lymphocytes as well as the survival of cells with oncogenic DNA lesions. MicroRNA-155 (miR-155) plays an important role in immune cell biology; miR-155 knock-out (KO) mice have a defective mature B cell development characterized by a decreased number of GC B cells, whereas the Eμ-miR-155 transgenic mouse model develops and oligoclonal proliferation which evolves to B cell lymphoma. These observations suggest that miR-155 may regulate B cell sensitivity and response to DNA damage, which could mechanistically explain the phenotypes observed in the gain and loss-of-function animal models. To examine this concept, we purified mature B cells from multiple pairs of miR-155 KO and WT littermates, and stimulated them with LPS and IL4 thus recapitulating the GC reaction in vitro. Next, we used immunohistochemistry to quantify γH2AX at the DNA double-strand breaks (DSBs) foci, western blot to evaluate the phospho-p53 (Ser15) levels, and real-time RT-PCR to quantify the expression of p53 target genes (p21, GADD45a, cdc25c, PCNA). In these assays, we found a significantly higher γH2AX staining in B cells null for miR-155 than in WT controls (number of foci per nucleus: 4.1±0.9 vs. 1.4±0.2, p<0.01), and a markedly elevated p53 activity, defined by its phospho-level and target genes expression. To confirm these observations in a more physiologic setting, we immunized miR-155 WT and KO mice with NP-CGG, collected spleens and purified mature B-cells. Quantification of subpopulation by FACS confirmed that miR-155 mice developed fewer GC B cells, and the examination of DSBs foci and p53 target genes expression supported our in vitro observation of a heightened sensitivity to DNA damage and p53 engagement in miR-155 null cells. We concluded that miR-155 deficiency in B cells associates with excessive DNA damage and p53 activity. To further isolate define the role of miR-155 in these events, we used a retrovirus system to rescue miR-155 expression in B cells derived from the KO mouse, and defined the pattern of DNA damage response. In these assays, cells transduced with a MSCV-miR-155 construct formed fewer DSBs foci than their control counterparts transduced with an empty vector (9.0±2.1 vs. 4.9±1.8, p<0.05) and showed significantly lower p53 activity, defined by target gene expression. To test if miR-155 controls the DNA damage induced by broad genotoxic stresses, we exposed miR-155 KO or WT B cells, thymocytes and bone marrow cells to 5Gy of ionizing radiation (IR) or etoposide (4µM). In these assays, albeit a robust induction of γH2AX foci formation and p53 activation were detected, there was no significant difference between WT and miR-155 KO mice, in any of the cell types analyzed. We concluded that the heightened sensitivity to genotoxic stress in miR-155 KO mice is specific to B cells undergoing the GC reaction. This observation suggests a potential role in this process for activation-induced cytidine deaminase (AID), a key regulator of the DNA damage inducing SHM and CSR, which is also a miR-155 target gene. We confirmed that AID expression is higher in miR-155 KO than in WT B cells, and to test its contribution to the excessive DSB and p53 activity found miR-155 null cells we used an RNAi approach. We found that the inhibition of AID levels in miR-155 KO B cells significantly reduced γH2AX foci formation and expression of p53 target genes. Together, our data highlight a hitherto unappreciated interaction between miR-155 and the p53 pathway, involving DNA lesions that are germane to the GC reaction and the control of AID expression/activity. The excessive engagement of p53 is this setting may explain, at least in part, the loss of normal GC B cells found in the miR-155 KO mice. Conversely, it is possible that cells expressing abnormally high miR-155 levels show a subpar activation of the DNA damage response thus leading to the accumulation of oncogenic mutations. This paradigm would provide a mechanistic explanation for the initial poly/oligoclonal proliferation reported in the Eμ-miR-155 mice, which eventually evolves into a B-cell lymphoma Disclosures: No relevant conflicts of interest to declare.

scholarly journals Constitutive activation of the DNA damage response pathway as a novel therapeutic target in diffuse large B-cell lymphoma

Oncotarget ◽  
2015 ◽  
Vol 6 (9) ◽  
pp. 6553-6569 ◽  
Author(s):  
Enrico Derenzini ◽  
Claudio Agostinelli ◽  
Enrica Imbrogno ◽  
Ilaria Iacobucci ◽  
Beatrice Casadei ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Constitutive Activation ◽  
Large B Cell Lymphoma ◽  
Damage Response ◽  
Dna Damage Response Pathway ◽  
Large B Cell ◽  
Novel Therapeutic

