FLT3 Receptors with Internal Tandem Duplications Promote Cell Viability and Proliferation by Signaling Through Foxo Proteins

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FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins

Blanca Scheijen1,2, Hai T Ngo1,2, Hyun Kang1,2 and James D Grifﬁn*,1,2

1Department of Medical Oncology, Dana-Farber Cancer Institute, Mayer 540, 44 Binney Street, Boston, MA 02115, USA; 2Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Mayer 540, 44 Binney Street, Boston, MA 02115, USA

In about 30% of the patients with acute myeloid leukemia, related to c-KIT/stem cell factor (SCF), macrophage activating FLT3 receptor mutations have been identified, colony-stimulating factor (M-CSF) and platelet-derived often as in-frame internal tandem duplications (ITD) at growth factor (PDGF) receptors (Scheijen and Griffin, the juxtamembrane domain of the receptor. FLT3-ITD 2002). FLT3 expression can be detected in gonads, placenta, receptors exhibit constitutive tyrosine kinase activity in peripheral and central nervous system, and on the surface the absence of FLT3 ligand (FL) binding, and when of hematopoietic stem cells (HSC), uncommitted lymphoid expressed in cytokine-dependent cell lines and primary and myeloid progenitors as well as CD14 þ monocytes hematopoietic cells suppress programmed cell death and (Rosnet et al., 1991; Lyman and Jacobsen, 1998). increase cell division. However, the signaling pathways Accumulating data indicate that FLT3 represents the important for transformation, in particular the nuclear most frequently mutated gene in human acute myeloid targets, are unknown. Here we demonstrate that FLT3- leukemia (AML), with approximately one-third of the ITD expression in Ba/F3 cells results in activation of Akt patients displaying somatic FLT3 gene alterations on and concomitant phosphorylation of the Forkhead family chromosome 13q12, which result in FLT3-ligand (FL)- member Foxo3a. Phosphorylation of Foxo proteins independent tyrosine kinase activation of the FLT3 through FLT3-ITD signaling promotes their translocation receptor (Gilliland and Griffin, 2002). In about three- from the nucleus into the cytoplasm, which requires the quarters of these patients, one or more internal tandem presence of conserved Akt phosphorylation sites in Fork- duplications (ITD) within the juxtamembrane (JM) head transcription factors and PI3K activity. Induction of region of the FLT3 receptor can be detected (Nakao Foxo3a phosphorylation by FLT3-ITD receptors in Ba/ et al., 1996), while point mutations, insertions or F3 cells correlates with the suppression of Foxo-target deletions involving codons 835/836 and 840/841 in the genes p27Kip1 and the proapoptotic Bcl-2 family member activation loop of the bipartite tyrosine kinase domain Bim. Specifically, FLT3-ITD expression prevents Foxo- are present in the remaining cases (Abu-Duhier et al., 3a-mediated apoptosis and upregulation of p27Kip1 and 2001; Yamamoto et al., 2001; Spiekermann et al., 2002; Bim gene expression. These data indicate that the Thiede et al., 2002). Interestingly, wild-type FLT3 oncogenic tyrosine kinase FLT3 can negatively regulate receptors are also often highly expressed in pre-B acute Foxo transcription factors, thereby promoting cell lymphoblastic leukemia (ALL) (Rosnet et al., 1993; survival and proliferation. DaSilva et al., 1994; Meierhoff et al., 1995; Carow et al., Oncogene (2004) 23, 3338–3349. doi:10.1038/sj.onc.1207456 1996), and in pediatric leukemias carrying rearrange- Published online 23 February 2004 ments involving the mixed-lineage leukemia (MLL) gene (Armstrong et al., 2002), suggesting that in vivo Keywords: Akt; Bim; FLT3; forkhead transcription proliferation of these leukemic blast cells is augmented factors; p27Kip1 through autocrine or paracrine FL stimulation. It has been shown that FLT3-ITD receptors prevent apoptosis and mediate oncogenic transformation of cytokine-dependent cell lines (Zhao et al., 2000; Levis et al., 2002), block myeloid differentiation (Zheng et al., Introduction 2002), and induce a lethal myeloproliferative disease in mice, employing a bone marrow transplant assay (Kelly FMS-like tyrosine kinase-3 (FLT3/CD135) belongs to the et al., 2002). Several studies have suggested that the type III tyrosine kinase receptor family, and is structurally signaling proteins STAT5, ERK and Akt are linked to the activation of FLT3 (Hayakawa et al., 2000; Mizuki et al., 2000; Zhang et al., 2000). The serine/threonine *Correspondence: JD Griffin, Department of Medical Oncology, kinase Akt, also termed protein kinase B (PKB), is Dana-Farber Cancer Institute, Mayer 540, 44 Binney Street, Boston, known to be a downstream target of phosphatidylino- MA 02115, USA; E-mail: James_Griffi[email protected] Received 21 April 2003; revised 14 December 2003; accepted 15 sitol 3-kinase (PI3K) pathway and involved in mediating December 2003; Published online 23 February 2004 insulin, survival and cell proliferation reponses (Datta FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3339 et al., 1999; Lawlor and Alessi, 2001). Full activation of we generated Ba/F3 cells expressing a FLT3-ITD Akt requires recruitment to the plasma membrane receptor under the control of a tetracycline-responsive through its N-terminal lipid-binding pleckstrin homol- promoter. To this end, one specific FLT3-ITD variant, ogy (PH) domain, where Ser473 phosphorylation occurs, termed ITD4 (FYVDFREYDEDFYVDFREY), with a followed by phosphoinositide-dependent kinase 1 hemagglutinin A (HA)-antigenic tag at the C-terminus (PDK1)-mediated phosphorylation of residue Thr308 (Figure 1a), was introduced by retroviral infection into (Scheid et al., 2002). the TonBaF.1 cell line (TonB) containing the reverse- Several important substrates of Akt have been identified Tet transactivator gene (Klucher et al., 1998), generating (Lawlor and Alessi, 2001), including members of the the polyclonal cell line TonB.FLT3-ITD4. Addition of Forkhead family of transcription factors (Brunet et al., 1999; Kops et al., 1999). In Caenorhabditis elegans,thereis strong genetic evidence implicating Forkhead transcription factor DAF-16 as a critical target of the insulin receptor/ PI3K/PDK1/Akt pathway (Ogg et al., 1997; Paradis and Ruvkun, 1998). The Foxo proteins Foxo1 (FKHR), Foxo3a (FKHRL1) and Foxo4 (AFX) represent the mammalian orthologues of DAF-16, and phosphorylation of these Forkhead transcription factors by Akt inhibits their nuclear translocation and hence their ability to transactivate transcriptional target genes (Brunet et al., 1999; Kops et al., 1999; Rena et al., 1999; Tang et al., 1999). In mammals, Foxo1 is involved in insulin inhibition of hepatic glucose production, stimulation of b cell proliferation and adipocyte differentiation (Nakae et al., 2002, 2003), while Foxo3a has been implicated as a regulator of ovarian follicular growth activation (Castrillon et al., 2003). Foxo proteins, in their nonphosphorylated form, contribute to apoptosis by their ability to activate FasL (Brunet et al., 1999), Bim (Dijkers et al., 2000a), TRAIL (Modur et al., 2002) and TRADD (Rokudai et al., 2002) gene expression. Furthermore, Foxo proteins are able to control different cell cycle checkpoints by modulating the expression of pRb family member p130 (Kops et al., 2002), cyclin-dependent kinase (CDK) inhibitor p27Kip1 (Medema et al., 2000), cyclin D (Ramaswamy et al., 2002; Schmidt et al., 2002), cyclin B and polo-like kinase (Plk) (Alvarez et al., 2001). Given the growing evidence that the PI3K/Akt pathway transmits important signals downstream of oncogenic tyrosine kinases, we have examined the potential involvement of Foxo proteins in mediating the growth- stimulating and antiapoptotic action of FLT3-ITD receptor signaling. Here, we demonstrate that activated FLT3 receptor signaling induces phosphorylation of Foxo3a in Ba/F3 cells. FLT3-ITD receptors suppress Figure 1 Doxycycline-dependent expression of FLT3-ITD4 in Kip1 Tet-On Ba/F3 (TonB) cell line. (a) Schematic representation of the Foxo3a-mediated apoptosis and induction of p27 and FLT3-ITD4-HA receptor that harbors one specific ITD (ITD4) in Bim gene expression. In addition, constitutive activation the JM domain and HA tag at the carboxy-terminus. The FLT3 of FLT3 signaling triggers nuclear exclusion of Foxo receptor contains five immunoglobulin-like domains in the extra- proteins and suppresses their transcriptional activity. cellular domain (ECD), followed by a transmembrane (T) region, a Thus, inhibition of Foxo protein function may con- short intracellular JM domain and a split tyrosine kinase (TK1 and TK2) domain. (b) FLT3-ITD4-HA was cloned in pRevTRE-Hyg, tribute to the oncogenic transformation of FLT3-ITD and stable transduced in TonB cells to generate the TonB.FLT3- receptors in hematopoietic malignancies. ITD4 cell line. TonB.FLT3-ITD4 cells were cultured for 24 h either in the absence of doxycycline (dox), FLT3 ligand (FL) and FLT3 tyrosine kinase inhibitor PKC412 (lane 1), in the presence of 2 mg/ ml doxycycline (lane 2), with 2 mg/ml doxycycline, and stimulated Results for 10 min with 100 ng/ml FL (lane 3), or in the presence of doxycycline and 10 nM PKC412 (lane 4). Cells were collected and subjected to immunoprecipitation (IP) with HA antibody (Ab) and Expression of FLT3-ITD4 is sufficient to promote cell analysed by immunoblotting (IB) using either FLT3 or phospho- survival and proliferation in IL-3-deprived Ba/F3 cells tyrosine (p-Tyr) MoAb. (c) TonB.FLT3-ITD4 cells were cultured in medium with doxycycline for 24 or 72 h and cell surface To examine the signaling events that are strictly expression of FLT3-ITD4 was analysed by flow cytometry using a regulated by activated FLT3 receptors harboring ITD, PE-conjugated FLT3/CD135 MoAb

