Molecular Mechanisms Underlying FIP1L1-PDGFRA– Mediated Myeloproliferation

Research Article

Miranda Buitenhuis,1 Liesbeth P. Verhagen,1 Jan Cools,2 and Paul J. Coffer1

1Molecular Immunology Lab, Department of Immunology, University Medical Center, Utrecht, the Netherlands and 2Department of Human Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), University of Leuven, Leuven, Belgium

Abstract between a previously uncharacterized gene, FIP1-like 1 (FIP1L1) a An interstitial deletion on chromosome 4q12 resulting in the and the platelet-derived growth factor receptor (PDGFRA; ref. 2). formation of the FIP1L1-PDGFRA fusion protein is involved in Sequencing of the fusion gene in several patients revealed that the the pathogenesis of imatinib-sensitive chronic eosinophilic breakpoint in the PDGFRA gene is conserved and is located in a leukemia. The molecular mechanisms underlying the devel- small region in exon 12, thereby deleting the extracellular and opment of disease are largely undefined. Human CD34+ transmembrane domain of the receptor. The breakpoint in FIP1L1, hematopoietic progenitor cells were used to investigate the however, is variable and spreads from exon 7 to exon 10 (2, 3), role of FIP1L1-PDGFRA in modulating lineage development. resulting in fusion proteins differing more than 120 amino acids in FIP1L1-PDGFRA induced both proliferation and differentia- length. The clinical implications of this variability are thus far unknown. tion of eosinophils, neutrophils, and erythrocytes in the absence of cytokines, which could be inhibited by imatinib. Chromosomal deletion resulting in the formation of FIP1L1- Whereas expression of FIP1L1-PDGFRA in hematopoietic stem PDGFRA has been observed in 14% to 60% of patients with cells and common myeloid progenitors induced the formation hypereosinophilic syndrome (2–7). These patients were diagnosed of multiple myeloid lineages, expression in granulocyte- with chronic eosinophilic leukemia (CEL) according to the WHO macrophage progenitors induced only the development of disease classification criteria and were treated with imatinib, which eosinophils, neutrophils, and myeloblasts. Deletion of amino results in complete remission in many patients with CEL (8–10). acids 30 to 233 in the FIP1L1 gene [FIP1L1(1–29)-PDGFRA] Imatinib is a 2-phenylaminopyrimidine derivative designed to gave rise to an intermediate phenotype, exhibiting a dramatic inhibit BCR-ABL by association with ATP-binding sites. Imatinib reduction in the number of erythrocytes. FIP1L1-PDGFRA and not only inhibits BCR-ABL, but also inhibits the activity of other kinases including c-Kit, c-fms, and PDGFR (2, 11–13). Although it FIP1L1(1–29)-PDGFRA both induced the activation of p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) in myeloid has been shown that FIP1L1-PDGFRA acts as a constitutively active progenitors, whereas signal transducers and activators of tyrosine kinase resulting in the activation of signal transducers and transcription 5 (STAT5) and protein kinase B/c-akt were activators of transcription 5 (STAT5; ref. 2), the molecular only activated by FIP1L1-PDGFRA. Dominant-negative mechanisms underlying FIP1L1-PDGFRA–mediated CEL, including STAT5 partially inhibited FIP1L1-PDGFRA–induced colony the relevance of STAT5 activation, are at the moment, incompletely formation, whereas combined inhibition of phosphatidylino- understood. sitol-3-kinase and ERK1/2 significantly reversed FIP1L1- Recent studies showed that transplantation of interleukin (IL)-5 PDGFRA–induced colony formation. Taken together, these transgenic mouse hematopoietic progenitors ectopically expressing results suggest that expression of FIP1L1-PDFGRA in human FIP1L1-PDGFRA induces a CEL-like phenotype in mice, including hematopoietic progenitors induce a myeloproliferative phe- tissue eosinophilia, as observed in humans (14). However, these notype via activation of multiple signaling molecules includ- studies focused on the effect of expression of the fusion protein on murine hematopoietic progenitors rather than human hematopoi- ing phosphatidylinositol-3-kinase, ERK1/2, and STAT5. [Cancer etic progenitors, and did not identify the molecular mechanisms Res 2007;67(8):3759–66] underlying the development of disease. We have therefore investigated whether the expression of Introduction FIP1L1-PDGFRA in primary human hematopoietic progenitors Idiopathic hypereosinophilic syndromes are a rare heterogenous was sufficient to induce hypereosinophilia. In addition, we have group of hematologic disorders characterized by an unexplained investigated which signal transduction pathways are aberrantly 9 persistent eosinophilia exceeding 1.5 Â 10 eosinophils per liter for regulated in primary human