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PDGFRβ inhibits self-renewal and directs myelomonocytic differentiation of ES cells
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Leukemia Research 32 (2008) 1554–1564
Tel/PDGFR inhibits self-renewal and directs myelomonocytic differentiation of ES cells E. Dobbin a , P.M. Corrigan a , C.P. Walsh a , M.J. Welham b , R.W. Freeburn a,∗ , H. Wheadon a a
Biomedical Sciences Research Institute, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland, UK b Department of Pharmacy and Pharmacology and Centre for Regenerative Medicine, University of Bath, Bath, UK Received 9 January 2008; received in revised form 4 February 2008; accepted 9 February 2008 Available online 19 March 2008
Abstract The leukemic oncogene Tel/PDGFR, was inducibly expressed in embryonic stem (ES) cells and the phenotypic and molecular changes occurring during hematopoietic differentiation investigated. Expression of Tel/PDGFR resulted in an inability of ES cells to self-renew and caused a significant increase in myelopoiesis with a corresponding decrease in erythropoiesis. Analysis of gene expression patterns indicated a dramatic alteration in the levels of genes associated with self-renewal and differentiation, especially myelomonocytic genes in Tel/PDGFRexpressing cells. This study indicates Tel/PDGFR drives myelopoiesis by altering expression of genes involved in hematopoiesis and demonstrates the potential of this stem cell system to study oncogene-induced pathogenesis. © 2008 Elsevier Ltd. All rights reserved. Keywords: Leukemia; Tyrosine kinase; Tel/PDGFR; Embryonic stem cell; Gene expression
1. Introduction Myeloproliferative neoplasms (MPN) are clonal stem cell diseases characterised by excess proliferation of one or more myeloid lineages [1,2]. The pathogenesis of MPN often involves deregulation of a tyrosine kinase, with BCR/ABL involvement in chronic myelogenous leukemia being the best characterised example [3,4]. The discovery of the JAK2 V617F mutation in patients presenting with Polycythemia Vera, Essential Thrombocytopenia and Myelofibrosis has further enhanced our understanding of the molecular aetiology of these classic myeloproliferative diseases and highlighted the critical role tyrosine kinases play in both normal and abnormal hematopoiesis [5]. In addition to the classic MPN other myeloid malignancies, such as the myelodysplastic syndromes (MDS), share these common characteristics of being stem cell-derived clonal diseases, with activated signal transduction pathways due to mutation of tyrosine kinases, ∗
Corresponding author. Tel.: +44 28703 24606; fax: +44 28703 24375. E-mail address: [email protected] (R.W. Freeburn).
0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.02.007
often involving an acquired chromosomal translocation [1,6]. Recently, fusion genes involving the platelet-derived growth factor receptor beta (PDGFRβ) or platelet-derived growth factor receptor alpha (PDGFRα) genes have been associated with a subgroup of these disorders. PDGFR, a class III receptor tyrosine kinase, rearranges with several gene/chromosome partners and identifies a subgroup of hematological diseases originally classified by the World Health Organization (WHO) as myeloproliferative disorders/myelodysplastic syndromes (MPD/MDS) [1]. These malignancies constitute a heterogeneous group of disorders, where despite advances in identifying the molecular origin, the pathogenic events occurring downstream of these tyrosine kinases are still poorly characterised. The first PDGFR rearrangement was identified as a chromosomal translocation, t(5:12) (q33;p13) in a subset of chronic myelomonocytic leukemia (CMML) patients [7]. The genes involved were later cloned and shown to fuse a novel gene called Tel (ETV6) at 12p13 to the PDGFRβ gene at 5q33, resulting in a constitutively active chimeric protein, Tel/PDGFR [8]. Currently at least 15 different transloca-
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tions leading to the activation of the tyrosine kinase PBGFR have been described and in the recent revision to the WHO classification, PDGFR and FGFR1 rearranged myeloid neoplasms associated with eosinophilia are now considered a separate category among chronic myeloid disorders [2,9,10]. Expression of Tel/PDGFR in a murine bone marrow transplantation model induces a myeloproliferative disease that closely resembles the clinical symptoms of CMML in humans, implicating Tel/PDGFR as the pathological oncoprotein in this disorder [11]. Similarly, transfection with Tel/PDGFR can transform cell lines to cytokineindependent proliferation, with the tyrosine kinase activity of PDGFR playing an essential role in activating several major signaling pathways important for mitogenesis, such as the JAK-STAT, PI3-K and Ras-ERK pathways [12–15]. However, the contribution of these signals to the process of transformation is still not fully resolved and despite recent work highlighting the importance of STAT5 gene dosage to the severity of the disease, very few of the downstream targets regulated by PDGFR fusion proteins have been characterized [16]. In order to investigate in more detail the effect of Tel/PDGFR-induced molecular signals during hematopoiesis, we have used tetracycline (Tet)-regulated expression of Tel/PDGFR in embryonic stem (ES) cells [17]. The ability to manipulate ES cells to differentiate into various hematological lineages means they are an ideal model system to study the molecular and cellular mechanisms involved in cell fate and lineage determination. Hematopoietic differentiation is one of the best characterized systems in ES cells with studies involving both gene expression and progenitor cell analysis demonstrating similarities to both primitive and more definitive hematopoietic commitment during hematopoietic development in the mouse embryo [18,19]. The use of this system provides an ideal in vitro model to monitor the effects of expressing Tel/PDGFR not only in primitive stem cells with pluripotent potential but also during embryonic hematopoiesis. In this study we report that expressing Tel/PDGFR in ES cells reduces the ability of ES cells to self-renew and drives hematopoietic differentiation along the myelomonocytic lineage by altering the level of expression of key genes involved in hematopoietic regulation and myeloid lineage commitment.
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tion and screening of clones were performed as previously described [20]. For induction of expression of Tel/PDGFR, TP clones were washed 3× with PBS and incubated in LIFcontaining media in the presence of 500 ng/mL Tet to prevent the expression of Tel/PDGFR, or no Tet to induce the expression of Tel/PDGFR. 2.2. Hematopoietic differentiation Primary embryoid body (EB) formation was carried out using a single cell suspension of ES cells plated at 1 × 104 /mL in low adhesion Petri dishes in the following basal media; 1% methylcellulose (Sigma), 1× IMDM (Invitrogen Life Technologies), 100 g/mL holo-transferrin, 10 g/mL insulin, 10−4 M 2-Mecaptoethanol, 50 g/mL Ascorbic acid (Sigma), 15% FCS (Invitrogen Life Technologies) supplemented with the following growth factors to promote good mesodermal differentiation; 10 ng/mL BMP-4 (R&D systems Ltd., Abingdon, Oxfordshire, UK), 2 ng/mL Activin A, 10 ng/mL basic FGF (PeproTechTM , London, UK). Following 3.75 days in culture EBs were harvested, washed 3× with PBS and treated with 0.25% trypsin EDTA for 3 min. Cells were replated at 1 × 105 /mL in basal media supplemented with the following growth factors to promote hemangioblast formation; 100 ng/mL SCF, 10 ng/mL IL-6 and 10 ng/mL VEGF (PeproTechTM ). 4 days later EBs and blast-like colonies (BC) were harvested as outlined and a single cell suspension replated at 2.5 × 104 /mL in basal media supplemented with 1% BSA (Invitrogen Life Technologies) and cytokines to promote both myeloid and erythroid colony formation; 25 ng/mL GM-CSF, 25 ng/mL G-CSF, 10 ng/mL SCF, 10 ng/mL IL3 (PeproTechTM ) and 2 U/mL EPO (R&D Systems Ltd.). 7 days later hematopoietic colonies were scored using a Nikon Eclipse TS100 microscope and digital camera system. 2.3. Self-renewal assays
2. Methods
Parental and TP ES cells were plated at 3 × 103 –1 × 104 cells per gelatin-coated 60 mm tissue culture dish and cultured using 2% FBS, LIF (0, 20, 500 or 1000 U/mL) with and without Tet, as indicated. To detect alkaline phosphatase expression, cells were washed, fixed in methanol and then stained for 15 min with 1 mg/mL Fast RedTM TR salt (Sigma) dissolved in 0.1 M Tris pH 9.2 containing 200 g/mL Napthol AS-MX phosphate [20].
