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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2009 Dec;175(6):2416–2429. doi: 10.2353/ajpath.2009.080953

Expression of Activated STAT5 in Neoplastic Mast Cells in Systemic Mastocytosis

Subcellular Distribution and Role of the Transforming Oncoprotein KIT D816V

Christian Baumgartner *, Sabine Cerny-Reiterer *, Karoline Sonneck *, Matthias Mayerhofer *†, Karoline V Gleixner *, Richard Fritz *, Marc Kerenyi ‡, Cedric Boudot §, Fabrice Gouilleux , Jan-Wilhelm Kornfeld ∥, Christian Sillaber *, Richard Moriggl ∥, Peter Valent *,**
PMCID: PMC2789628  EMSID: UKMS32184  PMID: 19893034

Abstract

Recent data suggest that the signal transducer and activator of transcription (STAT)5 contributes to differentiation and growth of mast cells. It has also been described that constitutively phosphorylated STAT5 (pSTAT5) plays a pro-oncogenic role in various myeloid neoplasms. We examined the expression of pSTAT5 in neoplastic mast cells in systemic mastocytosis and asked whether the disease-related oncoprotein KIT D816V is involved in STAT5 activation. As assessed by immunohistochemistry using the anti-pSTAT5 antibody AX1, neoplastic mast cells were found to display pSTAT5 in all SM patients examined (n = 40). Expression of pSTAT5 was also demonstrable in the KIT D816V-positive mast cell leukemia cell line HMC-1. Using various staining-protocols, pSTAT5 was found to be located in both the cytoplasmic and nuclear compartment of mast cells. To define the functional role of KIT D816V in STAT5-activation, Ba/F3 cells with doxycycline-inducible expression of KIT D816V were used. In these cells, induction of KIT D816V resulted in an increased expression of pSTAT5 without substantial increase in total STAT5. Moreover, the KIT D816V-targeting kinase-inhibitor PKC412 was found to counteract expression of pSTAT5 in HMC-1 cells as well as doxycycline-induced expression of pSTAT5 in Ba/F3 cells. Finally, a dominant negative STAT5-construct was found to inhibit growth of HMC-1 cells. Together, our data show that neoplastic mast cells express cytoplasmic and nuclear pSTAT5, that KIT D816V promotes STAT5-activation, and that STAT5-activation contributes to growth of neoplastic mast cells.

Systemic mastocytosis (SM) is a myeloid neoplasm characterized by abnormal growth and accumulation of neoplastic mast cells in one or more visceral organs.1,2,3 The clinical picture and course of the disease are variable.1,2,3 Whereas most patients have an indolent disease stable over decades,1,2,3,4,5,6,7 some of the patients may have aggressive SM (ASM), or even mast cell leukemia (MCL) with short survival time.6,7,8,9 The World Health Organization discriminates four major variants of SM, namely indolent SM, SM with an associated clonal hematological non-mast cell lineage disease, ASM, and MCL.6,7

In a majority of patients with SM, the somatic KIT mutation D816V is detectable.10,11,12,13 The gene product, KIT, is a tyrosine kinase (TK) receptor for stem cell factor (SCF), and is considered to serve as a key regulator of differentiation of normal mast cells. The KIT mutation D816V is associated with SCF-independent phosphorylation and activation of the protein product, the KIT TK receptor, and supposedly contributes to the autonomous differentiation and growth of neoplastic mast cells in SM.14,15 However, so far, little is known about downstream signaling pathways and molecules responsible for KIT D816V-dependent growth and survival of neoplastic mast cells.16,17,18

Previous, as well as more recent, data suggest that the signal transducer and activator of transcription-5 (STAT5) contributes to SCF-dependent growth of mast cells in mice.19,20,21,22 Likewise, STAT5 knock out mice, like KIT-deficient animals, exhibit mast cell deficiency.20,21 Moreover, it has been described that dimerization of KIT by SCF in mast cells is associated with STAT5 activation.19 Other studies have shown that constitutive active STAT5 can act as an oncoprotein inducing myeloid leukemias in mice, and that neoplastic cells in human leukemias often display phosphorylated STAT5 (pSTAT5).23,24,25,26,27 An interesting aspect is that pSTAT5 apparently is expressed in the cytoplasmic compartment of leukemic cells.27 More recently, it has been reported that neoplastic mast cells in mastocytosis also react with antibodies against STAT5.28,29 However, so far, little is known about the exact distribution of pSTAT5 in the various categories of SM, about the mechanisms of STAT5 activation in neoplastic mast cells, and about the functional role of pSTAT5 in the pathogenesis of SM.

The aims of the present study were to examine the expression of pSTAT5 in neoplastic mast cells in various categories of SM, to examine the subcellular localization of STAT5 in neoplastic mast cells, to define the role of the D816V-mutated variant of KIT in STAT5 activation, and to ask whether pSTAT5 contributes to growth and survival of neoplastic mast cells.

Materials and Methods

Patients

Forty patients with SM and four patients with cutaneous mastocytosis (without involvement of the bone marrow [BM]), and five control cases (staging of lymphomas or reactive BM) were examined. Mastocytosis was diagnosed according to World Health Organization criteria.6,7 In the SM-group, 27 patients had indolent SM, eight had SM with an associated clonal hematological non-mast cell lineage disease, two had ASM, and three patients had MCL. The patients’ characteristics are shown in Table 1. Routine staging included physical examination, ultrasound of abdomen, complete blood count, serum tryptase measurement, BM histology, and immunohistochemistry (tryptase and CD25), cytologic examination of BM cells on Wright-Giemsa–stained BM smears, flow cytometry for detection of CD2 and CD25 on BM mast cells,30,31 and analysis of BM cells for KIT D816V by reverse transcription-PCR and restriction fragment length polymorphism.12 BM mononuclear cells were enriched using Ficoll. Informed consent was obtained in each case.

Table 1.

Patients’ Characteristics (SM)

ISM (n = 27) ASM/MCL (n = 5) SM-AHNMD (n = 8*) All SM patients (n = 40)
F:M 12:15 1:4 2:6 15:25
Median age, years [range] 49 [32 to 73] 46 [21 to 74] 44 [17 to 70] 48 [17 to 74]
Median serum tryptase (ng/ml) [range] 350.3 [14.5 to 1090] 420.9 [53.6 to 796] 303.4 [89.3 to 615] 333.6 [14.5 to 1090]
Median BM MC infiltration (%) [range]† 19 [2 to 80] 30 [5 to 70] 21 [5 to 70] 21 [2 to 80]
D816V mutation status: +/−/n.d. 20/4/3 2/1/2 5/2/1 27/7/6
Median WBC count (G/L) [range] 7.4 [3.8 to 30.1] 8.0 [5.0 to 11] 14.6 [6.2 to 35.5] 8.8 [3.8 to 35.5]
Median hemoglobin (g/dL) [range] 14 [11 to 16.5] 10.9 [8.8 to 13.7] 9.8 [8.8 to 16.4] 12.9 [8.8 to 16.5]
Median platelet count (G/L) [range] 230 [128 to 405] 163 [28 to 269] 97 [9 to 287] 200 [9 to 405]
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ISM, indolent systemic mastocytosis; ASM, aggressive systemic mastocytosis; MCL, mast cell leukemia; SM-AHNMD, systemic mastocytosis with an associated clonal hematologic non mast cell lineage disease; f, female; m, male; BM MC, bone marrow mast cells; n.d., not determined; WBC; white blood cell count. 

