ETS1 Expression Is Upregulated in Chronic Myeloid Leukemia (CML) and the ETS1 Transcriptional Program Correlates with Disease Progression Towards Blast Crisis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 442-442
Author(s):  
Christophe Desterke ◽  
Maud Voldoire ◽  
Nathalie Sorel ◽  
Marie Laure Bonnet ◽  
Sarah Pagliaro ◽  
...  
Keyword(s):  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Leukemic Cells ◽  
P Value ◽  
Healthy Controls ◽  
Transcriptional Program ◽  
Chip Sequencing ◽  
Cd34 Cells

Abstract Chronic myeloid leukemia is a clonal myeloproliferative neoplasm characterized by the occurrence of the Ph1 chromosome in a primitive hematopoietic stem cell with a major amplification of myeloid compartment. Despite the major progress obtained by the use of tyrosine kinase inhibitors (TKI), resistances to these drugs occur with progression towards blast crisis (BC) during which there is an increase of BCR-ABL expression. To evaluate the potential targets of BCR-ABL in this context, a gene profiling analysis of the UT7-11 cell line overexpressing BCR-ABL fusion protein was performed using a whole transcriptome microarray. One of the highly upregulated genes was ETS1 with a fold change of +35.86. ETS1 proto-oncogene, the founder member of the ETS-domain family of transcription factors such as TEL and FLI1, is the human homolog of the avian erythroblastosis virus E26. They play a crucial role in stem cell biology, tumorigenesis and ETS1 has been shown to be involved in the regulation of granulocytic differentiation. Its role in CML pathophysiology has not been studied so far. We first showed by Western blots that ETS1 protein was highly increased in UT7-11 cells as compared to parental UT7 cells. Using a DOX-Inducible BCR-ABL system, we have shown that ETS1 expression was downregulated upon inhibition of BCR-ABL expression. ETS1 expression was a tyrosine kinase dependent event as its expression was reduced in the presence of TKI. To determine if ETS1 expression is upregulated in primary leukemic cells, we have analyzed leukemic cells of CML patients at diagnosis (n= 40) as compared to healthy controls (n= 30) showing an increase of ETS1 mRNA expression in primary CML cells in a highly significant manner (p < 0.0007). We then analyzed ETS1 targets using chip-sequencing in K562 cells, performed on HG19 human genome allowing to predict 3209 proximal promoters (-3000 upstream pb, + 50pb around Transcription Starting Sites) which represents a promoter enrichment of +33.68 (p-value=4.9E-324) as compared to the distribution of peaks in the whole genome. Chromosomes 19, 16, 17, 11, 12 and 1 were found over-represented with ETS1 bound events in Chip-sequencing. Integration of BCR-ABL transcriptome with ETS1 promoters identified by Chip-Sequencing led to the identification of 130 ETS1-targets activated by BCR-ABL, allowing reclassification of BCR-ABL transfected samples with transcriptome data by unsupervised classification. ETS1 transcriptional program regulated by BCR-ABL analysis performed on CD34+ from CML patients during progression of the disease (GSE47927) allowed discrimination of the different states of the progression (from the chronic phase to accelerated phase and BC p-value=1.63E-33). This analysis was also found significant by Gene Set Enrichment Analysis ETS1_BC specific geneset (NES=+1.52, p-value<0.0001). Among this ETS1 program specific of BC, we identified 2 targets that are discriminant for CD34+ cells of CML patients without cytogenetic response under Imatinib therapy. These two genes were Dynamin 3 (DNM3) and LIMS1/ PINCH1. Dynamin 3 is a member of the motor proteins implicated in cell motility and cytokinesis and it is implicated in megakaryocyte development whereas LIMS1 / PINCH1 is essentially involved in cell migration and adhesion. In order to validate ETS1 and its target expressions in a novel cohort of CML patients, we have analyzed the expression of DNM3 and PINCH1 in whole blood samples in CML patients ( n= 60 ) as compared to healthy controls (n= 32). We have found that, similar to ETS1, DNM3 and LIMS1 / PINCH1 mRNA were highly upregulated in primary CML and this increase was highly significant. In this new cohort of patients, DNM3 transcript levels were inversely correlated to WBC count (r=-0.32, p-value=0.011) and especially in male gender (r=-0.43, p-value=0.012). The expression of LIMS1 was inversely correlated with the age at diagnosis and the major molecular response at 12 months. Thus, our results show for the first time the major upregulation of ETS1 transcriptional program in CML. ETS1 transcriptional program has been found to correlate with the gene expression pattern involved in the progression of the disease in the transcriptomic analysis of CML CD34+ cells. The novel druggable targets that we identified in the BCR-ABL-activated transcriptional program is currently under investigation to determine their use in patients with resistance to targeted therapies. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Embryonic Program Activated during Blast Crisis of Chronic Myelogenous Leukemia (CML) Implicates a TCF7L2 and MYC Cooperative Chromatin Binding and Represents a Druggable Target

