scholarly journals UBR5 Mutations in Mantle Cell Lymphoma Lead to Increased Proliferation through a Cyclin D1-Dependent Mechanism

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2849-2849
Author(s):  
Olga Kutovaya ◽  
Stacy Hung ◽  
Elena Viganò ◽  
Adele Telenius ◽  
Bruce W Woolcock ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Gene Mutations ◽  
Non Hodgkin Lymphoma ◽  
Expression Of Genes ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is an aggressive type of non-Hodgkin lymphoma, with patient outcomes inferior to most other lymphoma subtypes. Recent progress in describing recurrent somatic gene mutations has led to a better understanding of MCL pathogenesis. However, the functional and clinical implications of many alterations remain to be elucidated. Here, to uncover the role of recurrent UBR5 gene mutations in lymphomagenesis, we studied a cohort of 248 MCL patients by targeted sequencing and performed genome editing of MCL-derived cell lines to investigate UBR5-mutation associated phenotypes in vitro. We identified deleterious UBR5 exon 58 hotspot mutations in 8% of MCL patients, all of which were mutually exclusive with CCND1 mutations. Proteomics analysis of Granta-519 and Jeko-1 cell lines with engineered UBR5 exon 58 indel mutations showed differential expression of genes involved in cell cycle and ubiquitination, and led to the discovery of decreased phosphorylation of CCND1 in the UBR5-mutated lines. Accordingly, in vitro studies of engineered genome-edited Granta-519, Jeko-1 and Mino cells revealed accumulation of cells in the S phase of the cell cycle, increased phosphorylation of retinoblastoma protein (Rb), and increased lymphoma cell proliferation. Our results demonstrate that UBR5 mutations, in addition to the hallmark t(11;14) translocation drive proliferation of MCL cells, potentially rendering mutation-carrying cells more sensitive to targeted therapies. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Roche: Research Funding; Celgene: Consultancy, Honoraria; Janssen: Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: patent holding; Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Tioma: Research Funding.

Enzastaurin Treatment Affects Multiple Regulatory Pathways at Transcriptome and Cellular Proteome Level of Mantle Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2893-2893
Author(s):  
Marc Weinkauf ◽  
Grit Hutter ◽  
Yvonne Zimmermann ◽  
Malte Rieken ◽  
Alessandro Pastore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Signal Pathways ◽  
Rna Expression ◽  
Cellular Functions ◽  
Mantle Cell ◽  
Canonical Pathways

Abstract Abstract 2893 Background: The protein kinase C beta inhibitor enzastaurin is one of the promising molecular targeted approaches currently investigated in mantle cell lymphoma (MCL), a disease still characterized by a dismal long term prognosis. Methods: Four well characterized MCL cell lines (Granta 519, HBL-2, Jeko-1 and Rec-1) as well as three patient samples were exposed to enzastaurin at a previously defined dose (10 μM). Cell viability as well as cell cycle activity were analyzed by tryphan blue exclusion test and flow cytometry, respectively, after 24 and 48 hours. To dissect the regulatory processes targeted by enzastaurin, the panel of MCL cell lines was screened on both protein and RNA expression levels (2D-gel electrophoresis and mass spectrometric peptide fingerprint analysis and Affymetrix microarray) after 4h enzastaurin treatment. Results: Enzastaurin in vitro resulted in a reduced viability and cell proliferation by 15–20% after 24h in cell lines and 9–20% in primary patient samples after 48h. This effect was related to a G2/M block of cell cycle and induction of apoptosis. Based on the proteome and transcriptome analysis of early alterations, only HSPD1 was affected on both regulation levels. Nonetheless, combined analysis of alterations on both, protein and RNA expression levels, resulted in identification of common signal pathways characterizing a more comprehensive network of affected molecular interactions mapping to distinct canonical pathways and defined cellular functions. Indicated canonical pathways included ‘calcium signalling', (CAMKK2, HDAC5, HDAC9, TP63) ‘calcium induced T-lymphocyte apoptosis' (MEF2D, NR4A1, PRKCG, TRA@), ‘NFkB signalling' (KRAS, MAP3K8, TNFAIP3, TNFRS17) and ‘molecular mechanisms of cancer' (APAF1, CDKN2D, FOS, PAK6), whereas the top ranking cellular functions were ‘cellular growth and proliferation' (CCNG2, EIF4E, PDIA3, TOP1, TPM1,), ‘cell death' (BCL6, EEF1D, PAK6, RAD50), ‘cell cycle'(AKAP9, BMF, CUL5, GADD45B, PDIA3), ‘cellular development' (APAF1, GAS7, ID1, PAX8) and ‘gene expression' (ABCG1, HOXB4, LMO4, PIM1). Alterations of these pathways were confirmed by Western Blot analysis of selected candidate proteins marker proteins of the regulated pathways. Conclusion: In summary, the combined approach of RNA and protein analysis revealed the targeted signal pathways after Enzastaurin exposure. These data will allow a more rationally designed combination of biologicals to finally improve the clinical outcome of MCL. Disclosures: Dreyling: Eli Lilly: Support of in vitro studies of Enzostaurin in MCL.

