Rictor Overexpression and mTORC2 Signaling in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3884-3884 ◽  
Author(s):  
Gwen Jordaan ◽  
Wei Liao ◽  
Joe Gera ◽  
Sanjai Sharma
Keyword(s):  
Western Blot ◽  
Mantle Cell Lymphoma ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Mantle Cell ◽  
Bcr Signaling ◽  
Mtorc2 Complex

Abstract Abstract 3884 Signaling via the B-cell receptor (BCR) stimulates growth and survival of CLL leukemic cells and inhibits apoptosis by phosphorylating immunoreceptor tyrosine based activation motifs. This signaling subsequently activates PI3 Kinase/AKT, mTOR, ERK and other pathways. Activation of Akt in turn requires phosphorylation by mTOR kinase, which assembles in two complexes mTORC1 and mTORC2 and it is the mTORC2 complex that phosphorylates and activates Akt. This phosphorylation of Akt in CLL specimen's upregulates anti-apoptotic proteins such as Mcl-1, Bcl-xl and XIAP. We have identified that Rictor, a component of mTORC2 complex is over-expressed in CLL specimens as compared to normal peripheral mononuclear B cells. This over-expression was noted by real time PCR that showed 1.5 to 4 fold upregulation (n=12). Western blot analysis also showed Rictor overexpression in all the twelve CLL specimens tested. Rictor overexpression was also seen in Mantle cell lymphoma cell lines and to study its role in BCR signaling, stable Mantle cell lymphoma lines with SiRNA mediated Rictor knockdown were established. Rictor knockdown resulted in a significant decrease in Akt activation as phosphorylation (phospho S473) of Akt both in unstimulated cells and when the cells were stimulated with BCR crosslinking was decreased. To determine the effect of Rictor and mTORC2 inhibition on CLL specimens, we tested the activity of three compounds isolated via yeast two hybrid drug screen designed to identify molecules that inhibit Rictor/mTOR interaction. When tested on CLL specimens in the presence of BCR crosslinking, these mTORC2 inhibitor compounds inhibited the downstream phosphorylation of Akt S473. Functionally the inhibitors also induced apoptosis in CLL cells with 40–60% of CLL cells undergoing apoptosis (1.0mM, cells treated for 48 hours). In comparison, rapamycin (mTORC1 inhibitor) and ppp242 (mTORC1, 2 inhibitor) were comparatively less active in CLL specimens as they were less effective in the induction of apoptosis. Western blot analysis of mTORC2 inhibitor treated cells also showed PARP cleavage and an increase in the pro-apoptotic protein BAD. Our data indicates that Rictor overexpression in CLL specimens is required for Akt phosphorylation activation and downstream BCR signaling. Inhibition of this pathway by mTORC2 inhibitors in CLL will be an effective therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

Novel CRM-1 Inhibitors for Therapy In Mantle Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2734-2734
Author(s):  
Kejie Zhang ◽  
Lan V Pham ◽  
Liang Zhang ◽  
Archito T. Tamayo ◽  
Zhishuo Ou ◽  
...  
Keyword(s):  
B Cells ◽  
Western Blot ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Mantle Cell ◽  
Dose Dependent

