Oncogenic Tyrosine Kinase of Malignant Hemopathy Targets ... · hematopoietic progenitor cells. Many MPDs result from a chromosomal translocation that creates a fusion gene encod-ing

Oncogenic Tyrosine Kinase of Malignant Hemopathy Targets

the Centrosome

Benedicte Delaval,1Sebastien Letard,

2Helene Lelievre,

1Veronique Chevrier,

3

Laurent Daviet,4Patrice Dubreuil,

2and Daniel Birnbaum

1

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

Abstract

Myeloproliferative disorders (MPD) are malignant diseases ofhematopoietic progenitor cells. Many MPDs result from achromosomal translocation that creates a fusion gene encod-ing a chimeric kinase. The fibroblast growth factor receptor 1(FGFR1)-MPD is characterized by the fusion of the FGFR1kinase with various partners, including FOP. We show herethat both normal FOP and FOP-FGFR1 fusion kinase localizeto the centrosome. The fusion kinase encounters substrates atthe centrosome where it induces strong phosphorylation ontyrosine residues. Treatment with FGFR1 kinase inhibitorSU5402 abolishes FOP-FGFR1-induced centrosomal phosphor-ylation and suppresses the proliferative and survival potentialsof FOP-FGFR1 Ba/F3 cells. We further show that FOP-FGFR1allows cells to overcome G1 arrest. Therefore, the FOP-FGFR1fusion kinase targets the centrosome, activates signalingpathways at this organelle, and sustains continuous entry inthe cell cycle. This could represent a potential new mechanismof oncogenic transformation occurring specifically at thecentrosome. (Cancer Res 2005; 65(16): 7231-40)

Introduction

Myeloproliferative disorders (MPD) are clonal malignant hemo-pathies that affect progenitor cells. MPD cells proliferatecontinuously but, in contrast to acute leukemia blasts, undergomaturation. The disease progresses towards an acute syndrome.Many MPDs are caused by a chromosome translocation thatproduces a fusion gene encoding a chimeric, constitutivelyactivated kinase protein. One of these oncogenic events occurs ina rare and aggressive MPD, the fibroblast growth factor receptor 1(FGFR1)-MPD. This MPD is also called stem cell MPD or 8p12MPD because both lymphoid and myeloid lineages are affectedfollowing activation of the FGFR1 tyrosine kinase, which isencoded by a gene on the p11-12 region of chromosome 8 (1).FGFR1-MPDs are characterized by fusion proteins (hereafterdesignated X-FGFR1) made of the FGFR1 catalytic domain fusedto a protein-protein interaction domain from several possiblepartners, including FOP/FGFR1OP (FGFR1 oncogene partner),CEP1 (centrosomal protein 1), ZNF198 (zing finger 198), andBCR (2–8). With the exception of BCR , none of the characterizedpartner genes has been found in a fusion involving another genethan FGFR1 . The same FGFR1 intracellular region, whichincludes the kinase domain, is preserved in all MPD-FGFR1

fusions. The receptor transmembrane region is not conserved inthe fusion. X-FGFR1 proteins promote cell survival throughsignaling pathways involving, among others, phospholipase Cg(PLCg), phosphoinositol-3 kinase (PI3K), AKT and STAT proteins(9–12). The disease has been reproduced in mouse bone marrowtransplantation models (13–15). The effects of X-FGFR1 proteinscan be abrogated by treatment with an inhibitor of the FGFR1kinase (7, 14, 15).The subcellular localization of X-FGFR1 proteins has been

studied, however, often only coarsely and in transfected cells withhigh levels of expression; fusion proteins are found predominantlyin the cytoplasm. Normal partners have been found at varioussubcellular localizations. ZNF198 was found predominantly in thenucleus (3, 6, 9, 11, 16). FOP was found in the cytoplasm (6);however, a recent observation is noteworthy: a list of centrosomalproteins established by proteomic analysis included FOP (17). CEP1is located in a specific domain at the open end of the centrosometube associated with maturation of a daughter centrosome in amother centrosome, and is required for centrosome function (18).The centrosome is an organelle important for nucleation and

organization of microtubules but is also essential for cell cycleprogression mostly during the G1-S transition (19–22). Thisparticular localization of FOP and CEP1 at the centrosome suggestedthat FGFR1 fusion partners may not only provide dimerizationdomains but also target oncogenic kinases to a specific area. Weshow here that FOP-FGFR1 is targeted to the centrosome where itactivates signaling pathways via tyrosine phosphorylation. Thisphosphorylation at the centrosome and the proliferative potential ofFOP-FGFR1-expressing cells are abolished after treatment with akinase inhibitor. We also show that FOP-FGFR1 is important duringG1-S transition to overcome G1 arrest and allow cells to sustaincontinuous cell cycle. This led us to hypothesize that FOP-FGFR1proteins may exert an oncogenic activity through dysregulation ofcell processes associated with the centrosome.

