Published OnlineFirst February 11, 2016; DOI: 10.1158/1541-7786.MCR-15-0497

Oncogenes and Tumor Suppressors Molecular Cancer Research Oncogenic Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation Katelyn N. Nelson1, April N. Meyer1, Asma Siari2, Alexandre R. Campos3, Khatereh Motamedchaboki3, and Daniel J. Donoghue1,4

Abstract

Fibroblast growth factor receptors (FGFR) are critical for cell phosphorylation of key activating FGFR3 tyrosine residues. proliferation and differentiation. Mutation and/or transloca- ThepresenceoftheTACCcoiled-coildomainleadstoincreased tion of FGFRs lead to aberrant signaling that often results in and altered levels of FGFR3 activation, fusion phos- developmental syndromes or cancer growth. As sequencing of phorylation, MAPK pathway activation, nuclear localization, human tumors becomes more frequent, so does the detection cellular transformation, and IL3-independent proliferation. of FGFR translocations and fusion . The research con- Introduction of K508R FGFR3 kinase-dead mutation abrogates ducted in this article examines a frequently identified fusion these effects, except for nuclear localization which is due solely protein between FGFR3 and transforming acidic coiled-coil to the TACC3 domain. containing protein 3 (TACC3), frequently identified in glio- blastoma, lung cancer, bladder cancer, oral cancer, head and Implications: These results demonstrate that FGFR3 kinase neck squamous cell carcinoma, gallbladder cancer, and cervical activity is essential for the oncogenic effects of the FGFR3- cancer. Using titanium dioxide–based phosphopeptide enrich- TACC3 fusion protein and could serve as a therapeutic target, ment (TiO2)-liquid chromatography (LC)-high mass accuracy but that phosphorylated tyrosine residues within the TACC3- tandem mass spectrometry (MS/MS), it was demonstrated that derived portion are not critical for activity. Mol Cancer Res; 14(5); the fused coiled-coil TACC3 domain results in constitutive 458–69. 2016 AACR.

Introduction their initial discovery in the late 1990s, the detection of these fusion proteins has steadily increased at a surprising rate. The A subset of the Receptor Tyrosine Kinase (RTK) family is the focus of this article is a fusion protein consisting of FGFR3 fused to fibroblast growth factor receptor (FGFR) family, which contains transforming acidic coiled-coil containing protein 3 (TACC3) that four homologous receptors: FGFR1, FGFR2, FGFR3, and FGFR4. has been identified in glioblastoma, lung cancer, bladder cancer, FGFR activation results in changes in cellular proliferation and oral cancer, head and neck squamous cell carcinoma, gallbladder migration, antiapoptosis, angiogenesis, and wound healing. All cancer, and cervical cancer (1, 2). The FGFR3-TACC3 fusion FGFRs contain three immunoglobulin-like (Ig) domains, a trans- protein is a consequence of a 70 kb tandem duplication at membrane (TM) domain, and a split tyrosine kinase (TK) 4p16.3 (3). This causes a reversal of the two , as TACC3 is domain. Binding of fibroblast growth factors (FGF) and heparin normally upstream of FGFR3. TACC3 is a member of the TACC sulfate proteoglycans (HSPG) to the extracellular Ig domains family, which consists of 3 known human proteins, TACC1, collectively induces FGFR activation through dimerization of TACC2, and TACC3, all of which are involved in key roles of receptor monomers and transautophosphorylation of kinase microtubule organization during mitosis. TACC3 is believed to domain activation loop tyrosine residues. Tyrosine phosphory- be essential for the stabilization of kinetochore fibers and the lation of the kinase domain initiates activation of RAS-MAPK, mitotic spindle. A particularly important domain of this family PI3K-AKT, and JAK/STAT pathways (1). is the C-terminal coiled coil domain (named TACC domain), Point mutations in FGFRs have been linked to numerous which is highly conserved in all family members. This domain is human cancers and somatic disorders, many of which have been believed to play an important role in localization of the protein extensively studied. More recently, FGFR fusion proteins have during mitosis (4). Although TACC2 has been shown to be been increasingly detected in various human cancers (1). Since dispensable for normal mouse development without affecting proliferation, cell-cycle progression, or centrosome numbers (5),

1 in contrast, knockdown of TACC3 has been shown to result Department of Chemistry and Biochemistry, University of California – San Diego, La Jolla, California. 2Universite Joseph Fourier Grenoble, in G2 M phase arrest, apoptosis, abnormal spindle assembly, Grenoble, France. 3Proteomics Facility, Sanford Burnham Prebys misalignment, and impaired microtubule assembly Medical Discovery Institute, La Jolla, California. 4Moores UCSD Cancer and nucleation (6–8). Center, University of California San Diego, La Jolla, California. The frequent occurrence of this fusion protein in many cancer Corresponding Author: Daniel J. Donoghue, University of California, San Diego, types raises important questions as to whether activation of the UH 4130, 9500 Gilman Drive, La Jolla, CA 92093-0367. Phone: 858-534-7146; FGFR3 kinase domain occurs as a result of the TACC3 fusion, Fax: 858-822-5999; E-mail: [email protected] and whether this domain contributes through its localization to doi: 10.1158/1541-7786.MCR-15-0497 the observed biologic effects. Using mass spectrometry to analyze 2016 American Association for Cancer Research. the phosphorylation of key tyrosine residues as our point of

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Tyrosine Phosphorylation of FGFR3-TACC3

