Oncogenic Tyrosine Kinase of Malignant Hemopathy Targets the Centrosome

Research Article

Be´ne´dicte Delaval,1 Se´bastien Le´tard,2 He´le`ne Lelie`vre,1 Ve´ronique Chevrier,3 Laurent Daviet,4 Patrice Dubreuil,2 and Daniel Birnbaum1

Laboratories of 1Molecular Oncology and 2Molecular Hematopoiesis, Marseille Cancer Institute, UMR599 Inserm and Institut Paoli-Calmettes, Marseilles, France; 3U366 Inserm, Grenoble; and 4Hybrigenics S.A., Paris, France

Abstract fusions. The receptor transmembrane region is not conserved in the fusion. X-FGFR1 proteins promote cell survival through Myeloproliferative disorders (MPD) are malignant diseases of g hematopoietic progenitor cells. Many MPDs result from a signaling pathways involving, among others, phospholipase C g chromosomal translocation that creates a fusion gene encod- (PLC ), phosphoinositol-3 kinase (PI3K), AKT and STAT proteins ing a chimeric kinase. The fibroblast growth factor receptor 1 (9–12). The disease has been reproduced in mouse bone marrow (FGFR1)-MPD is characterized by the fusion of the FGFR1 transplantation models (13–15). The effects of X-FGFR1 proteins kinase with various partners, including FOP. We show here can be abrogated by treatment with an inhibitor of the FGFR1 that both normal FOP and FOP-FGFR1 fusion kinase localize kinase (7, 14, 15). to the centrosome. The fusion kinase encounters substrates at The subcellular localization of X-FGFR1 proteins has been the centrosome where it induces strong phosphorylation on studied, however, often only coarsely and in transfected cells with tyrosine residues. Treatment with FGFR1 kinase inhibitor high levels of expression; fusion proteins are found predominantly in the cytoplasm. Normal partners have been found at various SU5402 abolishes FOP-FGFR1-induced centrosomal phosphorylation and suppresses the proliferative and survival potentials subcellular localizations. ZNF198 was found predominantly in the of FOP-FGFR1 Ba/F3 cells. We further show that FOP-FGFR1 nucleus (3, 6, 9, 11, 16). FOP was found in the cytoplasm (6); however, a recent observation is noteworthy: a list of centrosomal allows cells to overcome G1 arrest. Therefore, the FOP-FGFR1 fusion kinase targets the centrosome, activates signaling proteins established by proteomic analysis included FOP (17). CEP1 pathways at this organelle, and sustains continuous entry in is located in a specific domain at the open end of the centrosome the cell cycle. This could represent a potential new mechanism tube associated with maturation of a daughter centrosome in a of oncogenic transformation occurring specifically at the mother centrosome, and is required for centrosome function (18). The centrosome is an organelle important for nucleation and centrosome. (Cancer Res 2005; 65(16): 7231-40) organization of microtubules but is also essential for cell cycle progression mostly during the G -S transition (19–22). This Introduction 1 particular localization of FOP and CEP1 at the centrosome suggested Myeloproliferative disorders (MPD) are clonal malignant hemo- that FGFR1 fusion partners may not only provide dimerization pathies that affect progenitor cells. MPD cells proliferate domains but also target oncogenic kinases to a specific area. We continuously but, in contrast to acute leukemia blasts, undergo show here that FOP-FGFR1 is targeted to the centrosome where it maturation. The disease progresses towards an acute syndrome. activates signaling pathways via tyrosine phosphorylation. This Many MPDs are caused by a chromosome translocation that phosphorylation at the centrosome and the proliferative potential of produces a fusion gene encoding a chimeric, constitutively FOP-FGFR1-expressing cells are abolished after treatment with a activated kinase protein. One of these oncogenic events occurs in kinase inhibitor. We also show that FOP-FGFR1 is important during a rare and aggressive MPD, the fibroblast growth factor receptor 1 G1-S transition to overcome G1 arrest and allow cells to sustain (FGFR1)-MPD. This MPD is also called stem cell MPD or 8p12 continuous cell cycle. This led us to hypothesize that FOP-FGFR1 MPD because both lymphoid and myeloid lineages are affected proteins may exert an oncogenic activity through dysregulation of following activation of the FGFR1 tyrosine kinase, which is cell processes associated with the centrosome. encoded by a gene on the p11-12 region of chromosome 8 (1). FGFR1-MPDs are characterized by fusion proteins (hereafter designated X-FGFR1) made of the FGFR1 catalytic domain fused Materials and Methods to a protein-protein interaction domain from several possible Plasmids, cells, and reagents. Rat2 cells are fibroblastic cells. Ba/F3 are partners, including FOP/FGFR1OP (FGFR1 oncogene partner), murine hematopoietic cells that need to be cultured in the presence of CEP1 (centrosomal protein 1), ZNF198 (zing finger 198), and interleukin-3 (IL-3). FGFR1 is not expressed in native, nontransfected Ba/F3 BCR (2–8). With the exception of BCR, none of the characterized cells. FOP, FOP-FGFR1, FOP-FGFR1 kinase-defective (K259A), PLCg binding partner genes has been found in a fusion involving another gene site (Y511F) mutants, CEP1-FGFR1, wild-type FGFR1 (FGFR1wt) constructs, than FGFR1. The same FGFR1 intracellular region, which and corresponding clones of stably-transfected Rat2 or Ba/F3 cells used in includes the kinase domain, is preserved in all MPD-FGFR1 this study have been previously described (5, 6, 9, 10). The largest FOP protein (or FGFR1OP) has 399 amino acid residues; the FOP-FGFR1 fusion

(568 residues) joins the first 173 NH2-terminal residues of FOP to the intracellular region of FGFR1; the kinase-defective mutation is localized in Requests for reprints: Daniel Birnbaum, Laboratory of Molecular Oncology, the first FGFR1 kinase subdomain. The kinase-defective mutant has Marseille Cancer Institute, UMR599 Inserm, 27 Bd. Leı¨Roure, 13009 Marseilles, France. previously been characterized (10). BCR-FGFR1 construct is described in Phone: 33-49175-8407; Fax: 33-49126-0364; E-mail: [email protected]. I2005 American Association for Cancer Research. ref. (7) and was a kind gift from Dr. N.C. Cross. For FGFR1wt, two conditions doi:10.1158/0008-5472.CAN-04-4167 of stimulation were used: a short stimulation corresponding to 5 minutes of www.aacrjournals.org 7231 Cancer Res 2005; 65: (16). August 15, 2005