Materials and Methods

Plasmids, cells, and reagents. Rat2 cells are fibroblastic cells. Ba/F3 aremurine hematopoietic cells that need to be cultured in the presence of

interleukin-3 (IL-3). FGFR1 is not expressed in native, nontransfected Ba/F3

cells. FOP, FOP-FGFR1, FOP-FGFR1 kinase-defective (K259A), PLCg bindingsite (Y511F) mutants, CEP1-FGFR1, wild-type FGFR1 (FGFR1wt) constructs,

and corresponding clones of stably-transfected Rat2 or Ba/F3 cells used in

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

the first FGFR1 kinase subdomain. The kinase-defective mutant haspreviously been characterized (10). BCR-FGFR1 construct is described in

ref. (7) and was a kind gift from Dr. N.C. Cross. For FGFR1wt, two conditions

of stimulation were used: a short stimulation corresponding to 5 minutes of

Requests for reprints: Daniel Birnbaum, Laboratory of Molecular Oncology,Marseille Cancer Institute, UMR599 Inserm, 27 Bd. Leı Roure, 13009 Marseilles, France.Phone: 33-49175-8407; Fax: 33-49126-0364; E-mail: [email protected].

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-04-4167

www.aacrjournals.org 7231 Cancer Res 2005; 65: (16). August 15, 2005

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stimulation with 10 ng/mL FGF1 (P100-17A from AbCys, Paris, France) and10 Ag/mL heparin (H-0777 from Sigma, Saint Quentin Fallavier, France) and

a long stimulation corresponding to a 48-hour culture in the presence of

10 ng/mL FGF1 and 10 Ag/mL heparin. For inhibition experiments,

concentrations of 0.15, 1.5, and 15 Amol/L of the kinase inhibitor, ATP-competitor, SU5402 (Calbiochem, Merck Biosciences, Darmstadt, Germany)

and 0.1, 1, and 10 Amol/L of STI571 (a gift from Dr P. Manley, Novartis),

respectively, were used. The EOL-1 cell line, used to study the FIP1L1-PDGFRA

fusion (23), was a gift from Dr. B. Papp (Hopital St Louis, Paris, France).Antibodies. We used monoclonal anti-myc (9E10), polyclonal anti-

FGFR1 (C-15), polyclonal anti-PLCg (1,249), anti-GRB2 (C-23) from Santa

Cruz Biotechnology (Santa Cruz, CA), anti-phospho-STAT1 (Y701), anti-

phospho-STAT3 (Y705), anti-phospho-STAT5 (Y694) from Cell SignalingTechnology (Beverly, MA), anti-p27 (610241) from BD Biosciences (Pont de

Claix, France), anti-PI3K (06-195) from Upstate Biotechnology (Mundol-

sheim, France), anti-g-tubulin either monoclonal (GTU-88) or polyclonal(T3559) from Sigma, and anti-phosphotyrosine (anti-phosphotyrosine; 4G10;

ref. 10).

Immunofluorescence analyses. Immunofluorescence analyses were

done as previously described (24). Briefly, Rat2 or Ba/F3 cells either grownon glass coverslips or centrifuged on poly-L-lysine–coated coverlips,

respectively, were fixed in cold methanol for 5 minutes. After permeabiliza-

tion with 0.5% Triton X-100 for 5 minutes, cells were incubated at room

temperature for 60 minutes with the first antibody and then for 45 minuteswith the secondary antibody. Samples were then stained with the DNA-

specific 4V,6-diamino-2-phenylindole (DAPI; Sigma).

Most immunofluorescence images were recorded by a TCS-NT confocalMicroscope (Leica Microsystem, Mannheim, Germany) controlled by a Leica

software. Images shown after confocal acquisitions were pseudocolored

with Leica software, correspond to one confocal section and were notsubmitted to additional treatment. For immunofluorescence with DAPI

staining and on purified centrosomes, acquisitions were done using a Zeiss

Axiovert 200 microscope equipped with Cool Snap HQ camera (Ropper

Scientific, Evry, France) controlled by Metamorph software (UniversalImaging, Downingtown, PA). For immunofluorescence images containing

DAPI staining, Z stacks were acquired, deconvoluted and analyzed with

Metamorph software. Monochrome images were collected for each

appropriate channel and pseudocolored with Metamorph. Single opticalsections are presented.