Targeting Oxphos Pathway Against Ibrutinib Resistance to Mantle Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 290-290 ◽  
Author(s):  
Yang Liu ◽  
Taylor Bell ◽  
Hui Zhang ◽  
Yuting Sun ◽  
Carrie J Li ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Gene Set Enrichment Analysis ◽  
Pdx Model ◽  
Mantle Cell ◽  
Ibrutinib Resistance

Abstract Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially responsive but ultimately relapses to frontline therapy. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) has achieved 68% of overall response rate in relapsed/refractory mantle cell lymphoma (MCL) patients. However, the vast majority of MCL patients experience disease progression, demonstrating that standard-of-care approaches are failing and that a means for targeting ibrutinib resistant MCL is clinically needed. Our hypothesis is that the ibrutinib-resistant MCL may rely on the mitochondrial oxidative phosphorylation (OXPHOS) pathway to produce energy for tumor growth. In this study, we investigated the effects of IACS-010759, a small molecule mitochondrial complex I inhibitor discovered in MD Anderson Cancer Center which can block the OXPHOS pathway, to overcome ibrutinib resistance in MCL in vitro and in a patient-derived xenograft (PDX) model. Methods: The OXPHOS metabolic pathways were investigated by RNASeq in a panel of ibrutinib-sensitive and -resistant MCL samples. Cell growth inhibition assays were tested after 72-hour treatment with IACS-010759 in ibrutinib-resistant MCL cell lines, Z-138 and Maver-1, and ibrutinib-sensitive MCL cell lines, Rec-1, Mino, and Jeko-1, by CellTiter-Glo luminescent cell viability assay (Promega). Furthermore, an IBN-resistant MCL PDX model was established and the therapeutic effects and tolerability of IACS-010759 were investigated in the primary MCL-bearing PDX model. Results: We have done RNA sequencing (RNASeq) in 7 primary ibrutinib-resistant and 16 ibrutinib-sensitive MCL patient samples, and analyzed the data using Gene Set Enrichment Analysis (GSEA) software. The results demonstrated that the OXPHOS pathway was activated in the primary ibrutinib-resistant MCL cells but not ibrutinib-sensitive MCL cells. Based on the RNASeq data, we selected an OXPHOS inhibitor IACS-010759 to investigate its effects on both primary ibrutinib-resistant and ibrutinib-sensitive MCL cells in vitroand in PDX mice. IACS-010759 significantly inhibited cell proliferation in ibrutinib-resistant MCL cell lines, Z-138 and Maver-1, but not in ibrutinib-sensitive MCL cell lines, Rec-1, Mino, and Jeko-1, during a 72-hour incubation. Furthermore, the primary ibrutinib-resistant MCL PDX mice were administrated with 10 mg/kg IACS-10759 by oral gavage, for 28 days using a 5 on/2 off dosing schedule. Our data showed that IACS-010759 completely eradicated tumor growth in ibrutinib-resistant MCL PDX mice (n=5, p=0.045). All mice tolerated the treatment dose and no toxicity was found during 28 days of IACS-010759 treatment. Conclusions: The OXPHOS inhibitor IACS-010759 overcomes ibrutinib resistance both in vitro and in the PDX mouse model. The investigation of its mechanism-of-action is ongoing. IACS-010759 could have the potential for clinical use in ibrutinib-resistant relapsed/refractory MCL patients. Disclosures Wang: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Juno Therapeutics: Research Funding; Asana biosciences, Beigene, Celgene, Juno, Kite, Onyx, Pharmacyclics: Research Funding; Dava Oncology: Honoraria; BeiGene: Research Funding; Acerta: Consultancy, Research Funding.