*

AHNMD were: chronic myelomonocytic leukemia, n = 5; small lymphocytic lymphoma, n = 1; acute myeloid leukemia, n = 2. 

†

BM MC infiltration was determined by immunohistochemistry using an antibody against tryptase. 

Monoclonal Antibodies and Other Reagents

The anti-tryptase monoclonal antibody (mAb) G3 (IgG1) was purchased from Chemicon (Temecula, CA), the anti-pSTAT5a/b mAb AX1 from Advantex Bioreagents LLP (Conroe, TX), a polyclonal anti-pSTAT5 antibody from Cell Signaling (Danvers, MA), and Alexa Fluor 488-conjugated mAb against pSTAT5, anti-Phospho-STAT5(Y694):Alexa Fluor 488, an isotype-matched antibody (mIgG1-Alexa Fluor 488), and a polyclonal rabbit antibody against active caspase 3 from BD Biosciences Pharmingen (San Jose, CA). The fluorescein-isothiocyanate-labeled mAb 2A3 (CD25), the phycoerythrin-labeled mAb RPA-2.10 (CD2), the fluorescein-isothiocyanate–labeled mAb 581 (CD34), the phycoerythrin-labeled mAb 581 (CD34), and the PerCP-labeled mAb 2D1 (CD45) were from Becton Dickinson, and the allophycocyanin-labeled mAb 104D2D1 (CD117) from Beckman Coulter/Immunotech (Marseilles, France). Imatinib, midostaurin (PKC412),32 and nilotinib (AMN107)33 were kindly provided by Dr. Elisabeth Buchdunger, Dr. Paul Manley, and Dr. Doriano Fabbro (Novartis Pharma AG, Basel, Switzerland). Piceatannol was purchased from Sigma (St. Louis, MO). Stock solutions of midostaurin, nilotinib, and piceatannol were prepared by dissolving in dimethyl-sulfoxide (Merck, Darmstadt, Germany). Recombinant human SCF was from Strathmann Biotech (Hannover, Germany), RPMI 1640 medium, and fetal calf serum from PAA laboratories (Pasching, Austria), l-glutamine and Iscove′s modified Dulbecco′s medium from Gibco Life Technologies (Gaithersburg, MD), bovine serum albumin from Sigma, and recombinant human interleukin-4 from Peprotech (Rocky Hill, NJ). The Vybrant MTT Cell Proliferation Assay Kit was purchased from Invitrogen (Oregon).

Culture of HMC-1 Cells Expressing KIT D816V

The human mast cell line HMC-1,34 generated from a patient with MCL, was kindly provided by Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN). Unless otherwise stated, the subclone HMC-1.2, harboring the KIT mutation D816V, was used. HMC-1 cells were grown in Iscove′s modified Dulbecco′s medium supplemented with 10% fetal calf serum, l-glutamine, and antibiotics at 37°C and 5% CO2. HMC-1 cells were re-thawed from an original stock every 4 to 8 weeks and passaged weekly. As control of phenotypic stability, HMC-1 cells were periodically checked for i) the presence of metachromatic granules by Wright-Giemsa staining, ii) expression of KIT, and iii) the down-modulating effect of interleukin-4 (100 U/ml, 48 hours) on KIT-expression.35 In select experiments, a KIT D816V-negative subclone of HMC-1 (HMC-1.1)36 was used.

Ba/F3 Cells with Inducible Expression of Wild-Type KIT or KIT D816V

The generation of Ba/F3 cells with doxycycline-inducible expression of wild-type KIT (Ton.Kit.wt) or KIT D816V has recently been described.15,37 In brief, Ba/F3 cells expressing the reverse tet-transactivator15 were co-transfected with pTRE2 vector (Clontech, Palo Alto, CA) containing KIT D816V cDNA (or wild-type KIT cDNA, both kindly supplied by Dr. J. B. Longley, Columbia University, New York) and pTK-Hyg (Clontech) by electroporation. Stably transfected cells were selected by growing in hygromycin and cloned by limiting dilution. In this study, subclone Ton.Kit.D816V.2715,37 was used in all experiments. As assessed by Western blotting and conventional reverse transcriptase PCR, expression of KIT D816V can be induced in Ton.Kit.D816V.27 cells within 12 hours by exposure to doxycycline (1 μg/ml).15,37

Immunohistochemistry and Immunocytochemistry

Immunohistochemistry was performed on serial sections prepared from paraffin-embedded, formalin-fixed BM biopsy specimens using the indirect immunoperoxidase staining technique following established protocols.15,28,29,38,39,40 Endogenous peroxidase was blocked by methanol/H2O2. The following antibodies were applied: anti-pSTAT5 mAb AX1 and anti-tryptase antibody G3. Two different staining protocols were established, one for optimal detection of cytoplasmic pSTAT5 (protocol A), and one for optimal detection of nuclear pSTAT5 (protocol B) in neoplastic cells (Table 2). Before staining with anti-pSTAT5 mAb AX1, sections were pretreated either by microwave oven (“predominantly cytoplasmic” staining protocol A) or by autoclave (“predominantly nuclear” staining protocol B).

Table 2.

Immunohistochemical Staining Protocols for the Detection of Cytoplasmic and Nuclear pSTAT5 in Neoplastic Mast Cells Using the Anti-pSTAT5 Antibody AX1

Steps in procedure Predominantly cytoplasmic pSTAT5-staining protocol Predominantly nuclear pSTAT5-staining protocol
Antigen retrieval Microwave oven, citrate buffer Autoclave, citrate buffer
Peroxidase block Methanol/H2O2 Methanol/H2O2
First step antibody (Ab) Anti-pSTAT5 Ab, AX1 Anti-pSTAT5 Ab, AX1
Ab pre-diluted in TBS-BSA (5%), 1:1000 Renoir Red, 1:1000
Second step Ab Biotinylated goat-anti-mouse IgG, (diluted in TBS and horse serum) Biotinylated goat anti-mouse IgG
Reaction complex Biotin-peroxidase complex Streptavidin HRP-label
Chromogen and counterstain AEC + Hemalaun DAB + Hemalaun
Dehydration NA Ethanol + xylol dilution
Embedding Aquatex Eukitt
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TBS, Tris-buffered saline; BSA, bovines serum albumine; HRP, horseradish peroxidase; AEC, 3-amino-9-ethylcarbazole; DAB, 3,3-diaminobenzidine; NA, not applicable. 