2.1. Cell culture and generation of transfectants
2.4. Proliferation, cell viability and apoptosis assays
E14tg2a murine ES cells expressing the Tet-sensitive transactivator, tTA [17] were routinely cultured on tissue culture plates (Nunc) coated with 0.1% (v/v) porcine gelatin (Sigma) as described previously [20]. 1 × 107 cells were electroporated at 800 mV/3.0 F with 10 g linearised response plasmid encoding the Tel/PDGFR (TP) oncogene. Selec-
XTT bioreduction assays were used to assess growth of TP clones, in media containing LIF with and without Tet, as previously described [15]. For cell growth curve analyses, cells were plated at 1 × 104 cells per 60 mm dish and counted in duplicate at defined times on a Vi-CELL XR counter (Beckman Coulter), to determine viability and cell
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number. Apoptosis was determined by flow cytometry using the stain DiOC6 as previously described [15]. 2.5. Short-term and long-term expression of Tel/PDGFRβ and cell lysates TP clones were plated at 1 × 105 –1 × 106 cells per gelatincoated 100 mm tissue culture dish and cultured with and without Tet for 24, 48 or 72 h to induce the expression of Tel/PDGFR. Cells were harvested and washed 3× with icecold PBS prior to being lysed as described previously [15]. Protein concentrations were determined using a Bio-Rad protein assay kit. 2.6. Immunoblotting and antibodies For immunoblotting, 20 g of each cell lysate were fractionated by SDS-PAGE and blotted onto nitrocellulose. Primary antibodies were used at the following dilutions: 0.1 g/mL anti-phosphotyrosine 4G10 (Upstate Biotechnology, 05–321); 0.5 g/mL PDGFR antibody, which recognizes Tel/PDGFR (Cell Signaling Technology CST 3162), 1:2000 anti-SHP-2 (Santa Cruz, sc-293). Secondary antibodies, conjugated to horseradish peroxidase (Dako) were used at 1:10,000 dilution and blots developed using ECL (Amersham Pharmacia). 2.7. PCR analyses Total RNA was prepared using RNAeasy Plus extraction kit (Qiagen). RNA (1 g) was reverse-transcribed using Superscript reverse transcriptase and oligo dT primers (Invitrogen Life Technologies). PCR was performed using 2 L of cDNA and standard conditions with gene specific primers (Supplementary Table 1). 2.8. Lightcycler real-time PCR analysis Relative quantification between cDNA samples was carried out using the Quantitect Sybr Green PCR kit (Qiagen) on a Lightcycler Instrument (Roche). Lightcycler reactions were performed in 20 L total reaction volume as per the manufacturer’s instructions. The LightCycler program for each gene was denaturation (94 ◦ C, 15 min): PCR amplification and quantification (95 ◦ C, 15 s; 60 ◦ C, 15 s; 72 ◦ C, 20 s), with a single fluorescence measurement at the end of each of the 40 cycles. A single Lightcycler run contained four log dilutions of each cDNA sample in triplicate, with the number of runs ≥3 for each sample to ensure consistently accurate quantification. Data were quantified using RelQuant LightCycler analysis software. Second-derivative maximum analysis, arithmetic baseline adjustment, and polynomial calculation methods were used for the quantification. A standard curve using a calibrator sample (pooled ES cell samples) was prepared for each housekeeping gene and target gene and used to create a coefficient file prior to sample analysis.
Inclusion of this calibrator in each Lightcycler run enabled normalized calibration with efficiency correction between runs. The ratio (arbitory units) between TBP and/or GusB and the target gene was used as a level of mRNA gene expression.
3. Results 3.1. Generation of transfectants inducibly expressing Tel/PDGFRβ In this study we have used a tetracycline-regulated expression system in ES cells to investigate the effects of the Tel/PDGFR (TP) oncogene in a stem cell model with both self-renewal and hematopoietic differentiation ability. Following Tel/PDGFR gene transfer into E14tg2a-tTA ES cells, 31 clones were found to express the oncogene and four were selected for further functional analysis. Fig. 1A shows the levels of Tel/PDGFR protein expressed in the different clones (top panel) and an increase in tyrosine phosphorylation of cellular proteins was seen proportional to the level of TP protein expressed (middle panel), indicating functional tyrosine kinase activity of the oncogene. 3.2. Tel/PDGFRβ expression reduces ES cell self-renewal A fundamental property of all stem cells is their ability to undergo self-renewal. Constitutive expression of BCRABL in murine ES cells increased self-renewal both in the presence and absence of LIF [21]. We therefore investigated whether expressing Tel/PDGFR in ES clones would alter their growth or differentiation patterns. Self-renewal was quantified using clonal analysis and staining for the presence of alkaline phosphatase, an enzyme expressed by undifferentiated ES cells. Uninduced TP clones (+Tet) cultured for 2 days in the presence of tetracycline and LIF maintained their pluripotency, with on average 88–90% of cells staining positive with Fast RedTM (Fig. 1B). The percentage of undifferentiated cells fell to between 68 and 70% following 3 days in culture. Induction of TP expression (−Tet) significantly reduced the ability of LIF to maintain self-renewal of ES cells with only 18% (TP1) and 40% (TP2) staining positive for alkaline phosphatase following 2 days in culture, with a further reduction to either 9% (TP1) or 23% (TP2) occurring by 3 days. Culturing parental E14tg2a-tTA cells +/− Tet had no significant effect on their self-renewal potential. The morphology and proliferation rates of cells expressing TP also changed. Cells proliferated slower and lost their characteristic three-dimensional ES cell structures, appearing flatter and more epithelial and fibroblast-like in character, highly suggestive of a more differentiated phenotype (data not shown). The effects on self-renewal observed appear to be directly related to the level of Tel/PDGFR expressed, with clones expressing lower levels (TP3 and TP4) showing on average only a 25% reduction in self-renewing capacity P > 0.005,
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n = 4 (Supplementary data). For the remainder of our experiments we concentrated on the TP1 clone, verifying all our results in the TP2 clone. 3.3. Tel/PDGFRβ decreases cell proliferation but not viability in the presence of LIF Reduced proliferation is a property observed when ES cells undergo differentiation, we therefore investigated the proliferation rates of ES cells cultured in LIF and induced to express TP using cell counts on a Vi-CELL XR counter. There was a marked reduction in cell numbers in the population expressing Tel/PDGFR compared to controls (Fig. 2A). In order to determine whether cells were still viable and not apoptotic we measured their mitochondrial membrane integrity using DiOC6 and flow cytometry. The viability of ES cells expressing TP was 74.7 ± 6.3%, n = 4 following 72 h culture in the presence of LIF, with no significant differences observed to non-induced TP clones 75.9 ± 12.4%, n = 4 (Fig. 2B) or parental cells 72.2 ± 6.9%, n = 4 (+Tet) and 70.0 ± 9.9%, n = 4 (−Tet). 3.4. TP-induced differentiation of ES cells is enhanced by LIF removal
Fig. 1. Inducible expression of TEL/PDGFR reduces ES cell selfrenewal. (A) Variable levels of TEL/PDGFR protein in different clones. Immunoblotting analysis with protein extracts from parental ES cells and stable clones containing the TEL/PDGFR oncogene (TP1-4) after incubation for 24 h in the presence (+) or absence (−) of tetracycline (Tet). Upper panel: Immunoblotting (IB) with a polyclonal antibody that recognizes the TEL/PDGFR fusion; middle panel: an antibody which detects tyrosine-phosphorylated proteins (pTyr); lower panel: antibody that detects SHP2 (loading control). (B) Self-renewal decreases in the presence of high levels of the oncoprotein. Clones TP1 & TP2 were incubated for 2–3 days in 1000 U/mL LIF, +/− Tet. The percentage of cells positive for alkaline phosphatase is given (mean ± S.E.M.s, n = 4). Asterisks denote P < 0.005 by paired Student’s t-test. (C) Representative immunoblot using protein from cells depicted in panel B, showing TEL/PDGFR expression levels.
Our previous study, using Ba/F3 cells, demonstrated that dual signalling via Tel/PDGFR and IL-3 had a detrimental effect on cell proliferation and survival [15]. Therefore, we were interested to investigate whether LIF signalling, in conjunction with Tel/PDGFR contributed to the loss of ES pluripotency observed upon expression of Tel/PDGFR. Self-renewal assays were carried out in the absence of LIF or in the presence of 20, 500 or 1000 U/mL LIF. Fewer TP cells stained positive for alkaline phosphatase in the lower doses of LIF compared to the optimal concentration of 1000 U/mL (Fig. 2C) with no cells staining positive following 24 h of LIF removal (data not shown). These results indicate that unlike constitutive expression of BCR-ABL in ES cells, which promoted self-renewal both in the presence and absence of LIF by activating the Jak/STAT3 pathway [21], inducible expression of Tel/PDGFR is unable to maintain ES cell pluripotency, and appears to promote the differentiation potential of ES cells, with the removal of LIF enhancing this process. This data indicates that expressing Tel/PDGFR in ES cells activates signaling pathways, which promote ES cell differentiation with LIF signaling delaying this process. 3.5. Tel/PDGFRβ alters expression of regulatory genes involved in hematopoiesis To ascertain the mechanism by which Tel/PDGFR promotes the differentiation of ES cells, Tel/PDGFR was expressed over a 72-h period and RNA extracted from the TP and control cells. Semi-quantitative PCR (Fig. 3A) was used to analyse gene expression (for primer sequences see Supplementary tables) and the expression levels of certain
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Fig. 2. Inducible expression of TEL/PDGFR reduces ES cell proliferation and self-renewal, but not viability. (A) Reduced proliferation in the presence of the oncoprotein. Cell counts for TP1 clone following tetracycline removal (+TP expression) or in the presence of tetracycline (−TP expression), following culture for 0–60 h in 1000 U/mL LIF is given (mean ± S.E.M.s, n = 3). Results were verified using TP2 clone (data not shown). (B) Apoptosis is not increased in cells expressing the TEL/PDGFR oncogene. TP clones were cultured for 72 h in 1000 U/mL LIF, stained with 10 nM Di0C6 and fluorescence measured by flow cytometry. The average percentage of viable cells for each condition is given (mean ± S.E.M.s, n = 4). (C) Self-renewal decreases in induced cells following LIF removal. Alkaline phosphatase staining of TP1 clone +/− TP expression at 72 h in the presence of different amounts of LIF. The percentage of cells positive for AP is given (mean ± S.E.M.s, n = 3). Asterisk denotes P < 0.01 and cross denotes P < 0.005 by paired Student’s t-test.