Immunofluorescence on purified centrosomes. Centrosomes were

isolated from FOP-FGFR1 and FOP-FGFR1 kinase-defective cells aspreviously described (25). Centrosomes were sedimented on glass

coverslips at 24,000 � g , fixed with methanol and processed for

immunofluorescence as described (26). Antibodies used included anti-g-tubulin, anti-phosphotyrosine, and anti-a-tubulin YL1/2 (1/1,000; ref. 27),

goat anti-rabbit Alexa 488 (Molecular Probes, Invitrogen, Cergy Pontoise,

France), goat anti-mouse Cy3 and donkey anti-rat Cy5 ( Jackson

ImmunoResearch Laboratories, Cambridgeshire, United Kingdom).MPD mice. Mice developing MPD and kinase-defective controls have

been described previously (13). Briefly, bone marrow from 5-fluorouracil-

treated mice was enriched in early hematopoietic precursors by positive

selection using stem cell antigen-1 Sca-1. Sca-1+-cells were transduced withMSCVneo retroviral vectors with FOP-FGFR1 or kinase-defective. FOP-

FGFR1 transplanted mice but not kinase-defective mice developed a fatal

MPD within 4 weeks after transplantation, characterized by markedleukocytosis, hypercellular bone marrow, and hepatosplenomegaly indica-

tive of myeloid hyperplasia. Hematopoietic progenitors (spleen colony-

forming unit) from enlarged spleens of MPD mice were collected,

maintained in culture without cytokines and used for immunofluorescenceexperiments. Morphologic study of these cells showed a majority of mature

and immature granulocytes, in contrast to a normal spleen that contains a

majority of lymphocytes.

Cell lysis, immunoprecipitation, and Western blot. For inhibitorexperiments, 3 � 106 nontransfected Ba/F3 cells or Ba/F3 cells expressing

wild-type or fusion proteins cultivated in the presence or absence of IL-3,

respectively, were lysed in 200 AL as previously described (9). NP40 lysates(10) from 20 � 106 Ba/F3 cells were used for immunoprecipitation

experiments with anti-g-tubulin monoclonal antibody. Protein extracts andimmunoprecipitated complexes were separated by SDS-PAGE, transferred

onto membrane and probed with anti-phosphotyrosine antibody.

Cell survival and proliferation assays. The number of viable cells in

the presence or absence of inhibitors was measured by trypan blueexclusion. Cell proliferation was monitored by [3H]thymidine uptake. A total

of 5 � 103 Ba/F3 cells, and 2 � 104 FGFR1wt, FOP-FGFR1, CEP1-FGFR1,

BCR-FGFR1 Ba/F3 cells or EOL-1 cells were grown in duplicate in 96-well

plates in the presence or absence of IL-3, respectively. Cells were incubatedfor 48 hours at 37jC and pulsed with 0.5 ACi of [methyl-3H]thymidine

(Amersham Biosciences, Orsay, France) for the last 6 hours (Ba/F3 cells) or

24 hours (Ba/F3 transfected with FGFR1 fusions). Cells were then

transferred onto glass filters (Packard Instruments, Netherlands), andincorporation was measured using a B-counter Rack-h Compact 1212-411

(LKB, Uppsala, Sweden).

Cell cycle analysis. For cell cycle analysis, murine IL-3-dependent Ba/F3cells transfected or not with fusion proteins were cultured in the presence

or absence of IL-3. Cells presynchronized in G0/G1 by IL-3 overnight

withdrawal were irradiated (10 Gy) and immediately returned to 37jC,either in the presence or absence of IL-3 for 8 hours. Cells were thenharvested and DNA content was analyzed (24). Flow cytometry analysis

after propidium iodide incorporation revealed the presence of G0/G1, S, and

G2-M population. Sub-G1 population corresponds to dying cells.

Results

FOP and FOP-FGFR1 are centrosomal proteins. To determinewhen, during the cell cycle, FOP was present at the centrosome, wedid immunofluorescence experiments on stable Rat2 cell clonesoverexpressing myc-tagged FOP. FOP localizes to the centrosome(Fig. 1A) both in interphase before (Fig. 1Aa and Da) and aftercentrosome duplication (Fig. 1Ab and Db) and in dividing cells(Fig. 1Acd and Dcd). These results suggested that some fusionpartners may not only provide dimerization domains but couldalso determine the subcellular localization of the correspondingoncogenic kinase. Therefore, we wondered if FOP-FGFR1 fusionprotein was addressed to the centrosome. In Rat2 clonesexpressing myc-FOP-FGFR1, the fusion protein was localizedexclusively at the centrosome during the whole cell cycle, ininterphasic cells before (Fig. 1Ba and De) and after centrosomeduplication (Fig. 1Bb and Df ), and also during metaphase(Fig. 1Bc and Dg ) and cytokinesis (Fig. 1Bd and Dh). FOP-FGFR1centrosomal localization is detected with both myc (Fig. 1B andCa) and FGFR1 (Fig. 1Cab) antibodies. When it is not fused toFOP, FGFR1 kinase is not detected at the centrosome (Fig. 1Cd).Thus, an ectopic, activated fusion kinase of FGFR1-MPDs couldbe targeted to the centrosome.FOP-FGFR1 proteins can signal at the centrosome during

