Selective Cytotoxic Mechanism of GTP-14564, a Novel Tyrosine Kinase Inhibitor in Leukemia Cells Expressing a Constitutively Active Fms-like Tyrosine Kinase 3 (FLT3)

The receptor tyrosine kinase FLT3 is constitutively activated by an internal tandem duplication (ITD) mutation within the juxtamembrane domain in 20–30% of patients with acute myeloid leukemia. In this study, we identified GTP-14564 as a specific kinase inhibitor for ITD-FLT3 and investigated the molecular basis of its specificity. GTP-14564 inhibited the growth of interleukin-3-independent Ba/F3 expressing ITD-FLT3 at 1 μm, whereas a 30-fold higher concentration of GTP-14564 was required to inhibit FLT3 ligand-dependent growth of Ba/F3 expressing wild type FLT3 (wt-FLT3). However, this inhibitor suppressed the kinase activities of wt-FLT3 and ITD-FLT3 equally, suggesting that the signaling pathways for proliferation differ between wt-FLT3 and ITD-FLT3. Analysis of downstream targets of FLT3 using GTP-14564 revealed STAT5 activation to be essential for growth signaling of ITD-FLT3. In contrast, wt-FLT3 appeared to mainly use the MAPK pathway rather than the STAT5 pathway to transmit a proliferative signal. Further analysis demonstrated that the first two tyrosines in an ITD were critical for STAT5 activation and growth induction but that all of the tyrosines in the juxtamembrane region were dispensable in terms of the proliferation signals of wt-FLT3. These results indicate that an ITD mutation in FLT3 elicits an aberrant STAT5 activation that results in increased sensitivity to GTP-14564. Thus, FLT3-targeted inhibition is an attractive approach, with the potential for selective cytotoxicity, to the treatment of ITD-FLT3-positive acute myeloid leukemia. The receptor tyrosine kinase FLT3 is constitutively activated by an internal tandem duplication (ITD) mutation within the juxtamembrane domain in 20–30% of patients with acute myeloid leukemia. In this study, we identified GTP-14564 as a specific kinase inhibitor for ITD-FLT3 and investigated the molecular basis of its specificity. GTP-14564 inhibited the growth of interleukin-3-independent Ba/F3 expressing ITD-FLT3 at 1 μm, whereas a 30-fold higher concentration of GTP-14564 was required to inhibit FLT3 ligand-dependent growth of Ba/F3 expressing wild type FLT3 (wt-FLT3). However, this inhibitor suppressed the kinase activities of wt-FLT3 and ITD-FLT3 equally, suggesting that the signaling pathways for proliferation differ between wt-FLT3 and ITD-FLT3. Analysis of downstream targets of FLT3 using GTP-14564 revealed STAT5 activation to be essential for growth signaling of ITD-FLT3. In contrast, wt-FLT3 appeared to mainly use the MAPK pathway rather than the STAT5 pathway to transmit a proliferative signal. Further analysis demonstrated that the first two tyrosines in an ITD were critical for STAT5 activation and growth induction but that all of the tyrosines in the juxtamembrane region were dispensable in terms of the proliferation signals of wt-FLT3. These results indicate that an ITD mutation in FLT3 elicits an aberrant STAT5 activation that results in increased sensitivity to GTP-14564. Thus, FLT3-targeted inhibition is an attractive approach, with the potential for selective cytotoxicity, to the treatment of ITD-FLT3-positive acute myeloid leukemia. Fms-like tyrosine kinase 3 (FLT3), 1The abbreviations used are: FLT3, Fms-like tyrosine kinase; FL, FLT3 ligand; RTK, receptor