Ras-Pathway Inhibition With Targeted Therapies Abrogates Self-Renewal In Acute Myelogenous Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 819-819
Author(s):  
Rebecca S. LaRue ◽  
Hanh Nguyen ◽  
Karen Sachs ◽  
Nurul Azyan Mohd Hassan ◽  
Ernesto Diaz-Flores ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Stem Cell Maintenance ◽  
Acute Myelogenous ◽  
Primary Leukemia ◽  
Cell Maintenance

Abstract Hyperactivated Ras-pathways serve as oncogenic drivers in multiple human tumors including acute myelogenous leukemia (AML) (Ahearn et al. Nat Rev Mol Cell Biol 2011). The specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Recently, we have shown that NRASG12V–activated signaling pathways are critical to leukemia stem cell maintenance (Sachs et al. submitted). To elucidate which Ras-activated signaling molecules mediate self-renewal in AML, we employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V) and develops AML (Kim et al. Blood 2009). Primary leukemia cells were treated with therapeutic agents targeting Ras-activated signaling pathways. We used PD325901 to inhibit the Mek-Erk pathway, GDC0941 to inhibit the Pi3k pathway, and RAD001 to inhibit the mTor pathway. Using MTS assays, we identified the IC50 dose for each of these agents. Inhibitor-treated leukemia cells were submitted for RNA sequencing in order to investigate the effects of these agents on leukemia gene expression. Previously, we identified a list of NRASG12V responsive genes in our model. In these studies, we identified that PD325901-treatment most closely recapitulates the effect of NRASG12V inhibition on this comprehensive list of RAS-responsive genes. However, when we study the effects of these inhibitors on the subset of RAS-responsive genes that mediate leukemia self-renewal, we find that both PD325901 and RAD001 independently recapitulate the effects of NRASG12V withdrawal on this subset of genes implicating the Mek and mTor pathways in leukemia self renewal. Next, we treated primary leukemia cells with the IC50 dose of each drug and plated them in colony forming assays. We found that Mek or mTor inhibition, but not Pi3k inhibition, abrogated secondary colony formation corroborating our gene expression analyses and showing that, at doses that have equivalent effects on cell growth, only the Mek and mTor pathways are important for leukemia cell stem cell maintenance. These studies provide potential targets for leukemia stem cell-specific therapies. Disclosures: Sachs: Silicon Valley Biosystems: Consultancy. Bendall:DVS Sciences: Consultancy. Nolan:SAB for DVS Sciences and Nodality: Chairman Other; Cell Signalling Technologies and Becton Dickenson, Inc: Consultancy. Largaespada:Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.

scholarly journals Evolution Of Acute Myelogenous Leukemia Stem Cell Properties Following Treatment and Progression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 883-883 ◽  
Author(s):  
TzuChieh Ho ◽  
Mark W LaMere ◽  
Kristen O'Dwyer ◽  
Jason H. Mendler ◽  
Jane L. Liesveld ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Acute Myelogenous Leukemia ◽  
Cell Model ◽  
Phenotypic Diversity ◽  
Hierarchical Organization ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Leukemia Stem Cell ◽  
Acute Myelogenous

