MLN4924 An Investigational Inhibitor Of NEDD8 Activating Enzyme (NAE) Is Active In Pre-Clinical Models Of Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma

The results of the Collaborative trial in relapsed aggressive lymphoma (CORAL) study suggest that diffuse large B-cell lymphomas (DLBCL) that relapse or fail to respond to rituximab-chemotherapy in the front-line possess a more resistant disease and represent an emerging challenge for clinicians treating aggressive B-cell lymphomas. It also stresses the need to further study and define at the molecular level the mechanisms by which DLBCL are developing resistance to chemo-immunotherapy. We previously demonstrated that the ubiquitin-proteasome system (UPS) plays an important role in the development of rituximab-chemotherapy resistance. Targeting the UPS has become an important therapeutic strategy in relapsed/refractory DLBCL. MLN4924, a NAE inhibitor selectively blocks the UPS up-stream by preventing the activation of a subset of ubiquitin ligases known as cullin-ring ligases. We evaluated the activity of MLN4924 in a panel of rituximab-chemotherapy (RSCL) sensitive and resistant (RRCL) germinal center B-cell (GCB) and ABC-DLBCL cell lines. RSCL and RRCL were exposed to MLN4924 (0.5μM and 1.0μM) for 24-72 hrs. Changes in cell viability, cell cycle and expression of key regulatory proteins of the cell cycle, Bcl-2 family members, and the UPS were evaluated using the cell titer glo assay, flow cytometry and western blotting respectively. MLN4924 induced cell death in ABC-DLBCL cell lines (both RSCL and RRCL) and to a lesser degree in GCB-DLBCL cell lines. Anti-tumor activity plateau was seen after 48 hrs of drug exposure. In MLN4924 sensitive cells we consistently observed cell cycle arrest in G1 phase, down-regulation of Bcl-XL and PARP cleavage. We also observed down regulation of NEDD8 protein across all treated cell lines. Bcl-XL down-regulation appears to be regulated at the transcriptional level. MLN4924 exposure in vitro resulted in a decrease in Bcl-XL mRNA as determined by quantitative polymerase chain reaction (qPCR), perhaps due to the inhibition of NFkB activity as demonstrated in MLN4924-exposed cells by p65 co-localization studies using the imagestream technology. Our data suggests that MLN4924 is active in ABC-DLBCL by inducing cell cycle arrest in G1 phase and rendering lymphoma cells more susceptible to apoptosis. MLN4924 lowers the apoptotic threshold of lymphoma cells by negatively regulating Bcl-XL levels at the transcription level. Selective inhibition of the NFkB transcription factor most likely play a role in the down-regulation of Bcl-XL observed in all cell lines tested. Ongoing studies aimed to further define the molecular mechanisms of action of MLN4924 can potentially assist scientists and clinicians in the optimal design of clinical trials incorporating this agent in relapsed/refractory DLBCL patients. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures: Czuczman: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

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SYK inhibition and response prediction in diffuse large B-cell lymphoma

Blood ◽

10.1182/blood-2011-02-333773 ◽

2011 ◽

Vol 118 (24) ◽

pp. 6342-6352 ◽

Cited By ~ 69

Author(s):

Shuhua Cheng ◽

Greg Coffey ◽

X. Hannah Zhang ◽

Rita Shaknovich ◽

Zibo Song ◽

...

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Cycle Arrest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Cycle Arrest ◽

Large B Cell Lymphoma ◽

Bcr Signaling ◽

Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G1-S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLCγ2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLCγ2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors.

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Inhibition of phosphatidylinositol 3-kinase-mediated glucose metabolism coincides with resveratrol-induced cell cycle arrest in human diffuse large B-cell lymphomas

Biochemical Pharmacology ◽

10.1016/j.bcp.2006.08.009 ◽

2006 ◽

Vol 72 (10) ◽

pp. 1246-1256 ◽

Cited By ~ 45

Author(s):

Anthony C. Faber ◽

Fay J. Dufort ◽

Derek Blair ◽

Dean Wagner ◽

Mary F. Roberts ◽

...

Keyword(s):

Cell Cycle ◽

Glucose Metabolism ◽

B Cell ◽

Cell Cycle Arrest ◽

Phosphatidylinositol 3 Kinase ◽

B Cell Lymphomas ◽

Cycle Arrest ◽

Large B Cell ◽

Phosphatidylinositol 3

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Telomere-Based Pre-Clinical Therapy in a Murine Model of Non-Hodgkin’s Lymphoma of the Diffuse Large B Cell (DLCL)Type.

