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Originally published In Press as doi:10.1074/jbc.M411974200 on December 1, 2004

J. Biol. Chem., Vol. 280, Issue 7, 5361-5369, February 18, 2005
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FLT3/ITD Mutation Signaling Includes Suppression of SHP-1*

Peili Chen, Mark Levis, Patrick Brown, Kyu-Tae Kim, Jeffrey Allebach, and Donald Small{ddagger}

From the Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21231-1000

Received for publication, October 21, 2004 , and in revised form, November 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the FLT3 gene are the most common genetic alteration found in AML patients. FLT3 internal tandem duplication (ITD) mutations result in constitutive activation of FLT3 tyrosine kinase activity. The consequences of this activation are an increase in total phosphotyrosine content, persistent downstream signaling, and ultimately transformation of hematopoietic cells to factor-independent growth. The Src homology (SH)2 domain-containing protein-tyrosine phosphatase (SHP)-1 is involved in the down-regulation of a broad range of growth factor and cytokine-driven signaling cascades. Loss-of-function or deficiency of SHP-1 activity results in a hyperproliferative response of myelomonocytic cell populations to growth factor stimulation. In this study, we examined the possible role of SHP-1 in regulating FLT3 signaling. We found that transformation of TF-1 cells with FLT3/ITD mutations suppressed the activity of SHP-1 by ~3-fold. Suppression was caused by decreased SHP-1 protein expression, as analyzed at both the protein and RNA levels. In contrast, protein levels of SHP-2, a phosphatase that plays a stimulatory role in signaling through a variety of receptors, did not change significantly in FLT3 mutant cells. Suppressed SHP-1 protein levels in TF-1/ITD cells were partially overcome after cells were exposed to CEP-701, a selective FLT3 inhibitor. SHP-1 protein levels also increased in naturally occurring FLT3/ITD expressing AML cell lines and in primary FLT3/ITD AML samples after CEP-701 treatment. Furthermore, a small but reproducible growth/survival advantage was observed in both TF-1 and TF-1/ITD cells when SHP-1 expression was knocked down by RNAi. Taken together, these data provide the first evidence that suppression of SHP-1 by FLT3/ITD signaling may be another mechanism contributing to the transformation by FLT3/ITD mutations.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Deregulation of signaling through protein kinases has been identified as one of the most important mechanisms in human cancers (1, 2). Activating mutations of tyrosine kinases cause hyperphosphorylation of downstream targets and transformation of cells. c-Kit and Bcr-Abl are examples of kinases mutated in mastocytoma and gastrointestinal stromal tumors (GISTs) (3–5), and CML (6), respectively. FLT3 1 (FMS-like tyrosine kinase 3, Flk2, Stk-1), a member of the type III receptor-tyrosine kinase (RTK) family, is normally expressed in hematopoietic stem/progenitor cells (HSCs) (7). Wild-type FLT3 is activated following binding of the cognate ligand (FL) and plays a role in differentiation, proliferation, and survival of HSCs and dendritic cells (8). FLT3 is also expressed in ~90% of cases of acute myeloid leukemia (AML) and almost 100% of cases of B-cell lineage acute lymphoid leukemia (ALL) (9–11). Internal tandem duplication (ITD) mutations within the juxtamembrane domain of the FLT3 gene occur in 20–30% of patients with AML (12–15). Point mutations of FLT3 at D835 or nearby codons occur in an additional 7–9% of AML patients (16, 17). This makes FLT3 the most frequently mutated gene in AML (18, 19). FLT3-ITDs form homodimers or heterodimerize with wild-type FLT3 receptors in a ligand-independent manner (20). This leads to constitutive activation of the tyrosine kinase domain, which in turn leads to autophosphorylation of the receptor and subsequent phosphorylation of substrate proteins. This provides growth and survival signals for AML cells through either direct or indirect phosphorylation and activation of a wide range of signal transduction molecules including the p85 subunit of phosphatidylinositol 3-kinase, RAS/MAPK, Cbl, Shc, Vav, SHP-2, Src homology (SH)2 domain-containing inositol 5-phosphatase (SHIP), Stat5, and phospholipase C-{gamma} (21–26). Expression of FLT3/ITDs in murine factor-dependent cell lines, such as Ba/F3 and 32D (27, 28), results in factor-independent growth and development of leukemia phenotypes when the transfected cells are inoculated into mice (29–31). All of these observations suggest that FLT3/ITDs play an important role in leukemogenesis by triggering disturbed signaling pathways through protein phosphorylation.