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research stimulation with 10 ng/mL FGF1 (P100-17A from AbCys, Paris, France) and experiments with anti-g-tubulin monoclonal antibody. Protein extracts and 10 Ag/mL heparin (H-0777 from Sigma, Saint Quentin Fallavier, France) and immunoprecipitated complexes were separated by SDS-PAGE, transferred a long stimulation corresponding to a 48-hour culture in the presence of onto membrane and probed with anti-phosphotyrosine antibody. 10 ng/mL FGF1 and 10 Ag/mL heparin. For inhibition experiments, Cell survival and proliferation assays. The number of viable cells in concentrations of 0.15, 1.5, and 15 Amol/L of the kinase inhibitor, ATP- the presence or absence of inhibitors was measured by trypan blue competitor, SU5402 (Calbiochem, Merck Biosciences, Darmstadt, Germany) exclusion. Cell proliferation was monitored by [3H]thymidine uptake. A total and 0.1, 1, and 10 Amol/L of STI571 (a gift from Dr P. Manley, Novartis), of 5 Â 103 Ba/F3 cells, and 2 Â 104 FGFR1wt, FOP-FGFR1, CEP1-FGFR1, respectively, were used. The EOL-1 cell line, used to study the FIP1L1-PDGFRA BCR-FGFR1 Ba/F3 cells or EOL-1 cells were grown in duplicate in 96-well fusion (23), was a gift from Dr. B. Papp (Hoˆpital St Louis, Paris, France). plates in the presence or absence of IL-3, respectively. Cells were incubated Antibodies. We used monoclonal anti-myc (9E10), polyclonal anti- for 48 hours at 37jC and pulsed with 0.5 ACi of [methyl-3H]thymidine FGFR1 (C-15), polyclonal anti-PLCg (1,249), anti-GRB2 (C-23) from Santa (Amersham Biosciences, Orsay, France) for the last 6 hours (Ba/F3 cells) or Cruz Biotechnology (Santa Cruz, CA), anti-phospho-STAT1 (Y701), anti- 24 hours (Ba/F3 transfected with FGFR1 fusions). Cells were then phospho-STAT3 (Y705), anti-phospho-STAT5 (Y694) from Cell Signaling transferred onto glass filters (Packard Instruments, Netherlands), and Technology (Beverly, MA), anti-p27 (610241) from BD Biosciences (Pont de incorporation was measured using a B-counter Rack-h Compact 1212-411 Claix, France), anti-PI3K (06-195) from Upstate Biotechnology (Mundol- (LKB, Uppsala, Sweden). sheim, France), anti-g-tubulin either monoclonal (GTU-88) or polyclonal Cell cycle analysis. For cell cycle analysis, murine IL-3-dependent Ba/F3 (T3559) from Sigma, and anti-phosphotyrosine (anti-phosphotyrosine; 4G10; cells transfected or not with fusion proteins were cultured in the presence ref. 10). or absence of IL-3. Cells presynchronized in G0/G1 by IL-3 overnight Immunofluorescence analyses. Immunofluorescence analyses were withdrawal were irradiated (10 Gy) and immediately returned to 37jC, done as previously described (24). Briefly, Rat2 or Ba/F3 cells either grown either in the presence or absence of IL-3 for 8 hours. Cells were then on glass coverslips or centrifuged on poly-L-lysine–coated coverlips, harvested and DNA content was analyzed (24). Flow cytometry analysis respectively, were fixed in cold methanol for 5 minutes. After permeabiliza- after propidium iodide incorporation revealed the presence of G0/G1, S, and tion with 0.5% Triton X-100 for 5 minutes, cells were incubated at room G2-M population. Sub-G1 population corresponds to dying cells. temperature for 60 minutes with the first antibody and then for 45 minutes with the secondary antibody. Samples were then stained with the DNA- specific 4V,6-diamino-2-phenylindole (DAPI; Sigma). Results Most immunofluorescence images were recorded by a TCS-NT confocal FOP and FOP-FGFR1 are centrosomal proteins. To determine Microscope (Leica Microsystem, Mannheim, Germany) controlled by a Leica when, during the cell cycle, FOP was present at the centrosome, we software. Images shown after confocal acquisitions were pseudocolored with Leica software, correspond to one confocal section and were not did immunofluorescence experiments on stable Rat2 cell clones submitted to additional treatment. For immunofluorescence with DAPI overexpressing myc-tagged FOP. FOP localizes to the centrosome staining and on purified centrosomes, acquisitions were done using a Zeiss (Fig. 1A) both in interphase before (Fig. 1Aa and Da) and after Axiovert 200 microscope equipped with Cool Snap HQ camera (Ropper centrosome duplication (Fig. 1Ab and Db) and in dividing cells Scientific, Evry, France) controlled by Metamorph software (Universal (Fig. 1Acd and Dcd). These results suggested that some fusion Imaging, Downingtown, PA). For immunofluorescence images containing partners may not only provide dimerization domains but could DAPI staining, Z stacks were acquired, deconvoluted and analyzed with also determine the subcellular localization of the corresponding Metamorph software. Monochrome images were collected for each oncogenic kinase. Therefore, we wondered if FOP-FGFR1 fusion appropriate channel and pseudocolored with Metamorph. Single optical protein was addressed to the centrosome. In Rat2 clones sections are presented. expressing myc-FOP-FGFR1, the fusion protein was localized Immunofluorescence on purified centrosomes. Centrosomes were isolated from FOP-FGFR1 and FOP-FGFR1 kinase-defective cells as exclusively at the centrosome during the whole cell cycle, in previously described (25). Centrosomes were sedimented on glass interphasic cells before (Fig. 1Ba and De) and after centrosome coverslips at 24,000 Â g, fixed with methanol and processed for duplication (Fig. 1Bb and Df ), and also during metaphase immunofluorescence as described (26). Antibodies used included anti-g- (Fig. 1Bc and Dg) and cytokinesis (Fig. 1Bd and Dh). FOP-FGFR1 tubulin, anti-phosphotyrosine, and anti-a-tubulin YL1/2 (1/1,000; ref. 27), centrosomal localization is detected with both myc (Fig. 1B and goat anti-rabbit Alexa 488 (Molecular Probes, Invitrogen, Cergy Pontoise, Ca) and FGFR1 (Fig. 1Cab) antibodies. When it is not fused to France), goat anti-mouse Cy3 and donkey anti-rat Cy5 (Jackson FOP, FGFR1 kinase is not detected at the centrosome (Fig. 1Cd). ImmunoResearch Laboratories, Cambridgeshire, United Kingdom). Thus, an ectopic, activated fusion kinase of FGFR1-MPDs could MPD mice. Mice developing MPD and kinase-defective controls have be targeted to the centrosome. been described previously (13). Briefly, bone marrow from 5-fluorouracil- FOP-FGFR1 proteins can signal at the centrosome during treated mice was enriched in early hematopoietic precursors by positive selection using stem cell antigen-1 Sca-1. Sca-1+-cells were transduced with G1-S phase. In subsequent experiments, we used Rat2 cells to MSCVneo retroviral vectors with FOP-FGFR1 or kinase-defective. FOP- allow easy visualization of the centrosome before and after dupli- FGFR1 transplanted mice but not kinase-defective mice developed a fatal cation and Ba/F3 cells for functional studies. MPD within 4 weeks after transplantation, characterized by marked To exert its effects at the centrosome, the oncogenic fusion leukocytosis, hypercellular bone marrow, and hepatosplenomegaly indica- protein must induce tyrosine phosphorylation of downstream tive of myeloid hyperplasia. Hematopoietic progenitors (spleen colony- substrates at this subcellular site (Fig. 2A). Study of global forming unit) from enlarged spleens of MPD mice were collected, tyrosine phosphorylation in FOP-FGFR1 expressing Rat2 cells maintained in culture without cytokines and used for immunofluorescence revealed a strong centrosomal staining during interphase before experiments. Morphologic study of these cells showed a majority of mature (data not shown) and after (Fig. 2Ba) centrosome duplication. and immature granulocytes, in contrast to a normal spleen that contains a This staining could represent either FOP-FGFR1 autophosphor- majority of lymphocytes. Cell lysis, immunoprecipitation, and Western blot. For inhibitor ylation or the phosphorylation of its downstream substrates or experiments, 3 Â 106 nontransfected Ba/F3 cells or Ba/F3 cells expressing both. The staining was stronger in interphasic cells (Fig. 2BbI) wild-type or fusion proteins cultivated in the presence or absence of IL-3, than in mitotic cells (Fig. 2BbM). Because FOP-FGFR1 is present respectively, were lysed in 200 AL as previously described (9). NP40 lysates at the centrosome during the whole cell cycle, we think that (10) from 20 Â 106 Ba/F3 cells were used for immunoprecipitation phosphotyrosine staining at the centrosome in mitosis corresponds