hematopoietic progenitors expressing more than 6 months in combination with symptoms of organ FIP1L1-PDGFRA and whether these molecules are critical for the damage resulting from eosinophil infiltration (1). Recent studies, in transforming capacity of this fusion protein. Our data shows that a subgroup of patients with hypereosinophilic syndrome, identified expression of FIP1L1-PDGFRA induces the activation of multiple a specific interstitial 800 kb deletion on chromosome 4q12 signaling pathways including STAT5, extracellular signal-regulated [del(4)(q12g12)], resulting in the formation of a fusion protein kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and protein kinase B (PKB/c-akt), resulting in cytokine-independent colony formation. Deletion of amino acids 30 to 233 of FIP1L1 prevents the activation of PKB and STAT5, resulting in an Requests for reprints: Paul J. Coffer, Department of Immunology, KC02.085.2 intermediate phenotype, suggesting that FIP1L1 may play an University Medical Center, Lundlaan 6, 3584 EA Utrecht, the Netherlands. Phone: important role in the regulation of disease phenotype. These data 31-30-250-7674; Fax: 31-30-2504305; E-mail: [email protected]. I2007 American Association for Cancer Research. have implications for the development of novel therapies targeting doi:10.1158/0008-5472.CAN-06-4183 imatinib-resistant CEL. www.aacrjournals.org 3759 Cancer Res 2007; 67: (8). April 15, 2007

Materials and Methods erythrocyte/monocyte/megakaryocyte), CFU-GM (granulocyte/macrophage), CFU-E (erythrocyte), and CFU-Eo (eosinophil) were scored by Isolation and culture of human CD34+ cells. Mononuclear cells were May-Grunwald Giemsa staining of cells derived from individual colonies. isolated from human umbilical cord blood by density centrifugation over a Western blot analysis. Western blot analysis was done using standard Ficoll-Paque solution (density, 1.077 g/mL). MACS immunomagnetic cell techniques. In brief, differentiating granulocytes were lysed in Laemmli separation (Miltenyi Biotech, Auburn, CA) using a hapten-conjugated buffer [0.12 mol/L Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.05 Ag/AL antibody against CD34, which was coupled to beads, was used to isolate h + + bromophenol blue, and 35 mmol/L -mercaptoethanol], and boiled for CD34 cells. CD34 cells were cultured in Iscove’s modified Dulbecco’s 5 min. Equal amounts of total lysate were analyzed by 10% SDS-PAGE. medium (Life Technologies, Paisley, United Kingdom) supplemented Proteins were transferred to Immobilon-P and incubated with blocking A h with 8% FCS, 50 mol/L of -mercaptoethanol, 10 units/mL of penicillin, buffer (TBS/Tween 20) containing 5% low-fat milk for 16 h at 4jC before A 10 g/mL of streptomycin, and 2 mmol/L of glutamine at a density of incubating with antibodies against either PDGFRA (Santa Cruz, Inc., Santa Â 6 0.3 10 cells/mL. Cells were cultured either in the presence of stem cell Cruz, CA), phosphorylated PKB (Cell Signaling Technology, Danvers, MA), factor (SCF; 50 ng/mL), and FMS-related tyrosine kinase 3 (FLT-3) ligand phosphorylated ERK1/2 (Cell Signaling Technology), phosphorylated (50 ng/mL), or in the presence of SCF (50 ng/mL), FLT-3 ligand (50 ng/mL), STAT5a (Cell Signaling Technology), phosphorylated p38MAPK (Cell granulocyte macrophage colony-stimulating factor (GM-CSF; 0.1 nmol/L), Signaling Technology), or an antibody against tubulin (Sigma-Aldrich, IL-3 (0.1 nmol/L), and IL-5 (0.2 nmol/L) to induce eosinophil differentiation. Zwijndrecht, the Netherlands) overnight in the same buffer. Subsequently, Retroviral transduction experiments were done 2 days after isolation. Cord blots were incubated with peroxidase-conjugated secondary antibodies for blood samples were collected from healthy donors after informed consent 1 h. Enhanced chemiluminescence was used as a detection method was provided according to the Declaration of Helsinki. Protocols were according to the protocol of the manufacturer (Amersham Pharmacia, approved by the local ethics committee of the University Medical Center in Amersham, United Kingdom). Utrecht. Statistics. A Levene’s test for equality of variances was done in all + Viral transduction of CD34 cells. Bicistronic retroviral DNA experiments. Subsequently, an independent sample t test was done to constructs were used coexpressing enhanced green fluorescent protein compare the differences in colony numbers, between the controls and cells (eGFP) and either FIP1L1-PDGFRA, fusing the NH2-terminal 233 amino transduced with FIP1L1-PDGFRA or FIP1L1(1–29)-PDGFRA. P V 0.05 was acids of FIP1L1 to the COOH-terminal 523 amino acids of PDGFRA, or considered significant. FIP1L1(1–29)-PDGFRA, a fusion gene in which amino acids 30 to 233 of FIP1L1 were deleted (15). Retrovirus was produced by transient transfection Results of the retroviral packaging cell line, Phoenix-ampho, by calcium phosphate coprecipitation. Cells were plated in 6 cm dishes, 24 h before transfection. A FIP1L1-PDGFRA induces cytokine-independent prolifera- total of 10 Ag of DNA was used per transfection. Medium was refreshed 16 tion and differentiation. To determine whether expression of h after transfection. After an additional 24 h, viral supernatants were the FIP1L1-PDGFRA fusion gene is sufficient to recapitulate the collected and filtered through a 0.2-Am filter. CD34+ cells were transduced development of CEL in human hematopoietic progenitors, in 24-well dishes precoated with 10 Ag/cm2 of recombinant human bicistronic retroviral DNA constructs were used coexpressing eGFP RetroNectin (Takara, Otsu, Japan) for 2 h. Transduction was done with the and either FIP1L1-PDGFRA, or FIP1L1(1–29)-PDGFRA (15), in addition of 0.5 mL of viral supernatant to 0.5 mL of medium containing 0.5 which the amino acids 30 to 233 of FIP1L1 were deleted (2). Retro- Â 6 10 cells. Twenty-four hours after transduction, 0.7 mL of medium was virus was generated and used to transduce umbilical cord blood– removed from the cells and 0.5 mL of fresh virus supernatant was added derived CD34+ cells. Transduced cells were plated in CFU assays, together with 0.5 mL of fresh medium. and colony formation was analyzed after 12 days. The expression of Isolation of myeloid progenitors. Hematopoietic progenitors were + isolated as described by Manz et al. (16). In short, CD34+ cells were isolated FIP1L1-PDFGRA in human CD34 cells dramatically induced as described above and cultured for 1 day in the presence of SCF and FLT-3 colony formation in the absence of cytokines, whereas the addition ligand. Cells were subsequently washed and resuspended in PBS/5% FCS of IL-3 and IL-5, normally required to induce eosinophil differen- and incubated for 30 min on ice with a mixture of antibodies (all from tiation (17), did not further enhance colony numbers (Fig. 1A). The Becton Dickinson, Alphen a/d Rijn, the Netherlands). Lineage markers expression of both FIP1L1-PDGFRA and FIP1L1(1–29)-PDGFRA included CD2, CD3,CD4, CD7, CD8, CD14, and CD235a. Myeloid progenitors induced not only the development of eosinophils but also of À are negative for these lineage markers. The lineage negative (Lin ), CD34+, À À erythrocytes and neutrophils both in the absence of cytokines or in and CD38 populations consist of hematopoietic stem cells (HSC). Lin , B–D À the presence of IL-3 and IL-5 (Fig. 1 ), indicating that expression CD34+,CD38+, CD123+,andCD45RA cells are common myeloid À + + + + of the fusion protein itself induces a myeloproliferative phenotype progenitors (CMP), whereas Lin , CD34 , CD38 , CD123 , and CD45RA rather than CEL, this suggests that additional factors are required for cells are granulocyte-macrophage progenitors (GMP). HSCs, CMPs, and the development of the observed clinical phenotype. GMPs were sorted using a FACS ARIA (from Becton Dickinson). Isotype antibody staining was used to ensure sorting of the correct population. Although the expression of the fusion proteins rendered the cells Sorting of the different progenitor populations was confirmed by culture of cytokine-independent, the cells remained, at least in part, cytokine- the individual populations in the presence of SCF, FLT3L, GM-CSF, IL-3, responsive. Stimulation of cells expressing FIP1L1-PDGFRA with IL-5, granulocyte colony-stimulating factor, and erythropoietin to allow IL-3 and IL-5 induced the number of CFU-Eo colonies (Fig. 1D) differentiation of all lineages in a colony-forming assay as described below. without affecting total colony numbers (Fig. 1A), indicating that IL- Colony-forming unit assay. Retrovirally transduced cells were sorted 5 was still important for the induction of eosinophil development from not-transduced cells by flow cytometry and used in colony-forming in FIP1L1-PDGFRA–expressing cells. + unit (CFU) assays. CD34 cells were plated in Iscove’s modified Dulbecco’s The role of FIP1L1 in the regulation of FIP1L1-PDGFRA– medium (Life Technologies) supplemented with 35.3% FCS (Hyclone, Logan, mediated transformation is, at the moment, incompletely under- UT), 44.4% methylcellulose-based medium called Methocult (StemCell stood. Whereas previous studies show that fusion of the first 29 Technologies, Vancouver, Canada), 11.1 Amol/L of h-mercaptoethanol, 2.2 units/mL of penicillin, 2.2 Ag/mL of streptomycin, and 0.44 mmol/L of amino acids of FIP1L1 to the intracellular domain of the PDGFRA glutamine at a density of 1,250 cells/well. CFU assays were done either in gene is both necessary and sufficient to induce cytokine- the absence of cytokines or in the presence of SCF (50 ng/mL), FLT-3 ligand independent proliferation in Ba/F3 cells (2), recent studies suggest (50 ng/mL), GM-CSF (0.1 nmol/L), IL-3 (0.1 nmol/L), and IL-5 (0.2 nmol/L). that FIP1L1 is not required to induce cytokine-independent Colonies were scored after 12 days of culture. CFU-GEMM (granulocyte/ proliferation of these cells (15). To investigate whether expression