scholarly journals Pirtobrutinib Overcomes Ibrutinib and Venetoclax Resistance in Mantle Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1182-1182
Author(s):  
Yang Liu ◽  
Changying Jiang ◽  
Fangfang Yan ◽  
Joseph McIntosh ◽  
Alexa A Jordan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
Mantle Cell ◽  
Btk Inhibitor

Abstract Background Mantle cell lymphoma (MCL) is a rare and aggressive B-cell lymphoma characterized by poor prognosis. Although remarkable therapeutic advances have been made by covalent Bruton's tyrosine kinase (BTK) inhibition and CAR T cell therapy, therapeutic resistance inevitably occurs and leads to dismal clinical outcome. Pirtobrutinib (LOXO-305) is a next-generation, highly selective and non-covalent BTK inhibitor. A phase 1/2 BRUIN study showed that pirtobrutinib demonstrated promising efficacy in heavily pretreated MCL patients with or without prior covalent BTK inhibition. Here, we investigated the mechanism of action of pirtobrutinib in MCL cells in vitro and proposed the potential combination therapy in a venetoclax-resistant xenograft model. Methods MCL cell proliferation was monitored by trypan blue exclusion assay after 24-, 48- and 72-hour treatment with pirtobrutinib and ibrutinib. We performed Annexin V/PI staining to measure the apoptosis inductive effects. Cell cycle analysis using propidium iodide (PI) DNA staining was conducted to compare cell cycle progression kinetics between pirtobrutinib and ibrutinib. We performed RNAseq analysis in Z138 cells to compare differentially expressed genes (DEGs) between pirtobrutinib and ibrutinib treatment. Western blotting was utilized to detect specific signaling proteins. Mino-venetoclax-R cells were inoculated subcutaneously into NSG mice and used for in vivo drug efficacy determination. Results Compared to covalent BTK inhibitor ibrutinib, the novel non-covalent BTK inhibitor pirtobrutinib was more potent in inhibiting MCL cell proliferation in a panel of MCL cell lines, especially in ibrutinib/venetoclax resistant cell lines (pirtobrutinib vs. ibrutinib, p<0.01). Treatment with pirtobrutinib (10μM) for 24 hours induced higher levels of apoptosis than that by ibrutinib in all the MCL cell lines tested (p<0.05), which was also confirmed at the molecular level by stronger caspase-3 activation and PARP cleavage. To understand the mechanism of action, we performed whole transcriptomic profiling by RNAseq analysis using Z138 cells treated with/without pirtobrutinib or ibrutinib. Pirtobrutinib treatment resulted in upregulation of 137 genes and downregulation of 97 genes compared to the ibrutinib treatment (adjusted p<0.05). In addition to the downregulated MYC targets and PI3K/Akt pathway, gene set enrichment analysis (GSEA) revealed a significant enrichment for G2/M checkpoints and E2F targets signatures (key genes: PLK1, CDKN1A and CCNB1) in pirtobrutinib treated cells. Consistently, follow-up studies showed that γH2AX level was highly increased upon pirtobrutinib treatment. Pirtobrutinib treatment but not ibrutinib treatment resulted in G2/M cell cycle arrest. The blockade of cell cycle progression is positively correlated with decreased protein levels of critical regulators of S and G2/M phase transition such as cyclin B and CDC25C. BTK inhibitor (ibrutinib) in combination with venetoclax has shown great efficacy in preclinical models and in MCL patients. Therefore, here we assessed the in vivo efficacy of pirtobrutinib in combination with venetoclax with side-by-side comparison to ibrutinib & venetoclax in the Mino-venetoclax-R mouse model. Pirtobrutinib & venetoclax combination enhanced the efficacy of pirtobrutinib in restraining the tumor size (p<0.001) in the xenograft model. Notably, this novel combinatorial treatment exerted much higher potency than ibrutinib and venetoclax combination therapy (p<0.001). In addition, the pirtobrutinib & venetoclax combination was well tolerated and did not reduce overall mouse body weights compared with the vehicle treated mice. Conclusions Pirtobrutinib overcame both ibrutinib and venetoclax resistance in MCL cells in vitro and in vivo. G2/M checkpoints and E2F targets pathways were significantly enriched in both cases. Pirtobrutinib & venetoclax showed better in vivo efficacy in MCL models than combination of ibrutinib & venetoclax. Figure 1 Figure 1. Disclosures Wang: Genentech: Consultancy; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; CAHON: Honoraria; InnoCare: Consultancy, Research Funding; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Lilly: Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; OMI: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bayer Healthcare: Consultancy; Chinese Medical Association: Honoraria; Dava Oncology: Honoraria; Celgene: Research Funding; Mumbai Hematology Group: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; Newbridge Pharmaceuticals: Honoraria; CStone: Consultancy; BGICS: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Scripps: Honoraria; Epizyme: Consultancy, Honoraria; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; VelosBio: Consultancy, Research Funding; Anticancer Association: Honoraria.