In case of nuclear pSTAT5 staining, sections were stained with anti-pSTAT5 mAb AX1 diluted (1:1000) in Renoir Red Diluent (Biocare Medical, Walnut Creek, CA). Slides were incubated with AX1 mAb for 20 hours at room temperature. Then, slides were washed in Tris-buffered saline (TBS) and incubated with biotinylated goat anti-mouse IgG (Biocare Medical) for 30 minutes (room temperature), washed three times in TBS, and then were exposed to streptavidin-horseradish peroxidase complex for 30 minutes. 3,3-diaminobenzidine (DAB) was used as chromogen.

For cytoplasmic staining of pSTAT5, mAb AX1 was diluted (1:1000) in 0.05 M/L TBS (pH 7.5) plus 1% bovine serum albumin. Slides were incubated with AX1 mAb for 20 hours at room temperature. Then, slides were washed in TBS and incubated in biotinylated goat anti-mouse IgG (Vector, Burlingame, CA) for 30 minutes (room temperature), washed in TBS and then exposed to biotin-peroxidase complex for 30 minutes at room temperature; 3-amino-9-ethylcarbazole (Sigma) was used as chromogen. All slides were counterstained in Mayer′s Hemalaun.

In a series of 30 patients with SM, serial sections were stained for nuclear pSTAT5, cytoplasmic pSTAT5, or mast cell tryptase. For tryptase staining, the anti-tryptase antibody G3 diluted (1:5000) in 0.05 M/L TBS (pH 7.5) plus 1% bovine serum albumin, was used. G3 mAb was applied for 1 hour. Otherwise, the tryptase-staining protocol was identical to that used for detection of cytoplasmic pSTAT5.

Immunocytochemistry was performed on purified mast cells obtained from two patients with MCL (>20% mast cells in BM smears), normal BM mononuclear cells, and HMC-1 cells. Cells were spun on cytospin slides and then were incubated with AX1 mAb (standard dilution 1:1000) for 20 hours. Before AX1 staining, slides were incubated in citrate buffer (pH 6.0) at 95°C for 20 minutes. In a separate set of experiments, various antibody dilutions (1:1000 to 1:10,000) were applied. In select experiments, a polyclonal rabbit anti-pSTAT5 antibody (1:100) or a rabbit anti caspase 3 antibody (1:1000) was applied overnight. After incubation with antibodies, cells were washed, and thereafter exposed to biotinylated goat-anti-mouse IgG or goat anti-rabbit IgG for 30 minutes at room temperature. As chromogen, alkaline phosphatase complex (Biocare) was used. Antibody-reactivity was made visible by Neofuchsin (Nichirei, Tokyo, Japan). For detection of nuclear pSTAT5, slides were stained with AX1 diluted in Renoir Red. Slides were incubated with AX1 for 20 hours, washed, and incubated with biotinylated goat anti-mouse IgG (Biocare) for 30 minutes (room temperature). Then, slides were again washed and exposed to streptavidin-horseradish peroxidase complex for 30 minutes; 3,3-diaminobenzidine was used as chromogen.

In control experiments, AX1 was pre-incubated with control buffer or a STAT5-specific blocking peptide (1 μmol/L) for 1 hour before immunohistochemistry or immunocytochemistry experiments were performed. The STAT5-specific blocking peptide (sequence: KAVDG[phosphoY] VKPQIK) was produced at the Institute for Molecular Pathology (Vienna, Austria).

Flow Cytometry

Expression of cell surface antigens on primary neoplastic mast cells was analyzed by multicolor flow cytometry using antibodies against KIT, CD2, CD25, and CD34, as described.30,31 BM mast cells were defined as KIT++/CD34− cells. For flow cytometric detection of cytoplasmic pSTAT5, HMC-1 cells were incubated in control medium, midostaurin (0.05, 0.1, 0.5, or 1 μmol/L), nilotinib (1 μmol/L), or imatinib (1 μmol/L) for 4 hours. Then, cells were fixed in formaldehyde (1.6%), permeabilized by exposure to ice-cold methanol (−20°C, 10 minutes), washed in PBS containing 0.1% bovine serum albumin, and stained with an Alexa488-conjugated anti-pSTAT5 mAb or an isotype-matched control antibody for 15 minutes (room temperature). Cells were then washed and analyzed on a FACScan (BD Biosciences, Pharmingen, San Jose, CA).

Western Blot Analysis of Expression of pSTAT5

For Western blotting, HMC-1 cells were incubated in control medium (Iscove′s modified Dulbecco′s medium +10% FCS), midostaurin (1 μmol/L), nilotinib (1 μmol/L), or imatinib (1 μmol/L) at 37°C for 4 hours. Immunoprecipitation (IP) and Western blotting were performed as described.15,37 In brief, 107 cells were washed at 4°C and resuspended in 100 μl radioimmunoprecipitation assay buffer. After incubation in radioimmunoprecipitation assay buffer, supplemented with proteinase inhibitor cocktail (Roche, Basel, Switzerland) for 30 minutes at 4°C, lysates were centrifuged. For IP, lysates from 107 cells were incubated with a polyclonal anti-STAT5a antibody (Zymed, San Francisco, CA) and protein G Sepharose beads (Amersham, Buckinghamshire, UK) in IP buffer at 4°C overnight. Beads were then washed three times in IP buffer. Lysates and immunoprecipitates were separated under reducing conditions by 7.5% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Amersham) in buffer containing 25 mmol/L Tris, 192 mmol/L glycine, and 20% methanol, at 4°C. Membranes were blocked for 1 hour in 5% blocking reagent (Roche) and were then incubated with anti-STAT5 antibody (Transduction laboratories, Lexington, KY) (1:1000) or with anti-phospho-protein mAb 4G10 (1:1000) (Upstate Biotechnology, Lake Placid, NY) at 4°C overnight. Antibody reactivity was made visible by sheep anti-mouse IgG antibody and Lumingen PS-3 detection reagent (both from Amersham), with CL-Xposure film (Pierce Biotechnology, Rockford, IL).