tyrosine kinase; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; ITD, internal tandem duplication; ITD-YF, mutant in which Phe has been substituted for Tyr in the ITD domain; AML, acute myeloid leukemia; wt, wild type; TPO, thrombopoietin; IL-3, interleukin-3; mIL-3, mouse IL-3; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; FCS, fetal calf serum; STAT5A, signal transducers and activators of transcription 5A; dnSTAT5A, dominant negative STAT5A; IRES, internal ribosomal entry site; GFP, green fluorescent protein; JAK, Janus kinase. originally isolated as a hematopoietic progenitor cell-specific kinase, belongs to the class III receptor tyrosine kinase (RTK) family to which c-Fms, c-Kit, and the platelet-derived growth factor (PDGF) receptor also belong (1Matthew W. Jordan C.T. Wiegand G.W. Pardoll D. Lemischka I.R. Cell. 1991; 65: 1143-1152Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 2Rosnet O. Marchetto S. deLapeyriere O. Birnbaum D. Oncogene. 1991; 6: 1641-1650PubMed Google Scholar, 3Rosnet O. Schiff C. Pebusque M.J. Marchetto S. Tonnelle C. Torion Y. Birg F. Birnbaum D. Blood. 1993; 82: 1110-1119Crossref PubMed Google Scholar). Normal expression of FLT3 is restricted to hematopoietic progenitors (4Small D. Levenstein M. Kim E. Carow C. Amin S. Rockwell P. Witte L. Burrow C. Ratajczak M.Z. Gewirtz A.M. Civin C.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 459-463Crossref PubMed Scopus (376) Google Scholar, 5Zeigler F.C. Bennett B.D. Jordan C.T. Spencer S.D. Baumhueter S. Carroll K.J. Hooley J. Bauer K. Matthews W. Blood. 1994; 84: 2422-2430Crossref PubMed Google Scholar), whereas aberrantly expressed FLT3 is observed in acute leukemia cells (6Birg F. Courcoul M. Rosnet O. Bardin F. Pebusque M.J. Marchetto S. Tabilio A. Mannoni P. Birnbaum D. Blood. 1992; 80: 2584-2593Crossref PubMed Google Scholar, 7Carow C.E. Levenstein M. Kaufmann S.H. Chen J. Amin S. Rockwell P. Witte L. Borowitz M.J. Civin C.I. Small D. Blood. 1996; 87: 1089-1096Crossref PubMed Google Scholar). Recently, an internal tandem duplication (ITD) mutation within the juxtamembrane domain of the FLT3 gene has been found in leukemic cells from 20–30% of patients with acute myeloid leukemia (AML) and is closely associated with a poor prognosis in AML patients (8Yokota S. Kiyoi H. Nakao M. Iwai T. Misawa S. Okuda T. Sonoda Y. Abe T. Kashima K. Matsuo Y. Naoe T. Leukemia. 1997; 11: 1605-1609Crossref PubMed Scopus (390) Google Scholar, 9Fenski R. Flesch K. Serve S. Mizuki M. Oelmann E. Kratz-Albers K. Kienast J. Leo R. Schwartz S. Berdel W.E. Serve H. Br. J. Haematol. 2000; 108: 322-330Crossref PubMed Scopus (137) Google Scholar, 10Abu-Duhier F.M. Goodeve A.C. Wilson G.A. Gari M.A. Peake I.R. Rees D.C. Vandenberghe E.A. Winship P.R. Reilly J.T. Br. J. Haematol. 2000; 111: 190-195Crossref PubMed Scopus (268) Google Scholar, 11Meshinchi S. Woods W.G. Stirewalt D.L. Sweetser D.A. Buckley J.D. Tjoa T.K. Bernstein I.D. Radich J.P. Blood. 2001; 97: 89-94Crossref PubMed Scopus (427) Google Scholar). Also found in 7% of AML patients were activating mutations at aspartic acid 835 (Asp-835) in the kinase domain of FLT3; mutations are also associated with a poor prognosis (12Yamamoto Y. Kiyoi H. Nakano Y. Suzuki R. Kodera Y. Miyawaki S. Asou N. Kuriyama K. Yagasaki F. Shimazaki C. Akiyama H. Saito K. Nishimura M. Motoji T. Shinagawa K. Takeshita A. Saito H. Ueda R. Ohno R. Naoe T. Blood. 