Characterization of 4 Mantle Cell Lymphoma Cell Lines

2003 ◽  
Vol 127 (4) ◽  
pp. 424-431 ◽  
Author(s):  
Hesham M. Amin ◽  
Timothy J. McDonnell ◽  
L. Jeffrey Medeiros ◽  
Georgios Z. Rassidakis ◽  
Vasiliki Leventaki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
In Vitro Model ◽  
Cell Lymphoma ◽  
Cyclin D3 ◽  
Mantle Cell ◽  
Vitro Model

Abstract Context.—Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin lymphoma characterized by t(11;14)(q13;q32) and cyclin D1 overexpression. The pathogenesis of MCL has not been comprehensively studied, which can be attributed in part to the paucity of well-characterized MCL cell lines. Objectives.—We collected 4 previously developed MCL cell lines and performed extensive characterization, including the susceptibly of these cell lines to transduction by adenovirus vectors. Our aim was to facilitate the establishment of an in vitro model that can be reliably used to study the pathogenesis of MCL. Methods.—Standard techniques were used to compare the morphologic, immunophenotypic, and cytogenetic features of the 4 cell lines. In addition, Western blotting was used to investigate the presence of several cell cycle- and apoptosis-related proteins. TP53 DNA sequencing was also performed on the cell lines. The adenoviral transduction efficiency was assessed using an adenoviral vector carrying the gene encoding for the green fluorescence protein (Ad-GFP). Results.—All cell lines demonstrated evidence of t(11;14)(q13;q32) and overexpression of cyclin D1. Cyclin D2 was not detectable in all cell lines, whereas cyclin D3 was weakly expressed in JeKo-1 and SP-53. Other abnormalities of the cell cycle G1 phase regulatory pathway were detected, including loss of expression of p53 (JeKo-1) and p16INK4a (SP-53 and Granta 519), as well as TP53 mutation (Mino). All cell lines express high levels of cyclin E, c-Myc, Bcl-2, Bax, Bcl-xL, and Mcl-1. Retinoblastoma protein is hyperphosphorylated in all cell lines. With the exception of Mino, MCL cell lines are highly transducible with adenoviral vectors. Conclusion.—These cell lines are representative of MCL and can be used as an in vitro model to further explore the pathogenesis of this disease. The susceptibility of these cell lines to gene transfer provides opportunities to evaluate the importance of various oncogenes and tumor suppressor genes that may have an impact on developing effective therapeutic regimens for MCL.

scholarly journals BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

ESMO Open ◽  
2018 ◽  
Vol 3 (6) ◽  
pp. e000387 ◽  
Author(s):  
Chiara Tarantelli ◽  
Elena Bernasconi ◽  
Eugenio Gaudio ◽  
Luciano Cascione ◽  
Valentina Restelli ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Antitumour Activity ◽  
Single Agent ◽  
Treatment Strategies ◽  
Bet Inhibitor ◽  
Mantle Cell

BackgroundThe outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials.Materials and methodsThe activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines.ResultsBirabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines.ConclusionOur data provide the rationale to evaluate birabresib in patients affected by MCL.

