Erlotinib Effectively Inhibits JAK2V617F Activity and Polycythemia Vera Cell Growth

JAK2V617F, a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva™) is a potent inhibitor of JAK2V617F activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2V617F-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2V617F-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2V617F-positive PV and other myeloproliferative disorders. JAK2V617F, a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva™) is a potent inhibitor of JAK2V617F activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2V617F-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2V617F-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2V617F-positive PV and other myeloproliferative disorders. Protein-tyrosine kinases (PTKs) 3The abbreviations used are: PTK, protein-tyrosine kinase; PV, polycythemia vera; EGFR, epidermal growth factor receptor; EPO, erythropoietin; GST, glutathione S-transferase. are central regulators of signaling pathways and play a crucial role in controlling proliferation, differentiation, transformation, motility, and invasion. Perturbation of PTK signaling by mutations and other genetic alterations such as chromosomal translocation, interstitial deletion, and internal tandem duplication results in deregulated kinase activity and malignant transformation (1Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3161) Google Scholar). These mutant kinases are attractive therapeutic targets, as exemplified by the efficacy of imatinib mesylate (STI571, Gleevec) in BCR-ABL-positive chronic myelogenous leukemia and hypereosinophilia associated with activating alleles involving PDG-FRA (2Druker B.J. Talpaz M. Resta D.J. Peng B. Buchdunger E. Ford J.M. Lydon N.B. Kantarjian H. Capdeville R. Ohno-Jones S. Sawyers C.L. N. Engl. J. Med. 2001; 344: 1031-1037Crossref PubMed Scopus (4487) Google Scholar, 3Cools J. DeAngelo D.J. Gotlib J. Stover E.H. Legare R.D. Cortes J. Kutok J. Clark J. Galinsky I. Griffin J.D. Cross N.C. Tefferi A. Malone J. Alam R. Schrier S.L. Schmid J. Rose M. Vandenberghe P. Verhoef G. Boogaerts M. Wlodarska I. Kantarjian H. Marynen P. Coutre S.E. Stone R. Gilliland D.G. N. Engl. J. Med. 2003; 348: 1201-1214Crossref PubMed Scopus (1525) Google Scholar) or in the use of gefitinib (Iressa, ZD1839) and erlotinib (Tarceva) in the treatment of non-small cell lung cancer with mutation of the epidermal growth factor receptor (EGFR) (4Paez J.G. Janne P.A. Lee J.C. Tracy S. Greulich H. Gabriel S. Herman P. Kaye F.J. Lindeman N. Boggon T.J. Naoki K. Sasaki H. Fujii Y. Eck M.J. Sellers W.R. Johnson G.E. Meyerson M. Science. 2004; 304: 1497-1500Crossref PubMed Scopus (8538) Google Scholar, 5Lynch T.J. Bell D.W. Sordella R. Gurubhagavatula S. Okimoto R.A. Brannigan B.W. Harris P.L. Haserlat S.M. Supko J.G. Haluska F.G. Louis D.N. Christiani D.C. Settleman J. Haber D.A. N. Engl. J. Med. 2004; 350: 2129-2139Crossref PubMed Scopus (10094) Google Scholar). Recently, a somatic activating mutation in the JAK2 tyrosine kinase resulting from a valine to phenylalanine substitution within the regulatory pseudokinase domain (JAK2V617F) was identified in polycythemia vera (PV), essential thrombocythemia, and idiopathic myelofibrosis (6Baxter E.J. Scott L.M. Campbell P.J. East C. Fourouclas N. Swanton S. Vassiliou G.S. Bench A.J. Boyd E.M. Curtin N. Scott M.A. Erber W.N. Green A.R. Project Cancer Genome Lancet. 2005; 365: 1054-1061Abstract Full Text Full Text PDF PubMed Scopus (2300) Google Scholar, 7Levine R.L. Wadleigh M. Cools J. Ebert B.L. Wernig G. Huntly B.J. Boggon T.J. Wlodarska I. Clark J.J. Moore S. Adelsperger J. Koo S. Lee J.C. Gabriel S. Mercher T. D'Andrea A. Frohling S. Dohner K. Marynen P. Vandenberghe P. Mesa R.A. Tefferi A. Griffin J.D. Eck M.J. Sellers W.R. Meyerson M. Golub T.R. Lee S.J. Galliland D.G. Cancer Cell. 2005; 7: 387-397Abstract Full Text Full Text PDF PubMed Scopus (2484) Google Scholar, 8James C. Ugo V. Le Couedic J.P. Staerk J. Delhommeau F. Lacout C. Garcon L. Raslova H. Berger R. Bennaceur-Griscelli A. Villeval J.L. Constantinescu S.N. Casadevall N. Vainchenker W. Nature. 