Oncogenic Tyrosine Kinase of Malignant Hemopathy Targets the Centrosome
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Research Article Oncogenic Tyrosine Kinase of Malignant Hemopathy Targets the Centrosome 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 phosphor- ylation 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.