Cell Cycle Regulation by Iron: Novel Approaches to Mantle Cell Lymphoma Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2515-2515 ◽  
Author(s):  
Heather Gilbert ◽  
John Cumming ◽  
Josef T. Prchal
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Iron Chelation ◽  
Lymphoma Cell ◽  
Protein Levels ◽  
Mantle Cell

Abstract Abstract 2515 Poster Board II-492 Mantle cell lymphoma is a well defined subtype of B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the BCL1 gene on chromosome 11q13 (which encodes cyclin D1) next to the immunoglobulin heavy chain gene promoter on chromosome 14q32. The result is constitutive overexpression of cyclin D1 (CD1) resulting in deregulation of the cell cycle and activation of cell survival mechanisms. There are no “standard” treatments for MCL. Despite response rates to many chemotherapy regimens of 50% to 70%, the disease typically progresses after treatment, with a median survival time of approximately 3-4 years. Mantle cell lymphoma represents a small portion of malignant lymphomas, but it accounts for a disproportionately large percentage of lymphoma-related mortality. Novel therapeutic approaches are needed. In 2007, Nurtjaha-Tjendraputra described how iron chelation causes post-translational degradation of cyclin D1 via von Hippel Lindau protein-independent ubiquitinization and subsequent proteasomal degradation (1). Nurtjaha-Tjendraputra demonstrated that iron chelation inhibits cell cycle progression and induces apoptosis via proteosomal degradation of cyclin D1 in various cell lines, including breast cancer, renal carcinoma, neuroepithelioma and melanoma. Our preliminary data show similar findings in mantle cell lymphoma. To establish whether iron chelation can selectively inhibit and promote apoptosis in mantle cell derived cell lines, the human MCL cell lines Jeko-1, Mino, Granta and Hb-12; the Diffuse Large B cell lymphoma line SUDHL-6; and the Burkitt's Lymphoma lines BL-41 and DG75 were grown with media only, with two different iron chelators (deferoxamine (DFO) and deferasirox) at various concentrations (10, 20, 40, 100 and 250 μM), and with DMSO as an appropriate vehicle control. Cells were harvested at 24, 48 and 72 hours. For detection of apoptotic cells, cell-surface staining was performed with FITC-labeled anti–Annexin V antibody and PI (BD Pharmingen, San Diego, CA). Cell growth was analyzed using the Promega MTS cytotoxicity assay. CD1 protein levels were assessed using standard Western blot techniques. At 24, 48 and 72 hours of incubation with iron chelators, the mantle cell lymphoma cell lines showed significantly increased rates of apoptosis compared to the non-mantle cell lymphoma cell lines (p<0.0001 for all time points). DFO and deferasirox inhibted cell growth with an IC50 of 18 and 12 μM respectively. All of the mantle cell lines had measurable cyclin D1 levels at baseline. None of the non-mantle cell lines expressed baseline measurable cyclin D1. In the mantle cell lines, cyclin D1 protein levels were no longer apparent on western blot after 24 hours of incubation with chelation. We then added ferrous ammonium sulfate (FAS) to DFO in a 1:1 molarity ratio and to deferasirox in a 2:1 ratio, and then treated the same lymphoma cell lines with the FAS/chelator mixture and with FAS alone for 72 hours. Adding iron to the chelators completely negated all the pro-apoptotic effects that were seen with iron chelation treatment. Treating with FAS alone had no effect on cell growth or apoptosis. Iron chelation therapy with both DFO and deferasirox results in decreased cell growth, increased cellular apoptosis, and decreased cyclin D1 protein levels in vitro in mantle cell lymphoma. The cytotoxic effects are prevented by coincubation with ferrous ammonium citrate, confirming that the effects are due to iron depletion. Proposed future research includes further defining the molecular basis of iron chelation effects; studying these therapies in combination with other cancer treatments both in vitro and in vivo; and studying iron chelation therapy in mantle cell lymphoma patients. 1. Nurtjahja-Tjendraputra, E., D. Fu, et al. (2007). “Iron chelation regulates cyclin D1 expression via the proteasome: a link to iron deficiency-mediated growth suppression.” Blood109(9): 4045–54. Disclosures: No relevant conflicts of interest to declare.

