Abstract
FLT3 is a member of the type III receptor tyrosine kinase (RTK) family. These receptors all contain an intrinsic tyrosine kinase domain that is critical to signaling. Aberrant expression of the FLT3 gene has been documented in both adult and childhood leukemias including AML, ALL and CML. In addition, 17–27% of pediatric and adult patients with AML have small internal tandem duplication mutations in FLT3. Patients expressing the mutant form of the receptor have been shown to have a decreased chance for cure. Our previous study, using a constitutively activated FLT3, demonstrated transformation of Ba/F3 cells and leukemic development in an animal model. Thus, there is accumulating evidence for a role for FLT3 in human leukemias. This has prompted us to search for inhibitors of FLT3 as a possible therapeutic approach in these patients. AG1296 is a compound of the tyrphostin class that is known to selectively inhibit the tyrosine kinase activity of the PDGF and KIT receptors. Since FLT3 is a close relative of KIT, we wanted to test the possible inhibitory activity of AG1296 on FLT3. In transfected Ba/F3 cells, AG1296 selectively and potently inhibited autophosphorylation of FL-stimulated wild-type and constitutively activated FLT3. Treatment by AG1296 abolished IL-3-independent proliferation of Ba/F3 cells expressing the constitutively activated FLT3 and thus, reversed the transformation mediated by activated FLT3. Inhibition of FLT3 activity by AG1296 in cells transformed by activated FLT3 resulted in apoptotic cell death, with no deleterious effect on their parental counterparts. Addition of IL-3 rescued the growth of cells expressing activated FLT3 in the presence of AG1296. This demonstrates that the inhibition is specific to the FLT3 pathway in that it leaves the kinases of the IL-3 pathway and other kinases further downstream involved in proliferation intact. Several proteins phosphorylated by the activated FLT3 signaling pathway, including STAT 5A, STAT 5B and CBL, were no longer phosphorylated when these cells were treated with AG1296. The activity against FLT3 suggests a potential therapeutic application for AG1296 or similar drugs in the treatment of leukemias involving deregulated FLT3 tyrosine kinase activity and as a tool for studying the biology of FLT3.
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References
D'Andrea A . Hematopoietic growth factors and the regulation of differentiative decisions Curr Opin Cell Biol 1994 6: 804–805
Miyajima A, Kitamura T, Haroda N, Yokota T, Arai K-I . Cytokine receptors and signal transduction Annu Rev Immunol 1992 10: 295–331
Metcalf D . Cellular hematopoiesis in the twentieth century Semin Hematol 1999 36: 5–12
Yarden Y, Ullrich A . Growth factor receptor tyrosine kinases Ann Rev Biochem 1988 57: 443–478
Hanks SK, Quinn AM, Hunter T . The protein kinase family conserved features and deduced phylogeny of the catalytic domains Science 1988 241: 42–52
Rosnet O, Birnbaum D . Hematopoietic receptors of class III receptor-type tyrosine kinases Crit Rev Oncog 1993 4: 595–613
Small D, Levenstein M, Kim E, Carow C, Amin S, Rockwell P, Witte L, Burrow C, Ratajczak MZ, Gewirtz AM, Civin CI . STK-1, the human homolog of Flk-2/FLT-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells Proc Natl Acad Sci USA 1994 91: 459–463
Rappold I, Ziegler BL, Kohler I, Marchetto S, Rosnet O, Birnbaum D, Simmons PJ, Zannettino ACW, Hill B, Neu S, Knapp W, Alitalo R, Alitalo K, Ullrich A, Kanz L, Buhring HJ . Functional and phenotypic characterization of cord blood and bone marrow subsets experessing FLT3 (CD135) receptor tyrosine kinase Blood 1997 90: 1111–1125
