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.

scholarly journals The Role of UBR5 Mutations in the Pathogenesis of Mantle CELL Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4124-4124
Author(s):  
Olga Kutovaya ◽  
Stacy Hung ◽  
Hughes Christopher ◽  
Randy D Gascoyne ◽  
Morin Gregg ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Cycle ◽  
Dna Damage ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Interacting Proteins ◽  
Mantle Cell ◽  
Damage Response

Abstract Intro: Mantle cell lymphoma (MCL) accounts for 6% of non-Hodgkin lymphomas and represents a particularly challenging disease with patient outcomes inferior to most other lymphoma subtypes. Using targeted capture sequencing of MCL biopsy samples, we recently reported frequent mutations (18%) in UBR5, a gene encoding an E3 ubiquitin-protein ligase that has not been previously implicated in lymphomagenesis. All mutations were clustered within 100bp in or around exon 58 of UBR5 and truncate the reading frame or change a key lysine residue. These mutations are predicted to result in the loss of the conserved cysteine residue in the HECT-domain, which is responsible for binding the ubiquitin co-factor. The recurrence and clustering of UBR5 mutations suggest their critical pathogenic involvement in a subgroup of MCL that might be therapeutically targetable. The aim of this study is to determine UBR5 mutation-associated proteome changes and altered cell signaling. Methods: As seen in MCL patients, mutations in exon 58 of UBR5 were introduced to three MCL cell lines (Granta-519, Jeko-1, and Mino) using the CRISPR-Cas9 genome engineering tool. First, mass spectrometry-based immunoprecipitation proteomics (IP-MS) was employed to identify differences in UBR5 interacting partners between UBR5 mutant and wildtype (WT) cells. Candidate UBR5 interacting proteins were validated by flow cytometry, western blotting, co-immunoprecipitation, and immunofluorescence. Next, global proteomes of UBR5 mutants and WT were analyzed by Tandem Mass Tag (TMT)-based mass spectrometry quantification to identify proteins with differential expression due to the UBR5 mutations. Results: The IP-MS analysis of WT vs UBR5 mutants revealed histone and cell cycle control proteins as candidate differential UBR5 interacting proteins (p<0.05). Particularly, histones H1, H4, and H2AFX, as well as the cell cycle genes CDC5L, BUB3, MAP4, RAD50 and CDK11B were identified as candidate UBR5 interacting partners. The global proteome analysis identified a set of differentially expressed genes (mutant vs wt; p<0.05) that are common among the MCL cell lines with the same direction of change. Gene ontology analysis of this set revealed DNA damage response, chromosome organization, and cell cycle response pathways as the predominant pathways affected. Moreover, our preliminary functional studies indicate constitutive phosphorylation of H2AFX in UBR5 mutants vs WT in line with the role of UBR5 in DNA damage response. Conclusions: Our results are consistent with UBR5 functioning as a key regulator of cell signalling and strongly suggest UBR5 as a novel regulator of histone modifications and DNA damage response. These findings provide an experimentally valid platform for further functional investigation and testing of target therapies for MCL harbouring UBR5 mutations. Disclosures No relevant conflicts of interest to declare.

A Synthetic Lethal RNA Interference Screen Identifies Components of Genotoxic Stress Response As Targets for Anti-CCND1 Therapy in Mantle Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2430-2430
Author(s):  
Suchismita Mohanty ◽  
Natalie Sandoval ◽  
Charles Warden ◽  
Vu N Ngo
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Genotoxic Stress ◽  
Rnai Screen ◽  
Synthetic Lethal ◽  
Mantle Cell ◽  
Damage Response

