Erlotinib Effectively Inhibits JAK2V617F Activity and Polycythemia Vera Cell Growth
2006; Elsevier BV; Volume: 282; Issue: 6 Linguagem: Inglês
10.1074/jbc.c600277200
ISSN1083-351X
AutoresZhe Li, Mingjiang Xu, Shu Xing, Wan‐Ting Ho, Takefumi Ishii, Qingshan Li, Xueqi Fu, Zhizhuang Joe Zhao,
Tópico(s)Chronic Myeloid Leukemia Treatments
ResumoJAK2V617F, 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. 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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 Q anion-exchange column. The JAK2 protein was eluted at around 0.3 m NaCl, and we were able to detect the activity of this partially purified JAK2 by using GST-JAKS as a substrate. As shown in supplemental Fig. S1, JAK2 was also inhibited by erlotinib but with an IC50 value beyond 20 μm. This suggests that the enzyme is less sensitive to the inhibitor. However, the data should not be overinterpreted since the data may only reflect the fact that wild type JAK2 stays in an inactive state. Inhibition of PV Hematopoietic Progenitor Cell Growth by Erlotinib—The effectiveness of erlotinib in inhibiting the growth of JAK2V617F-positive PV hematopoietic progenitor cells was further analyzed with a cell-based system. For this purpose, CD34+ hematopoietic cells from normal and PV blood samples were cultured in a semisolid colony assay medium supplemented with erythropoietin to support the growth of erythroid cells or with a mixture of six growth factors/cytokines to stimulate growth of granulocytes/macrophages and megakaryocyte as well. Fig. 3 shows representative results obtained with one normal blood and two JAK2V617F-positive PV blood samples. Erlotinib effectively inhibited the growth of PV hematopoietic progenitor cells with an IC50 of ∼5 μm, while it hardly affected the growth of normal cells at double that concentration. It should be pointed out that the reported IC50 values of erlotinib for inhibition of the EGF receptor and various mutants were at submicromolar to micromolar according to cell-based assays (20Greulich H. Chen T.H. Feng W. Janne P.A. Alvarez J.V. Zappaterra M. Bulmer S.E. Frank D.A. Hahn W.C. Sellers W.R. Meyerson M. PLoS Med. 2005; 2: 1167-1176Crossref Scopus (578) Google Scholar). In addition, imatinib mesylate, a highly effective drug for treatment of BCR-ABL-positive chronic myelogenous leukemia, inhibited the in vitro growth of BCR-ABL-positive cells with an IC50 value at the submicromolar to micromolar range (21Druker B.J. Tamura S. Buchdunger E. Ohno S. Segal G.M. Fanning S. Zimmermann J. Lydon N.B. Nat. Med. 1996; 2: 561-566Crossref PubMed Scopus (3167) Google Scholar, 22Deininger M.W. Goldman J.M. Lydon N. Melo J.V. Blood. 1997; 90: 3691-3698Crossref PubMed Google Scholar). Enhanced Sensitivity of JAK2V617F-positive Cells to Erlotinib—To verify the selectivity of erlotinib toward JAK2V617F-positive cells, genomic DNAs were extracted from hematopoietic cell colonies randomly collected from the culture plates, and then a highly sensitive nested allele-specific PCR method was employed to identify whether these colonies were JAK2V617F positive or not. Representative data are shown in Fig. 4. All the PV samples used were heterogeneous for the JAK2V617F mutation. When cultured in the absence of erlotinib, these cells gave rise to both JAK2V617F-positive and -negative colonies at certain percentages. In the presence of 5 μm erlotinib, percentages of JAK2V617F-positive colonies were significantly reduced while those of the JAK2V617F-negative colonies increased. Each colony theoretically represents a single hematopoietic progenitor cell seeded in the original culture plate. These data thus demonstrate that JAK2V617F-positive hematopoietic progenitor cells were more sensitive to the inhibition by erlotinib than JAK2V617F-negative cells, providing evidence that erlotinib inhibits PV hematopoietic cell growth by acting on JAK2V617F. Inhibition of a JAK2V617F-postive Cell Line by Erlotinib—We also analyzed the effects of erlotinib on the human erythroleukemia HEL cell line, which is known to contain the JAK2V617F mutation (23Quentmeier H. MacLeod R.A. Zaborski M. Drexler H.G. Leukemia. 