Abstract Abstract 2734 Chromosomal Region Maintenance 1 (CRM1) overexpression has been associated with cancer progression and mortality in several human cancers, suggesting that activation of nuclear export may play a role in human neoplasia and may serve as a novel target for the treatment of cancers. This overexpression of CRM1 may be related to the export of most tumor suppressor and growth regulatory proteins out of the nucleus, thereby functionally inactivating them. Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma that is not yet curable. The objective of our study was to investigate the status of CRM1 in MCL, both in MCL cell lines and primary MCL cells, in comparison to normal B cells, and to evaluate the therapeutic efficiency of CRM1 inhibition in MCL in vitro and in vivo, and to elucidate the mechanism of CRM1 inhibitor-mediated MCL cell apoptosis. We used 8 established MCL cell lines and primary cells from 4 patients with relapsed/refractory MCL. KPT185 and KPT276 are novel, highly selective, drug-like small molecular CRM1 inhibitors. Western Blot analysis showed that CRM1 was expressed in both the cytoplasm and nuclei of 8 MCL cell lines. CRM1 was mainly detected in nuclei of normal resting B cells; In contrast, CRM1 was primarily detected in the cytoplasm of freshly isolated primary MCL cells from patients with relapsed/refractory MCL. In 3H-thymidine incorporation assays, inhibition of CRM1 by KPT185 resulted in a significant dose-dependent growth inhibition of 8 MCL cells, with IC50 values range between 10 nM to 120 nM. The blastoid-variant MCL cell lines (Z-138 and Rec-1) were significantly more sensitive to KPT185 than the non-blastoid variant MCL cell lines. Flow cytometry analysis with fluorescence-labeled Annexin V and propidium iodide showed that KPT185 induced MCL cells apoptosis in both time- and dose-dependent manners, but had no effect on cell cycle arrest. MCL cells treated with KPT185 for 12 hours showed caspase 3 activation and PARP cleavage. As shown in Western blot and confocal microscopy, blocking CRM1 activity by KPT185 in MCL cells up-regulated the protein expression of p53, a known CRM1-mediated export protein, and also induced CRM1 translocation to the nucleus and decreased CRM1 expression. In severe combined immunodeficient (SCID) mice bearing palpable Z-138 tumors, treatment with KPT-276 (similar structure to KPT-185 but improved animal pharmacokinetics), 50mg/kg or 150 mg/kg PO QDx5 each week, or cyclophosphamide 100 mg/kg on days 1–3, was initiated. Tumor growth was significantly inhibited (>75%) in all of treatment groups compared with vehicle control. Neutropenia and other cytotoxic-agent specific effects have not been observed in treated animals. In conclusion, CRM1 inhibitors inhibited growth of MCL cells in vitro and in vivo, and induced apoptosis of MCL cells via inhibition of CRM1 expression and blockage of its translocation with functional nuclear proteins. Our data suggest that novel CRM1 inhibitors provide a potential therapy for patients with relapsed/refractory MCL. Disclosures: No relevant conflicts of interest to declare.

Histone 3 Methyltransferase (EZH2) Inhibition Enhances TRAIL-Induced Apoptosis In Mantle Cell Lymphoma Cells By Accelerated cFLIP Degradation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4425-4425 ◽  
Author(s):  
Frank K Braun ◽  
Rohit Mathur ◽  
Lalit Sehgal ◽  
Zuzana Berkova ◽  
Felipe Samaniego
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Caspase 8 ◽  
Apoptosis Resistance ◽  
Lymphoma Cells ◽  
Mantle Cell ◽  
Trail Sensitivity

Introduction Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma that is characterized by the t(11:14)(q13:p32) translocation. MCL cells have altered cyclinD1 levels, impaired cell cycle regulation, DNA damage response, and likely defects in apoptosis signaling. Furthermore, up-regulated anti-apoptotic mediators such as the target of NF-κB c-FLIP were correlated with decreased apoptosis signaling. Also many cancer cells and malignant tumors show a prevalent resistance to apoptosis induction by TRAIL. Thus, by understanding the underpinnings of apoptosis resistance, we will be in a better position to develop strategies that improve TRAIL-induced killing of lymphoma cells. Methods/Results MCL cell lines (Mino, JeKo-1, JVM-2 and Z-138) were treated with DZNep (3-Deazaneplanocin A; 0.2-5µM) for 24 h followed by incubation with TRAIL (10-20ng/ml, 6-16h). Cell death, DNA fragmentation, and mitochondrial membrane potential (Δψm) were determined by calcein staining, subG1 analysis, and TMRM staining, respectively. Neither DZnep alone nor in combination with TRAIL showed a significant induction of necrosis as determined by LDH-release levels, but DZNep alone showed strong antiproliferative properties at higher concentrations (Promega CellTiter 96 assay). Activation of the caspase signaling cascade (caspase-8, -9, -3, Bid, and PARP cleavage) was analyzed by Western blotting. TRAIL-induced signaling was significantly increased and caspase-8 processing enhanced in DZNep pretreated cells indicating a regulation at the TRAIL/DISC. Although a reduced expression of DR5 in total cell lysates of DZNep treated cell was observed, the surface receptor levels were not altered. Interestingly, downregulation of the well-known caspase inhibitor, cFLIP, correlated with the DZNep-induced increased TRAIL sensitivity in MCL cell lines. However, it appears that cFLIP levels are not reduced due to blocked NF-kB signaling but rather by an accelerated ubiquitin-mediated degradation. Conclusions This study reveals that inhibition of histone methyltransferase (EZH2) activity by DZNep has a profound positive impact on TRAIL signaling; it enhances TRAIL sensitivity by promoting processing of caspase-8 through enhanced cFLIP degradation. The capacity of DZNep to target stability of cFLIP, which represents a center piece in DISC regulation underscores its potential for enhancing efficacy of TRAIL-based cancer therapies. Disclosures: No relevant conflicts of interest to declare.