GST-n and Nitric Oxide: A Novel Approach to Mantle Cell Lymphoma Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3729-3729
Author(s):  
Heather Gilbert ◽  
John Cumming ◽  
Josef T. Prchal ◽  
Michelle Kinsey ◽  
Paul Shami
Keyword(s):  
Nitric Oxide ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
Malignant Cells ◽  
Mantle Cell ◽  
Tumor Viability

Abstract Abstract 3729 Poster Board III-665 Mantle cell lymphoma (MCL) is a well defined B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the BCL1 gene on chromosome 11q13, which encodes cyclin D1 (CD1), next to the immunoglobulin heavy chain gene promoter on chromosome 14. The resulting constitutive overexpression of CD1 leads to a deregulated cell cycle and activation of cell survival mechanisms. In addition, the gene which encodes GST-n, an enzyme that has been implicated in the development of cancer resistance to chemotherapy, is also located on chromosome 11q13 and is often coamplified along with the BCL1 gene in MCL (1). These two unique biological features of MCL - the overproduction of cyclin D1 and GST-n – may be involved in the carcinogenesis, tumor growth and poor response of this disease to treatment, and they offer potential mechanisms for targeted anti-cancer therapy. Nitric oxide (NO) is a biologic effector molecule that contributes to a host's immune defense against microbial and tumor cell growth. Indeed, NO is potently cytotoxic to tumor cells in vitro (2–4). However, NO is also a potent vasodilator and induces hypotension, making the in vivo administration of NO very difficult. To use NO in vivo requires agents that selectively deliver NO to the targeted malignant cells. A new compound has recently been developed that releases NO upon interaction with glutathione in a reaction catalyzed by GST-n. JS-K seeks to exploit known GST-n upregulation in malignant cells by generating NO directly in cancer cells, and it has been shown to decrease the growth and increase apoptosis in vitro in AML cell lines, AML cells freshly isolated from patients, multiple myeloma cell lines, hepatoma cells and prostate cancer cell lines (3, 5–7). JS-K also decreases tumor burden in NOD/SCID mice xenografted with AML and multiple myeloma cells (5, 7). Importantly, JS-K has been used in cytotoxic doses in the mouse model without significant hypotension. To evaluate whether JS-K treatment has anti-tumor activity in MCL, the human MCL cell lines Jeko1, Mino, Granta and Hb-12 were grown with media only, with JS-K at varying concentrations and with DMSO as an appropriate vehicle control. For detection of apoptotic cells, cell-surface staining was performed with FITC-labeled anti–Annexin V and PI. Cell growth was evaluated using the Promega MTS cytotoxicity assay. Results show that JS-K (at concentrations up to 10 μM) inhibits the growth of MCL lines compared to untreated controls, with an average IC50 of 1 μM. At 48 hours of incubation, all cell lines showed a significantly greater rate of apoptosis than untreated controls. A human MCL xenograft model was then created by subcutaneously injecting two NOD/SCID IL2Rnnull mice with luciferase-transfected Hb12 cells. Seven days post-injection, one of the mice was treated with JS-K at a dose of 4 μmol/kg (expected to give peak blood levels of around 17 mM in a 20 g mouse). Injections of JS-K were given intravenously through the lateral tail vein 3 times a week. The control mouse was injected with an equivalent volume of micellar formulation (vehicle) without active drug. The Xenogen bioluminescence imaging clearly showed a difference in tumor viability, with a significantly decreased signal in the JS-K treated mouse. Our studies demonstrate that JS-K markedly decreases cell proliferation and increases apoptosis in a concentration- and time-dependent manner in mantle cells in vitro. In a xenograft model of mantle cell lymphoma, treatment with JS-K results in decreased tumor viability. Proposed future research includes further defining the molecular basis of these treatment effects; using this therapy in combination with other cancer treatments both in vitro and in vivo; and studying JS-K treatment in MCL patients. Disclosures: Shami: JSK Therapeutics: Founder, Chief Medical Officer, Stockholder.

