Centrosomal Targeting of Tyrosine Kinase Activity Does Not Enhance Oncogenicity in Chronic Myeloproliferative Disorders

Centrosomal targeting of tyrosine kinase activity does not enhance oncogenicity in chronic myeloproliferative disorders

T Bochtler1,2, M Kirsch1,2, B Maier1, J Bachmann3, U Klingmu¨ller3, S Anderhub1,ADHo2 and A Kra¨mer1,2

1Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center (DKFZ) and Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany; 2Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany and 3Division Systems Biology of Signal Transduction, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany

Constitutive tyrosine kinase activation by reciprocal chromo- translocation partner with its oligodimerization domains is fused somal translocation is a common pathogenetic mechanism in to the C-terminal part of the tyrosine kinase with its preserved chronic myeloproliferative disorders. Since centrosomal pro- catalytic domain. Different translocation partners fused to the teins have been recurrently identified as translocation partners of tyrosine kinases FGFR1, JAK2, PDGFRa and PDGFRb in same tyrosine kinase at identical breakpoints can induce subtly these diseases, a role for the centrosome in oncogenic different disease phenotypes. This suggests that the translocation transformation has been hypothesized. In this study, we partners have a specific pathogenetic role, which goes beyond addressed the functional role of centrosomally targeted simply providing oligodimerization motifs.3,6 tyrosine kinase activity. First, centrosomal localization was Markedly, many of the translocation partner proteins not routinely found for all chimeric fusion proteins tested. identified to date are localized to the centrosome, a cell Second, targeting of tyrosine kinases to the centrosome by organelle, which contributes to cell-cycle control and organizes creating artificial chimeric fusion kinases with the centrosomal 7–18 targeting domain of AKAP450 failed to enhance the oncogenic the mitotic spindle apparatus. As the primary microtubule transforming potential in both Ba/F3 and U2OS cells, although organizing center of most eukaryotic cells, the centrosome phospho-tyrosine-mediated signal transduction pathways were ensures symmetry and bipolarity of the cell division process, a initiated at the centrosome. We conclude that the centrosomal function that is essential for accurate chromosome segrega- localization of constitutively activated tyrosine kinases does tion.19 Furthermore, cell-cycle progression into mitosis is not contribute to disease pathogenesis in chronic myeloproli- initiated and controlled by cell-cycle regulatory proteins ferative disorders. 20,21 Leukemia (2012) 26, 728–735; doi:10.1038/leu.2011.283; localized to the centrosome. In addition, centrosome published online 21 October 2011 amplification occurs frequently in both solid tumors and hematological malignancies including chronic myeloid leuke- Keywords: chronic myeloproliferative disorder; tyrosine kinase; mia and is thought to contribute to the development of centrosome; PDGFR chromosomal abnormalities in these disorders.22–25 An overview on translocation partners of tyrosine kinases in Introduction CMD that localize to centrosomes is given in Table 1. The large number of centrosomally localized translocation partners has Chronic myeloproliferative disorders (CMDs) are clonal hemato- led to the hypothesis that centrosomal targeting of the tyrosine poietic stem cell diseases characterized by an increased kinase contributes to the oncogenic potential of fusion kinases in 4,26–28 proliferation of myeloid cells with a retained capacity for CMD. Although the frequent fusion of tyrosine kinases to differentiation.1 Typically, disease progression in CMD is translocation partner proteins with a centrosomal localization in reflected by an increase of immature cells and growing CMD is widely recognized, FOP-FGFR1 so far is the only 27,29 chromosomal instability. Constitutive activation of tyrosine chimeric fusion with a confirmed centrosomal localization. kinases ABL, FGFR1, JAK2, PDGFRa or PDGFRb has been FOP-FGFR1 has been shown to activate PI-3K, PLC-g, AKT and identified as a common underlying pathomechanism in CMD.1– STAT pathways at the centrosome and to promote cell-cycle 27,28 5 Structurally, with the exception of ABL these tyrosine kinases progression. However, the functional role of centrosomal belong to the receptor tyrosine kinase family and consist of an localization for the oncogenic potential of tyrosine kinases in extracellular ligand binding domain, a transmembrane domain, CMD is still poorly understood. In this study, we therefore an often autoinhibitory cytoplasmic juxtamembranous domain addressed this issue by artificially targeting tyrosine kinases to and a catalytically active C-terminal domain. centrosomes by fusion with the centrosomal targeting domain of Tyrosine kinase activation is either due to a single point AKAP450 (PACT, Pericentrin-AKAP450 Centrosomal Target- 21,30 31 mutation as in JAK2-V617F or to fusion of the tyrosine kinase ing) or the C-terminus of CEP164. with an oligodimerization domain-rich translocation partner. As exemplified by the reciprocal translocation t(9;22)(q34;q11), which results in a BCR-ABL fusion, the N-terminal portion of the Materials and methods