genes confirmed by real-time PCR (Fig. 3B). Initially, genes known to be involved in ES cell self-renewal and those altered during the early stages of ES cell differentiation were analyzed (Fig. 3A, panel i). Decreased expression of the self-renewal marker Nanog, was observed and could be a contributing factor to the loss of self-renewal observed upon expression of TP. An increase in the mesodermal gene Brachyury suggests that differentiation towards mesoderm formation maybe being enhanced. However no change was seen in the self-renewal marker Oct4, or the mesodermal markers associated with muscle (MyoD1) and endothelial/hematopoietic differentiation (BMP4), suggesting TP is not inducing global differentiation of ES cells into mesoderm during this short-time frame. Several important genes associated with hematopoietic stem cell formation were also up-regulated, including Sca-1, HoxB4 and CDX4, following TP expression (Fig. 3A, panel ii). The notable exception being c-kit, the receptor for stem cell factor, which was down-regulated by TP expression, however these findings are supported by a previous study, which indicates that c-kit is highly expressed by ES cells and becomes down-regulated following differentiation induction [22]. Examination of genes associated with committed hematopoietic progenitors showed the greatest differences (panel iii). Expression levels of the genes BMP2, GATA2, GATA3, and to a lesser extent GATA4, were much higher following TP induction. The erythroid specific factor GATA1 was not affected, although interestingly its cofactor Fog1 did increase with TP expression. Analysis of hematopoietic lineage-specific genes showed induction of the embryonic globin chain (Hbb-bH1) by 72 h, indicating TP is driving primitive hematopoietic differentiation. However, no expression of the more definitive erythroid lineage genes Hbb-b1, KLF1 and EPO R was seen (data not shown). The myeloid transcription factor MafB showed a slight increase with TP expression but no expression was seen for PU.1, which directs differentiation towards the common lymphoid–myeloid progenitor (Fig. 3A panel iv). Several genes important for monocytic development, Egr-1, Egr-2 and Nab2 were also up-regulated following TP induction. Quantitative analysis of HoxB4, GATA2 and MafB expression confirmed the accuracy of the changes identified during the semi-quantitative analysis (Fig. 3B). Overall results from the gene expression analyse strongly suggest that in this system, Tel/PDGFR expression has the maximum impact on genes associated with initiating hematopoietic differentiation with a clear induction of genes linked to early myeloid and monocytic development. 3.6. Tel/PDGFRβ promotes myeloid differentiation of ES cells Results from the gene expression analysis indicate Tel/PDGFR may influence several stages of hematopoietic differentiation. To investigate the events involved in leukemic transformation by this oncogene we expressed TP throughout the hematopoietic differentiation of ES cells using a modi-
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Fig. 3. Induction of Tel/PDGFR in ES cells alters gene expression towards hematopoietic differentiation, primarily the myeloid lineage. (A) RT-PCR analysis of mRNA levels for TP1 clone cultured +/− TP expression for 24, 48, and 72 h. The housekeeping genes Gus-B and TBP were used as controls. Panel (i) Genes associated with self-renewal and differentiation status of ES cells. (ii) Genes involved in early hematopoietic regulation. (iii) Genes associated with hematopoiesis. (iv) Genes associated with lineage restriction. (B) Changes to expression levels seen by RT-PCR were confirmed for selected genes by quantitative PCR using a Lightcycler. Representative gel images are shown for each gene n = 3.
fied three-stage differentiation approach (Fig. 4A, panel i). This system is based on the original work of Keller et al. [18] and uses a wide range of cytokines (EPO, G-CSF, GM-CSF, IL-3, SCF) during the final stage of differentiation to induce both myeloid and erythroid lineage commitment. ES cells expressing Tel/PDGFR were capable of undergoing all three stages of the differentiation process. However, differences were observed in the first round of embryoid body formation and at the final stage of hematopoietic colony formation.
There was on average a 33% reduction in total cell number in the primary EB cultures induced to express TP compared to the control cultures. The average area of these EBs when measured using Nikon NIS-Elements imaging software, was also decreased (Fig. 4A, ii). However, when used in the second phase of differentiation these cells efficiently formed both secondary EBs and blast-like colonies (BC), comparable to control cultures (−TP) (Fig. 4A, iii). BC are diffuse clusters of cells, which contain hemangioblasts [23,24], early precur-
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Fig. 4. Tel/PDGFR increases myeloid differentiation of ES cells. (A) (i) Schematic diagram of the three stages involved in the hematopoietic differentiation of ES cells. The time-scales involved in the formation of primary embryoid bodies (1◦ EB), secondary embryoid bodies/blast-like colonies (2◦ EB/BC) and hematopoietic colonies (Haem) are indicated and represent when morphological changes and RNA samples were analysed. (ii) Expressing Tel/PDGFR reduces the size of primary embryoid bodies. The areas of the primary embryoid bodies were measured following the first round of differentiation. 100 embryoid body measurements were taken from three independent experiments. (iii) Photographs of representative primary embryoid bodies, secondary embryoid bodies/blastlike colonies, taken at 10× magnification. Scale bar equals 100 m. (iv) Percentage of each type of hematopoietic colony formed by day 15 of differentiation with and without induction of Tel/PDGFR expression. Colonies were defined as follows; myeloid (including macrophage and granulocyte/macrophage colonies), erythroid or mixed lineage (including erythroid-macrophage colonies and multi-lineage colonies). Data represents 500 hematopoietic colonies scored per experiment, for each condition (±S.E.M.s, n = 3). (v) Photographs of representative hematopoietic colonies, at 10× magnification, scale as above.
sor cells with both endothelial and hematopoietic potential, that have been shown to be involved in establishing definitive hematopoiesis in the mouse embryo [25]. Cells from second phase cultures were then assessed for their ability to generate mature hematopoietic cells using hematopoietic colony assays, supplemented with cytokines to enhance both myelopoiesis and erythropoiesis. Here we observed a significant increase in myeloid colony formation, with cultures expressing the oncogene (+TP) producing on average 20% more myeloid colonies compared to control cells (−TP). Colony size also altered with the average area of the colonies being six times larger (16,970 m2 −TP compared to 113,000 m2 +TP, n = 20). The ability of TP-expressing cells to form erythroid and mixed lineage colonies also diminished with, on average, a 41% reduction in erythroid colonies and a 66% reduction in mixed colonies being observed in the
cultures. The erythroid colonies were considerably smaller in area than those observed in the control cultures (15,362 m2 −TP compared to 7438 m2 +TP, n = 20), with no difference in the size of the mixed colonies being observed (Fig. 4A, iv and v). These results indicate that TP drives myelopoiesis and suppresses erythropoiesis. 3.7. Tel/PDGFRβ-induced changes in gene expression during hematopoietic differentiation of ES cells To examine alterations in the hematopoietic gene expression profile during lineage commitment, RNA was extracted from cells either induced to express Tel/PDGFR (+TP, no tet in culture) or without Tel/PDGFR expression (−TP, tet present in culture) during each phase of hematopoietic differentiation. As seen previously, Nanog expression was low
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Fig. 5. Analysis of gene changes during hematopoietic differentiation of ES cells +/− TP expression. (A) RT-PCR analysis of mRNA levels for TP1 clone cultured +/− TP expression during hematopoietic differentiation system, samples taken at the time points indicated in Fig. 4A (i). Panel (i) Genes associated with self-renewal and differentiation status of ES cells. (ii) Genes involved in early hematopoietic regulation. (iii) Genes associated with hematopoiesis. (iv) Genes associated with lineage restriction. (B) Changes to expression levels seen by RT-PCR were confirmed for selected genes by quantitative PCR using a Lightcycler. Representative gel images are shown for each gene n = 3.