Combined Epigenetic Therapy with the Novel Histone Methyl Transferase EZH2 Inhibitor 3-Deazaneplanocin and Histone Deacetylase Inhibitor Panobinostat Exerts Synergistic Activity against Human Mantle Cell Lymphoma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3622-3622
Author(s):  
Warren Fiskus ◽  
Yongchao Wang ◽  
Anand Jillella ◽  
Pace Johnston ◽  
Rajeshree Joshi ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mantle Cell Lymphoma ◽  
Histone Deacetylase ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Epigenetic Therapy ◽  
Synergistic Activity ◽  
Gene Promoters ◽  
Core Proteins ◽  
Mantle Cell

Abstract Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p16 and JunB. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces tri-methylation (3Me) of lysine (K)-27 on histone H3, as well as promotes cell proliferation and aggressiveness of neoplastic cells. EZH2 is preferentially overexpressed in proliferating but not resting Mantle Cell Lymphoma (MCL) cells. In the present studies we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) dose-dependently (500 nM to 2.0 uM) depletes EZH2, SUZ12 and EED levels, as well as inhibits 3Me K27 on H3 while inducing K27 H3 acetylation. DZNep treatment also induces the levels of p21, p27, JunB and FBXO32, while depleting cyclin D1 and cyclin E levels in the cultured human MCL Jeko-1, MO2058 and Z138 cells and in primary patient-derived MCL cells. Treatment with DZNep induces PARP cleavage activity of the caspases and apoptosis in the cultured and primary MCL cells. DZNep promoted proteasomal degradation of EZH2 and SUZ12, since co-treatment with bortezpmib significantly restored EZH2 and SUZ12 levels in the MCL cells. We had previously reported that treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) (LBH589, Novartis Pharmaceutical Corp) depletes the levels of EZH2, SUZ12 and EED in cultured and primary AML cells (Mol Cancer Ther.2006; 5:3096). Within the PRC2 complex, EZH2 bound and recruited the DNA methyltransferases DNMT1, and treatment with PS also disrupted the interaction of EZH2 with DNMT1, attenuated DNMT1 levels and its binding to the EZH2-targeted gene promoters, e,g, JunB. Here, we also demonstrate that, PS treatment depletes DNMT1 levels and induces JunB levels in cultured MCL cells. As compared to treatment with either agent alone, co-treatment with DZNep and PS caused more depletion of EZH2 and SUZ12, but not of DNMT1, more induction of JunB, p21 and p27, as well as synergistically induced apoptosis of cultured MCL cells (combination indices < 1.0). Taken together, these findings indicate that DZNep and PS mediated targeting of EZH2 and the PRC2 complex is an effective epigenetic therapy of MCL, which also results in undermining several molecular determinants of MCL cell proliferation and survival. Additionally, combined epigenetic therapy with DZNep and PS exerts synergistic in vitro activity against human MCL cells, suggesting that this combination may be a promising novel treatment for MCL.