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1632-1632
Author(s):  
Christophe Desterke ◽  
Sarah Pagliaro ◽  
Patricia Hugues ◽  
Annelise Bennaceur-Griscelli ◽  
Ali G Turhan
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Embryonic Stem ◽  
K562 Cells ◽  
The United States ◽  
Myelogenous Leukemia ◽  
P Value ◽  
Chip Sequencing

Chronic myeloid leukemia (CML) is characterized by an inherent genetic instability, which contributes to the progression of the disease towards accelerated and blast crisis (BC). The occurrence of the latter has been hampered by the use of tyrosine kinase inhibitors (TKI), which changed this natural progression, but BC still occurs in patients resistant to TKI. Several cytogenetic (major and minor routes) and genomic (TP53 mutation, p16/INK4A deletions, DNA repair abnormalities such as BRCA1, DNA-PKcs, hnRNP metabolism) events have been reported in the progression towards BC. Previous data have also suggested the involvement of embryonic stem cell program activated in BC cells such as Lin28A. In this work, we have taken advantage of the previously reported gene profiling of BC in a large cohort of patients (Radich et al. 2006) and found a correlation between blast numbers and the involvement of the Transcription Factor 7 like 2 (TCF7L2) in BC. TFC7L2 is a member of the TCF family of proteins that are known to activate WNT target genes such as Cyclin D1. TCF7L2 has been shown to be overexpressed in acute myeloid leukemia (AML) and represents a druggable target (Saenz et al Leukemia 2019). The involvement of TCF7L2 in CML-BC and its interaction with the epigenetic regulators has not been studied so far. The gene correlation study that we have performed using the blast numbers and the expression of TCF7L2 in CD34+ CML cells was found to be highly significant (Pearson test, r = 0.56, p-value=5.2e-4) (Fig.1A). TCF7L2 promoter was classed as active in K562 with ChromHMM Functional genomic analysis. K562 epigenetics peaks of TCF7L2 CHIP-seq were found principally mapped in proximal promoters (39% of the peaks, -3000pb upstream Transcription Starting Sites (TSS), Fig. 1B) and 183 unique peaks matched with promoter of 144 unique genes found to be correlated to the blast number in blood of the CML patients during BC (Fig 1C). This TCF7L2-dependent BC program was characterized to be active because promoters were also found positive for H3K27Ac and negative for H3K27Me3 histone marks, and functionally enriched with binding sites for MYC/MAX interactions (p=1.15e-6). The analysis of CHIP-sequencing of MYC revealed a significant overlapping of TCF7L2 epigenetic program with MYC (fold enrichment: 20.81, p < 2.2e-16). Surprisingly, the TCF7L2 program was found independent of RUNX1 and GATA2 transcriptional program. To determine these potential interactions, we have designed experiments in K562 cell line using the b-catenin activator Lithium Chloride (LiCL2) and the Myc/Max dimerization inhibitor 10058-F4. K562 cells were cultured in the presence of LiCL2 (10mM & 24hours) and the compound 10058-F4 (64µM & 48hours) and the expression of three epigenetic targets was analyzed by Q-RT-PCR in the presence of DMSO controls. The three targets chosen were protein arginine N-methyltransferase (PRMT1), the ATPase/Helicase RUVBL1 and the WD-repeat containing protein WDR77. As expected, after culture with LiCL2, the expression of PRMT1 was increased x 6.3 fold (p=8.49e-13) , that of RUVBL1 by x 1.66 Fold (p=1.67e-6) and that of WDR77 by x 2 fold (p=4.97e) (Fig.1D). On the contrary, the culture of K562 cells in the presence of MYC/MAX inhibitor 10058-F4, decreased the expression of 3 targets as compared to DMSO controls (x 1.6 fold for PRMT1, p=6.52e-5; x2 fold reduction for RUVBL1, p-value=2.71e-5; and x 1.4 fold for WDR77, p =0.0000643). These results show for the first time a cooperative role of TCF7L2 and MYC during blast crisis of CML and provide mechanistic insights into the interactions for the role of MYC in CML blast crisis. In addition they strengthen previous data showing a possible embryonic footprint in the blast development over the hematopoietic differentiation program during progression of the disease and provide a rationale for the pharmacological targeting of BC by the use of MYC/MAX inhibitors such as 10058-F4. Experiments are underway to evaluate the role of these factors and the MYC/MAX inhibitors in primary CML samples. Reference : Radich JP, Dai H, Mao M, Oehler V, Schelter J, Druker B, Sawyers C, Shah N, Stock W, Willman CL, Friend S, Lindsey PS.(2006) :Gene Expression Changes Associated with Progression and Response in Chronic Myeloid Leukemia. Proceedings of the National Academy of Sciences of the United States of America 103 (8): 2794-99. Disclosures Turhan: Incyte: Consultancy, Honoraria; novartis: Honoraria, Research Funding.