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Figure 1. Ectopic expression of FIP1L1-PDGFRA and FIP1L1(1–29)-PDGFRA induces cytokine-independent proliferation and differentiation of eosinophils, neutrophils, and erythrocytes. CD34+ cells were cultured either in the absence of cytokines or in the presence of IL-3 and IL-5 to induce eosinophil differentiation. Cells were retrovirally transduced to ectopically express FIP1L1-PDGFRA or FIP1L1(1–29)-PDGFRA. A, transduced cells (1,250) were plated into methylcellulose and colony formation was analyzed after 12 d of culture. The total number of colonies was scored. The total number of erythrocyte colonies (CFU-E; B), granulocyte-macrophage colonies (CFU-GM; C), and eosinophil colonies (CFU-Eo; D) were scored. of the FIP1L1(1–29)-PDGFRA fusion protein in human CD34+ cells order to investigate whether imatinib inhibits FIP1L1-PDGFRA– gives rise to a similar phenotype compared with the expression of mediated induction of colony formation, CD34+ cells were FIP1L1-PDGFRA, CD34+ cells were retrovirally transduced to retrovirally transduced to ectopically express FIP1L1-PDGFRA. ectopically express either FIP1L1(1–29)-PDGFRA or FIP1L1- Transduced cells were plated into CFU assays either in the absence PDGFRA. Transduced cells were plated in CFU assays, and colony or presence of imatinib, and colony formation was analyzed after formation was analyzed after 12 days. Although expression of 12 days. Treatment of human hematopoietic progenitors express- FIP1L1(1–29)-PDGFRA in primary human hematopoietic progen- ing FIP1L1-PDGFRA with imatinib completely blocked colony itors also induced colony formation in the absence of cytokines, the formation in the absence of cytokines, but not in the presence of number of colonies was reduced compared with cells expressing IL-3 and IL-5 (Fig. 2A). Normal eosinophil development was FIP1L1-PDGFRA (Fig. 1A), which was predominantly caused by observed in FIP1L1-PGFRA expressing cells cultured in the a significant reduction in the number of erythrocyte colonies presence of IL-3, IL-5, and imatinib compared with control cells (Fig. 1B). No significant difference was observed in both CFU-GM cultured under the same conditions (Fig. 2D). This indicates that and CFU-Eo numbers (Fig. 1C and D). imatinib treatment, rather than inducing apoptosis of transduced Partial deletion of FIP1L1 resulted in reduced erythrocyte cells, enables cells to normally respond to cytokines. Surprisingly, development compared with FIP1L1-PDGFRA, suggesting that the formation of granulocyte-macrophage colonies from CD34+ FIP1L1 itself is important in the development of the myeloprolif- cells was also inhibited upon treatment with imatinib, suggesting erative phenotype observed. that long-term treatment of patients might also affect normal ImatinibinhibitsFIP1L1-PDGFRA–induced colony forma- hematopoiesis. tion, enabling cells to undergo normal differentiation. Patients Expression of FIP1L1-PDGFRA in a specific myeloid pro- diagnosed with FIP1L1-PDGFRA–mediated CEL are currently genitor population induces lineage-specific myeloprolifera- treated with imatinib, often resulting in complete remission. In tion. The expression of FIP1L1-PDGFRA in human CD34+ www.aacrjournals.org 3761 Cancer Res 2007; 67: (8). April 15, 2007

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Cancer Research hematopoietic progenitors induces a myeloproliferative phenotype numbers of erythrocyte colonies, whereas expression of the fusion À À rather than a phenotype resembling CEL. The cell surface protein in a Lin , CD34+, CD38+, CD45RA , CD123lo GMP (16) sialomucin-like adhesion molecule CD34 is expressed on early population only resulted in high numbers of myeloblasts, hematopoietic cells and is generally used as a marker for HSCs (18). neutrophils, and eosinophils (Fig. 3B). This supports the idea that However, CD34 is not exclusively expressed on HSCs, but is also for FIP1L1-PDGFRA to induce CEL, the translocation is more likely expressed in more committed cells such as CMP, GMP, and to occur in a more committed population such as the GMP. megakaryocyte-erythrocyte progenitors (16). To investigate wheth- FIP1L1-PDGFRA–induced activation of multiple signal er the expression of FIP1L1-PDGFRA in different hematopoietic transduction pathways. The observed