Bendamustine for Mantle Cell Lymphoma: Retrospective Analysis of the Spanish Experience

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4942-4942
Author(s):  
Ana García-Noblejas ◽  
Belén Navarro Matilla ◽  
Carolina Da Silva Rodriguez ◽  
Raquel De Oña Navarrete ◽  
María José Ramirez Sánchez ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Mantle Cell Lymphoma ◽  
Retrospective Analysis ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Alkylating Agents ◽  
Stage Iv ◽  
Progression Free Survival ◽  
Median Number ◽  
Mantle Cell

Abstract Abstract 4942 INTRODUCTION. Patients with Mantle cell lymphoma (MCL) have an adverse outcome after relapse due to chemorefractory disease with conventional treatments. Bendamustine, a nitrogen mustard compound chemically related to the alkylating agents, has demonstrated high efficacy with a low toxicity profile in reported clinical trials. AIM. To analyze the Spanish experience in patients with relapsed/refractory MCL treated with Bendamustine. METHODS. Retrospective analysis of spanish experience in relapsed/refractory MCL treated with Bendamustine alone or in combination. This study has been approved by local ethical committees. RESULTS. Currently, there are 36 patients registered and 28 are available for this analysis. Patients'characteristics: 69% male, median age 65 years old (range 41–81), 87% ECOG≤ 1, 83% Ann Arbor stage IV, 37% high risk MIPI and 9% blastic variant. Previous regimens were CHOP or CHOP like ± R in 42.5%, HyperCVAD/MtxAraC ± R in 42.5%, R-CVP in 9% and other regimens in 6%. Median number of previous treatments were 2.6 (range 1–6), all patients had received prior Rituximab and 73% had chemosensitive disease to the last treatment. Bendamustine regimen was R-B (R-375mg/m2 D1, B-90 mg/m2 D1-2) in 78% patients, R-B with B-70 mg/m2 in 8%, B alone in 3%, R-B-Bortezomib in 3% and R-B plus consolidation (SCT, Y90Ibritumomab-tiuxetan) in 8%. Median number of cycles was 4.61 (range 1–7). G- CSF support was administered in 43% of cycles. Response: Overall response rate was 73%, with 43% CR & uCR and 30% PR. Survival: Median overall survival from diagnosis is 8,26 years (range: 1.6–11,6 years) without plateau. Median progression free survival (PFS) after Bendamustine treatment was 16 months (95% CI: 11.7–20.4), data that compares favourably with patients' PFS to previous therapy (12 months, 95% CI: 6.5–17.5). Median PFS for patients who achieved CR/uCR is 32.6 months (95% CI: 19.9–45.4) versus 11 months in patients with PR (95% CI: 3.9–18.8). With a median follow-up for surviving patients of 12 months since Bendamustine treatment, the estimated OS at 3 years is 47% (+ SD 14%). Toxicity: No treatment related mortality has been described so far. Over 152 cycles, only 10 hospitalizations due to febrile neutropenia were reported. No one case of lysis tumoral syndrome has been reported. CONCLUSION. Bendamustine plus Rituximab is a good rescue treatment in non selected pretreated patients with mantle cell lymphoma. CR rate and duration of response seem to reproduce in current clinical practice the good data reported in previous clinical trials and compares favourably with other available treatments. Disclosures: No relevant conflicts of interest to declare.

Combination Of Ibrutinib With ABT-199, a BCL-2 Pathway Inhibitor: Effective Therapeutic Strategy In a Novel Mantle Cell Lymphoma Cell Line Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 645-645 ◽  
Author(s):  
Xiaoxian Zhao ◽  
Juraj Bodo ◽  
Danyu Sun ◽  
Jeffrey J. Lin ◽  
Lisa Durkin ◽  
...  
Keyword(s):  
Mouse Model ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Therapeutic Strategy ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Down Regulation ◽  
Mantle Cell ◽  
Bcr Signaling ◽  
Nsg Mouse

