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• W2127135280 abstract "We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)V617F myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2V617F MPN compared to JAK2 wild-type myeloid leukemia cell lines. By global gene expression analysis, we observed that at 6 hours, GVS modulated 293 common genes in the JAK2V617F cell lines HEL and UKE1, of which 19 are implicated in cell cycle regulation and 33 in hematopoiesis. In particular, the hematopoietic transcription factors NFE2 and C-MYB were downmodulated by the drug specifically in JAK2V617F cells at both the RNA and protein level. GVS also inhibited JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 phosphorylation, but modulation of NFE2 and C-MYB was JAK2-independent, as shown using the JAK2 inhibitor TG101209. GVS had a direct effect on the NFE2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NFE2 was also observed in freshly isolated CD34+ cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 inhibition and downmodulation of NFE2 and C-MYB transcription. We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)V617F myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2V617F MPN compared to JAK2 wild-type myeloid leukemia cell lines. By global gene expression analysis, we observed that at 6 hours, GVS modulated 293 common genes in the JAK2V617F cell lines HEL and UKE1, of which 19 are implicated in cell cycle regulation and 33 in hematopoiesis. In particular, the hematopoietic transcription factors NFE2 and C-MYB were downmodulated by the drug specifically in JAK2V617F cells at both the RNA and protein level. GVS also inhibited JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 phosphorylation, but modulation of NFE2 and C-MYB was JAK2-independent, as shown using the JAK2 inhibitor TG101209. GVS had a direct effect on the NFE2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NFE2 was also observed in freshly isolated CD34+ cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 inhibition and downmodulation of NFE2 and C-MYB transcription. Philadelphia-negative myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell diseases characterized by deregulated proliferation and differentiation in several myeloid lineages. JAK2 mutations (at position 617 or within exon 12) are detected in most MPN patients of the polycythemia vera subgroup (95% of cases), and in about 50% of essential thrombocythemia (ET) and primary myelofibrosis patients [1Levine R.L. Gilliland D.G. Myeloproliferative disorders.Blood. 2008; 112: 2190-2198Crossref PubMed Scopus (244) Google Scholar, 2Kota J. Caceres N. Constantinescu S.N. Aberrant signal transduction pathways in myeloproliferative neoplasms.Leukemia. 2008; 22: 1828-1840Crossref PubMed Scopus (55) Google Scholar]. JAK2V617F confers to hematopoietic progenitor cells growth factor−independent proliferation [3Ugo V. Marzac C. Teyssandier I. et al.Multiple signaling pathways are involved in erythropoietin-independent differentiation of erythroid progenitors in polycythemia vera.Exp Hematol. 2004; 32: 179-187Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar]. In addition, it induces a transplantable myeloproliferative disease resembling human polycythemia vera or ET in vivo in transgenic or knock in animals [4Tiedt R. Hao-Shen H. Sobas M.A. et al.Ratio of mutant JAK2-V617F to wild type JAK2 determines the MPD phenotypes in transgenic mice.Blood. 2008; 111: 3931-3940Crossref PubMed Scopus (353) Google Scholar, 5Mullally A. Lane S.W. Ball B. et al.Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells.Cancer Cell. 2010; 17: 584-596Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 6Li J. Spensberger D. Ahn J.S. et al.JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia.Blood. 2010; 116: 1528-1538Crossref PubMed Scopus (165) Google Scholar, 7Marty C. Lacout C. Martin A. et al.Myeloproliferative neoplasm induced by constitutive expression of JAK2V617F in knock-in mice.Blood. 2010; 116: 783-787Crossref PubMed Scopus (127) Google Scholar, 8Akada H. Yan D. Zou H. Fiering S. Hutchison R.E. Mohi M.G. Conditional expression of heterozygous or homozygous Jak2V617F from its endogenous promoter induces a polycythemia vera-like disease.Blood. 