Oncogene FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3340 the tetracycline analog doxycycline induced expression doxycycline treatment had no significant additional of both the 135 and 160 kDa isoforms of FLT3-ITD4, effect on the level of ERK and STAT5 phosphorylation, with virtually undetectable FLT3-ITD4 protein levels in but phosphorylation of Akt on Ser473 continued to its absence (Figure 1b). Flow-cytometric analysis con- firmed cell surface expression of FLT3-ITD receptors in the presence of doxycycline, with more selective out- growth of higher FLT3-ITD-expressing cells 72 h after the addition of doxycycline (Figure 1c). As expected from previous studies, the constitutive tyrosine kinase activity of FLT3-ITD4 was readily inhibited by the addition of 10 nM PKC412, a potent small-molecule tyrosine kinase inhibitor of both wild-type and mutant FLT3 receptors (Weisberg et al., 2002). TonB cells, like the parental Ba/F3 cell line, require IL-3 both for proliferation as well as to overcome the default apoptotic program. As measured by annexin-V staining, cell viability of the parental TonB cell line decreased progressively following removal of IL-3, both in the presence and absence of doxycycline (Figure 2a). In contrast, apoptosis was significantly reduced in TonB cells expressing FLT3-ITD4, especially after prolonged IL-3 deprivation (Figure 2a). Similar results were obtained with FACS analysis on propidium iodide- labeled cells, where induction of FLT3-ITD4 expression was sufficient to reduce the fraction of apoptotic cells (sub-G1 content) (Figure 2b). Furthermore, cell division was actively stimulated by FLT3-ITD4 receptor signaling with more TonB.FLT3-ITD4 cells in S phase in the presence of doxycycline (Figure 2b), accompanied by an exponential increase in cell numbers (Figure 2c). These results demonstrate that the expression of FLT3-ITD4 receptors is sufficient to rescue IL-3-deprived Ba/F3 cells from programmed cell death and stimulate their cell division.

FLT3 receptors with ITD4 mutation activate ERK, STAT5 and Akt signaling pathways We initially analysed the activation of several known signaling targets of FLT3, including ERK (MAP kinase), STAT5 and Akt. FLT3-ITD4 expression could be detected in IL-3-starved TonB.FLT3-ITD4 cells 5 h after the addition of doxycycline, and this was accompanied by phosphorylation and activation of ERK1/2, STAT5 and Akt (Figure 3a). Further increase in FLT3-ITD4 expression levels with prolonged

Figure 2 Induction of FLT3-ITD4 expression is sufficient to promote survival and cell proliferation in IL-3-deprived Ba/F3 cells. (a) IL-3-dependent parental Tet-On Ba/F3 (TonB) and TonB.FLT3-ITD4 cells were grown in the absence of IL-3 with or without 2 mg/ml doxycycline (dox). After 0, 16 and 64 h, cells were harvested and the percentage of Annexin-V positive cells was determined by flow-cytometric analysis. (b) TonB.FLT3-ITD4 cells were cultured in the absence of IL-3 with or without doxycycline. After 16 and 40 h, cells were collected, fixed in ethanol, and labeled with propidium iodide to determine their DNA profiles by flow cytometry. (c)1Â 105 TonB.FLT3-ITD4 cells were cultured without IL-3 in the absence or presence of 2 mg/ml doxycycline for 4 days, and each day the viable cell count was determined by trypan blue exclusion

Oncogene FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3341 increase. PKC412 treatment strongly inhibited the apoptotic action of FLT3-ITD4 expression correlated activation of ERK, STAT5 and Akt (Figure 3a). with the status of Foxo3a phosphorylation. Indeed, These data confirm previous findings that FLT3 immunoblot analysis indicated an increased level of receptors with ITD mutations efficiently trigger the Foxo3a phosphorylation on residue Thr32, as shown by activation of ERK, STAT5 and Akt in the absence of the appearance of additional slower-migrating phospho- FL stimulation. species immediately after induction of FLT3-ITD4 expression (Figure 3a). To further verify that FLT3-ITD receptor signaling Phosphorylation of Foxo3a is induced after FLT3-ITD4 promotes Foxo3a phosphorylation, Ba/F3 cells were receptor expression transiently transfected with expression vectors encoding Akt is thought to mediate critical signals from several FLT3-ITD4, wild-type FLAG-Foxo3a or triple mutant oncogenic tyrosine kinases, including BCR/ABL and (TM) FLAG-Foxo3a, where all the three Akt phos- TEL/platelet-derived growth factor receptor b (Skorski phorylation sites (Thr32, Ser253 and Ser315) of Foxo3a et al., 1997; Dierov et al., 2002). In turn, although Akt have been mutated to Ala residues. Phosphorylation of has a number of substrates, genetic studies in C. elegans Thr32 was monitored after immunoprecipitation with have implicated the Forkhead transcription factors of FLAG antibody in IL-3-deprived Ba/F3 cells. In the particular importance for growth and viability down- absence of FLT3-ITD expression, FLAG-Foxo3a was stream of the insulin receptor. Furthermore, negative not phosphorylated on residue Thr32 (Figure 3b, lane 1). regulation of Foxo3a has been implicated in cytokine- However, coexpression of FLT3-ITD4 induced the mediated survival signaling downstream of PI3K/Akt phosphorylation of wild-type FLAG-Foxo3a pathway (Dijkers et al., 2000b, 2002), and phosphoryla- (Figure 3b, lane 3), but not FLAG-Foxo3a-TM tion of Foxo3a on residue Thr32 by Akt is critical for (Figure 3b, lane 4). These data show that FLT3-ITD4- interaction with 14-3-3 proteins and its cytoplasmic induced stimulation of the Akt pathway is accompanied retention (Brunet et al., 1999). Thus, we examined by enhanced Foxo3a phosphorylation in Ba/F3 cells whether the unscheduled cell proliferation and anti- deprived from IL-3.