Abstract Background Mantle cell lymphoma (MCL) is an aggressive subtype of Non-Hodgkin Lymphoma associated with poor prognosis. Constitutive activation of B-cell receptor (BCR) signaling plays an essential role for the survival and proliferation of malignant B-cells. Targeting Bruton’s tyrosine kinase (BTK), a component of BCR signaling pathway, with ibrutinib is a promising strategy. As a single agent, complete and partial response rates of 21% and 47%, respectively, were observed in a phase 2 study for relapsed or refractory MCL. Simultaneous inhibition of multiple biologic pathways has the potential to result in a synergism. We combined ibrutinib with ABT-199, a BH3 mimetic that selectively targets the BCL-2 pathway, and tested their in vitroefficacy against MCL. Experimental design A novel MCL cell line, CCMCL1, and four other MCL cell lines (Jeko-1, Mino, JVM2, Rec-1) were used for flow cytometry-based apoptosis and cell cycle analyses to evaluate the combinational effect of ibrutinib with ABT-199 (ChemieTek. Indianpolis. IN). The interaction between drugs was examined with Calcusyn software and combination index values served to determine the combined effect as synergistic (<1), additive (=1), or antagonistic (>1). Immunoblotting was performed to investigate signaling pathways of MCL cells exposed to these agents. Results CCMCL1 was derived from primary leukemic MCL cells. Cells were initially directly injected via tail vein into an NSG mouse. Engrafted cells were then isolated at 10 weeks from spleen and placed into routine cell culture. Immunophenotyping showed CCMCL1 cells have similar characteristics as the primary patient MCL cells, which expressed CD5, CD19, CD20, FMC7 and monotypic kappa light chain. Immunohistochemical staining of engrafted mouse spleen tissue showed expression of cyclin D1 and SOX11. The karyotype is highly complex with an IGH@/CCND1 fusion by metaphase FISH. In addition to spleen, MCL cell infiltration was observed in mouse liver, bone marrow, blood, brain, lung, kidney and intestine. In vitro cultured CCMCL1 cells underwent apoptosis upon expose to ibrutinib and ABT-199 as single agents. Combination of these two drugs resulted in synergistic induction of apoptosis (Table 1). Synergism was also observed with Jeko-1, Mino, JVM-2 and Rec-1 cells. Immunoblotting showed CCMCL1 cells have constitutive expression of cyclin D1, SOX11, PAX5 and MCL1. Ibrutinib as a single agent induced a rapid down-regulation of SOX11 and MCL1, while combination of ibrutinib with ABT-199 further enhanced down regulation of SOX11, followed by down-regulation of PAX5 at a later time point. We are currently testing the in vivo efficacy of combining these two drugs in a CCMCL1 NSG mouse model. Conclusion Our CCMCL1/NSG mouse model is a new model for pre-clinical assessment of MCL treatment approaches. Combination of ibrutinib with ABT-199 has synergistic effect of apoptotic induction in CCMCL1 as well as four other MCL cell lines tested. Ibrutinib or ibruitnib/ABT-199 combination induced apoptosis of MCL is associated with down-regulation of SOX11 and PAX5. Simultaneous down regulation of MCL1 via ibrutinib and targeting of BCL2 may contribute to the in vitrosynergism observed. These data support further investigation of this novel therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

Human Mesenchymal Stem Cells Promote Resistance to Death Signals By Potentiating FGF Signaling in Mantle Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5119-5119
Author(s):  
Lalit Sehgal ◽  
Rohit Mathur ◽  
Frank K Braun ◽  
Zuzana Berkova ◽  
Sattva Neelapu ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Death Receptor ◽  
Treatment Strategies ◽  
Human Mesenchymal Stem Cells ◽  
Pcr Analysis ◽  
Tumor Initiating Cells ◽  
Autocrine Loop ◽  
Mantle Cell