Correspondence: Professor A Kra¨mer, Clinical Cooperation Unit Plasmid cloning Molecular Hematology/Oncology, German Cancer Research Center The chimeric fusion kinases ninein-PDGFRb and NDE1- (DKFZ) and Department of Internal Medicine V, University of PDGFRb were obtained by cloning the respective ninein32 and Heidelberg, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany. E-mail: [email protected] NDE1 cDNAs (Gene Bank Accession numbers AY 515727 and Received 21 June 2011; revised 5 August 2011; accepted 9 August NM_017668, respectively) together with PDGFRb (accession 2011; published online 21 October 2011 number NM_002609) into the pEGFP-C1 vector (accession Centrosomes in myeloproliferative disorders T Bochtler et al 729 Table 1 Overview on translocation partners with proposed centrosomal localization in chronic myeloproliferative disorders

Tyrosine kinase Translocation partner Chromosomal translocation Reference

FGFR1 FOP t(6;8)(q27;p11) Guasch et al.7 CEP110 t(8;9)(p11;q33) Guasch et al.8 MYO18A t(8;17)(p11;q23) Walz et al.9 JAK2 PCM1 t(8;9)(p21–23;p23–24) Reiter et al.,10 Murati et al.,11 Bousquet et al.12 PDGFRa CDK5RAP2 ins(9;4)(q33;q12q25) Walz et al.13 PDGFRb PDE4DIP t(1;5)(q23;q33) Wilkinson et al.14 NINEIN t(5;14)(q33;q24) Vizmanos et al.15 TRIP11 t(5;14)(q33;q32) Abe et al.16 TPE53BP1 t(5;15)(q33;q22) Grand et al.17 NDE1 t(5;16)(q33;p13) La Starza et al.18 number U55763) (Clontech, Saint-Germain-en-Laye, France). were transiently transfected using the calcium-phosphate CDK5RAP2-PDGFRa was provided as a full-length cDNA.13 method to produce retroviral particles. Ba/F3 cells were then Full-length ninein, NDE1 and CDK5RAP2 (accession number transduced with retroviral particle-containing supernatants in a BX 537421) cDNAs as well as their respective breakpoint two-step procedure. First, the pMOWSIN-TREt and in a second variants, which consist of the respective cDNA fragments step the pMOWS-rtTA35 vector containing the tetracycline conserved in the fusion protein, were also cloned into pEGFP- transactivator protein were transduced. Subsequently, clones C1. To generate tyrosine kinase-PACT-FLAG fusions, the were selected using G418 and puromycin. C-terminal portion of the respective tyrosine kinases preserved in the leukemic fusion proteins as well as the PACT domain Immunofluorescence comprising the C-terminal amino acids 3643–3808 of AKAP450 U2OS cells grown on glass coverslips were fixed in À20 1C (accession number AJ131693.1) were cloned into the pCMV-Tag methanol/acetone (1:1) for 7 min. HeLa cells employed for 4A vector (Stratagene, Santa Clara, CA, USA).21,30 For CEP164 CDK5RAP2 subcellular localization experiments were permea- fusion constructs, C-terminal amino acids 1201–1461 of CEP164 bilized in PHEM buffer with 0.5% Triton X-100 before fixation (accession number NM_014956) were used for centrosomal with À20 1C methanol/acetone (1:1) for 10 min. Indirect im- targeting instead of PACT.31 Kinase-dead (KD) variants of munofluorescence staining was performed using the following PDGFRb-PACT-FLAG and PDGFRa-PACT-FLAG were obtained mouse monoclonal antibodies and rabbit polyclonal antibodies: by introducing lysine to arginine mutations in the respective anti-GFP (sc-9996, Santa Cruz, Heidelberg, Germany), anti-FLAG catalytic tyrosine kinase domainsFK634R in PDGFRb and M2 (F1804, Sigma, Munich, Germany), anti-g-tubulin (TU-30, K627R in PDGFRa, respectively, by site-directed mutagenesis Exbio, Prague, Czech Republic), anti-a-tubulin (T6199, Sigma), (Quick Change Site Directed Mutagenesis Kit; Stratagene).33 anti-pericentrin (ab4448, Abcam, Cambridge, UK), anti-PRC-1 (sc-8356, Santa Cruz) and anti-phospho-tyrosine (05-321X, Cell culture Upstate, Waltham, MA, USA). Secondary antibodies were from Molecular Probes (Darmstadt, Germany); (Alexa Fluor 488, U2OS cellsFa human osteosarcoma-derived cell line with a highly cross-adsorbed) or from Jackson (ImmunoResearch, complex aberrant karyotype and wild-type (WT) p53 expression Newmarket, UK) (Cy3 conjugated). Cells were mounted in (ATCC, Wesel, Germany)Fwere maintained in Dulbecco’s Vectashield with DAPI (H 1200; Vector Laboratories, Lo¨rrach, modified Eagle’s medium supplemented with 10% fetal calf Germany). Fluorescence images were captured and processed serum. Ba/F3 cellsFa murine interleukin-3 (IL-3)-dependent using a Zeiss Axiovert 200 M microscope (Zeiss, Go¨ttingen, cell line with a near diploid karyotype and 33% polyploidy Germany) and Axiovison Software (Zeiss). (DSMZ, Braunschweig, Germany)Fwere cultured in RPMI- 1640 with 10% fetal calf serum. Except for growth factor withdrawal experiments, Ba/F3 cells, in addition, received 10% Results conditioned medium from WEHI cells. Transient transfection of plasmid constructs into U2OS cells was performed by lipo- Subcellular localization of chimeric fusion proteins fection using FuGENE (Roche, Basel, Switzerland). Imatinib To assess the subcellular localization of leukemic fusion (Roche) was used at a concentration of 20 mM. proteins, GFP- or FLAG-tagged versions of three fusion proteins (ninein-PDGFRb, NDE1-PDGFRb and CDK5RAP2-PDGFRa) were transiently transfected into U2OS cells. As controls, the Generation of stable tetracycline-responsive cell lines respective full-length centrosomal proteins and their N-terminal Fusion kinases with a C-terminal FLAG tag were cloned into parts conserved in the leukemic fusionsFreferred to as break- pBI-3,34 a vector with a tetracycline-responsive promoter. point variantsFwere transfected as well. Following linearization, these constructs were co-transfected As shown in Figure 1a, full-length ninein, ninein-PDGFRb and with a linearized resistance marker (Clontech, Cat. No. 631625) the ninein breakpoint variant localize to the centrosome as into a U2OS Tet-On Cell Line (Clontech, Cat. No. 630919) at a revealed by colocalization with g-tubulin as a centrosomal molar ratio of 20:1 using FuGENE (Roche). Stable clones were marker. To the contrary, both FLAG- and GFP-tagged versions of selected using G418 and hygromycin. For the establishment of NDE1-PDGFRb and GFP-CDK5RAP2-PDGFRa displayed a stable Ba/F3 clones retroviral transduction was used. The inserts diffuse cytoplasmic fluorescence but did not localize to the were cloned into pMOWSIN-TREt,35 a retroviral vector with a centrosome (Figure 1b and data not shown) whereas a GFP- tetracycline-responsive promoter. Phoenix-eco packaging cells tagged full-length version of CDK5RAP2, which was used as a