Figure 3 Activation of FLT3 receptor signaling induces Akt and Foxo3a phosphorylation. (a) Distinct signaling pathways are activated by FLT3-ITD receptors. TonB.FLT3-ITD4 cells were deprived of IL-3 for 16 h, and thereafter 2 mg/ml doxycycline (dox) was added to the medium. Cells were collected 0, 5, 8 and 11 h after the addition of dox in the absence or presence of 20 nM PKC412. Parallel polyacrylamide gels loaded with 50 mg of protein extracts were blotted and probed with phospho-ERK, phospho-STAT5, phospho-Serine 473 (pS473)-Akt and phospho-threonine 32 (pT32)-Foxo3a Ab. Blots were stripped and reprobed with anti-ERK, anti-STAT5, anti-Akt and anti-Foxo3a, respectively. Equal protein loading was veriﬁed by analysing actin expression. (b) FLT3-ITD- mediated Foxo3a phosphorylation requires intact Akt phosphorylation sites. Ba/F3 cells were electroporated with expression vectors encoding FLT3-ITD4, FLAG-tagged wild-type Foxo3a (FLAG-Foxo3a), or FLAG-tagged triple mutant Foxo3a, where each of the three Akt phosphorylation sites (Thr32, Ser253, Ser315) have been mutated to Ala (FLAG-Foxo3a-TM). After 20 h of IL-3 deprivation, cells were harvested and subjected to immunoprecipitation (IP) with FLAG Ab. Whole-cell lysates were probed with FLT3 Ab, while IPs were probed with pT32-Foxo3a and Foxo3a Ab. (c) FL stimulates Akt and Foxo3a phosphorylation in Ba/F3 cells expressing wild-type FLT3 receptors. TonB, TonB.FLT3-WT and TonB.FLT3-ITD4 cell lines were cultured for 20 h in the absence of IL-3 and serum and in the presence of 2 mg/ml doxycycline. Cells were harvested after incubation with or without 200 ng/ml FL for 5 or 10 min, and analysed by Western blot analysis using pS473-Akt, Akt, pT32-Foxo3a and Foxo3a antibodies

Oncogene FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3342 FL stimulation of wild-type FLT3 receptors promotes Akt and Foxo3a phosphorylation in IL-3- and serum-starved Ba/F3 cells It has been demonstrated that FLT3-ITD but not ligand-bound wild-type FLT3 receptors activate STAT5 signaling (Hayakawa et al., 2000; Mizuki et al., 2000; Spiekermann et al., 2003), and upregulate Bcl-XL expression (Minami et al., 2003), implying qualitative differences between wild-type FLT3 and mutant FLT3- ITD signaling. Therefore, we assessed whether stimulation of wild-type FLT3 receptors with FL was able to trigger Akt activation and inhibitory Foxo3a phosphorylation similar to FLT3-ITD receptors. To this end, TonB.FLT3-WT cells were generated that expressed equal levels of FLT3 protein as TonB.FLT3-ITD4 cells (Figure 3c). Only in the absence of exogenous IL-3 and serum, parental TonB and TonB.FLT3-WT cells dis- played complete reduction of phospho-Akt and phospho-Foxo3a levels. In the presence of 10% FCS, there was apparently sufficient FL in the medium, to trigger wild-type FLT3 receptor signaling (data not shown). In starved TonB.FLT3-WT cells, the addition of 200 ng/ml FL for 5 or 10 min resulted in similar phospho-Akt and phospho-Foxo3a levels, as observed in TonB.FLT3- ITD4 cells in the absence of FL (Figure 3c). Addition of exogenous FL to TonB.FLT3-ITD4 cells did not result in a further increase of Akt or Foxo3a phosphorylation. In conclusion, these findings indicate that stimulation of wild-type FLT3 receptors by FL and mutant FLT3-ITD receptor signaling both induce Akt activation and inhibitory phosphorylation of Foxo3a to the same Figure 4 Cytoplasmic retention of EGFP-Foxo3a by FLT3-ITD extent in Ba/F3 cells. receptor expression. (a–f) COS7 cells were transfected with 2.5 mg of wild-type EGFP-Foxo3a (a, c and e) or mutant EGFP-Foxo3a- TM (b, d and f) and 2.5 mg pEBB empty vector DNA (a and b)or pEBB-FLT3-ITD4-HA (c–f). Directly after transfection, cells were ITD mutations in FLT3 receptors regulate serum-starved by culturing them in the absence of FBS. At 22 h nuclear-cytoplasmic shuttling of EGFP-Foxo3a post-transfection, cells were treated with DMSO (a–d)or10mM LY294002 (e and f) for 5 h. Images were taken on living cells with Each of the Foxo protein family members contains three an inverted microscope Akt consensus phosphorylation sites (Arg–Xaa–Arg– Xaa–Xaa–Ser/Thr), which play distinct roles in controlling intracellular localization of Forkhead factors for 5 h with the specific PI3K inhibitor LY294002 (Brownawell et al., 2001; Rena et al., 2001, 2002). To abrogated nuclear-cytoplasmic shuttling of EGFP-Fox- determine the regulation of Foxo protein subcellular o3a by FLT3-ITD4 signaling (Figure 4e). Similar results localization by constitutively active FLT3 signaling, we were obtained for EGFP-Foxo1 and EGFP-Foxo1-TM analysed the intracellular localization of a fusion protein (data not shown). We conclude, therefore, that cyto- composed of enhanced green fluorescence protein plasmic retention of Foxo proteins is actively induced by (EGFP) and Foxo3a (EGFP-Foxo3a), as well as FLT3-ITD4 receptor expression and requires signaling EGFP-Foxo3a-TM, where the Akt phosphorylation through the PI3K/Akt pathway. sites Thr24, Ser256 and Ser319 of Foxo3a have been mutated to Ala. COS7 cells were transfected with either Transcription activation by Foxo transcription factors is pEGFP-Foxo3a or pEGFP-Foxo3a-TM and cultured inhibited by FLT3-ITD4 signaling for 24 h in the absence of serum. Under these conditions, both EGFP-Foxo3a and EGFP-Foxo3a-TM were loca- Foxo proteins show strong transcription activation of a lized in the nucleus (Figure 4a and b). However, co- minimal promoter element containing three tandem expression of FLT3-ITD4 resulted in a significant copies of alternating IRS-A (CAAAACAA) and IRS-B re-localization of EGFP-Foxo3a from the nucleus into (TTATTTTG) sequences derived from IGFBP-1 pro- the cytoplasm (Figure 4c), which required the presence moter (3 Â IRS). The observation that FLT3-ITD of intact Akt sites in Foxo3a, as cytoplasmic retention promotes Foxo3a phosphorylation, along with the was not observed with EGFP-Foxo3a-TM (Figure 4d). localization data obtained with EGFP-Foxo3a, suggests Nuclear exclusion of EGFP-Foxo3a was dependent on that FLT3-ITD signaling could regulate transcription PI3K activity, since treatment of transfected COS7 cells activation by Foxo proteins. To test this hypothesis,