Abstract Introduction Mantle cell lymphoma (MCL) represents an aggressive, incurable form of non-Hodgkin’s lymphoma (NHL). The health complications associated with advanced age of MCL patients further restrict treatment with intense chemotherapy. Translocation t(11;14), responsible for overexpression of cyclin-D1 is the hallmark of MCL. An intense analysis of primary MCL had been delayed until development of a tissue culture system, using human mesenchymal stromal cells (hMSC), suitable for propagation of primary MCL cells. We hypothesized that tumor-initiating cells are responsible for MCL relapse and chemoresistance and thus, identification of survival signals responsible for survival and maintenance of MCL-initiating cells (MCL-ICs) is essential for design of successful treatment strategies. Methods Isolates of primary MCL cells (n=24) were co-cultured with hMSC and content of MCL-ICs was analyzed by flow-cytometry based on marker expression profile; CD34-CD3-CD45+CD19-. Cytokine array was used to identify the soluble factors enriched in the cocultures and the expression of these factors was confirmed by RT-PCR analysis. The signaling pathways employed by the newly-identified factors were blocked in 3 MCL cell lines (JVM2, Mino, Z138) and cell survival was monitored. Results Co-cultures of primary MCL isolates with hMSCs supported the growth of MCL cells for over 4 weeks with continued presence of MCL-ICs (CD34-CD3-CD45+CD19-) representing about 1% of MCL cells. We found IL-6 triggered a FGF/FGFR autocrine loop in primary MCL in cocultures. Patients with MCL showing high FGFR levels in MCL tissues compared to lymphocytes, and the highest FGFR expressing cohort of patients showed a lower overall survival rated compared to those with low FGFR levels. Blockage of FGF/FGFR rendered MCL cells dead and also sensitized the cells to the killing effects of death receptor ligands. Conclusion We establish that primary MCL use an FGFR autocrine loop for propagation in cocultures with hMSCs. We defined the factors supporting MCL and MCL-ICs survival; such knowledge is essential for targeting MCL and MCL-ICs. Disclosures No relevant conflicts of interest to declare.

The synergistic effect of BCR signaling inhibitors combined with an HDAC inhibitor on cell death in a mantle cell lymphoma cell line

APOPTOSIS ◽  
2015 ◽  
Vol 20 (7) ◽  
pp. 975-985 ◽  
Author(s):  
Kazumi Hagiwara ◽  
Shinji Kunishima ◽  
Hiroatsu Iida ◽  
Yasuhiko Miyata ◽  
Tomoki Naoe ◽  
...  
Keyword(s):  
Cell Death ◽  
Synergistic Effect ◽  
Mantle Cell Lymphoma ◽  
Hdac Inhibitor ◽  
Cell Lymphoma ◽  
Lymphoma Cell Line ◽  
Mantle Cell Lymphoma Cell ◽  
Mantle Cell ◽  
Bcr Signaling ◽  
Signaling Inhibitors

scholarly journals Acetylshikonin Suppresses Diffuse Large B-Cell Lymphoma Cell Growth By Targeting The TOPK Signaling Pathway

2021 ◽  
Author(s):  
Jieke Cui ◽  
Rong Guo ◽  
Yingjun Wang ◽  
Yue Song ◽  
Xuewen Song ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Growth ◽  
Western Blot ◽  
B Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is one of the most common causes of cancer death worldwide, and responds badly to the existing treatment. Thus, identifying the novel therapeutic targets of DLBCL are urgent. Methods and results: In this study, we found that the T-lymphokine-activated killer cell-originated protein kinase (TOPK) was highly expressed in DLBCL cells and tissues. The TOPK expression were analyzed by bioinformatics analysis, immunohistochemistry (IHC) and western blot analysis. TOPK knockdown inhibited cell growth and induced apoptosis of DLBCL cells with MTS and flow cytometry. Further experiments demonstrated that acetylshikonin, the targeted compound of TOPK, could attenuate the cell growth and aggravate the cell apoptosis through TOPK/extra cellular signal-regulated kinase (ERK)-1/2 signaling using MTS, flow cytometry and western blot analysis. In addition, we demonstrated that TOPK overexpression significantly reduced the acetylshikonin effect on cell proliferation and apoptosis in U2932 and OCI-LY8 cells using MTS, flow cytometry and western blot analysis. Conclusions: Taken together, the present study suggests that the targeted inhibition of TOPK by acetylshikonin may be a promising approach to the treatment of DLBCL.