Leukemia Centrosomes in myeloproliferative disorders T Bochtler et al 730

Figure 1 Subcellular localization of chimeric fusion proteins. (a) Subcellular localization of ninein constructs. Full-length GFP-ninein, the GFP- ninein breakpoint variant, and the ninein-PDGFRb-FLAG fusion localize to the centrosome. (b) Subcellular localization of CDK5RAP2 constructs. Whereas full-length GFP-CDK5RAP2 and the GFP-CDK5RAP2 breakpoint variant localize to the centrosome, the GFP-CDK5RAP2-PDGFRa fusion does not show centrosomal localization. Images were obtained 24 h after transient transfection of U2OS (a) or HeLa (b) cells using lipofection. Cells were co-immunostained using an antibody against g-tubulin for detection of centrosomes. The scale bars denote 20 mm.

control, did show centrosomal localization. Also, the under the control of a tetracyline-responsive promoter could CDK5RAP2 breakpoint variant comprising its 494 N-terminal be established. Effective centrosomal targeting was conﬁrmed amino acids localized to the centrosome. by colocalization with g-tubulin (Figure 2). Whereas stable transfection experiments yielded a total of 15 (KD)PDGFRa- PACT-FLAG clones with centrosomal localization of the fusion Generation of stable cell lines expressing centrosomally protein, none out of 58 clones analyzed revealed centrosomal targeted tyrosine kinases targeting of the (WT)PDGFRa-PACT-FLAG fusion. Centrosomal To address a possible functional role of tyrosine kinases at targeting of (WT)PDGFRa-PACT-FLAG was also not enabled by centrosomes, the C-terminal parts of tyrosine kinases PDGFRa substituting the PACT domain by the C-terminus of CEP164 and PDGFRb preserved in the respective leukemic fusions were (data not shown). targeted to the centrosome by fusion either with the PACT domain of AKAP450(ref.30)Fan immobile protein of the pericentriolar matrixFor with the centrosomal targeting do- In U2OS cells, centrosomally targeted tyrosine kinase main of CEP164, containing its C-terminal 260 amino acids.31 activity induces tyrosine phosphorylation at the U2OS clones conditionally expressing KD versions of centrosome, but confers no proliferative advantage PDGFRa-PACT-FLAG and PDGFRb-PACT-FLAG as well as Immunostaining with a pan-phospho-tyrosine antibody revealed (WT)PDGFRb-PACT-FLAG but not (WT)PDGFRa-PACT-FLAG that expression of (WT)PDGFRb-PACT-FLAG induced tyrosine

Leukemia Centrosomes in myeloproliferative disorders T Bochtler et al 731

Figure 2 Conditional expression of tyrosine kinases targeted to the centrosome by fusion with the PACT domain of AKAP450 in U2OS cells. Like the GFP-PACT positive control, KD versions of both PDGFRa-PACT-FLAG and PDGFRb-PACT-FLAG as well as (WT)PDGFRb-PACT-FLAG were effectively targeted to the centrosome. Images were obtained 48 h after inducing the respective U2OS clones with doxycyclin. The scale bar denotes 20 mm.