differences in FIP1L1- progenitor populations gives rise to different phenotypes, hema- PDGFRA and FIP1L1(1–29)-PDGFRA–mediated colony formation topoietic progenitors, including HSCs, CMPs, and GMPs were and lineage development suggests differences in activation of sorted from CD34+ cells by flow cytometry (16), and subsequently intracellular signal transduction pathways. It has previously been retrovirally transduced to ectopically express FIP1L1-PDGFRA. shown that FIP1L1-PDGFRA induces the activation of STAT5 in Transduced cells were plated into CFU assays without any Ba/F3 cells, whereas ERK1/2 did not seem to be a downstream cytokines and colony formation was analyzed after 12 days target of FIP1L1-PDGFRA in this cell line (2). To investigate which (Fig. 3A). Sorting of the different progenitor populations was signal transduction pathways are aberrantly regulated in FIP1L1- confirmed by culturing the individual populations in the presence PDGFRA expressing human CD34+ cells, and to better understand of SCF, FLT3L, GM-CSF, IL-3, IL-5, granulocyte colony-stimulating the molecular mechanisms underlying the observed differences in factor, and erythropoietin to allow differentiation of all lineages in lineage development caused by the deletion of amino acids 30 to a colony-forming assay (data not shown). The expression of 233 in FIP1L1, CD34+ hematopoietic progenitors were retrovirally FIP1L1-PDGFRA in HSCs and CMPs resulted in the formation of all transduced to ectopically express FIP1L1-PDGFRA or FIP1L1(1– myeloid lineages in the absence of cytokines including high 29)-PDGFRA. Transduced cells were sorted from nontransduced

Figure 2. Imatinib inhibits FIP1L1-PDGFRA–induced colony formation, and enables cells to undergo a normal program of differentiation. CD34+ cells were cultured either in the absence of cytokines or in the presence of IL-3 and IL-5 to induce eosinophil differentiation. Cells were retrovirally transduced to ectopically express FIP1L1-PDGFRA. A, transduced cells (1,250) were plated into methylcellulose and cultured either in the absence or in the presence of 5 Amol/L of imatinib, and colony formation was analyzed after 12 d of culture. The total number of colonies was scored. The total number of erythrocyte colonies (CFU-E; B), granulocyte-macrophage colonies (CFU-GM; C), and eosinophil colonies (CFU-Eo; D) were scored.

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in the absence of cytokines. Constitutive activation of STAT5a induced the number of colonies (Fig. 5B), including erythrocyte colonies (Fig. 5C). Colony size, however, was dramatically reduced compared with FIP1L1-PDGFRA–expressing colonies (data not shown), suggesting that although STAT5a can play a role in FIP1L1- PDGFRA–induced colony formation, the activation of additional signaling molecules is required for the myeloproliferative phenotype observed. Combined inhibition of phosphatidylinositol-3-kinase and ERK1/2 blocks FIP1L1-PDGFRA–induced colony formation. To investigate whether inhibition of additional signaling pathways could rescue the FIP1L1-PDGFRA–mediated phenotype, CD34+ cells were retrovirally transduced to express FIP1L1-PDGFRA. Transduced cells were plated in methylcellulose medium either in the absence or in the presence of LY294002, SB203580, and U0126, pharmacologic inhibitors of phosphatidylinositol-3-kinase (PI3K), p38MAPK, and ERK1/2, respectively. Whereas colony formation of FIP1L1-PDGFRA–expressing cells was unaffected by the inhibition of p38MAPK, inhibition of either PI3K or ERK1/2 modestly decreased colony numbers both in presence or absence (Fig. 6B) of IL-3 and IL-5 (Fig. 6A). Importantly, combined inhibition of both signaling pathways dramatically reduced colony formation, suggesting that combined activation of PI3K, ERK1/2, and STAT5a is Figure 3. Ectopic expression of FIP1L1-PDGFRA in specific myeloid progenitor population induces lineage-specific myeloproliferation. Hematopoietic critical for FIP1L1-PDGFRA–mediated cytokine-independent line- progenitors were sorted from CD34+ cells by flow cytometry. A, HSCs, CMPs, age development. and GMPs were cultured in the presence of SCF and FLT-3. Cells were retrovirally transduced to ectopically express FIP1L1-PDGFRA. Transduced cells were plated into methylcellulose in the absence of cytokines and colony Discussion formation was analyzed after 12 d of culture. The total number of colonies was scored. B, lineage development in the percentage of CFU-E, CFU-GEMM, In this study, we have investigated for the first time, the CFU-GM, and CFU-Eo (pie charts). molecular mechanisms underlying FIP1L1-PDGFRA transforming ability in human hematopoietic progenitor cells. Our data show that expression of FIP1L1-PDGFRA in human hematopoietic