scholarly journals Genome-Wide Analysis of BRD4-Targets Reveals the Therapeutic Relevance of Simultaneous Targeting of BCR Pathway and IKZF-MYC Axis in Mantle Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4100-4100
Author(s):  
Junya Kuroda ◽  
Taku Tsukamoto ◽  
Shingo Nakahata ◽  
Kazuhiro Morishita ◽  
Ryuichi Sato ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Research Funding ◽  
Target Genes ◽  
Cell Lymphoma ◽  
Genome Wide ◽  
Mantle Cell ◽  
Bcr Signaling ◽  
Dose Dependent

Abstract Mantle cell lymphoma (MCL) has been mostly incurable, and there is an urgent need to identify targetable molecules for development of a more effective treatment strategy. Bromodomain and extraterminal domain (BET) proteins associate with acetylated histones and facilitate transcription of target genes, and bromodomain-containing 4 (BRD4), a member of BET proteins, recruits the P-TEFb complex to genomic lesions in chromatin and thereby activates RNA Pol II at specific promoter sites of target genes. In addition, super-enhancers have been recognized as regulatory regions with a high level of acetylated histones, mediator complexes and BRD4, and super-enhancers in cancer cells are enriched at oncogenes. Recent studies have shown that BRD4 promotes expression of pivotal molecules in disease development, maintenance and progression in various cancers, including lymphoma. Given, we in this study examined the effect of BRD4 inhibition on human MCL-derived cell lines, Jeko-1, JVM-2, MINO and Z138, and performed broad screening of BRD4-regulated molecules using genome-wide approaches to identify therapeutic targets for MCL. As the results, treatment with a BRD4 inhibitor I-BET151 for 72 h showed a dose-dependent inhibitory effect on cell proliferation in all four cell lines, with half maximal inhibitory concentrations (IC50s) of 15.6 nM, 3.6 nM, 2.6 nM and 3.0 nM in Jeko-1 cells, JVM2 cells, MINO cells and Z138 cells, respectively, which was accompanied by G1/S cell cycle arrest and the induction of apoptosis. Next, we performed comprehensive gene expression profile (GEP) analysis for JVM2 and Z138 cells with or without I-BET151 treatment, and BRD4 chromatin immunoprecipitation sequencing (ChIP-Seq) in JVM2 cells treated with 10 nM I-BET151 or DMSO. Accordingly, GEP analyses revealed that more than 600 genes were commonly upregulated by more than 1.5-fold and downregulated by less than 0.67-fold, respectively, in JVM2 and Z138 cells treated by I-BET151, while ChIP-Seq showed that 7988 BRD4-binding regions were dysregulated by I-BET151, with most of these sites in enhancer regions, and 547 BRD4-binding regions were characterized as super-enhancers. Integrated analysis using the Reactome Pathway Database and the results of GEP and ChIP-Seq showed that a series of genes involved in the B cell receptor (BCR) signaling pathway and IKZF-MYC axis are regulated by BRD4 in MCL cells. To confirm whether each BRD4 target contributes to survival and proliferation of MCL cells, we focused on several candidate targets: the BCR pathway, IKZF and MYB. However, ibrutinib, a Bruton kinase inhibitor, suppressed cell growth in only two of the four cell lines (MINO and JVM2) in a dose-dependent manner, while lenalidomide, an inhibitor of the IKZF family, did not affect cell survival, despite its potency in decreasing IKZF1 and IKZF3 proteins. MYB silencing using shMYB did not decrease cell proliferation in any of the four MCL cell lines. In conclusion, our study disclosed that BRD4 regulates transcription of multiple genes by binding to enhancer region, partly involving super-enhancers and multiple known pathways, such as BCR signaling and the IKZF-MYC axis, which play essential roles in survival of MCL cells. While the efficacy of single targeting of BCR-signaling, IKZF, or MYB was limited, I-BET151 concomitantly inactivated the BCR pathway and IKZF and had a high growth inhibitory efficacy in MCL cells. These results suggest that simultaneous targeting of multiple molecules involved in the BCR pathway and IKZF-MYC axis may overcome resistance to ibrutinib and/or lenalidomide in MCL, and that BRD4 inhibitors are promising candidates for MCL treatment. Disclosures Kuroda: Chugai Pharma: Honoraria, Research Funding. Taniwaki:Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.,: Research Funding; Astellas Pharma Inc,: Research Funding.