Figure 3 Tyrosine phosphorylation at the centrosome as surrogate marker for activation of centrosomal downstream signaling pathways. Kinase active centrosomally targeted PDGFRb induces tyrosine phosphorylation at the centrosome; images were obtained 48 h after doxycyclin induction of cells conditionally expressing (WT)PDGFRb-PACT-FLAG. (WT)PDGFRb-PACT-FLAG cells treated with 20 mM imatinib 24 h before ﬁxation as well as (KD)PDGFRb-PACT-FLAG cells were used as controls. The scale bar denotes 20 mm. phosphorylation at the centrosome in 81.3±3.5% of cells, However, tyrosine phosphorylation at the centrosome in which could be abrogated by concomitant treatment with the (WT)PDGFRb-PACT-FLAG expressing U2OS cells did not tyrosine kinase inhibitor imatinib (Figure 3). Tyrosine phosphor- translate into a proliferative advantage. Mitotic indices as ylation was neither detectable in (WT)PDGFRb-PACT-FLAG determined by immunoﬂuorescence microscopy in cells in the absence of doxycylin nor in U2OS cells expressing (WT)PDGFRb-PACT-FLAG versus (KD)PDGFRb-PACT-FLAG (KD)PDGFRb-PACT-FLAG as a control. expressing U2OS cells were virtually identical (1.73% versus

Leukemia Centrosomes in myeloproliferative disorders T Bochtler et al 732

Figure 4 Conditional expression of tyrosine kinases targeted to the centrosome by fusion with the PACT domain of AKAP450 in Ba/F3 cells. Whereas (KD)PDGFRa-PACT and (KD)PDGFRb-PACT localized to the centrosome, only a minority of (WT)PDGFRb-PACT expressing cells displayed centrosomal targeting of the construct. (WT)PDGFRa-PACT did not localize to the centrosome at all. The scale bar denotes 20 mm.

1.60%, P ¼ 0.66). In addition, targeting of (WT)PDGFRb tyrosine PACT-FLAG clearly localized to centrosomes, effective centro- kinase to the centrosome did not lead to significant centrosome somal localization of (WT)PDGFRb-PACT-FLAG was found in amplification nor did it induce the formation of aberrant mitotic only a fraction of Ba/F3 cells. spindles. As judged by co-immunostaining with an antibody to With IL-3 supplementation, all cell lines showed similar g-tubulin, expression of (WT)PDGFRb-PACT-FLAG led to proliferation and cell-cycle characteristics after doxycyclin centrosome amplification in 11±1.6%, while (KD)PDGFRb- induction of construct expression (data not shown). Upon IL-3 PACT-FLAG induced aberrant centrosomes in 7±2.8% (P ¼ 0.2) withdrawal, the original leukemic fusion proteins FIP1L1- of U2OS cells 14 days after transgene induction. PDGFRa and TEL-PDGFRb were able to maintain growth factor-independent proliferation as determined by counting viable cells using trypan blue exclusion (Figure 5). Similarly, Targeting of tyrosine kinases to the centrosome does not (WT)PDGFRa-PACT-FLAG as well as (WT)PDGFRa-FLAG with- lead to growth factor independence in Ba/F3 cells out PACT domainFboth containing breakpoint-truncated To test whether tyrosine kinase activity targeted to centrosomes PDGFRaFdid confer growth factor independence. Expectedly, confers growth factor-independent proliferation of myeloid both (KD)PDGFRa-PACT-FLAG and (KD)PDGFRb-PACT-FLAG cells, we established Ba/F3 cell lines conditionally expressing failed to induce IL-3-independent proliferation. (WT)PDGFRa-PACT-FLAG and (WT)PDGFRb-PACT-FLAG. In Markedly, however, the (WT)PDGFRb-PACT-FLAG fusion addition, Ba/F3 lines expressing KD versions targeted to that contains breakpoint-truncated PDGFRb fused to the centrosomes ((KD)PDGFRa-PACT-FLAG and (KD)PDGFRb- centrosomal targeting domain of AKAP450 failed to induce IL- PACT-FLAG), WT tyrosine kinase breakpoint variants without 3-independent growth, although the cells retained their viability. the PACT domain ((WT)PDGFRa-FLAG and (WT)PDGFRb- Ba/F3 cells expressing (WT)PDGFRb-FLAG without centrosomal FLAG) and the respective original leukemic fusion kinases targeting domain were not viable. FIP1L1-PDGFRa and Tel-PDGFRb were established as controls. As in U2OS cells, centrosomal localization of the PACT domain-containing constructs in Ba/F3 cells was verified by Discussion colocalization of FLAG and g-tubulin signals. Again, effective centrosomal targeting could only consistently be demonstrated In this study, we addressed the question whether the recurrent for the KD version of PDGFRa-PACT-FLAG, but not for the occurrence of centrosomal proteins as translocation partners of respective WT fusion (Figure 4). Also, whereas (KD)PDGFRb- tyrosine kinases in CMD is due to a specific interaction with