Brasel K, Escobar S, Anderberg R, de Vries P, Gruss H-J, Lyman SD . Expression of the Flt3 receptor and its ligand on hematopoietic cells Leukemia 1995 9: 1212–1218
Gotze KS, Ramirez M, Tabor K, Small D, Matthews W, Civin CI . Flt3high and Flt3low CD34+ progenitor cells isolated from human bone marrow are functionally distinct Blood 1997 91: 1947–1958
Gabbianelli M, Pelosi E, Montesoro E, Valtieri M, Luchetti L, Samoggia P et al. Multi-level effects of Flt3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors Blood 1995 86: 1661–1670
Shah AJ, Smogorzewska EM, Hannum C, Crooks GM . Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38− cells and maintains progenitor cells in vitro Blood 1996 87: 3563–3570
Hirayama F, Lyman SD, Clark SC, Ogawa M . The flt3 ligand supports proliferation of lymphohematopoietic progenitors and early B-lymphoid progenitors Blood 1995 85: 1762–1768
Hunte BE, Hudak S, Campbell D, Xu Y, Rennick D . Flk2/Flt3 ligand is a potent cofactor for the growth of primitive B cell progenitors J Immunol 1996 156: 489–496
Mackaretschian K, Hardin JD, Moore KA, Boast S, Goff SP, Lemischka IR . Targeted disruption of the Flk2/Flt3 gene leads to deficiencies in primitive hematopoietic progenitors Immunity 1995 3: 146–161
Hannum C, Culpepper J, Campbell D, McClanahan T, Zurawski S, Bazan JF, Kastelein R, Hudak S, Wagner J, Mattson J, Luh J, Duda G, Martina N, Peterson D, Menon S, Shanafelt A, Muench M, Keiner G, Namikawa R, Rennick D, Roncarolo MG, Zlotnik A, Rosnet O, Dubreuil P, Birnbaum D, Lee F . Ligand for FLT3/FLK2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant RNAs Nature 1994 368: 643–648
McKenna HJ, de Vries P, Brasel K, Lyman SD, Williams DE . Effect of Flt3 ligand on the ex vivo expansion of human CD34+ hematopoietic progenitor cells Blood 1995 86: 3414–3420
Lyman SD, Jacobsen SEW . c-kit ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities Blood 1998 91: 1101–1134
van der Geer P, Hunter T, Lindberg R . Receptor protein-tyrosine kinases and their signal transduction pathways Annu Rev Cell Biol 1994 10: 251–337
Heldin C . Dimerization of cell surface receptors in signal transduction Cell 1995 80: 213–223
Turner AM, Lin NL, Issarachai S, Lyman SD, Broudy VC . FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells Blood 1996 88: 3383–3390
Dosil M, Wang S, Lemischka I . Mitogenic signalling and substrate specificity of the Flk2/Flt3 receptor tyrosine kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 1993 13: 6572–6585
Rottapel R, Turck CW, Casteran N, Liu X, Birnbaum D, Pawson T, Dubreuil P . Substrate specificities and identification of a putative binding site for PI3K in the carboxy tail of the murine Flt3 receptor tyrosine kinase Oncogene 1994 9: 1755–1765
Casteran N, Rottapel R, Beslu N, Lecocq E, Birnbaum D, Dubreuil P . Analysis of the mitogenic pathway of the FLT3 receptor and characterization in its C terminal region of a specific binding site for the PI3 kinase Cell Mol Biol 1994 40: 443–456
Lavagna-Sevenier C, Marchetto S, Birnbaum D, Rosnet O . FLT3 signaling in hematopoietic cells involves CBL, SHC and an unknown P115 as prominent tyrosine-phosphorylated substrates Leukemia 1998 12: 301–310
Zhang S, Fukuda S, Lee Y, Hangoc G, Cooper S, Spolski R, Leonard WJ, Broxmeyer HE . Essential role of signal transducer and activator of transcription STAT5A but not STAT5B for FLT3-dependent signaling J Exp Med 2000 5: 719–728
Tse K-F, Mukherjee G, Small D . Constitutive activation of FLT3 stimulates multiple intracellular signal transducers and results in transformation Leukemia 2000 14: 1766–1776