G1-S phase. In subsequent experiments, we used Rat2 cells toallow easy visualization of the centrosome before and after dupli-cation and Ba/F3 cells for functional studies.To exert its effects at the centrosome, the oncogenic fusion

protein must induce tyrosine phosphorylation of downstreamsubstrates at this subcellular site (Fig. 2A). Study of globaltyrosine phosphorylation in FOP-FGFR1 expressing Rat2 cellsrevealed a strong centrosomal staining during interphase before(data not shown) and after (Fig. 2Ba) centrosome duplication.This staining could represent either FOP-FGFR1 autophosphor-ylation or the phosphorylation of its downstream substrates orboth. The staining was stronger in interphasic cells (Fig. 2BbI)than in mitotic cells (Fig. 2BbM). Because FOP-FGFR1 is presentat the centrosome during the whole cell cycle, we think thatphosphotyrosine staining at the centrosome in mitosis corresponds

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Cancer Res 2005; 65: (16). August 15, 2005 7232 www.aacrjournals.org



only to FOP-FGFR1 autophosphorylation, whereas phosphotyr-osine staining during interphase represents phosphorylation ofboth FOP-FGFR1 and its substrates. This result shows that FOP-FGFR1 encounters substrates with tyrosine phosphorylation sitesat the centrosome. It also suggests that phosphotyrosine signalingis important for G1-S events, when centrosome duplicationoccurs. Phosphotyrosine staining was absent in cells expressinga kinase-defective FOP-FGFR1 K259A mutant although themutant protein also localized to the centrosome (Fig. 2Bc).Because FGFR1wt is not targeted to the centrosome (Fig. 1Cd)vesicular cytoplasmic but not centrosomal phosphotyrosinestaining was detected in Rat2 cells overexpressing FGFR1wt(Fig. 2Bd).A very strong phosphotyrosine staining was similarly detected at

the centrosome of interphasic Ba/F3 cells expressing myc-FOP-FGFR1 (Fig. 2Ca) but not in mitotic cells (data not shown) or incells expressing the kinase-defective mutant (Fig. 2Cb), even if bothproteins localized to the centrosome (Fig. 2Ccd). Immunofluores-cence on purified centrosomes confirmed this result (Fig. 2Ce-l);phosphotyrosine staining colocalized with pericentriolar material

and centrioles stained with g-tubulin and tyrosylated a-tubulin,respectively.Expression of FOP-FGFR1 in primary bone marrow cells induced

by retroviral transduction generates a fatal MPD in micecharacterized by myeloid hyperplasia and hepatosplenomegaly(13). We used cells isolated from the spleen of such transplantedFOP-FGFR1 mice to investigate FOP-FGFR1 activity in conditionsclose to those of the natural disease. Hematopoietic progenitors(spleen colony-forming unit) from spleens of FOP-FGFR1 micewere collected and maintained in culture without cytokines for >2months, showing that these cells have a proliferative potential.Immunofluorescence on cultured cells showed strong FOP-FGFR1(Fig. 2Da) and phosphotyrosine (Fig. 2Db) signals at thecentrosome. Thus, FOP-FGFR1 is targeted to the centrosome andsignals at this organelle, bringing tyrosine phosphorylation, bothin vitro and in vivo .FOP-FGFR1 protein encounters, recruits, and phosphory-

lates substrates at the centrosome. We next wondered whichwere the substrates activated by FOP-FGFR1 at the centrosome. Wefirst 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 cyclewere 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 Leicaconfocal (D). Bar , 10 Am.

Centrosome Targeting by Oncogenic Kinase




STAT1, -3, -5 proteins, PLCg, PI3K, AKT and p70S6K proteins (10).We found p70S6K and phospho-AKT at the centrosome ininterphasic and mitotic Rat2 cells, respectively (data not shown).PLCg, which was present at the spindle pole during mitosis inRat2 cells transfected (Fig. 3Aa) or not with FOP-FGFR1, wasrecruited to the centrosome in interphase before and aftercentrosome duplication in FOP-FGFR1 (Fig. 3Abc) but not inFOP-FGFR1 kinase-defective cells (Fig. 3Ade ) or in cellsexpressing FOP-FGFR1 Y511F mutant, which lacks the PLCgbinding site (Fig. 3Afg ). In Ba/F3 cells, PLCg was also recruitedto the centrosome in the presence of FOP-FGFR1 (Fig. 3Babc,arrow) but neither in its absence (Fig. 3abc, arrowhead) nor inthe presence of kinase-defective mutant (Fig. 3Bdef ). We havepreviously shown that PLCg interacts and is phosphorylated byFOP-FGFR1 (10); we show here that this could occur at thecentrosome during the G1-S phase of the cell cycle.