Abstract Abstract 2430 Background. Almost all cases of mantle cell lymphoma (MCL) harbor the t(11:14) chromosomal translocation resulting in overexpression of the cell cycle regulatory protein cyclin D1 (CCND1), which promotes cell proliferation and poor survival. Targeting CCND1 in vitro and clinically, however, is not sufficient to cause tumor cell death, suggesting that additional mechanisms compensate for MCL growth and survival. Unraveling these additional signals will help identify novel targets for rational combination therapies in MCL. Previously, we developed a novel functional genomics tool using an inducible RNA interference (RNAi) library, which can simultaneously assess the role of thousands of genes in cell viability in tumor cell lines. Here we apply the inducible RNAi screen to identify synthetic lethal interactions with CCND1 in MCL. The screen uncovered several components of the DNA damage response as potential new combination targets for anti-CCND1 therapy in mantle cell lymphoma. Methods. The RNAi library was previously constructed using a retroviral vector that inducibly expresses small-hairpin RNA (shRNA). Each shRNA vector contains a unique 60-mer bar code that can hybridize to a corresponding complementary sequence spotted on a custom Agilent oligonucleotide microarray. To perform a synthetic lethal RNAi screen for CCND1, we first established a stable MCL line (UPN-1) that express an inducible CCND1 or control shRNA, transduced these lines with the pooled shRNA library, selected for transduced cells with puromycin, and induced shRNA expression for eight days. Genomic DNA containing bar code sequences was then amplified by PCR, fluorescently labeled, and hybridized onto microarrays. Each screen was repeated four times to enable statistical analysis. Candidate shRNAs obtained from the screen were validated for synergistic killing of MCL cells when combined with CCND1 knockdown. We evaluated genotoxic stress response triggered by DNA damage following CCND1 inactivation in MCL lines by Western blots. DNA damage and repair were assessed by comet assays and immuno-fluorescent staining of DNA repair proteins including phospho-H2AX, RAD51 and 53BP1. Results. The RNAi screen uncovers multiple shRNAs targeting RIPK1, RIPK3, NEMO, and TAK1, which sensitize MCL cells to CCND1 inhibition. RIPK1, NEMO, and TAK1 have been shown to play an essential role in cells undergoing genotoxic stress by linking DNA damage-induced ATM activation and NF-kB activity. We demonstrated that silencing CCND1 in the MCL cell lines UPN-1, JEKO-1, Z138, and Granta-519 up-regulates RIPK1 mRNA and protein expression, in addition to increased phosphorylation of DNA damage response proteins such as ATM, CHEK1/2 and H2AX. We observed a two-fold increase of DNA damage levels in CCND1 knockdown cells as assessed by comet assays. We also detected cell cycle-independent increase of DNA double strand break (DSB) foci in CCND1 knockdown cells by staining with fluorescently labeled anti-phospho-H2AX antibody. Knockdown of RIPK1 in MCL lines (UPN-1 and JEKO-1) resulted in apoptotic cell death and these RIPK1 shRNA-transduced cells are hypersensitive to irradiation and DNA damaging agents, indicating RIPK1 plays a protective role against DNA damage-induced apoptosis. The survival role of RIPK1 in MCL cells may correlate with the DNA repair function as demonstrated by the inability of RIPK1 knockdown cells to efficiently resolve etoposide-induced DNA DSB foci over time. Furthermore, we also found that RIPK1 knockdown cells failed to down-regulate the G2/M cell cycle checkpoint protein CDC25B and to up-regulate ATM phosphorylation and Ku86 protein expression in response to genotoxic stress. Blocking these RIPK1-dependent responses could sensitize MCL cells to CCND1 knockdown-induced DNA damage. Similar analyses of the other hits from the RNAi screen are on going. Conclusions. There are few viable treatment options for mantle cell lymphoma. We have identified the receptor interacting protein kinase 1 (RIPK1), of the DNA damage response pathway, as a potential therapeutic target whose downregulation sensitizes MCL cells to anti-CCND1 treatment, possibly by promoting insurmountable genotoxic stress. Successful implementation of our functional genetic screens for genes that sensitize MCL cells to anti-CCND1 treatment could define novel targets suitable for effective combination therapies. Disclosures: No relevant conflicts of interest to declare.

Combined Targeting of Chromatin Modifying Enzymes LSD1, EZH2 and Histone Deacetylases (HDACs) Has Superior Efficacy Against Human Mantle Cell Lymphoma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2429-2429
Author(s):  
Warren Fiskus ◽  
Sunil Sharma ◽  
Rekha Rao ◽  
Ramesh Balusu ◽  
Sreedhar Venkannagari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Core Protein ◽  
Cell Lymphoma ◽  
Amine Oxidase ◽  
Combined Treatment ◽  
Reversible Inhibitor ◽  
Mantle Cell

Abstract Abstract 2429 PRC (polycomb repressive complex) 2 contains three core protein components, i.e., EZH2, SUZ12 and EED, of which EZH2 has the SET domain with its intrinsic histone methyltransferase activity that mediates the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive chromatin mark for gene expression. We have previously reported that treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep as well as treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharma) deplete PRC2 complex proteins. LSD1 (KDM1A) is a demethylase of H3K4Me2, and inhibiton of LSD1 leads to increase in H3K4Me3-a permissive mark for gene expression. In the present studies, we determined the chromatin-modifying and cytotoxic effects of LSD1 inhibition alone and in combination with PS or DZNep in cultured (JeKo-1 and Z138C) and primary human Mantle Cell Lymphoma (MCL) cells. Treatment with the non-amine oxidase reversible inhibitor of LSD1 CIT0665 (250 to 1000 nM), or the more potent analogue HCI2509 (20 to 250 nM), dose-dependently increased the levels of H3K4Me2 & Me3, p21 and p27, while decreasing the levels of cyclin D1, which was associated with inhibition of cell proliferation and accumulation of the MCL cells in the G1 phase of the cell cycle. Abrogation of LSD1 by a specific shRNA treatment also induced similar chromatin, cell cycle and growth inhibitory effects. Exposure to CIT0665 or HCI2509 disrupted the binding of LSD1 with the co-repressor CoREST and HDAC1, without affecting the levels of these proteins. As noted above, treatment with PS (10 to 50 nM) dose-dependently depleted the levels of not only EZH2, SUZ12 and the PRC1 complex protein BMI1, but also of LSD1 in MCL cells. PS treatment alone also depleted the levels of AKT, cRAF, CDK4 and cyclin D1, as well as induced cell cycle growth inhibition and apoptosis of MCL cells. Co-treatment with PS enhanced the chromatin modifying effects of CIT0665 or HCI2509. The combination synergistically induced apoptosis of the cultured MCL cells (combination indices, CI <1.0). This was associated with greater induction of p27 and depletion of cyclin D1. Treatment with PS and HCI2509 also synergistically induced loss of viability of primary MCL cells (CI <1.0). We have previously reported that DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression, inhibited H3K27Me3 levels, induced p21, p27 and FBXO32 (muscle atrophy F-box protein, also called atrogin-1) levels in cultured and primary MCL cells. Here, we determined the effects of co-treatment with HCI2509 and DZNep in MCL cells. Combined treatment with HCI2509, although not synergistic, enhanced the apoptosis of MCL cells induced by DZNep. Taken together these findings indicate that combined targeted depletion of the level and activity of LSD1 by PS and CIT0665 or HCI2509 along with PS-mediated depletion of PRC2 proteins, BMI and HDACs exerts superior activity against MCL cells. These studies also support the in vivo testing of combined epigenetic therapies in the treatment of MCL. Disclosures: Sharma: Novartis: Research Funding.