2001; 97: 2434-2439Crossref PubMed Scopus (977) Google Scholar). Both types of FLT3 mutations result in constitutive activation of FLT3 kinase activity and induce autonomous proliferation of factor-dependent hematopoietic cell lines (13Hayakawa F. Towatari M. Kiyoi H. Tanimoto M. Kitamura T. Saito H. Naoe T. Oncogene. 2000; 19: 624-631Crossref PubMed Scopus (475) Google Scholar, 14Mizuki M. Fenski R. Halfter H. Matsumura I. Schmidt R. Muller C. Gruning W. Kratz-Albers K. Serve S. Steur C. Buchner T. Kienast J. Kanakura Y. Berdel W.E. Serve H. Blood. 2000; 96: 3907-3914Crossref PubMed Google Scholar). Targeted inhibition of a mutant tyrosine kinase might be an attractive therapeutic strategy for hematological malignancies, just as the BCR-ABL tyrosine kinase inhibitor STI571 has been effectively used for the treatment of chronic myelogenous leukemia harboring gain-of-function mutations in the c-ABL tyrosine kinase (15Druker B.J. Lydon N.B. J. Clin. Invest. 2000; 105: 3-7Crossref PubMed Scopus (809) Google Scholar, 16Druker 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 (4457) Google Scholar). Thus, FLT3 is considered to be a rational target for therapeutic intervention aimed at the commonly mutated gene in AML. For this purpose, we screened a small molecular compound library and identified 11 compounds that preferentially inhibited the autonomous growth of Ba/F3 cells expressing ITD-FLT3 (BaF/ITD-FLT3), as compared with Ba/F3 cells expressing wild type FLT3 (BaF/wt-FLT3). Among them, GTP-14564 had the most potent and specific inhibitory activity against BaF/ITD-FLT3. GTP-14564 inhibited the growth of BaF/ITD-FLT3 at 1 μm, whereas a 30-fold higher concentration of GTP-14564 was required for the inhibition of BaF/wt-FLT3 growth. We anticipated that the difference in sensitivity to GTP-14564 between BaF/wt-FLT3 and BaF/ITD-FLT3 was caused by more potent suppression of the kinase activity of ITD-FLT3 than that of wt-FLT3 by GTP-14564. However, we found that GTP-14564 inhibited the kinase activities of wt-FLT3 and ITD-FLT3 equally. This raises the possibility that the selective cytotoxic potency of GTP-14564 is due to differences in growth signaling mechanisms between BaF/wt-FLT3 and BaF/ITD-FLT3. To clarify the molecular basis of the selective cytotoxicity of GTP-14564, we analyzed the signaling pathways down-stream from wt-FLT3 and ITD-FLT3. Reagents—Recombinant human FLT3 ligand (FL), PDGF-BB, stem cell factor (SCF), thrombopoietin (TPO), vascular endothelial growth factor (VEGF), and mouse interleukin-3 (mIL-3) were obtained from R 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2751) Google Scholar), was from Calbiochem-Novabiochem. The anti-phosphotyrosine antibody 4G10 and anti-phospho MAPK antibody were purchased from Upstate Biotechnology and Promega, respectively. Cells—A mouse pro-B cell line, Ba/F3, was maintained in RPMI 1640 containing 10% fetal calf serum (FCS) and 1 ng/ml mIL-3. Ba/F3 cells expressing c-kit or PDGFRβ were grown in RPMI 1640 supplemented with 10% FCS, 1 ng/ml mIL-3, and 0.4 mg/ml G418 (Nacalai Tesque, Inc., Tokyo, Japan). Human leukemia cell lines, including Jurkat, K562, HL-60, THP-1, and MOLM13, were cultured in RPMI 1640 with 10% FCS. Murine myeloid leukemia M-NFS-60 cells were maintained in RPMI 1640 with 10% FCS and 100 ng/ml macrophage colony-stimulating factor (18Nakoinz I. Lee M.-T. Weaver J.F. Ralph P. J. Immunol. 