Abstract Acute Myelogenous Leukemia (AML) is a disease that clinically evolves over time as many patients who are responsive to therapy upfront acquire resistance to the same agents when applied in the relapse setting. The stem cell model for AML has been invoked to explain primary resistance to standard therapy; the leukemia stem cell (LSC) population representing a therapy-refractory reservoir for relapse. There have been no prospective efforts to formally assess the evolution of the LSC population during patients’ clinical course. We performed a prospective characterization of specimens from a well-defined cohort of patients with AML at diagnosis and relapse to assess the frequency and phenotype of functionally defined LSCs. Methods Primary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Pre-therapy and post-relapse LSC frequencies were assessed using xenotransplantation limiting dilution analyses (LDA). We assessed the frequencies of CD45RA, CD32, TIM-3, CD96, CD47, and CD97 expressing populations that have been previously published to possess LSC activity. Functionally validated pre-therapy and post-relapse LSC populations were identified using fluorescent labeled cell sorting and NSG xenotransplantation. LSC activity was confirmed for each population using secondary xenotransplantation. Gene expression analysis of highly enriched LSC populations from pre-therapy and post-relapse samples was performed using ABI TILDA qPCR analyses following pre-amplification. Results We demonstrated by LDA an 8 to 42-fold increase in LSC frequency between diagnosis and relapse in paired primary patient samples. The increase in LSC activity was not associated with an increase in frequency for phenotypically-defined populations previously reported to possess LSC activity. Rather, we found that LSC activity expanded at relapse to immunophenotypic populations of leukemic cells that did not possess LSC activity prior to treatment. Moreover, in all patients, the number of phenotypically distinct LSC populations (as defined by CD34 and CD38 or CD32 and CD38) detectable at relapse was dramatically expanded. Further, while the majority of the LSC populations’ gene expression profile remained stable between diagnosis and relapse, a subset of genes were enriched in defined LSC populations at relapse including IL3-receptor alpha and IL1-RAP, both previously demonstrated to play a role in LSC biology. Conclusions This study is the first to characterize the natural evolution of LSCs in vivo following treatment and relapse. We demonstrate an increase in LSC activity and greatly increased phenotypic diversity of the LSC population, suggesting a loss of hierarchical organization following relapse. These findings demonstrate that treatment of AML patients with conventional chemotherapy regimens can promote quantitative and qualitative expansion of the LSC compartment. Further, the data indicate that surface antigen immune-phenotype is not predictive of function in relapse and suggest a major limitation to efforts targeting specific surface antigens in the relapse setting. Understanding the mechanisms by which LSC expansion occurs and how to target it will likely improve our currently poor treatment options for patients who relapse. Disclosures: Becker: Millenium: Research Funding.

MLL-Fusion Proteins Induce a Stem Cell Program in Committed Progenitors To Generate Leukemia Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 465-465
Author(s):  
Andrei V. Krivstov ◽  
David Twomey ◽  
Zhaohui Feng ◽  
Matthew C. Stubbs ◽  
Todd R. Golub ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Cell Lines ◽  
Fusion Proteins ◽  
Expression Profiles ◽  
Cellular Reprogramming ◽  
Gene Set Enrichment Analysis ◽  
Myelogenous Leukemia ◽  
Self Renewal ◽  
Leukemia Stem Cell

Abstract Leukemias are composed of a hierarchy of cells only a fraction of which have stem cell like properties, and are capable of self-renewal. MLL fusion proteins produced by translocations involving the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 confer stem cell-like properties on committed hematopoietic progenitors. This provides an opportunity to determine if global cellular reprogramming is necessary for leukemia stem cell (LSC) generation from committed progenitors or if induction of a more limited self-renewal signature in committed progenitors is sufficient. We transduced murine IL-7R− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIhi granulocyte macrophage progenitors (GMPs) with retroviruses encoding the MLL-AF9 fusion protein, which led to the development of acute myelogenous leukemia. From the leukemias we isolated a population of IL-7R− Lin− Sca-1− c-Kit+ CD34int. FcγRII/IIIint. LSCs which can transplant the disease when fewer than 20 cells are injected into secondary recipients. We used hierarchical clustering, K-means clustering and principal component analysis to compare gene expression profiles of the LSC population to the normal lin− sca-1+ c-kit+ HSC-enriched population, IL-7R− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIlo common myeloid progenitors (CMPs), IL-7R− Lin− Sca-1− c-Kit+ CD34− FcγRII/III− megakaryocyte erythroid progenitors (MEPs) and GMPs and found that the global gene expression profile most resembles the normal GMP from which they arose. However, a leukemia self-renewal signature was identified that shows significant overlap with a group of genes normally highly expressed in HSCs whose expression decreases during the transition to normal committed progenitors. Supervised analysis and gene set enrichment analysis (GSEA) demonstrated approximately 300 genes in the leukemia self-renewal signature. This is only a subset of the approximately 1500 genes that are highly expressed in the normal HSC-enriched population that show decreased expression in CMPs, MEPs, and GMPs. Next, we determined if this 300-gene leukemia stem cell signature is directly regulated by MLL-AF9 or if there is a hierarchy of gene expression. Assessment of gene expression changes 48 hours after MLL-AF9 expression in isolated GMPs demonstrated increased expression of 23/300 genes in the leukemia self-renewal signature. Of interest, there is a high degree of similarity between the 23 MLL immediate response genes and human MLL-rearranged AMLs including HOXA5, HOXA7, HOXA9, HOXA10, MEIS1 and genes not previously known to have a role in MLL-mediated leukemogenesis such as myocyte enhancer factor 2C (MEF2C). Detailed loss-of-function studies using shRNA and dominant negative mutants show inhibition of MEF2C reduces LSC colony formation and serial replating in semi-solid culture to less than 20% of control. Furthermore shRNA mediated inhibition of MEF2C has a significant impact on proliferation of human MLL-AF9 dependent leukemia cell lines, but not cell lines from other subtypes of AML. These data demonstrate LSCs can be generated from committed progenitors without widespread reprogramming of gene expression, and a leukemia self-renewal signature is activated in the process. We have used this program to identify MEF2C as playing a role in MLL-AF9 induced AML. Identification of this program provides an opportunity to further assess its importance in normal tissue homeostasis and neoplastic self-renewal/proliferation, and defines the progression from normal hematopoietic progenitor to leukemia stem cell.