Roussel MF, Downing JR, Sherr CJ . Transforming activities of human CSF-1 receptors with different point mutations at codon 301 in their extracellular domains Oncogene 1990 5: 25–30
Kitayama H, Kanakura Y, Furitsu T, Tsujimura T, Oritani K, Ikeda H, Sugahra H, Mitsui H, Kanayama Y, Kitamura Y, Matsuzawa Y . Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines Blood 1995 85: 790–798
Shtivelman E, Lifshitz B, Gale R, Canaani E . Fused transcript of abl and bcr genes in chronic myelogenous leukaemia Nature 1985 315: 550–4
Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DG, Saltman L, Look AT . Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma Science 1994 263: 1281–1284
Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, Mauchauffe M, Berthou C, Lesssrd M, Berger R, Ghysdael J, Bernard OA . A TEL-JAK2 fusion protein with constitutive kinase activity in human leukemia Science 1997 278: 1309–1312
Golub TR, Barker GF, Lovett M, Gilliland DG . Fusion of PDGF receptor β to a novel ETS-like gene, TEL, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation Cell 1994 77: 307–316
Carroll M, Tomasson MH, Barker GF, Golub TR, Gilliland DG . The TEL/platelet-derived growth factor β receptor (PDGFβR) fusion in chronic meylomonocytic leukemia is a transforming protein that self-associates and activates PDGFBR kinase-dependent signaling pathways Proc Natl Acad Sci USA 1996 93: 14845–14850
Horiike S, Yokota S, Nakao M, Iwaai T, Sasai Y, Kaneko H, Taniwaki M, Kashima K, Fujii H, Abe T, Misawa S . Tandem duplications of the FLT3 receptor gene are associated with leukemic transformation of myelodysplasia Leukemia 1997 11: 1442–1446
Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, Sonoda Y, Fujimoto T, Missawa S . Internal tandem duplication of the Flt3 gene found in acute myeloid leukemia Leukemia 1996 10: 1911–1918
Iwai T, Yokota S, Nakao M, Okamoto T, Taniwaki M, Onodera N, Watanabe A, Kikuta A, Tanaka A, Asami K, Sekine I, Mugishima H, Nishimura Y, Koizumi S, Horikoski Y, Mimaya J, Ohta S, Nishikawa K, Iwai A, Shimokawa T, Nakayama M, Kawakami K, Gushiken T, Hyakuna N, Katano N, Tsurusawa M, Fujimoto T . Internal tandem duplication of the FLT3 gene and clinical evaluation in childhood acute myeloid leukemia Leukemia 1999 13: 38–43
Xu F, Taki T, Yang HW, Hanada R, Hongo T, Ohnishi H, Kobayashi M, Bessho F, Yanagisawa M, Hayashi Y . Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children Br J Haematol 1999 105: 155–162
Meshinchi S, Woods W, Stirewalt D, Sweetser D, Buckley J, Tjoa T, Bernstein I, Radich J . Prevalence and prognostic significance of flt3 internal tandem duplication in pediatric acute myeloid leukemia Blood 2001 97: 89–94
Kondo M HK, Takahashi Y, Matsumoto K, Fukuda M, Inaba J, Kato K, Kojima S, Matsuyama T . Prognostic value of internal tandem duplication of the FLT3 gene in childhood acute myelogenous leukemia Med Pediatr Oncol 1999 33: 525–529
Kiyoi H, Towatari M, Yokota S, Hamaguchi M, Ohno R, Saito H, Naoe T . Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product Leukemia 1998 12: 1333–1337
Hayakawa F TM, Kiyoi H, Tanimoto M, Kitamura T, Saito H, Naoe T . Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines Oncogene 2000 19: 624–631
Mizuki 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, Serve H . FLT3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways Blood 2000 96: 3907–3914
Pietsch T, Kyas U, Yakisan E, Hadam M, Ludwig W, Zsebo K et al. Effects of human stem cell factor (c-kit ligand) on proliferation of myeloid leukemia cells: heterogeneity in response and synergy with other hematopoietic growth factors Blood 1992 80: 1199–1206