scholarly journals Stem Cell Factor as a Single Agent Induces Selective Proliferation of the Philadelphia Chromosome Positive Fraction of Chronic Myeloid Leukemia CD34+ Cells

Blood ◽  
1998 ◽  
Vol 92 (7) ◽  
pp. 2461-2470 ◽  
Author(s):  
Sarah Moore ◽  
David N. Haylock ◽  
Jean-Pierre Lévesque ◽  
Louise A. McDiarmid ◽  
Leanne M. Samels ◽  
...  
Keyword(s):  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Stem Cell Factor ◽  
Myeloid Leukemia ◽  
Single Agent ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Leukemic Cell ◽  
Proliferative Effect ◽  
Cd34 Cells

Abstract The interaction between p145c-KIT and p210bcr-abl in transduced cell lines, and the selective outgrowth of normal progenitors during long-term culture of chronic myeloid leukemia (CML) cells on stroma deficient in stem-cell factor (SCF) suggests that the response of CML cells to SCF may be abnormal. We examined the proliferative effect of SCF(100 ng/mL), provided as the sole stimulus, on individual CD34+ cells from five normal donors and five chronic-phase CML patients. Forty-eight percent of isolated single CML CD34+ cells proliferated after 6 days of culture to a mean of 18 cells, whereas only 8% of normal CD34+ cells proliferated (mean number of cells generated was 4). SCF, as a single agent, supported the survival and expansion of colony-forming unit–granulocyte-macrophage (CFU-GM) from CML CD34+CD38+ cells and the more primitive CML CD34+CD38− cells. These CFU-GM colonies were all bcr-abl positive, showing the specificity of SCF stimulation for the leukemic cell population. Coculture of CML and normal CD34+ cells showed exclusive growth of Ph+cells, suggesting that growth in SCF alone is not dependent on secretion of cytokines by CML cells. SCF augmentation of β1-integrin–mediated adhesion of CML CD34+cells to fibronectin was not increased when compared with the effect on normal CD34+ cells, suggesting that the proliferative and adhesive responses resulting from SCF stimulation are uncoupled. The increased proliferation may contribute to the accumulation of leukemic progenitors, which is a feature of CML.