Phosphorylation of protein on tyrosine residues is a critical mechanism for signaling pathways involved in cellular proliferation, differentiation, and apoptosis. This process is regulated by the opposing activities of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). These enzymes maintain a dynamic balance of protein phosphorylation and thereby set the duration and magnitude of the response to extracellular stimuli (32). Phosphatase activity is often up-regulated by activated kinases, providing a feedback loop to fine-tune the regulation of protein-tyrosine phosphorylation (33, 34). Any deviation in this balance caused by either increased PTK activity or decreased PTP activity can trigger accumulation of tyrosine-phosphorylated proteins, which in turn can lead to abnormal cell proliferation and differentiation. An example of the consequence of loss of this regulation is the constitutive activation of Akt seen with loss of PTEN activity. Both loss of expression and mutations within the conserved catalytic domain in PTEN occur in a wide spectrum of cancers including glioblastomas, breast, and prostate carcinomas, and small cell lung cancers (35). Two SH2 domain-containing PTPs, SHP-1 and SHP-2 are widely involved in regulating the signaling pathways of a variety of cytokine and growth factor receptors involved in cell proliferation, differentiation, and survival (36). Increasing evidence has also implicated these phosphatases in the pathogenesis of lymphoma, leukemia, and other cancers (37, 38). Despite a high degree of homology with SHP-1, SHP-2 has a completely distinct expression profile and function. In most circumstances, SHP-2 plays a positive role in activation of the ERK/MAPK pathway by growth factors and cytokines (39–41). Gain of function mutations of SHP-2 have been reported in 34% of juvenile myelomonocytic leukemia (JMML) and in some cases of myelodysplastic syndrome (MDS) and AML (42). The SHP-2 mutations found in JMML, D61Y and E76K, exhibit elevated phosphatase activity compared with wild-type SHP-2. Expression of these mutants in COS-7 cells causes prolonged activation of ERK2 in response to epidermal growth factor stimulation and is associated with increased cell proliferation (42). More recent studies have also shown that Ba/F3 cells expressing those same mutants demonstrate enhanced growth factor-independent survival (43). These experimental data suggested that the SHP-2 mutations found in JMML might contribute to leukemogenesis by activating Ras/ERK signaling pathways and might explain the characteristic GM-CSF hypersensitivity of JMML hematopoietic progenitors. In contrast to SHP-2, which is widely expressed, SHP-1 is predominantly expressed in hematopoietic cells. It mainly functions as a negative regulator of a wide variety of signaling pathways involving cytokines and growth factors that include erythropoietin (Epo) (44), interleukin (IL)-3 (45), interferon (IFN)-{alpha}, and -{beta} (46), colony-stimulating factor-1 (CSF-1) (47), stem cell factor (SCF) (48), and antigen receptors on B cells and T cells (49). The important roles of SHP-1 in development and function of myeloid cells has been demonstrated by the studies of two naturally occurring loss-of-function SHP-1 gene mutations in motheaten (me/me) and viable motheaten (mev/mev) mice (49). These mice express either no SHP-1 or a catalytically defective SHP-1 protein, respectively. The hematopoietic abnormalities are characterized by hyperproliferation and abnormal activation of myeloid/monocytic cells. Infiltration of macrophages into the lungs causes a hemorrhagic pneumonitis and eventual death of the mice. Bone marrow progenitor cells from me/me and mev/mev mice have enhanced clonogenic and/or proliferative responses to multiple growth factors and cytokines, including granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), Epo, and IFNs (50, 51). Studies examining SHP-1 expression demonstrate decreases in both SHP-1 mRNA and SHP-1 protein in many leukemia and lymphoma samples as well as in various leukemia/lymphoma cell lines (52, 53). In vitro studies have also shown that SHP-1 is up-regulated when leukemic cell lines like K562, 32D, and HT93 are induced to differentiate (54–56). Forced expression of wild-type SHP-1 in these cell lines leads to enhanced differentiation and decreased proliferation while introduction of a dominant negative mutation results in increased proliferation and a delay of differentiation. In a mast cell line expressing a c-Kit activation loop mutant, increased degradation of SHP-1 through a ubiquitin-dependent proteolytic pathway has been reported (57). These observations together provide strong support that SHP-1 functions as a tumor suppressor during myelopoiesis. This prompted us to investigate whether SHP-1 is involved in cell transformation and leukemogenesis mediated by FLT3/ITD signaling. In this study, we report for the first time that SHP-1 activity is suppressed by FLT3/ITD mutations and that this suppression is associated with a cell growth and a survival advantage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Monoclonal mouse anti-phosphotyrosine antibody 4G10 and recombinant protein A-agarose beads were purchased from Upstate Biotechnology (Lake Placid, NY), monoclonal antibody to SHP-1 and SHP-2 from BD Pharmingen (San Diego, CA), and polyclonal antibodies to FLT3, SHP-1, SHP-2, and actin from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies and the enhanced chemiluminescence (ECL) detection system were from Amersham Biosciences. Recombinant human GM-CSF and FL were obtained from Pepro Tech (Rocky Hill, NJ). CEP-701 was kindly provided by Cephalon Inc. (West Chester, PA). RNeasy total RNA purification and QuantiTect SYBR Green RT-PCR kits were obtained from Qiagen (Valencia, CA). Dicer small interfering RNA (siRNA) generation kit was obtained from Gene Therapy Systems, Inc. (San Diego, CA).