2010; 115: 3589-3597Crossref PubMed Scopus (193) Google Scholar].The pan-histone deacetylase inhibitor (HDACi) Givinostat (GVS, ITF2357) has antiproliferative and proapoptotic activity against several cancer cells, including acute myelogenous leukemia and multiple myeloma cells in vitro and in vivo [9Golay J. Cuppini L. Leoni F. et al.The histone deacetylase inhibitor ITF2357 has anti-leukemic activity in vitro and in vivo and inhibits IL-6 and VEGF production by stromal cells.Leukemia. 2007; 21: 1892-1900Crossref PubMed Scopus (96) Google Scholar]. Recently, GVS has been discovered to inhibit colony formation of cells from MPN patients at doses at least 10 times lower than those required to affect cells bearing wild-type (wt) JAK2 (JAK2wt) [10Guerini V. Barbui V. Spinelli O. et al.The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).Leukemia. 2008; 22: 740-747Crossref PubMed Scopus (122) Google Scholar]. Furthermore exposure to low doses of GVS allows the preferential expansion of JAK2wt over JAK2 mutated cells in colony assays using MPN patients’ cells [10Guerini V. Barbui V. Spinelli O. et al.The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).Leukemia. 2008; 22: 740-747Crossref PubMed Scopus (122) Google Scholar]. The mechanism for the higher sensitivity to GVS of JAK2V617F cells is unclear, although the drug has been observed to downmodulate phosphorylated JAK2 and signal transducer and activator of transcription-5 (STAT-5) in MPN cells [10Guerini V. Barbui V. Spinelli O. et al.The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).Leukemia. 2008; 22: 740-747Crossref PubMed Scopus (122) Google Scholar]. These findings have led to the initiation of a phase I/II clinical studies of the drug, alone or in combination with hydroxyurea, in JAK2V617F MPN patients [11Rambaldi A. Dellacasa C.M. Finazzi G. et al.A pilot study of the histone-deacetylase inhibitor Givinostat in patients with JAK2V617F positive chronic myeloproliferative neoplasms.Br J Haematol. 2010; 150: 446-455PubMed Google Scholar] (www.clinicaltrials.gov).In this report, we analyzed in more detail the biological activities of GVS in JAK2V617F cells using a panel of JAK2V617F or JAK2wt cell lines [12Quentmeier H. MacLeod R.A. Zaborski M. Drexler H.G. JAK2 V617F tyrosine kinase mutation in cell lines derived from myeloproliferative disorders.Leukemia. 2006; 20: 471-476Crossref PubMed Scopus (124) Google Scholar], as well as CD34+ cells isolated from MPN patients, measuring, in particular, proliferation, apoptosis, and differentiation along the erythroid lineage. By global gene expression profiling (GEP), we demonstrate that GVS rapidly modulates important hematopoietic transcription factors (TF), including C-MYB and, most interestingly NFE2, a TF overexpressed in MPN and crucial for erythromyeloid lineage differentiation [13Goerttler P.S. Kreutz C. Donauer J. et al.Gene expression profiling in polycythaemia vera: overexpression of transcription factor NF-E2.Br J Haematol. 2005; 129: 138-150Crossref PubMed Scopus (94) Google Scholar, 14Guglielmelli P. Zini R. Bogani C. et al.Molecular profiling of CD34+ cells in idiopathic myelofibrosis identifies a set of disease-associated genes and reveals the clinical significance of Wilms' tumor gene 1 (WT1).Stem Cells. 2007; 25: 165-173Crossref PubMed Scopus (104) Google Scholar, 15Kralovics R. Teo S.S. Buser A.S. et al.Altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of Jak2.Blood. 2005; 106: 3374-3376Crossref PubMed Scopus (153) Google Scholar].Materials and methodsDrugs and cell linesThe pan-HDAC inhibitor GVS (ITF2357) was a kind gift from Italfarmaco, Milano, Italy [10Guerini V. Barbui V. Spinelli O. et al.The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).Leukemia. 2008; 22: 740-747Crossref PubMed Scopus (122) Google Scholar]. The JAK2 inhibitor TG101209 was from Axon Medchem (Groningen, The Netherlands).The erythroleukemia cell line HEL, the ET cell line SET2, and the chronic myeloid leukemia cell line KU812 were from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The ET cell line UKE1 was a kind gift of Dr. Walter Fiedler (Eppendorf Hospital, Hamburg, Germany) [16Fiedler W. Henke R.P. Ergun S. et al.Derivation of a new hematopoietic cell line with endothelial features from a patient with transformed myeloproliferative syndrome: a case report.Cancer. 