Centrosome Aberrations in Chronic Myeloid Leukemia (CML) Are Correlated with Disease Progression and Chromosome Instability.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2949-2949
Author(s):  
Michelle Giehl ◽  
Alice Fabarius ◽  
Chun Zheng ◽  
Oliver Frank ◽  
Andreas Hochhaus ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Chromosome Instability ◽  
Chronic Phase ◽  
Normal Karyotype ◽  
Benign Tumors ◽  
Preneoplastic Lesions ◽  
Cd34 Cells

Abstract Purpose: Numerical and structural centrosome abnormalities are hallmarks of a variety of cancers and have been implicated in chromosome missegregation, chromosomal instability, and aneuploidy. These phenomena already occur in preneoplastic lesions like oral leukoplakia, early cervical neoplasias, and small benign tumors of colon and breast. Moreover, deviations from normal karyotype seem to increase as tumors enlarge and become malignant. Genetic instability is a common feature in chronic myeloid leukemia (CML). We sought to establish a relationship between centrosome abnormalities and cytogenetic aberrations in CD34+ cells from CML patients at diagnosis (chronic phase - CP) and in blast crisis (BC). Methods: Diagnosis of CML was established by hematologic, cytogenetic and molecular parameters. Treatment was performed according to the protocols of the German CML study group (www.kompetenznetz-leukaemie.de). CD34+ cells from ten umbilical cord blood specimens served as negative controls. Centrosome number and morphology were analyzed by immunofluorescence microscopy. In brief, CD34+ cells from ficollized peripheral blood samples were concentrated by magnetic cell sorting (MACS) and cytospun onto coated slides. After methanol fixation cells were incubated with antibodies directed to centrosomal proteins Pericentrin and gamma-Tubulin. Antibody-antigen complexes were stained by incubation with FITC- and Cy3-conjugated secondary antibodies. Results: CML CP samples tested at initial diagnosis (n=20) already displayed numerical and structural centrosome aberrations (30.0% +/−2.3) as compared with corresponding normal control cells (n=10) (2.3% +/−1.1). In BC samples (n=10) an increase of centrosome aberrations was observed (58.0% +/−2.0). Conclusion: The findings suggest that centrosome defects in CML occur early and are already present at primary diagnosis. Centrosome defects may contribute to disease progression by generation of further chromosome instability leading to accumulation of alleles carrying pro-oncogenic mutations and loss of alleles containing normal tumor suppressor genes and thus accelerating complex genomic changes associated with CML BC.

Evidence for CD34+ Hematopoietic Progenitor Cell Involvement in Acute Myeloid Leukemia with NPM1 Gene Mutation: Implications for the Cell of Origin

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 307-307 ◽  
Author(s):  
Maria Paola Martelli ◽  
Valentina Pettirossi ◽  
Elisabetta Bonifacio ◽  
Federica Mezzasoma ◽  
Nicla Manes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Hox Genes ◽  
Leukemic Cells ◽  
Mutant Protein ◽  
Cell Of Origin ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Acute Myeloid