Isolation of Cytoplasmic and Nuclear Fractions of HMC-1 Cells

HMC-1 cells were lysed in hypotonic buffer (20 mmol/L Hepes, 10 mmol/L KCl, 1 mmol/L EDTA, 0.2% NP40, 10% glycerol, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mmol/L phenylmethylsulfonide fluoril, and 1 mmol/L Na2VO4) as described.29 Supernatants (cytoplasmic fraction) were frozen at −70°C. Pelleted nuclei were resuspended in hypertonic buffer (hypotonic buffer plus 350 mmol/L NaCl), and protein extracts prepared by agitation (30 minutes, 4°C). After debris was removed by centrifugation, nuclear extracts were frozen at −70°C. Expression of pSTAT5 was determined by Western blotting and quantified by densitometry as reported.29 Fractionation of subcellular compartments was controlled by applying anti-Raf-1 (cytoplasmic) and anti-topoisomerase-1 (nuclear) antibodies in parallel.29 All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).

Analysis of STAT5 Phosphorylation by Electromobility Shift Assay

Before electromobility shift assay (EMSA), doxycycline-exposed (12 hours, 1 μg/ml) Ton.Kit.D816V.27 cells were incubated in control medium, midostaurin (1 μmol/L), or imatinib (1 μmol/L) at 37°C for 6 hours. For STAT5 analysis, the proximal STAT-binding element (5′-AGATTTCTAGGAATTCAAATC-3′) of the bovine β-casein promoter was used.15,26 Binding reactions were performed by incubating 10,000 cpm of radiolabeled probe with cell lysates (20 μg) for 30 minutes. For supershift reactions of STAT-containing complexes, 2 μg of antibodies specific for the C-terminal transactivation-domains of STAT1 (M22; Santa Cruz Biotechnologies), STAT3, and STAT5 (C-17; Santa Cruz) were added before the EMSA was performed. Samples were separated by electrophoresis through 6% native polyacrylamide gels and analyzed by autoradiography using BioMax sensitive films (Kodak, Rochester, NY).

Evaluation of Effects of a Dominant Negative STAT5 Mutant

To investigate the role of STAT5 in KIT D816V-dependent growth, HMC-1 cells were retrovirally transduced with a dominant negative (dn) STAT5 construct (STAT5bΔ754)41 or with the empty vector. Transfection was performed essentially as described.15 Retroviruses were produced by transient transfection of HEK-293FT cells (Invitrogen) with pMSCV-dnSTAT5-IRES-green fluorescent protein (GFP), pVSV, and pGAG-Pol, using Lipofectamine2000 (Invitrogen) according to the instructions of the manufacturer. To determine the relative growth advantage of pSTAT5-expressing HMC-1 cells, as compared with HMC-1 cells in which pSTAT5 was blocked by the STAT5 dn construct, mixtures of nontransfected HMC-1 cells and STAT5 dn-transfected cells and of nontransfected HMC-1 cells and vector control-transfected cells were cultured for various time periods (1, 3, 5, and 7 days) and then were examined for the presence (percentage) of GFP-positive cells by flow cytometry on a FACS Calibur (Becton Dickinson).

Evaluation of Effects of Piceatannol

To further determine the functional role of STAT5 in neoplastic mast cells, we applied piceatannol, an enzyme inhibitor that has been reported to block a number of signaling molecules including Syk, ZAP70, and STAT5.42,43 In these experiments, HMC-1 cells were incubated with increasing concentrations of piceatannol (1 to 100 μmol/L) in 96-well culture plates (TPP, Trasadingen, Switzerland) at 37°C for 48 hours. Cell proliferation was determined using the Vybrant MTT assay. In brief, after incubation with piceatannol, cell supernatants were discarded and 100 μl MTT (12 mmol/L) were added to each well. Cells were incubated for another 4 hours at 37°C. Then, 100 μl of a HCl-SDS solution were added and cells were incubated overnight at room temperature in humidified chambers. Thereafter, absorbance (570 nm) was measured in a microplate reader. For evaluation of apoptosis, HMC-1 cells were incubated in control medium or in various concentrations of piceatannol (1, 50, and 100 μmol/L) in 24-well culture plates (TPP) at 37°C for 24 hours. Thereafter, the percentage of apoptotic cells was quantified on cytospin preparations stained with Wright-Giemsa by microscopy. Apoptosis was defined according to conventional cytomorphologic criteria.44 In a separate set of experiments, piceatannol-exposed HMC-1 cells were stained with an antibody against active caspase 3 by immunocytochemistry, to confirm apoptosis-induction by a second (more objective) approach. All experiments were performed in triplicate.

Results

Primary Neoplastic Mast Cells in Patients with SM Express pSTAT5

As assessed by immunohistochemistry, primary neoplastic mast cells were found to react with the anti-pSTAT5a/b antibody AX1 in all SM variants and in all patients with SM examined (n = 40) using either the predominantly cytoplasmic staining protocol (protocol A) or the predominantly nuclear staining protocol (protocol B) (Table 3, Figure 1, A–L). In particular, we found that pSTAT5 is expressed in both the nuclear compartment and cytoplasmic compartment of neoplastic mast cells in all donors tested (Figure 1, Table 3). Interestingly, even in staining protocol B, the anti-pSTAT5 antibody AX1 was found to react with the cytoplasm of neoplastic mast cells in all patients examined, whereas on the other hand, the nuclei of neoplastic mast cells were not labeled when applying the protocol A, suggesting that cytoplasmic pSTAT5 may be expressed in excess over nuclear pSTAT5 in neoplastic mast cells. Pre-incubation of mAb AX1 with a pSTAT5-specific blocking peptide resulted in a negative stain in both staining protocols (Figure 1, M–P). No significant differences in nuclear or cytoplasmic pSTAT5 expression in mast cells were detected when comparing indolent SM patients with patients suffering from advanced SM (SM with an associated clonal hematological non-mast cell lineage disease, ASM, MCL) or when comparing KIT D816V-positive cases with KIT D816V-negative patients. To confirm AX1 staining results, a second (polyclonal) anti-pSTAT5 antibody was applied. Using this antibody, identical staining results were obtained in SM when compared with the AX1 antibody (not shown). All in all, these data show that pSTAT5 is expressed in both the cytoplasmic and nuclear compartments of neoplastic mast cells in SM.

Table 3.

Expression of pSTAT5 in Neoplastic MC in Patients with SM

Expression of pSTAT5 in mast cells
Patient number Diagnosis KIT D816V Nuclear expression Cytoplasmic expression
#01 ISM − + +
#02 ISM n.d. + +
#03 ISM + + +
#04 ISM n.d. + +
#05 ISM + n.d. +
#06 ISM + + +
#07 ISM + + +
#08 ISM + n.d. +
#09 ISM + + +
#10 ISM − n.d. +
#11 ASM + + +
#12 ISM − + +
#13 ISM + + +
#14 ISM + + +
#15 ISM + n.d. +
#16 ISM + + +
#17 ISM + + +
#18 ISM + + +
#19 ISM + n.d. +
#20 ISM + + +
#21 ISM + + +
#22 ISM + + +
#23 ISM − + +
#24 ISM n.d. + +
#25 ISM + + +
#26 ISM + + +
#27 ISM + + +
#28 ASM n.d. + +
#29 ISM + + +
#30 MCL + + +
#31 MCL n.d. + +
#32 MCL − + +
#33 SM-AHNMD + n.d. +
#34 SM-AHNMD + + +
#35 SM-AHNMD + n.d. +
#36 SM-AHNMD + n.d. +
#37 SM-AHNMD − + +
#38 SM-AHNMD n.d. + +
#39 SM-AHNMD + n.d. +
#40 SM-AHNMD − n.d. +
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ISM, indolent systemic mastocytosis; ASM, aggressive systemic mastocytosis; MCL, mast cell leukemia; SM-AHNMD, systemic mastocytosis with an associated clonal hematologic non mast cell lineage disease; n.d., not determined. 