departure, this work investigates the FGFR3-TACC3 fusion pro- buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% tein with regard to its oncogenicity, activation of downstream TritionX-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/L signaling, altered subcellular localization, and induction of cell NaF, 1 mmol/L sodium orthovanadate, 1 mmol/L PMSF, and 10 transformation. mg/mL aprotinin]. Bradford assay or Lowry assay was used to measure total protein concentration. Antibodies were added to Materials and Methods lysates for overnight incubation at 4C with rocking, followed by immunoprecipitation, as described previously (10). Samples DNA constructs were separated by 10% or 12.5% SDS-PAGE and transferred to The TACC3 gene was purchased from Sino Biological Inc Immobilon-P membranes (Millipore). Membranes were blocked (pMD-TACC3) and was subcloned into pcDNA3. FGFR3, in 3% milk/TBS/0.05% Tween 20 or 3% bovine serum albumin FGFR3(K650E), and FGFR3(K508R) were developed as described (BSA)/TBS/0.05% Tween 20 (for anti-phosphotyrosine, anti- previously (9). To construct FGFR3-TACC3 fusion gene, a unique phospho-STAT1, and anti-phospho-STAT3 blots). Immunoblot- ClaI site was introduced by PCR-based site-directed mutagenesis ting was performed as described previously (12). after residue 758 in FGFR3 and before residue 648 in TACC3. This unique site was used to subclone TACC3 30 of FGFR3 in pcDNA3, creating a fusion breakpoint of FGFR3 exon 18 to TACC3 exon 11 Mass spectrometry sample preparation with a 9 basepair linker, containing the ClaI site, and encoding the HEK293T cells were plated one day prior to transfection at 3.0 residues ASM. Fragments containing K650E or K508R mutations 106 cells per 15-cm tissue culture plate. Ten plates per sample were were subcloned into the FGFR3-TACC3 fusion gene. Single and transfected by calcium phosphate precipitation with 9 mgof multiple tyrosine mutations in the TACC3 region (Y798F, Y853F, FGFR3 or FGFR3-TACC3 derivatives. Eighteen hours after trans- Y867F, Y878F) were introduced by PCR-based site-directed muta- fection, cells were serum starved overnight and then treated with genesis. DNA constructs were subcloned into pLXSN vector (10) 10 mmol/L MG132 for 4 to 6 hours, and then washed once in 1 for focus, proliferation, and 3-(4, 5-dimethylthiazol-2-yl)-2,5- PBS þ 1 mmol/L Na3VO4 before being lysed in RIPA. Clarified diphenyltetrazolium bromide (MTT) assays. All clones were con- lysates were immunoprecipitated with FGFR3 antisera overnight firmed by DNA sequencing. at 4C with rocking. Immune complexes were collected with Pierce protein A/G magnetic beads as per manufacturer's Cell culture directions. HEK293, HEK293T, and NIH3T3 cells were maintained in Following immunoprecipitation, proteins were digested DMEM plus 10% FBS and 1% penicillin/streptomycin in 10% directly on-beads using Trypsin/Lys-C mix. Briefly, samples CO2 at 37 C. MCF7 cells were maintained at 5% CO2 in DMEM were washed with 50 mmol/L ammonium bicarbonate, and plus 10% FBS and 1% penicillin/streptomycin in 37C. 32D clone then resuspended with 8 mol/L urea, 50 mmol/L ammonium 3 (ATCC CRL-11346) cells were maintained in RPMI1640 medi- bicarbonate, and cysteine disulfide bonds were reduced with um with 10% FBS, 1% penicillin/streptomycin, and 5 ng/mL 10 mmol/L tris(2-carboxyethyl)phosphine (TCEP) at 30 Cfor mouse IL3 in 5% CO2 at 37 C. 60 minutes followed by cysteine alkylation with 30 mmol/L iodoacetamide (IAA) in the dark at room temperature for 30 Antibodies and reagents minutes. Following alkylation, urea was diluted to 1 mol/L urea Antibodies were obtained from the following sources: FGFR3 using 50 mmol/L ammonium bicarbonate. The samples were fi (B-9), mSin3A (K-20), b-tubulin (H-235), STAT1 (E-23), STAT3 nally subjected to overnight digestion with mass spectrometry (C-20) from Santa Cruz Biotechnology; phosphotyrosine (4G10) grade Trypsin/Lys-C mix (Promega). Finally, peptides were from Millipore; TACC3 C-terminal (SAB4500103) from Sigma; collectedintoanewtube,andthe magnetic beads were washed phospho-STAT1 (Tyr701; 9171), phospho-STAT3 (Tyr705; once with 50 mmol/L ammonium bicarbonate to increase D3A7), phospho-p44/42 MAPK (Erk1/2; T202/Y204; E10), peptide recovery. The digested samples were partially dried to p44/42 MAPK (Erk1/2; 9102) from Cell Signaling Technology; approximately 50% of the total volume and desalted using a horseradish peroxidase (HRP) anti-mouse, HRP anti-rabbit, and C18 TopTip (PolyLC) according to the manufacturer's recom- Enhanced chemiluminence (ECL and Prime-ECL) reagents were mendations, and dried. from GE Healthcare. MG132, aFGF, and recombinant mouse IL3 Aliquots were resuspended in 80% acetonitrile, 5% tri- fl were obtained from R&D Systems. Heparin was from Sigma, uoroacetic acid in 1 mol/L glycolic acid and incubated with Geneticin (G418) was from Gibco, and Lipofectamine 2000 TiO2 magnetic beads (GE) for 30 minutes at room temper- Reagent was from Invitrogen. ature. Unbound peptides were removed from magnetic beads with two washes of 80% acetonitrile, 5% trifluoroacetic acid. Finally, phosphopeptides were eluted with 5% NH OH and Transfection, immunoprecipitation, and immunoblot analysis 4 dried. HEK293 were plated at a density of 1 106 cells/100-mm plate and transfected with 3 mg plasmid DNA using calcium phosphate transfection in 3% CO2 as described previously (11). Twenty to 24 LC-MS/MS and proteomics analysis hours after transfection, media were changed to DMEM with 0% Samples were analyzed by LC-MS/MS using a 0.180 20 mm FBS. Cells were starved for 20 hours before collecting and lysis. C18 trap Symmetry column (Waters) connected to an analytical Transfected HEK293 cells were collected, washed once in PBS, C18 BEH130 PicoChip column (0.075 100 mm, 1.7 mm and lysed in 1% NP40 Lysis Buffer [20 mmol/L Tris-HCl (pH 7.5), particles; NewObjective) mounted on a nanoACQUITY Ultra 137 mmol/L NaCl, 1% Nonidet P-40, 5 mmol/L EDTA, 50 mmol/ Performance Liquid Chromatography system (Waters), directly L NaF, 1 mmol/L sodium orthovanadate, 1 mmol/L phenyl- coupled to an Orbitrap Velos Pro mass spectrometer (Thermo methylsulfonylfluoride (PMSF), and 10 mg/mL aprotinin] or RIPA Fisher Scientific). The peptides were separated with a 90-minute