cells and cell lysates were prepared. Expression of FIP1L1-PDFGRA progenitors induces not only eosinophil differentiation, but also induced phosphorylation of STAT5a, ERK1/2, p38MAPK, and PKB the development of other myeloid lineages including erythrocytes in the absence of cytokines (Fig. 4). Interestingly, whereas and neutrophils, resembling a myeloproliferative phenotype. expression of FIP1L1(1–29)-PDGFRA resulted in the activation of ERK1/2 and p38MAPK, STAT5a and PKB were not, or were undetectably phosphorylated, providing a potential mechanism for the differences observed in colony formation between both fusion proteins. STAT5 is necessary but not sufficient for FIP1L1-PDGFRA– induced colony formation. To investigate whether activation of STAT5a in CD34+ cells ectopically expressing FIP1L1-PDGFRA plays an important role in the induction of cytokine-independent colony formation, CD34+ cells expressing FIP1L1-PDGFRA were retrovirally transduced with STAT5aD750, a dominant-negative STAT5a (19). FIP1L1-PDGFRA–mediated colony formation was inhibited upon the expression of STAT5aD750 (Fig. 5A), however, dominant-negative STAT5a was insufficient to completely block colony formation, suggesting that other signal transduction pathways may play a critical role. This is supported by the observation that whereas FIP1L1(1–29)-PDGFRA does not activate STAT5, it is still able to induce some degree of myeloproliferation. Several studies have shown that activation of STAT5a was critical for the induction of differentiation in various hematopoietic lineages including eosinophils, neutrophils (20), and erythrocytes (21). To investigate whether constitutive activation of STAT5a was Figure 4. Activation of multiple signaling pathways by FIP1L1-PDGFRA. CD34+ indeed sufficient to induce cytokine-independent colony formation cells were cultured in the presence of SCF and FLT-3L for 2 d. Cells were + from hematopoietic progenitors, CD34 cells were retrovirally retrovirally transduced to ectopically express FIP1L1-PDGFRA or FIP1L1(1–29)- transduced to ectopically express constitutively active STAT5a PDGFRA, and cultured in the absence of cytokines. Protein lysates were made from transduced cells after 14 d of culture and Western blot analysis was done with (STAT5a1*6; ref. 22) or eGFP as a control. Transduced cells were antibodies against PDGFRA, tubulin phosphorylated PKB, phosphorylated sorted from the nontransduced cells and plated in methylcellulose STAT5, phosphorylated ERK1/2, and phosphorylated p38MAPK. www.aacrjournals.org 3763 Cancer Res 2007; 67: (8). April 15, 2007

the presence of IL-5 (Fig. 1F), suggesting that IL-5 may help induce the phenotype, but is not sufficient. Besides prolonged hypereosinophilia resulting in end-organ damage, patients with CEL also show increased serum tryptase levels and increased levels of atypical mast cells, distinct from systemic mastocytosis. In addition, distinct features including anemia, thrombocytopenia, neutrophilia, splenomegaly, marrow hypercellularity, and early myeloid precursors in peripheral blood smear have been observed in these patients (23, 24). Our experiments show that expression of FIP1L1-PDGFRA in HSCs and CMPs results in the formation of all myeloid lineages, whereas À À expression of the fusion protein in a Lin , CD34+, CD38+, CD45RA , and CD123lo GMP (16) population only resulted in high numbers of myeloblasts, neutrophils, and eosinophils without inducing erythrocyte development (Fig. 3). This supports the idea that the leukemic stem cell for CEL resides in a more committed population such as the GMP. However, it has recently been described that the FIP1L1-PDGFRA fusion gene can be found in both myeloid and

Figure 5. STAT5 is necessary but not sufficient to induce the FIP1L1-PDGFRA– mediated colony formation. A, CD34+ cells were cultured in the presence of IL-3 and IL-5 to induce eosinophil differentiation for 2 d. Cells were retrovirally transduced to ectopically express FIP1L1-PDGFRA. eGFP-positive cells were retrovirally transduced to ectopically express STAT5aD750 or DNGFR as a control. Transduced cells were again sorted from nontransduced cells and plated into methylcellulose in the presence of IL-3 and IL-5, and colony formation was analyzed after 12 d of culture. The total number of colonies was scored. B, CD34+ cells were cultured in the presence of SCF and FLT-3L for 2 d. Cells were retrovirally transduced to ectopically express STAT5a(1*6). Transduced cells (1,250) were plated into methylcellulose and cultured in the absence of cytokines and colony formation was analyzed after 12 d of culture. The total number of colonies was scored. C, lineage development in the percentage of CFU-E, CFU-GEMM, CFU-GM, and CFU-Eo (pie charts).