2000; 88: 344-351Crossref PubMed Scopus (34) Google Scholar]. The chronic myeloid leukemia cell line K562 and acute myelogenous leukemia cell line KG1 have been described previously [10Guerini V. Barbui V. Spinelli O. et al.The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).Leukemia. 2008; 22: 740-747Crossref PubMed Scopus (122) Google Scholar]. HEL, UKE1, and SET2 all bear the JAK2V617F mutation [12Quentmeier H. MacLeod R.A. Zaborski M. Drexler H.G. JAK2 V617F tyrosine kinase mutation in cell lines derived from myeloproliferative disorders.Leukemia. 2006; 20: 471-476Crossref PubMed Scopus (124) Google Scholar], as confirmed by polymerase chain reaction (PCR) in our laboratory (data not shown). K562 and KU812 carry the bcr-abl translocation. Cell lines were maintained in RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 2 mM glutamine (Euroclone, Wetherby, West Yorkshire, UK), 110 μM gentamycin (PHT Pharma, Milano, Italy), and 10% fetal calf serum (HEK, K562, KG1; Euroclone), 20% fetal calf serum (SET2, KU812) or, for the UKE1 cell line, 10% fetal calf serum plus 10% horse serum (GIBCO Invitrogen Corp., Paisley, UK), 1 μM hydrocortisone, and 25 mM HEPES buffer solution (Sigma-Aldrich, Milan, Italy).Peripheral blood from MPN patients was obtained after informed consent as approved by the Internal Ethical Committee. CD34+ cells were purified to ≥70% purity by antibody-based magnetic bead separation (Miltenyi, Bergsch Gladbach, Germany).Colony-forming assayFive-thousand cells were plated in methylcellulose medium (HSC-CFU containing recombinant human erythropoietin [rh-EPO; Miltenyi Biotech]) for 10 days at 37°C 5% CO2 [10Guerini V. Barbui V. Spinelli O. et al.The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).Leukemia. 2008; 22: 740-747Crossref PubMed Scopus (122) Google Scholar].Cytotoxicity assaysCytotoxicity assays were performed using the alamar blue vital dye (AbD Serotec, Kidlington, UK), as described [10Guerini V. Barbui V. Spinelli O. et al.The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).Leukemia. 2008; 22: 740-747Crossref PubMed Scopus (122) Google Scholar].Proliferation and cell deathCells were cultured at 0.5–1 × 105 cell/mL in complete medium and absolute cell number and cell death were determined at different times by flow cytometry using calibration beads (Bright Count Microspheres; IQ Products, Groningen, The Netherlands) and 7-aminoactinomycin D (BD Biosciences, Buccinasco, Italy), respectively, on a FACSCalibur Instrument (BD Biosciences).Erythroid differentiationCD34+ cells were cultured at 7 × 104 cell/mL in StemSpan Serum-Free Expansion Medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 50 ng/mL recombinant human stem cell factor (rhSCF), 1 IU/mL rhEPO, 10 ng/mL rh interleukin-3 (all from Sigma Aldrich), 40 ng/mL human low-density lipoprotein (Stem Cell Technologies). Absolute total and CD34+ cell numbers were determined at different times by single-platform flow cytometric CD34+ cell count analysis using CD34-phycoerythrin and CD45-fluorescein isothiocyanate antibodies (BD Biosciences) according to the guidelines of the International Society of Hematotherapy and Graft Engineering [17Keeney M. Chin-Yee I. Weir K. Popma J. Nayar R. Sutherland D.R. Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering.Cytometry. 1998; 34: 61-70Crossref PubMed Scopus (409) Google Scholar]. For evaluation of erythroid differentiation, cells were stained with phycoerythrin-conjugated anti-CD235a/GpA and allophycocyanin-conjugated anti-CD36 antibodies (BD Biosciences) and analyzed on a FACScan (BD Biosciences).Western blottingCells were lysed in M-PER extraction reagent (Pierce, Rockford, IL, USA). Equivalent amounts of protein were analyzed by standard Western blotting. The following antibodies were used: JAK2, STAT5, TAL1, and NFE2 (Santa Cruz Biotechnology, Inc., Heidelberg, Germany); phosphorylated JAK2 (Tyr1007/1008) and STAT5 (Tyr694) (Cell Signaling Technology, Danvers, MA, USA), and c-myb clone CB10018 [18Arsura M. Luchetti M.M. Erba E. Golay J. Rambaldi A. Introna M. Dissociation between p93B-myb and p75c-myb expression during the proliferation and differentiation of human myeloid cell lines.Blood. 