Whole Transcriptome Sequencing Reveals Recurrent NOTCH1 Mutations in Mantle Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 436-436 ◽  
Author(s):  
Robert Kridel ◽  
Barbara Meissner ◽  
Sanja Rogic ◽  
Merrill Boyle ◽  
Adele Telenius ◽  
...  
Keyword(s):  
Overall Survival ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
The Other ◽  
Mantle Cell ◽  
Whole Transcriptome ◽  
Notch1 Mutations

Abstract Abstract 436 Background: Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma that is characterized by the hallmark t(11;14)(q13;q32) translocation, as well as a high number of secondary chromosomal alterations. Further, a small number of genes such as TP53, ATM and CCND1 have been reported to be recurrently mutated in MCL, but do not fully explain the biology and do not adequately account for the wide spectrum of clinical manifestations, response to treatment and prognosis. The aim of this study was to discover new somatic mutations that could contribute to our understanding of the pathogenesis of MCL. Methods: In our discovery cohort, we sequenced the transcriptomes of 18 clinical samples (11 diagnostic and 7 progression biopsies) and 2 mantle cell lymphoma-derived cell lines (Mino and Jeko-1). For this purpose, whole transcriptome shotgun sequencing was performed on RNA extracted from fresh frozen tissue. We assembled an extension cohort of 103 diagnostic patient samples and 4 additional cell lines (Rec-1, Z-138, Maver-1, JVM-2), and performed Sanger sequencing of NOTCH1 exons 26, 27 and 34 on genomic DNA. We further exposed the 6 cell lines to 1 μM of the γ-secretase inhibitor XXI (compound E) for 7 days and measured cellular proliferation with an EdU incorporation assay. Survival analysis was carried out in the 113 patients with diagnostic biopsies and available outcome data. Results: NOTCH1 mutations were found in 14 out of 121 patient samples (11.6%) and in 2 out of 6 cell lines, Mino and Rec-1 (33.3%). The majority of these mutations (12 out of 14) lie in exon 34 that encodes the PEST domain of NOTCH1 and consist of either small frameshift-causing indels (10 cases) or nonsense mutations (2 cases). These mutations are predicted to cause truncations of the C-terminal PEST domain. To gain further insight into functional relevance, we treated 6 cell lines with compound E, an inhibitor of the γ-secretase complex that plays a critical role in the release of the intracellular domain of NOTCH1 after ligand-induced activation. In Rec-1, that harbours a NOTCH1 mutation, we observed a significant decrease in proliferation (mean percentage of cells in culture incorporating EdU decreasing from 47.5% to 1.4%, p<.001). No effect of compound E was observed in Mino, the other cell line with a NOTCH1 mutation, nor in the 4 cell lines that are wild type for NOTCH1. Outcome correlation analysis showed that NOTCH1 mutations are associated with poor overall survival (1.56 versus 3.86 years respectively, p=.001), but not with significantly shortened progression-free survival (0.88 versus 1.73 years respectively, p=.07). Discussion: We have identified recurrent mutations in NOTCH1 in a subset of patients with MCL (11.6%). The frequency and the pattern of mutations are strikingly similar to what has recently been reported in chronic lymphocytic leukemia, the other major CD5 positive B-cell malignancy (Nature, 2011 Jun 5, 475:101–105 and J Exp Med, 2011 Jul 4, 208:1389–1401). NOTCH1 mutations are associated with adverse prognosis as evidenced by shortened overall survival. This latter finding, however, should ideally be validated in a larger and uniformly treated cohort. Finally, the sensitivity of the Rec-1 cell line to compound E suggests that NOTCH1 mutations could serve as the target for tailored therapy in mantle cell lymphoma. Disclosures: Sehn: Roche/Genentech: Consultancy, Honoraria, Research Funding. Connors:Roche: Research Funding.