Torres H, Dubrueil P, Falzetti F, Courcoul M, Lopez M, Falcinelli F, Birg F, Tabilio A, Mannoni P . C-fms expression in acute leukemias with complex phenotypes Leukemia 1990 4: 673–677
Parwaresch MR, Kriepe H, Felgner J, Heidorn K, Jaquet K, Bodewadt-Radzun S, Radzun HJ . M-CSF and M-CSF receptor gene expression in acute myelomonocytic leukemias Leuk Res 1990 14: 27–37
Carow CE, Levenstein M, Kaufmann SH, Chen J, Amin S, Rockwell P, Witte L, Borowitz MJ, Civin CI, Small D . Expression of the hematopoietic growth factor receptor FLT3 (STK-1/Flk2) in humanleukemias Blood 1996 87: 1089–1096
Birg F, Courcoul M, Rosnet O, Bardin F, Pebusque M, Marchetto S, Tabilio A, Mannoni P, Birnbaum D . Expression of the FMS/KIT-like gene FLT3 in human acute leukemias of the myeloid and lymphoid lineages Blood 1992 80: 2584–2593
Rosnet O, Buhring HJ, Marchetto S, Rappold I, Lavagna C, Sainty D, Arnoulet C, Chabannon C, Kanz L, Hannum C, Birnbaum D . Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells Leukemia 1996 10: 238–248
Meierhoff G, Dehmel U, Gruss H-J, Rosnet O, Birnbaum D, Quentmeier H, Dirks W, Drexler HG . Expression of FLT3 receptor and FLT3-ligand in human leukemia-lymphoma cell lines Leukemia 1995 9: 1368–1372
Metcalf D . The roles of stem cell self-renewal and autocrine growth factor production in the biology of myeloid leukemia Cancer Res 1989 49: 2305–2311
Hawley R . Interleukin-6-type cytokines in myeloproliferativedisease Ann NY Acad Sci 1995 762: 294–307
Carroll M, Ohno-Jones S, Tamura S, Buchdunger E, Zimmermann J, Lydon N, Gilliand D, Druker B . CGP 57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, TEL-ABL, and TEL-PDGFR fusion proteins Blood 1997 90: 4947–4952
Buchdunger E, Zimmermann J, Mett H, Meyer T, Muller M, Regenass U, Lydon NB . Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class Proc Natl Acad Sci USA 1995 92: 2558–2562
Gazit A, Yaish P, Gilon C, Levitzki A . Tyrphostins I: synthesis and biological activity of protein tyrosine kinase inhibitors J Med Chem 1989 32: 2344–2352
Gazit A, App H, McMahon G, Chen J, Levitzki A, Bohmer F . Tyrphostins. 5. Potent inhibitors of platelet-derived growth factor receptor tyrosine kinase: structure-activity relationships in quinoxalines, quinolines, and indole tyrphostins J Med Chem 1996 39: 2170–2177
Kovalenko M, Gazit A, Bohmer A, Rorsman C, Ronnstrand L, Heldin C, Waltenberger J, Bohmer F, Levitzki A . Selective platelet-derived growth factor receptor kinase blockers reverse sis-transformation Cancer Res 1994 54: 6106–6114
Osherov N, Levitzki A . Epidermal-growth-factor-dependent activation of the src-family kinases Eur J Biochem 1994 225: 1047–1053
Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, Leeder, JS, Freeman M, Cohen A, Gazit A, Levitzki A, Roifman CM . Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor Nature 1996 379: 645–648
Hara T, Miyajima A . Function and signal transduction mediated by the interleukin 3 receptor system in hematopoiesis Stem Cells 1996 14: 605–618
Darnell JE, Kerr IM, Stark G . Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins Science 1994 264: 1415–1420
Fukazawa T, Reedquist K, Trub T, Soltoff S, Panchamoorthy G, Druker B, Cantley L, Shoeslon SE, Band H . The SH3 domain-binding T cell tyrosyl phosphoprotein p120. Demonstration of its identity with the c-cbl protooncogene product and in vivo complexes with Fyn, Grb2, and phosphatidylinositol 3-kinase J Biol Chem 1995 270: 19141–19150