Oncogene FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3343 U2-OS cells were transfected with a luciferase reporter plasmid containing 3 Â IRS promoter element (3 Â IRS- Luc), together with increasing concentrations of expression vectors encoding HA-tagged Foxo3a, FLT3- ITD4-HA or HA-tagged Akt containing a myristolated amino-terminus (HA-Myr-Akt). Under serum-starved conditions, Foxo3a transfection resulted in a dose-dependent increase in transcription of the 3 Â IRS-Luc reporter (Figure 5a), whereas no enhancement was observed with a reporter plasmid lacking IRS elements (data not shown). Importantly, the transcriptional activity of Foxo3a was strongly reduced after cotransfection of FLT3-ITD4 (Figure 5a), indicat- ing that FLT3-ITD receptor signaling signiﬁcantly inhibits the ability of Foxo3a to act as a transcriptional activator. In the presence of constitutive active Akt through transfection of HA-Myr-Akt, Foxo3a-mediated transcription activation was almost completely abol- ished, as has been reported before (Brunet et al., 1999; Dijkers et al., 2000b). Similarly, transcriptional activity of Foxo1 (data not shown) and Foxo4 (Figure 5b) were diminished by co-expression of FLT3-ITD4. These results demonstrate that constitutively active FLT3 signaling through acquired ITD mutations negatively regulates Foxo protein function by prohibiting their ability to activate transcriptional target genes.

Downregulation of Foxo target genes p27Kip1 and Bim upon induction of FLT3-ITD4 expression Cytokine-mediated proliferation and survival has been shown to correlate with phosphorylation of Foxo3a and suppression of p27Kip1 and Bim gene expression (Dijkers et al., 2000a, b; Stahl et al., 2002). High p27Kip1 levels are linked to cell cycle arrest in G0/G1 through interaction with CDK-cyclin complexes (Toyoshima and Hunter, 1994), while the proapoptotic Bcl-2 family member Bim acts as an important death activator in hematopoietic cells (Bouillet et al., 1999; Shinjyo et al., 2001; Villunger et al., 2003). Thus, we investigated whether activation of FLT3-ITD receptor signaling may regulate p27Kip1 and Bim expression in cytokine-starved Ba/F3 cells. Multiple isoforms of Bim have been identiﬁed (O’Connor et al., 1998; U et al., 2001), but in IL-3-deprived TonB.FLT3- ITD4 cells we detected predominantly BimEL and BimL expression by immunoblot analysis (Figure 6a). Doxy- cycline treatment resulted in a progressive induction of FLT3-ITD4 expression and simultaneous downregula- Kip1 tion of p27 and BimEL protein levels (Figure 6a). Figure 5 FLT3-ITD receptor signaling regulates transcriptional Importantly, this decrease in protein expression was activity of Foxo proteins. (a and b) U2-OS cells were transfected preceded by repression of p27Kip1 2.2 kb and Bim 5.7 kb with 2 mg3Â IRS-Luciferase reporter plasmid and increasing concentrations of pSG5-HA-Foxo3a (a) or pMT2-HA-Foxo4 (b) mRNA transcripts (Figure 6b), arguing that FLT3-ITD (0.4, 1 or 2 mg of plasmid DNA) in the absence or presence of either signaling contributes to transcriptional inhibition of 2 mg pEBB-FLT3-ITD4-HA or 0.5 mg pcDNA3.1-HA-Myr-Akt. p27Kip1 and Bim gene expression. Immediately after transfection, cells were cultured in 0.1% FCS and 36 h later luciferase activity was analysed. Relative luciferase activity indicates luciferase values corrected for transfection Foxo3a-mediated induction of p27Kip1 and Bim expression efﬁciency, and is representative of two independent experiments. is abrogated by FLT3 receptors with internal tandem Five percent of the lysates used to measure luciferase activity were duplications loaded on 8% polyacrylamide gel, blotted and probed with HA MoAb In addition to Foxo proteins, c-Myc (Yang et al., 2001) and BRCA1 (Williamson et al., 2002) have been

Oncogene FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3344 Foxo3a-mediated transcriptional regulation of p27Kip1 and Bim. To this end, doxycycline-inducible Ba/F3 cells were generated, which expressed FLAG-Foxo3a (TonB.Foxo3a). These cells were subsequently infected with empty pMSCV-IRES-GFP retrovirus (TonB.Foxo- 3a/control cells) or pMSCV-IRES-GFP encoding FLT3-ITD4-HA (TonB.Foxo3a/FLT3-ITD4 cells). Treatment of IL-3-deprived TonB.Foxo3a/control cells for 24 h with doxycycline resulted in a signiﬁcant induction of Foxo3a expression, with moderate upregu- Kip1 lation of p27 and BimEL protein levels (Figure 7a), as well as p27Kip1 and Bim mRNA (Figure 7b). However, in the presence of FLT3-ITD4, induction of p27Kip1 and Bim transcription by Foxo3a was signiﬁcantly diminished (Figure 7c), even though Foxo3a protein levels were more abundant in the presence of FLT3-ITD4 Kip1 expression. Downregulation of p27 and BimEL protein levels correlated with abrogation of Foxo3a- mediated apoptosis by FLT3-ITD4 receptor signaling (Figure 7d). In summary, these data indicate that, in hematopoietic cells, FLT3-ITD receptors inhibit Foxo- 3a-dependent transcriptional activation of p27Kip1 and Bim, which in turn are likely to play a major role in regulating proliferation and cell viability.

Discussion

Activating mutations in the tyrosine kinase receptor FLT3 have been detected in about one-third of patients with AML (Gilliland and Griffin, 2002), but the mechanisms of oncogenic transformation by these constitutively active FLT3 receptors have not yet been examined in detail. In this study, we have analysed the biological consequences of FLT3-ITD4 expression employing a tetracycline-inducible promoter in the IL- 3-dependent pre-B cell line Ba/F3 (TonB cells). Induc- tion of FLT3-ITD4 expression is sufficient to inhibit apoptosis and promote cell cycle entry of IL-3-deprived TonB.FLT3-ITD4 cells, and leads to the activation of ERK, STAT5 and Akt. It is well established that Akt kinase activity is stimulated by growth factor-induced Figure 6 Induction of FLT3-ITD receptor signaling results in PI3K activation (Franke et al., 1995; Datta et al., 1996) Kip1 downregulation of p27 and Bim expression levels. (a) and activation of PI3K/Akt pathway is essential for TonB.FLT3-ITD4 cells were cultured for 16 h in the absence of IL-3 and subsequently treated for increasing time periods with 2 mg/ transformation by oncogenic tyrosine kinases BCR/ ml doxycycline. Cells were harvested at the indicated time points ABL (Skorski et al., 1997) and TEL/platelet-derived and 2 Â 107 cells were lysed for protein analysis. Parallel growth factor receptor b (Dierov et al., 2002). In various polyacrylamide gels were loaded with 50 mg protein extracts and systems, antiapoptotic signaling by Akt has been shown probed with FLT3, pT32-Foxo3a, p27Kip1, Bim and actin antibodies. (b) RNA was extracted from 6 Â 107 TonB.FLT3-ITD4 to occur both through inhibitory phosphorylation of cells collected at the same time points as described in (a), and Bad (del Peso et al., 1997), caspase-9 (Cardone et al., subjected to Northern blot analysis using murine p27Kip1, Bim and 1998), or Foxo subfamily of Forkhead transcription b-actin cDNA probes factors (Brunet et al., 1999; Kops et al., 1999; Rena et al., 1999; Tang et al., 1999). Here, we provide evidence that Foxo transcription factors are likely to play important roles as mediators of cell proliferation and viability as implicated in controlling p27Kip1 gene expression, while part of the PI3K/Akt pathway downstream of activated Bim transcription is also regulated through the JNK and FLT3 receptor signaling. Raf/MEK/ERK pathway (Harris and Johnson, 2001; The Foxo subclass of Forkhead transcription factors Shinjyo et al., 2001; Whitfield et al., 2001). Therefore, represents the vertebrate orthologues of C. elegans we asked if FLT3-ITD signaling specifically altered DAF-16, which include Foxo1, Foxo3a and Foxo4