Blood ◽

10.1182/blood.v106.11.607.607 ◽

2005 ◽

Vol 106 (11) ◽

pp. 607-607 ◽

Cited By ~ 2

Author(s):

Harold Longe ◽

Douglas V. Faller ◽

Gerald V. Denis

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Cycle Arrest ◽

Cyclin A ◽

Common Type ◽

Lymphoma Cells ◽

Basal Transcription ◽

Cycle Arrest ◽

Novel Approach ◽

Large B Cell

Abstract The dual bromodomain Brd2 is closely related to the basal transcription factor TAFII250, which is essential for cyclin A transactivation and mammalian cell cycle progression. Constitutive expression of BRD2 (under Eμ control) in the lymphoid lineage of transgenic mice elevates basal transcription of cyclin A, destabilizes the cell cycle and leads to B cell leukemias and lymphomas that are monoclonal, morphologically uniform, transplantable and highly malignant. The surface immunophenotype of the lymphoma cells is: B220+, CD19+, sIgM+, CD5+, CD9+; B7-1 and B7-2 elevated, CD23low; CD11b-, Ly-6G- and CD49b-, supporting lymphoid-restricted lineage; CD117- (c-kit) and CD127- (interleukin-7 receptor α-chain), consistent with mature cells; CD25-, syndecan-1-, and CD69- and exhibit a high IgM/low IgD ratio, which taken together identify a B-1 cell type. Malignant, proliferating cells infiltrate lymph nodes, liver, lung and kidney, and at late stages, cause anemia and a fatal peripheral leukemia over a 14-day time course from tumor cell transplantation to death. Genome-wide transcriptional expression profiling of these lymphoma cells reveals a transcriptional fingerprint that is most similar to human diffuse large B cell lymphoma (DLCL) with an “activated B cell” signature, consistent with histopathology, and establishes a novel murine DLCL model. DLCL is the most common type of non-Hodgkin’s lymphoma (NHL) in humans; all lymphomas combined are the fifth most common type of cancer diagnosed and the sixth most common cause of death in the United States. We treated syngeneic transplanted mice with CHOP (i.e. cyclophosphamide 40 mg/kg i.v., doxorubicin 3.3 mg/kg i.v., vincristine 0.5 mg/kg i.v. and predisone 0.2 mg/kg p.o. every day for 14 days) and monitored individual lymphoma cells, tagged with human-CD4+, by flow cytometry of lymphoid and non-lymphoid organs. In a novel approach, we supplemented CHOP with DNA oligonucleotides that mimic the chromosomal telomere, which we call a “T-oligo.” Normal cells exposed to this drug in vitro undergo transient cell cycle arrest, but DLCL cells undergo p53-dependent apoptosis. The mechanism immediately suggests a novel method of chemotherapy for leukemia and lymphoma to supplement CHOP. In mice treated systemically with CHOP and T-oligo, we observed major reductions in the leukemic burden in peripheral blood, reduced lymphadenopathy, reduced leukemic infiltrates of non-lymphoid organs and splenomegaly in the combined (“CHOP+T”) regimen over CHOP alone. We also confirmed in normal B lymphocytes that T-oligo causes only cell cycle arrest, not apoptosis. Mice likewise showed low toxicity to T-oligo at the effective doses, opening the way to a more extensive pre-clinical trial of this novel approach to NHL therapy.

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Interleukin-21 Induces Cell Cycle Arrest and Apoptosis of Diffuse Large B-Cell Lymphomas (DLBCL) Via Activation of STAT3 and Upregulation of C-Myc

Blood ◽

10.1182/blood.v112.11.601.601 ◽

2008 ◽

Vol 112 (11) ◽

pp. 601-601 ◽

Cited By ~ 1

Author(s):

Kristopher A Sarosiek ◽

Hovav Nechushtan ◽

Eli Avisar ◽

Izidore S Lossos

Keyword(s):