2005; 434: 1144-1148Crossref PubMed Scopus (2939) Google Scholar, 9Kralovics R. Passamonti F. Buser A.S. Teo S.S. Tiedt R. Passweg J.R. Tichelli A. Cazzola M. Skoda R.C. N. Engl. J. Med. 2005; 352: 1779-1790Crossref PubMed Scopus (2997) Google Scholar, 10Zhao R. Xing S. Li Z. Fu X. Li Q. Krantz S.B. Zhao Z.J. J. Biol. Chem. 2005; 280: 22788-22792Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Infrequent occurrence of this unique mutation has also been reported in chronic myelomonocytic leukemia, in atypical or unclassified myeloproliferative disorders, myelodysplastic syndrome, systemic mastocytosis, chronic neutrophilic leukemia, and acute myeloid leukemia (11Steensma D.P. Dewald G.W. Lasho T.L. Powell H.L. McClure R.F. Levine R.L. Gilliland D.G. Tefferi A. Blood. 2005; 106: 1207-1209Crossref PubMed Scopus (415) Google Scholar, 12Jones A.V. Kreil S. Zoi K. Waghorn K. Curtis C. Zhang L. Score J. Seear R. Chase A.J. Grand F.H. White H. Zoi C. Loukopoules D. Terpos E. Vervessou E.C. Schultheis B. Emig M. Ernst T. Lengfelder E. Hehlmann R. Hochhaus A. Oscier D. Silver R.T. Reiter A. Cross N.C. Blood. 2005; 106: 2162-2168Crossref PubMed Scopus (739) Google Scholar, 13Scott L.M. Campbell P.J. Baxter E.J. Todd T. Stephens P. Edkins S. Wooster R. Stratton M.R. Futreal P.A. Green A.R. Blood. 2005; 106: 2920-2921Crossref PubMed Scopus (133) Google Scholar, 14Jelinek J. Oki Y. Gharibyan V. Bueso-Ramos C. Prchal J.T. Verstovsek S. Beran M. Estey E. Kantarjian H.M. Issa J.P. Blood. 2005; 106: 3370-3373Crossref PubMed Scopus (321) Google Scholar, 15Levine R.L. Loriaux M. Huntly B.J. Loh M.L. Beran M. Stoffregen E. Berger R. Clark J.J. Willis S.G. Nguyen K.T. Flores N.J. Estey E. Gattermann N. Armstrong S. Look A.T. Griffin J.D. Bernard O.A. Heinrich M.C. Gilliland D.G. Druker B. Deininger M.W. Blood. 2005; 106: 3377-3379Crossref PubMed Scopus (321) Google Scholar). The mutant enzyme possesses enhanced tyrosine kinase activity and, when expressed in cells, causes a constitutive activation of signal transduction pathways and growth factor/cytokine-independent cell growth (8James C. Ugo V. Le Couedic J.P. Staerk J. Delhommeau F. Lacout C. Garcon L. Raslova H. Berger R. Bennaceur-Griscelli A. Villeval J.L. Constantinescu S.N. Casadevall N. Vainchenker W. Nature. 2005; 434: 1144-1148Crossref PubMed Scopus (2939) Google Scholar, 10Zhao R. Xing S. Li Z. Fu X. Li Q. Krantz S.B. Zhao Z.J. J. Biol. Chem. 2005; 280: 22788-22792Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Furthermore, its expression in murine bone marrow transplant models results in a PV-like phenotype (16Wernig G. Mercher T. Okabe R. Levine R.L. Lee B.H. Gilliland D.G. Blood. 2006; 107: 4274-4281Crossref PubMed Scopus (410) Google Scholar, 17Lacout C. Pisani D.F. Tulliez M. Gachelin F.M. Vainchenker W. Villeval J.L. Blood. 2006; 108: 1652-1660Crossref PubMed Scopus (355) Google Scholar). Because of its pathogenicity, JAK2V617F represents an obvious potential target for therapeutic drug development. This study was initiated to identify an effective inhibitor of the mutated enzyme. Materials—Polyclonal anti-JAK2 and 4G10 monoclonal anti-phosphotyrosine antibodies were from Santa Cruz Biotechnology and Upstate Biotechnology, Inc., respectively. Erlotinib, imatinib mesylate, and gefitinib were purchased from a local pharmacy. Tyrphostin AG490 was purchased from LC Laboratories, and 1,2,3,4,5,6-hexabromocyclohexane (C6H6Br6) was requested from the NCI Developmental Therapeutics Program. Collection of Peripheral Blood and Purification of Human CD34+ Cells—Phlebotomized units of blood were obtained from patients who met the World Health Organization diagnostic criteria for PV and were treated with phlebotomy only. Normal peripheral blood samples were obtained from healthy donors after blood mobilization with granulocyte colony-stimulating factor. Institutional Review Board approvals have been obtained for the procedures, and each donor was consented. A CD34+ cell population was isolated from low density mononuclear cells of the blood by using the magnetic