Selective Inhibition of Activated B-Cell Lymphoma Cells by a Novel Small Molecule Inhibitor of NF-κB

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2498-2498
Author(s):  
John T ◽  
Tomoko Hayashi ◽  
Raymond P. Wu ◽  
Howard Cottam ◽  
Dennis A. Carson
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Line ◽  
Nuclear Translocation ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Constitutive Activation ◽  
Structure Activity

Abstract Abstract 2498 Diffuse large B-cell lymphomas (DLBCL) account for approximately 40% of lymphomas in adults, with the activated B-cell lymphoma (ABC) subtype being the least curable. ABC lymphoma cells display the phenotype of activated B-cells, which is induced by constitutive activation of the transcription factor Nuclear Factor-kappa B (NF-κB). NF-κB activation in ABC lymphoma can result from several different mutations or abnormal expression of proteins upstream of NF-κB nuclear translocation. No matter the mechanism of NF-κB constitutive activation, the resulting gene expression pattern confers an anti-apoptotic and pro-survival advantage to B-cells, hence driving the oncogenesis and supporting the survival of ABC lymphomas. Therefore, inhibiting NF-κB activity is an attractive strategy for the treatment of ABC lymphomas. Using a cell line with a reporter for NF-κB transcriptional activity in a high throughput screen of 180,000 compounds we discovered 12 compounds that inhibited NF-κB activation, suggesting these compounds negatively regulate NF-κB. Since ABC lines are generally more dependent on NF-κB signalling for cell survival than are the other types of B-cell lymphoma, the hits from the primary screen were tested in a secondary screen for the ability to selectively inhibit the growth of ABC lymphoma cells. Of the 12 hits, two compounds belonging to the same quinolone chemotype were found to selectively inhibit the growth of an ABC line compared to a non-ABC line and normal human peripheral blood mononuclear cells (PBMCs). Three more structurally related quinolones were obtained to investigate a possible limited structure activity relationship for this class of molecules, designated here as Quinolone Inhibitors of NF-κB (QINs). QIN 1 was significantly more potent (Figure A and B) and selective (Figure B and C) relative to other QINs. The limited structure activity relationship suggests that two structural regions of the chemotype may be important for potency and for ABC selectivity, hence providing impetus to further investigate QINs as possible ABC lymphoma drugs. QIN1 is more potent in inhibiting the growth of ABC lines than a known inhibitor of NF-κB activity, a commercially available selective IKK inhibitor (CAS 507475-17-4). Active IKK causes degradation of IκBα, the natural inhibitor of NF-κB, hence IKK inhibitors prevent NF-κB activation by preventing degradation of IκBα. In addition to being more potent, QIN1 retains similar ABC selectivity as the IKK inhibitor (Figure C). QIN1 and the IKK inhibitor both cause apoptosis at a similar rate and both cause G1 arrest in ABC lines at equi-toxic concentrations, initially suggesting similar mechanisms of action. Subsequently, QIN1was found to inhibit NF-κB in several different assays using the IKK inhibitor as a positive control. First, QIN1 inhibited the activation of a transcription based NF-κB reporter cell line in response to LPS, an NF-κB activator. Supporting these results, QIN1 inhibited cytokine release from human PBMCs stimulated with LPS. In addition, QIN1 prevented degradation of IκBα in response to NF-κB activating stimuli, further demonstrating the ability of QIN1 to inhibit NF-κB activity. Finally, QIN1 inhibited nuclear translocation of active NF-κB, thus preventing pro-survival gene expression. The QINs studied here all contain an alpha-beta unsaturated ketone, an electrophilic chemical moiety known to interact with cysteine thiols. Compounds containing similar electrophilic groups are known to bind a cysteine thiol in IKK, preventing its activity and hence inhibiting NF-κB activation. This suggests that this electrophilic moiety in QINs maybe responsible for the NF-κB inhibition observed in these cell lines. The presence of this electrophilic group is not necessarily detrimental to normal cells as we have shown with QIN1 in vitro (Figure A-C) and in vivo (large daily doses of QIN1 cause no observable toxicities in mice). Overall, our data indicate QIN1 inhibits IKK, a kinase immediately upstream of NF-κB nuclear translocation, allowing the majority of ABC lymphomas to be treated irrespective of the upstream mechanism of pathway activation. The selectivity for inhibition of ABC lymphoma cells by QIN1 and our in vitro data showing synergy with an inhibitor of B-cell receptor signalling, which also promotes ABC survival, provides the impetus for further preclinical development of QINs. Disclosures: No relevant conflicts of interest to declare.

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