1994; 83: 1778-1790Crossref PubMed Google Scholar]. Detection was performed using horseradish peroxidase−labeled secondary antibodies (Santa Cruz) and Super Signal West Pico Chemiluminescent Substrate (Pierce).Gene expression profilingHEL and UKE1 cells were cultured in presence or absence of 250 nM GVS in triplicate flasks for 6 hours and lysed in TRIzol Reagent (Life Technologies, Inc., Rockville, MD, USA). Total RNA was purified from each using the RNeasy total RNA Isolation Kit (Qiagen, Valencia, CA, USA). Preparation of DNA single-stranded sense target, hybridization to GeneChip Gene 1.0 ST Array (Affymetrix Inc., Santa Clara, CA, USA) and scanning of the chips (7G Scanner; Affymetrix Inc.) were carried out according to manufacturer’s protocols. Signal intensities were converted to expression values by probe set summarization, robust multiarray average normalization, and log2 transformation procedures using the Expression Console Software (Affymetrix Inc.). Probe sets mapping on the same gene (according to the hugene-1_0-st-v1_na30_hg19 Affymetrix Annotation file) were considered as a single series of expression values by median summarization, resulting in 20,072 unique genes.The gene expression analyses were performed using the RankProd R package (http://www.bioconductor.org/) [19Breitling R. Armengaud P. Amtmann A. Herzyk P. Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments.FEBS Lett. 2004; 573: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1201) Google Scholar]. RankProd utilizes the nonparametric method rank product to identify upregulated or downregulated genes. The algorithm detects genes consistently highly ranked in a two-condition experiments comparison [19Breitling R. Armengaud P. Amtmann A. Herzyk P. Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments.FEBS Lett. 2004; 573: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1201) Google Scholar]. Two statistical filters on percentage of false-positive predictions (pfp < 0.05) and on the difference in gene expression values (fold change ≥2) were applied to the ranked list of genes. Only the matching list (293 genes) of the differentially expressed genes in both HEL and UKE1 was considered for functional annotation.The functional analysis of the selected genes was performed by means of NetAffx (Affymetrix at https://www.affymetrix.com/analysis/netaffx/) and the Database for Annotation, Visualization and Integrated Discovery Tool 6.7 (http://david.abcc.ncifcrf.gov/). [20Huang da W. Sherman B.T. Lempicki R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.Nucleic Acids Res. 2009; 37: 1-13Crossref PubMed Scopus (10008) Google Scholar, 21Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat Protoc. 2009; 4: 44-57Crossref PubMed Scopus (24693) Google Scholar]. The Functional Annotation Clustering tool of Database for Annotation, Visualization and Integrated Discovery measures relationships among the annotation terms in order to group the similar and redundant annotation contents from different sources into a single annotation cluster. The Enrichment Score for each annotation term is calculated as the minus log geometric mean of all the enrichment p values associated with the gene members included in each group. Only the Gene Ontology Biological Process and Molecular Function terms were chosen as annotation categories and “medium” classification stringency was set for the analysis. The clusters with an Enrichment Score >1.3 (equivalent to an average p value of 0.05) [20Huang da W. Sherman B.T. Lempicki R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.Nucleic Acids Res. 2009; 37: 1-13Crossref PubMed Scopus (10008) Google Scholar, 21Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat Protoc. 2009; 4: 44-57Crossref PubMed Scopus (24693) Google Scholar] were selected among the 65 annotation clusters globally identified.Real-time PCRTotal RNA was extracted from three independent experiments, different from those used for microarray, using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). Complementary DNA was synthesized using the SuperScript III First-strand Synthesis System for reverse transcription PCR (Invitrogen). PCR was performed with specific primers (Supplementary Table E1; online only, available at www.exphem.org) on the LightCycler 2.0 Instruments (Roche Diagnostics, Indianapolis, IN, USA) using LightCycler FastStart DNA MasterPLUS SYBR Green I mix (Roche Diagnostics) and the RPL13A ribosomal gene as the normalization control. Relative quantification was performed using the comparative Ct (2−ΔΔCt) method, where ΔΔCt = mean ΔCt gene – mean ΔCt housekeeping.Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) assays were performed as described previously [22Donati G. Imbriano C. Mantovani R. Dynamic recruitment of transcription factors and epigenetic changes on the ER stress response gene promoters.Nucleic Acids Res. 2006; 34: 3116-3127Crossref PubMed Scopus (66) Google Scholar]. Briefly, cross-linked chromatin from treated and untreated UKE1 and SET2 cells was sonicated and immunoprecipitated with 3 μg of antibodies specific for the following: histone H3 either unmodified (H3; Abcam, Cambridge MA) or acetylated at lysine 9 (H3K9ac) or 14 (H3K14ac) (both from Active Motif, La Hulpe, Belgium) and Thioredoxin as control (Genespin, Milan, Italy). The primers used to amplify NFE2 promoters, control AGTR1 promoter and satellite centromeric region (SAT_CEN) are listed in Supplementary Table E1 (online only, available at www.exphem.org). We perform control amplifications of centromeric regions as a routine step to verify that adequate immunoprecipitation has taken place. The satellite sequences are amplified from the immunoprecipitates obtained with anti-histone H3 antibody, but not anti-acetylated histone antibodies, because histones are not significantly acetylated in these satellite regions [23Ceribelli M. Dolfini D. Merico D. et al.The histone-like NF-Y is a bifunctional transcription factor.Mol Cell Biol. 2008; 28: 2047-2058Crossref PubMed Scopus (94) Google Scholar]. RTQ-PCR was performed using SYBR Green Buffer (Genespin) in the iCycler IQ detection system (Bio-Rad, Hercules, CA, USA). The relative sample enrichment, normalized at first on a satellite centromeric region, was calculated with the following formula: 2ΔCtx − 2ΔCtb, where ΔCtx = Ct input − Ct sample, and ΔCtb = Ct input − Ct control antibody (TRX). Each value of acetylation of lysine 9 and 14 has been normalized on the total amount of immunoprecipitated unmodified H3 [24Gatta R. Mantovani R. NF-Y substitutes H2A-H2B on active cell-cycle promoters: recruitment of CoREST-KDM1 and fine-tuning of H3 methylations.Nucleic Acids Res. 2008; 36: 6592-6607Crossref PubMed Scopus (30) Google Scholar].Statistical analysesDifferences between treated and untreated or mutated and wt cell lines were analyzed by unpaired Student’s t test. Colony assay curves were analyzed using the nonparametric Mann-Whitney test. The effect of GVS during time on proliferation and differentiation was analyzed by analysis of variance for repeated measures.ResultsJAK2-mutated cell lines are more sensitive to GVS than JAK2wt cell linesWe have previously shown that the JAK2V617F-positive cell line HEL (homozygous) showed an IC50 in response to GVS in cytotoxicity assays about threefold lower than JAK2wt myeloid cell lines [10Guerini V. Barbui V. Spinelli O. et al.The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).Leukemia. 2008; 22: 740-747Crossref PubMed Scopus (122) Google Scholar]. In order to extend these observations to a larger panel of cell lines, we analyzed the effect of the drug on UKE1 (homozygous) and SET2 (heterozygous) in alamar blue assays. As shown in Figure 1A and B, the JAK2-mutated cell lines responded to GVS with an IC50 of 60 to 100 nM, whereas an approximately threefold higher IC50 was observed with JAK2wt cell lines (KU812, THP1, and K562) (p < 0.001; Fig. 1B). Similarly in colony assays, the response to GVS of HEL and UKE1 (JAK2V617F) was significantly different than that of K562 and THP1 (JAK2wt) with ED50 of about 40 nM vs 80 to 220 nM, respectively (p < 0.01; Fig. 2A and B). The difference was significant also when each JAK2V617F cell line was compared singly to each JAK2wt line (p < 0.05). The other two cell lines did not form clear colonies (data not shown).Figure 2Cell lines bearing JAK2V617F are more sensitive to GVS than JAK2wt cells in