activated cell sorting CD34+ isolation kit (Miltenyi Biotec, Auburn, CA). Colony-forming Cell Assays—CD34+ cells (1000 cells) were cultured in 1 ml of semisolid medium (Stem Cell Technologies, Vancouver, British Columbia, Canada) containing α-minimal essential medium, 0.9% methylcellulose, 30% fetal bovine serum, 1% bovine serum albumin, 0.05 mm 2-mercaptoethanol, and 0–50 μm erlotinib supplemented with 2 units/ml EPO alone or a mixture of six growth factors/cytokines (2 units/ml EPO, 100 ng/ml stem cell factor, 10 ng/ml interleukin 3, 100 ng/ml interleukin 6, 10 ng/ml granulocyte colony-stimulating factor, and 100 ng/ml thrombopoietin). All cultures were performed in triplicate and various colony types enumerated using an inverted microscope at day 12–14 of culture according to the standard criteria. DNA Extraction and PCR Amplification—Individual hematopoietic cell colonies were taken out from the semisolid phase culture media and diluted into 1 ml of α-minimal essential medium supplemented with 10% fetal bovine serum. After spin down, genomic DNAs were isolated from the pelleted cells by using the Extract-N-Amp™ blood PCR kit from Sigma. The JAK2V617F mutation was detected by using nested allele-specific PCR as described previously (18Xu X. Zhang Q. Luo J. Xing S. Li Q. Krantz S.B. Fu X. Zhao Z.J. Blood. 2007; 109: 339-342Crossref PubMed Scopus (127) Google Scholar). Generation of a Protein Substrate for JAK2 Kinase Activity Assays—A peptide fragment with a sequence of PQDKEYYKVKE derived from the autophosphorylation sites of human JAK2 was expressed as a GST fusion protein by using the pGex-2T vector. The fusion protein designated GST-JAKS was expressed in Escherichia coli cells and then purified by using a glutathione-Sepharose column. JAK2 Kinase Activity Assays—COS7 cells were transfected with pCDNA3 constructs carrying JAK2 or JAK2V617F as described previously (10Zhao R. Xing S. Li Z. Fu X. Li Q. Krantz S.B. Zhao Z.J. J. Biol. Chem. 2005; 280: 22788-22792Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Cells were lysed in a buffer containing 25 mm β-glycerophosphate (pH 7.3), 5 mm EDTA, 2 mm EGTA, 5 mm β-mercaptoethanol, 1% Triton X-100, 0.1 m NaCl, and a protease inhibitor mixture (Roche Applied Science). Cell extracts and anti-JAK2 immunoprecipitates were used for kinases assays in a buffer system containing 25 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 0.2 mm ATP, 2 mm dithiothreitol, and 0.2 mg/ml GST-JAKS. The reactions were allowed to proceed at room temperature for 20 min and then stopped by addition of the SDS gel sample buffer. Tyrosine phosphorylation of GST-JAKS was determined by Western blotting analysis with an anti-phosphotyrosine antibody. Capture of Western blot images and quantification of band signals were carried out by using FluorChem SP imaging system from Alpha Innotech. Development of an Assay for Kinase Activity of JAK2V617F—Wild type JAK2 and the mutant JAK2V617F were expressed in COS7 cells. The overexpressed recombinant proteins were then immunopurified by using a specific anti-JAK2 antibody immobilized onto protein-A beads. Activity assays were carried out with substrate GST-JAKS, a GST fusion protein containing a C-terminal segment of JAK2 where the autophosphorylation sites reside. While JAK2V617F underwent strong autophosphorylation and caused marked phosphorylation of the added substrate, wild type JAK2 showed essentially no activity (Fig. 1, left panel). That GST-JAKS serves as a highly specific substrate for JAK2V617F was further demonstrated by the fact that when the tyrosine kinase activity assays were performed directly with crude cell extracts, only JAK2V617F-transfected cells gave rise to a phosphorylation of the substrate, while cells transfected with the control vector or wild type JAK2 showed no activity (Fig. 1, right panel). Note that tyrosine phosphorylation by JAK2V617F caused a slight mobility shift of GST-JAKS. As expected, the phosphorylation reaction occurred in the JAK2 portion of the fusion protein because GST alone was not phosphorylated (data not shown). The