1990; 145: 860-864PubMed Google Scholar). NIH3T3 cells expressing human kinase insert domain-containing receptor (KDR)/Flk-1 were maintained as described (19Takahashi T. Shibuya M. Oncogene. 1997; 14: 2079-2089Crossref PubMed Scopus (273) Google Scholar). An ecotropic retrovirus packaging cell line, Plat-E (20Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus Google Scholar), was maintained in containing 10% FCS, 1 and Co., Tokyo, Japan). of FLT3 and the of Ba/F3 wt-FLT3 or human wt-FLT3 and ITD-FLT3 were the expression containing a gene and Ba/F3 cells using a Gene ITD-FLT3 was from leukemic cells harboring a acid tandem duplication (8Yokota S. Kiyoi H. Nakao M. Iwai T. Misawa S. Okuda T. Sonoda Y. Abe T. Kashima K. Matsuo Y. Naoe T. Leukemia. 1997; 11: 1605-1609Crossref PubMed Scopus (390) Google Scholar). These cells were in the of mg/ml G418 for to BaF/wt-FLT3 and BaF/ITD-FLT3. An and an were and downstream from the ITD in the acid and of tyrosine to mutations were wt-FLT3 or ITD-FLT3. Ba/F3 expressing wt-FLT3 or ITD-FLT3 as as were maintained in RPMI 1640 containing 10% FCS and 1 ng/ml in the of 0.4 mg/ml G418 that BaF/ITD-FLT3 was maintained in the of mIL-3. growth was by a using an R. J. Immunol. 1994; PubMed Scopus Google as described M. T. Misawa K. D. M. A. Kitamura T. Cell. Biol. 1998; PubMed Scopus Google Scholar). inhibition of by E. J. H. T. Muller M. B.J. Lydon N.B. 1996; Google Scholar), D.A. P. M. PubMed Scopus Google Scholar), L. A.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and B. S. S. PubMed Scopus Google Scholar), Lee 1998; PubMed Scopus Google Scholar), growth factor receptor K. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), human growth factor receptor C.I. R.A. PubMed Scopus Google Scholar), protein kinase A PubMed Scopus Google Scholar), protein kinase T. Nakano H. Biotechnology. 1990; PubMed Scopus Google Scholar), and K. H. E. B. Lydon N.B. 1992; PubMed Scopus Google was as described were by the of GTP-14564 receptor by ligand cells were for in of the inhibitor to with macrophage colony-stimulating factor stem cell factor vascular endothelial growth factor or for respectively. of protein of cell were analyzed by using the anti-phosphotyrosine and were as in and and were as described M. T. Misawa K. D. M. A. Kitamura T. Cell. Biol. 1998; PubMed Scopus Google Scholar). The antibody was using the A the The dominant negative its R. R. S. C. L. A. B. F. Cell. Biol. 1996; PubMed Scopus Google was the and of the ribosomal entry fluorescent protein T. T. Misawa K. K. Kitamura T. J. PubMed Scopus Google to This was Plat-E (20Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus Google to the using to the of a for we cells by wt-FLT3 or ITD-FLT3 Ba/F3 The BaF/wt-FLT3 and expressed of FLT3 BaF/wt-FLT3 in the of FL, whereas BaF/ITD-FLT3 growth. a for cell we screened a library of small molecular compounds and identified 11 compounds that inhibited the growth of BaF/ITD-FLT3 at than required for inhibition of growth of Among 11 GTP-14564 had the most potent and specific inhibitory BaF/ITD-FLT3 GTP-14564 is a compound originally to a for F. J. Chem. Scopus Google and is from and Inc. the has with the of that protein kinase by for by GTP-14564 of GTP-14564 to inhibit or receptor was against a of The concentration of GTP-14564 in a of kinase activity or a in the of receptor tyrosine kinase was in I. GTP-14564 inhibited of class III including FLT3, c-Fms, c-Kit, and PDGFRβ with of as in In contrast, GTP-14564 inhibit