scholarly journals Six1 regulates leukemia stem cell maintenance in acute myeloid leukemia

2019 ◽  
Vol 110 (7) ◽  
pp. 2200-2210 ◽  
Author(s):  
Yajing Chu ◽  
Yangpeng Chen ◽  
Mengke Li ◽  
Deyang Shi ◽  
Bichen Wang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Leukemia Stem Cell ◽  
Stem Cell Maintenance ◽  
Cell Maintenance ◽  
Acute Myeloid

scholarly journals The control of hematopoietic stem cell maintenance, self-renewal, and differentiation by Mysm1-mediated epigenetic regulation

Blood ◽  
2013 ◽  
Vol 122 (16) ◽  
pp. 2812-2822 ◽  
Author(s):  
Tao Wang ◽  
Vijayalakshmi Nandakumar ◽  
Xiao-Xia Jiang ◽  
Lindsey Jones ◽  
An-Gang Yang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Histone Modifications ◽  
Epigenetic Regulation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Key Points ◽  
And Function ◽  
Cell Maintenance

Key Points Mysm1 is required to maintain the quiescence and pool size of HSC, and its deletion severely impairs the survival and function of HSC. Mysm1 controls HSC homeostasis by regulating Gfi1 expression via modulating histone modifications and transcriptional factors recruitment.

Inhibition of Chronic Myelogenous Leukemia Stem Cells with Novel Wnt Antagonists.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 238-238 ◽  
Author(s):  
Edward Kavalerchik ◽  
Jason Gotlib ◽  
Ifat Geron ◽  
Annelie Abrahamsson ◽  
Wolfgang Wrasidlo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myelogenous Leukemia ◽  
Reporter Gene ◽  
Myelogenous Leukemia ◽  
Beta Catenin ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Wnt Inhibitors

Abstract Introduction A growing proportion of chronic myelogenous leukemia (CML) patients show evidence of disease progression. Recent research suggests that leukemia stem cells (LSC) that share phenotypic characteristics with granulocyte-macrophage progenitors (GMP) are involved in CML progression. These LSC have aberrantly gained self-renewal capacity as a result of enhanced Wnt/beta-catenin signaling. We assayed the capacity of novel Wnt/beta-catenin antagonists to inhibit CML LSC. Methods To assay the efficacy of a novel Wnt inhibitor, MC-001, HEK293 cells were transfected with a Wnt-dependent reporter gene and expression plasmid for Dsh. After 16h, the cells were treated for 24 h with MCC-001, a novel marine sponge derived inhibitor, at varying concentrations and the reporter gene activity was measured. All cells were also transfected with a b-gal reporter gene to control for transfection efficiency. To assess the effects of MCC-001 and other Wnt inhibitors on Wnt/beta-catenin induced self-renewal, hematopoietic stem cells (HSC), GMP and lineage positive cells from normal (n=8) and advanced phase CML (n=8) peripheral blood and marrow (n=8) were clone sorted with the aid of a FACS Aria into methocult media (Stem Cell Technologies) with or without Wnt inhibitors including recombinant Dkk1, lentiviral axin or MCC-001. On day 10, individual colonies were plucked and replated in new methylcellulose and the replating efficiency determined at day 10. To establish an in vivo CML LSC model, HSC, GMP and lineage positive cells were transduced with a lentiviral luciferase GFP for 48 hours and transplanted intrahepatically into newborn immunocompromised mice (RAG2−/−gamma−/−) mice that facilitate high levels of human hematopoietic progenitor engraftment. Results The HEK293 beta-catenin reporter assay revealed that the MC-001 IC50 was 2.1 microM. In comparative Wnt inhibitor replating assays (n=8), recombinant Dkk1 did not inhibit CML HSC (n=8) while lentiviral axin and MCC-001 (at 2 and 10 microM) inhibited both CML HSC and CML GMP at doses that spared normal HSC replating (Figure 1). Transplantation of CML HSC, GMP and lineage positive cells into RAG2−/−gamma−/− mice demonstrated that only CML GMP provided serial transplantation potential and thus, were enriched for the LSC population (Figure 2). Conclusions Selective Wnt/beta-catenin inhibition with a marine sponge derived beta-catenin antagonist, MCC-001, blocks in vitro replating capacity of CML LSC at doses that spare normal HSC. Current experiments focus on in vivo inhibition of LSC self-renewal with novel Wnt inhibitors in a robust CML LSC bioluminescent imaging model (Figure 2). Figure 1. Chronic Myelogenous Leukemia Stem Cell Inhibition with MCC-001: A novel β-catenin Inhibitor Figure 1. Chronic Myelogenous Leukemia Stem Cell Inhibition with MCC-001: A novel β-catenin Inhibitor Figure 2. Bioluminescent Chronic Myelogenous Leukemia Stem Cell Transplantation Model. Figure 2. Bioluminescent Chronic Myelogenous Leukemia Stem Cell Transplantation Model.