Cell Cycle ◽

Cell Death ◽

B Cell ◽

Cell Cycle Arrest ◽

Signaling Pathway ◽

Cell Lines ◽

Striking Difference ◽

B Cell Lymphomas ◽

Primary Tumors ◽

Cycle Arrest

Abstract IL-21, a recently discovered member of the IL-2 cytokine family, has been shown to have diverse regulatory effects on B-cells including the induction of antibody secretion, differentiation, or apoptosis depending on the cellular milieu and activation status. However, the effects of IL-21 on B-cell neoplasms such as DLBCL are largely unknown. Our research has uncovered the widespread expression of the IL-21 receptor (IL-21R) in B-cell lymphomas including DLBCL. Our results confirmed reports that IL-21R stimulation results in potent phosphorylation of STAT-1 and -3 and weak activation of STAT-5. However, our findings also show that treatment of DLBCL cell lines with IL-21 induces cell cycle arrest and apoptosis. The cell death is caspase-dependent and evident in a majority of DLBCL cell lines. To further examine the potential therapeutic applicability of IL-21, we assessed the effects of IL-21 on primary DLBCL tumor cells and in vivo DLBCL mice models. In primary tumors, IL-21 induced apoptosis in five of five DLBCLs compared to two of three follicular lymphomas, and two of seven chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B-lymphocytes. In xenograft DLBCL tumors, in situ IL-21 injections induced tumor regression and dramatically extended the overall survival of mice (P<0.001). To elucidate the mechanism of IL-21-induced cell death, microarray analysis was performed on endogenously sensitive or resistant DLBCL cell lines as well as an RCK8 cell line that acquired IL-21 resistance following chronic exposure to the cytokine. The most striking difference between IL-21 sensitive and resistant cell lines was observed in the expression of c-Myc, a known target of STAT-3, which was only induced in cell lines undergoing apoptosis upon IL-21 treatment. We have shown that the cell death associated with IL-21 treatment was prevented by utilizing c-Myc specific siRNAs and shRNA. Interestingly, c-Myc upregulation and cell death was also prevented upon knockdown of STAT-3 with siRNAs. Stimulation of the STAT-3-cMYC signaling pathway led to decreases in levels of anti-apoptotic proteins Bcl-XL and Bcl-2, which are bone-fide targets of c-Myc. Our results delineate a new IL-21-induced pro-apoptotic signaling pathway involving STAT-3 and c-Myc, which are usually considered as anti-apoptotic effectors in cancer cells. Furthermore, our findings demonstrate that IL-21 is a highly potent anti-DLBCL agent in vitro and in xenograft DLBCL models and warrant initiation of clinical studies in patients.

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Pterostilbene induces apoptosis and cell cycle arrest in diffuse large B-cell lymphoma cells

Scientific Reports ◽

10.1038/srep37417 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 26

Author(s):

Yuanyuan Kong ◽

Gege Chen ◽

Zhijian Xu ◽

Guang Yang ◽

Bo Li ◽

...

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Cycle Arrest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Cycle Arrest ◽

Large B Cell Lymphoma ◽

Large B Cell

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Abstract 45: BTM-3528 potently induces G1/G0 cell cycle arrest and is efficacious in preclinical models of diffuse large B-cell lymphoma

10.1158/1557-3265.hemmal17-45 ◽

2017 ◽

Author(s):

Matthew Kostura ◽

Jedd Levine ◽

Vibha Oza ◽

Alan Cooper ◽

Meztli Arguello ◽

...

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Cycle Arrest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Preclinical Models ◽

Cycle Arrest ◽

Large B Cell Lymphoma ◽

Large B Cell

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MicroRNA-Mediated Down-Regulation of the Tumor Suppressor Gene PRDM1/Blimp-1 in Diffuse Large B-Cell Lymphomas.

Blood ◽

10.1182/blood.v110.11.3187.3187 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3187-3187

Author(s):

Kui Nie ◽

Hatim Allawi ◽

Victor Lyamichev ◽

Mario F. Gomez ◽

Yifang Liu ◽

...

Keyword(s):