data further demonstrate that JAK2V617F is a hyperactive tyrosine kinase and provide a good assay system to screen for inhibitors of the mutated enzyme. Identification of Erlotinib as a Potent Inhibitor of JAK2V617F—Cell extracts obtained from JAK2V617F-transfected cells were employed to analyze the inhibitory effects of potential inhibitors. Included in our screening were the aforementioned anti-cancer drugs imatinib mesylate, gefitinib, and erlotinib and two other known inhibitors of JAK2, namely, AG490 and 1,2,3,4,5,6-hexabromocyclohexane. AG490, a putative JAK2 and EGFR inhibitor, had been shown to inhibit the growth of PV cells in vitro (8James C. Ugo V. Le Couedic J.P. Staerk J. Delhommeau F. Lacout C. Garcon L. Raslova H. Berger R. Bennaceur-Griscelli A. Villeval J.L. Constantinescu S.N. Casadevall N. Vainchenker W. Nature. 2005; 434: 1144-1148Crossref PubMed Scopus (2939) Google Scholar), and 1,2,3,4,5,6-hexabromocyclohexane was recently reported to be a specific JAK2 inhibitor as well (19Sandberg E.M. Ma X. He K. Frank S.J. Ostrov D.A. Sayeski P.P. J. Med. Chem. 2005; 48: 2526-2533Crossref PubMed Scopus (54) Google Scholar). The data illustrated in Fig. 2 demonstrate that, among these chemicals, erlotinib was by far the most potent inhibitor. It displayed an IC50 value of 4 μm, where IC50 represents the concentration of compounds required to achieve a 50% reduction in the phosphorylation of the exogenous substrate. Imatinib mesylate, gefitinib, and AG490 also showed some inhibitory effects but only at much higher concentrations with IC50 values in submillimolar to millimolar ranges. In contrast, 1,2,3,4,5,6-hexabromocyclohexane exhibited virtually no inhibitory effect on JAK2V617F. We also analyzed the inhibitory effects of erlotinib on wild type JAK2. However, as shown in Fig. 1, JAK2 displayed essentially no kinase activity in comparison with the JAK2V617F mutant when cell extracts or immunoprecipitates were employed for assays. For this reason, we enriched the enzyme from extracts of JAK2-transfected COS7 cells by using a Mono column. The JAK2 protein was at m NaCl, and we were to the activity of this purified JAK2 by using GST-JAKS as a substrate. As shown in Fig. JAK2 was also by erlotinib but with an IC50 value 20 This that the enzyme is to the inhibitor. However, the data not be the data may only the fact that wild type JAK2 in an of PV Cell by of erlotinib in the growth of JAK2V617F-positive PV hematopoietic progenitor cells was further analyzed with a For this CD34+ hematopoietic cells from normal and PV blood samples were cultured in a semisolid colony assay medium supplemented with to the growth of cells or with a mixture of six growth factors/cytokines to growth of and as Fig. shows results obtained with normal blood and two JAK2V617F-positive PV blood Erlotinib effectively the growth of PV hematopoietic progenitor cells with an IC50 of μm, while the growth of normal cells at that It be out that the reported IC50 values of erlotinib for of the receptor and various were at to according to assays H. W. Janne P.A. M. S.E. Frank D.A. Sellers W.R. Meyerson M. Med. 2005; Scopus Google Scholar). In imatinib mesylate, a highly effective drug for treatment of BCR-ABL-positive chronic myelogenous leukemia, the in vitro growth of BCR-ABL-positive cells with an IC50 value at the to B.J. S. Buchdunger E. S. S. J. Lydon N.B. Med. PubMed Scopus Google Scholar, M.W. J.M. Lydon N. Blood. PubMed Google Scholar). of JAK2V617F-positive Cells to the of erlotinib JAK2V617F-positive cells, genomic DNAs were from hematopoietic cell colonies from the culture and then a highly nested allele-specific PCR was employed to identify these colonies were JAK2V617F or data are shown in Fig. All the PV samples used were for the JAK2V617F cultured in the of these cells gave rise to JAK2V617F-positive and colonies at In the of 5 μm of JAK2V617F-positive colonies were while of the colonies colony represents a hematopoietic progenitor cell in the culture These data demonstrate that JAK2V617F-positive hematopoietic progenitor cells were to the by erlotinib