The HDACi Romidepsin Markedly Synergizes With Inhibition Of ATM By KU60019 In Mantle Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3066-3066 ◽  
Author(s):  
Luigi Scotto ◽  
Kelly Zullo ◽  
Xavier Jirau Serrano ◽  
Laura K Fogli ◽  
Owen A. O'Connor
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Cdk Inhibitor ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is a disease characterized by gross cell cycle dysregulation driven by the constitutive overexpression of cyclin D1. The identification of a “proliferation signature” in MCL, underscores the necessity of new therapeutic approaches aimed at lowering the proliferative signature of the disease, theoretically shifting the prognostic features of the disease. Romidepsin, an HDAC inhibitor (HDACi) approved for the treatment of relapsed T-cell lymphoma, is thought to induce cell cycle arrest and apoptosis. Central to the block of cell proliferation is the up-regulation of the cdk inhibitor p21Cip1/Waf1. However up-regulation of p21Cip1/Waf1 has also been shown to reduce sensitivity to romidepsin. HDACi activates p21Cip1/Waf1 expression via ATM and KU60019, a specific ATM inhibitor, has been shown to decrease the p21Cip1/Waf1 protein levels in a concentration dependent manner. We sought to explore the effect of the combination of romidepsin and KU60019 in inducing cell death in MCL. Analysis of romidepsin treated Jeko-1 cell extracts showed a marked effect on the expression of proteins involved in cell cycle regulation. Decrease expression of Emi1, a mitotic regulator required for the accumulation of the APC/C substrates was observed. Emi1 is also responsible for the stability of the E3 ubiquitin ligase Skp2 that specifically recognizes and promotes the degradation of phosphorylated cdk inhibitor p27. However, decrease in Emi1 protein levels, upon addition of romidepsin, was not followed by an increased expression of the cdk inhibitor p27. On the other end, increased expression of the cdk inhibitor p21Cip1/Waf1, was a common feature of all romidepsin treated MCL lines analyzed. Cell cycle analysis via Fluorescent Activated Cell Sorting (FACS) of romidepsin treated Jeko-1 cells showed an accumulation of romidepsin treated cells in the G2/M phase when compared to the control suggesting a p21Cip1/Waf1 induced cell cycle arrest. For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-GloTM followed by acquisition on a Biotek Synergy HT and IC50s calculated using the Calcusyn software. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR). Synergy analyses were performed using Jeko-1, Maver-1 and Z-138 cells treated with different concentrations of romidepsin corresponding to IC10-20 in combination with KU60019 at a concentration of 2.5, 5.0, 7.5 and 15 umol/L for 24, 48 and 72 hours. A synergistic cytotoxic effect was observed in all MCL cell lines when the HDACi was combined with KU60019 throughout the range of all concentrations. The RRR analysis showed a strong synergism at 48 and 72 hours in virtually all combinations of HDACi and KU60019 in all three cell lines. The results of drug:drug combination in two of the three cell lines are shown below. Protein expression analysis of Jeko-1 and Maver-1cells treated with single agents or combinations for 48 hours revealed changes in a host of proteins known to be involved in cell cycle control and apoptosis. The increased p21 protein expression upon addition of romidepsin, was not observed when the romidepsin treatment was combined with the KU60019. Increased activation of the programmed cell death proteins Caspase 8, induced by Fas, and Caspase 3 was observed upon combinations of the single agents in all three cell lines, resulting in an increased cleavage of Poly (ADP-ribose) polymerase (PARP-1). Finally, the abundance of the anti-apoptotic proteins Bcl-XL and BCL-2 showed a significant decrease after treatment with romidepsin plus increase concentrations of KU60019 when compared with their abundance in the presence of the single agents. Cell cycle analysis of Jeko-1 cells treated for 24 hours with single agents and combination suggests that the increased apoptosis is the result of inhibition of the p21Cip1/Waf1 induced G2/M cell cycle arrest by KU60019. Overall, these data demonstrated that the combination of romidepsin and KU60019 was synergistically effective in inhibiting the in vitro growth of the mantle cell lymphoma lines. Jeko-1 Maver-1 Disclosures: O'Connor: Celgene: Consultancy, Research Funding.

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