PI3K was present at the centrosome of interphasic cellstransfected (Fig. 3Bghi, arrow) or not (Fig. 3Bghi, arrowhead) withFOP-FGFR1. PI3K is important for centrosome duplication (28).Because PI3K colocalizes with FOP-FGFR1 (Fig. 3Bghi), we suspectthat phosphorylation of PI3K by FOP-FGFR1 occurs at thecentrosome. It may play a role when centrosome duplicationoccurs at the G1-S transition.Study of phosphotyrosine STAT1, STAT3, and STAT5 subcellular

localization in Rat2 cells showed, in addition to their knowncytoplasmic and nuclear localizations, a strong phosphotyrosinestaining at the centrosome of interphasic cells, before (data notshown) and after (Fig. 3Cabc, arrow) centrosome duplication. Thisphosphorylation in FOP-FGFR1 but not in kinase-defective mutantcells (Fig. 3Cdef ) could facilitate subsequent STAT activation (10).FOP-FGFR1 Ba/F3 cells, in which the STAT3 pathway is activated(10), showed the same result (Fig. 3Cg, arrow). No phospho-STAT3

Figure 2. FOP-FGFR1 induces tyrosine phosphorylation at the centrosome during the G1-S transition of the cell cycle. A, schematic representation of FOP-FGFR1protein. K259 belongs to the ATP-binding site necessary for the kinase activity of the fusion protein (TK, tyrosine kinase subdomains); it is mutated in thekinase-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 ofRat2 cells expressing FOP-FGFR1 (a and b) but not in Rat2 cells expressing FOP-FGFR1 kinase-defective mutant (c ). Phosphotyrosine staining of interphasiccells (a and bI ) is strong compared with mitotic cells (bM ). Staining with anti-phosphotyrosine (red ) and anti-g-tubulin (green ) on Rat2 expressing FGFR1wt showsabsence of detectable phosphorylation at the centrosome (d ). Bar , 10 Am. C, costaining using anti-phosphotyrosine (red) and anti-g-tubulin (green ) revealsphosphorylation 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 , 5 Am. Costaining using anti-g-tubulin (green ), phosphotyrosine (red ), and antityrosylated a-tubulin (blue) on purified centrosomes from Ba/F3 cellsexpressing FOP-FGFR1 (e-h) or kinase-defective mutant (i-l ). Scale , 10 Am (3.4 cm). D, presence of FOP-FGFR1 (a ) and phosphotyrosine (b ) at the centrosome ofcolony-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 , 10 Am.

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signal was detected in untransfected (Fig. 3Cg, arrowhead) orkinase-defective mutant (Fig. 3Ch) cells.To further show that FOP-FGFR1-activated substrates were

associated with the centrosome, we immunoprecipitated proteinsassociated with the pericentriolar material using anti-g-tubulinantibody. We found many proteins phosphorylated on tyrosineresidues bound to g-tubulin in FOP-FGFR1 Ba/F3 lysates (e.g., redasterisks), which were absent in kinase-defective mutant lysates(Fig. 3D). These proteins can either be signaling moleculesphosphorylated at the centrosome or intrinsic centrosomalproteins. Several proteins of high molecular mass in particular(red asterisks) could be centrosomal proteins directly phosphor-ylated by FOP-FGFR1; however, they remain to be characterized. Inconclusion, FOP-FGFR1 protein constitutively activates substratesat the centrosome.Phosphorylation at the centrosome and proliferation in

different clones. We next studied if other kinases were targeted tothe centrosome and/or induced phosphorylation at this site. We

used proliferating Ba/F3 cells expressing several FGFR1wt or fusionproteins and the EOL-1 cell line (23) expressing FIP1L1-PDGFRAfusion (Fig. 4A). Ba/F3 cells normally need IL-3 for survival andproliferation. Cell proliferation experiments using [3H]thymidineincorporation indicated that FOP-FGFR1 Ba/F3 cells not onlysurvived as previously shown (10), but even proliferated in theabsence of IL-3, although to a lesser degree than in the presence ofIL-3 (data not shown). Kinase-defective mutant cells did notsurvive in the absence of IL-3. FGFR1wt cells cultivated in thepresence of FGF1 and heparin were used as controls. To determineif the phosphorylation pattern obtained with FOP-FGFR1 was dueto its centrosomal localization we compared (a) phosphorylationprofiles on Western blot and (b) global phosphotyrosine localiza-tion induced by different fusion proteins and FGFR1wt.Western blot analysis after IL-3 starvation for 8 hours showed a

phosphorylation profile specific for FOP-FGFR1 (Fig. 4A, redasterisk). Some substrates, probably corresponding to the onesimmunoprecipitated 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 bykinase-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 cellsexpressing 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 ). PhosphotyrosineY705-STAT3 staining in FOP-FGFR1 (g ) Ba/F3 cells but not kinase-defective (h ). Bar , 10 Am. D, Western blot with anti-phosphotyrosine shows phosphorylatedFOP-FGFR1 (black asterisk) and its substrates (red asterisk ) in FOP-FGFR1 but not kinase-defective mutant lysates or after immunoprecipitation with anti-g-tubulin.