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nonlinear gradient of 2%–35% solvent B (acetonitrile) at a flow cytometer and Trypan blue exclusion on days 2, 4, 6, and 7. Media rate of 400 nL/minute. The mass spectrometer was operated in were added to cultures when cell numbers reached approximately positive data-dependent acquisition mode. MS1 spectra were 1 106 cells/mL during the assays to maintain at viable con- measured with a resolution of 60,000, an AGC target of 106, and centrations. For metabolic assays, MTT assays were performed. A a mass range from 350 to 1,400 m/z. Up to 5 MS2 spectra per duty stock solution of 5 mg/mL in PBS of MTT (Sigma) was added at a cycle were triggered, and each precursor was fragmented twice by ratio of 1:10 to the cultures. After incubation at 37 C, 5% CO2 for collision-induced dissociation (CID) and electron transfer disso- approximately 4 hours, equal volume of 0.04 mol/L HCl in ciation (ETD), and acquired in the ion trap with an AGC target of isopropanol was added and mixed well and incubated again for 104, an isolation window of 2.0 m/z, and a normalized collision at least 30 minutes (15). Cultures were transferred to microfuge energy of 35. Dynamic exclusion was set to 5 seconds to allow tubes, spun for 30 seconds at room temperature, and supernatant multiple fragmentation of phosphopeptides. absorbance was measured at 570 nm. A total of 5 104 cells per All mass spectra from were analyzed with MaxQuant software well were plated in triplicate in 24-well plates in the presence or version (13). Briefly, MS/MS spectra were searched against the absence of IL3 and 1 nmol/L aFGF and 30 mg/mL heparin and cRAP protein sequence database (http://www.thegpm.org/crap/) assayed 3 days later. The cell viability at day 7 was measured using indexed with the mutant FGFR3 or FGFR3-TACC3 sequences cultures from the proliferation assay. In triplicate, 0.5 mL of the described here. Precursor mass tolerance was set to 20 ppm and cultures were transferred to 24-well plates and treated with the 4.5 ppm for the first search where initial mass recalibration was MTT reagent. completed and for the main search, respectively. Product ions were searched with a mass tolerance 0.5 Da. The maximum Fractionation precursor ion charge state used for searching was 7. Carbamido- MCF7 cells were plated at a density of 1.5 106 cells/100-mm methylation of cysteines was searched as a fixed modification, plates 24 hours before transfection. Immediately prior to trans- while phosphorylation of serines, threonines, and tyrosines, and fection, media was changed to DMEM 0% FBS with no antibiotic. oxidation of methionines was searched as variable modifications. Cells were transfected with 8 mg of plasmid DNA using Lipofec- Enzyme was set to trypsin in specific mode and a maximum of two tamine 2000 Reagent. Twenty-three hours after transfection, cells missed cleavages was allowed for searching. The target-decoy- were collected in PBS and 1 mmol/L EDTA for fractionation as based false discovery rate (FDR) filter for spectrum and protein described previously (16). Total protein concentrations of sepa- identification was set to 1%. Modification site decoy fraction was rated fractions were analyzed by Bradford assay, separated by 10% also set to 1% and minimum score and delta score for modified SDS-PAGE, and transferred to Immobilon-P membrane for West- peptides were set to 40 and 6, respectively, to assure high con- ern Blot analysis. fidence phosphopeptide identification. All phosphorylation sites reported here were identified with a localization site of at least 0.75. Second peptide mode of MaxQuant software was also Results enabled. Spectra presented were produced in MaxQuant and Constitutive phosphorylation of FGFR3-TACC3 fusion protein manually annotated using Canvas Draw. Peak assignments were In the FGFR3-TACC3 fusion protein, tyrosine kinase domain confirmed using software provided by UCSF ProteinProspector dimerization and autophosphorylation may be elevated by the (http://prospector.ucsf.edu/prospector/). presence of the TACC3 coiled-coil domain, which could be crucial to cancer progression. To investigate changes in phosphorylation Focus assay and biologic activity, various FGFR3-TACC3 derivatives were Focus assays were performed using NIH3T3 cells plated at a constructed. All fusion constructs contain the breakpoint between 5 density of 2 10 cells/60-mm plates in DMEM with 10% FBS exon 18 of FGFR3 to exon 11 of TACC3 as shown in Fig. 1A, 24 hours before transfection. Cells were transfected by Lipofecta- chosen due to the high occurrence of this particular fusion break- mine 2000 Reagent with 10 mg plasmid DNA. Between 22 and 24 point (3, 17). This fusion is predicted to contain the extracellular, hours after transfection, cells were re-fed with DMEM 10% FBS. transmembrane, and intracellular kinase domains of FGFR3 Cells were split 1:12 onto duplicate 100-mm plates between 22 fused 50 to the coiled-coil domain of TACC3 (18). Constitutively and 24 hours later. Foci were scored at 12 to 14 days, fixed in activated FGFR3 clones were produced by the K650E mutation. methanol, stained with Geimsa stain, and photographed. Effi- This mutation, originally identified as the cause of the lethal ciency of transfection was determined by Geneticin (G418, 0.5 skeletal disorder Thanatophoric Dysplasia type II (TDII), results mg/mL)-resistant colonies plated on duplicate plates at a dilution in profound FGFR3 kinase activation (1). The kinase activity of of 1:240. FGFR3 was abrogated by the mutation K508R, known as the "kinase-dead" mutant (Fig. 1A). IL3-independent growth in 32D cells To examine the phosphorylation of each fusion construct A total of 1 106 exponentially growing 32D cells were compared with FGFR3 WT, FGFR3(K650E) and FGFR3(K508R) electroporated (1,500 V, 10 ms, 3 pulse) by the Neon Transfection were expressed in HEK293 cells, collected, and immunoprecipi- System (Invitrogen) using 30 mg of FGFR3, FGFR3-TACC3 or PR/ tated with an N-terminal FGFR3 antibody (Fig. 1B, top). HEK293 neu derivatives in pLXSN in triplicate. Twenty-four hours after cells were chosen as they have been used previously for studies of transfection, cells were selected with 1.5 mg/mL Geneticin (G418) protein phosphorylation, downstream signaling, and protein– sulfate for 10 days to generate stable cell lines. For IL3-indepen- protein interactions (11, 12, 19). An increase in tyrosine phos- dent proliferation assays, 2 105 cells/well were seeded in 12-well phorylation was seen in FGFR3-TACC3 compared with FGFR3 WT plates in the absence of IL3 or 6-well plates in the presence of IL3. (lanes 2 and 6). No phosphorylation signal could be detected for The media also contained 1 nmol/L aFGF and 30 mg/mL heparin the kinase-dead FGFR3 with or without the fused TACC3 (Fig. 1B). (14). Cell numbers were determined in triplicate, with a hemo- These results show that tyrosine phosphorylation was increased