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Centrosome Targeting by Oncogenic Kinase only to FOP-FGFR1 autophosphorylation, whereas phosphotyr- and centrioles stained with g-tubulin and tyrosylated a-tubulin, osine staining during interphase represents phosphorylation of respectively. both FOP-FGFR1 and its substrates. This result shows that FOP- Expression of FOP-FGFR1 in primary bone marrow cells induced FGFR1 encounters substrates with tyrosine phosphorylation sites by retroviral transduction generates a fatal MPD in mice at the centrosome. It also suggests that phosphotyrosine signaling characterized by myeloid hyperplasia and hepatosplenomegaly is important for G1-S events, when centrosome duplication (13). We used cells isolated from the spleen of such transplanted occurs. Phosphotyrosine staining was absent in cells expressing FOP-FGFR1 mice to investigate FOP-FGFR1 activity in conditions a kinase-defective FOP-FGFR1 K259A mutant although the close to those of the natural disease. Hematopoietic progenitors mutant protein also localized to the centrosome (Fig. 2Bc). (spleen colony-forming unit) from spleens of FOP-FGFR1 mice Because FGFR1wt is not targeted to the centrosome (Fig. 1Cd) were collected and maintained in culture without cytokines for >2 vesicular cytoplasmic but not centrosomal phosphotyrosine months, showing that these cells have a proliferative potential. staining was detected in Rat2 cells overexpressing FGFR1wt Immunofluorescence on cultured cells showed strong FOP-FGFR1 (Fig. 2Bd). (Fig. 2Da) and phosphotyrosine (Fig. 2Db)signalsatthe A very strong phosphotyrosine staining was similarly detected at centrosome. Thus, FOP-FGFR1 is targeted to the centrosome and the centrosome of interphasic Ba/F3 cells expressing myc-FOP- signals at this organelle, bringing tyrosine phosphorylation, both FGFR1 (Fig. 2Ca) but not in mitotic cells (data not shown) or in in vitro and in vivo. cells expressing the kinase-defective mutant (Fig. 2Cb), even if both FOP-FGFR1 protein encounters, recruits, and phosphory- proteins localized to the centrosome (Fig. 2Ccd). Immunofluores- lates substrates at the centrosome. We next wondered which cence on purified centrosomes confirmed this result (Fig. 2Ce-l); were the substrates activated by FOP-FGFR1 at the centrosome. We phosphotyrosine staining colocalized with pericentriolar material first focused on known FOP-FGFR1 substrates, which include

Figure 1. Both FOP and FOP-FGFR1 chimeric protein are localized at the centrosome during the cell cycle. Immunofluorescence experiment with anti-myc antibody (green) shows the localization of myc-FOP (A) and myc-FOP-FGFR1 (B) in various phases of the cell cycle of Rat2 cells (a-d). Colocalization with g-tubulin (red) at the centrosome of interphasic cells before (a) and after (b) centrosome duplication, and during metaphase (c) and cytokinesis (d) is shown. Stages of the cell cycle were determined using DAPI staining (blue). C, controls: colocalization of anti-myc (red) and anti-FGFR1 (green) staining on myc-FOP-FGFR1 transfected cells (a), FGFR1 staining (green) at the centrosome (g-tubulin, red) of myc-FOP-FGFR1 cells (b), no myc staining on nontransfected cells (c), no signal at the centrosome (g-tubulin, red) but vesicular staining of FGFR1 antibody (green) in FGFR1-transfected cells (d). D, visualization of centrosomal localization of myc-FOP (a-d) and myc-FOP-FGFR1 (e-h) using a confocal microscope. Images were acquired with a Zeiss microscope and treated with Metamorph software (A-C) or with a Leica confocal (D). Bar,10Am. www.aacrjournals.org 7233 Cancer Res 2005; 65: (16). August 15, 2005

Figure 2. FOP-FGFR1 induces tyrosine phosphorylation at the centrosome during the G1-S transition of the cell cycle. A, schematic representation of FOP-FGFR1 protein. K259 belongs to the ATP-binding site necessary for the kinase activity of the fusion protein (TK, tyrosine kinase subdomains); it is mutated in the kinase-defective mutant (K259A). Y511 is the PLCg-binding site; it is mutated in the PLCg-binding mutant. LisH, lissencephaly type-1–like homology motif. Arrow, breakpoint fusion. B, costaining using anti-phosphotyrosine (phosphotyrosine, red) and anti-FGFR1 (green) reveals phosphorylation on tyrosine at the centrosome of Rat2 cells expressing FOP-FGFR1 (a and b) but not in Rat2 cells expressing FOP-FGFR1 kinase-defective mutant (c). Phosphotyrosine staining of interphasic cells (a and bI) is strong compared with mitotic cells (bM). Staining with anti-phosphotyrosine (red) and anti-g-tubulin (green) on Rat2 expressing FGFR1wt shows absence of detectable phosphorylation at the centrosome (d). Bar,10Am. C, costaining using anti-phosphotyrosine (red) and anti-g-tubulin (green) reveals phosphorylation on tyrosine at the centrosome of Ba/F3 cells expressing FOP-FGFR1 (a) but not kinase-defective mutant (b). Immunofluorescence with anti-myc (red) antibody shows localization of tagged FOP-FGFR1 (c) and FOP-FGFR1 kinase-defective mutant (d) at the centrosome of Ba/F3 cells stained with anti-g-tubulin (green). Bar,5Am. Costaining using anti-g-tubulin (green), phosphotyrosine (red), and antityrosylated a-tubulin (blue) on purified centrosomes from Ba/F3 cells expressing FOP-FGFR1 (e-h) or kinase-defective mutant (i-l). Scale,10Am (3.4 cm). D, presence of FOP-FGFR1 (a) and phosphotyrosine (b) at the centrosome of colony-forming cells isolated from spleens of transplanted FOP-FGFR1 mice revealed by immunofluorescence using anti-myc (green), anti-phosphotyrosine (green) and anti-g-tubulin (red). Bar,10Am.