Cell Culture, Transfection, and Treatment—Primary AML samples were collected through an institutional review board-approved protocol. Frozen aliquots of AML samples were thawed and incubated for 12 h followed by centrifugation over Ficoll to eliminate cells dying from the freeze-thaw process. All cells were grown at 37 °C in a humidified incubator with 5% CO2. MV4-11 cells were maintained in Iscoves modified Dulbecco medium (IMDM) (Invitrogen) with 20% heat-inactivated fetal bovine serum (Gemini BioProducts, Woodland, CA). All other cells (including primary AML samples) were maintained in RPMI 1640 medium (Invitrogen) with 10% fetal bovine serum. Medium was supplemented with penicillin/streptomycin, and 2 ng/ml GM-CSF for TF-1 cells (American Type Culture Collection (ATCC), Manassas, VA). TF-1 cells were stably transfected with pCI-neo vectors (Promega, Madison, WI) containing full-length cDNA coding for either a wild-type FLT3 or a FLT3/ITD by electroporation, as described previously (21). Transfected cells were selected in 1 mg/ml G418 (Invitrogen) for 2 weeks and subcloned by limiting dilution. Cells were maintained in log-phase growth for experiments. Pervanadate was freshly prepared by mixing equal volumes of 0.1 M H2O2 and 0.1 M sodium orthovanadate and incubated for 10–20 min before use. When CEP-701 (stored as a 4 mM stock solution in dimethyl sulfoxide (Me2SO) at –20 °C) was used to treat cells, the corresponding control samples also contained identical amounts of Me2SO (<0.25%).

MTT Proliferation Assay—Cells were aliquoted into 96-well plates at an initial density of 1–5 x 104/ml in duplicate or triplicate. Plates were incubated at 37 °C, 5% CO2 for the indicated period of time. The MTT assays (Roche Applied Science) were performed according to the manufacturer's instructions. Plates were read at 562 nm in a microplate reader (Molecular Devices, Sunnyvale, CA).

Immunoprecipitation and Immunoblotting—Cells were washed with ice-cold phosphate-buffered saline and lysed on ice for 30 min in lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 100 mM NaF, 10% glycerol, 10 mM EDTA) containing protease and phosphatase inhibitors (2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml antipain, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). To immunoprecipitate specific proteins, clarified whole cell lysates were incubated with corresponding antibodies followed by binding to protein A-agarose beads overnight at 4 °C. Immunoprecipitates were resolved by SDS-PAGE and transferred onto immobilon membranes (Millipore, Bedford, MA). Immunoblotting assays were performed with the designated antibodies and immunoreactive bands were visualized using chemiluminescence (ECL, Amersham Biosciences). For reprobing, blots were stripped with a buffer containing 50 mM Tris-HCl, pH 6.8, 2% SDS, and 0.1 M 2-mercaptoethanol. Quantitation of immunoblots by densitometry was performed with NIH Image 1.62.

p-Nitrophenyl Phosphate (p-NPP) Hydrolysis Assay—SHP-1 and SHP-2 were immunoprecipitated and washed three times with lysis buffer, three times with lysis buffer without phosphatase inhibitors, and twice with p-NPP assay buffer (50 mM Tris-HCl, pH 7.4, 5 mM dithiothreitol). Phosphatase catalytic activity was assayed at 37 °C for 15 min in 200 µl of p-NPP buffer containing 20 mM p-NPP. Optical density was determined at 405 nm in a microplate reader.

RNA Purification and Northern Blot Analysis—Total RNA was extracted from 1 x 107 cells using RNeasy total RNA purification kits. The RNA samples (15 µg/lane) were separated on 1% formaldehyde-denaturing agarose gels and transferred to nylon membranes (PerkinElmer Life Sciences). Full-length SHP-1 cDNA labeled with [32P]dCTP by random primer labeling (Stratagene) was used as a probe. Hybridization was performed in 5x SSC (1x SSC = 0.15 M NaCl, 0.15 M sodium citrate), 50% deionized formamide, 5x Denhardt's solution, 1% SDS, 10% dextran sulfate (Mr 500,000), 0.1 mg/ml salmon sperm DNA at 42 °C for 16–24 h. The membrane was washed under monitoring and then exposed to XAR film (Kodak, Rochester, NY) at –80 °C.

SHP-1 RNA Interference—SHP-1 siRNA was prepared with Dicer siRNA generation kit according to the manufacturer's instructions. Briefly, T7 promoters were added to both ends of selected SHP-1 sequence (5'-GTCAGGGTGGGGGATCAGGTG... GCCAAGGCTGGCTTCTGGGAG-3') by means of PCR. The resultant DNA templates were used for an in vitro transcription reaction with T7 RNA polymerase to generate double strand (ds) RNA, which was digested into small interference RNA fragments with recombinant Dicer enzyme. GFP siRNA was prepared simultaneously from gWIZ/GFP control plasmid as a control. The SHP-1 siRNA transfected TF-1 cells were incubated overnight and aliquoted for MTT proliferation assays or for determination of SHP-1 mRNA levels and protein levels by quantitative real time RT-PCR and immunoblotting assays at intervals of 24 h.