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HEK293T cells expressing FGFR3 or FGFR3-TACC3 derivatives. HEK293T cells provide a successful model for phosphorylation, signal transduction, and protein–protein interactions, and thus are useful for mass spectrometry studies (11, 19). Significant results are presented in Fig. 2A and B. As potential negative controls, duplicate samples of kinase-dead derivatives FGFR3 (K508R) and FGFR3(K508R)-TACC3 were analyzed by mass spectrometry, and no phosphorylated tyrosine residues were detected (data not shown). All tyrosine phosphorylation sites detected on the fusion proteins are indicated in Fig. 2C. Figure 2A presents the spectral intensity of each phosphotyr- osine site normalized against internal constitutive phosphoryla- tion sites pS424 and pS444, which were found to be phosphor- ylated in all samples, even kinase-dead samples, as background cellular phosphorylation with no known function. Figure 2B presents another view of the same data, presenting the intensity of each phosphotyrosine site as a percentage of the total phos- photyrosine signal in that sample. In the FGFR3 WT samples, a very low level of phosphorylation was observed on the activation loop residues, Y647 and Y648 (Fig. 2A, top), with each site accounting for 50% of the phosphotyrosine in that sample (Fig. 2B, top). By comparing nonfused FGFR3 to FGFR3-TACC3, the effect of the coiled-coil domain on receptor phosphorylation and activation can be seen. The addition of the TACC3 domain leads to a modest increase in the overall phosphotyrosine (Fig. 2A, second panel), but surprisingly leads to more promiscuous phos- phorylation as additional sites such as Y577, Y599, Y607, and Y798 are now observed (Fig. 2B, second panel). Introduction of the activating mutation K650E into the FGFR3 background leads to an overall increase in tyrosine phosphorylation (Fig. 2A, third panel); furthermore, many Figure 1. Increase in tyrosine phosphorylation by introduction of the TACC domain. additional phosphorylation sites are now observed, such as A, schematic of FGFR3-TACC3 fusion protein. The N-terminal extracellular Y107, Y226, Y278, Y305, Y599, Y607, and Y724, in addition ligand-binding domain, transmembrane (TM), kinase, and kinase insert (KI) to the activation loop residues Y647 and Y648 (Fig. 2B, third domains of FGFR3 are followed by a 3 amino acid linker (residues ASM), and panel). Finally, the introduction of the activating K650E muta- fused to TACC3 starting at exon 11, which contains a coiled-coil domain. The tion into the FGFR3-TACC3 background, creating FGFR3 location of the kinase-dead mutation K508R and the kinase-active mutation (K650E)-TACC3, leads to massive activation of tyrosine phos- K650E are shown. B, various mutation and fusions with FGFR3 were expressed in HEK293 cells, immunoprecipitated with FGFR3 antibody, and phorylation (Fig. 2A and B, fourth panel), including all sites immunoblotted with phosphotyrosine antibody (top). Expression of the observed in the preceding samples, but with pronounced phos- constructs was visualized in lysates by immunoblotting with FGFR3 antisera phorylation of the site Y798 in the TACC3 domain. (middle) and TACC3 antisera (bottom). C, quantification of tyrosine Representative spectra of phosphorylated peptides are shown phosphorylation showing the SEM for 3 independent repeats, normalized to in Fig. 3. A commonly identified FGFR3 WT peptide containing FGFR3(K650E). double phosphorylation of Y647 and Y648 in the activation loop is shown in panel A. In FGFR3-TACC3 fusion protein constructs, by fusion of the TACC3 coiled-coil domain (lane 6). Furthermore, this double phosphorylated peptide becomes less frequent, with relative to FGFR3-TACC3, introduction of the K650E mutation detection of peptides containing single Y647 phosphorylation fi (lane 7) resulted in a signi cant increase in tyrosine phosphor- becoming more common (Fig. 2, Fig. 3B and C). Also shown ylation. Quantitation of phosphorylation levels shows a 2-fold are spectra containing primary phosphorylation sites Y577, increase in tyrosine phosphorylation on the FGFR3-TACC3 fusion Y798, and Y867 in FGFR3-TACC3 and FGFR3(K650E)-TACC3 protein compared with FGFR3 WT (Fig. 1C). (Fig. 3D–F). There are four tyrosine residues in the TACC3 portion of the LC-MS/MS analysis identifies elevated and novel FGFR3-TACC3 fusion protein: Y798, Y853, Y867, and Y878, phosphorylation sites corresponding to residues Y684, Y739, Y753, and Y764 in TACC3 The strong increase in tyrosine phosphorylation seen by immu- WT. In FGFR3-TACC3, it was previously unknown whether these noblotting leads to the question of whether TACC3 leads to a tyrosine residues were also phosphorylated, possibly by the fused constitutively phosphorylated FGFR3 kinase and whether addi- kinase domain, and whether they play a role in cancer develop- tional or novel phosphorylation sites exist on the fusion protein. ment. Through MS/MS analysis, significant phosphorylation was Following immunoprecipitation with the FGFR3 N-terminal anti- observed at Y798 and Y867 in FGFR3-TACC3, with trace amounts body and on-bead tryptic digestion, titanium dioxide–based detected at Y853 (Fig. 2C). Phosphorylation was not detected at phosphopeptide enrichment (TiO2)-liquid chromatography Y878, although this may be due to the small predicted tryptic (LC)-high mass accuracy MS/MS was used with samples from peptide containing this residue. Increasing receptor activation by

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Figure 2. Phosphorylated tyrosine residues in FGFR3, FGFR3(K650E), FGFR3-TACC3, and FGFR3(K650E)-TACC3 identified by mass spectrometry analysis. A, the spectral intensity of each phosphotyrosine residue detected is presented, normalized against two phosphoserine residues (pS424 and pS444) which are constitutively phosphorylated across all samples including the kinase-dead samples (not shown). Duplicate, independent samples are averaged here after mass spectrometry analysis. Quantitation was based on MS spectral intensity as determined by MaxQuant for each identified peptide peak, normalized against the total pS424þpS444 intensity for that individual sample. The intensity of the pY798 peak in the FGFR3(K650E)-TACC3 sample was set arbitrarily at 100%, although the actual spectral intensity of this peak averaged 23% of the total intensity of all phosphopeptides containing pS242 þ pS444. B, for each phosphotyrosine residue detected, the percentage of intensity within the total protein is presented, with the total spectral intensity for all phosphotyrosines in each samples set to 100%. C, schematic of FGFR3-TACC3 with the location of all tyrosine phosphorylation sites identified by LC-MS/MS.

K650E mutation led to an increase in intensity levels of TACC3 these sites is unclear and these residues are not conserved in the tyrosine phosphorylation (Fig. 2A, second and fourth panels). TACC family (20, 21). However, Y867 is a conserved tyrosine Of the phosphorylation sites detected in the TACC3 portion of residue in the TACC family and our data identify it as a novel the fusion protein, Y798 and Y853 have been previously identi- phosphorylation site for the FGFR3(K650E)-TACC3 fusion fied as a phosphorylation sites in TACC3 WT. The function of protein.