STAT1, -3, -5 proteins, PLCg, PI3K, AKT and p70S6K proteins (10). PI3K was present at the centrosome of interphasic cells We found p70S6K and phospho-AKT at the centrosome in transfected (Fig. 3Bghi, arrow) or not (Fig. 3Bghi, arrowhead) with interphasic and mitotic Rat2 cells, respectively (data not shown). FOP-FGFR1. PI3K is important for centrosome duplication (28). PLCg, which was present at the spindle pole during mitosis in Because PI3K colocalizes with FOP-FGFR1 (Fig. 3Bghi), we suspect Rat2 cells transfected (Fig. 3Aa) or not with FOP-FGFR1, was that phosphorylation of PI3K by FOP-FGFR1 occurs at the recruited to the centrosome in interphase before and after centrosome. It may play a role when centrosome duplication centrosome duplication in FOP-FGFR1 (Fig. 3Abc) but not in occurs at the G1-S transition. FOP-FGFR1 kinase-defective cells (Fig. 3Ade)orincells Study of phosphotyrosine STAT1, STAT3, and STAT5 subcellular expressing FOP-FGFR1 Y511F mutant, which lacks the PLCg localization in Rat2 cells showed, in addition to their known binding site (Fig. 3Afg). In Ba/F3 cells, PLCg was also recruited cytoplasmic and nuclear localizations, a strong phosphotyrosine to the centrosome in the presence of FOP-FGFR1 (Fig. 3Babc, staining at the centrosome of interphasic cells, before (data not arrow) but neither in its absence (Fig. 3abc, arrowhead) nor in shown) and after (Fig. 3Cabc, arrow) centrosome duplication. This the presence of kinase-defective mutant (Fig. 3Bdef ). We have phosphorylation in FOP-FGFR1 but not in kinase-defective mutant previously shown that PLCg interacts and is phosphorylated by cells (Fig. 3Cdef ) could facilitate subsequent STAT activation (10). FOP-FGFR1 (10); we show here that this could occur at the FOP-FGFR1 Ba/F3 cells, in which the STAT3 pathway is activated centrosome during the G1-S phase of the cell cycle. (10), showed the same result (Fig. 3Cg, arrow). No phospho-STAT3

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Centrosome Targeting by Oncogenic Kinase signal was detected in untransfected (Fig. 3Cg, arrowhead)or used proliferating Ba/F3 cells expressing several FGFR1wt or fusion kinase-defective mutant (Fig. 3Ch) cells. proteins and the EOL-1 cell line (23) expressing FIP1L1-PDGFRA To further show that FOP-FGFR1-activated substrates were fusion (Fig. 4A). Ba/F3 cells normally need IL-3 for survival and associated with the centrosome, we immunoprecipitated proteins proliferation. Cell proliferation experiments using [3H]thymidine associated with the pericentriolar material using anti-g-tubulin incorporation indicated that FOP-FGFR1 Ba/F3 cells not only antibody. We found many proteins phosphorylated on tyrosine survived as previously shown (10), but even proliferated in the residues bound to g-tubulin in FOP-FGFR1 Ba/F3 lysates (e.g., red absence of IL-3, although to a lesser degree than in the presence of asterisks), which were absent in kinase-defective mutant lysates IL-3 (data not shown). Kinase-defective mutant cells did not (Fig. 3D). These proteins can either be signaling molecules survive in the absence of IL-3. FGFR1wt cells cultivated in the phosphorylated at the centrosome or intrinsic centrosomal presence of FGF1 and heparin were used as controls. To determine proteins. Several proteins of high molecular mass in particular if the phosphorylation pattern obtained with FOP-FGFR1 was due (red asterisks) could be centrosomal proteins directly phosphor- to its centrosomal localization we compared (a) phosphorylation ylated by FOP-FGFR1; however, they remain to be characterized. In profiles on Western blot and (b) global phosphotyrosine localiza- conclusion, FOP-FGFR1 protein constitutively activates substrates tion induced by different fusion proteins and FGFR1wt. at the centrosome. Western blot analysis after IL-3 starvation for 8 hours showed a Phosphorylation at the centrosome and proliferation in phosphorylation profile specific for FOP-FGFR1 (Fig. 4A, red different clones. We next studied if other kinases were targeted to asterisk). Some substrates, probably corresponding to the ones the centrosome and/or induced phosphorylation at this site. We immunoprecipitated with g-tubulin (see Fig. 3D), were detected

Figure 3. FOP-FGFR1 encounters and phosphorylates signaling substrates at the centrosome. A, PLCg (green) localization during mitosis (a). PLCg staining (green) shows the recruitment of endogenous PLCg to the centrosome of Rat2 cells by FOP-FGFR1, before (b) and after (c) centrosome duplication, but neither by kinase-defective (d and e) nor by PLCg binding site mutant (f and g). Red, anti-g-tubulin stains the centrosomes. B, PLCg or PI3K staining (green) in Ba/F3 cells expressing tagged FOP-FGFR1 or kinase-defective mutant (red). Arrows and arrowheads, centrosomes of transfected cells and nontransfected cells, respectively. C, phosphotyrosine Y701-STAT1 (a), Y705-STAT3 (b), and Y694-STAT5 (c) staining (green) detects the phosphorylated protein (P-STAT) at the centrosome (red) of interphasic FOP-FGFR1 but not kinase-defective (d-f) Rat2 cells. STAT5 nuclear staining is also detected after centrosome duplication (c). Phosphotyrosine Y705-STAT3 staining in FOP-FGFR1 (g) Ba/F3 cells but not kinase-defective (h). Bar,10Am. D, Western blot with anti-phosphotyrosine shows phosphorylated FOP-FGFR1 (black asterisk) and its substrates (red asterisk) in FOP-FGFR1 but not kinase-defective mutant lysates or after immunoprecipitation with anti-g-tubulin. www.aacrjournals.org 7235 Cancer Res 2005; 65: (16). August 15, 2005