exclusively after FOP-FGFR1 direct centrosomal activation but notafter FGFR1wt activation (whether long or short), which is notdirectly targeted to the centrosome. This indicates that FOP-FGFR1-specific targeting to the centrosome can directly phosphor-ylate additional centrosomal proteins. Because of its molecularmass, CEP1-FGFR1, the other fusion protein localizing to thecentrosome, did not allow the detection of this pattern. Westernblots were also useful to control the expression of the differentfusion proteins (Fig. 4A, black asterisk).Phosphotyrosine staining (Fig. 4B), which allows the detection

of the fusion protein and its substrates, showed that FOP-FGFR1 was the most specifically and exclusively targeted to thecentrosome where it induces tyrosine phosphorylation (Fig. 4Bd).Ba/F3 cells expressing CEP1-FGFR1 also displayed strong centro-somal staining, suggesting that the fusion protein is also targetedto the centrosome. However, this localization seemed less exclusivethan that of FOP-FGFR1 and some cytoplasmic fusion proteincould be detected, perhaps due to a high level of expression

(Fig. 4A). FGFR1wt, BCR-FGFR1, and FIP1L1-PDGFRA were notdirectly targeted to the centrosome and the phosphotyrosine signalwas detected in the cytoplasm (Fig. 4Bg-j). However, somephosphorylation staining was detected at the centrosome of someproliferating cells (Fig. 4Bg-j, arrow). Similarly, in the presence ofIL-3, phosphotyrosine staining was, in some cells, also detected atthe centrosome (Fig. 4Ba, arrow). This could correspond todownstream substrates common to both IL-3 and FGFR1activation pathways (e.g., STAT5). These results suggest that anyof these signalings indirectly activate substrates at the centrosome,at least at some specific time of the cell cycle. No centrosomalphosphorylation could be detected in nonproliferating cells, Ba/F3and kinase-defective mutant cells without IL-3 (Fig. 4Bbc). Wepropose that bringing phosphorylation directly to the centrosomeis sufficient to allow cells to enter a proliferating state.Centrosomal phosphorylation and proliferation induced by

FOP-FGFR1 are inhibited by FGFR1 kinase inhibitor. To furthershow that centrosomal staining is required for survival and

Figure 4. Phosphotyrosine profiles on Western blot and centrosomal tyrosine phosphorylation localization induced by various oncogenic kinases. A, Western blotanalysis shows differential phosphotyrosine profiles of various cells: Ba/F3, FOP-FGFR1, FOP-FGFR1 K259A mutant, FGFR1wt, BCR-FGFR1, CEP1-FGFR1transfected Ba/F3, or EOL-1 (FIP1L1-PDGFRA) cells. Fusion proteins and substrates are indicated by black and red asterisks, respectively. B, immunofluorescenceexperiment shows the corresponding localization of tyrosine phosphorylation (red ; s. stim and l. stim, short and long stimulation of wild-type FGFR1 by FGF andheparin). Arrows , phosphotyrosine staining at the centrosome. Bar , 10 Am.

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proliferation we used the kinase inhibitor SU5402. SU5402 interactsdirectly with FGFR1 catalytic domain and can inhibit [3H]thymi-dine incorporation of cells stimulated by FGF1 (29). Phosphoryla-tion at the centrosome of FOP-FGFR1 Ba/F3 cells was specificallyabolished after SU5402 treatment (Fig. 5A). A strong phosphotyr-osine staining was detected at the centrosome of Ba/F3 cells in thepresence of a low, inefficient concentration (0.15 Amol/L) ofSU5402 (Fig. 5Aac), or with STI571 (data not shown), an inhibitorwith a different specificity known to be inefficient on FGFR1kinase. In contrast, treatment with 15 Amol/L of SU5402 for 90minutes reduced phosphotyrosine staining at the centrosome ofBa/F3 cells expressing FOP-FGFR1 (data not shown). After 15 hoursof SU5402 treatment, most cells were dying (data not shown). Theremaining cells showed no or very low levels of phosphotyrosinestaining at the centrosome (Fig. 5Abd). This result indicatesthat centrosomal phosphorylation is required for survival andproliferation.Indeed, loss of phosphorylation at the centrosome observed after

SU5402 treatment correlated with loss of proliferation measuredusing [3H]thymidine incorporation: FOP-FGFR1-induced prolifera-tion was inhibited by increasing concentrations of SU5402 (Fig. 5B).Because SU5402 is cytotoxic (29) this loss of proliferation wasassociated with a loss of cell survival verified by cell count aftertrypan blue exclusion (data not shown). In contrast, proliferation

induced by FOP-FGFR1 was not abolished by STI571 (Fig. 5B).SU5402 also showed an inhibitory effect on the proliferation ofFGFR1wt, CEP1-FGFR1, BCR-FGFR1 Ba/F3 cells, and EOL-1 cells,but not on untransfected Ba/F3 cells (Fig. 5B). SU5402 is known toinhibit FGFR1 and platelet-derived growth factor receptor tyrosinekinase activity. The results of SU5402 and STI571 treatments wereverified in Western blot experiments; in agreement with cellproliferation experiments, SU5402, but not STI571, induced loss oftyrosine phosphorylation in FOP-FGFR1 cells (Fig. 5C).In conclusion, phosphorylation at the centrosome in the

presence of FOP-FGFR1, which is inhibited by SU5402, is essentialfor survival and proliferation of Ba/F3 FOP-FGFR1.FOP-FGFR1 has an effect on the cell cycle. To reveal FOP-

