Transformation by Oncogenic Mutants and Ligand-Dependent Activation of FLT3Wild-type Requires theTyrosine

Purpose: Mutations in the receptor tyrosine kinase FLT3 are found in up to 30% of acute myelogenous leukemia patients and are associated with an inferior prognosis. In this study, we characterized critical tyrosine residues responsible for the transforming potential of active FLT3-receptor mutants and ligand-dependent activation of FLT3-WT. Experimental Design: We performed a detailed structure-function analysis of putative autophosphorylation tyrosine residues in the FLT3-D835Ytyrosine kinase domain (TKD)mutant. All tyrosine residues in the juxtamembrane domain (Y566,Y572,Y589,Y591,Y597, and Y599), interkinase domain (Y726 andY768), andCOOH-terminal domain (Y955 andY969) of the FLT3D835Y construct were successively mutated to phenylalanine and the transforming activity of these mutants was analyzed in interleukin-3-dependent Ba/F3 cells. Tyrosine residues critical for the transforming potential of FLT3-D835Y were also analyzed in FLT3 internal tandem duplicationmutants (FLT3-ITD)and the FLT3 wild-type (FLT3-WT) receptor. Result:The substitution of the tyrosine residues by phenylalanine in the juxtamembrane, interkinase, and COOH-terminal domains resulted in a complete loss of the transforming potential of FLT3-D835Y-expressing cells which can be attributed to a significant reduction of signal tranducer and activator of transcription 5 (STAT5) phosphorylation at the molecular level. Reintroduction of single tyrosine residues revealed the critical role ofY589 andY591in reconstituting interleukin-3-independent growth of FLT3-TKD-expressing cells. Combined mutation of Y589 and Y591 to phenylalanine also abrogated ligand-dependent proliferation of FLT3-WTand the transforming potential of FLT3-ITD-with a subsequent abrogation of STAT5 phosphorylation. Conclusion:We identified two tyrosine residues,Y589 andY591, in the juxtamembrane domain that are critical for the ligand-dependent activation of FLT3-WTand the transforming potential of oncogenic FLT3 mutants. FLT3 is a member of the class III protein receptor tyrosine kinase family (RTK) that is characterized by five extracellular immunoglobulin-like domains, a juxtamembrane domain (JM), and two protein tyrosine kinase domains (TKD) split by an interkinase domain (IK; ref. 1). The class III receptors also include KIT, FMS, platelet-derived growth factor receptor-a (PDGFRA), and platelet-derived growth factor receptor-h (PDGFRB). Binding of FLT3 ligand (FL) to its receptor induces dimerization, phosphorylation, and subsequent activation of downstream signaling pathways such as signal tranducer and activator of transcription 5 (STAT5), Ras/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase/AKT (2–6). FLT3 has been shown to play an important role in normal hematopoiesis and is highly expressed in CD34 hematopoietic progenitor cells (2, 7–9). Activating mutations of FLT3 are found in 30% of patients with acute myelogenous leukemia (AML) and are associated with an inferior clinical outcome (10–12). FLT3 internal tandem duplications (FLT3-ITD) represent one of the most frequent genetic alterations and occur in f20% to 25% of patients. These mutations have a variable length resulting in an elongated FLT3 protein with constitutive kinase activity and are associated with higher leukocyte counts at diagnosis (13). A second class of FLT3 mutations primarily occurs at the highly conserved residue D835 in the TKD and is present in 7% to 8% of all patients with AML (14, 15). Human Cancer Biology Authors’ Affiliations: Clinical Cooperative Group ‘‘Leukemia,’’ GSF-National Research Center for Environment and Health; and Department of Medicine III and Laboratory for Leukemia Diagnostics, University of Munich-Grosshadern, Munich, Germany Received 7/31/07; revised1/11/08; accepted1/24/08. Grant support: Deutsche Forschungsgemeinschaft SFB 684/1: Project A12. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). S.Vempati, C. Reindl, T.M. Kohl, andK. Spiekermann contributed equally to thiswork. Requests for reprints: Karsten Spiekermann, Clinical Cooperative Group ‘‘Leukemia,’’ GSF-National Research Center for Environment and Health, Marchioninistr. 25, 81377 Munich, Germany. Phone: 49-89-7099-417/425; Fax: 49-89-7099-400; E-mail: [email protected]. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-1873 www.aacrjournals.org Clin Cancer Res 2008;14(14) July15, 2008 4437 Research. on April 9, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from We and others have recently shown that FLT3-ITD mutants enhance the proliferative potential of hematopoietic progenitor cells and collaborate with fusion oncogenes, such as the AML1-ETO or PML-RARa oncoproteins, to induce AML in vivo (16, 17). FLT3 selective tyrosine kinase inhibitors are currently in clinical trials for combined treatment with conventional chemotherapy (18, 19). Nevertheless, the underlying mechanism of transformation that is exerted by constitutively activated FLT3 remains elusive. In the present study, we performed a detailed structurefunction analysis of FLT3-TKD and FLT3-ITD receptor mutants to characterize the molecular mechanisms of FLT3-induced transformation. We identified two tyrosine residues, 589 and 591, in the juxtamembrane region of FLT3 that are indispensable for the transforming potential of both FLT3-ITD and FLT3-TKD mutants and ligand-dependent activation of FLT3 wild-type (FLT3-WT). Materials andMethods Reagents and cell lines. Low-passage murine Ba/F3 cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) and maintained in RPMI 1640 with 10% fetal bovine serum and 10% WEHI conditioned Fig. 1. SuccessiveY!F substitutions in the FLT3-TKDmutant indicate the critical role ofY589 andY591for the transforming potential of mutated FLT3. A, overview of Y!Fmutations created in the FLT3-TKD-D835Yconstruct.B, Ba/F3 cells stably transducedwith FLT3-WT, FLT3-D835Y, D835Y-KD, D835Y-F1to D835Y-F8were seeded at a density of 4 10/mL in the absence or presence of IL-3.Viable cells were counted after 72 h by trypan blue exclusion.The proliferation of cells in the presence of IL-3 was defined as100% (control). SE of three independent experiments are shown.C, long-termproliferation assay of Ba/F3 cells expressing FLT3-WT, FLT3-D835Y, D835Y-F1 to D835Y-F8, D835Y-4F. Cells (4 10/mL) were seeded and counted every 3 d for13 d.To avoid overgrowth, cells were split every 3 d.The proliferation of FLT3-TKDexpressing cells was defined as100% (control). SE of three independent experiments are shown.The FLT3-D835Y-kinase dead (KD) mutant was used as a negative control. Human Cancer Biology www.aacrjournals.org Clin Cancer Res 2008;14(14) July15, 2008 4438 Research. on April 9, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from medium as a source of murine interleukin-3 (IL-3) when indicated. Recombinant human FL was purchased from Promokine and recombinant murine IL-3 from Biosource (Solingen, Germany). DNA constructs and vectors. The FLT3-ITD-W51 construct contains a 7–amino acid duplicated sequence (REYEYDL) inserted between amino acids 601 and 602 of human FLT3-WT, and the FLT3-ITDNPOS contains a 28–amino acid duplicated sequence (CSSDNEYFYVDFREYEYDLKWEFPRENL) inserted between amino acids 611 and 612 of FLT3-WT(17). The FLT3-TKD carries substitution (point mutation) of aspartic acid to tyrosine at position 835 of FLT3-WT. All FLT3 constructs were subcloned in the MSCV-IRES-EYFP retroviral expression vector (kindly provided by R.K. Humphries, The Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada). In vitro site directed mutagenesis, DNA sequencing, and nomenclature. Point mutations were introduced into FLT3-WT cDNA, FLT3-ITD (FLT3-W51 and FLT3-NPOS), and FLT3-TKD by site-directed mutagenesis using the QuickChange kit from Stratagene as described previously (20). The correct sequence of all constructs was confirmed by complete nucleotide sequencing. Mutants with successive single tyrosine residues at the FLT3-D835Y background were named F1 to F8 (Fig. 1A), the mutant 4F contains four tyrosine-to-phenylalanine (Y!F) mutations in the JM domain (YF589/591/597/599). FLT3D835Y-kinase dead (KD) indicates the mutant containing the K644R mutation leading to the complete loss of kinase activity as described previously (16). Mutations in the JM domain of FLT3-W51 and FLT3NPOS were named according to the modified positions. All modifications of FLT3-ITD constructs were done in the wild-type, not the duplicated, DNA stretch (Fig. 3A). Expression of CD135 by flow cytometry. Determination of FLT3 expression by FACS analysis was carried out as described previously