Figure 4. Phosphotyrosine profiles on Western blot and centrosomal tyrosine phosphorylation localization induced by various oncogenic kinases. A, Western blot analysis shows differential phosphotyrosine profiles of various cells: Ba/F3, FOP-FGFR1, FOP-FGFR1 K259A mutant, FGFR1wt, BCR-FGFR1, CEP1-FGFR1 transfected Ba/F3, or EOL-1 (FIP1L1-PDGFRA) cells. Fusion proteins and substrates are indicated by black and red asterisks, respectively. B, immunofluorescence experiment shows the corresponding localization of tyrosine phosphorylation (red; s. stim and l. stim, short and long stimulation of wild-type FGFR1 by FGF and heparin). Arrows, phosphotyrosine staining at the centrosome. Bar,10Am. exclusively after FOP-FGFR1 direct centrosomal activation but not (Fig. 4A). FGFR1wt, BCR-FGFR1, and FIP1L1-PDGFRA were not after FGFR1wt activation (whether long or short), which is not directly targeted to the centrosome and the phosphotyrosine signal directly targeted to the centrosome. This indicates that FOP- was detected in the cytoplasm (Fig. 4Bg-j). However, some FGFR1-specific targeting to the centrosome can directly phosphor- phosphorylation staining was detected at the centrosome of some ylate additional centrosomal proteins. Because of its molecular proliferating cells (Fig. 4Bg-j, arrow). Similarly, in the presence of mass, CEP1-FGFR1, the other fusion protein localizing to the IL-3, phosphotyrosine staining was, in some cells, also detected at centrosome, did not allow the detection of this pattern. Western the centrosome (Fig. 4Ba, arrow). This could correspond to blots were also useful to control the expression of the different downstream substrates common to both IL-3 and FGFR1 fusion proteins (Fig. 4A, black asterisk). activation pathways (e.g., STAT5). These results suggest that any Phosphotyrosine staining (Fig. 4B), which allows the detection of these signalings indirectly activate substrates at the centrosome, of the fusion protein and its substrates, showed that FOP- at least at some specific time of the cell cycle. No centrosomal FGFR1 was the most specifically and exclusively targeted to the phosphorylation could be detected in nonproliferating cells, Ba/F3 centrosome where it induces tyrosine phosphorylation (Fig. 4Bd). and kinase-defective mutant cells without IL-3 (Fig. 4Bbc). We Ba/F3 cells expressing CEP1-FGFR1 also displayed strong centro- propose that bringing phosphorylation directly to the centrosome somal staining, suggesting that the fusion protein is also targeted is sufficient to allow cells to enter a proliferating state. to the centrosome. However, this localization seemed less exclusive Centrosomal phosphorylation and proliferation induced by than that of FOP-FGFR1 and some cytoplasmic fusion protein FOP-FGFR1 are inhibited by FGFR1 kinase inhibitor. To further could be detected, perhaps due to a high level of expression show that centrosomal staining is required for survival and

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Centrosome Targeting by Oncogenic Kinase proliferation we used the kinase inhibitor SU5402. SU5402 interacts induced by FOP-FGFR1 was not abolished by STI571 (Fig. 5B). directly with FGFR1 catalytic domain and can inhibit [3H]thymi- SU5402 also showed an inhibitory effect on the proliferation of dine incorporation of cells stimulated by FGF1 (29). Phosphoryla- FGFR1wt, CEP1-FGFR1, BCR-FGFR1 Ba/F3 cells, and EOL-1 cells, tion at the centrosome of FOP-FGFR1 Ba/F3 cells was specifically but not on untransfected Ba/F3 cells (Fig. 5B). SU5402 is known to abolished after SU5402 treatment (Fig. 5A). A strong phosphotyr- inhibit FGFR1 and platelet-derived growth factor receptor tyrosine osine staining was detected at the centrosome of Ba/F3 cells in the kinase activity. The results of SU5402 and STI571 treatments were presence of a low, inefficient concentration (0.15 Amol/L) of verified in Western blot experiments; in agreement with cell SU5402 (Fig. 5Aac), or with STI571 (data not shown), an inhibitor proliferation experiments, SU5402, but not STI571, induced loss of with a different specificity known to be inefficient on FGFR1 tyrosine phosphorylation in FOP-FGFR1 cells (Fig. 5C). kinase. In contrast, treatment with 15 Amol/L of SU5402 for 90 In conclusion, phosphorylation at the centrosome in the minutes reduced phosphotyrosine staining at the centrosome of presence of FOP-FGFR1, which is inhibited by SU5402, is essential Ba/F3 cells expressing FOP-FGFR1 (data not shown). After 15 hours for survival and proliferation of Ba/F3 FOP-FGFR1. of SU5402 treatment, most cells were dying (data not shown). The FOP-FGFR1 has an effect on the cell cycle. To reveal FOP- remaining cells showed no or very low levels of phosphotyrosine FGFR1 potential, we studied the consequence on the cell cycle of staining at the centrosome (Fig. 5Abd). This result indicates a stable overexpression of FOP-FGFR1 at the centrosome in Ba/ that centrosomal phosphorylation is required for survival and F3 cells. Ba/F3 cells transfected with an empty vector or the proliferation. kinase-defective mutant were used as control. Two different Indeed, loss of phosphorylation at the centrosome observed after conditions of stress, IL-3 withdrawal and irradiation, were used SU5402 treatment correlated with loss of proliferation measured (Fig. 6A and B). 3 using [ H]thymidine incorporation: FOP-FGFR1-induced prolifera- As expected, Ba/F3 cells transiently arrested in G0/G1 after IL-3 tion was inhibited by increasing concentrations of SU5402 (Fig. 5B). withdrawal, did not enter S phase (Fig. 6A) and died (data not Because SU5402 is cytotoxic (29) this loss of proliferation was shown). The ratio G1/(S + G2) is used to represent cells blocked at associated with a loss of cell survival verified by cell count after the G1-S checkpoint. The lack of G1 arrest in FOP-FGFR1 cells was trypan blue exclusion (data not shown). In contrast, proliferation revealed by the decrease of this ratio, as compared with control or

Figure 5. Phosphorylation at the centrosome and proliferation induced by FOP-FGFR1 are inhibited by SU5402 in Ba/F3 cells. A, phosphotyrosine staining (red)in Ba/F3 cells expressing FOP-FGFR1 after SU5402 treatment: 0.15 Amol/L for 15 hours (a), or 15 Amol/L for 15 hours (b). Colocalization of phosphotyrosine (red) with g-tubulin (green) shows inhibition of phosphotyrosine staining at the centrosome after 15 Amol/L SU5402 treatment (d) but not 0.15 Amol/L (c). Bar,10Am. B, proliferation of Ba/F3 and FGFR1wt, FOP-FGFR1, BCR-FGFR1, CEP1-FGFR1 transfected Ba/F3 or EOL-1 cells in the presence or absence of IL-3, respectively, assessed by [3H]thymidine incorporation. Cell proliferation is assessed in the presence of increasing concentrations of SU5402 but not STI571. Results are shown in percentage: 100% represents the mean proliferation rate obtained without addition of drug. Columns, mean; bars, F SD. Similar results were obtained in three independent experiments. C, Western blot with anti-phosphotyrosine antibody shows FOP-FGFR1 phosphotyrosine inhibition (top) 90 minutes after treatment with increasing concentrations of SU5402 but not of STI571. Total cell lysates were probed with anti-GRB2 antibody to compare the amount of proteins in the lysates (bottom). www.aacrjournals.org 7237 Cancer Res 2005; 65: (16). August 15, 2005