scholarly journals Role of UBR5 mutations in DNA damage response in mantle cell lymphoma

2017 ◽  
Vol 28 ◽  
pp. v583
Author(s):  
O. Kutovaya ◽  
S. Hung ◽  
E. Vigano ◽  
R. Gascoyne ◽  
G.B. Morin ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Mantle Cell ◽  
Damage Response

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2

2019 ◽  
Vol 105 (3) ◽  
pp. 839-853
Author(s):  
Aglaia Kyrilli ◽  
David Gacquer ◽  
Vincent Detours ◽  
Anne Lefort ◽  
Frédéric Libert ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Iodide Uptake ◽  
Transcriptomic Response ◽  
Uptake Inhibition ◽  
Γ Radiation ◽  
Damage Response

Abstract Background The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. Methods Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (β radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. Results Overall, the thyrocyte responses following exposure to β or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. β and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to β/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. Conclusions TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

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.

FTY720 Demonstrates Promising in-Vitro and in-Vivo Pre-Clinical Activity by Down-Modulation of Cyclin D1 and Pakt in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3728-3728
Author(s):  
Lapo Alinari ◽  
Qing Liu ◽  
Ching-Shih Chen ◽  
Fengting Yan ◽  
James T Dalton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lymphoma ◽  
Tumor Burden ◽  
Time Dependent ◽  
Control Group ◽  
Mantle Cell

Abstract Abstract 3728 Poster Board III-664 Over-expression of Cyclin D1 and constitutive phosphorylation of Akt has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Here we describe FTY720 (fingolimod), an immunosuppressive agent currently being explored in phase III studies in renal transplantation and multiple sclerosis patients, to mediate time- and dose-dependent cell death in primary MCL cells (6 patients) and MCL cell lines, Jeko and Mino. FTY720-induced apoptosis was associated with reactive oxygen species (ROS) generation, Bax up-regulation but not associated with caspase 3 activation in MCL. FTY720 treatment resulted in time-dependent down-modulation of Cyclin D1 and phospho Akt (p-Akt) protein level, two critical disease-relevant molecules in the pathogenesis of MCL. Consistent with the modulation of Cyclin D1, FTY720-induced cell cycle arrest with accumulation of cells in G0/G1 and G2/M phases of the cell cycle with concomitant decrease in S phase entry. Importantly, FTY720 treatment was also associated with a time-dependent phospho Erk (p-Erk) induction in Mino and Jeko cells. To determine the in vivo efficacy of FTY720, we developed a preclinical, in vivo xenograft model of human MCL where MCL cell lines (Jeko, Mino and SP53) were engrafted into severe combined immune deficient (SCID) mice. Cell dose titration trials identified 4 × 107 Mino or Jeko cells injected intravenously via tail vein to result in consistent engraftment and fatal tumor burden in all mice. All mice engrafted with 4 × 107 Jeko cells developed a disseminated disease within 3 weeks and had a median survival of 28 days (compared to 43 days for Mino and 51 days for SP53). Because the Jeko cell line was established from the peripheral blood of a patient with blastic variant MCL and demonstrated a more resistant phenotype to several immuno-chemoterapeutic compounds, this cell line was chosen to create a more stringent in vivo preclinical model. SCID mice were treated with the monoclonal antibody TMβ1 to deplete murine NK cells, engrafted with 4 × 107 Jeko cells and observed daily for signs of tumor burden. Ten mice/group were treated starting at day 15 post-engraftment with intraperitoneal injection of 100 μl of saline or FTY720 (5 mg/kg resuspended in 100 μl of saline), every day, for two weeks. The median survival for FTY720-treated mice (N=10) was 38 days (95% CI:30-39) compared to 26.5 days (95% CI: 26-27 days) for the control group mice (N=10). The results from the log-rank test indicated an overall statistical significant difference in survival functions between the FTY720 treatment and the control group (p=0.001). These results provide the first evidence for a potential use of FTY720 in targeting key pathways that are operable in the pathogenesis of MCL and warrant the further investigation of FTY720 in combination with other agents in clinical trials treating patients with MCL. Disclosures: No relevant conflicts of interest to declare.

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