Oncogene FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3345

Figure 7 FLT3-ITD expression inhibits Foxo3a-mediated activation of p27Kip1 and Bim gene expression and apoptosis. (a) Doxycycline-inducible Ba/F3 cells expressing FLAG-Foxo3a (TonB.Foxo3a) were generated and infected with either empty or FLT3- ITD4-HA-encoding pMSCV-IRES-GFP retroviral vector. After GFP sorting, stable TonB.Foxo3a/control and TonB.Foxo3a/FLT3- ITD4 cells were obtained. TonB.Foxo3a/control and TonB.Foxo3a/FLT3-ITD4 cells were cultured for 24 h without IL-3 in the presence or absence of 2 mg/ml doxycycline (dox). Cells were collected, protein lysates of 2 Â 107 cells were obtained and subjected to Western blot analysis using FLT3, Foxo3a, p27Kip1, Bim and actin antibodies. (b) Identical samples as in (a) were harvested to extract RNA and analysed by Northern blot analysis using Foxo3a, p27Kip1, Bim and b-actin cDNA probes. (c) Densitometric quantification of results as depicted in (b), where intensities were obtained with radiolabeled cDNA probes, were corrected for b-actin signal. (d) TonB.Foxo3a (TonB.F3) and FLT3-ITD4/TonB.Foxo3a cells (FLT3-ITD4 TonB.F3) were cultured without IL-3 and in the absence or presence of 2 mg/ml doxycycline for either 16 or 40 h. Thereafter, the cells were collected, fixed in ethanol, and labeled with propidium iodide to determine their DNA profiles by flow cytometry

(Kaestner et al., 2000). PI3K- and PDK1-dependent Our data demonstrate that both ligand-bound wild- activation of Akt induces phosphorylation of Foxo type FLT3 and FLT3-ITD signaling promotes Akt proteins on conserved serine and threonine residues, activation as well as Thr32-Foxo3a phosphorylation in which inhibits their ability to act as transcriptional cytokine- and serum-starved Ba/F3 cells, which relies on regulators. Recent studies have shown that in hemato- the presence of the consensus Akt phosphorylation sites poietic cells phosphorylation of Foxo3a is a downstream in Foxo3a. We observed a basal level of Thr32-Foxo3a event of the PI3K/Akt pathway in IL-2 (Stahl et al., phosphorylation in IL-3-starved TonB.FLT3-ITD4 2002), IL-3 (Dijkers et al., 2000b), Kit ligand (Engstrom cells, but not in IL3-deprived Ba/F3 cells. This ﬁnding et al., 2003), erythropoietin and thrombopoietin (TPO) suggests that the very low level of FLT3-ITD4 expres- signaling (Kashii et al., 2000; Uddin et al., 2000; Tanaka sion that is present in the absence of doxycycline et al., 2001), where the activity of this transcription treatment may be sufﬁcient to induce detectable Foxo3a factor has been linked to the induction of apoptosis. phosphorylation but not detectable Ser473-Akt

Oncogene FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3346 phosphorylation. This differential sensitivity for Foxo3a To further elucidate the mechanism by which FLT3- phosphorylation has also been noted in TPO signaling, ITD signaling inhibits apoptosis in hematopoietic cells, where saturated Thr32-Foxo3a phosphorylation levels we investigated the potential involvement of the are achieved with at least 10-fold lower concentration of previously described Foxo target gene Bim (Dijkers TPO than Ser473-Akt phosphorylation (Tanaka et al., et al., 2000a). Bim encodes an BH3-only pro-apoptotic 2001). Bcl-2 family member, which acts as an allosteric The nonphosphorylated forms of Foxo proteins inhibitor of the antiapoptotic proteins Bcl-2 and Bcl- localize to the nucleus, but Akt-mediated phosphoryla- xL. Activation of Bim does not require caspase cleavage, tion promotes their interaction with 14-3-3 proteins and as observed for Bid (Li et al., 1998), and Bim is not exclusion from the cell nucleus, prohibiting their action regulated by phosphorylation, like Bad (del Peso et al., as transcription factors (Brunet et al., 1999; Brownawell 1997; U et al., 2001). Instead, induction of Bim activity et al., 2001; Rena et al., 2001). Our data indicate that occurs mainly at the transcriptional level and Bim expression of FLT3-ITD receptors in serum-deprived mRNA gives rise to multiple alternatively spliced COS7 cells triggers cytoplasmic retention of EGFP- isoforms (O’Connor et al., 1998). We found that both Foxo1 and EGFP-Foxo3a, which requires PI3K activity BimEL protein and Bim mRNA levels decrease after and intact Akt phosphorylation sites of Foxo proteins. induction of FLT3-ITD receptor signaling in IL-3- Furthermore, FLT3-ITD signaling in U2-OS cells starved TonB.FLT3-ITD4 cells. Moreover, FLT3-ITD4 strongly suppresses transcriptional activation of Foxo prohibits Foxo3a-mediated induction of Bim gene transcription factors in a reporter assay. Moreover, expression. The fact that Bim-deficient bone marrow FLT3-ITD diminishes Foxo3a-induced apoptosis in Ba/ HSC and granulocytes are relatively resistant to F3 cells. Thus, through its ability to induce inhibitory apoptosis after cytokine withdrawal or PI3K inhibition phosphorylation of Forkhead transcription factors and (Dijkers et al., 2002) suggests that inhibition of Bim their exclusion from cell nucleus, FLT3-ITD signaling expression is an important downstream event of FLT3- rescues the hematopoietic cells from Foxo3a-induced ITD/Akt signaling pathway in hematopoietic cells. apoptosis. In addition, we analysed whether FLT3-ITD controls At present, we have not firmly established whether expression of the important regulator of cell cycle Akt is the kinase that phosphorylates and negatively progression p27Kip1, which can be transcriptionally regulates Forkhead transcription factors after activation induced by Foxo proteins (Medema et al., 2000). The of FLT3-ITD signaling. In insulin receptor-deficient CDK-inhibitor p27Kip1 mainly acts as a negative hepatocytes, residues Thr24 and Ser319 of Foxo1 are regulator of cell division through its ability to inhibit not phosphorylated by constitutively activated Akt cyclin E-associated CDK2, thereby preventing pRb (Nakae et al., 2000, 2001), and dominant-negative phosphorylation at the G1/S phase of the cell cycle Akt fails to inhibit Thr32 of Foxo3a in HEK293 (Brunet (Toyoshima and Hunter, 1994). Furthermore, high et al., 2001), and only partially prohibits Foxo4 p27Kip1 levels sensitize Ba/F3 cells to programmed cell phosphorylation after insulin stimulation in A14 cells death, while p27Kip1-deficient HSC display increased (Kops et al., 1999). Indeed, other members of the survival upon cytokine-starvation (Dijkers et al., 2000b; AGC kinase family, which, besides Akt, include the Parada et al., 2001). We found that FLT3-ITD4 serum- and glucocorticoid-inducible kinase 1 (SGK1) downregulates p27Kip1 and the presented data support and related kinase SGK3/CISK, are able to directly the notion that FLT3-ITD signaling mainly reduces phosphorylate Foxo3a (Liu et al., 2000; Brunet et al., p27Kip1 protein levels through repression of transcrip- 2001). tion, although potential alternative mechanisms, includ- Moreover, AGC kinase-independent signaling path- ing proteosome-dependent degradation of p27Kip1, have ways have been implicated in the regulation of Foxo not been excluded. Specifically, FLT3-ITD4 could subclass of Forkhead transcription factors. Phosphor- prevent Foxo3a-induced upregulation of p27Kip1 in Ba/ ylation of Foxo1 on Ser329 by dual-specificity tyrosine- F3 cells. Thus, inhibition of p27Kip1 expression presents phosphorylated regulated kinase 1A (DYRK1A) reg- an important way for FLT3-ITD to induce mitogenic ulates basal nuclear localization and transactivation signaling and provide protection against apoptosis as (Woods et al., 2001). Furthermore, Ser322 and Ser325 of well. Foxo1 are substrates for casein kinase 1 (CK1), after In conclusion, the studies reported here identify for Akt-catalysed phosphorylation of Ser319, regulating the the first time downstream targets of the Akt pathway nuclear exclusion of Foxo1 (Rena et al., 2002). In that are regulated by oncogenic FLT3 receptors. FLT3- addition, the Ras/Ral pathway promotes Foxo4 phos- ITD signaling promotes inhibitory phosphorylation of phorylation on Thr447 and Thr451, which inhibits Foxo4 Foxo Forkhead transcription factors and repression of Kip1 transcription activation (Kops et al., 1999; De Ruiter p27 and Bim gene expression, thereby bypassing G1 et al., 2001). Thus, although activation of Akt by FLT3- cell cycle checkpoint and rescuing hematopoietic cells ITD correlates closely with phosphorylation of Foxo from default programmed cell death. Similarly, BCR/ proteins, future analysis has to provide more insight as ABL regulates phosphorylation of Foxo3a in chronic to which intermediate signaling molecules play a critical myeloid leukemia (CML) cell lines and Foxo3a regulates role in the regulation of Foxo protein function upon p27Kip1 expression downstream of BCR/ABL-signaling FLT3 receptor activation during normal hematopoiesis in CML cells (Komatsu et al., 2003). Thus, an emerging and in myeloid leukemia. theme is that constitutively activated tyrosine kinases in