Figure 6. FOP-FGFR1 induces continuous S phase entry of BaF3 cells. A, G0/G1 arrest after IL-3 withdrawal and irradiation (10 Gy) visualized by the following ratio: % of living cells in G0/G1/% of living cells in S-G2-M. Graphical data represent the average of three independent experiments F mean deviation. B, cell cycle analysis focusing on living cells for FOP-FGFR1 (a-d) and kinase-defective mutant (e-h). The percentage of cells in G0/G1 corresponds to cells with 2 N DNA content. C, Western blot using anti-p27 (top) on total lysates illustrates S phase entry of different proliferating Ba/F3 cells. Total cell lysates were probed with anti-a-tubulin antibody to compare the amount of proteins in the lysates (bottom). D, diagram synthesizing the results.

kinase-defective mutant cells (Fig. 6A). Accordingly, FOP-FGFR1 p27 is an inhibitor of the G1-S transition. When cells enter S cells were protected from cell death and 26% of cells even entered phase, p27 degradation is induced after CDK2/cyclin E activation the cell cycle in the absence of IL-3 (Fig. 6Bb) as compared with 9% (30) and decrease of p27 is observed in proliferating cells (Fig. 6C). for kinase-defective mutant cells (Fig. 6Bf ). The presence of the Decrease of p27 expression in FOP-FGFR1 Ba/F3 cells revealed the constitutively active oncogenic protein at the centrosome was thus proliferative potential of cells entering S phase (Fig. 6C). SU5402 enough to bypass a restriction point and sustain continuous entry treatment abolished this effect. This result is in favor of FOP- in S phase in the absence of IL-3. FGFR1 inducing S phase entry. Similarly, Ba/F3 cells underwent G1 arrest but either survived or rapidly died when irradiated in the presence or absence of IL-3, respectively. FOP-FGFR1 protected irradiated cells from death, Discussion although less than the mere presence of IL-3, and overcame G1 We have shown that FOP and FOP-FGFR1, the oncogenic fusion arrest after irradiation (Fig. 6A). Indeed, the ratio G1/(S + G2) was kinase of an FGFR1-MPD, are addressed to the centrosome. reduced in irradiated FOP-FGFR1 cells as compared with control Centrosomal addressing of the fusion protein occurs not only in cells (Fig. 6A). The marked increase of the G2-M population transfected cultured cells but also in hematopoietic cells of an MPD (Fig. 6Bcd) also revealed the lack of G1 arrest after irradiation mouse model. Our search for FOP partners by two-hybrid screen in compared with kinase-defective mutants (Fig. 6Bgh). This potential yeast identified further centrosomal proteins such as CAP350, depended on tyrosine kinase activity because kinase-defective cells confirming these observations. FOP has only one identified domain reacted exactly like Ba/F3 control cells (Fig. 6A). The PLCg mutant called LisH, which it shares with other proteins such as PAFAH1B1, had a moderate effect on the G1 arrest (data not shown). TCOF1, and OFD1. LisH proteins are mutated in Miller-Dieker

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Centrosome Targeting by Oncogenic Kinase lissencephaly, Treacher Collins syndrome, oral-facial-digital type 1 ectopic oncogenic kinases, those that directly target the centro- and contiguous syndrome ocular albinism with late onset some (or at least the Golgi/centrosome area), and those that do sensorineural deafness (31). LisH motifs contribute to the not. A non–kinase oncogene may also abnormally function at the regulation of microtubule dynamics, either by mediating dimer- centrosome (45). ization, or else by binding cytoplasmic dynein heavy chain or What are the effects of the oncogenic kinase at the centrosome? microtubules directly. However, mutation of the LisH domain of It is likely that FOP-FGFR1 exerts its oncogenic activity through OFD1 did not abrogate centrosomal localization (32). Therefore, dysregulation of cell processes associated with the centrosome. the region of FOP-FGFR1 needed for its centrosomal localization Centrosomes nucleate microtubules and contribute to mitotic remains to be determined. spindle organization and function. They also participate in We have further shown that the oncogenic fusion kinase cytokinesis and cell cycle progression. The first type of alteration encounters or recruits substrates (e.g., PLCg) at the centrosome associated with centrosome defect is aneuploidy. However, the where it induces strong phosphorylation on tyrosine residues, both karyotype of X-FGFR1-positive cells, either in patients or after in vitro and in vivo. FOP-FGFR1 substrates at the centrosome could transfection of chimeric genes, does not show a particularly high either be signaling molecules phosphorylated at the centrosome degree of aneuploidy as compared with other types of hemopa- (e.g., STAT) or intrinsic centrosomal proteins. Comparison of thies or cancers. There is no amplification of the centrosomes in phosphorylation patterns of other fusion proteins or FGFR1 wild- cells overexpressing FOP-FGFR1. Experiments in Ba/F3 cells type shows that FOP-FGFR1 and CEP-FGFR1 are the most directly showed that FOP-FGFR1 interferes with the G1 checkpoint. We targeted to the centrosome. This further suggests that any of these may have uncovered a mechanism of oncogenic transformation signaling pathways seem to activate relays at the centrosome, at associated with a defect of centrosome function but not of least at some periods of the cell cycle. Treatment with SU5402 centrosome number; this mechanism will need to be further abolishes kinase-induced centrosomal phosphorylation and sup- delineated. presses the proliferative potential of FOP-FGFR1 Ba/F3 cells. The centrosome is important for the cell cycle; it influences cell Finally, we have shown that FOP-FGFR1 at the centrosome allows shape, polarity, and motility; it is also linked to DNA repair (19–21). cells to proliferate and overcome G1 arrest. The results are Thus, abnormal activation of the FGFR1 tyrosine kinase at the summarized in Fig. 6D. centrosome may affect several processes by disrupting the We therefore propose that targeting an oncogenic, constitutively regulation of various molecular complexes that remain to be active kinase to the centrosome is enough to overcome cell cycle identified. We have shown here that components of the FGFR1 arrest, overcome a restriction point during the G -S transition 1 cascade which interact with the chimeric X-FGFR1 proteins are when centrosome duplication occurs, and force entry in the cell localized at the centrosome either during interphase and mitosis or cycle. It is the first time that an oncogenic product of a human both. The PI3K-AKT/PKB pathway is particularly interesting in this disease is shown to be addressed to the centrosome. This raises at context. It regulates G cyclins (D1 and E), is involved in least two questions. 1 centrosome duplication, and is well-known to be associated with How general is the phenomenon? Both FOP-FGFR1 and CEP1- cell survival and cell proliferation, which are two cell processes FGFR1 localize to the centrosome. This localization and stability predominantly affected in MPD (28, 46, 47). Similarly, a recent seem sufficient for these kinases to exert their effects. However, centrosomal localization may not be necessary to trigger the report has also pointed to the role of STAT3 in centrosome disease. Indeed, the study of ZNF198-FGFR1 (3, 9, 11) and BCR- duplication (48). Downstream targets of AKT and STAT in FGFR1 (this work), two other well-characterized fusion kinases of centrosome regulation remain unidentified. FGFR1-MPDs have not alluded to a potential centrosomal We hypothesize that the centrosome, which is linked to the localization. However, we have shown here that even if BCR- microtubules, close to the nucleus, and connected to the Golgi FGFR1 is not targeted to the centrosome, some relay of the apparatus and the proteasome, could be an integrating place for oncogenic signal may take place at the centrosome. Conversely, some of the multiple signaling pathways controlling cell division, FOP-FGFR1 and CEP1-FGFR1 may not be the only oncogenic cell migration, and cell fate (49). In embryogenesis and normal kinases to target the centrosome. A new FGFR1-MPD with processes of proliferation and differentiation, different types of FGFR1OP2-FGFR1 fusion has been described recently (33). signaling are associated with centrosome duplication and function FGFR1OP2 has coiled-coil motifs. These motifs are frequently and centrosomal activity is linked to cell division (50, 51). Like viral found in centrosomal proteins. Centrosomal targeting of oncogenic proteins, an oncogenic protein would prey upon these normal kinases may even occur in other malignancies than FGFR1-MPDs. A signals and use them to its profit. Abnormal kinase activity at the case of MPD has been described in which the platelet-derived centrosome should be an efficient way to pervert cell division in growth factor receptor B kinase is fused to ninein, a centrosomal malignancy. protein with CEP1-like structure and function (34). Several other cases of MPD have been described with rearrangements involving Acknowledgments platelet-derived growth factor receptor B and numerous partners (35–41). Some of these partners may be localized at the Received 11/22/2004; revised 5/21/2005; accepted 5/25/2005. Grant support: Institut National de la Sante et de la Recherche Medicale, Institut centrosome (42, 43). Finally, we and others have evidence for Paoli-Calmettes, Cance´ropoˆle, and Ligue Nationale Contre le Cancer. B. Delaval has fusion of JAK2 kinase with centrosomal protein PCM1 in MPD been successively supported by the Ministry of Research, the Ligue Nationale Contre le 5 Cancer, and the Socie´te´Franc¸aised’He´matologie. with t(8;9) translocation (44). Thus, there might be two classes of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We thank N.C.P. Cross, A. Ferrand, J.R. Galindo, M. Goldfarb, G. Guasch, D. Isnardon, A. Murati, J. Nunes, V. Ollendorff, B. Papp, M.J. Pe´busque,and C. Popovici for 5 Murati et al., unpublished. discussions, help, and/or reagents. www.aacrjournals.org 7239 Cancer Res 2005; 65: (16). August 15, 2005