Recently, it has been shown that transplantation of mouse hematopoietic progenitors from IL-5 transgenic mice ectopically expressing FIP1L1-PDGFRA induces hypereosinophilia in mice including organ infiltration (14). In contrast, transplantation of normal mouse hematopoietic progenitors expressing FIP1L1- PDGFRA induces a myeloproliferative phenotype, suggesting that Figure 6. Combined inhibition of PI3K and ERK1/2 blocks FIP1L1-PDGFRA– induced colony formation. CD34+ cells were cultured either in the presence of IL-5 is critical for induction of CEL. In contrast to these IL-3 and IL-5 (A) to induce eosinophil differentiation or in the absence of observations, although the addition of IL-5 induced eosinophil cytokines (B). Cells were retrovirally transduced to ectopically express differentiation of human CD34+ cells expressing FIP1L1-PDGFRA in FIP1L1-PDGFRA. Cells (1,250) were plated into methylcellulose in the absence or presence of 10 Amol/L of LY294002, 10 Amol/L of SB20538, or 10 Amol/L of our experiments, the total number of eosinophil colonies was U0126, and colony formation was analyzed after 12 d of culture. The total actually slightly decreased compared with control cells cultured in number of colonies was scored.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. FIP1L1-PDGFRA Induces Myeloproliferation lymphoid cells (25), indicating that the initial mutation resulting in expression of FIP1L1-PDGFRA and FIP1L1(1–29)-PDGFR, whereas the expression of FIP1L1-PDGFRA may also occur in HSCs. Our STAT5a and PKB are only activated upon the expression of FIP1L1- experiments suggest that chromosomal translocation resulting in PDGFRA but not FIP1L1(1–29)-PDGFRA (Fig. 4). the expression of FIP1L1-PDGFRA would not be sufficient to Interestingly, a recent study showed that truncation of the first induce CEL in the absence of secondary factors or mutations. tryptophan residue in the juxtamembrane domain of PDGFRA was Interestingly, others have shown that in a mouse transplantation sufficient to induce cytokine-independent proliferation of Ba/F3 model, serial transplantation of the FIP1L1-PDGFRA–mediated cells, suggesting that FIP1L1 does not play a role in FIP1L1- disease was only successful using high cell numbers, suggesting PDGFRA–mediated transformation (15). This seems to be in that the expression of FIP1L1-PDGFRA does not induce self- contrast with earlier studies showing that deletion of amino acids 6 renewal of hematopoietic progenitors (14). This suggests that to 233 of FIP1L1 results in the expression of a protein unable to although the initial mutation in patients may occur in HSCs, induce cytokine-independent transformation of Ba/F3 cells (2). In secondary mutations resulting in increased self-renewal capacity addition, whereas the expression of a PDGFRA deletion mutant À À should occur in Lin , CD34+, CD38+, CD45RA , and CD123lo GMP consisting of one tryptophan residue was sufficient to induce cells or even more committed eosinophil progenitors. Interestingly, cytokine-independent proliferation, the level of proliferation of Krivtsov et al. recently described that expression of the MLL-AF9 these cells was reduced compared with cells expressing FIP1L1- fusion protein in a committed GMP progenitor was sufficient to PDGFRA. This suggests that truncation of the juxtamembrane induce leukemia in mice. Although retaining their normal GMP domain is involved in the induction of transformation, but that phenotype, the self-renewal capacity of these cells was induced on FIP1L1 may be critical for disease pathogenesis in CEL. the expression of MLL-AF9, suggesting that reactivation of self- Furthermore, although it is evident that the breakpoint in PDGFRA renewal in committed progenitors is both possible and sufficient to is located in the juxtamembrane region of most patients, deleting induce leukemia (26). one tryptophan residue, one patient has been described with a Patients diagnosed with FIP1L1-PDGFRA–mediated CEL are breakpoint located before the juxtamembrane domain leaving both currently treated with imatinib. Although imatinib results in tryptophan residues intact (3), suggesting that truncation of complete remission in many patients (8–10), clinical observations PDFGRA is not always sufficient to induce CEL. Interestingly, a suggest that imatinib also affects normal hematopoiesis. For novel t(4;17)(q12;q21) translocation has recently been discovered in example, grades 3 to 4 neutropenia has been observed in 14% of a case of juvenile myelomonocytic leukemia resulting in the Philadelphia chromosome–positive patients treated with imatinib, formation of a FIP1L1-RARa fusion protein (32). Because the whereas thrombocytopenia and anemia has been observed in 8% dimerization capacity of previously described RARa fusion and 3% of these patients, respectively (27). Several in vitro studies partners seems to be critical for the induction of leukemic confirm that imatinib can affect normal hematopoiesis. For transformation, it is likely that FIP1L1 also plays an important role example, both differentiation