colony assays. The indicated cell lines were plated in methylcellulose in the absence or presence of increasing concentrations of GVS (1−250 nM) and colonies counted after 10 days. (A) The dose−response curves for each cell line and (B) ED50 values for each JAK2wt (striped bars) or JAK2V617F cell lines (black bars). Results are the mean percentages and standard deviations of treated relative to untreated controls from three independent experiments for each cell line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next wondered whether the different sensitivity to GVS was at the level of proliferation or apoptosis. Live cell counts at different time points showed that GVS started to inhibit expansion of the UKE1 and SET2 cell lines already at 50 nM, and of HEL at 100 nM, whereas a 250 nM dose was required to inhibit to a similar extent K562, KU812, or THP1 cells (Supplementary Figure E1; online only, available at www.exphem.org). These data were confirmed in standard cell cycle experiments, showing a 3% to 5% reduction in S-phase cells and 6% to 7% increased G1 phase cells after 48 hours treatment in HEL and UKE1 cells treated with 100 nM GVS (p < 0.05), and no significant change observed in the K562, THP1, and KG1 cell lines at the same dose (Supplementary Figure E2; online only, available at www.exphem.org).As far as apoptosis is concerned, 100 nM and 250 nM GVS induced more apoptosis of JAK2V617F than wt cells at days 3 and 1, respectively (Fig. 3A and B). The difference was statistically significant when the JAK2V617F vs JAK2 wt cell lines were globally compared with each other (p < 0.01 and <0.001 at day 3 and 1, respectively). Differences were also significant when the single JAK2V617F cell lines were compared with each JAK2wt line (p at least < 0.05, except for HEL vs KG1 at day 1, where the difference was not statistically significant) (Supplementary Table E2; online only, available at www.exphem.org). The original dot plots and histograms of a representative experiment are shown in Supplementary Figure E3A and B (online only, available at www.exphem.org).Figure 3The JAK2V617F cell lines are more sensitive to GVS than JAK2wt cells in apoptosis assays. The indicated JAK2V617F (black bars) or JAK2wt cell lines (striped bars) were plated in absence or presence of 100 nM (A) or 250 nM GVS (B). After 1 day (B) or 3 days (A), the percentage of 7-aminoactinomycin D + cells was measured by flow cytometry. Results shown are the means and standard deviations of three independent experiments for each cell line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Global gene expression profiling in JAK2V617F cell linesIn order to analyze the mechanism of action of GVS in JAK2V617F cells at a molecular level, we performed GEP. The HEL and UKE1 cell lines were cultured in triplicate flasks for 6 hours in presence or absence of 250 nM GVS. RNA was extracted and probes prepared for hybridization to the Affymetrix GeneChip Gene 1.0 ST array (Affymetrix) representing >20,000 human genes. Genes significantly up- or downmodulated at least twofold were identified using the nonparametric rank product method. Seven hundred and sixteen genes were modulated by the drug in HEL (258 down and 458 up) and 863 genes in UKE1 (430 down and 433 up). A total of common 293 genes were modulated in both cell lines, of which 114 down and 179 up and these are listed in Supplementary Table E3 (online only, available at www.exphem.org). The complete microarray original data have been deposited in the Gene Expression Omnibus repository (GSE27615).Hierarchical clustering analysis was performed on the 293 common genes modulated by GVS at 6 hours, revealing cell cycle as the functional category with the highest enrichment score (1.48) (Supplementary Table E4; online only, available at www.exphem.org), with 19 genes belonging to this category. The single cell cycle−associated genes are listed in Supplementary Figure E4 (online only, available at www.exphem.org). Of particular interest is the induction of RGS2, CCNG2 (cyclin G2), NOTCH2, and MLF1 and downmodulation of HCFC1, all of which can contribute to the inhibition of proliferation. Other annotation clusters included 14 genes involved in cellular amino acid and derivative