Quantitative Real-time RT-PCR—Total RNAs were purified as above. One-step real-time reverse transcription-polymerase chain reaction (qPCR) was performed according to the manufacturer's instructions (Qiagen) on an iCycler iQ Real-time PCR System (Bio-Rad). Primers used in the reactions were as follow: SHP-1 FP, 5'-ATGCAGAGACCCTGCTCAAG-3', SHP-1 RP, 5'-ACCAGCTCTGTCAGAGTCG-3'; GAPDH FP, 5'-CCTCAACGACCACTTTGTCA-3', GAPDH RP, 5'-GGTGGTCCAGGGGTCTTACT-3'. Serially diluted {beta}-actin cDNA and primers were included as a standard curve.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FLT3/ITD Induces Factor-independent Growth and Increases in Phosphotyrosine Levels in TF-1 Cells—TF-1 is a Philadelphia chromosome-negative (Ph–), CD34+, FLT3-negative human GM-CSF-dependent myeloid leukemia-derived cell line (58). It is capable of differentiating upon treatment with differentiation agents, with {delta}-aminolevulinic acid inducing erythroid differentiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) inducing macrophage differentiation. In this study, TF-1 cells were transfected by electroporation with pCI-neo vectors expressing either wild-type FLT3 or FLT3/ITD. After selection of stable transfectants in G418 + GM-CSF for 2 weeks, the ability of the cells to survive in the absence of GM-CSF was determined by trypan blue exclusion. Wild-type FLT3-expressing TF-1 cells (TF-1/FLT3) still required GM-CSF to maintain normal growth. In contrast, FLT3/ITD-expressing TF-1 cells (TF-1/ITD) grew in the absence of GM-CSF (data not shown). The same results have been observed when BaF3 and 32D cells are transfected with wild-type FLT3 or FLT3/ITDs (29, 59). The phosphotyrosine levels of cell extracts from TF-1, TF-1/FLT3, and TF-1/ITD cells were examined by immunoblotting with anti-phosphotyrosine antibody, 4G10. TF-1/ITD cells were notable for significantly increased levels of phosphotyrosine proteins when compared with TF-1 and TF-1/FLT3 cells (Fig. 1, compare lane 5 to lanes 1 and 3). In addition, after a brief treatment with pervanadate, a potent membrane-permeable PTP inhibitor, the phosphotyrosine level in TF-1/ITD cells was further dramatically increased (Fig. 1, lane 6). This contrasts with the minimal to moderate increases observed in TF-1 and TF-1/FLT3 cells after pervanadate treatment, respectively. Total phosphotyrosine protein levels are the result of the balance between protein-tyrosine kinase and phosphatase activities. The increased kinase activity of constitutively activated FLT3/ITD potentially could be counteracted by increased levels of phosphatase activity. The fact that this is not seen in TF-1/ITD cells even in the absence of pretreatment with pervanadate implies that sufficient levels of phosphatases are not induced to counterbalance FLT3/ITD kinase activity. The augmentation of phosphoprotein levels with pervanadate treatment raised the possibility that an additional way for FLT3/ITDs to increase their signal would be to suppress the expression or activity of at least some phosphatases.



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  		FIG. 1.
Enhanced protein-tyrosine phosphorylation in TF-1/ITD cells is further augmented by pervanadate treatment. TF-1, TF-1/FLT3, and TF-1/ITD cells were cultured in growth medium containing 2 ng/ml of recombinant human GM-CSF in the absence (–) or presence (+) of 20 µM freshly prepared pervanadate for 30 min prior to cell lysis. Total cell extracts were subjected to immunoblotting assays with the anti-phosphotyrosine antibody, 4G10.

 
SHP-1 Activity and Expression Is Down-regulated in TF-1/ITD Cells—SHP-1 has been implicated in negative regulation of a wide spectrum of growth factor and cytokine-mediated signaling pathways in hematopoietic cells. This includes involvement in the regulation of two other type-III RTK family members closely related to FLT3: c-Kit and c-FMS (47, 48). To examine the functional status of SHP-1 in the context of FLT3 signaling, SHP-1 proteins were immunoprecipitated from TF-1, TF-1/FLT3, and TF-1/ITD cells. The immunoprecipitated SHP-1 was then subjected to a phosphatase activity assay as measured by hydrolysis of p-NPP. We found that SHP-1 activity in TF-1/ITD cells was ~3-fold lower than in TF-1 cells (Fig. 2a). TF-1/FLT3 cells showed an intermediate level of SHP-1 activity. The decreased SHP-1 activity in TF-1/ITD cells observed here seems unlikely to be a result of a lack of activation as SHP-1 is primarily activated by conformational changes induced by binding to phosphotyrosine-containing proteins. This binding disrupts the intramolecular autoinhibition of SHP-1 activity by overcoming the interaction between the N-terminal SH2 domain (N-SH2) and the catalytic domain. One possible explanation for the decreased phosphatase activity is decreased protein expression.



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  		FIG. 2.
Expression and activity of SHP-1 and SHP-2 in TF-1 cells. SHP-1 or SHP-2 was immunoprecipitated from equal amounts of whole cell protein extracts from TF-1, TF-1/FLT3, and TF-1/ITD cells. SHP-1 (a) and SHP-2 (c) phosphatase activity was measured as p-NPP hydrolysis at 37 °C for 15 min. The OD values were determined at 405 nm. b, immunoprecipitated (IP) SHP-1 from 15 mg of total cell lysates was resolved on SDS-PAGE followed by Coomassie Blue staining (upper panel), or aliquots of the samples were subjected to immunoblotting (IB) with antibody to SHP-1 (lower panel). d, immunoprecipitated SHP-2 was subjected to immunoblotting with antibody to SHP-2.