protein in this study, including kinase insert domain-containing growth factor human growth factor receptor and as protein kinase and These demonstrated that GTP-14564 is a potent and specific inhibitor of class III of inhibition of protein by tyrosine in a GTP-14564 the selective cytotoxicity of GTP-14564 against we the of GTP-14564 the proliferation of and Ba/F3 Also in this was a Ba/F3 expressing a mutant of FLT3 with a to mutation This mutant also autonomous growth in Ba/F3 in GTP-14564 inhibited the proliferation of BaF/ITD-FLT3 at 1 the a 30-fold higher concentration of GTP-14564 was to inhibit growth of The sensitivity of to GTP-14564 was between of BaF/ITD-FLT3 and We the of GTP-14564 inhibition of FLT3 the cell growth of human leukemia cell Among the cell lines proliferation of expressing ITD-FLT3 (8Yokota S. Kiyoi H. Nakao M. Iwai T. Misawa S. Okuda T. Sonoda Y. Abe T. Kashima K. Matsuo Y. Naoe T. Leukemia. 1997; 11: 1605-1609Crossref PubMed Scopus (390) Google was inhibited by 1 GTP-14564. However, cell lines, including Jurkat, expressing expressing and HL-60, were at higher of GTP-14564 proliferation of Ba/F3 cells was also to GTP-14564 These results revealed GTP-14564 to be a specific inhibitor of ITD-FLT3. GTP-14564 the of Both wt-FLT3 and ITD-FLT3 with a demonstrated that cells were more to GTP-14564 than we GTP-14564 the kinase activity of ITD-FLT3 more than that of wt-FLT3 by using receptor as an To we found that GTP-14564 suppressed the of wt-FLT3 and ITD-FLT3 at that suppressed the kinase activities of wt-FLT3 and ITD-FLT3. These results the possibility that the selective growth inhibitory potency of GTP-14564 is due to a difference in signaling pathways between wt-FLT3 and ITD-FLT3. we investigated the of two signaling and MAPK Small D. Leukemia. 2000; 14: PubMed Scopus Google Scholar, S. S. Lee Y. S. R. W.J. J. Med. 2000; PubMed Scopus Google Scholar), of which are to be for cell downstream from the wt-FLT3 and ITD-FLT3 by using GTP-14564. ITD-FLT3 but wt-FLT3 in Ba/F3 an by GTP-14564 Among of the Janus kinase and but were expressed in Ba/F3 of and was by wt-FLT3 or ITD-FLT3 was activated in BaF/ITD-FLT3 but in BaF/wt-FLT3 In the activation of in BaF/ITD-FLT3 was inhibited by GTP-14564 However, GTP-14564 inhibition of activation is to be of the we is activated by in Ba/F3 cells expressing M. Y. L. S. A. Kitamura T. Blood. 1996; PubMed Google this activation cell growth was inhibited by GTP-14564 STAT5 to GTP-14564 than was constitutively in BaF/ITD-FLT3 and was activated in BaF/wt-FLT3 cells factor by with The activation of was inhibited by of GTP-14564 in BaF/ITD-FLT3 as compared with with BaF/wt-FLT3 and inhibition of were the as of was that STAT5A, but was activated by wt-FLT3 S. S. Lee Y. S. R. W.J. J. Med. 2000; PubMed Scopus Google Scholar). However, in the activation of and by wt-FLT3 as as ITD-FLT3 was by or including the used in the A for this is that STAT5 activation in human cells but in hematopoietic cells S. S. Lee Y. S. R. W.J. J. Med. 2000; PubMed Scopus Google Scholar). of and was by wt-FLT3 or ITD-FLT3 and in but of and was as in analysis in BaF/wt-FLT3 cells factor by and this activation was suppressed by GTP-14564 of and was in BaF/ITD-FLT3 To the of and activation in growth signals of wt-FLT3 and we the inhibitor U0126 (17Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2751) Google with to inhibition of the growth of in U0126 the growth of the autonomous growth of BaF/ITD-FLT3. and are activated by ITD-FLT3 in Ba/F3 as as cell line, M. Fenski R. Halfter H. Matsumura I. Schmidt R. Muller C. Gruning W. Kratz-Albers K. Serve S. Steur C. Buchner T. Kienast J. Kanakura Y. Berdel W.E. Serve H. Blood. 