metabolic process (1.42 enrichment score) and 7 genes related to methyltransferase activity (1.39 enrichment score) (Supplementary Table E4; online only, available at www.exphem.org).Given the cellular context under study, we specifically searched, among the 293 commonly modulated genes, those known to play a role in hematopoiesis. We found 33 modulated genes that are directly or indirectly relevant for hematopoiesis and these are listed in Figure 4A. They include 8 genes related to migration and adhesion: CX3CR1, sphingosine-1 phosphate lyase 1 (involved in lymphocyte egress from thymus), STAT4 (a TF downstream from CXCR4), cyclic adenosine monophosphate response element binding protein 3 (implicated in monocyte migration), all upregulated by GVS, as well as selectin P ligand, chemokine (C-X-C motif) ligand 2, CD84 (SLAM family member 5), and CD244 (natural killer cell receptor 2B4), all downmodulated by the drug. The other 25 hematopoietic genes included many TF and signaling molecules. In order to further define which genes might be of major interest in our cellular context, on the basis of an extensive search of the literature, we were able to locate 23 of these 25 genes in specific points along the hematopoietic differentiation dendogram, as illustrated in Figure 4B. Most of these genes are thought to be involved directly or indirectly in the control of either multipotent stem cells, the erythrothrombopoietic-myeloid lineage, the lymphoid lineage, or in some cases in more than one differentiation steps (Fig. 4B)Figure 4GVS modulates 33 genes involved in hematopoiesis. (A) The 33 hematopoiesis-associated genes modulated by GVS in both HEL (gray bars) and UKE1 (black bars) are shown. (B) The major steps along the hematopoiesis differentiation dendogram where 25 of the GVS modulated genes are known to be involved are indicated. B = mature B cell; CLP = common lymphoid progenitor; CMP = common myeloid progenitor; E =" @default.
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• W2127135280 date "2012-08-01" @default.
• W2127135280 modified "2023-10-18" @default.
• W2127135280 title "The HDAC inhibitor Givinostat modulates the hematopoietic transcription factors NFE2 and C-MYB in JAK2V617F myeloproliferative neoplasm cells" @default.
• W2127135280 cites W1562891803 @default.
• W2127135280 cites W1844168922 @default.
• W2127135280 cites W1963679004 @default.
• W2127135280 cites W1964551219 @default.
• W2127135280 cites W1964885501 @default.
• W2127135280 cites W1964991616 @default.
• W2127135280 cites W1977046269 @default.
• W2127135280 cites W1980642919 @default.
• W2127135280 cites W1980915232 @default.
• W2127135280 cites W1984329234 @default.
• W2127135280 cites W1999047431 @default.
• W2127135280 cites W2003535009 @default.
• W2127135280 cites W2007567598 @default.
• W2127135280 cites W2007713145 @default.
• W2127135280 cites W2015535501 @default.
• W2127135280 cites W2026973552 @default.
• W2127135280 cites W2029318743 @default.
• W2127135280 cites W2031750072 @default.
• W2127135280 cites W2035421116 @default.
• W2127135280 cites W2037841266 @default.
• W2127135280 cites W2038195476 @default.
• W2127135280 cites W2039907721 @default.
• W2127135280 cites W2054523011 @default.
• W2127135280 cites W2054792541 @default.
• W2127135280 cites W2055096888 @default.
• W2127135280 cites W2066372442 @default.
• W2127135280 cites W2069151092 @default.
• W2127135280 cites W2075214128 @default.
• W2127135280 cites W2086551569 @default.
• W2127135280 cites W2104427970 @default.
• W2127135280 cites W2107344580 @default.
• W2127135280 cites W2121458987 @default.
• W2127135280 cites W2133465414 @default.
• W2127135280 cites W2140936513 @default.
• W2127135280 cites W2142945941 @default.
• W2127135280 cites W2143789585 @default.
• W2127135280 cites W2148256187 @default.
• W2127135280 cites W2151094120 @default.
• W2127135280 cites W2151821895 @default.
• W2127135280 cites W2156694334 @default.
• W2127135280 cites W2158217645 @default.
• W2127135280 cites W2165156559 @default.
• W2127135280 cites W2253277805 @default.
• W2127135280 cites W2320125856 @default.
• W2127135280 cites W2417923273 @default.
• W2127135280 cites W4229780401 @default.
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