 
The expression level of SHP-1 was examined by immunoprecipitation followed by either Coomassie Blue staining of the SDS-PAGE gel or immunoblotting with anti-SHP-1 antibody (Fig. 2b). Consistent with the decreased phosphatase activity determined by p-NPP hydrolysis assays, SHP-1 protein levels in TF-1/ITD cells were reduced compared with TF-1 cells (Fig. 2b, compare lane 3 to lane 1). An intermediate level of SHP-1 was observed in TF-1/FLT3 cells. The stability of SHP-1 protein in TF-1 and TF-1/ITD cells was also examined after cycloheximide treatment, and no difference in SHP-1 half-life was detected (data not shown). These data indicate that the down-regulated SHP-1 phosphatase activity seen in TF-1/ITD cells is caused by decreased protein expression.

SHP-2 Expression Levels and Activity Is Unaffected by FLT3/ITD Expression—To determine whether down-regulation of SHP-1 in TF-1/ITD cells is simply a reflection of a general down-regulation of phosphatases in general, we also examined SHP-2 protein levels in these same cells. In contrast to the decreases in SHP-1 activity and protein levels observed in TF-1/ITD cells, neither activity (Fig. 2c) nor expression levels (Fig. 2d) of SHP-2 decreased. These results indicate that FLT3/ITD signaling does not evoke a generalized decrease in PTP expression and activity, but specifically leads to an inhibition of SHP-1 protein expression and activity.

CEP-701 Treatment Inhibits FLT3/ITD Phosphorylation and Proliferation in TF-1/ITD Cells—An indolocarbazole derivative, CEP-701, has been previously demonstrated to be a highly potent, selective inhibitor of FLT3 (29). It does not show any activity against KIT, FMS, or PDGFR at concentrations >10-fold higher than the IC50 (50% inhibitory concentration) for FLT3. CEP-701 inhibits proliferation and survival of FLT3/ITD-expressing cell lines as well as primary cells from most AML patients expressing FLT3/ITD mutations. The inhibition occurs in a dose-dependent fashion that parallels the inhibition of FLT3 phosphorylation and phosphorylation of downstream signaling molecules in the FLT3 pathway including STAT5 and MAPK. CEP-701 also overcomes the block to G-CSF-mediated differentiation of 32D cells caused by FLT3/ITD expression (59, 60). In TF-1/ITD cells, CEP-701 potently inhibited the phosphorylation of FLT3/ITD with an IC50 < 5 nM (Fig. 3a). The effect of CEP-701 on cell proliferation was evaluated by the MTT assays after TF-1/ITD cells were exposed to increasing concentrations of CEP-701 for 48 h. At 10 nM, CEP-701 inhibited the proliferation of TF-1/ITD cells by ~50%, but showed very little effect on TF-1 cells (Fig. 3b). At higher concentrations, CEP-701 did slow the proliferation of TF-1 cells, most likely through inhibition of other targets. However, the inhibition does not reach an IC50 even at 100 nM, >10-fold higher than the IC50 for TF-1/ITD cells. Furthermore, the addition of GM-CSF reversed the inhibitory effect of CEP-701on the growth of TF-1/ITD cells. While it is unknown what other kinases or substrates are inhibited by higher concentrations of CEP-701, these results imply that at concentrations that inhibit FLT3/ITD signaling efficiently, CEP-701 does not interfere with GM-CSF-mediated signaling pathways which are critical for proliferation and survival of TF-1 cells.



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  		FIG. 3.
CEP-701 treatment inhibits proliferation and FLT3/ITD phosphorylation in TF-1/ITD cells. a, TF-1/ITD cells were incubated with the indicated concentrations of CEP-701 for 6 h. Whole cell lysates were prepared and immunoprecipitated (IP) with anti-human FLT3 antibody, followed by immunoblotting (IB) with the anti-phosphotyrosine antibody, 4G10 (upper panel). The membrane was then stripped and reprobed with anti-FLT3 antibody (lower panel). b, triplicate samples of 5 x 104 cells were incubated in 96-well plates with increasing concentrations of CEP-701. TF-1 cells were cultured with 2 ng/ml of GM-CSF (open diamond), whereas TF-1/ITD cells were cultured with (solid circle) or without GM-CSF (solid triangle). After 48 h of incubation, cell proliferation was assessed with the MTT assay. Results, in the form of mean OD, were plotted as percent of untreated controls. Error bars represent S.E.

 
CEP-701 Treatment Inhibits Total Tyrosine Phosphorylation and Partially Overcomes the FLT3/ITD-mediated Suppression of SHP-1—The effect of CEP-701 treatment on total cellular phosphotyrosine levels in TF-1/ITD cells was examined. As shown in Fig. 4a, a significant reduction of total cellular protein tyrosine phosphorylation levels was observed in TF-1/ITD cells treated with ≥ 5 nM CEP-701. This parallels the inhibition of FLT3/ITD phosphorylation seen in Fig. 3a. In addition to the inhibition of FLT3/ITD kinase activity and other kinases activated downstream of FLT3/ITD, the decrease of total phosphotyrosine levels could also be a result of induction and activation of protein-tyrosine phosphatases. Therefore, to further evaluate the relationship of FLT3/ITD signaling to SHP-1 activity, the effects of CEP-701 treatment on SHP-1 protein levels were examined. TF-1/ITD cells were treated with increasing concentrations of CEP-701 for 8 h. SHP-1 and SHP-2 proteins were then immunoprecipitated and subjected to immunoblotting assays (Fig. 4b). SHP-1 protein levels were increased by treatment with 5–20 nM CEP-701. In contrast, there was no change observed in the level of SHP-2 upon treatment with CEP-701. At higher concentrations of CEP-701, there was a decrease of SHP-1 levels toward TF-1/ITD baseline, which likely reflects the induction of apoptosis in these cells.