in the TP expressing cells during the initial stages of differentiation (Fig. 5A panel i, 24 h and 1◦ EB) and this low level of expression was sustained into the blast-like colony stage (2◦ BC) unlike control cells (−TP) where it was no longer detected by this stage. Similarly sustained expression of both Oct4 and Brachyury were also seen at this later 2◦ BC stage in the TP samples compared to controls. A modest increase in HoxB4 expression was seen at all stages following TP expression and confirmed by quantitative PCR (Fig. 5A, ii and b) whereas other stem cell markers such as, c-kit was reduced at the early stages (24 h and 1◦ EB) but increased
at the later stages (2◦ BC and Haem) and Sca-1 decreased at the hematopoietic colony stage. The regulatory genes involved in early hematopoietic commitment showed the greatest changes especially during the earlier stages of differentiation (24 h and 1◦ EB) with GATA2 expression increased and GATA1/Fog1 decreased in TP cells, suggesting an early bias towards myeloid differentiation following induction of TP (Fig. 5A, iii and B). Subtle changes were also seen in genes associated with specific lineage commitment. The erythroid genes KLF1 and EPO R showed a slight decrease in expression at the 2◦ BC
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stage, whereas, the key myeloid regulator PU.1 was upregulated favouring monopoiesis over granulopoiesis [26]. The monocytic genes Egr1, Egr2 and Nab2 were also increased especially very early during the differentiation and at the final hematopoietic colony stage when TP was expressed (Fig. 5A, iv). Surprisingly, the myelomoncytic regulator MafB appears to be up-regulated at the early stages of differentiation by TP then down-regulated by the oncogene at later stages of differentiation. This gene is normally expressed during the later but not early stages of monocytic development [27], a progression, which was deregulated by expressing TP. One interesting observation from this gene expression profiling is the inverse relationship between expression levels seen at 24 h and the hematopoietic colony stages for certain genes. Genes up-regulated within 24 h of TP expression (HoxB4, Sca1, GATA2, MafB) appear to be down-regulated (or unaltered) by the hemopoietic colony stage, and viceversa (Nanog, Oct4, brachyury, c-kit) suggesting that the context and phase of gene expression is altered during differentiation by expressing Tel/PDGFR in stem cells. Overall this data confirms that this activated tyrosine kinase fusion protein activates transcription factors and genes important for driving myelopoiesis.
4. Discussion The receptor tyrosine kinase PDGFR rearranges with several oncogenic partners to act as a key pathogenic factor in a subgroup of hematological diseases with both myeloproliferative and myelodysplastic features [1,2,9,10]. In vivo studies support a role for Tel/PDGFR, in the generation of human and murine leukemia, but the direct mechanisms by which this oncogene manipulates the self-renewal, proliferation and differentiation decisions made by stem cells are still not clearly defined [11]. To investigate TP-induced functional responses, we have used tetracycline-regulated expression in ES cells to control the expression of TP in pluripotent primitive stem cells with hematopoietic potential. Overall, our investigations revealed that TP is sufficient to change the growth and differentiation properties of primitive ES cells and enhance their myeloid differentiation by altering the balance of key developmental genes. We demonstrate that unlike BCR-ABL, which promotes the self-renewal of ES cells [21], expressing TP resulted in an increase in the differentiation of ES cells. The level of expression of the oncogene played a critical role in promoting differentiation while the removal of LIF the key cytokine involved in ES cell self-renewal, resulted in the TP-expressing cells differentiating faster, indicating that this effect was TP-mediated and not due to combined signaling. Analysis of gene expression changes induced by TP expression showed major changes in the balance between genes associated with ES cell self-renewal and those involved in hematopoietic differentiation. Expression of TP for up to 72 h
was able to diminish Nanog expression while increasing the expression of the differentiation marker Brachyury, suggesting a rapid initiation of differentiation when TP is expressed. Nanog has been shown in conjunction with other transcription factors to maintain pluripotency through repression of differentiation programs [28,29]. Interestingly, while Nanog is initially down-regulated after TP expression, its expression, along with Oct4 and Brachyury, appears to be sustained by TP during the blast-like and hemopoietic colony stages of hematopoietic differentiation. This suggests that in addition to promoting the initial differentiation of ES cells, TP may also maintain a population of cells with self-renewal potential in the later stages of hematopoietic development, akin to the clonal ‘leukemic’ stem cells with abnormal tyrosine kinase activity identified in many MPN [3–6]. These results indicate that unlike BCR-ABL, Tel/PDGFR exerts its effects in a later stem cell/progenitor population and could explain why, bone marrow cells retrovirally transformed with Tel/PDGFR are unable to induce disease when transplanted into secondary recipients [11,30]. There appears to be a critical balance between the levels of different transcription factors, which determines the stem cells decision either to self-renew or differentiate. The overall effect of the signals activated by TP was a loss in ES cell pluripotency and an increase in differentiation, highlighted by a reduction in alkaline phosphatase staining and levels of self-renewal genes. This was accompanied by a rapid and dramatic increase in the expression of genes associated with early hematopoietic differentiation (Sca1, HoxB4, CDX4) and hematopoietic regulators (BMP-2, GATA2, 3, 4, Fog1). Increased HoxB4 and CDX4 expression are known to enhance hematopoiesis during ES cell differentiation [31] while over-expression of GATA-2 and differentiation analysis using GATA2 null ES cells, indicates that it plays a central role in the proliferation and survival of immature hematopoietic progenitors [32,33]. Combined with the induction of genes associated with monocytic development (Egr1, 2, Nab2) [27], these findings provide compelling evidence for a shift in the balance of the transcription machinery towards myelomonocytic differentiation when TP is expressed. Tel/PDGFR was also able to influence the differentiation of ES cells throughout in vitro hematopoietic development, with the most notable physiological differences being observed at the initial and final stages of the differentiation process. Cells expressing the oncogene were able to form primary embryoid bodies but they were smaller compared to control colonies. This could either be due to a reduction in the overall proliferation or through an incomplete formation of all three germ layers, and although slight reductions in expression of Nanog, Brachyury and Flk-1 were seen when TP was expressed at this stage, further studies are required to determine the exact mechanism by which TP is mediating its effects during this phase of the differentiation process. Overall TP expression resulted in enhanced myelopoiesis and suppressed erythropoiesis, reflected in both
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the increased number (20% increase) and size (6 times larger) of myeloid colonies seen at the hematopoietic colony stage. These findings indicate that expressing the oncogene alters the balance of key transcription factors involved in erythroid and myeloid development. It is clear that transcription factors work as intricate networks often dependent on each other as well as cofactor expression for lineage restriction of the cell [34]. Although many transcription factors are regarded as lineage specific they are frequently expressed at lower levels in uncommitted hematopoietic progenitors with changes in their levels often being sufficient to cause changes in lineage commitment [35]. Interactions between the later more lineage-specific transcription factors also plays an important role in determining the fate of hematopoietic cells [34] and results from this study show that changes mediated by TP are likely to alter lineage commitment. TP induction during hematopoietic differentiation caused a reduction in gene expression of the erythroid regulator GATA1, its cofactor Fog1 and also the erythropoietin receptor (EPO R) at the primary embryoid body and/or blast-like colony stages, consistent with the decreased number of erythroid colonies seen at the final hematopoietic colony stage. Interestingly, in a transformed myelomonocytic cell line, forced over expression of GATA1 suppressed the myelomonocytic phenotype converting them into cells resembling erythroblasts while lower level of GATA1 converted the cells to an eosinophil-like phenotype [35]. Given that Fog1 is downregulated following induction of eosinophil differentiation [36] while increased expression of GATA2 is known to block erythroid differentiation in vitro [32,33], it is clear that TP shifts transcription factor expression to very much favour myelomonocytic and even eosinophilic differentiation. Additional work to further analyse the altered balance between these factors combined with changes to the differentiation system to promote the eosinophil lineage, such as addition of IL-5 to the hematopoietic colony stage, might provide valuable insight into the eosinophilia often associated with PDGFR fusion genes [37,38]. In summary, this study reports the establishment of a stem cell model to inducibly express the leukemic oncogene Tel/PDGFR. Expressing Tel/PDGFR rapidly induced differentiation towards hematopoietic precursors and favoured the establishment of myeloid in preference to erythroid lineages. Tel/PDGFR initially induced these changes by decreasing the levels of transcription factors associated with pluripotency while increasing the expression of factors known to increase hematopoietic stem cell proliferation. Hematopoietic lineage commitment genes were also altered resulting in TP increasing myeloid cell production during in vitro hematopoietic differentiation. This work demonstrates that this system can be used as a powerful tool to study the molecular changes induced by leukemic oncogenes, allowing changes directly attributed to expression of the oncogene to be examined not only during the initiating stages but also during the more definitive stages of hematopoiesis.
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Acknowledgements This study was supported by the Medical Research Council, Department of Education and Learning and Northern Ireland Leukaemia Research Fund. We would like to thank the following people: Dr. M. Carroll, University of Pennsylvania, for the pcCDN3 Tel/PDGFR construct and Dr. O. Witte, UCLA for the E14tg2a mouse embryonic stem cells expressing the Tetsensitive transactivator, tTA. Contributions. Ms. Dobbin and Mrs. Corrigan performed experiments and data analysis. Prof. Welham and Dr. Walsh provided vital reagents, support and advice during the project. Dr. Freeburn performed some of the experiments and helped prepare the manuscript. Dr. Wheadon designed and coordinated the experiments and helped prepare the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. leukres.2008.02.007.