References protein, involved in rearrangement in myeloproliferative encoding a CEP110-like centrosomal protein, is fused to disease, forms complexes with the DNA repair-associ- PDGFRB in a patient with a t(5;14)(q33;q24) and an 1. Cross NC, Reiter A. Tyrosine kinase fusion genes in ated HHR6A/6B and RAD18 proteins. Oncogene 2003; imatinib-responsive myeloproliferative disorder. Cancer chronic myeloproliferative diseases. Leukemia 2002;16: 22:3417–23. Res 2004;64:2673–6. 1207–12. 17. Andersen JS, Wilkinson CJ, Mayor T, et al. Proteomic 35. Abe A, Emi N, Tanimoto M, et al. Fusion of the 2. Popovici C, Ade´laı¨de J, Ollendorff V, et al. Fibroblast characterization of the human centrosome by protein platelet-derived growth factor receptor h to a novel growth factor receptor 1 is fused to FIM in stem-cell correlation profiling. Nature 2003;426:570–4. gene CEV14 in acute myelogenous leukemia after clonal myeloproliferative disorder with t(8;13). Proc Natl Acad 18. Ou YY, Mack GJ, Zhang M, Rattner JB. CEP110 and evolution. Blood 1997;90:4271–7. Sci U S A 1998;95:5712–7. ninein are located in a specific domain of the centrosome 36. Golub TR, Barker GF, Lovett M, Gilliland DG. Fusion 3. Xiao S, Nalabolu SR, Aster JC, et al. FGFR1 is fused associated with centrosome maturation. J Cell Sci 2002; of PDGF receptor h to a novel ets-like gene, tel, in with a novel zinc-finger gene, ZNF198, in the t(8;13) 115:1825–35. chronic myelomonocytic leukemia with t(5;12) chromo- leukaemia/lymphoma syndrome. Nat Genet 1998;18: 19. Rieder CL, Faruki S, Khodjakov A. The centrosome in somal translocation. Cell 1994;77:307–16. 84–7. vertebrates: more than a microtubule-organizing center. 37. Kulkarni S, Heath C, Parker S, et al. Fusion of H4/ 4. Smedley D, Hamoudi R, Clark J, et al. The Trends Cell Biol 2001;11:413–9. D10S170 to the platelet-derived growth factor receptor t(8;13)(p11;q11–12) rearrangement associated with an 20. Ou Y, Rattner JB. The centrosome in higher h in BCR-ABL-negative myeloproliferative disorders atypical myeloproliferative disorder fuses the fibroblast organisms: structure, composition, and duplication. Int with a t(5;10)(q33;q21). Cancer Res 2000;60:3592–8. growth factor receptor 1 gene to a novel gene RAMP. Rev Cytol 2004;238:119–82. 38. Morerio C, Acquila M, Rosanda C, et al. HCMOGT-1 Hum Mol Genet 1998;7:637–42. 21. Khodjakov A, Rieder CL. Centrosomes enhance the is a novel fusion partner to PDGFRB in juvenile myelo- 5. Popovici C, Zhang B, Gre´goire MJ, et al. The fidelity of cytokinesis in vertebrates and are required for monocytic leukemia with t(5;17)(q33;p11.2). Cancer Res t(6;8)(q27;p11) translocation in a stem cell myeloprolif- cell cycle progression. J Cell Biol 2001;153:237–42. 2004;64:2649–51. erative disorder fuses a novel gene, FOP, to fibroblast 22. Hinchcliffe EH, Miller FJ, Cham M, Khodjakov A, 39. Ross TS, Bernard OA, Berger R, Gilliland DG. Fusion growth factor receptor 1. Blood 1999;93:1381–9. Sluder G. Requirement of a centrosomal activity for cell of Huntingtin interacting protein 1 to platelet-derived h h 6. Guasch G, Mack GJ, Popovici C, et al. FGFR1 is fused cycle progression through G1 into S phase. Science 2001; growth factor receptor (PDGFR ) in chronic myelo- to the centrosome-associated protein CEP110 in the 291:1547–50. monocytic leukaemia with t(5;7)(q33;q11.2). Blood 1998; 8p12 stem cell myeloproliferative disorder with 23. Cools J, Quentmeier H, Huntly BJ, et al. The EOL-1 91:4419–26. t(8;9)(p12;q33). Blood 2000;95:1788–96. cell line as an in vitro model for the study of FIP1L1- 40. Wilkinson K, Velloso ER, Lopes LF, et al. Cloning of 7. Demiroglu A, Steer EJ, Heath C, et al. The t(8;22) in PDGFRA-positive chronic eosinophilic leukemia. Blood the t(1;5)(q23;q33) in a myeloproliferative disorder chronic myeloid leukemia fuses BCR to FGFR1: trans- 2004;103:2802–5. associated with eosinophilia: involvement of PDGFRB forming activity and specific inhibition of FGFR1 fusion 24. Delaval B, Ferrand A, Conte N, et al. Aurora B-TACC1 and response to imatinib. Blood 2003;102:4187–90. proteins. Blood 2001;98:3778–83. protein complex in cytokinesis. Oncogene 2004;23: 41. MagnussonMK,MeadeKE,BrownKE,etal. 8. Fioretos T, Panagopoulos I, Lassen C, et al. Fusion of 4516–22. Rabaptin-5 is a novel fusion partner to platelet-derived the BCR and the fibroblast growth factor receptor-1 25. Moudjou M, Bornens M. Isolation of centrosomes growth factor h receptor in chronic myelomonocytic (FGFR1) genes as a result of t(8;22)(p11;q11) in a from cultured animal cells. In: Celis JE, editor. Cell leukemia. Blood 2001;98:2518–25. myeloproliferative disorder: the first fusion gene involv- Biology: A Laboratory Handbook. New York: Academic 42. Infante C, Ramos-Morales F, Fedriani C, Bornens M, ing BCR but not ABL. Genes Chromosomes Cancer Press; 1994. p. 595–604. Rios RM. GMAP-210, a cis-Golgi network-associated 2001;32:302–10. 