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  		FIG. 4.
CEP-701 inhibits total phosphotyrosine levels and partially overcomes suppression of SHP-1 protein. TF-1/ITD cells were treated with the indicated concentrations of CEP-701 for 8 h and then lysed. a, total tyrosine phosphorylation levels were determined by immunoblotting with the anti-phosphotyrosine antibody, 4G10. b, SHP-1 (upper panel) and SHP-2 (lower panel) levels were determined by immunoblotting (IB) following immunoprecipitation (IP) from equal amounts of cell extracts as described under "Materials and Methods."

 
CEP-701 Treatment Increases SHP-1 Expression in AML Cell Lines and Primary AML Samples—To study the effects of inhibition of FLT3/ITD signaling on SHP-1 expression in other cell lines and primary cells, two AML cell lines, MOLM-14 and MV4-11, and 4 primary AML samples all expressing FLT3/ITD mutations, were treated with CEP-701. Both AML cell lines have naturally occurring ITD mutations in the FLT3 exon 14; MV4-11 expresses a 30-bp ITD in homozygous form and MOLM-14 is heterozygous with a 21-bp ITD and a FLT3 wild-type allele. FLT3/ITD expression in primary AML samples was verified by reverse transcript PCR followed by sequencing as described previously (61). SHP-1 expression was examined by immunoprecipitation followed by immunoblotting after treatment with CEP-701 for 6–8 h at the indicated concentrations. An increase in the SHP-1 protein levels was observed in both AML cell lines and in 3 of 4 primary AML samples (Fig. 5, a and b). In each of the three samples that showed increase in SHP-1 in response to CEP-701, the cells showed expression of constitutively phosphorylated FLT3 that was inhibited by the drug. The only sample that did not respond with an increase of SHP-1 expression did not express detectable constitutively phosphorylated FLT3 (Fig. 5c).



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  		FIG. 5.
CEP-701 induces expression of SHP-1 in FLT3/ITD expressing AML cell lines and primary AML samples. a, SHP-1 expression in MOLM-14 and MV4-11 cells after treatment with the indicated concentrations of CEP-701 for 8 h. Immunoblotting (IB) of immunoprecipitated (IP) SHP-1 was performed as outlined under "Materials and Methods." Lysate aliquots were electrophoresed on a duplicate gel, and the resultant blot was probed with anti-actin antibody to verify equal protein loading. b, response of SHP-1 to CEP-701-mediated inhibition of FLT3/ITD in primary AML cells. Cells were treated with the indicated concentrations of CEP-701 for 6–8 h, and SHP-1 proteins levels were detected as stated in b. c, FLT3 phosphorylation in primary samples was detected by immunoblotting following immunoprecipitation (1 mg of protein extract per sample) as described in the legend to Fig. 3 (upper panel). The membrane was stripped and reprobed with anti-FLT3 antibody to demonstrate FLT3 protein levels (lower panel).

 
FLT3/ITD Signaling Suppresses SHP-1 at the RNA Level—To explore the mechanism(s) by which FLT3/ITD signaling down-regulates SHP-1, SHP-1 mRNA levels were determined by Northern blotting. Consistent with the observed down-regulation of protein level, SHP-1 mRNA was significantly decreased in TF-1/ITD cells compared with TF-1 and TF-1/FLT3 cells (Fig. 6). This suppression was at least partially overcome in the TF-1/ITD cells by inhibiting FLT3/ITD signaling with CEP-701. These results indicate that FLT3/ITD signaling suppresses SHP-1 expression at the level of RNA.



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  		FIG. 6.
SHP-1 RNA levels are suppressed in TF-1/ITD cells. Cells were treated with CEP-701 at the indicated concentrations for 8 h or left untreated. a, total RNA was extracted for Northern blotting assays with 32P-labeled full-length SHP-1 cDNA probe. b, SHP-1 bands from a were quantitated and normalized to 28 S bands by densitometry using NIH image 1.62.