Leukemia Centrosomes in myeloproliferative disorders T Bochtler et al 733 partner as well as the addition of tyrosine kinase domains instead. In detail, the ninein-PDGFRb fusion, in which 2037 of the 2107 amino acids of ninein are conserved including its centrosomal targeting domain,36 does clearly localize to the centrosome. To the contrary, CDK5RAP2-PDGFRaFan inframe fusion between the N-terminal 494 out of 1205 amino acids of CDK5RAP2 and truncated exon 12 of PDGFRa, cloned from a patient with chronic eosinophilic leukemia13–displayed a homogenous cytoplasmic localization without enrichment at centrosomes, although centrosomal localization of full-length CDK5RAP2 itself was confirmed in our study.37,38 Similarly, NDE1-PDGFRb18 also localized to the cytoplasm without centrosomal enrichment. In previous studies, FOP-FGFR1 was shown to localize to the centrosome, whereas the centrosomal localization of CEP110-FGFR1 was rather equivocal.8,27 In conclusion, the subcellular localization is variable among individual CMD chimeric fusion proteins. Centrosomal localization, therefore, does not appear to be necessary for the effects conferred by constitutive tyrosine kinase activation. To test for the oncogenic potential of individual tyrosine kinases at centrosomes we designed artificial chimeric fusion kinases, which substituted the centrosomal translocation partner by a mere centrosomal targeting domain derived from either AKAP450 (PACT) or CEP164 proteins. Using these constructs enabled us to test whether centrosomal targeting of tyrosine kinases by itself is sufficient to confer a proliferative advantage. After expression of these constructs into U2OS cells, we could demonstrate (i) effective targeting of PDGFRb- but not PDGFRa- associated tyrosine kinase activity to the centrosome and (ii) subsequent phosphorylation of proteins at centrosomes which could be abrogated by the tyrosine kinase inhibitor imatinib for PDGFRb. Similar findings have been reported for FOP-FGFR1 earlier.27 However, phosphorylation of centrosomal substrates did not translate into a proliferative advantage of myeloid Ba/F3 cells, which are commonly used to assess growth factor-independent proliferation as a measure of oncogenic transformation. Specifically, as expected the original leukemic fusion proteins FIP1L1-PDGFRa and TEL-PDGFRb conferred IL-3-independent proliferation in this system. In full accordance with a study by Stover et al.,39 truncated PDGFRa, bereft of its autoinhibitory WW domain, was constitutively active alike. However, its transforming potential was not further enhanced by centrosomal localization, as Ba/F3 cells showing effective centrosomal Figure 5 Proliferation kinetics of Ba/F3 cells conditionally expressing targeting of this construct could never be obtained. tyrosine kinase constructs. The respective Ba/F3 cell clones were Unlike PDGFRa, the truncated form of PDGFRb, which induced with doxycyclin at day À1. At day 0, the WEHI conditioned retains its autoinhibitory juxtamembranous region, displays no medium was withdrawn. Cells were resuspended in RPMI-1640 plus 40 10% fetal calf serum without WEHI at 2 Â 105 cells/ml. Viable cells oncogenic transforming potential by itself and depends on were subsequently counted on days 2, 4 and 6 by trypan blue self-association by domains of the translocation partner for exclusion. Subsequently, cell concentrations were again adjusted to signaling. Accordingly, in contrast to PDGFRa, clones expres- 5 2 Â 10 /ml for proliferating clones. The proliferation kinetics are sing (WT)PDGFRb-PACT with centrosomal localization of the displayed on a logarithmical scale. The error bars indicate the s.d. of construct could be generated, although with low efficacy. the assay, which was performed three times in triplicate each. Whereas the leukemic fusions FIP1L1-PDGFRa and TEL-PDGFRb as However, although preserving cell viability, possibly due to low well as breakpoint-truncated PDGFRa confer growth factor-indepen- efficacy oligomerization brought about by the PACT domain, it dent proliferation, Ba/F3 cells expressing breakpoint-truncated did not trigger growth factor-independent growth. We conclude PDGFRb or KD constructs died. (WT)PDGFRb-PACT confers growth that centrosomal localization by itself does not promote factor-independent viability though not proliferation. PDGFRa or PDGFRb oncogenicity. As shown by the superiority of the TEL-PDGFRb fusion over our artificial PDGFRb-PACT construct, centrosomal localization by itself cannot compensate centrosomal function or whether it is merely a reflection of their for the loss of the translocation partner with its oligodimeriza- wide expression and their oligodimerization domain-rich motifs. tion domains. In a first step, we showed that centrosomal localization of the One downside of our experimental setting needs to be translocation partner does not necessarily entail centrosomal discussed. Whereas clones expressing kinase-inactive variants localization of the entire fusion protein. This might be due to the of centrosomally targeted kinases PDGFRa and PDGFRb were loss of C-terminal domains of the centrosomal translocation easily obtained for both U2OS and Ba/F3 cells, centrosomal