2000; 96: 3907-3914Crossref PubMed Google Scholar, H. Towatari M. S. M. Ohno R. Saito H. Naoe T. Leukemia. 1998; PubMed Scopus Google Scholar), activation of and was than suggesting that were activated but in We obtained a result in an using inhibitor, L. A.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google of STAT5 a for by and that of STAT5 as dominant negative R. R. S. C. L. A. B. F. Cell. Biol. 1996; PubMed Scopus Google Scholar, H. T. Kitamura T. A. J. 1996; Scopus Google Scholar). To activation of STAT5 is critical for growth signals by we expressed R. R. S. C. L. A. B. F. Cell. Biol. 1996; PubMed Scopus Google in BaF/wt-FLT3 and BaF/ITD-FLT3 by using a retrovirus T. T. Misawa K. K. Kitamura T. J. PubMed Scopus Google Scholar). the of BaF/wt-FLT3 and BaF/ITD-FLT3 cells were cultured in the or of FL, and cells cells expressing wt-FLT3 or were at to the growth inhibitory of The cell growth was by the of the as a negative growth of BaF/ITD-FLT3 was suppressed by the expression of whereas growth of BaF/wt-FLT3 was by the expression of These results that STAT5 activation is essential for cell growth signals by ITD-FLT3. of ITD-FLT3 for STAT5 and the of STAT5 activation in cell we a of ITD-FLT3 harboring of tyrosine in in the ITD domain we to of tyrosine to STAT5 activation and growth all tyrosines in the ITD region were by this mutant in activated STAT5 induce autonomous growth of Ba/F3 cells To the tyrosine required for STAT5 activation and growth we the of to induce STAT5 activation and cell growth. the mutant in in which the first tyrosines were by was to the in mutant in which the tyrosines were by STAT5 The mutant in but the mutant of ITD-FLT3 in activated that the first two tyrosines are critical for STAT5 We also found that of tyrosines was to induce STAT5 in an using the mutant in or the mutant in in which the first or the tyrosine was respectively. The potential to STAT5 with the potential to induce autonomous cell proliferation in a of the of STAT5 activation in cell growth. the the mutant in of wt-FLT3 in which all tyrosines in the juxtamembrane domain of wt-FLT3 were by induce STAT5 activation but induce cell growth in the of FL, of and by was observed in BaF/wt-FLT3 and Ba/F3 cells expressing the mutant of wt-FLT3 and These results the MAPK pathway was and the STAT5 pathway was dispensable for wt-FLT3 growth In a for of the mutant of FLT3, we a in which two cell lines were used as cell is which in the of FL, and the is which autonomous cell growth. two lines, we identified a kinase inhibitor, GTP-14564, that has and for ITD-FLT3. GTP-14564 has a ITD-FLT3 than its wild type the kinase activities of wt-FLT3 and ITD-FLT3 were suppressed by GTP-14564, suggesting that the selective cytotoxicity of GTP-14564 the difference in the growth signals by wt-FLT3 and ITD-FLT3. including and been in FLT3 signaling Small D. Leukemia. 