of dendritic cells from mobilized in the development of disease. peripheral blood–derived CD34+ cells (28), as well as proliferation We and others have previously shown that STAT5 plays an of T cells (29), are inhibited by imatinib. The normal expansion of important role in the regulation of the development of various CD34+ cells is also inhibited upon treatment with imatinib (30, 31). myeloid lineages (20). In this study, we have shown that STAT5 is Imatinib, in our experiments, does not induce the apoptosis of sufficient to induce the development of granulocyte-macrophage FIP1L1-PDGFRA–expressing cells, but apparently inactivates the colonies, erythrocyte colonies, and eosinophil colonies in the constitutively active tyrosine kinase, enabling cells to undergo absence of cytokines (Fig. 5). Colony size, however, was normal differentiation (Fig. 2). This is important for current clinical dramatically reduced compared with FIP1L1-PDFGRA–expressing therapies and indicates that imatinib treatment should be cells, indicating that other signal transduction pathways play an continued on remission to prevent relapse of the disease. However, important role in the regulation of progenitor expansion. Indeed, we also observed that the formation of granulocyte-macrophage combined inhibition of the PI3K and ERK1/2 pathway dramat- colonies from normal CD34+ cells was inhibited on treatment with ically reduced FIP1L1-PDGRFA–induced colony numbers (Fig. 6), imatinib (Fig. 2), suggesting that long-term treatment of patients suggesting that these pathways play an important role in with CEL using imatinib may ultimately result in the development controlling FIP1L1-PDGFRA–mediated progenitor expansion. Our of additional hematological disorders in these patients. data suggests that although activation of STAT5 may result in Expression of FIP1L1(1–29)-PDGFRA in primary human hema- cytokine-independent differentiation, PI3K and ERK1/2 are topoietic progenitors induced colony formation in the absence of needed for the expansion of transformed colonies. Indeed, both cytokines, however, the number of erythrocyte colonies was PI3K-PKB and ERK1/2 have been clearly shown to play critical reduced (Fig. 1). No differences were observed in granulocyte- roles in the regulation of proliferation in a variety of cell systems macrophage and eosinophil colony formation. This indicates that (33, 34). FIP1L1 plays a role in cytokine-independent development of some, Taken together, our results show that an interstitial deletion on but not all, myeloid lineages. Previous studies showing that FIP1L1 chromosome 4q12, resulting in the expression of FIP1L1-PDGFRA was dispensable for cytokine-independent proliferation were done in human hematopoietic progenitors is sufficient to induce in Ba/F3 cells, a mouse pro-B cell line (15). cytokine-independent myeloproliferation in human CD34+ progen- The function of FIP1L1 is incompletely understood. It could be itors. Investigation of the molecular mechanisms underlying hypothesized that FIP1L1 plays an important role in the regulation FIP1L1-PDGFRA–mediated lineage development revealed that of dimerization of the intracellular PDGFRA domain, normally combined activation of multiple signaling molecules including required for activation, resulting in constitutively active fusion PI3K, ERK1/2, and STAT5a plays an important role in the induction protein. Dimerization effects, however, are unlikely because a lack of of cytokine-independent colony formation. dimerization would reduce the activation of all signaling pathways. Imatinib inactivates FIP1L1-PDGFRA, enabling cells to undergo We have shown that both ERK1/2 and p38MAPK are activated upon normal differentiation, indicating that imatinib treatment should www.aacrjournals.org 3765 Cancer Res 2007; 67: (8). April 15, 2007

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Cancer Research be continued upon remission to prevent relapse of the disease. imatinib-resistant CEL and may reduce the potential side effects However, colony formation of control cells was also inhibited by of long-term imatinib treatment. imatinib, suggesting that long-term treatment of patients with CEL using imatinib affects normal hematopoiesis, which may ultimately result in the development of additional hematologic disorders in Acknowledgments these patients. It is therefore important to develop alternatives for Received 11/13/2006; revised 1/12/2007; accepted 2/2/2007. treatment with imatinib. Because cancer cells become highly Grant support: Dutch Cancer Society research grant UU2001-2491 and UU2005- dependent on aberrantly regulated intracellular signaling pathways, 3659. The costs of publication of this article were defrayed in part by the payment of page inhibition of the FIP1L1-PDGFRA–induced signaling pathways charges. This article must therefore be hereby marked advertisement in accordance might provide future alternative therapies for patients with with 18 U.S.C. Section 1734 solely to indicate this fact.

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