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Tel/PDGFR inhibits self-renewal and directs myelomonocytic differentiation of ES cells E. Dobbin a , P.M. Corrigan a , C.P. Walsh a , M.J. Welham b , R.W. Freeburn a,∗ , H. Wheadon a a
Biomedical Sciences Research Institute, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland, UK b Department of Pharmacy and Pharmacology and Centre for Regenerative Medicine, University of Bath, Bath, UK Received 9 January 2008; received in revised form 4 February 2008; accepted 9 February 2008 Available online 19 March 2008
Abstract The leukemic oncogene Tel/PDGFR, was inducibly expressed in embryonic stem (ES) cells and the phenotypic and molecular changes occurring during hematopoietic differentiation investigated. Expression of Tel/PDGFR resulted in an inability of ES cells to self-renew and caused a significant increase in myelopoiesis with a corresponding decrease in erythropoiesis. Analysis of gene expression patterns indicated a dramatic alteration in the levels of genes associated with self-renewal and differentiation, especially myelomonocytic genes in Tel/PDGFRexpressing cells. This study indicates Tel/PDGFR drives myelopoiesis by altering expression of genes involved in hematopoiesis and demonstrates the potential of this stem cell system to study oncogene-induced pathogenesis. © 2008 Elsevier Ltd. All rights reserved. Keywords: Leukemia; Tyrosine kinase; Tel/PDGFR; Embryonic stem cell; Gene expression
1. Introduction Myeloproliferative neoplasms (MPN) are clonal stem cell diseases characterised by excess proliferation of one or more myeloid lineages [1,2]. The pathogenesis of MPN often involves deregulation of a tyrosine kinase, with BCR/ABL involvement in chronic myelogenous leukemia being the best characterised example [3,4]. The discovery of the JAK2 V617F mutation in patients presenting with Polycythemia Vera, Essential Thrombocytopenia and Myelofibrosis has further enhanced our understanding of the molecular aetiology of these classic myeloproliferative diseases and highlighted the critical role tyrosine kinases play in both normal and abnormal hematopoiesis [5]. In addition to the classic MPN other myeloid malignancies, such as the myelodysplastic syndromes (MDS), share these common characteristics of being stem cell-derived clonal diseases, with activated signal transduction pathways due to mutation of tyrosine kinases, ∗
Corresponding author. Tel.: +44 28703 24606; fax: +44 28703 24375. E-mail address: [email protected] (R.W. Freeburn).
0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.02.007
often involving an acquired chromosomal translocation [1,6]. Recently, fusion genes involving the platelet-derived growth factor receptor beta (PDGFRβ) or platelet-derived growth factor receptor alpha (PDGFRα) genes have been associated with a subgroup of these disorders. PDGFR, a class III receptor tyrosine kinase, rearranges with several gene/chromosome partners and identifies a subgroup of hematological diseases originally classified by the World Health Organization (WHO) as myeloproliferative disorders/myelodysplastic syndromes (MPD/MDS) [1]. These malignancies constitute a heterogeneous group of disorders, where despite advances in identifying the molecular origin, the pathogenic events occurring downstream of these tyrosine kinases are still poorly characterised. The first PDGFR rearrangement was identified as a chromosomal translocation, t(5:12) (q33;p13) in a subset of chronic myelomonocytic leukemia (CMML) patients [7]. The genes involved were later cloned and shown to fuse a novel gene called Tel (ETV6) at 12p13 to the PDGFRβ gene at 5q33, resulting in a constitutively active chimeric protein, Tel/PDGFR [8]. Currently at least 15 different transloca-
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tions leading to the activation of the tyrosine kinase PBGFR have been described and in the recent revision to the WHO classification, PDGFR and FGFR1 rearranged myeloid neoplasms associated with eosinophilia are now considered a separate category among chronic myeloid disorders [2,9,10]. Expression of Tel/PDGFR in a murine bone marrow transplantation model induces a myeloproliferative disease that closely resembles the clinical symptoms of CMML in humans, implicating Tel/PDGFR as the pathological oncoprotein in this disorder [11]. Similarly, transfection with Tel/PDGFR can transform cell lines to cytokineindependent proliferation, with the tyrosine kinase activity of PDGFR playing an essential role in activating several major signaling pathways important for mitogenesis, such as the JAK-STAT, PI3-K and Ras-ERK pathways [12–15]. However, the contribution of these signals to the process of transformation is still not fully resolved and despite recent work highlighting the importance of STAT5 gene dosage to the severity of the disease, very few of the downstream targets regulated by PDGFR fusion proteins have been characterized [16]. In order to investigate in more detail the effect of Tel/PDGFR-induced molecular signals during hematopoiesis, we have used tetracycline (Tet)-regulated expression of Tel/PDGFR in embryonic stem (ES) cells [17]. The ability to manipulate ES cells to differentiate into various hematological lineages means they are an ideal model system to study the molecular and cellular mechanisms involved in cell fate and lineage determination. Hematopoietic differentiation is one of the best characterized systems in ES cells with studies involving both gene expression and progenitor cell analysis demonstrating similarities to both primitive and more definitive hematopoietic commitment during hematopoietic development in the mouse embryo [18,19]. The use of this system provides an ideal in vitro model to monitor the effects of expressing Tel/PDGFR not only in primitive stem cells with pluripotent potential but also during embryonic hematopoiesis. In this study we report that expressing Tel/PDGFR in ES cells reduces the ability of ES cells to self-renew and drives hematopoietic differentiation along the myelomonocytic lineage by altering the level of expression of key genes involved in hematopoietic regulation and myeloid lineage commitment.
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tion and screening of clones were performed as previously described [20]. For induction of expression of Tel/PDGFR, TP clones were washed 3× with PBS and incubated in LIFcontaining media in the presence of 500 ng/mL Tet to prevent the expression of Tel/PDGFR, or no Tet to induce the expression of Tel/PDGFR. 2.2. Hematopoietic differentiation Primary embryoid body (EB) formation was carried out using a single cell suspension of ES cells plated at 1 × 104 /mL in low adhesion Petri dishes in the following basal media; 1% methylcellulose (Sigma), 1× IMDM (Invitrogen Life Technologies), 100 g/mL holo-transferrin, 10 g/mL insulin, 10−4 M 2-Mecaptoethanol, 50 g/mL Ascorbic acid (Sigma), 15% FCS (Invitrogen Life Technologies) supplemented with the following growth factors to promote good mesodermal differentiation; 10 ng/mL BMP-4 (R&D systems Ltd., Abingdon, Oxfordshire, UK), 2 ng/mL Activin A, 10 ng/mL basic FGF (PeproTechTM , London, UK). Following 3.75 days in culture EBs were harvested, washed 3× with PBS and treated with 0.25% trypsin EDTA for 3 min. Cells were replated at 1 × 105 /mL in basal media supplemented with the following growth factors to promote hemangioblast formation; 100 ng/mL SCF, 10 ng/mL IL-6 and 10 ng/mL VEGF (PeproTechTM ). 4 days later EBs and blast-like colonies (BC) were harvested as outlined and a single cell suspension replated at 2.5 × 104 /mL in basal media supplemented with 1% BSA (Invitrogen Life Technologies) and cytokines to promote both myeloid and erythroid colony formation; 25 ng/mL GM-CSF, 25 ng/mL G-CSF, 10 ng/mL SCF, 10 ng/mL IL3 (PeproTechTM ) and 2 U/mL EPO (R&D Systems Ltd.). 7 days later hematopoietic colonies were scored using a Nikon Eclipse TS100 microscope and digital camera system. 2.3. Self-renewal assays
2. Methods
Parental and TP ES cells were plated at 3 × 103 –1 × 104 cells per gelatin-coated 60 mm tissue culture dish and cultured using 2% FBS, LIF (0, 20, 500 or 1000 U/mL) with and without Tet, as indicated. To detect alkaline phosphatase expression, cells were washed, fixed in methanol and then stained for 15 min with 1 mg/mL Fast RedTM TR salt (Sigma) dissolved in 0.1 M Tris pH 9.2 containing 200 g/mL Napthol AS-MX phosphate [20].