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As mentioned above, mass spectrometry of HEK293T cells (14), select fusion proteins were examined in an alternate IL3- expressing FGFR3(K508R)-TACC3 (kinase dead mutation) dependent cell line, 32D. Both are murine-derived cell revealed no tyrosine phosphorylated peptides within the FGFR3 lines dependent on IL3 for viability and growth and have been or TACC3 domains. This indicates that an active FGFR3 kinase extensively used as a model system for studying transforming domain is required for tyrosine phosphorylation of the fusion properties of oncogenes (27–31). The choice of the 32D cells over proteins and the TACC domain is most likely phosphorylated by Ba/F3 cells was due to their more established origin (32–34) and the FGFR3 kinase domain, not another tyrosine kinase. availability. FGFR3 WT, FGFR3-TACC3, FGFR3(K650E), FGFR3 (K650E)-TACC3, FGFR3-TACC3(4xYF), and PR/neu were electroporated into the 32D cell line and selected as described Cell transforming ability of FGFR3-TACC3 by focus assay in Materials and Methods. As seen in Fig. 5A, in the absence of IL3, To examine the transforming activity of FGFR3-TACC3 and all the clones expressed were able to stimulate IL3-independent subsequent mutants, focus-forming assays with NIH3T3 cells growth indicating their transforming potential. Interestingly, the were performed. NIH3T3 cell transformation assays represent FGFR3-TACC3(4xYF) clone had the highest proliferation even one of the original assays for the identification and characteriza- without the activating K650E mutation. This could support the tion of oncogenes, having been used for analysis of activated RAS, suggestion of the TACC3 tyrosine residues as being inhibitory. In MUC4, AKT, and many other oncogenes (22–25). This assay relies addition, even in the presence of IL3 (Fig. 5B), the expression of on the oncogenic growth of transfected cells to overcome the some of the clones enhanced the proliferation of the 32D cells contact-inhibited monolayer of NIH3T3 cells and establish a compared with nonexpressing cells. The MTT metabolic assays focus of proliferating cells. FGFR3-TACC3 and FGFR3(K650E)- performed on days 3 and 7 shown in Fig. 5C support the cell TACC3 produced extremely high foci formation and cell trans- population assay results. All transfected constructs display formation compared with FGFR3 WT or FGFR3(K650E) (Fig. 4). cell viability, whereas 32D control cells do not, indicating that Expression of PR/neu, a focus assay positive control, displayed FGFR3-TACC3 and other constructs promote cell proliferation. less transformation than FGFR3-TACC3, which also consistently produced much larger foci. PR/neu is a platelet-derived growth factor receptor, beta (PDGFR-b) with a Neu receptor transmem- FGFR3-TACC3 displays nuclear localization brane domain with the activating V664E mutation (p185neu ; The presence of the TACC3 domain, derived from a nuclear ref. 26). Despite the previously demonstrated elevated activation localizing protein (4), raises the question whether the fusion of PR/neu, its transforming ability was dwarfed by the foci protein FGFR3-TACC3 relocalizes to the nucleus as a result of formation seen by FGFR3(K650E)-TACC3. As a result, samples this domain. MCF7 cells have been previously used for fraction- were normalized to FGFR3(K650E)-TACC3 (Fig. 4). Expression of ation studies with high success (16, 35, 36). Indeed, fractionation FGFR3(K508R)-TACC3 (kinase-dead mutation) and TACC3 WT of MCF7 cells expressing FGFR3 WT, FGFR3(K650E), FGFR3 in NIH3T3 cells did not produce significant foci formation, (K508R), and their fusion counterparts displayed a clear differ- indicating that an active FGFR3 kinase domain is essential for ence in localization (Fig. 6A). All three fusions, FGFR3-TACC3, cell transforming ability of FGFR3-TACC3. FGFR3(K650E)-TACC3, and FGFR3(K508R)-TACC3 (nuclear Within the coiled-coil domain in FGFR3-TACC3, there are fraction, lanes 5, 6, and 7) displayed strong nuclear localization. four tyrosine residues. Three of these residues were shown to be The nonfused tyrosine kinase domains (lanes 2, 3, and 4) were phosphorylated by MS/MS analysis, as discussed above. To present mainly in the cytoplasmic fraction, which also includes assess the importance of these FGFR3-TACC3 phosphorylation the cell membrane fraction. Perinuclear localization of FGFR3 sites, all four TACC3 tyrosine residues were mutated to phe- (K650E) has been demonstrated previously (37), but fusion of nylalanine (Y798F, Y853F, Y867F, Y878F) with and without FGFR3(K650E) to TACC3 dramatically increased nuclear locali- the activating FGFR3 K650E mutation by site-directed muta- zation. These results indicate the presence of the TACC3 coiled- genesis and analyzed for focus forming ability. NIH3T3 cells coil domain is responsible for nuclear localization of the FGFR3 expressing the fusion constructs with all four tyrosine muta- kinase, regardless of receptor activation. Immunoblotting for tions, FGFR3-TACC3 4xYF and FGFR3(K650E)-TACC3 4xYF, nuclear localizing mSin3A and cytoplasmic b-tubulin confirmed displayed high levels of focus formation when compared with separation of nuclear and cytoplasmic fractions (Fig. 6A). FGFR3-TACC3 or FGFR3(K650E)-TACC3 with no additional mutations (Fig. 4). Downstream signaling activation by FGFR3-TACC3 To assess the effects of each individual phosphorylation site, It has been shown previously that FGFR3 WT and FGFR3 single Y to F mutants were made in combination with activating (K650E) activate the STAT pathway and MAPK pathway, but it mutation K650E. As shown in Fig. 4, three of the mutations is not clear how this activation compares with FGFR3-TACC3 or (Y853F, Y867F, Y878F) appeared to increase focus formation to FGFR3(K650E)-TACC3 fusions. HEK293 cells, previously used in a slightly higher level than K650E mutation alone, and the FGFR3 FGFR signal transduction studies (12, 16), expressing these (K650E)-TACC3(Y798F) appeared to result in a slightly lower fusions and their nonfused counterparts were analyzed for STAT1 transformation ability than FGFR3(K650E)-TACC3. However, and STAT3 activation. FGFR3 expression resulted in undetectable these differences were not statistically significant in a Student t or slight phosphorylation of STAT1 and STAT3, whereas the test. activated mutant FGFR3(K650E) resulted in significant phos- phorylation of both STAT1 and STAT3 (Fig. 6B, lanes 2 and 3). FGFR3-TACC3 promotes IL3 independent cell growth In comparison, the addition of the TACC3 domain in either Because of the role of activated FGFR3 mutants in multiple FGFR3-TACC3 or FGFR3(K650E)-TACC3 did not lead to a sig- myeloma and previous studies showing their oncogenic and nificant change (Fig. 6B, lanes 6 and 7). However, MAPK phos- proliferative potential in the IL3-dependent cell line, Ba/F3 phorylation was strongly elevated by FGFR3-TACC3 and FGFR3

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Figure 3. Representative spectra of selected peptides. MS/MS spectra are presented for several key phosphorylation sites. In the fragmentation patterns, spectral peak evidence was observed for all indicated fragments and, furthermore, fragmentation evidence is present for all phosphorylation sites shown here. As noted, due to space constraints, all identified fragment ions are not individually labeled on the spectra. Peptides presented are as follows: A, from a sample of FGFR3 WT, an ETD (electron transfer dissociation) spectrum of the doubly phosphorylated peptide DVHNLDpYpYKK including pY647 and pY648 within the activation loop. B, from a sample of FGFR3-TACC3, a CID (Collision Induced Dissociation) spectrum of the phosphorylated peptide DVHNLDpYYKK including pY647 within the activation loop. In this spectrum, the following fragment ions were identified but not labeled: y6, m/z 909.41; z8, 1144.49. C, from a sample of FGFR3(K650E)- TACC3, an ETD spectrum of the phosphorylated peptide DVHNLDpYYKETTNGRLPVK including pY647 within the activation loop. In this spectrum, the z3 fragment ion with m/z 372.22 was identified but not labeled on the spectrum. (Continued on the following page.)

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Figure 4. Transformation of NIH3T3 cells by FGFR3 and FGFR3-TACC3 derivatives. Representative plates from a focus assay are shown, with transfected constructs indicated. Number of foci were scored, normalized by transfection efficiency, and quantitated relative to FGFR3(K650E)-TACC3 SEM. PR/neu is a positive control. Assays were performed a minimum of three times per DNA construct.