Abstract Acute myeloid leukemia expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM2005;352:254–266] is a new entity of WHO classification that shows distinctive biological and clinical features, including a unique molecular signature characterized by downregulation of CD34 and upregulation of most HOX genes [Falini B et al, Blood2007;109:874–885]. Involvement of HOX genes in the maintenance of the stem-cell phenotype strongly suggest that AML with mutated NPM1 originates from a multipotent hematopoietic progenitor (HSC). This view is also supported by immunohistological findings showing that AML with mutated NPM1 frequently displays multilineage involvement [Pasqualucci L et al, Blood2006;108:4146–4155]. On the other hand, the frequent negativity of NPMc+ AML for the HSC-associated antigen CD34 raises the question of whether the mutation event occurs in a CD34-negative HSC (these cells have been identified in mice) or whether a minimal pool of CD34-positive NPM1-mutated leukemic cells does exist. Currently, the hierarchical level of stem cell involvement in NPMc+ AML is unknown. To address this issue, we purified CD34+ cells from NPMc+ AML patients and detected NPM1 mutant protein in the sorted population by Western blot with anti-NPM mutant specific antibodies [Martelli MP et al, Leukemia 2008] (Figure 1A). We investigated 6 NPMc+ AML patients presenting at diagnosis with 0.12%, 0.14%, 0.38%, 5%, 22%, and 28% of CD34+ cells in the peripheral blood. In all cases, CD34+ fractions (purity &gt;90%) harboured NPM1 mutant protein, indicating they belong to the leukemic clone (Figure 1B). The percentage of most undifferentiated CD34+/CD38− cells in the CD34+ fractions ranged from 5 to 97%. Notably, in at least one case, all CD34+ NPM1-mutated leukemic cells were CD38−negative. Moreover in all cases, CD34+ NPM1-mutated leukemic cells appeared to express CD123 (IL-3 receptor), considered a marker of the leukemic stem cell and target of potential therapy. Double staining of bone marrow biopsies with anti-CD34 and anti-NPM antibodies revealed that the rare CD34+ cells expressed NPM1 aberrantly in the cytoplasm. Inoculation of CD34+ NPM1-mutated AML cells into sublethally irradiated NOD/SCID mice resulted into leukemia engrafment in various body sites, especially bone marrow, spleen, lung and liver. Preliminary results showed that CD34+ leukemic cells reacquired the same leukemic phenotype as the original patient’s, including CD34-negativity of the leukemic bulk in spite of any lack of differentiation. This finding suggests that NPM1 mutant protein may be involved in downregulation of CD34 antigen, while keeping a gene expression profile typical of the hematopoietic stem cell. These findings suggest the CD34+ fraction contains the SCID-leukemia initiating cells (SL-IC) and point to CD34+/CD38− HSC as the cell of origin of AML with mutated NPM1. Figure Figure

A Bio-Integrative Approach Identifies an Inflammatory Signature in Chronic Myeloid Leukemia (CML) Stem Cells That Is Highly Perturbed in CML Blast Crisis and Involves REL transcription Factor

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4017-4017
Author(s):  
Christophe Desterke ◽  
Ludovic Marie-Sainte ◽  
Amine Sbitti ◽  
Ali Naama ◽  
Annelise Bennaceur-Griscelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Integrative Approach ◽  
Hematopoietic Stem ◽  
Gene Set