 
SHP-1 Suppression by RNAi Promotes Cell Growth and Survival—To confirm the roles of SHP-1 in cell proliferation and survival, SHP-1 expression was knocked-down, in both TF-1 and TF-1/ITD cells, using RNAi technology. Down-regulation of SHP-1 in transfected cells was verified by quantitative real-time RT-PCR and/or by immunoblotting assays performed at 24-h intervals (Fig. 7). In RNAi-treated TF-1 cells SHP-1 mRNA levels began to decrease significantly by 48 h and reached minimal levels at 72 h after transfection (Fig. 7a). SHP-1 protein levels also displayed a similar pattern of decrease, and at 72 h dropped to ~28% of untreated levels (densitometric analysis of the immunoblot shown in Fig. 7b). Cells were subjected to MTT proliferation assays with or without GM-CSF at the same time points. SHP-1 RNAi treated TF-1 cells grew faster (+GM-CSF) and survived better (–GM-CSF), and reached a slightly higher saturation density than the control cells (Fig. 7, c and d). In RNAi-treated TF-1/ITD cells, SHP-1 mRNA levels were ~20% of untreated levels at 96 h, and knockdown of SHP-1 expression also stimulated cell growth and survival (Fig. 8). These findings indicate that SHP-1 suppression is associated with a promotion of cell growth and survival in TF-1 cells. As inhibition of SHP-1 expression contributes to cell growth and survival, the suppression of SHP-1 by FLT3/ITD mutations is likely part of the pathway by which FLT3/ITD signaling contributes to leukemogenesis. The limited changes in growth and survival between SHP-1 RNAi-treated cells and control cells likely reflects the fact that some SHP-1 protein remains and that in TF-1/ITD cells SHP-1 is already fairly suppressed by FLT3/ITD signaling.



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  		FIG. 7.
Effects of SHP-1 knockdown on proliferation and survival of TF-1 cells. SHP-1 siRNA fragments were generated and transfected into TF-1 cells as described under "Materials and Methods." Control cells were treated with GFP RNAi. After overnight incubation (time 0), the transfected cells were aliquoted and cultured for experiments described below at intervals of 24 h. a, SHP-1 RNA levels were determined by qPCR with primers specific for SHP-1 and GAPDH shown under "Materials and Methods." SHP-1 RNA levels were normalized to the corresponding GAPDH RNA levels and expressed as the levels relative to time 0, which was given a value of 1. b, same cells were lysed for SHP-1 immunoprecipitation. SHP-1 expression was analyzed by immunoblotting with anti-SHP-1 antibody. c and d, MTT assays of SHP-1 RNAi-treated cells and control cells were performed as described previously in the absence (c) or presence (d) of GM-CSF. The results, expressed as mean ± S.E., were plotted as the levels relative to time 0, which was given a value of 1.

 



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  		FIG. 8.
Effects of SHP-1 knockdown on proliferation and survival of TF-1/ITD cells. a, RNAi experiments were performed as described in Fig. 7 except that SHP-1 RNA levels were only determined at 96 h. b, MTT assays of SHP-1 RNAi-treated cells and control cells were performed as outlined in Fig. 7.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we show that transfection of TF-1 cells with mutated, constitutively activated FLT3 results in increased phosphotyrosine levels and factor-independent growth. Expression of FLT3/ITD induces down-regulation of SHP-1 expression and activity. Activation of kinases normally induces increased phosphatase activity in response in order to maintain a dynamic equilibrium. The suppression of phosphatase activity we observed appears to be one mechanism by which mutated, constitutively activated kinases defeat the normal balance of protein phosphorylation. Treatment of FLT3/ITD-expressing cells, cell lines and primary AML cells, with a selective FLT3 inhibitor, CEP-701, results in inhibition of proliferation and up-regulation of SHP-1 expression. The suppression of SHP-1 by FLT3/ITD signaling is selective because SHP-2 protein expression and activity is unaffected by FLT3/ITD expression or treatment with CEP-701. Knockdown of SHP-1 by RNAi in both TF-1 and TF-1/ITD cells results in a growth/survival advantage. Therefore, the results presented here implicate SHP-1 as a possible tumor suppressor in the process of leukemogenesis activated by FLT3/ITD mutations.

Phosphatases have been previously implicated in the regulation of cellular proliferation, differentiation and survival. Some of the best evidence for these roles comes from the study of two naturally occurring mutations in the SHP-1 gene in motheaten (me/me) and viable motheaten (mev/mev) mice (49). These mice develop multilineage hemopoietic disorders involving hyperproliferation and abnormal activation of myeloid/monocytic cells with severe immunodeficiency and autoimmunity as well. Bone marrow progenitor cells from me/me and mev/mev mice are hypersensitive to multiple growth factors and cytokines, such as GM-CSF, G-CSF, Epo, and IFNs. This suggests that SHP-1 plays an important negative regulatory role in hematopoiesis downstream of multiple receptors. These observations, together with the findings that expression of SHP-1 is induced during forced differentiation of certain leukemia cells, including K562 and 32D cells (54, 55), implies that suppression of SHP-1 activity might be important in the pathogenesis of leukemia.

Analogous to the results observed in this study, the inhibition of SHIP by constitutively activated kinase Bcr-Abl has been reported in Philadelphia chromosome-positive hematopoietic cell lines and primary cells from patients (62). Bcr-Abl inhibits the expression and phosphatase activity of SHIP by decreasing the half-life and, to lesser extent, down-regulating the transcription of the gene. Since SHIP negatively regulates the activity of the phosphatidylinositol 3-kinase signaling pathway, direct inhibition of SHIP by Bcr-Abl results in increased myeloid proliferation and contributes to cell transformation (63, 64). Treatment of Bcr-Abl expressing cell lines with the Abl-selective tyrosine kinase inhibitor, STI571, results in cellular growth inhibition, differentiation, apoptosis, and up-regulation of SHIP (62). Recent studies have also shown possible roles for a loss-of-function mutation of SHIP within its conserved motif in the development of AML (65). This mutation conferred cells with enhanced and prolonged Akt phosphorylation and a subsequent growth/survival advantage. These findings indicate suppression of phosphatase activity is an important mechanism that facilitates leukemogenesis.