2.1. Cell culture and generation of transfectants
2.4. Proliferation, cell viability and apoptosis assays
E14tg2a murine ES cells expressing the Tet-sensitive transactivator, tTA [17] were routinely cultured on tissue culture plates (Nunc) coated with 0.1% (v/v) porcine gelatin (Sigma) as described previously [20]. 1 × 107 cells were electroporated at 800 mV/3.0 F with 10 g linearised response plasmid encoding the Tel/PDGFR (TP) oncogene. Selec-
XTT bioreduction assays were used to assess growth of TP clones, in media containing LIF with and without Tet, as previously described [15]. For cell growth curve analyses, cells were plated at 1 × 104 cells per 60 mm dish and counted in duplicate at defined times on a Vi-CELL XR counter (Beckman Coulter), to determine viability and cell
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number. Apoptosis was determined by flow cytometry using the stain DiOC6 as previously described [15]. 2.5. Short-term and long-term expression of Tel/PDGFRβ and cell lysates TP clones were plated at 1 × 105 –1 × 106 cells per gelatincoated 100 mm tissue culture dish and cultured with and without Tet for 24, 48 or 72 h to induce the expression of Tel/PDGFR. Cells were harvested and washed 3× with icecold PBS prior to being lysed as described previously [15]. Protein concentrations were determined using a Bio-Rad protein assay kit. 2.6. Immunoblotting and antibodies For immunoblotting, 20 g of each cell lysate were fractionated by SDS-PAGE and blotted onto nitrocellulose. Primary antibodies were used at the following dilutions: 0.1 g/mL anti-phosphotyrosine 4G10 (Upstate Biotechnology, 05–321); 0.5 g/mL PDGFR antibody, which recognizes Tel/PDGFR (Cell Signaling Technology CST 3162), 1:2000 anti-SHP-2 (Santa Cruz, sc-293). Secondary antibodies, conjugated to horseradish peroxidase (Dako) were used at 1:10,000 dilution and blots developed using ECL (Amersham Pharmacia). 2.7. PCR analyses Total RNA was prepared using RNAeasy Plus extraction kit (Qiagen). RNA (1 g) was reverse-transcribed using Superscript reverse transcriptase and oligo dT primers (Invitrogen Life Technologies). PCR was performed using 2 L of cDNA and standard conditions with gene specific primers (Supplementary Table 1). 2.8. Lightcycler real-time PCR analysis Relative quantification between cDNA samples was carried out using the Quantitect Sybr Green PCR kit (Qiagen) on a Lightcycler Instrument (Roche). Lightcycler reactions were performed in 20 L total reaction volume as per the manufacturer’s instructions. The LightCycler program for each gene was denaturation (94 ◦ C, 15 min): PCR amplification and quantification (95 ◦ C, 15 s; 60 ◦ C, 15 s; 72 ◦ C, 20 s), with a single fluorescence measurement at the end of each of the 40 cycles. A single Lightcycler run contained four log dilutions of each cDNA sample in triplicate, with the number of runs ≥3 for each sample to ensure consistently accurate quantification. Data were quantified using RelQuant LightCycler analysis software. Second-derivative maximum analysis, arithmetic baseline adjustment, and polynomial calculation methods were used for the quantification. A standard curve using a calibrator sample (pooled ES cell samples) was prepared for each housekeeping gene and target gene and used to create a coefficient file prior to sample analysis.
Inclusion of this calibrator in each Lightcycler run enabled normalized calibration with efficiency correction between runs. The ratio (arbitory units) between TBP and/or GusB and the target gene was used as a level of mRNA gene expression.
3. Results 3.1. Generation of transfectants inducibly expressing Tel/PDGFRβ In this study we have used a tetracycline-regulated expression system in ES cells to investigate the effects of the Tel/PDGFR (TP) oncogene in a stem cell model with both self-renewal and hematopoietic differentiation ability. Following Tel/PDGFR gene transfer into E14tg2a-tTA ES cells, 31 clones were found to express the oncogene and four were selected for further functional analysis. Fig. 1A shows the levels of Tel/PDGFR protein expressed in the different clones (top panel) and an increase in tyrosine phosphorylation of cellular proteins was seen proportional to the level of TP protein expressed (middle panel), indicating functional tyrosine kinase activity of the oncogene. 3.2. Tel/PDGFRβ expression reduces ES cell self-renewal A fundamental property of all stem cells is their ability to undergo self-renewal. Constitutive expression of BCRABL in murine ES cells increased self-renewal both in the presence and absence of LIF [21]. We therefore investigated whether expressing Tel/PDGFR in ES clones would alter their growth or differentiation patterns. Self-renewal was quantified using clonal analysis and staining for the presence of alkaline phosphatase, an enzyme expressed by undifferentiated ES cells. Uninduced TP clones (+Tet) cultured for 2 days in the presence of tetracycline and LIF maintained their pluripotency, with on average 88–90% of cells staining positive with Fast RedTM (Fig. 1B). The percentage of undifferentiated cells fell to between 68 and 70% following 3 days in culture. Induction of TP expression (−Tet) significantly reduced the ability of LIF to maintain self-renewal of ES cells with only 18% (TP1) and 40% (TP2) staining positive for alkaline phosphatase following 2 days in culture, with a further reduction to either 9% (TP1) or 23% (TP2) occurring by 3 days. Culturing parental E14tg2a-tTA cells +/− Tet had no significant effect on their self-renewal potential. The morphology and proliferation rates of cells expressing TP also changed. Cells proliferated slower and lost their characteristic three-dimensional ES cell structures, appearing flatter and more epithelial and fibroblast-like in character, highly suggestive of a more differentiated phenotype (data not shown). The effects on self-renewal observed appear to be directly related to the level of Tel/PDGFR expressed, with clones expressing lower levels (TP3 and TP4) showing on average only a 25% reduction in self-renewing capacity P > 0.005,
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n = 4 (Supplementary data). For the remainder of our experiments we concentrated on the TP1 clone, verifying all our results in the TP2 clone. 3.3. Tel/PDGFRβ decreases cell proliferation but not viability in the presence of LIF Reduced proliferation is a property observed when ES cells undergo differentiation, we therefore investigated the proliferation rates of ES cells cultured in LIF and induced to express TP using cell counts on a Vi-CELL XR counter. There was a marked reduction in cell numbers in the population expressing Tel/PDGFR compared to controls (Fig. 2A). In order to determine whether cells were still viable and not apoptotic we measured their mitochondrial membrane integrity using DiOC6 and flow cytometry. The viability of ES cells expressing TP was 74.7 ± 6.3%, n = 4 following 72 h culture in the presence of LIF, with no significant differences observed to non-induced TP clones 75.9 ± 12.4%, n = 4 (Fig. 2B) or parental cells 72.2 ± 6.9%, n = 4 (+Tet) and 70.0 ± 9.9%, n = 4 (−Tet). 3.4. TP-induced differentiation of ES cells is enhanced by LIF removal
Fig. 1. Inducible expression of TEL/PDGFR reduces ES cell selfrenewal. (A) Variable levels of TEL/PDGFR protein in different clones. Immunoblotting analysis with protein extracts from parental ES cells and stable clones containing the TEL/PDGFR oncogene (TP1-4) after incubation for 24 h in the presence (+) or absence (−) of tetracycline (Tet). Upper panel: Immunoblotting (IB) with a polyclonal antibody that recognizes the TEL/PDGFR fusion; middle panel: an antibody which detects tyrosine-phosphorylated proteins (pTyr); lower panel: antibody that detects SHP2 (loading control). (B) Self-renewal decreases in the presence of high levels of the oncoprotein. Clones TP1 & TP2 were incubated for 2–3 days in 1000 U/mL LIF, +/− Tet. The percentage of cells positive for alkaline phosphatase is given (mean ± S.E.M.s, n = 4). Asterisks denote P < 0.005 by paired Student’s t-test. (C) Representative immunoblot using protein from cells depicted in panel B, showing TEL/PDGFR expression levels.
Our previous study, using Ba/F3 cells, demonstrated that dual signalling via Tel/PDGFR and IL-3 had a detrimental effect on cell proliferation and survival [15]. Therefore, we were interested to investigate whether LIF signalling, in conjunction with Tel/PDGFR contributed to the loss of ES pluripotency observed upon expression of Tel/PDGFR. Self-renewal assays were carried out in the absence of LIF or in the presence of 20, 500 or 1000 U/mL LIF. Fewer TP cells stained positive for alkaline phosphatase in the lower doses of LIF compared to the optimal concentration of 1000 U/mL (Fig. 2C) with no cells staining positive following 24 h of LIF removal (data not shown). These results indicate that unlike constitutive expression of BCR-ABL in ES cells, which promoted self-renewal both in the presence and absence of LIF by activating the Jak/STAT3 pathway [21], inducible expression of Tel/PDGFR is unable to maintain ES cell pluripotency, and appears to promote the differentiation potential of ES cells, with the removal of LIF enhancing this process. This data indicates that expressing Tel/PDGFR in ES cells activates signaling pathways, which promote ES cell differentiation with LIF signaling delaying this process. 3.5. Tel/PDGFRβ alters expression of regulatory genes involved in hematopoiesis To ascertain the mechanism by which Tel/PDGFR promotes the differentiation of ES cells, Tel/PDGFR was expressed over a 72-h period and RNA extracted from the TP and control cells. Semi-quantitative PCR (Fig. 3A) was used to analyse gene expression (for primer sequences see Supplementary tables) and the expression levels of certain
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Fig. 2. Inducible expression of TEL/PDGFR reduces ES cell proliferation and self-renewal, but not viability. (A) Reduced proliferation in the presence of the oncoprotein. Cell counts for TP1 clone following tetracycline removal (+TP expression) or in the presence of tetracycline (−TP expression), following culture for 0–60 h in 1000 U/mL LIF is given (mean ± S.E.M.s, n = 3). Results were verified using TP2 clone (data not shown). (B) Apoptosis is not increased in cells expressing the TEL/PDGFR oncogene. TP clones were cultured for 72 h in 1000 U/mL LIF, stained with 10 nM Di0C6 and fluorescence measured by flow cytometry. The average percentage of viable cells for each condition is given (mean ± S.E.M.s, n = 4). (C) Self-renewal decreases in induced cells following LIF removal. Alkaline phosphatase staining of TP1 clone +/− TP expression at 72 h in the presence of different amounts of LIF. The percentage of cells positive for AP is given (mean ± S.E.M.s, n = 3). Asterisk denotes P < 0.01 and cross denotes P < 0.005 by paired Student’s t-test.