Investigations of Murine Embryonic Stem Cell Maintenance by Analyses of Culture Variables and Gene Expression

2007 ◽  
pp. 185-189
Author(s):  
James M. Piret ◽  
Clive H. Glover ◽  
Michael Marin ◽  
Mohammed A. S. Chaudhry ◽  
Connie J. Eaves ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Stem Cell Maintenance ◽  
Murine Embryonic Stem Cell ◽  
Cell Maintenance

STEM-14. THE WRAD COMPLEX REPRESENTS A THERAPEUTIC TARGET FOR CANCER STEM CELLS IN GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi23-vi24
Author(s):  
Kelly Mitchell ◽  
Joseph Alvarado ◽  
Christopher Goins ◽  
Steven Martinez ◽  
Jonathan Macdonald ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Cancer Stem Cells ◽  
Molecular Mechanisms ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Self Renewal ◽  
Stem Cell Maintenance ◽  
Cell Maintenance

Abstract Glioblastoma (GBM) progression and resistance to conventional therapies is driven in part by cells within the tumor with stem cell properties including quiescence, self-renewal and drug efflux potential. It is thought that eliminating these cancer stem cells (CSCs) is a key component to successful clinical management of GBM. However, currently, few known molecular mechanisms driving CSCs can be exploited for therapeutic development. Core transcription factors such as SOX2, OLIG2, OCT4 and NANOG maintain the CSC state in GBM. Our laboratory recently uncovered a self-renewal signaling axis involving RBBP5 that is necessary and sufficient for CSC maintenance through driving expression of these core stem cell maintenance transcription factors. RBBP5 is a component of the WRAD complex, which promotes Lys4 methylation of histone H3 to positively regulate transcription. We hypothesized that targeting RBBP5 could be a means to disrupt epigenetic programs that maintain CSCs in stemness transcriptional states. We found that genetic and pharmacologic inhibition of the WRAD complex reduced CSC growth, self-renewal and tumor initiation potential. WRAD inhibitors partially dissembled the WRAD complex and reduced H3K4 trimethylation both globally and at the promoters of key stem cell maintenance transcription factors. Using a CSC reporter system, we demonstrated that WRAD complex inhibition decreased growth of SOX2/OCT4 expressing CSCs in a concentration-dependent manner as quantified by live imaging. Overall, our studies assess the function of the WRAD complex and the effect of WRAD complex inhibitors in preclinical models and specifically on the stem cell state for the first time in GBM. Studying the functions of the WRAD complex in CSCs may improve understanding of GBM pathogenesis and elucidate how CSCs survive despite aggressive chemotherapy and radiation. Our ongoing studies aim to develop brain penetrant inhibitors targeting the WRAD complex as an anti-CSC strategy that could potentially synergize with standard of care treatments.