Tumor Suppressor ◽

Protein Expression ◽

B Cell ◽

Cell Lines ◽

Gene Mutations ◽

Terminal Differentiation ◽

B Cell Lymphomas ◽

Down Regulation ◽

Translation Repression ◽

Large B Cell

Abstract PR (PRDI-BF1-RIZ-homology) domain zinc finger protein 1 (PRDM1) is a master regulator in plasma cell differentiation recently identified as a tumor suppressor target for inactivation in diffuse large B-cell lymphomas (DLBCL) of the activated B-cell (ABC) type, implying interference of B-cell terminal differentiation as a pathogenetic mechanism in DLBCL. Besides deleterious gene mutations, it has been suggested that PRDM1 may also be inactivated in DLBCL by an epigenetic mechanism. In this study, we examined the hypothesis of microRNA (miRNA)-mediated down-regulation of PRDM1 in DLBCL. 10 DLBCL cell lines and 25 clinical DLBCL samples were analyzed for PRDM1α (PRDM1 functional isoform) RNA and protein expression by quantitative real-time reverse transcriptase-PCR, Western blotting and immunohistochemistry. The clinical samples included 5 ABC-DLBCLs with PRDM1 gene deletions and inactivating mutations (Group I), 12 ABC-DLBCLs without PRDM1 mutations (Group II) and 8 germinal center B-cell (GCB) type (Group III). The myeloma cell line U266, which expresses relatively abundant PRDM1α mRNA and protein, was used as a reference standard (arbitrarily set as 1). These expression studies identify desynchrony in PRDM1α mRNA and protein expression. PRDM1α is weakly expressed or undetectable in DLBCL cell lines regardless of levels of PRDM1α transcripts. For the primary DLBCL cases, the mean levels of PRDM1α mRNA in groups I, II and III were: 2.21+0.53 (p<0.05 vs. II & III), 0.84+0.19, and 0.43+0.24, respectively. However, immunohistochemistry demonstrated that in all three DLBCL groups, including Group II which has relatively high PRDM1α mRNA and harbors no PRDM1 mutations, an average of only <5% (range: 0 to 10%) of the neoplastic B cells weakly expressed PRDM1. These results suggest epigenetic down-regulation of PRDM1 protein expression in some DLBCLs. Several lines of evidence support a role for miRNA let-7 in mediating translation repression of PRDM1 in DLBCLs: (1) let-7a levels in DLBCL cell lines and primary cases, as determined by quantitative modified Invader assays, are higher (∼2 to 30 fold) than in U266; (2) The lowest (let-7a)/(PRDM1α mRNA) ratio is found in those ABC-DLBCLs harboring PRDM1 mutations; (3) Enforced expression of let-7a caused binding site-dependent reduction in reporter gene activities of at least 50%. This reduction is due to translation repression; (4) Enforced let-7a expression reduces PRDM1α levels by ∼ 50% in U266 cell lines, suggesting functional in vivo interaction of let-7a with PRDM1 mRNA. In conclusion, PRDM1 protein levels correlate poorly with PRDM1 mRNA expression in DLBCLs. Our studies suggest miRNA-mediated down-regulation as a mechanism of lowering PRDM1 activity in DLBCL, apart from genetic mutations and transcription repression. In ABC-DLBCLs without PRDM1 gene mutations, PRDM1 inactivation is likely mediated at least in part via translation repression of PRDM1 transcripts by high levels of let-7. Those ABC-DLBCLs with PRDM1 gene mutations might have “escaped” let-7-mediated down-regulation, for example, via higher levels of induction of PRDM1 transcripts or some other mechanisms. let-7 may be considered a potential target for therapeutic inhibition to restore terminal differentiation in DLBCL cells.

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Repression of Mir-106b Family Members Does Not Alter Cell Cycle Progression in Hodgkin Lymphoma

Blood ◽

10.1182/blood.v112.11.4722.4722 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4722-4722

Author(s):

Johan H Gibcus ◽

Lu Ping Tan ◽

Rikst Nynke Schakel ◽

Geert Harms ◽

Peter Moeller ◽

...

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Cycle Arrest ◽

Hodgkin Lymphoma ◽

Cell Lines ◽

Target Genes ◽

Family Members ◽

Luciferase Reporter ◽

P21 Protein ◽

Cycle Arrest

Abstract MicroRNAs (miRNAs) are 19–25 nucleotide long RNA molecules derived from precursor genes that inhibit the expression of target genes by binding to their 3′ UTR region. Expression of miRNAs is often tissue specific and miRNA profiling has shown specific miRNA expression patterns in both B-cell development and lymphomagenesis. Hodgkin lymphoma is derived from pre-apoptotic germinal center B-cells, although a general loss of B cell phenotype is noted. Using quantitative RT-PCR and miRNA microarray, we determined the miRNA profile of HL and compared this with the profile of a panel of B-cell non-Hodgkin lymphomas (NHL). The two methods showed a very good correlation for the expression levels of the individual miRNAs. Using a large panel of cell lines, we confirmed differential expression between HL and other B-cell lymphoma derived cell lines for 27 miRNAs. The HL specific miRNAs included miR-155, miR-21 and miR-106b seed family members miR-17-5p, miR-20a, miR-93, miR-106a and miR- 106b. Next, we performed target gene validation of predicted target genes for miR-17-5p, which is highly expressed in HL. Using luciferase reporter assays with stabilized anti-sense miR17-5p oligonucleotides, we showed that GPR137B, RAB12 and RBJ are likely miR-17-5p target genes in two different HL cell lines. Previous publications indicated that miR-106b seed family members negatively regulate the cyclin-dependent kinase inhibitor 1A (p21/CIP1) resulting in cell cycle arrest at G1. Consistent with these findings, we show that the miR-106b family members are highly expressed in L428, whereas p21 is not. However, inhibition of the miR-106b seed family members in L428 does not result in elevated p21 protein expression. Furthermore, there is no cell cycle arrest, growth reduction or increase in cell death and apoptosis after inhibition of the miR-106b seed family members. Thus, we conclude that blocking of the miR-106b seed family members does not necessarily lead to indiction of p21 protein. This suggests an additional regulatory layer of p21 expression in L428 cells.

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