genes confirmed by real-time PCR (Fig. 3B). Initially, genes known to be involved in ES cell self-renewal and those altered during the early stages of ES cell differentiation were analyzed (Fig. 3A, panel i). Decreased expression of the self-renewal marker Nanog, was observed and could be a contributing factor to the loss of self-renewal observed upon expression of TP. An increase in the mesodermal gene Brachyury suggests that differentiation towards mesoderm formation maybe being enhanced. However no change was seen in the self-renewal marker Oct4, or the mesodermal markers associated with muscle (MyoD1) and endothelial/hematopoietic differentiation (BMP4), suggesting TP is not inducing global differentiation of ES cells into mesoderm during this short-time frame. Several important genes associated with hematopoietic stem cell formation were also up-regulated, including Sca-1, HoxB4 and CDX4, following TP expression (Fig. 3A, panel ii). The notable exception being c-kit, the receptor for stem cell factor, which was down-regulated by TP expression, however these findings are supported by a previous study, which indicates that c-kit is highly expressed by ES cells and becomes down-regulated following differentiation induction [22]. Examination of genes associated with committed hematopoietic progenitors showed the greatest differences (panel iii). Expression levels of the genes BMP2, GATA2, GATA3, and to a lesser extent GATA4, were much higher following TP induction. The erythroid specific factor GATA1 was not affected, although interestingly its cofactor Fog1 did increase with TP expression. Analysis of hematopoietic lineage-specific genes showed induction of the embryonic globin chain (Hbb-bH1) by 72 h, indicating TP is driving primitive hematopoietic differentiation. However, no expression of the more definitive erythroid lineage genes Hbb-b1, KLF1 and EPO R was seen (data not shown). The myeloid transcription factor MafB showed a slight increase with TP expression but no expression was seen for PU.1, which directs differentiation towards the common lymphoid–myeloid progenitor (Fig. 3A panel iv). Several genes important for monocytic development, Egr-1, Egr-2 and Nab2 were also up-regulated following TP induction. Quantitative analysis of HoxB4, GATA2 and MafB expression confirmed the accuracy of the changes identified during the semi-quantitative analysis (Fig. 3B). Overall results from the gene expression analyse strongly suggest that in this system, Tel/PDGFR expression has the maximum impact on genes associated with initiating hematopoietic differentiation with a clear induction of genes linked to early myeloid and monocytic development. 3.6. Tel/PDGFRβ promotes myeloid differentiation of ES cells Results from the gene expression analysis indicate Tel/PDGFR may influence several stages of hematopoietic differentiation. To investigate the events involved in leukemic transformation by this oncogene we expressed TP throughout the hematopoietic differentiation of ES cells using a modi-
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Fig. 3. Induction of Tel/PDGFR in ES cells alters gene expression towards hematopoietic differentiation, primarily the myeloid lineage. (A) RT-PCR analysis of mRNA levels for TP1 clone cultured +/− TP expression for 24, 48, and 72 h. The housekeeping genes Gus-B and TBP were used as controls. Panel (i) Genes associated with self-renewal and differentiation status of ES cells. (ii) Genes involved in early hematopoietic regulation. (iii) Genes associated with hematopoiesis. (iv) Genes associated with lineage restriction. (B) Changes to expression levels seen by RT-PCR were confirmed for selected genes by quantitative PCR using a Lightcycler. Representative gel images are shown for each gene n = 3.
fied three-stage differentiation approach (Fig. 4A, panel i). This system is based on the original work of Keller et al. [18] and uses a wide range of cytokines (EPO, G-CSF, GM-CSF, IL-3, SCF) during the final stage of differentiation to induce both myeloid and erythroid lineage commitment. ES cells expressing Tel/PDGFR were capable of undergoing all three stages of the differentiation process. However, differences were observed in the first round of embryoid body formation and at the final stage of hematopoietic colony formation.
There was on average a 33% reduction in total cell number in the primary EB cultures induced to express TP compared to the control cultures. The average area of these EBs when measured using Nikon NIS-Elements imaging software, was also decreased (Fig. 4A, ii). However, when used in the second phase of differentiation these cells efficiently formed both secondary EBs and blast-like colonies (BC), comparable to control cultures (−TP) (Fig. 4A, iii). BC are diffuse clusters of cells, which contain hemangioblasts [23,24], early precur-
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Fig. 4. Tel/PDGFR increases myeloid differentiation of ES cells. (A) (i) Schematic diagram of the three stages involved in the hematopoietic differentiation of ES cells. The time-scales involved in the formation of primary embryoid bodies (1◦ EB), secondary embryoid bodies/blast-like colonies (2◦ EB/BC) and hematopoietic colonies (Haem) are indicated and represent when morphological changes and RNA samples were analysed. (ii) Expressing Tel/PDGFR reduces the size of primary embryoid bodies. The areas of the primary embryoid bodies were measured following the first round of differentiation. 100 embryoid body measurements were taken from three independent experiments. (iii) Photographs of representative primary embryoid bodies, secondary embryoid bodies/blastlike colonies, taken at 10× magnification. Scale bar equals 100 m. (iv) Percentage of each type of hematopoietic colony formed by day 15 of differentiation with and without induction of Tel/PDGFR expression. Colonies were defined as follows; myeloid (including macrophage and granulocyte/macrophage colonies), erythroid or mixed lineage (including erythroid-macrophage colonies and multi-lineage colonies). Data represents 500 hematopoietic colonies scored per experiment, for each condition (±S.E.M.s, n = 3). (v) Photographs of representative hematopoietic colonies, at 10× magnification, scale as above.
sor cells with both endothelial and hematopoietic potential, that have been shown to be involved in establishing definitive hematopoiesis in the mouse embryo [25]. Cells from second phase cultures were then assessed for their ability to generate mature hematopoietic cells using hematopoietic colony assays, supplemented with cytokines to enhance both myelopoiesis and erythropoiesis. Here we observed a significant increase in myeloid colony formation, with cultures expressing the oncogene (+TP) producing on average 20% more myeloid colonies compared to control cells (−TP). Colony size also altered with the average area of the colonies being six times larger (16,970 m2 −TP compared to 113,000 m2 +TP, n = 20). The ability of TP-expressing cells to form erythroid and mixed lineage colonies also diminished with, on average, a 41% reduction in erythroid colonies and a 66% reduction in mixed colonies being observed in the
cultures. The erythroid colonies were considerably smaller in area than those observed in the control cultures (15,362 m2 −TP compared to 7438 m2 +TP, n = 20), with no difference in the size of the mixed colonies being observed (Fig. 4A, iv and v). These results indicate that TP drives myelopoiesis and suppresses erythropoiesis. 3.7. Tel/PDGFRβ-induced changes in gene expression during hematopoietic differentiation of ES cells To examine alterations in the hematopoietic gene expression profile during lineage commitment, RNA was extracted from cells either induced to express Tel/PDGFR (+TP, no tet in culture) or without Tel/PDGFR expression (−TP, tet present in culture) during each phase of hematopoietic differentiation. As seen previously, Nanog expression was low
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Fig. 5. Analysis of gene changes during hematopoietic differentiation of ES cells +/− TP expression. (A) RT-PCR analysis of mRNA levels for TP1 clone cultured +/− TP expression during hematopoietic differentiation system, samples taken at the time points indicated in Fig. 4A (i). Panel (i) Genes associated with self-renewal and differentiation status of ES cells. (ii) Genes involved in early hematopoietic regulation. (iii) Genes associated with hematopoiesis. (iv) Genes associated with lineage restriction. (B) Changes to expression levels seen by RT-PCR were confirmed for selected genes by quantitative PCR using a Lightcycler. Representative gel images are shown for each gene n = 3.