Abstract Chronic myeloid leukemia is a clonal myeloproliferative neoplasm defined by the presence of BCR-ABL fusion gene. This oncogenic event occurs in a hematopoietic stem cell (HSC) involved in CML initiation, maintenance, relapse and progression. Several evidences suggest that inflammatory pathways may participate to the pathophysiology of CML as well as disease progression to blast crisis. It has been shown that NFKB/REL pathway is constitutively activated both in BCR-ABL positive leukemic cell lines as well as in primary blast cells from CML-BC patients. More recent works identified IL6 as key cytokine acting on CML multipotent progenitors and their normal bystander counterpart to favor their differentiation toward the myeloid lineage. In addition, high levels of autocrine TNFα secretion by quiescent CML stem/progenitor cells activate NFKB pathway and promote their survival. Although all of these observations are linked to inflammatory processes, a focused analysis of inflammatory pathways in primary CML stem cells has not been performed so far. In the present study we undertook a text-mining strategy using pubmed e-querying to generate an exhaustive set of genes linked to inflammation. Then we integrated transcriptome analysis of highly purified CML stem cells to evaluate the contribution of these genes in CML development and progression. Methods : We queried 6 key words (Inflammation, macrophages, inflammatory response, chemokines, leukocytes and interleukins) that returned a total of 332000 hits in Pubmed. A raw gene set of 918 genes was found significantly associated (p<0.05) with these hits. Using R-package, we applied a false discovery rate correction that decreased the set to 588 relevant genes. The expression level of this gene set was then analyzed in previously reported microarray data (GEO accession: GSE47927) of highly purified normal cord blood CD34+CD38-CD90+ HSCs (CB; n=3), chronic phase (CP; n= 6), accelerated phase (AP; n =4) and Blast crisis (BC; n=2) CML cells. Results: Among the 588 genes related to inflammation we found 70 genes differentially expressed between the four groups (normal, CP, AP and BC, p<0.01; ANOVA test). Enrichment analysis confirmed 29 up regulated genes (NES = 2.12; p<0.0001) among which IL-6, PARP1, IL1R2, IRF5, IRF8, IL20. 39 genes such as STAT3, STAT4, CD47, CXCR4 IL-11, IL15, TLR-1, were down-regulated in CML CD34+CD38-CD90+ (all phases) as compared with normal HSCs (NES = -2,58; p<0.0001). Using principal component analysis on the 70 inflammatory deregulated genes we identified 10 genes such as IRAK1, IL1R2, VEGF and ESAM that discriminate "all phase" CML samples from normal HSCs (Dim 2 = 22.7%). Another inflammatory gene subset (n=26 genes) comprising IL6, REL, CXCR4, CXCL2, IL11, TLR1, IL1R2, PPARA highly separated CML stem cells according to the disease phase. The later gene set highly separates CP and AP-CML stem cells from BC-CML stem cell (Dim 1 = 50.3%). We next performed a random forest analysis with machine learning (1000 trees) and found that the inflammatory transcript level that best predicted CML phase was REL transcription factor. The expression of 413 genes were found positively correlated with REL expression in CP, AP and BC-CML CD34+CD38-CD90+ cells (r>0.75 and p-value <0.001). A search using JASPAR and TRANSFAC database identified a significant enrichment of NFKB1 and RELA binding motif in the promoter regions of these 413 genes (p<0.00001) among which several regulatory factors of hematopoietic stem cell biology. Conclusion : Using a bio-integrative approach we identified a specific inflammatory signature in CD34+CD38-CD90+ CML stem cells. This inflammatory network is highly altered in blast crisis suggesting its contribution to disease evolution. We identified REL overexpression as a good predictor for disease progression to blast crisis and found NFKB1and RELA (p=3.2x10-13) as the best REL target candidates. RELA/NFKB1 was previously shown to be constitutively activated in CML and Ph+ ALL and this analysis suggests that this may also take place in the most primitive subset of CML cells although REL may be the main partner of NFKB in CML stem cells. These results which are currently validated using functional assays, could lead to identification of novel therapeutic strategies. Disclosures Turhan: Bristol Myers Squibb: Consultancy; Novartis: Research Funding.

scholarly journals Vitamin E in Chronic Myeloid Leukemia (CML) Prevention

2021 ◽  
Author(s):  
Lyudmyla Shvachko ◽  
Michael Zavelevich ◽  
Daniil Gluzman ◽  
Gennadii Telegeev
Keyword(s):  
Alkaline Phosphatase ◽  
Vitamin E ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Blast Cells ◽  
Differentiation Block

The resistance to inhibitors of tyrosine kinase necessitates novel approaches to the therapy of chronic myeloid leukemia (CML). The progression of CML to blast crisis is associated with down-regulation of C/EBP-alpha being involved in the differentiation block in leukemic blast cells. Moreover, lowered C/EBP-alpha expression correlates with resistance to imatinib in CML. We have demonstrated that vitamin E up-regulates expression of C/EBP-alpha and down-regulates expression of Snail transcription factor in K562 cells in vitro contributing to the putative recovery of myeloid differentiation potential. In parallel with increased CEBP alpha expression, Vitamin E treatment results in the decreasing expression of placental-like alkaline phosphatase and increasing expression of tissue non-specific alkaline phosphatase. We suggest that vitamin E could be used as the plausible biological modulator to prevent the progression to blast crisis and to overcome drug resistance of leukemic cells in CML.

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