Figure 1.

Figure 1

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Immunohistochemical detection of phosphorylated (p) STAT5 in neoplastic mast cells in the bone marrow of patients with systemic mastocytosis (SM). Serial sections prepared from paraffin-embedded bone marrow (iliac crest) of a patient with indolent SM (A–F) and a patient with aggressive SM (G–L) were stained with anti-tryptase antibody G3 (A, D, G, J), and the anti-pSTAT5 antibody AX1 using two staining protocols, one ′predominantly cytoplasmic′ staining protocol (B, E, H, K), and a second ′predominantly nuclear′ staining protocol (C, F, I, L). Serial section-staining revealed that almost all of the tryptase-positive neoplastic mast cells co-expresssed cytoplasmic pSTAT5, and many of these cells also exhibited nuclear pSTAT5 (C, F, I, L). The lower panels (D, E, F, and J, K, L) represent higher magnifications (×100) of the respective upper panels (A, B, C, and G, H, I) (×40). In separate experiments, AX1 was pre-incubated with control buffer (M, O) or a STAT5-specific blocking peptide (N, P) before being applied, which resulted in a negative stain in both the predominantly cytoplasmic staining protocol (M, N) and predominantly nuclear staining protocol (O,P).

Expression of pSTAT5 in Other BM Cells

Apart from neoplastic mast cells, megakaryocytes and myeloid (neutrophil) precursor cells were also found to stain positive for pSTAT5 in the BM sections examined (Table 4), confirming previous data.28 By contrast, pSTAT5 was not detectable in erythroid progenitors, mature neutrophilic granulocytes, or eosinophil granulocytes (Table 4). This staining pattern was found in all categories of SM (all patients), as well as in BM sections obtained from controls, ie, patients with cutaneous mastocytosis (no BM mast cells infiltrates) and in the normal/reactive BM. When taking together all results obtained from the two staining protocols, we found that pSTAT5 is expressed in both cell compartments in all antibody-reactive cells including megakaryocytes, independent of diagnosis and disease variant, ie, in all forms of SM as well as in control BM sections (cutaneous mastocytosis or normal/reactive BM). In all samples, cytoplasmic pSTAT5 was detected using both staining protocols, whereas nuclear pSTAT5 was only detectable in the predominantly nuclear staining protocol.

Table 4.

Cellular Distribution of pSTAT5 in Bone Marrow Sections in Mastocytosis and Controls

Cell type Predominantly cytoplasmic pSTAT5 staining protocol
Predominantly nuclear pSTAT5 staining protocol
Normal/reactive BM CM ISM ASM/MCL SM-AHNMD Normal/reactive BM CM ISM ASM/MCL SM-AHNMD
Megakaryocytes ++ ++ ++ ++ ++ +** +** +** +** +**
Myeloid progenitors + + + + + +** +** +** +** +**
Neutrophil granulocytes − − − − − − − − − −
Eosinophil granulocytes − − − − − − − − − −
Erythroid progenitors − − − − − − − − − −
Mast cells n.d.* n.d.* + + + n.d.* n.d.* + + +
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Bone marrow sections were stained with antibody AX1 as described in the text. 

Score: −, negative; +, positive; ++, strongly reactive; BM, bone marrow; CM, cutaneous mastocytosis; ISM, indolent systemic mastocytosis; ASM, aggressive systemic mastocytosis; MCL, mast cell leukemia; SM-AHNMD, systemic mastocytosis with an associated clonal hematologic non MC-lineage disease; n.d., not determined. 

*

In these cases, only a few diffusely spread mast cells were found. 

**

In most patients and controls, pSTAT5 was found to be expressed in the cytoplasmic compartment in the ’predominantly nuclear’ staining protocol, often exceeding the nuclear staining-reaction. 

Detection of pSTAT5 in Isolated Leukemic Mast Cells and in the Mast Cell Line HMC-1

As assessed by immunocytochemistry, isolated primary leukemic mast cells obtained from a patient with MCL and the MCL-derived human mast cell line HMC-1 were found to express pSTAT5 (Figure 2, A–D). As expected, pSTAT5 was found to be expressed primarily in the cytoplasmic compartment in these cells. To confirm this assumption, we applied the two staining protocols (predominantly cytoplasmic versus predominantly nuclear) in parallel using serial antibody dilutions and HMC-1 cells. In these experiments, the cytoplasm of HMC-1 cells stained positive for pSTAT5 in most antibody-dilutions in both staining protocols, whereas nuclear pSTAT5 was only detectable in the predominantly nuclear staining protocol and disappeared rapidly when antibody AX1 was diluted (Figure 2, E and F). Interestingly, as assessed by dilution experiments, HMC-1.2 cells appeared to contain similar amounts of pSTAT5 when compared with HMC-1.1 cells. In both staining protocols, pre-incubation of the anti-pSTAT5 antibody AX1 with a pSTAT5-specific blocking peptide resulted in a negative stain (Figure 2, G–J). We were also able to confirm expression of cytoplasmic pSTAT5 in HMC-1 cells by flow cytometry using the Alexa Fluor 488-labeled anti-pSTAT5 mAb Y694. In particular, as visible in Figure 2, K and L, HMC-1 cells were found to react with the anti-pSTAT5 antibody. Interestingly, the KIT D816V+ subclone HMC-1.2 was found to express slightly higher levels of pSTAT5 compared with HMC-1.1 cells (Figure 2, K and L).

Figure 2.