2000; 14: PubMed Scopus Google Scholar, S. S. Lee Y. S. R. W.J. J. Med. 2000; PubMed Scopus Google Scholar). To clarify the molecular the selective inhibition of cell growth by GTP-14564, we compared signaling downstream of wt-FLT3 and ITD-FLT3 and to GTP-14564. we for the critical of STAT5 activation in but cell growth. A 30-fold higher concentration of GTP-14564 was required to inhibit STAT5 activation in BaF/wt-FLT3 than in BaF/ITD-FLT3. This difference is with the difference in of BaF/ITD-FLT3 and BaF/wt-FLT3 to GTP-14564 in growth is the that cell proliferation by but was inhibited by In the two tyrosine at the of the domain of ITD-FLT3 were required for STAT5 activation as as for the induction of cell growth. the the inhibitor U0126 preferentially inhibited cell proliferation as compared with cell for the MAPK pathway in growth signaling by wt-FLT3. We also found that all of the tyrosine in the juxtamembrane domain of wt-FLT3 were dispensable for cell the mutant of wt-FLT3 growth activation of activation of and by wt-FLT3 was inhibited by GTP-14564 at 3 μm, which suppressed STAT5 activation at These the that the proliferative signal wt-FLT3 mainly the activation of MAPK rather than is that activation of pathways is required for of cell growth. was activated by ITD-FLT3 but and this activation was inhibited by GTP-14564. The inhibition of by GTP-14564 was GTP-14564 inhibit the activation of in the Ba/F3 Thus, GTP-14564 suppressed activation by an most ITD-FLT3. is that ITD-FLT3 STAT5 or STAT5 of was that activation cell growth STAT5 activation R. M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). To is in cell growth STAT5 is required to a dominant negative of STAT5 activation and cell growth. To an dominant negative of has been that ITD-FLT3 in a I.R. C.L. Blood. PubMed Scopus Google Scholar). We are a mouse leukemia in which ITD-FLT3 is progenitor cells of or to critical in by ITD-FLT3. PDGFR, a of class III was to induce of and activation of D. M. Cell. Biol. 1996; PubMed Scopus Google Scholar). However, is expressed in cells and is in the ITD-FLT3 signal as of is most that GTP-14564 inhibition of STAT5 activation and cell growth is due to the suppression of ITD-FLT3 by GTP-14564, and are the targets of GTP-14564. FLT3 including and been to inhibit the proliferation of leukemic cells and expressing ITD-FLT3 E. C. P. D. T. J.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. B.D. E. Small D. Blood. Scopus Google Scholar, M. J. R. B.D. S. B. C. Small D. Blood. PubMed Scopus Google Scholar, C.L. M. J. I. N. D. A. S. Abe K. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). and to be for mouse leukemia by ITD-FLT3. However, were more ITD-FLT3 than wt-FLT3 is in this GTP-14564 is more ITD and of FLT3 than wt-FLT3. This is an the of molecular We also revealed the molecular basis the of GTP-14564 the of Thus, constitutive activation of STAT5 a in growth and suppressed cell proliferation by to the that STAT5 activation in ITD-FLT3 sensitivity to GTP-14564. In we identified GTP-14564, a potent and specific kinase inhibitor for ITD-FLT3. In the of study, with the of the selective cytotoxicity of GTP-14564, we identified differences in signaling pathways by wt-FLT3 and ITD-FLT3 for cell growth of STAT5 a in autonomous cell whereas activation of and was to be more in cell growth of Ba/F3 cells We that ITD mutation in the FLT3 receptor and constitutive STAT5 activation and that GTP-14564 preferentially cell STAT5 activation is more to the inhibitor than is an FLT3-targeted inhibitor strategy with the potential for selective cytotoxicity, as GTP-14564, is a treatment strategy for AML patients harboring ITD-FLT3. We O. T. Naoe for wt-FLT3 and ITD-FLT3 S. for the with kinase and M. for