Sequence-Based Evidence for Antigen Selection in Mantle Cell Lymphoma: Remarkable Immunoglobulin Gene Repertoire Biases, Stereotyped Antigen-Binding Sites and Recurrent Hypermutations in Certain Subsets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1933-1933 ◽  
Author(s):  
Anastasia Hadzidimitriou ◽  
Andreas Agathagelidis ◽  
Fiona Murray ◽  
Marie-Helene Delfau-Larue ◽  
Nikos Darzentas ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
Antigen Binding ◽  
Gene Repertoire ◽  
Immunoglobulin Gene ◽  
Antigen Binding Site ◽  
Ighv Gene ◽  
Mantle Cell

Abstract Abstract 1933 Poster Board I-956 According to the 2008 WHO Classification, mantle cell lymphoma (MCL) most likely derives from neoplastic transformation of a peripheral B cell of the inner mantle zone, mostly of naïve pre-germinal center type. Analysis of the immunoglobulin (IG) repertoire in MCL has revealed a bias in IG heavy variable (IGHV) gene usage and a predominance of unmutated rearrangements, with a variable minor proportion of cases carrying mutated IGHV genes (according to the 98% identity cutoff). However, limited knowledge exists regarding antigen-binding site sequence restrictions or specific somatic hypermutation (SHM) patterns in MCL. We examined the productive IGHV-D-J rearrangements of 651 MCL cases (including 398 cases from the European MCL Network), the largest series to date. We confirm and significantly extend previous observations that the IGHV gene repertoire is remarkably biased, with only three genes accounting for 40.3% of cases (IGHV3-21, 16.7%; IGHV4-34, 15.3%; IGHV1-8: 8.3%). Using bioinformatics approaches previously applied to CLL, biased associations of certain IGHV, IGHD and IGHJ genes with restricted (stereotyped) heavy complementarity-determining regions (HCDR3s) were identified in 68/651 cases (10.4%). Overall, 24 subsets of cases with stereotyped HCDR3s were recognized. Eight of 24 subsets included 3-7 cases each (“confirmed”), while the remaining 16 subsets included two cases each and were considered “provisional”. Stereotyped HCDR3s were found predominantly amongst rearrangements utilizing the IGHV3-21 and IGHV4-34 genes and the IGHJ6 gene. Notably, the MCL HCDR3 stereotypes identified here were distinct from those previously reported in CLL. Based on SHM analysis, the sequences were divided into three groups: (i) truly unmutated (100% germline identity, GI): 189/651 sequences (29.2%); (ii) minimally/borderline mutated (98-99.9% GI): 306/651 sequences (47%); and (iii) mutated (<98% GI): 155/651 sequences (23.8%), of which 93 had a GI<97%. In keeping with previous reports, the IGHV gene repertoire of the three identity groups differed considerably. For instance, the IGHV3-21 gene was used by 7% of rearrangements with <98% identity vs. 22.8% of rearrangements with 100% identity. In contrast, the IGHV3-23 gene was over-represented among mutated rearrangements (26.4%). Shared (“stereotyped”) amino acid (AA) changes across the entire IGHV gene sequence were identified for certain groups of sequences, especially those utilizing the IGHV1-8, IGHV3-21 and IGHV3-23 genes. A comparison to published series from other entities, in particular CLL, revealed that several stereotyped mutations identified in MCL were “disease-biased”. Importantly, stereotyped AA changes were also observed in the borderline or minimally mutated groups, indicating that even a low level of mutations may be functionally relevant. In conclusion, MCL is characterized by a highly distinctive IG gene repertoire with restricted HCDR3s and very precisely targeted and, probably, functionally driven SHM, strongly implying a role for antigen-driven selection of the clonogenic progenitors. Based on the evidence presented here, an antigen-driven origin of MCL could be envisaged, at least for subsets of cases. Disclosures: No relevant conflicts of interest to declare.

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