Leukemia Centrosomes in myeloproliferative disorders T Bochtler et al 734 targeting of the respective WT kinases appeared to be toxic to CDK5RAP2-PDGFRa plasmid and M Bornens and Y Hong for both cell types, thereby impeding the retrieval of the respective ninein plasmids. The pBI-3 vector was kindly provided by H clones. We could rule out a PACT domain-specific phenomen- Bujard. We are grateful to the Deutsche Jose´ Carreras Leuka¨mie- on, since the same difficulties were encountered when choosing Stiftung for financial support (DJCLS R 06/04). the centrosomal targeting domain of CEP164 as an alternative strategy. Therefore, this problem seems inherent to tyrosine kinase activity. We believe that the vulnerability of cells to References centrosomal targeting of tyrosine kinases argues against a pivotal role of centrosomally localized constitutive tyrosine kinase 1 Delhommeau F, Pisani DF, James C, Casadevall N, Constantinescu activity in CMD. Tight regulation of tyrosine kinase activity has S, Vainchenker W. Oncogenic mechanisms in myeloproliferative also been proven crucial for maintaining centrosomal integrity: disorders. Cell Mol Life Sci 2006; 63: 2939–2953. both constitutive BCR-ABL activation and abrogation of tyrosine 2 De Keersmaecker K, Cools J. Chronic myeloproliferative disorders: kinase activity by specific inhibitors have been shown to lead to a tyrosine kinase tale. Leukemia 2006; 20: 200–205. centrosomal aberrations.25,41–43 3 Macdonald D, Cross NC. Chronic myeloproliferative disorders: the F role of tyrosine kinases in pathogenesis, diagnosis and therapy. We do not regard our conclusion drawn from a functional Pathobiology 2007; 74: 81–88. point of viewFas contradictory to previous studies. These have 4 Reiter A, Invernizzi R, Cross NC, Cazzola M. Molecular basis of hypothesized a functional role of centrosomal tyrosine kinase myelodysplastic/myeloproliferative neoplasms. Haematologica activity based on the centrosomal localization of fusion proteins 2009; 94: 1634–1638. and have particularly relied on the detection of centrosomal 5 Tefferi A, Gilliland DG. Oncogenes in myeloproliferative signal transduction.26–28 The centrosomal phospho-tyrosine disorders. Cell Cycle 2007; 6: 550–566. 6 Roumiantsev S, Krause DS, Neumann CA, Dimitri CA, Asiedu F, staining pattern following expression of centrosomally targeted Cross NC et al. Distinct stem cell myeloproliferative/T lymphoma PDGFRb in U2OS cells in our study clearly recapitulates syndromes induced by ZNF198-FGFR1 and BCR-FGFR1 fusion tyrosine phosphorylation of proteins at the centrosome.26–28 genes from 8p11 translocations. Cancer Cell 2004; 5: 287–298. However, in our study phospho-tyrosine signaling did not 7 Guasch G, Ollendorff V, Borg JP, Birnbaum D, Pebusque MJ. 8p12 translate into a proliferative advantage. This questionFwhether stem cell myeloproliferative disorder: the FOP-fibroblast growth the transforming property depends on centrosomal targeting of factor receptor 1 fusion protein of the t(6;8) translocation induces F cell survival mediated by mitogen-activated protein kinase and the fusion kinase has never been addressed before. phosphatidylinositol 3-kinase/Akt/mTOR pathways. Mol Cell Biol If centrosomal targeting of tyrosine kinases does not contribute 2001; 21: 8129–8142. to a proliferative advantage, what then are the decisive 8 Guasch G, Mack GJ, Popovici C, Dastugue N, Birnbaum D, characteristics of translocation partner proteins with centrosomal Rattner JB et al. FGFR1 is fused to the centrosome-associated localization? Previous studies have demonstrated the relevance of protein CEP110 in the 8p12 stem cell myeloproliferative disorder coiled-coil or self-association motifs within translocation partners with t(8;9)(p12;q33). Blood 2000; 95: 1788–1796. 