The down-regulation of SHP-1 at the transcriptional level by FLT3/ITD signaling is an additional mechanism for regulating its catalytic activity. This regulation is in the opposite direction to the posttranslational mechanisms that normally stimulate its phosphatase activity in response to increased kinase activity. Stimulation of growth factor and cytokine receptors results in activation of tyrosine kinases either intrinsic to the receptor, such FMS and c-Kit, or extrinsic cytoplasmic kinases, such as Src and JAK kinases. The phosphorylation of tyrosine residues on substrate proteins creates docking sites for SHP-1 association and the engagement of its N-SH2 domain releases the inhibitory effect it has when bound to the phosphatase catalytic region. However, SHP-1 activity is also subject to regulatory mechanisms associated with direct phosphorylation. Tyrosine phosphorylation of SHP-1 by a variety of tyrosine kinases, such as c-Abl (66), Lyn (67), and insulin receptor (68) stimulates SHP-1 activity. In contrast, phosphorylation of SHP-1 on serine residues by protein kinase C is associated with the inhibition of its activity (69). Furthermore, both SHP-1 RNA and protein expression are induced by differentiating agents in K562 cells, which do not express detectable SHP-1 under normal culture conditions (54). Finally, activating mutations of the c-Kit receptor, a close relative of FLT3, appear to induce the down-regulation of SHP-1 through a ubiquitin-dependent proteolytic pathway (57).

Thus, SHP-1 phosphatase activity appears to be regulated by a number of mechanisms. Intramolecular suppression maintains its activity at a relatively low level in resting cells. Upon extracellular stimuli, kinases are activated and phosphorylated substrate proteins bind the N-SH2 domain of SHP-1 releasing the intramolecular suppression. Feedback control, in either a positive or negative direction, results from direct phosphorylation of SHP-1 at specific sites by specific kinases. Finally, SHP-1 expression levels can be induced by differentiating signals or suppressed by oncogenic signaling.

Because SHP-1 also regulates signaling from a number of hematopoietic receptors, the down-regulation of SHP-1 by FLT3/ITD might also make cells more sensitive to other growth factors and cytokines, further tipping the balance in favor of phosphoproteins. Therefore, overcoming the suppression of SHP-1 activity might be one of the mechanisms by which CEP-701 is able to inhibit proliferation and overcomes the block to differentiation in FLT3/ITD-transfected cells.

The exact mechanism(s) by which FLT3/ITD suppresses SHP-1 transcription warrants further study. Silencing of tumor suppressor genes like p15INK4b by hypermethylation is a common event in AML and ALL (70). Some leukemogenic translocations such as PML-RAR (71) and Bcr-Abl (72) induce hypermethylation and thereby silencing of related genes. Silencing of the SHP-1 gene by aberrant methylation has been recently reported in many leukemia/lymphoma samples and cell lines (73), and in some cell lines SHP-1 mRNA expression is induced by treatment with 5-deoxyazacytidine, a demethylation agent. Whether hypermethylation related SHP-1 gene silencing is the mechanism in the FLT3/ITD-mediated SHP-1 suppression remains to be examined. Also, alternative transcripts of SHP-1 have been identified in both normal peripheral blood mononuclear cells and some leukemia/lymphoma cell lines (74). Those "abnormal" versions of transcripts are noncoding per se or encode a truncated version of SHP-1 that may compete with wild-type SHP-1 for substrate interaction. This could also function as an inhibitory mechanism of SHP-1 expression at the transcriptional level by reducing production of wild-type SHP-1 protein. The myriad levels of control point to the critical role of this, and other phosphatases, in maintaining the proper balance of protein phosphorylation to prevent transformation.


   FOOTNOTES
 
* This work was supported by Grants CA 90668 and CA 91177 from the NCI, National Institutes of Health, the Leukemia and Lymphoma Society of America, and the Burroughs Wellcome Fund. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} The Douglas Kroll Research Foundation Translational Researcher of the Leukemia and Lymphoma Society and the Kyle Haydock Professor of Oncology. To whom correspondence should be addressed: Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Bunting-Blaustein Cancer Research Bldg., Rm. 253, 1650 Orleans St., Baltimore, MD 21231-1000. Fax: 410-955-8897; E-mail: donsmall{at}jhmi.edu.

1 The abbreviations used are: FLT3, FMS-like tyrosine kinase 3; ITD, internal tandem duplication; AML, acute myeloid leukemia; JMML, juvenile myelomonocytic leukemia; IFN, interferon; IL, interleukin; Epo, erythropoietin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SH2, Src homology domain 2; SHIP, SH2 domain-containing inositol 5-phosphatase; SHP, SH2 domain-containing protein-tyrosine phosphatase; PTP, protein-tyrosine phosphatase; GFP, green fluorescent protein; p-NPP, p-nitrophenyl phosphate; MAPK, mitogen-activated protein kinase; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We thank Bruce Ruggeri and Susan Jones-Bolin of Cephalon Inc. for providing CEP-701.



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