(K650E)-TACC3 (Fig. 6C, lanes 6 and 7), compared with non- activation and phosphorylation of key residues in FGFR3. Clearly, fused FGFR3 WT and FGFR3(K650E) (lanes 2 and 3), indicating the presence of the TACC domain leads to an increase of total that FGFR3-TACC3 induces MAPK pathway activation. The kinase activity, as well as increased promiscuity of phosphoryla- kinase-dead FGFR3(K508R)-TACC3 did not display this activa- tion sites, as shown by the additional phosphorylation sites tion (lane 8), indicating that FGFR3 kinase activity in the fusion detected by LC-MS/MS (Fig. 2). Activation by this coiled coil protein is essential to downstream signaling activation. domain has a more severe impact on cell transformation and downstream signaling than the activating K650E mutation alone. The oncogenic potential of the fusion protein was demonstrated Discussion by focus formation assay (Fig. 4), IL3-independent proliferation We extensively analyzed the FGFR3-TACC3 fusion protein by assay (Fig. 5), and metabolic activity assay (Fig. 5). The absence of tyrosine residue phosphorylation changes and the impacts on biologic activity and the lack of tyrosine phosphorylation of the oncogenic potential. We demonstrate that introduction of a fusion protein shown by FGFR3(K508R)-TACC3 kinase-dead C-terminal TACC3 coiled-coil domain results in constitutive mutant indicates that kinase activity is required for gain of

(Continued.) D, from a sample of FGFR3-TACC3, an ETD spectrum of the phosphorylated peptide RPPGLDpYSFDTCKPPEEQLTFK including pY577. In this spectrum, the following fragment ions were identified but not labeled: y3, m/z 395.23; y4, 508.31; y7, 894.46. Note that the Cys in this peptide was carbamidomethylated. E, from the FGFR3-TACC3 sample, a CID spectrum of the phosphorylated peptide IMDRFEEVVpYQAMEEVQK including pY798 within the TACC3 domain. In this spectrum, the following fragment ions were identified but not labeled: y2-NH3, m/z 258.14; y3-NH3, 357.21; y11, 1403.63; y13-H2O, 1643.7; a2, 217.14; b11, 1490.64; b12, 1561.68; b13, 1692.72. F, from the FGFR3(K650E)-TACC3 sample, a CID spectrum of the phosphorylated peptide KCVEDpYLAR including pY867 within the TACC3 domain. In this spectrum, the following fragment ions were identified but not labeled: y1, m/z 175.12; y5-H2O, 699.29; 2þ y5-NH3, 700.27, y6 , 423.67; y8-H2O, 1087.43; b2-NH3, 272.11; b3-NH3, 371.17; b4-NH3, 500.22; b6-NH3, 858.27. Note that the Cys in this peptide was carbamidomethylated.

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Figure 5. IL3-independent growth and MTT metabolic assay in 32D cells expressing FGFR3 or FGFR3-TACC3 derivatives. PR/neu is a positive control. A, 32D cells selectively expressing FGFR3, FGFR3-TACC3, FGFR3(K650E), FGFR3 (K650E)-TACC3, FGFR3-TACC3(4xYF), or PR/neu were cultured in the absence of IL3. The total number of viable cells were determined by Trypan blue exclusion. Experiments were performed in triplicate, SD is shown. B, cell counts of cultures in A in the presence of IL3. Experiments were performed in triplicate, SD is shown. Inset of growth from A without IL3 is shown for comparison. C, cell viability as determined by MTT assay by three independent repeats on days 3 and 7. Relative absorbance was obtained by ratio of IL3 to þIL3 absorbances read at 570 nm. SD is shown.

function and cancer progression, but not required for nuclear ways utilized by FGFR3 (14, 39–43). However, the K650E muta- localization of the fusion protein, as shown by cellular fraction- tion has not yet been identified together with the FGFR3-TACC3 ation (Fig. 6). oncogenic gene fusion in human cancer, and thus represents an The K650E mutation in FGFR3, originally identified as the artificial construct not yet found in nature. Nonetheless, our use of cause of the neonatal lethal skeletal malformation syndrome this construct here allowed us to compare different routes of Thanatophoric Dysplasia type II (38), has been identified fre- activation of FGFR3. quently as a somatic mutation in many different human cancers Examining the nonfused FGFR3 proteins, the analysis by LC- such as bladder cancer and multiple myeloma (1). This mutation MS/MS indicates key FGFR3 residues are being phosphorylated, has been exploited in many studies to examine signaling path- primarily residues Y647 and Y648 as part of the YYKK activation

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Figure 6. Localization and signaling of FGFR3-TACC3 fusions. A, fractionation of MCF7 cells expressing FGFR3 or FGFR3-TACC3 derivatives. Cells were separated into cytoplasmic (left) and nuclear (right) fractions. Immunoblotting with FGFR3 antibody shows nuclear localization of FGFR3- TACC3 fusions (top). Immunoblotting for mSin3A and b-Tubulin confirm fractionation (second and third panels). B, lysates of HEK293 cells expressing FGFR3 or FGFR3-TACC3 derivatives were immunoblotted for phospho- STAT1 (Y701; top), STAT1 (second panel), phospho-STAT3 (Y705; third panel), STAT3 (fourth panel), and FGFR3 (bottom). C, HEK293 cell lysates expressing FGFR3 or FGFR3-TACC3 derivatives were immunoblotted for phospho-MAPK (T202/Y204; top), MAPK (second panel), and FGFR3 (bottom).

loop motif essential to FGFR kinase activity (43). Activation due Also deleted as a result of the FGFR3-TACC3 fusion breakpoint to the K650E mutation reveals an increase in total phosphoryla- are the Aurora-A phosphorylation sites on TACC3. Aurora-A has tion as well as phosphorylation at additional sites, including Y724 been shown to phosphorylate TACC3 WT at S552 and S558 which (Fig. 2). Residue Y724 has been shown to be critical for activation is required for the localization of a TACC3-chTOG- com- of downstream signaling pathways, such as MAPK, STAT, and plex to mitotic spindle microtubules and spindle poles (7, 18, 45). PI3K, as well as cell transformation (40). In the fusion protein Localization of TACC3 to kinetochore fibers in complex with FGFR3-TACC3, Y647 was the major site of phosphorylation chTOG and clathrin is believed to assist with stabilization and within the activation loop, and phosphorylation of additional formation of the mitotic spindle (45). However, previous studies sites such as Y577, Y599, Y607, and Y798 was also observed. have found the FGFR3-TACC3 fusion protein localized only to the Introduction of the K650E mutation into the fusion protein mitotic spindle poles during mitosis, and relocated during late FGFR3(K650E)-TACC3 resulted in increased phosphorylation of stage mitosis to the midbody. A mechanism for this change in all the sites seen in the FGFR3-(K650E) as well as the sites seen in recruitment and the role of FGFR3-TACC3 during interphase the FGFR3-TACC3 sample, with the additional appearance of remains unclear (46). phosphorylation at Y867 in the TACC3 domain. Although not analyzed in regards to cell cycle, we demonstrate a The fusion breakpoint of exon 18 in FGFR3 excludes the strong indication of nuclear localization for the fusion protein. In binding site for PLCg (Y760), thus PLCg is no longer recruited addition, localization of FGFR3-TACC3 to the nucleus is not by FGFR3-TACC3, as previously shown (17). In addition, Y760 dependent on kinase activity as shown by K508R mutation, may contribute to maximal STAT activation (40). The removal of indicating that this localization is solely due to the fused TACC this site from FGFR3-TACC3 may be contributing to the absence domain. As the Aurora A phosphorylation sites are no longer of STAT activation. However, significant increase of downstream present in the fusion protein, there must be another nuclear signaling activation was seen in the MAPK pathway independent recruitment mechanism occurring. This delocalized kinase could of FGF ligand stimulation (Fig. 6C), which correlates with previ- be interacting with novel proteins that lead to cancer progression. ous findings and further indicates ligand-independent activation The detection of phosphorylated TACC3-derived residues in and cell growth (3, 17, 44). FGFR3-TACC3 (Y798, Y853 and Y867 corresponding to Y684,

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Nelson et al.