Donovan J, Wange R, Langdon W, Samelson L . The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor J Biol Chem 1994 269: 22921–22924
Langdon WY, Hartley JW, Klinken SP, Ruscetti SK, Morse HC . v-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas Proc Natl Acad Sci USA 1989 86: 1168–1172
Barber DL, Mason JM, Fukazawa T, Reedquist KA, Druker BJ, Band H, D'Andrea AD . Erythropoietin and interleukin-3 activate tyrosine phosphorylation of CBL and association with CRK adaptor proteins Blood 1997 89: 3166–3174
Solanilla A, Grosset C, Lemervier C, Dupouy M, Mahon F, Schweitzer K, Reiffers J, Weksler B, Ripoche J . Expression of Flt3-ligand by the endothelial cell Leukemia 2000 14: 153–162
Lisovsky M, Braun M, Ge Y, Takahira H, Lu L, Savchenko VG, Lyman SD, Broxmeyer HE . Flt3-ligand production by human bone marrow stromal cells Leukemia 1996 10: 1012–1018
Lisovsky M, Estrov Z, Zhang X, Consoli U, Sanchez-Williams G, Snell V, Munker R, Goodacre A, Savchenko V, Andreeff M . Flt3 ligand stimulates proliferation and inhibits apoptosis of acute myeloid leukemia cells: regulation of Bcl-2 and Bax Blood 1996 88: 3987–3997
Zhao M, Kiyoi H, Yammamoto Y, Ito M, Towatari M, Omura S, Kitamura T, Ueda T, Saito H, Naoe T . In vivo treatment of mutant FLT3-transformed murine leukemia with tyrosine kinase inhibitor Leukemia 2000 14: 374–378
Bryckaert M, Eldor A, Fontenay M, Gazit A, Osherov N, Gilon C, Levitzki A, Tobelem G . Inhibition of platelet-derived growth factor-induced mitogenesis and tyrosine kinase activity in cultured bone marrow fibroblasts by tyrphostins Exp Cell Res 1992 199: 255–261
Bilder G, Krawiec J, McVety K, Gazit A, Gilon C, Lyall R, Ziberstein A, Levitzki A, Perrone M, Schreiber A . Tyrphostins inhibit PDGF-induced DNA sythesis and associated early events in smooth muscle cells Am J Physiol 1991 260: 721–730
Kovalenko M, Ronnstrand L, Heldin C-H, Loubtchenkov M, Gazit A, Levitzki A, Bohmer FD . Phosphorylation site-specific inhibition of platelet-derived growth factor B-receptor autophosphorylation by the receptor blocking tyrphostin AG1296 Biochemistry 1997 36: 6260–6269
Druker B, Ttamura S, Buchdunger E, Ohno S, Segal G, Fanning S . Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells Nat Med 1996 2: 561–566
Kravtsov V, Greer J, Whitlock J, Koury M . Use of the microculture kinetic assay of apoptosis to determine chemosensitivities of leukemias Blood 1998 92: 968–980
Koopman G, Reutelingsperger C, Kuijten G, Keehnen R, Pals S, van Oers M . Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis Blood 1994 84: 1415–1420
Martin S, Reutelingsperger C, McGahon A, Rader J, van Schie R, LaFace D, Green D . Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl J Exp Med 1995 182: 1545–1556
Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, Sawyers CL . Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia N Engl J Med 2001 344: 1031–1037
Levis M, Tse K-F, Smith BD, Garrett E, and Small D . A FLT3 tyrosine kinase inhibitor is selectively cytotoxic to AML blasts harboring FLT3/ITD mutations Blood 2001 98 (in press):
Acknowledgements
This work was supported by grants from the National Institute of Health (CA70970, DS, CIC), Leukemia and Lymphoma Society (DS), Children's Cancer Foundation (DS), and the Alexander and Margaret Stewart Trust (DS).
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Tse, KF., Novelli, E., Civin, C. et al. Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor. Leukemia 15, 1001–1010 (2001). https://doi.org/10.1038/sj.leu.2402199
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DOI: https://doi.org/10.1038/sj.leu.2402199
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