Figure 2

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Detection of phosphorylated (p) STAT5 in the cytoplasm of primary neoplastic mast cells and in the mast cell leukemia cell line HMC-1. Bone marrow mast cells obtained from a patient with mast cell leukemia (A, B) and HMC-1 cells (C, D) were spun on cytospin slides and then were stained with the anti-pSTAT5 antibody AX1 (A, C). Wright-Giemsa staining confirmed the presence of immature mast cells (B, D). E, F: KIT D816V-negative HMC-1.1 cells (E) and KIT D816V+ HMC-1.2 cells (F) were stained with serial dilutions of AX1 antibody as indicated using two different staining protocol, ie, one conventional protocol (protocol A: left panels in E and F), and one protocol for optimal visualization of nuclear pSTAT5 (protocol B: right panels in E and F). In both staining protocols, the reactivity of the cytoplamic compartment (black bars) and nuclear compartment (open bars) with AX1 antibody was determined separately. Results are expressed as percentage of reactive cells in one typical experiment (the same results were obtained in a second independent experiment). G–J: The AX1 antibody was pre-incubated with control buffer (G, I) or a STAT5-specific blocking peptide (H, J) for 1 hour, and then was applied to HMC-1 cells using two different staining protocol, ie, one conventional staining protocol (G, H), and one for optimal visualization of nuclear pSTAT5 (I, J). K, L: Flow cytometry was performed with an Alexa488-conjugated anti-pSTAT5 antibody (shaded plot) or an isotype-matched control antibody (unshaded plot). As visible, the antibody was found to react with pSTAT5 in KIT D816V-positive HMC-1.2 cells (K), as well as in KIT D816V-negative HMC-1.1 cells (L).

Comparison of pSTAT5 Expression in Subcellular Compartments of Neoplastic Mast Cells by Western Blotting and Densitometry

To confirm that pSTAT5 is primarily expressed in the cytoplasm of neoplastic mast cells, we fractionated HMC-1 cell extracts, ie, into a nuclear and a cytoplasmic fraction, and performed Western blot experiments followed by densitometry. In these experiments, we found that cytoplasmic HMC-1 extracts express pSTAT5 in excess over nuclear pSTAT5 extracts in all three experiments (Figure 3, A–B). These data strongly suggest that cytoplasmic pSTAT5 is the predominant form of pSTAT5 detectable in neoplastic mast cells, confirming our immunostaining results (Figure 1, and Figure 2, G–J) and recently published data.27,29

Figure 3.

Figure 3

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Quantitative analysis of cytoplasmic and nuclear pSTAT5 in HCM-1 cells. Expression of pSTAT5 in cytoplasmic extracts (CE) and nuclear extracts (NE) of HMC-1 cells was determined in three independent experiments (#1, #2, #3) by Western blotting. For control purpose, extracts were also examined for expression of Raf-1 (cytoplamic marker) and topoisomerase-1 (nuclear marker antigen). A shows results from Western blotting, and B provides a densitometric evaluation of the data obtained with cytoplamic extracts (open bars) and nuclear extracts (black bars) in the three experiments (#1, #2, #3).

KIT D816V Induces Activation of STAT5 in Ba/F3 Cells

To define the role of the SM-related oncoprotein KIT D816V in STAT5 activation, Ton.Kit.D816V.27, a Ba/F3 clone in which KIT D816V can conditionally be expressed on exposure to doxycyline,15 was used. When these cells were exposed to doxycycline to express KIT D816V, a huge increase in pSTAT5 was observed (Figure 4). By contrast, the wild-type form of KIT did not induce comparable phosphorylation of STAT5 in Ba/F3 cells (Figure 4). The KIT D816V-induced activation of STAT5 was completely blocked by the KIT D816V-targeting drug midostaurin (PKC412), whereas no effect was seen with imatinib, a TK inhibitor that is unable to counteract KIT D816V TK activity (Figure 4). These data suggest that KIT D816V contributes to STAT5 activation in neoplastic cells.

Figure 4.

Figure 4

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Induction of STAT5 activity by KIT D816V in Ba/F3 cells. STAT DNA-binding acitivity was analyzed using extracts of unstimulated Ton.Kit.D816V cells (co, without KIT D816V) and extracts of Ton.Kit.D816V cells induced to express KIT D816V by exposure to doxycycline (+Doxycycline). Before being exposed to doxycycline, cells were incubated with control medium or medium containing imatinib (1 μmol/L) or midostaurin (1 μmol/L) at 37°C for 4 hours. Cell extracts were analyzed using blunt-ended annealed oligonucleotides. For STAT5 analysis in electrophoretic mobility shift assays (EMSA), the proximal STAT-binding element of the bovine β-casein promoter was used. Binding reactions were performed by incubating the radiolabeled probe with cell-lysates (20 μg) for 30 minutes. In supershift-reactions of STAT-containing complexes, 2 μg of antibodies specific for the C-terminal transactivation-domains of STAT1, STAT3, and STAT5 were added before EMSA was performed. Samples were separated by electrophoresis. As shown, the KIT D816V-induced phosphorylation of STAT5 was inhibited by PKC412/midostaurin.

Effects of KIT-Targeting Drugs on Expression of pSTAT5 in HMC-1 Cells

In a next step, we examined the mechanism of STAT5 activation in HMC-1 cells using KIT-targeting pharmacological inhibitors. As assessed by Western blotting and flow cytometry, unstimulated HMC-1 cells were found to display pSTAT5. In Western blot experiments, incubation of HMC-1 cells with midostaurin (PKC412) was followed by a decrease in expression of pSTAT5, and a slight effect was also seen with AMN107 (nilotinib), whereas no effect was produced by imatinib (Figure 5A). Moreover, we were able to show by flow cytometry that both midostaurin and nilotinib down-regulate the expression of cytoplasmic pSTAT5 in HMC-1 cells (Figure 5, B–D). Interestingly, in both assays (Western blotting and flow cytometry), PKC412 was found to be the more potent inhibitor compared with nilotinib (AMN107), which is in line with the more potent growth-inhibitory effect of PKC412 on neoplastic mast cells.37 In control experiments, PKC412 did not block expression of other key signaling molecules in neoplastic mast cells, including Btk and Lyn (data not shown). All in all, these data suggest that KIT D816V contributes to STAT5 activation in neoplastic mast cells in SM.

Figure 5.

Figure 5

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Effects of KIT TK inhibitors on pSTAT5 expression in HMC-1 cells. A: Western blot analysis of KIT D816V-positive HMC-1.2 cells using antibodies against pSTAT5. Before immunoprecipitation (IP) and Western blotting, cells were incubated in control medium, imatinib (1 μmol/L), midostaurin/PKC412 (1 μmol/L), or nilotinib/AMN107 (1 μmol/L) at 37°C for 4 hours. Western blotting and IP were performed as described in the text. IP was conducted using a polyclonal anti-STAT5a antibody. Western blotting was performed using the anti-phospho-tyr antibody 4G10 for detection of pSTAT5, and anti-STAT5a antibody for detection of total STAT5. B–D: Flow cytometric assessement of expression of pSTAT5 in HMC-1 cells. B, C: Cells were incubated with control medium (open graphs) or with KIT inhibitors (gray graphs), ie, AMN107/nilotinib, 1 μmol/L (B) and PKC412/midostaurin, 1 μmol/L (C) at 37°C for 4 hours. D: Dose-dependent effect of PKC412 on expression of pSTAT5 in HMC-1 cells. Cells were incubated in control medium (co) or various concentrations of PKC412 as indicated (4 hours). Then, cells were permeabilized by methanol and subjected to flow cytometry using an antibody to pSTAT5 as described in the Materials and Methods.