Y739, and Y753 in native TACC3) could indicate the ability of a study showed that the HSP90 inhibitor ganetespib exerted pleio- highly activated kinase to self-phosphorylate the TACC domain tropic effects on mitogenic and survival pathways in cells expres- and potentially lead to increased downstream signaling. Our sing oncogenic gene rearrangements of FGFR3, and provided an results, in which mutation to phenylalanine of all four tyrosine alternative therapeutic approach in addition to the pan-FGFR residues within the TACC3 domain leads to increased focus tyrosine kinase inhibitor BGJ398 (47). Further study of novel formation and IL3-independent cell proliferation, leads to the pathways activated by the FGFR3-TACC3 fusion protein, and a conclusion that phosphorylation at these sites, while it does deeper understanding of the molecular details exploited by occur, fails to contribute significantly to the oncogenic potential FGFR3-TACC3 to achieve its biologic potency, can be expected of the FGFR3-TACC3 fusion oncogene. lead to novel therapeutic paradigms. Recently, it has been shown that chTOG (colonic and hepatic tumor overexpressed gene), a centrosomal localizing protein, Disclosure of Potential Conflicts of Interest recruits TACC3 to microtubule plus-ends during interphase. This No potential conflicts of interest were disclosed. localization is dependent on chTOG, not TACC3, and is inde- pendent of Aurora A phosphorylation (45). TACC3 residues 672– Authors' Contributions 688 contain the binding site of ch-TOG and are present in the – Conception and design: K.N. Nelson, A.N. Meyer, D.J. Donoghue FGFR3-TACC3 fusion protein (at residues 786 802). Within this Development of methodology: K.N. Nelson, A.N. Meyer, A.R. Campos, region is Y798, which we found to be highly phosphorylated D.J. Donoghue in the FGFR3-TACC3 and FGFR3(K650E)-TACC3 fusion proteins. Acquisition of data (provided animals, acquired and managed patients, In the K650E background, when the phosphorylation site is provided facilities, etc.): K.N. Nelson, A.N. Meyer, A.R. Campos, removed by the mutation Y798F, we did not observe a significant K. Motamedchaboki, D.J. Donoghue change in biologic activity, suggesting that the specific phosphor- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K.N. Nelson, A.N. Meyer, A.R. Campos, ylation of Y798 occurs adventitiously and is extrinsic to the K. Motamedchaboki, D.J. Donoghue biological properties of the FGFR3-TACC3 fusion. Writing, review, and/or revision of the manuscript: K.N. Nelson, A.N. Meyer, Introduction of Tyr-to-Phe mutations at all of the retained A. Siari, A.R. Campos, K. Motamedchaboki, D.J. Donoghue TACC3-derived residues Y798, Y853, Y867, or Y878 resulted in Administrative, technical, or material support (i.e., reporting or organizing changes in biologic activity that were statistically insignificant, data, constructing databases): K.N. Nelson, D.J. Donoghue again indicating that the phosphorylation we observed at these Study supervision: D.J. Donoghue Other (working on 32D cell line): A. Siari sites is biologically inconsequential. An inhibitory phosphoryla- tion site has been shown to occur in FGFR3 WT at Y770 which, Acknowledgments upon phosphorylation, inhibits cell transformation (40). Inter- The authors thank all the laboratory members particularly Leandro Gallo and estingly, the residue Y770 has been removed from the FGFR3- fi Juyeon Ko for advice and encouragement, Annie Chang for assistance with DNA TACC3 fusion, but the signi cance of this change was not construction, and Kenneth Pomeroy from Sanford Burnham Prebys (SBP) explored here. Medical Discovery Institute for help with proteomics sample preparation. We have presented overwhelming evidence of the significant Support to the SBP Proteomics Facility from grant P30 CA030199 from the oncogenic potential of the FGFR3-TACC3 fusion protein. The NIH is gratefully acknowledged. The authors also thank Dr. Manquing Li and presence of the TACC coiled-coil domain leads to increased and Prof. Michael David (UCSD, La Jolla, CA) for assistance with electroporation. The costs of publication of this article were defrayed in part by the payment of altered levels of FGFR3 activation, fusion protein phosphoryla- page charges. This article must therefore be hereby marked advertisement in tion, downstream signaling, cellular transformation, prolifera- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tion, and viability. The existence of FGFR3-TACC3 fusions in human cancers creates additional challenges and opportunities Received December 22, 2015; revised January 12, 2016; accepted February 3, for identifying effective treatment strategies. For instance, a recent 2016; published OnlineFirst February 11, 2016.

References 1. Gallo LH, Nelson KN, Meyer AN, Donoghue DJ. Functions of Fibroblast 7. Fu W, Tao W, Zheng P, Fu J, Bian M, Jiang Q, et al. Clathrin recruits Growth Factor Receptors in cancer defined by novel translocations and phosphorylated TACC3 to spindle poles for bipolar spindle assembly and mutations. Cytokine Growth Factor Rev 2015;26:425–49. chromosome alignment. J Cell Sci 2010;123:3645–51. 2. Carneiro BA, Elvin JA, Kamath SD, Ali SM, Paintal AS, Restrepo A, et al. 8. Singh P, Thomas GE, Gireesh KK, Manna TK. TACC3 protein regulates FGFR3-TACC3: a novel gene fusion in cervical cancer. Gynecol Oncol Rep microtubule nucleation by affecting gamma-tubulin ring complexes. J Biol 2015;13:53–6. Chem 2014;289:31719–35. 3. Parker BC, Annala MJ, Cogdell DE, Granberg KJ, Sun Y, Ji P, et al. The 9. Webster MK, Donoghue DJ. Enhanced signaling and morphological tumorigenic FGFR3-TACC3 gene fusion escapes miR-99a regulation in transformation by a membrane-localized derivative of the fibroblast glioblastoma. J Clin Invest 2013;123:855–65. growth factor receptor 3 kinase domain. Mol Cell Biol 1997;17: 4. Gergely F, Karlsson C, Still I, Cowell J, Kilmartin J, Raff JW. The TACC 5739–47. domain identifies a family of centrosomal proteins that can 10. Bell CA, Tynan JA, Hart KC, Meyer AN, Robertson SC, Donoghue DJ. interact with microtubules. Proc Natl Acad Sci U S A 2000;97: Rotational coupling of the transmembrane and kinase domains of the Neu 14352–7. receptor tyrosine kinase. Mol Biol Cell 2000;11:3589–99. 5. Schuendeln MM, Piekorz RP, Wichmann C, Lee Y, McKinnon PJ, Boyd K, 11. Gallo LH, Meyer AN, Motamedchaboki K, Nelson KN, Haas M, Donoghue et al. The centrosomal, putative tumor suppressor protein TACC2 is DJ. Novel Lys63-linked ubiquitination of IKKbeta induces STAT3 signal- dispensable for normal development, and deficiency does not lead to ing. Cell Cycle 2014;13:3964–76. cancer. Mol Cell Biol 2004;24:6403–9. 12. Meyer AN, McAndrew CW, Donoghue DJ. Nordihydroguaiaretic acid 6. Yim EK, Tong SY, Ho EM, Bae JH, Um SJ, Park JS. Anticancer effects on inhibits an activated fibroblast growth factor receptor 3 mutant and blocks TACC3 by treatment of paclitaxel in HPV-18 positive cervical carcinoma downstream signaling in multiple myeloma cells. Cancer Res 2008;68: cells. Oncol Rep 2009;21:549–57. 7362–70.