Oncogene FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3347 leukemia signal through Foxo proteins and these plete), and selected for 7 days in the presence of 700 mg/ml transcription factors may play a key role in transforma- hygromycin. Signal transduction studies were performed with tion of myeloid cells. polyclonal TonB.FLT3-ITD4 cells by growing them for 3–5 days in 75 cm2 flasks in RPMI complete. Cells were washed once with PBS and resuspended in 10% FBS/RPMI (in the absence of IL-3). After 16 h cytokine starvation, 2 Â 107 cells Materials and methods were harvested for the first time point (0 h) and 2 mg/ml doxycycline was added to the remaining TonB.FLT3-ITD4 Antibodies and reagents cells for subsequent time points. For transient transfections, FLT3 (C-20), phosphotyrosine (PY20) and actin (C-11) Ba/F3 cells were electroporated (0.29 kV; capacitance 960 mF) antibodies were purchased from Santa Cruz Biotechnology with a total amount of 25 mg of the expression vectors pEBB or (Santa Cruz, CA, USA). Akt, ERK1/2 antibodies and pEBB-FLT3-ITD4-HA and 25 mg of pcDNA3.1-FLAG- phospho-antibodies for Thr202/Tyr204-ERK1/2 (E10), Ser473- Foxo3a or pcDNA3.1-FLAG-Foxo3a-TM, together with Akt and Tyr694-STAT5 were obtained from Cell Signaling 500 ng H2B-GFP to monitor the transfection efficiency. Technology (Beverley, MA, USA). Foxo3a and phospho- Thr32-Foxo3a were purchased from Upstate (Lake Placid, NY, Western blotting and immunoprecipitations USA), Bim antibodies from StressGen (Victoria, Canada) and Cell lysates were prepared in ice-cold lysis buffer (200 mM HA (HA.11) antibodies from Covance (Richmond, CA, USA). NaCl, 20 mM Tris.Cl. pH 8.0, 1% NP-40, 100 mM NaF, 5 mM STAT5 and p27Kip1 antibodies were obtained from BD EDTA and 1 mM Na3VO4) supplemented with Completet Transduction Laboratories (San Diego, CA, USA). FLAG protease inhibitors (Roche Applied Science, Indianapolis, IN, (M2) antibodies, doxycycline, LY294002, PMSF and poly- US) and 1mM PMSF. Protein concentration was determined brene were purchased from Sigma-Aldrich (St Louis, MO, with Bradford and equal amounts of protein extracts were USA). Human recombinant FLT3 ligand (FL) was obtained loaded on SDS-polyacrylamide gel. For immunoprecipita- from PeproTech (Rocky Hill, NJ, USA). PKC412 (N-benzoyl tions, 1 mg of protein extract was incubated with 5 mgof staurosporine) was kindly provided by Novartis Pharma AG antibody and 40 ml protein A-sepharose beads for 16 h at 41C. (Basel, Switzerland). Beads were washed four times with lysis buffer and subjected to SDS–PAGE. After SDS–PAGE, proteins were transferred DNA constructs in blotting buffer for 2 h to immobilon-P membranes (Milli- pore, Billerica, MA, USA). Blots were blocked for 1 h at room FLT3-ITD4 and wild-type FLT3 cDNA were a kind gift from temperature in 4% nonfat dry milk in Tris-buffered saline- Dr Tomoki Naoe (Nagoya University School of Medicine, Tween 20 (TBST: 0.15 M NaCl, 0.01 M Tris-HCl pH 7.4, 0.05% Nagoya, Japan). The expression vector pEBB-FLT3-ITD4- Tween 20), and incubated overnight at 41C with primary HA was created by exchanging the amino-terminal signal antibodies diluted 1 : 2000 in 1% non-fat dry milk/TBST. After sequence of FLT3 (1–27 aa) with the signal sequence of c-FMS washing, blots were incubated with 1 : 5000 dilution of (1–22 aa), providing FLT3-ITD4 with a carboxy-terminal horseradish peroxidase-conjugated secondary antibody hemagglutin A (HA) tag at 30 end of the open reading frame (Amersham Biosciences, Piscataway, NJ, USA) in 1% non- by PCR and cloning the assembled cDNA fragment into fat dry milk/TBST for 45 min, followed by four times 10 min BamHI–NotI restriction sites of pEBB. pMSCV-FLT3-ITD4 washes with TBST. Enhanced chemiluminescence was per- was generated by cloning FLT3-ITD4-HA BamHI–NotI/blunt formed according to the manufacturer’s instructions in restriction sites BglII–HpaI of pMSCV-IRES-GFP (pMIG). (Perkin-Elmer Life Sciences, Boston, MA, USA). Plasmids pcDNA3.1-FLAG-Foxo3a, pcDNA3.1-FLAG- Foxo3a-TM, pcDNA3.1-HA-Myr-Akt, pSG5-HA-Foxo3a, pMT2-HA-Foxo4 and pGL2-3 Â IRS-Luc were generously Flow-cytometric analyses provided by Dr William Sellers (Harvard Medical School, To determine FLT3 cell surface expression, 2 Â 106 cells were Boston, USA). pEGFP-Foxo3a and pEGFP-Foxo3a-TM were harvested, washed with PBS and incubated on ice with 100 ml obtained by cloning Foxo3a and Foxo3a-TM cDNAs in CD135-PE solution (Immunotech). Cells were washed, resus- pEGFP-C3 (BD Biosciences Clontech, Palo Alto, CA, USA). pended in PBS and analysed on a FACSscan using CellQuest H2B-GFP was a kind gift from Dr Reuven Agami and CMV- software (BD Biosciences). For annexin-V staining, cells were Renilla from Dr Rene´ Bernards (Netherlands Cancer Institute, washed with PBS and incubated for 15 min in 100 ml Amsterdam, Netherlands). fluorescein isothiocyanate (FITC)-conjugated annexin-V incubation buffer (Roche Applied Science) containing propi- Generation doxycycline-inducible cell lines and transient dium iodide. Cells were washed, resuspended in 1 ml binding transfection by electroporation buffer and immediately analysed by flow cytometry. For cell cycle analysis, the harvested cells were washed with PBS and FLT3-ITD4-HA, FLT3-WT-HA and FLAG-Foxo3a were fixed in 70% ethanol for at least 3 h on ice. Cells were cloned in pRevTRE-Hyg, and 10 mg of each retroviral vector centrifuged for 5 min at 480 Â g, treated with 200 mg/ml 6 was transfected with Superfect (Qiagen) in 5 Â 10 phoenix RNAase A at 371C for 40 min, washed with PBS, resuspended cells that were plated on a 10-cm dish and cultured in DMEM in 20 mg/ml propidium iodide in PBS, incubated for 10 min in supplemented with 10% (v/v) fetal bovine serum (FBS), the dark and analysed by flow cytometry using ModFit 50 mg streptomycin/ml and 50 U penicillin/ml. The virus- software. containing medium was collected after 24 and 48 h and added to 2 Â 106 TonBaF.1 (TonB) cells (Klucher et al., 1998), Luciferase reporter assays together with 10% (v/v) conditioned medium from WEHI-3B cells as a source of interleukin-3 (IL-3) and 8 mg/ml polybrene. Transfections for reporter assays were carried out in U2-OS After 24 h, virus-infected TonB cells were resuspended in cells plated on 6-cm tissue culture dishes using Superfect RPMI 1640 medium with 10% FBS, 10% WEHI-3B, (Qiagen). In each transfection, 20 ng cytomegalovirus (CMV)- 50 mg streptomycin/ml and 50 U penicillin/ml (RPMI com- driven Renilla luciferase plasmid was included as an internal