Effect of a dn STAT5 Construct on Growth of HMC-1 Cells

To define a role for activated STAT5 (pSTAT5) in growth of neoplastic mast cells, we expressed a dn STAT5 mutant in HMC-1 cells. Expression of this dn STAT5 construct resulted in a decrease in expression of pSTAT5 in HMC-1 cells (not shown) as well as in reduced growth of these cells compared with the vector control (Figure 6). In particular, the number (percentage) of GFP-positive cells decreased significantly over time in a mixture of dn STAT5-transfected (GFP-labeled) cells and nontransfected (unlabled) HMC-1 cells, whereas no decrease in GFP-positive cells was seen when mixtures of control vector-transfected (GFP-labeled) cells and control HMC-1 cells were examined over time (Figure 6). These data suggest that the knock-down of STAT5 is associated with reduced survival of mast cells.

Figure 6.

Figure 6

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Effect of dominant negative (dn) STAT5 on growth of HMC-1 cells. HMC-1 cells were transduced with a dn STAT5 construct (MSCV-STAT5BΔ754-IRES-GFP) (gray bars) or with a GFP-labeled vector control (black bars) as described in the text. Then, mixtures of transfected and nontransfected HMC-1 cells were prepared and cultured for various time periods. The number (percentage) of GFP-positive cells (relative to all viable cells) was determined by flow cytometry after various time intervals as indicated. Results represent the mean ± SD of three experiments performed in parallel.

Effects of Piceatannol on Growth and Survival of Neoplastic Mast Cells

Since piceatannol has been described to block STAT5 activation,43 we were interested to learn whether this agent would interfere with growth of neoplastic mast cells. To address this question, HMC-1 cells were incubated with increasing concentrations of piceatannol. As visible in Figure 7A, piceatannol was found to inhibit growth of HMC-1 cells in a dose-dependent manner. In addition, we found that piceatannol dose-dependently increases the number of apoptotic cells and the number of active caspase 3-positive cells in both HMC-1 subclones (Figure 7, B and C), with clear drug effects seen at 50 and 100 μmol/L, doses reportedly blocking the activation of STAT5 completely.43 The apoptosis-inducing effect of piceatannol on HMC-1 cells could also be demonstrated by staining for active caspase-3. In particular, exposure of HMC-1 cells to piceatannol (50 or 100 μmol/L) was followed by a significant increase in the number of active caspase 3-positive cells, as compared with control medium (Figure 7C). As expected, piceatannol was also found to inhibit STAT5 DNA-binding activity in HMC-1 cells in EMSA experiments (not shown).

Figure 7.

Figure 7

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Effects of piceatannol on growth and viability of HMC-1 cells. HMC-1.1 cells (lacking KIT D816V) and HMC-1.2 cells (expressing KIT D816V) were incubated in control medium (co) or with various concentrations of piceatannol as indicated at 37°C for 48 hours (A) or 24 hours (B, C). A: To determine cell growth, cultured cells were subjected to MTT assay as described in the text. Results are expressed as percentage of control and represent the mean ± SD of four independent experiments. B: After incubation with piceatannol, the numbers (percentage) of apoptotic cells were determined by light microscopy. Results show the percentage of apoptotic cells and represent the mean ± SD of three independent experiments. C: Numbers (percentage) of active caspase 3-positive HMC-1.2 cells after incubation with various concentrations of piceatannol as indicated. Expression of active caspase 3 was determined by immunocytochemistry as described in the text. Results show the percentage of caspase 3-positive cells and represent the mean ± SD of three independent experiments. *P < 0.05.

Discussion

STAT5 has recently been implicated in malignant cell growth in various hematological neoplasms.23,24,25,26,27,45,46,47,48 It has also been described that leukemia-specific oncoproteins such as BCR/ABL induce activation of STAT5 and thereby contribute to malignant transformation.45,46,47,48 The results of our study show that neoplastic mast cells express nuclear and cytoplasmic pSTAT5 in a constitutive manner, and that the SM-related oncoprotein KIT D816V promotes STAT5 activation. Furthermore, our data show that a knock-down of pSTAT5 is associated with reduced growth of neoplastic mast cells. Together, these data suggest that the STAT5-pathway of signaling contributes to KIT D816V-dependent growth of mast cells in SM, which is in line with previous studies.15,29

Several different variants of SM have been described, including indolent SM, ASM, and MCL.1,2,3,4,5,6,7,8,9 The transforming KIT mutant D816V is detectable in a vast majority of patients, independent of the type of SM.10,11,12,13 In the present study, we found that pSTAT5 is expressed in neoplastic mast cells in all variants of SM, including high grade SM disorders, ie, ASM and MCL. Moreover, pSTAT5 was found to be expressed in virtually all neoplastic mast cells in BM mast cell infiltrates, independent of the variant of disease, and without the formation of STAT5-negative subclones. In addition, there was no correlation between pSTAT5 expression in mast cells and clinical symptoms recorded in patients with SM. These data are best explained by the fact that KIT D816V is a primary and strong trigger of STAT5 activation in neoplastic mast cells. In line with this assumption, we were able to show that the KIT D816V-positive mast cell line HMC-1 contains substantial amounts of pSTAT5.

Interestingly, pSTAT5 was not only detectable in KIT D816V-positive mast cells in SM, but was also found in mast cells in SM patients in whom KIT D816V was not expressed. In addition, we found that pSTAT5 is expressed in a KIT D816V-negative subclone of HMC-1 cells (HMC-1.1) known to display KIT V560G, although by flow cytometry, the levels of pSTAT5 were slightly lower in these cells, as compared with the HMC-1.2 subclone. These observations may be explained by the fact that not only KIT D816V, but also other KIT mutants such as KIT V560G in HMC-1.1 cells, can contribute to STAT5 activation in neoplastic mast cells. This observation is also in line with the notion that not only KIT D816V but also KIT V560G and also the corresponding murine Kit mutants, V559G and D814V, lead to constitutive phosphorylation of KIT, growth-factor independence, and tumorigenicity in mice.49,50,51 An alternative explanation for the detection of pSTAT5 in KIT D816V-negative neoplastic mast cells would be that other KIT-independent (pro-oncogenic) molecules and pathways can also trigger activation of STAT5 in neoplastic mast cells. The possibility that the KIT mutation was missed in the BM samples examined seems unlikely as the detection assays exhibit sufficient sensitivity and precision.12