A Role for IL1RAP in Acute Myelogenous Leukemia Stem Cells Following Treatment and Progression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4266-4266 ◽  
Author(s):  
Tzu-Chieh Ho ◽  
Craig T Jordan ◽  
Mark W. LaMere ◽  
John M. Ashton ◽  
Kristen O'Dwyer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Cell Lines ◽  
Small Molecule ◽  
Acute Myelogenous Leukemia ◽  
Research Funding ◽  
Myelogenous Leukemia ◽  
Molecule Inhibitor ◽  
Acute Myelogenous ◽  
Pre Treatment

Abstract Background Acute Myelogenous Leukemia (AML) evolves as many patients who are responsive to therapy upfront are resistant to the same agents when applied at relapse. We previously reported the results of our prospective efforts to formally assess the evolution of the leukemia stem cell (LSC) population(s) during patients' clinical courses. We identified a 9-90 fold increase in LSC activity and greatly increased phenotypic diversity of the LSC population. To identify the potential mechanisms underlying these changes we further characterized functionally-defined LSC populations from paired diagnosis and relapse samples. Methods Primary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy as well as normal donors. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Transcriptional profiling of highly enriched LSC populations from seven patients was performed using ABI TaqMan® Low Density Array (TLDA) qPCR analyses following pre-amplification using a novel 153 gene expression platform. Protein expression levels of interleukin-1 receptor accessory protein (IL1RAP) on bulk leukemia cells and LSC populations from 25 patients were assessed by flow cytometry. The impact of loss of IL1RAP was assessed using lentiviral based shRNA targeting all IL1RAP isoforms followed by assessment of proliferation, apoptosis, colony forming unit (CFU) activity and NSG engraftment capacity in human cell lines as well as in primary patient samples. Downstream signaling events for IL1RAP were probed using a small molecule inhibitor approach. Results While the majority of the LSC populations' gene expression profile remained stable, twelve genes were differentially expressed between pre-treatment and relapsed LSC populations including IL1RAP. Flow cytometric analyses confirmed that IL1RAP is overexpressed on both bulk leukemia populations as well as LSC populations at diagnosis and relapse in comparison to normal hematopoietic stem cell (HSC) populations. Targeting ILRAP1 using shRNA in both cell lines and primary AML samples resulted in impaired proliferation, increased apoptosis, a marked loss of CFU capacity and impaired NSG engraftment. IL1 signaling is known to involve both the MAPkinase and NFKappB pathways. To determine which pathways are involved in IL1RAP mediated LSC survival, we performed a small molecule inhibitor screen targeting elements in both signaling cascades. Established inhibitors of the NFKappaB pathway resulted in loss in loss of leukemic cell function while MAPK signaling inhibition had minimal to no effect. Conclusions We identified IL1RAP as being overexpressed in both bulk leukemia and functionally defined LSC populations from pre-treatment and relapsed AML samples. Loss of IL1RAP was associated with a marked decline in LSC function. Preliminary studies support a primary role for the NF Kappa B pathway in LSC function. Our findings support a critical role for IL1RAP in LSC function and support its development as a target for AML therapy in both the upfront and relapse setting. Disclosures Wang: Immunogen: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties. Becker:Millenium: Research Funding.

scholarly journals Fine-Tuning of GLI Activity through Arginine Methylation: Its Mechanisms and Function

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1973
Author(s):  
Yoshinori Abe ◽  
Nobuyuki Tanaka
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Cancer Stem Cell ◽  
Target Gene ◽  
Arginine Methylation ◽  
Protein Stabilization ◽  
Fine Tuning ◽  
Target Gene Expression ◽  
Stem Cell Maintenance ◽  
Cell Maintenance

The glioma-associated oncogene (GLI) family consists of GLI1, GLI2, and GLI3 in mammals. This family has important roles in development and homeostasis. To achieve these roles, the GLI family has widespread outputs. GLI activity is therefore strictly regulated at multiple levels, including via post-translational modifications for context-dependent GLI target gene expression. The protein arginine methyl transferase (PRMT) family is also associated with embryogenesis, homeostasis, and cancer mainly via epigenetic modifications. In the PRMT family, PRMT1, PRMT5, and PRMT7 reportedly regulate GLI1 and GLI2 activity. PRMT1 methylates GLI1 to upregulate its activity and target gene expression. Cytoplasmic PRMT5 methylates GLI1 and promotes GLI1 protein stabilization. Conversely, nucleic PRMT5 interacts with MENIN to suppress growth arrest-specific protein 1 expression, which assists Hedgehog ligand binding to Patched, indirectly resulting in downregulated GLI1 activity. PRMT7-mediated GLI2 methylation upregulates its activity through the dissociation of GLI2 and Suppressor of Fused. Together, PRMT1, PRMT5, and PRMT7 regulate GLI activity at multiple revels. Furthermore, the GLI and PRMT families have strong links with various cancers through cancer stem cell maintenance. Therefore, PRMT-mediated regulation of GLI activity would have important roles in cancer stem cell maintenance.

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