2006; 20: 471-476Crossref PubMed Scopus (128) Google Scholar). Erlotinib effectively inhibited growth of HEL with an IC50 of 2 μm but hardly affected murine erythroleukemia MEL and T cell leukemia Jurkat cells at that concentration (see supplemental Fig. S2). In fact, it took at least 40 μm of erlotinib to achieve a 40% inhibition with the later two cell lines. Our additional studies revealed that JAK2 is intact in MEL cells, while HEL cells contain homozygous JAK2V617F mutation. These results provide further evidence that erlotinib specifically targets JAK2V617F-positive cells. By performing biochemical assays with a highly specific substrate, we have found that erlotinib acts a potent inhibitor of JAK2V617F with an IC50 value of around 4 μm when analyzed in the presence of 0.2 mm ATP. Most importantly, our in vitro cell-based assays demonstrated that erlotinib preferentially inhibited the growth of JAK2V617F-positive PV hematopoietic progenitor cells with an IC50 of around 5 μm. This concentration is at close range of the reported values for inhibition of BCR-ABL-positive cells by imatinib mesylate (21Druker B.J. Tamura S. Buchdunger E. Ohno S. Segal G.M. Fanning S. Zimmermann J. Lydon N.B. Nat. Med. 1996; 2: 561-566Crossref PubMed Scopus (3167) Google Scholar, 22Deininger M.W. Goldman J.M. Lydon N. Melo J.V. Blood. 1997; 90: 3691-3698Crossref PubMed Google Scholar). The latter 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 might be useful for the treatment of PV or other myeloproliferative disorders that bear an activation mutation of JAK2. Erlotinib (trade marked Tarceva) is an oral anti-cancer drug developed by OSI Pharmaceuticals, Genentech, and Roche Diagnostics. It is defined as an inhibitor of the EGFR PTK, but our study indicates that it also effectively inhibits JAK2V617F. Although it may also display some inhibitory activity toward wild type JAK2 and other members of the JAK family of tyrosine kinases, we believe that it can selectively inhibit JAK2V617F-positive cells because these cells have become "addicted" to the hyperactivity of the mutated enzyme. Such a phenomenon has been well demonstrated with imatinib mesylate, which selectively suppresses the growth of BCR-Abl-positive cells, although it also inhibits Abl and the platelet-derived growth factor receptor family of PTKs 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 approved by the United States Food and Drug Administration for the treatment of non-small cell lung cancer and, in combination with gemcitabine, for the treatment of locally advanced, inoperable or metastatic pancreatic cancer. It has a well established safety profile. The most common side effects in patients with non-small cell lung cancer receiving erlotinib monotherapy are mild to moderate rash and diarrhea. Considering its effectiveness in inhibiting JAK2V617F activity and JAK2V617F-positive PV cell growth, clinical trials of erlotinib for the treatment of PV and other related diseases appears to be well warranted. So far, there is no effective cure for the diseases. Our study also included another EGFR inhibitor, namely, gefitinib, but this drug was found to be poorly effective. Likewise, AG490 and imatinib mesylate were found to be only moderate inhibitors of JAK2V617F despite the fact that previous studies had shown that both AG490 and imatinib mesylate inhibited autonomous erythropoiesis 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, 24Oehler L. Jaeger E. Eser A. Sillaber C. Gisslinger H. Geissler K. Blood. 2003; 102: 2240-2242Crossref PubMed Scopus (34) Google Scholar), an effect possibly attributable to their inhibition of the JAK2V617F activity. Imatinib mesylate at a concentration of 1 μm suppressed autonomous erythroid burst-forming unit growth in the absence of growth factors with a mean inhibition of 73% (24Oehler L. Jaeger E. Eser A. Sillaber C. Gisslinger H. Geissler K. Blood. 2003; 102: 2240-2242Crossref PubMed Scopus (34) Google Scholar). Without a defined molecular target, this drug has been used in investigational treatment of PV and was shown to reduce phlebotomy requirements in polycythemia vera patients (25Silver R.T. Leukemia. 2003; 17: 1186-1187Crossref PubMed Scopus (42) Google Scholar). Although imatinib mesylate-treated patients remained positive for JAK2V617F, there was a significant reduction in the percentage of mutant alleles that correlated with hematologic responses (26Jones A.V. Silver R.T. Waghorn K. Curtis C. Kreil S. Zoi K. Hochhaus A. Oscier D. Metzgeroth G. Lengfelder E. Reiter A. Chase A.J. Cross N.C. Blood. 2006; 107: 3339-3341Crossref PubMed Scopus (100) Google Scholar). We believe that erlotinib should be a more promising drug than AG490 and imatinib mesylate for the treatment of PV patients because: (a) it is a much more potent and selective inhibitor of JAK2V617F and (b) it inhibits growth of PV cells even in the presence of optimal concentrations of growth factors and cytokines. By analyzing the structures of the JAK2 kinase domain and existing tyrosine kinase inhibitors, we should be able to modify erlotinib to produce more potent and selective JAK2 inhibitors, which should serve as more effective drugs to treat diseases associated with the JAK2V617F mutation. We are grateful to Dr. Edmond H. Fischer for his careful reading of the manuscript. Download .pdf (.17 MB) Help with pdf files
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