The transforming capacity of STAT5 is considered to be associated primarily with its role as a nuclear transcription factor.23 Correspondingly, most studies have described nuclear expression of pSTAT5 in neoplastic cells.23,24,25 More recent data suggest, however, that pSTAT5 may also be expressed as functionally active molecule in the cytoplasm of neoplastic cells in myeloid leukemias.27 In the present study, we were able to show that pSTAT5 is expressed in both the cytoplasmic and nuclear compartment of neoplastic mast cells in all SM patients examined. This result seems to be in contrast to a recently published report by Zuluaga Toro et al.28 They reported that pSTAT5 is expressed primarily (selectively) in the nucleus of neoplastic mast cells.28 The discrepancy is best explained by the different staining protocols applied. In fact, whereas Zuluaga Toro et al only presented a “predominantly nuclear” staining protocol,28 we applied two different protocols, one for optimal visualization of nuclear STAT5, and a second conventional protocol for visualization of cytoplasmic STAT5. Using these two protocols in parallel, we found that STAT5 is localized in both cellular compartments. However, whereas the predominantly nuclear staining protocol revealed expression of pSTAT5 in both the nuclei and cytoplasm in neoplastic mast cells, the predominantly cytoplasmic staining protocol revealed cytoplasmic pSTAT5, but did not reveal expression of nuclear pSTAT5, which would argue for predominant expression of pSTAT5 in the cytoplasm. Indeed, as assessed by antibody-dilution experiments and Western blotting results obtained with purified nuclear and cytoplasmic extracts, cytoplasmic pSTAT5 levels clearly exceed nuclear pSTAT5 levels in neoplastic mast cells (HMC-1). The cytoplasmic localization of pSTAT5 in neoplastic mast cells would also be in line with the observation that pSTAT5 forms a signaling complex with PI3-kinase in myeloid leukemias27 and murine mast cells.29,52 Furthermore, it has been described that the activated form of KIT interacts physically with STAT5 and other STAT proteins during STAT-activation,29,52 which makes a selective nuclear expression of activated STAT5 unlikely.

The transforming KIT mutant D816V is considered essential for neoplastic cell growth in SM.14,15,16 In fact, KIT D816V provides ligand-independent autonomous phosphorylation of the receptor (KIT), and thus SCF-independent growth of cells.14 We were therefore interested to learn whether KIT D816V is involved in STAT5 activation in neoplastic mast cells. The results of our study show that KIT D816V leads to a substantial activation of STAT5 in Ba/F3 cells. Moreover, the KIT D816V-targeting drug midostaurin (PKC412) was found to down-regulate KIT D816V-induced STAT5 activation in Ba/F3 cells, as well as expression of pSTAT5 in HMC-1 cells. All in all, these data suggest that KIT D816V induces activation of STAT5 in neoplastic mast cells.

Another interesting aspect of the study was that pSTAT5 was not only detectable in neoplastic mast cells, but also in megakaryocytes and myeloid progenitor cells in the same tissue sections. This may reflect involvement of multiple myeloid lineages and stem cells in SM. Notably, it is well known that in SM, KIT D816V is not only expressed in mast cells but also in other myeloid cells and sometimes even in CD34+ stem cells.53,54 On the other hand, pSTAT5 was also found to be expressed in megakaryocytes and myeloid progenitors in the normal/reactive bone marrow. Therefore, we believe that STAT5 activation may also be triggered by physiological stimuli in these cells.

Recent data suggest that STAT5 may play an important role as a pro-oncogenic signaling molecule in myeloid neoplasms.23,24,25,26,27 In the present study, we asked whether activated STAT5 contributes to growth and survival of neoplastic mast cells, ie, HMC-1 cells. To address this question, a dominant negative (dn) STAT5 construct was applied. In these experiments, we were able to show that the dn-induced knock down of STAT5 is associated with reduced growth of neoplastic mast cells. These data suggest that pSTAT5 plays an important role in growth and survival of neoplastic mast cells in SM. The observation is also in line with the notion that pSTAT5 is a critical survival factor and KIT-downstream signaling molecule supporting the (SCF-mediated) survival of normal MC,19 and with the observation that STAT5 knock out mice display mast cell deficiency.20,21,22 Finally, we were able to show that piceatannol, a drug the reportedly blocks STAT5 activation,43 inhibits growth and survival of neoplastic mast cells. An interesting aspect is that piceatannol may act on several different targets in neoplastic (mast) cells, such as Syk, ZAP70, or STAT3. Notably, HMC-1 cells also display pSTAT3, and piceatannol blocks not only the expression of pSTAT5, but also expression of pSTAT3 and pSTAT3 DNA binding activity in HMC-1 cells (unpublished data). Whether STAT3 is indeed an additional relevant target promoting growth or survival in neoplastic mast cells in SM is currently under investigation.

Several KIT D816V-targeting drugs have recently been shown to counteract growth and survival of neoplastic mast cells.37,55,56,57,58,59 In the present study, we show that these drugs, ie, midostaurin and nilotinib, counteract expression of pSTAT5 in HMC-1 cells. These observations provide further evidence that KIT D816V is involved in STAT5 activation in neoplastic mast cells. However, neither PKC412 nor nilotinib are specific for KIT, but also interact and block other kinase targets in neoplastic cells. Likewise, PKC412 is known to inhibit mutant forms of PDGFR as well as FLT3 mutants. Although these targets are usually not expressed in neoplastic mast cells, and other key kinase targets that play a role in mast cell growth may not inhibited by PKC412, it was of importance to demonstrate the role of KIT D816V in STAT5 activation by another more direct approach, ie, be introducing a dn STAT5 construct. Indeed, the dn STAT5 construct was found to block the growth of neoplastic mast cells. Based on these data and data obtained with piceatannol, it is tempting to speculate that STAT5 is indeed a key regulator of growth of neoplastic mast cells and thus a novel interesting target in SM.

In summary, our data show that neoplastic mast cells in SM exhibit pSTAT5 in their cytoplasm and use this signaling molecule as an essential growth/survival factor. Our data also show that the KIT D816V-targeting drug midostaurin down-regulates pSTAT5 expression in neoplastic mast cells, and that the drug-induced or dn STAT5-induced knock down of STAT5 is associated with reduced growth of neoplastic mast cells. Targeting of pSTAT5 may be a novel interesting approach to counteract malignant cell growth in ASM and MCL.

Acknowledgments

We thank Harald Herrmann and Emir Hadzijusufovic for skillful technical assistance.

Footnotes

Address reprint requests to Peter Valent, M.D., Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna and Ludwig Boltzmann Cluster Oncology, Währinger Gürtel 18-20, A-1090 Vienna, Austria, E-mail: peter.valent@meduniwien.ac.at.

Supported by the Austrian Science Fund (FWF) grants #P21173-B13 and #F01820. R.M. was supported by the FWF grant SFB F28.

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