9 Walz C, Chase A, Schoch C, Weisser A, Schlegel F, Hochhaus A for oligodimerization both in vitro and in vivo in murine bone et al. The t(8;17)(p11;q23) in the 8p11 myeloproliferative 40,44–46 marrow transplantation models. Beyond that a specific role syndrome fuses MYO18A to FGFR1. Leukemia 2005; 19: of individual translocation partners is suggested by their impact on 1005–1009. the disease phenotype. For example, in FGFR1-related disorders, 10 Reiter A, Walz C, Watmore A, Schoch C, Blau I, Schlegelberger B patients with an underlying t(8;13), which involves ZNF198, et al. The t(8;9)(p22;p24) is a recurrent abnormality in chronic and present with T-lymphoblastic lymphoma, whereas an underlying acute leukemia that fuses PCM1 to JAK2. Cancer Res 2005; 65: 2662–2667. t(8;22), which involves BCR, predisposes to a CML-like pheno- 11 Murati A, Gelsi-Boyer V, Adelaide J, Perot C, Talmant P, Giraudier 47 type. The finding that two different translocation partners fused S et al. PCM1-JAK2 fusion in myeloproliferative disorders and to the same tyrosine kinase at its same breakpoint can induce two acute erythroid leukemia with t(8;9) translocation. Leukemia 2005; distinct disease phenotypes was also confirmed in mouse bone 19: 1692–1696. marrow transplantation models and argues in favor of a specific 12 Bousquet M, Quelen C, De Mas V, Duchayne E, Roquefeuil B, role of the translocation partner but against centrosomal targeting Delsol G et al. The t(8;9)(p22;p24) translocation in atypical 6 chronic myeloid leukaemia yields a new PCM1-JAK2 fusion gene. as a common mechanism of action. At the molecular level, Oncogene 2005; 24: 7248–7252. specific downstream phosphorylation targets have been identified 13 Walz C, Curtis C, Schnittger S, Schultheis B, Metzgeroth G, Schoch for the ZNF-FGFR1 leukemic fusion as opposed to WT FGFR1.48 C et al. Transient response to imatinib in a chronic eosinophilic In conclusion, the fusion of a tyrosine kinase to a centrosomal leukemia associated with ins(9;4)(q33;q12q25) and a CDK5RAP2- translocation partner is a recurring phenomenon in CMD. PDGFRA fusion gene. Genes Chromosomes Cancer 2006; 45: However, the translocation partner does not regularly target the 950–956. 14 Wilkinson K, Velloso ER, Lopes LF, Lee C, Aster JC, Shipp MA et al. chimeric fusion to the centrosome. When addressed to Cloning of the t(1;5)(q23;q33) in a myeloproliferative disorder centrosomes, tyrosine kinase activity initiates localized signal associated with eosinophilia: involvement of PDGFRB and transduction, but fails to ignite transforming potential, which response to imatinib. Blood 2003; 102: 4187–4190. appears to rather depend on oligodimerization domains brought 15 Vizmanos JL, Novo FJ, Roman JP, Baxter EJ, Lahortiga I, Larrayoz about by translocation partner domains. MJ et al. NIN, a gene encoding a CEP110-like centrosomal protein, is fused to PDGFRB in a patient with a t(5;14)(q33;q24) and an imatinib-responsive myeloproliferative disorder. Cancer Res 2004; Conflict of interest 64: 2673–2676. 16 Abe A, Emi N, Tanimoto M, Terasaki H, Marunouchi T, Saito H. The authors declare no conflict of interest. Fusion of the platelet-derived growth factor receptor beta to a novel gene CEV14 in acute myelogenous leukemia after clonal evolution. Blood 1997; 90: 4271–4277. 17 Grand FH, Burgstaller S, Kuhr T, Baxter EJ, Webersinke G, Thaler J Acknowledgements et al. p53-Binding protein 1 is fused to the platelet-derived growth factor receptor beta in a patient with a t(5;15)(q33;q22) and We thank D Gilliland and E Stover for gently providing an imatinib-responsive eosinophilic myeloproliferative disorder. Tel-PDGFRb and FIP1L1-PDGFRa plasmids, A Reiter for the Cancer Res 2004; 64: 7216–7219.