than cells, that erlotinib PV hematopoietic cell growth by on JAK2V617F. of a Cell by also analyzed the effects of erlotinib on the human erythroleukemia HEL cell is known to the JAK2V617F mutation H. R.A. M. 2006; PubMed Scopus Google Scholar). Erlotinib effectively growth of HEL with an IC50 of 2 μm but murine erythroleukemia and cell leukemia cells at that concentration Fig. In at μm of erlotinib to achieve a with the two cell revealed that JAK2 is in cells, while HEL cells JAK2V617F These results provide further that erlotinib JAK2V617F-positive cells. assays with a highly specific substrate, we have found that erlotinib a potent inhibitor of JAK2V617F with an IC50 value of 4 μm when analyzed in the of 0.2 mm our in vitro assays demonstrated that erlotinib the growth of JAK2V617F-positive PV hematopoietic progenitor cells with an IC50 of 5 This concentration is at of the reported values for of BCR-ABL-positive cells by imatinib mesylate B.J. S. Buchdunger E. S. S. J. Lydon N.B. Med. PubMed Scopus Google Scholar, M.W. J.M. Lydon N. Blood. PubMed Google Scholar). The drug has been found to be highly effective for treatment of BCR-ABL-positive chronic myelogenous leukemia (2Druker B.J. Talpaz M. Resta D.J. Peng B. Buchdunger E. Ford J.M. Lydon N.B. Kantarjian H. Capdeville R. Ohno-Jones S. Sawyers C.L. N. Engl. J. Med. 2001; 344: 1031-1037Crossref PubMed Scopus (4487) Google Scholar). As a potent inhibitor of JAK2V617F, erlotinib be for the treatment of PV or other myeloproliferative that an activation mutation of JAK2. Erlotinib marked is an anti-cancer drug by and It is as an inhibitor of the EGFR PTK, but our study that also effectively JAK2V617F. may also some inhibitory activity wild type JAK2 and other of the of tyrosine we that inhibit JAK2V617F-positive cells because these cells have to the of the mutated enzyme. a has been well demonstrated with imatinib mesylate, suppresses the growth of cells, also and the growth factor receptor of in vitro (2Druker B.J. Talpaz M. Resta D.J. Peng B. Buchdunger E. Ford J.M. Lydon N.B. Kantarjian H. Capdeville R. Ohno-Jones S. Sawyers C.L. N. Engl. J. Med. 2001; 344: 1031-1037Crossref PubMed Scopus (4487) Google Scholar). Erlotinib has been by the and for the treatment of non-small cell lung cancer and, in with for the treatment of or It has a well The most effects in patients with non-small cell lung cancer erlotinib are to and its in JAK2V617F activity and JAK2V617F-positive PV cell of erlotinib for the treatment of PV and other to be well is no effective for the study also EGFR inhibitor, namely, gefitinib, but this drug was found to be AG490 and imatinib mesylate were found to be only inhibitors of JAK2V617F the fact that had shown that AG490 and imatinib mesylate of PV cells in vitro (8James C. Ugo V. Le Couedic J.P. Staerk J. Delhommeau F. Lacout C. Garcon L. Raslova H. Berger R. Bennaceur-Griscelli A. Villeval J.L. Constantinescu S.N. Casadevall N. Vainchenker W. Nature. 2005; 434: 1144-1148Crossref PubMed Scopus (2939) Google Scholar, L. E. A. C. H. K. Blood. 2003; PubMed Scopus Google Scholar), an effect to their of the JAK2V617F activity. Imatinib mesylate at a concentration of 1 μm growth in the of growth with a of L. E. A. C. H. K. Blood. 2003; PubMed Scopus Google Scholar). a this drug has been used in treatment of PV and was shown to phlebotomy in polycythemia vera patients R.T. 2003; PubMed Scopus Google Scholar). imatinib patients for JAK2V617F, was a reduction in the of mutant alleles that with A.V. Silver R.T. Waghorn K. Curtis C. Kreil S. Zoi K. Hochhaus A. Oscier D. G. Lengfelder E. Reiter A. Chase A.J. Cross N.C. Blood. 2006; 107: PubMed Scopus Google Scholar). We that erlotinib be a drug than AG490 and imatinib mesylate for the treatment of PV patients is a much potent and inhibitor of JAK2V617F and growth of PV cells in the of concentrations of growth and the of the JAK2 kinase domain and tyrosine kinase we be to erlotinib to potent and JAK2 as effective drugs to associated with the JAK2V617F We are to H. for of the with

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.