Oncogene FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3348 standard. Immediately after transfection, cells were cultured in membrane (Amersham). Membranes were successively hybri- 0.1% FCS/DMEM medium and analysed 36 h later. Cells were dized in Quick Hyb (Stratagene) at 681C with 32P-dCTP lysed on ice for 20 min in 1 Â reporter lysis buffer (Promega) radiolabeled p27Kip1, Bim and b-actin mouse cDNA probes supplemented with Completet protease inhibitors (Roche generated by random primed labeling. Filters were exposed to Applied Science). Cleared lysates were used for quantification Kodak Biomax MS films and densitometric analysis was of luciferase activities with a dual-luciferase reporter assay performed with Kodak Digital Science 1D Image Analysis system in accordance with the manufacturer’s instructions Software. (Promega). Acknowledgements Analyses of mRNA expression We wish to thank Tomoki Naoe, William Sellers and George Total mRNA was extracted from frozen cell pellets with RNA Daley for reagents. This work was supported by a Leukemia Trizol, as specified by the manufacturer (Invitrogen), and 15 mg and Lymphoma Society SCOR grant and NIH grant R01 RNA was subjected to paraformaldehyde-containing agarose CA66996. BS is the recipient of a Fellowship of the Dutch gel electrophoresis and transferred to Hybond-N þ nylon Cancer Society (KWF/NKB).

References

Abu-Duhier FM, Goodeve AC, Wilson GA, Care RS, Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Peake IR and Reilly JT. (2001). Br. J. Haematol., 113, Morrison DK, Kaplan DR and Tsichlis PN. (1995). Cell, 81, 983–988. 727–736. Alvarez B, Martinez AC, Burgering BM and Carrera AC. Gilliland DG and Grifﬁn JD. (2002). Blood, 100, 1532–1542. (2001). Nature, 413, 744–747. Harris CA and Johnson Jr EM. (2001). J. Biol. Chem., 276, Armstrong SA, Staunton JE, Silverman LB, Pieters R, den 37754–37760. Boer ML, Minden MD, Sallan SE, Lander ES, Golub TR Hayakawa F, Towatari M, Kiyoi H, Tanimoto M, Kitamura and Korsmeyer SJ. (2002). Nat. Genet., 30, 41–47. T, Saito H and Naoe T. (2000). Oncogene, 19, 624–631. Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, Kaestner KH, Knochel W and Martinez DE. (2000). Genes Kontgen F, Adams JM and Strasser A. (1999). Science, 286, Dev., 14, 142–146. 1735–1738. Kashii Y, Uchida M, Kirito K, Tanaka M, Nishijima K, Brownawell AM, Kops GJ, Macara IG and Burgering BM. Toshima M, Ando T, Koizumi K, Endoh T, Sawada K, (2001). Mol. Cell. Biol., 21, 3534–3546. Momoi M, Miura Y, Ozawa K and Komatsu N. (2000). Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Blood, 96, 941–949. Anderson MJ, Arden KC, Blenis J and Greenberg ME. Kelly LM, Liu Q, Kutok JL, Williams IR, Boulton CL and (1999). Cell, 96, 857–868. Gilliland DG. (2002). Blood, 99, 310–318. Brunet A, Park J, Tran H, Hu LS, Hemmings BA and Klucher KM, Lopez DV and Daley GQ. (1998). Blood, 91, Greenberg ME. (2001). Mol. Cell. Biol., 21, 952–965. 3927–3934. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke Komatsu N, Watanabe T, Uchida M, Mori M, Kirito K, TF, Stanbridge E, Frisch S and Reed JC. (1998). Science, Kikuchi S, Liu Q, Tauchi T, Miyazawa K, Endo H, Nagai T 282, 1318–1321. and Ozawa K. (2003). J. Biol. Chem., 278, 6411–6419. Carow CE, Levenstein M, Kaufmann SH, Chen J, Amin S, Kops GJ, de Ruiter ND, De Vries-Smits AM, Powell DR, Bos Rockwell P, Witte L, Borowitz MJ, Civin CI and Small D. JL and Burgering BM. (1999). Nature, 398, 630–634. (1996). Blood, 87, 1089–1096. Kops GJ, Medema RH, Glassford J, Essers MA, Dijkers PF, Castrillon DH, Miao L, Kollipara R, Horner JW and DePinho Coffer PJ, Lam EW and Burgering BM. (2002). Mol. Cell. RA. (2003). Science, 301, 215–218. Biol., 22, 2025–2036. DaSilva N, Hu ZB, Ma W, Rosnet O, Birnbaum D and Lawlor MA and Alessi DR. (2001). J. Cell. Sci., 114, Drexler HG. (1994). Leukemia, 8, 885–888. 2903–2910. Datta K, Bellacosa A, Chan TO and Tsichlis PN. (1996). Levis M, Allebach J, Tse KF, Zheng R, Baldwin BR, Smith J. Biol. Chem., 271, 30835–30839. BD, Jones-Bolin S, Ruggeri B, Dionne C and Small D. Datta SR, Brunet A and Greenberg ME. (1999). Genes Dev., (2002). Blood, 99, 3885–3891. 13, 2905–2927. Li H, Zhu H, Xu CJ and Yuan J. (1998). Cell, 94, 491–501. De Ruiter ND, Burgering BM and Bos JL. (2001). Mol. Cell. Liu D, Yang X and Songyang Z. (2000). Curr. Biol., 10, Biol., 21, 8225–8235. 1233–1236. del Peso L, Gonzalez-Garcia M, Page C, Herrera R and Nunez Lyman SD and Jacobsen SE. (1998). Blood, 91, 1101–1134. G. (1997). Science, 278, 687–689. Medema RH, Kops GJ, Bos JL and Burgering BM. (2000). Dierov J, Xu Q, Dierova R and Carroll M. (2002). Blood, 99, Nature, 404, 782–787. 1758–1765. Meierhoff G, Dehmel U, Gruss HJ, Rosnet O, Birnbaum D, Dijkers PF, Birkenkamp KU, Lam EW, Thomas NS, Quentmeier H, Dirks W and Drexler HG. (1995). Leukemia, Lammers JW, Koenderman L and Coffer PJ. (2002). 9, 1368–1372. J. Cell. Biol., 156, 531–542. Minami Y, Yamamoto K, Kiyoi H, Ueda R, Saito H and Dijkers PF, Medema RH, Lammers JW, Koenderman L and Naoe T. (2003). Blood, 102, 2969–2975. Coffer PJ. (2000a). Curr. Biol., 10, 1201–1204. Mizuki M, Fenski R, Halfter H, Matsumura I, Schmidt R, Dijkers PF, Medema RH, Pals C, Banerji L, Thomas NS, Lam Muller C, Gruning W, Kratz-Albers K, Serve S, Steur C, EW, Burgering BM, Raaijmakers JA, Lammers JW, Buchner T, Kienast J, Kanakura Y, Berdel WE and Serve H. Koenderman L and Coffer PJ. (2000b). Mol. Cell. Biol., (2000). Blood, 96, 3907–3914. 20, 9138–9148. Modur V, Nagarajan R, Evers BM and Milbrandt J. (2002). Engstrom M, Karlsson R and Jonsson JI. (2003). Exp. J. Biol. Chem., 277, 47928–47937. Hematol., 31, 316–323. Nakae J, Barr V and Accili D. (2000). EMBO J., 19, 989–996.