in the TP expressing cells during the initial stages of differentiation (Fig. 5A panel i, 24 h and 1◦ EB) and this low level of expression was sustained into the blast-like colony stage (2◦ BC) unlike control cells (−TP) where it was no longer detected by this stage. Similarly sustained expression of both Oct4 and Brachyury were also seen at this later 2◦ BC stage in the TP samples compared to controls. A modest increase in HoxB4 expression was seen at all stages following TP expression and confirmed by quantitative PCR (Fig. 5A, ii and b) whereas other stem cell markers such as, c-kit was reduced at the early stages (24 h and 1◦ EB) but increased
at the later stages (2◦ BC and Haem) and Sca-1 decreased at the hematopoietic colony stage. The regulatory genes involved in early hematopoietic commitment showed the greatest changes especially during the earlier stages of differentiation (24 h and 1◦ EB) with GATA2 expression increased and GATA1/Fog1 decreased in TP cells, suggesting an early bias towards myeloid differentiation following induction of TP (Fig. 5A, iii and B). Subtle changes were also seen in genes associated with specific lineage commitment. The erythroid genes KLF1 and EPO R showed a slight decrease in expression at the 2◦ BC
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stage, whereas, the key myeloid regulator PU.1 was upregulated favouring monopoiesis over granulopoiesis [26]. The monocytic genes Egr1, Egr2 and Nab2 were also increased especially very early during the differentiation and at the final hematopoietic colony stage when TP was expressed (Fig. 5A, iv). Surprisingly, the myelomoncytic regulator MafB appears to be up-regulated at the early stages of differentiation by TP then down-regulated by the oncogene at later stages of differentiation. This gene is normally expressed during the later but not early stages of monocytic development [27], a progression, which was deregulated by expressing TP. One interesting observation from this gene expression profiling is the inverse relationship between expression levels seen at 24 h and the hematopoietic colony stages for certain genes. Genes up-regulated within 24 h of TP expression (HoxB4, Sca1, GATA2, MafB) appear to be down-regulated (or unaltered) by the hemopoietic colony stage, and viceversa (Nanog, Oct4, brachyury, c-kit) suggesting that the context and phase of gene expression is altered during differentiation by expressing Tel/PDGFR in stem cells. Overall this data confirms that this activated tyrosine kinase fusion protein activates transcription factors and genes important for driving myelopoiesis.
4. Discussion The receptor tyrosine kinase PDGFR rearranges with several oncogenic partners to act as a key pathogenic factor in a subgroup of hematological diseases with both myeloproliferative and myelodysplastic features [1,2,9,10]. In vivo studies support a role for Tel/PDGFR, in the generation of human and murine leukemia, but the direct mechanisms by which this oncogene manipulates the self-renewal, proliferation and differentiation decisions made by stem cells are still not clearly defined [11]. To investigate TP-induced functional responses, we have used tetracycline-regulated expression in ES cells to control the expression of TP in pluripotent primitive stem cells with hematopoietic potential. Overall, our investigations revealed that TP is sufficient to change the growth and differentiation properties of primitive ES cells and enhance their myeloid differentiation by altering the balance of key developmental genes. We demonstrate that unlike BCR-ABL, which promotes the self-renewal of ES cells [21], expressing TP resulted in an increase in the differentiation of ES cells. The level of expression of the oncogene played a critical role in promoting differentiation while the removal of LIF the key cytokine involved in ES cell self-renewal, resulted in the TP-expressing cells differentiating faster, indicating that this effect was TP-mediated and not due to combined signaling. Analysis of gene expression changes induced by TP expression showed major changes in the balance between genes associated with ES cell self-renewal and those involved in hematopoietic differentiation. Expression of TP for up to 72 h
was able to diminish Nanog expression while increasing the expression of the differentiation marker Brachyury, suggesting a rapid initiation of differentiation when TP is expressed. Nanog has been shown in conjunction with other transcription factors to maintain pluripotency through repression of differentiation programs [28,29]. Interestingly, while Nanog is initially down-regulated after TP expression, its expression, along with Oct4 and Brachyury, appears to be sustained by TP during the blast-like and hemopoietic colony stages of hematopoietic differentiation. This suggests that in addition to promoting the initial differentiation of ES cells, TP may also maintain a population of cells with self-renewal potential in the later stages of hematopoietic development, akin to the clonal ‘leukemic’ stem cells with abnormal tyrosine kinase activity identified in many MPN [3–6]. These results indicate that unlike BCR-ABL, Tel/PDGFR exerts its effects in a later stem cell/progenitor population and could explain why, bone marrow cells retrovirally transformed with Tel/PDGFR are unable to induce disease when transplanted into secondary recipients [11,30]. There appears to be a critical balance between the levels of different transcription factors, which determines the stem cells decision either to self-renew or differentiate. The overall effect of the signals activated by TP was a loss in ES cell pluripotency and an increase in differentiation, highlighted by a reduction in alkaline phosphatase staining and levels of self-renewal genes. This was accompanied by a rapid and dramatic increase in the expression of genes associated with early hematopoietic differentiation (Sca1, HoxB4, CDX4) and hematopoietic regulators (BMP-2, GATA2, 3, 4, Fog1). Increased HoxB4 and CDX4 expression are known to enhance hematopoiesis during ES cell differentiation [31] while over-expression of GATA-2 and differentiation analysis using GATA2 null ES cells, indicates that it plays a central role in the proliferation and survival of immature hematopoietic progenitors [32,33]. Combined with the induction of genes associated with monocytic development (Egr1, 2, Nab2) [27], these findings provide compelling evidence for a shift in the balance of the transcription machinery towards myelomonocytic differentiation when TP is expressed. Tel/PDGFR was also able to influence the differentiation of ES cells throughout in vitro hematopoietic development, with the most notable physiological differences being observed at the initial and final stages of the differentiation process. Cells expressing the oncogene were able to form primary embryoid bodies but they were smaller compared to control colonies. This could either be due to a reduction in the overall proliferation or through an incomplete formation of all three germ layers, and although slight reductions in expression of Nanog, Brachyury and Flk-1 were seen when TP was expressed at this stage, further studies are required to determine the exact mechanism by which TP is mediating its effects during this phase of the differentiation process. Overall TP expression resulted in enhanced myelopoiesis and suppressed erythropoiesis, reflected in both
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the increased number (20% increase) and size (6 times larger) of myeloid colonies seen at the hematopoietic colony stage. These findings indicate that expressing the oncogene alters the balance of key transcription factors involved in erythroid and myeloid development. It is clear that transcription factors work as intricate networks often dependent on each other as well as cofactor expression for lineage restriction of the cell [34]. Although many transcription factors are regarded as lineage specific they are frequently expressed at lower levels in uncommitted hematopoietic progenitors with changes in their levels often being sufficient to cause changes in lineage commitment [35]. Interactions between the later more lineage-specific transcription factors also plays an important role in determining the fate of hematopoietic cells [34] and results from this study show that changes mediated by TP are likely to alter lineage commitment. TP induction during hematopoietic differentiation caused a reduction in gene expression of the erythroid regulator GATA1, its cofactor Fog1 and also the erythropoietin receptor (EPO R) at the primary embryoid body and/or blast-like colony stages, consistent with the decreased number of erythroid colonies seen at the final hematopoietic colony stage. Interestingly, in a transformed myelomonocytic cell line, forced over expression of GATA1 suppressed the myelomonocytic phenotype converting them into cells resembling erythroblasts while lower level of GATA1 converted the cells to an eosinophil-like phenotype [35]. Given that Fog1 is downregulated following induction of eosinophil differentiation [36] while increased expression of GATA2 is known to block erythroid differentiation in vitro [32,33], it is clear that TP shifts transcription factor expression to very much favour myelomonocytic and even eosinophilic differentiation. Additional work to further analyse the altered balance between these factors combined with changes to the differentiation system to promote the eosinophil lineage, such as addition of IL-5 to the hematopoietic colony stage, might provide valuable insight into the eosinophilia often associated with PDGFR fusion genes [37,38]. In summary, this study reports the establishment of a stem cell model to inducibly express the leukemic oncogene Tel/PDGFR. Expressing Tel/PDGFR rapidly induced differentiation towards hematopoietic precursors and favoured the establishment of myeloid in preference to erythroid lineages. Tel/PDGFR initially induced these changes by decreasing the levels of transcription factors associated with pluripotency while increasing the expression of factors known to increase hematopoietic stem cell proliferation. Hematopoietic lineage commitment genes were also altered resulting in TP increasing myeloid cell production during in vitro hematopoietic differentiation. This work demonstrates that this system can be used as a powerful tool to study the molecular changes induced by leukemic oncogenes, allowing changes directly attributed to expression of the oncogene to be examined not only during the initiating stages but also during the more definitive stages of hematopoiesis.
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Acknowledgements This study was supported by the Medical Research Council, Department of Education and Learning and Northern Ireland Leukaemia Research Fund. We would like to thank the following people: Dr. M. Carroll, University of Pennsylvania, for the pcCDN3 Tel/PDGFR construct and Dr. O. Witte, UCLA for the E14tg2a mouse embryonic stem cells expressing the Tetsensitive transactivator, tTA. Contributions. Ms. Dobbin and Mrs. Corrigan performed experiments and data analysis. Prof. Welham and Dr. Walsh provided vital reagents, support and advice during the project. Dr. Freeburn performed some of the experiments and helped prepare the manuscript. Dr. Wheadon designed and coordinated the experiments and helped prepare the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. leukres.2008.02.007.
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