26. Chevrier V, Komesli S, Schmit AC, Vantard M, protein, is a minus end microtubule-binding protein. 9. Ollendorff V, Guasch G, Isnardon D, et al. Character- Lambert AM, Job D. A monoclonal antibody, raised J Cell Biol 1999;145:83–98. ization of FIM-FGFR1, the fusion product of the against mammalian centrosomes and screened by 43. Verde I, Pahlke G, Salanova M, et al. Myomegalin is a myeloproliferative disorder-associated t(8;13) transloca- recognition of plant microtubule organizing centers, novel protein of the golgi/centrosome that interacts tion. J Biol Chem 1999;274:26922–30. identifies a pericentriolar component in different cell with a cyclic nucleotide phosphodiesterase. J Biol Chem 10. Guasch G, Ollendorff V, Borg JP, Birnbaum D, types. J Cell Sci 1992;101:823–35. 2001;276:11189–98. Pe´busque MJ. 8p12 stem cell myeloproliferative disor- 27. Wehland J, Willingham MC, Sandoval IV. A rat 44. Reiter A, Walz C, Watmore A, et al. The der: the FOP-fibroblast growth factor receptor 1 fusion monoclonal antibody reacting specifically with the t(8;9)(p22;p24) is a recurrent abnormality in chronic protein of the t(6;8) translocation induces cell survival tyrosylated form of a-tubulin. Biochemical character- and acute leukemia that fuses PCM1 to JAK2. Cancer mediated by mitogen-activated protein kinase and ization, effects on microtubule polymerisation in vitro, Res 2005;65:2662–7. phosphatidylinositol 3-kinase/Akt/mTOR pathways. and microtubule polymerization and organization 45. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic Mol Cell Biol 2001;21:8129–42. in vivo. J Cell Biol 1983;97:1467–75. nucleophosmin in acute myelogenous leukemia with 11. Baumann H, Kunapuli P, Tracy E, Cowell JK. The 28. Wang Q, Hirohashi Y, Furuuchi K, et al. The a normal karyotype. N Engl J Med 2005;352:254–66. oncogenic fusion protein-tyrosine kinase ZNF198/fibro- centrosome in normal and transformed cells. DNA Cell 46. De Nadai C, Huitorel P, Chiri S, Ciapa B. Effect blast growth factor receptor-1 has signaling function Biol 2004;23:475–589. of wortmannin, an inhibitor of phosphatidylinositol comparable with interleukin-6 cytokine receptors. J Biol 29. Mohammadi M, McMahon G, Sun L, et al. Structures 3-kinase, on the first mitotic divisions of the fer- Chem 2003;278:16198–208. of the tyrosine kinase domain of fibroblast growth fac- tilized sea urchin egg. J Cell Sci 1998;111:2507–18. 12. Heath C, Cross NC. Critical role of STAT5 activation tor receptor in complex with inhibitors. Science 1997; 47. Chang F, Lee JT, Navolanic PM, et al. Involvement of in transformation mediated by ZNF198-FGFR1. J Biol 955–60. PI3K/Akt pathway in cell cycle progression, apoptosis, Chem 2004;279:6666–73. 30. Bloom J, Pagano M. Deregulated degradation of the and neoplastic transformation: a target for cancer 13. Guasch G, Delaval B, Arnoulet C, et al. FOP-FGFR1 cdk inhibitor p27 and malignant transformation. Semin chemotherapy. Leukemia 2003;17:590–603. tyrosine kinase, the product of a t(6;8) translocation, Cancer Biol 2003;13:41–7. 48. Metge B, Ofori-Acquah S, Stevens T, Balczon R. Stat3 induces a fatal myeloproliferative disease in mice. Blood 31. Emes RD, Ponting CP. A new sequence motif linking activity is required for centrosome duplication in 2004;103:309–12. lissencephaly, Treacher Collins and oral-facial-digital Chinese hamster ovary cells. J Biol Chem 2004;279: 14. Roumiantsev S, Krause DS, Neumann CA, et al. type 1 syndromes, microtubule dynamics and cell 41801–6. Distinct stem cell myeloproliferative/T lymphoma migration. Hum Mol Genet 2001;10:2813–20. 49. Heldin CH. Signal transduction: multiple pathways, syndromes induced by ZNF198-FGFR1 and BCR-FGFR1 32. Romio L, Fry AM, Winyard PJ, et al. OFD1 is a multiple options for therapy. Stem Cells 2001;19:295–303. fusion genes from 8p11 translocations. Cancer Cell 2004; centrosomal/basal body protein expressed during mes- 50. Kaplan DD, Meigs TE, Kelly P, Casey PJ. Identification 5:287–98. enchymal-epithelial transition in human nephrogenesis. of a role for h-catenin in the establishment of a bipolar 15. Chen J, Deangelo DJ, Kutok JL, et al. PKC412 inhibits J Am Soc Nephrol 2004;15:2556–68. mitotic spindle. J Biol Chem 2004;279:10829–32. the zinc finger 198-fibroblast growth factor receptor 1 33. Grand EK, Grand FH, Chase AJ, et al. Identification of 51. Rawe VY, Payne C, Navara C, Schatten G. WAVE1 fusion tyrosine kinase and is active in treatment of stem a novel gene, FGFR1OP2, fused to FGFR1 in 8p11 mye- intranuclear trafficking is essential for genomic and cell myeloproliferative disorder. Proc Natl Acad Sci U S A loproliferative syndrome. Genes Chromosomes Cancer cytoskeletal dynamics during fertilization: cell-cycle- 2004;101:14479–84. 2004;40:78–83. dependent shuttling between M-phase and interphase 16. Kunapuli P, Somerville R, Still IH, Cowell JK. ZNF198 34. Vizmanos JL, Novo FJ, Roman JP, et al. NIN, a gene nuclei. Dev Biol 2004;276:253–67.

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