FGFR1 potential, we studied the consequence on the cell cycle ofa stable overexpression of FOP-FGFR1 at the centrosome in Ba/F3 cells. Ba/F3 cells transfected with an empty vector or thekinase-defective mutant were used as control. Two differentconditions of stress, IL-3 withdrawal and irradiation, were used(Fig. 6A and B).As expected, Ba/F3 cells transiently arrested in G0/G1 after IL-3

withdrawal, did not enter S phase (Fig. 6A) and died (data notshown). The ratio G1/(S + G2) is used to represent cells blocked atthe G1-S checkpoint. The lack of G1 arrest in FOP-FGFR1 cells wasrevealed 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 ) inBa/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) withg-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 , 10 Am. 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 inpercentage: 100% represents the mean proliferation rate obtained without addition of drug. Columns , mean; bars , F SD. Similar results were obtained in threeindependent experiments. C, Western blot with anti-phosphotyrosine antibody shows FOP-FGFR1 phosphotyrosine inhibition (top ) 90 minutes after treatment withincreasing 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 ).





kinase-defective mutant cells (Fig. 6A). Accordingly, FOP-FGFR1cells were protected from cell death and 26% of cells even enteredthe cell cycle in the absence of IL-3 (Fig. 6Bb) as compared with 9%for kinase-defective mutant cells (Fig. 6Bf ). The presence of theconstitutively active oncogenic protein at the centrosome was thusenough to bypass a restriction point and sustain continuous entryin S phase in the absence of IL-3.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,although less than the mere presence of IL-3, and overcame G1

arrest after irradiation (Fig. 6A). Indeed, the ratio G1/(S + G2) wasreduced in irradiated FOP-FGFR1 cells as compared with controlcells (Fig. 6A). The marked increase of the G2-M population(Fig. 6Bcd) also revealed the lack of G1 arrest after irradiationcompared with kinase-defective mutants (Fig. 6Bgh). This potentialdepended on tyrosine kinase activity because kinase-defective cellsreacted exactly like Ba/F3 control cells (Fig. 6A). The PLCg mutanthad a moderate effect on the G1 arrest (data not shown).

p27 is an inhibitor of the G1-S transition. When cells enter Sphase, p27 degradation is induced after CDK2/cyclin E activation(30) and decrease of p27 is observed in proliferating cells (Fig. 6C).Decrease of p27 expression in FOP-FGFR1 Ba/F3 cells revealed theproliferative potential of cells entering S phase (Fig. 6C). SU5402treatment abolished this effect. This result is in favor of FOP-FGFR1 inducing S phase entry.

Discussion

We have shown that FOP and FOP-FGFR1, the oncogenic fusionkinase of an FGFR1-MPD, are addressed to the centrosome.Centrosomal addressing of the fusion protein occurs not only intransfected cultured cells but also in hematopoietic cells of an MPDmouse model. Our search for FOP partners by two-hybrid screen inyeast identified further centrosomal proteins such as CAP350,confirming these observations. FOP has only one identified domaincalled LisH, which it shares with other proteins such as PAFAH1B1,TCOF1, and OFD1. LisH proteins are mutated in Miller-Dieker

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 cycleanalysis 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-tubulinantibody to compare the amount of proteins in the lysates (bottom ). D, diagram synthesizing the results.

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lissencephaly, Treacher Collins syndrome, oral-facial-digital type 1and contiguous syndrome ocular albinism with late onsetsensorineural deafness (31). LisH motifs contribute to theregulation of microtubule dynamics, either by mediating dimer-ization, or else by binding cytoplasmic dynein heavy chain ormicrotubules directly. However, mutation of the LisH domain ofOFD1 did not abrogate centrosomal localization (32). Therefore,the region of FOP-FGFR1 needed for its centrosomal localizationremains to be determined.We have further shown that the oncogenic fusion kinase

encounters or recruits substrates (e.g., PLCg) at the centrosomewhere it induces strong phosphorylation on tyrosine residues, bothin vitro and in vivo . FOP-FGFR1 substrates at the centrosome couldeither be signaling molecules phosphorylated at the centrosome(e.g., STAT) or intrinsic centrosomal proteins. Comparison ofphosphorylation patterns of other fusion proteins or FGFR1 wild-type shows that FOP-FGFR1 and CEP-FGFR1 are the most directlytargeted to the centrosome. This further suggests that any of thesesignaling pathways seem to activate relays at the centrosome, atleast at some periods of the cell cycle. Treatment with SU5402abolishes kinase-induced centrosomal phosphorylation and sup-presses the proliferative potential of FOP-FGFR1 Ba/F3 cells.Finally, we have shown that FOP-FGFR1 at the centrosome allowscells to proliferate and overcome G1 arrest. The results aresummarized in Fig. 6D .We therefore propose that targeting an oncogenic, constitutively

active kinase to the centrosome is enough to overcome cell cyclearrest, overcome a restriction point during the G1-S transitionwhen centrosome duplication occurs, and force entry in the cellcycle. It is the first time that an oncogenic product of a humandisease is shown to be addressed to the centrosome. This raises atleast two questions.How general is the phenomenon? Both FOP-FGFR1 and CEP1-