Oncogene FLT3-ITD receptors inhibit Foxo protein function B Scheijen et al 3349 Nakae J, Biggs III WH, Kitamura T, Cavenee WK, Wright CV, Spiekermann K, Bagrintseva K, Schwab R, Schmieja K and Arden KC and Accili D. (2002). Nat. Genet., 32, 245–253. Hiddemann W. (2003). Clin. Cancer Res., 9, 2140–2150. Nakae J, Kitamura T, Kitamura Y, Biggs WH, Arden KC and Stahl M, Dijkers PF, Kops GJ, Lens SM, Coffer PJ, Burgering Accili D. (2003). Dev. Cell, 4, 119–129. BM and Medema RH. (2002). J. Immunol., 168, 5024–5031. Nakae J, Kitamura T, Ogawa W, Kasuga M and Accili D. Tanaka M, Kirito K, Kashii Y, Uchida M, Watanabe T, Endo (2001). Biochemistry, 40, 11768–11776. H, Endoh T, Sawada K, Ozawa K and Komatsu N. (2001). Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima J. Biol. Chem., 276, 15082–15089. K, Sonoda Y, Fujimoto T and Misawa S. (1996). Leukemia, Tang ED, Nunez G, Barr FG and Guan KL. (1999). J. Biol. 10, 1911–1918. Chem., 274, 16741–16746. O’Connor L, Strasser A, O’Reilly LA, Hausmann G, Adams Thiede C, Steudel C, Mohr B, Schaich M, Schakel U, JM, Cory S and Huang DC. (1998). EMBO J., 17, 384–395. Platzbecker U, Wermke M, Bornhauser M, Ritter M, Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum Neubauer A, Ehninger G and Illmer T. (2002). Blood, 99, HA and Ruvkun G. (1997). Nature, 389, 994–999. 4326–4335. Parada Y, Banerji L, Glassford J, Lea NC, Collado M, Rivas Toyoshima H and Hunter T. (1994). Cell, 78, 67–74. C, Lewis JL, Gordon MY, Thomas NS and Lam EW. U M, Miyashita T, Shikama Y, Tadokoro K and Yamada M. (2001). J. Biol. Chem., 276, 23572–23580. (2001). FEBS Lett., 509, 135–141. Paradis S and Ruvkun G. (1998). Genes Dev., 12, 2488–2498. Uddin S, Kottegoda S, Stigger D, Platanias LC and Wickrema Ramaswamy S, Nakamura N, Sansal I, Bergeron L and Sellers A. (2000). Biochem. Biophys. Res. Commun., 275, 16–19. WR. (2002). Cancer Cell., 2, 81–91. Villunger A, Scott C, Bouillet P and Strasser A. (2003). Blood, Rena G, Guo S, Cichy SC, Unterman TG and Cohen P. 101, 2393–2400. (1999). J. Biol. Chem., 274, 17179–17183. Weisberg E, Boulton C, Kelly LM, Manley P, Fabbro D, Rena G, Prescott AR, Guo S, Cohen P and Unterman TG. Meyer T, Gilliland DG and Griffin JD. (2002). Cancer Cell, (2001). Biochem. J., 354, 605–612. 1, 433–443. Rena G, Woods YL, Prescott AR, Peggie M, Unterman TG, Whitfield J, Neame SJ, Paquet L, Bernard O and Ham J. Williams MR and Cohen P. (2002). EMBO J., 21, 2263–2271. (2001). Neuron, 29, 629–643. Rokudai S, Fujita N, Kitahara O, Nakamura Y and Tsuruo T. Williamson EA, Dadmanesh F and Koeffler HP. (2002). (2002). Mol. Cell. Biol., 22, 8695–8708. Oncogene, 21, 3199–3206. Rosnet O, Marchetto S, deLapeyriere O and Birnbaum D. Woods YL, Rena G, Morrice N, Barthel A, Becker W, Guo S, (1991). Oncogene, 6, 1641–1650. Unterman TG and Cohen P. (2001). Biochem. J., 355, Rosnet O, Schiff C, Pebusque MJ, Marchetto S, Tonnelle C, 597–607. Toiron Y, Birg F and Birnbaum D. (1993). Blood, 82, Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, 1110–1119. Miyawaki S, Asou N, Kuriyama K, Yagasaki F, Shimazaki Scheid MP, Marignani PA and Woodgett JR. (2002). Mol. C, Akiyama H, Saito K, Nishimura M, Motoji T, Shinagawa Cell. Biol., 22, 6247–6260. K, Takeshita A, Saito H, Ueda R, Ohno R and Naoe T. Scheijen B and Griffin JD. (2002). Oncogene, 21, 3314–3333. (2001). Blood, 97, 2434–2439. Schmidt M, de Mattos SF, van der Horst A, Klompmaker R, Yang W, Shen J, Wu M, Arsura M, FitzGerald M, Suldan Z, Kops GJ, Lam EW, Burgering BM and Medema RH. Kim DW, Hofmann CS, Pianetti S, Romieu-Mourez R, (2002). Mol. Cell. Biol., 22, 7842–7852. Freedman LP and Sonenshein GE. (2001). Oncogene, 20, Shinjyo T, Kuribara R, Inukai T, Hosoi H, Kinoshita T, 1688–1702. Miyajima A, Houghton PJ, Look AT, Ozawa K and Inaba Zhang S, Fukuda S, Lee Y, Hangoc G, Cooper S, Spolski R, T. (2001). Mol. Cell. Biol., 21, 854–864. Leonard WJ and Broxmeyer HE. (2000). J. Exp. Med., 192, Skorski T, Bellacosa A, Nieborowska-Skorska M, Majewski 719–728. M, Martinez R, Choi JK, Trotta R, Wlodarski P, Perrotti D, Zhao M, Kiyoi H, Yamamoto Y, Ito M, Towatari M, Omura Chan TO, Wasik MA, Tsichlis PN and Calabretta B. (1997). S, Kitamura T, Ueda R, Saito H and Naoe T. (2000). EMBO J., 16, 6151–6161. Leukemia, 14, 374–378. Spiekermann K, Bagrintseva K, Schoch C, Haferlach T, Zheng R, Friedman AD and Small D. (2002). Blood, 100, Hiddemann W and Schnittger S. (2002). Blood, 100, 3423–3425. 4154–4161.

Oncogene