Leukemia Centrosomes in myeloproliferative disorders T Bochtler et al 735 18 La Starza R, Rosati R, Roti G, Gorello P, Bardi A, Crescenzi B et al. 35 Pfeifer AC, Kaschek D, Bachmann J, Klingmuller U, Timmer J. A new NDE1/PDGFRB fusion transcript underlying chronic Model-based extension of high-throughput to high-content data. myelomonocytic leukaemia in Noonan Syndrome. Leukemia BMC Syst Biol 2010; 4: 106. 2007; 21: 830–833. 36 Chen CH, Howng SL, Cheng TS, Chou MH, Huang CY, Hong YR. 19 Luders J, Stearns T. Microtubule-organizing centres: a re-evalua- Molecular characterization of human ninein protein: two distinct tion. Nat Rev Mol Cell Biol 2007; 8: 161–167. subdomains required for centrosomal targeting and regulating 20 Doxsey S, Zimmerman W, Mikule K. Centrosome control of the signals in cell cycle. Biochem Biophys Res Commun 2003; 308: cell cycle. Trends Cell Biol 2005; 15: 303–311. 975–983. 21 Kramer A, Mailand N, Lukas C, Syljuasen RG, Wilkinson CJ, 37 Bond J, Roberts E, Springell K, Lizarraga SB, Scott S, Higgins J et al. Nigg EA et al. Centrosome-associated Chk1 prevents premature A centrosomal mechanism involving CDK5RAP2 and CENPJ activation of cyclin-B-Cdk1 kinase. Nat Cell Biol 2004; 6: 884–891. controls brain size. Nat Genet 2005; 37: 353–355. 22 Nigg EA. Centrosome aberrations: cause or consequence of cancer 38 Fong KW, Choi YK, Rattner JB, Qi RZ. CDK5RAP2 is a progression? Nat Rev Cancer 2002; 2: 815–825. pericentriolar protein that functions in centrosomal attachment of 23 Pihan GA, Purohit A, Wallace J, Knecht H, Woda B, Quesenberry the gamma-tubulin ring complex. Mol Biol Cell 2008; 19: 115–125. P et al. Centrosome defects and genetic instability in malignant 39 Stover EH, Chen J, Folens C, Lee BH, Mentens N, Marynen P et al. tumors. Cancer Res 1998; 58: 3974–3985. Activation of FIP1L1-PDGFRalpha requires disruption of the 24 Neben K, Giesecke C, Schweizer S, Ho AD, Kramer A. juxtamembrane domain of PDGFRalpha and is FIP1L1-indepen- Centrosome aberrations in acute myeloid leukemia are correlated dent. Proc Natl Acad Sci USA 2006; 103: 8078–8083. with cytogenetic risk proﬁle. Blood 2003; 101: 289–291. 40 Carroll M, Tomasson MH, Barker GF, Golub TR, Gilliland DG. The 25 Giehl M, Fabarius A, Frank O, Hochhaus A, Hafner M, Hehlmann TEL/platelet-derived growth factor beta receptor (PDGF beta R) R et al. Centrosome aberrations in chronic myeloid leukemia fusion in chronic myelomonocytic leukemia is a transforming correlate with stage of disease and chromosomal instability. protein that self-associates and activates PDGF beta R kinase- Leukemia 2005; 19: 1192–1197. dependent signaling pathways. Proc Natl Acad Sci USA 1996; 93: 26 Delaval B, Lelievre H, Birnbaum D. Myeloproliferative disorders: 14845–14850. the centrosome connection. Leukemia 2005; 19: 1739–1744. 41 Giehl M, Fabarius A, Frank O, Erben P, Zheng C, Hafner M et al. 27 Delaval B, Letard S, Lelievre H, Chevrier V, Daviet L, Dubreuil P Expression of the p210BCR-ABL oncoprotein drives centrosomal et al. Oncogenic tyrosine kinase of malignant hemopathy targets hypertrophy and clonal evolution in human U937 cells. Leukemia the centrosome. Cancer Res 2005; 65: 7231–7240. 2007; 21: 1971–1976. 28 Lelievre H, Chevrier V, Tassin AM, Birnbaum D. Myeloprolifera- 42 Fabarius A, Giehl M, Frank O, Spiess B, Zheng C, Muller MC et al. tive disorder FOP-FGFR1 fusion kinase recruits phosphoinositide-3 Centrosome aberrations after nilotinib and imatinib treatment in kinase and phospholipase Cgamma at the centrosome. Mol Cancer vitro are associated with mitotic spindle defects and genetic 2008; 7: 30. instability. Br J Haematol 2007; 138: 369–373. 29 Mikolajka A, Yan X, Popowicz GM, Smialowski P, Nigg EA, Holak 43 Fabarius A, Giehl M, Rebacz B, Kramer A, Frank O, Haferlach C TA. Structure of the N-terminal domain of the FOP (FGFR1OP) et al. Centrosome aberrations and G1 phase arrest after in vitro and protein and implications for its dimerization and centrosomal in vivo treatment with the SRC/ABL inhibitor dasatinib. Haema- localization. J Mol Biol 2006; 359: 863–875. tologica 2008; 93: 1145–1154. 30 Gillingham AK, Munro S. The PACT domain, a conserved 44 He Y, Wertheim JA, Xu L, Miller JP, Karnell FG, Choi JK et al. The centrosomal targeting motif in the coiled-coil proteins AKAP450 coiled-coil domain and Tyr177 of bcr are required to induce a and pericentrin. EMBO Rep 2000; 1: 524–529. murine chronic myelogenous leukemia-like disease by bcr/abl. 31 Graser S, Stierhof YD, Lavoie SB, Gassner OS, Lamla S, Le Clech M Blood 2002; 99: 2957–2968. et al. Cep164, a novel centriole appendage protein required for 45 Bischof D, Pulford K, Mason DY, Morris SW. Role of the primary cilium formation. J Cell Biol 2007; 179: 321–330. nucleophosmin (NPM) portion of the non-Hodgkin’s lymphoma- 32 Delgehyr N, Sillibourne J, Bornens M. Microtubule nucleation and associated NPM-anaplastic lymphoma kinase fusion protein in anchoring at the centrosome are independent processes linked by oncogenesis. Mol Cell Biol 1997; 17: 2312–2325. ninein function. J Cell Sci 2005; 118 (Pt 8): 1565–1575. 46 Xiao S, McCarthy JG, Aster JC, Fletcher JA. ZNF198-FGFR1 33 Schwaller J, Anastasiadou E, Cain D, Kutok J, Wojiski S, transforming activity depends on a novel proline-rich ZNF198 Williams IR et al. H4(D10S170), a gene frequently rearranged oligomerization domain. Blood 2000; 96: 699–704. in papillary thyroid carcinoma, is fused to the platelet-derived 47 Jackson CC, Medeiros LJ, Miranda RN. 8p11 myeloproliferative growth factor receptor beta gene in atypical chronic syndrome: a review. Hum Pathol 2010; 41: 461–476. myeloid leukemia with t(5;10)(q33;q22). Blood 2001; 97: 48 Kasyapa C, Gu TL, Nagarajan L, Polakiewicz R, Cowell JK. 3910–3918. Phosphorylation of the SSBP2 and ABL proteins by the ZNF198- 34 Baron U, Freundlieb S, Gossen M, Bujard H. Co-regulation of two FGFR1 fusion kinase seen in atypical myeloproliferative disorders gene activities by tetracycline via a bidirectional promoter. as revealed by phosphopeptide-speciﬁc MS. Proteomics 2009; 9: Nucleic Acids Res 1995; 23: 3605–3606. 3979–3988.

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