FGFR1 localize to the centrosome. This localization and stabilityseem sufficient for these kinases to exert their effects. However,centrosomal localization may not be necessary to trigger thedisease. Indeed, the study of ZNF198-FGFR1 (3, 9, 11) and BCR-FGFR1 (this work), two other well-characterized fusion kinases ofFGFR1-MPDs have not alluded to a potential centrosomallocalization. However, we have shown here that even if BCR-FGFR1 is not targeted to the centrosome, some relay of theoncogenic signal may take place at the centrosome. Conversely,FOP-FGFR1 and CEP1-FGFR1 may not be the only oncogenickinases to target the centrosome. A new FGFR1-MPD withFGFR1OP2-FGFR1 fusion has been described recently (33).FGFR1OP2 has coiled-coil motifs. These motifs are frequentlyfound in centrosomal proteins. Centrosomal targeting of oncogenickinases may even occur in other malignancies than FGFR1-MPDs. Acase of MPD has been described in which the platelet-derivedgrowth factor receptor B kinase is fused to ninein, a centrosomalprotein with CEP1-like structure and function (34). Several othercases of MPD have been described with rearrangements involvingplatelet-derived growth factor receptor B and numerous partners(35–41). Some of these partners may be localized at thecentrosome (42, 43). Finally, we and others have evidence forfusion of JAK2 kinase with centrosomal protein PCM1 in MPDwith t(8;9) translocation (44).5 Thus, there might be two classes of

ectopic oncogenic kinases, those that directly target the centro-some (or at least the Golgi/centrosome area), and those that donot. A non–kinase oncogene may also abnormally function at thecentrosome (45).What are the effects of the oncogenic kinase at the centrosome?

It is likely that FOP-FGFR1 exerts its oncogenic activity throughdysregulation of cell processes associated with the centrosome.Centrosomes nucleate microtubules and contribute to mitoticspindle organization and function. They also participate incytokinesis and cell cycle progression. The first type of alterationassociated with centrosome defect is aneuploidy. However, thekaryotype of X-FGFR1-positive cells, either in patients or aftertransfection of chimeric genes, does not show a particularly highdegree of aneuploidy as compared with other types of hemopa-thies or cancers. There is no amplification of the centrosomes incells overexpressing FOP-FGFR1. Experiments in Ba/F3 cellsshowed that FOP-FGFR1 interferes with the G1 checkpoint. Wemay have uncovered a mechanism of oncogenic transformationassociated with a defect of centrosome function but not ofcentrosome number; this mechanism will need to be furtherdelineated.The centrosome is important for the cell cycle; it influences cell

shape, polarity, and motility; it is also linked to DNA repair (19–21).Thus, abnormal activation of the FGFR1 tyrosine kinase at thecentrosome may affect several processes by disrupting theregulation of various molecular complexes that remain to beidentified. We have shown here that components of the FGFR1cascade which interact with the chimeric X-FGFR1 proteins arelocalized at the centrosome either during interphase and mitosis orboth. The PI3K-AKT/PKB pathway is particularly interesting in thiscontext. It regulates G1 cyclins (D1 and E), is involved incentrosome duplication, and is well-known to be associated withcell survival and cell proliferation, which are two cell processespredominantly affected in MPD (28, 46, 47). Similarly, a recentreport has also pointed to the role of STAT3 in centrosomeduplication (48). Downstream targets of AKT and STAT incentrosome regulation remain unidentified.We hypothesize that the centrosome, which is linked to the

microtubules, close to the nucleus, and connected to the Golgiapparatus and the proteasome, could be an integrating place forsome of the multiple signaling pathways controlling cell division,cell migration, and cell fate (49). In embryogenesis and normalprocesses of proliferation and differentiation, different types ofsignaling are associated with centrosome duplication and functionand centrosomal activity is linked to cell division (50, 51). Like viralproteins, an oncogenic protein would prey upon these normalsignals and use them to its profit. Abnormal kinase activity at thecentrosome should be an efficient way to pervert cell division inmalignancy.

Acknowledgments

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

Paoli-Calmettes, Canceropole, and Ligue Nationale Contre le Cancer. B. Delaval hasbeen successively supported by the Ministry of Research, the Ligue Nationale Contre leCancer, and the Societe Francaise d’Hematologie.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 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. Pebusque, and C. Popovici fordiscussions, help, and/or reagents.5 Murati et al., unpublished.





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2005;65:7231-7240. Cancer Res Bénédicte Delaval, Sébastien Létard, Hélène Lelièvre, et al. the CentrosomeOncogenic Tyrosine Kinase of Malignant Hemopathy Targets

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