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13. Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M. Accurate proteome- 31. Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, Miyawaki S, et al. wide label-free quantification by delayed normalization and maximal Activating mutation of D835 within the activation loop of FLT3 in human peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 2014;13: hematologic malignancies. Blood 2001;97:2434–9. 2513–26. 32. Greenberger JS, Sakakeeny MA, Humphries RK, Eaves CJ, Eckner RJ. 14. Chen J, Williams IR, Lee BH, Duclos N, Huntly BJ, Donoghue DJ, et al. Demonstration of permanent factor-dependent multipotential (ery- Constitutively activated FGFR3 mutants signal through PLC{gamma}- throid/neutrophil/basophil) hematopoietic progenitor cell lines. dependent and -independent pathways for hematopoietic transformation. Proc Natl Acad Sci U S A 1983;80:2931–5. Blood 2005;106:328–37. 33. Migliaccio G, Migliaccio AR, Kreider BL, Rovera G, Adamson JW. Selection 15. Mosmann T. Rapid colorimetric assay for cellular growth and survival: of lineage-restricted cell lines immortalized at different stages of hemato- application to proliferation and cytotoxicity assays. J Immunol Methods poietic differentiation from the murine cell line 32D. J Cell Biol 1983;65:55–63. 1989;109:833–41. 16. Meyer AN, Drafahl KA, McAndrew CW, Gilda JE, Gallo LH, Haas M, et al. 34. Ahmed N, Berridge MV. Regulation of glucose transport by interleukin-3 in Tyrosine phosphorylation allows integration of multiple signaling inputs growth factor-dependent and oncogene-transformed bone marrow- by IKKbeta. PLoS One 2013;8:e84497. derived cell lines. Leuk Res 1997;21:609–18. 17. Williams SV, Hurst CD, Knowles MA. Oncogenic FGFR3 gene fusions in 35. Wei W, Liu W, Cassol C, Zheng W, Asa SL, Ezzat S. The breast cancer bladder cancer. Hum Mol Genet 2013;22:795–803. susceptibility gene FGFR2 serves as a scaffold for regulation of NF-kappa- 18. Thakur HC, Singh M, Nagel-Steger L, Kremer J, Prumbaum D, Fansa EK, beta signaling. Mol Cell Biol 2012;32:4662–73. et al. The centrosomal adaptor TACC3 and the microtubule polymerase 36. Johnston CL, Cox HC, Gomm JJ, Coombes RC. Fibroblast growth factor chTOG interact via defined C-terminal subdomains in an Aurora-A kinase- receptors (FGFRs) localize in different cellular compartments. A splice variant independent manner. J Biol Chem 2014;289:74–88. of FGFR-3 localizes to the nucleus. J Biol Chem 1995;270:30643–50. 19. Lin YC, Boone M, Meuris L, Lemmens I, Van Roy N, Soete A, et al. Genome 37. Ronchetti D, Greco A, Compasso S, Colombo G, Dell'Era P, Otsuki T, et al. dynamics of the human embryonic kidney 293 lineage in response to cell Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): biology manipulations. Nat Commun 2014;5:4767. comparative analysis of Y373C, K650E and the novel G384D mutations. 20. Hornbeck PV, Kornhauser JM, Tkachev S, Zhang B, Skrzypek E, Murray Oncogene 2001;20:3553–62. B, et al. PhosphoSitePlus: a comprehensive resource for investigating 38. Tavormina PL, Shiang R, Thompson LM, Zhu YZ, Wilkin DJ, Lachman RS, the structure and function of experimentally determined post-transla- et al. Thanatophoric dysplasia (types I and II) caused by distinct mutations tional modifications in man and mouse. Nucleic Acids Res 2012;40: in fibroblast growth factor receptor 3. Nat Genet 1995;9:321–8. D261–70. 39. Naski MC, Wang Q, Xu J, Ornitz DM. Graded activation of fibroblast 21. Olsen JV, Vermeulen M, Santamaria A, Kumar C, Miller ML, Jensen LJ, et al. growth factor receptor 3 by mutations causing achondroplasia and tha- Quantitative phosphoproteomics reveals widespread full phosphorylation natophoric dysplasia. Nat Genet 1996;13:233–7. site occupancy during mitosis. Sci Signal 2010;3:ra3. 40. Hart KC, Robertson SC, Donoghue DJ. Identification of tyrosine residues in 22. Shih C, Shilo BZ, Goldfarb MP, Dannenberg A, Weinberg RA. Passage of constitutively activated fibroblast growth factor receptor 3 involved in phenotypes of chemically transformed cells via transfection of DNA and mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation. chromatin. Proc Natl Acad Sci U S A 1979;76:5714–8. Mol Biol Cell 2001;12:931–42. 23. Padua RA, Carter G, Hughes D, Gow J, Farr C, Oscier D, et al. RAS mutations 41. Lievens PM, Liboi E. The thanatophoric dysplasia type II mutation hampers in myelodysplasia detected by amplification, oligonucleotide hybridiza- complete maturation of FGF receptor 3, which activates STAT1 from the tion, and transformation. Leukemia 1988;2:503–10. endoplasmic reticulum. J Biol Chem 2003;278:17344–9. 24. Bafna S, Singh AP, Moniaux N, Eudy JD, Meza JL, Batra SK. MUC4, a 42. Krejci P, Murakami S, Prochazkova J, Trantirek L, Chlebova K, Ouyang Z, multifunctional transmembrane glycoprotein, induces oncogenic trans- et al. NF449 is a novel inhibitor of fibroblast growth factor receptor formation of NIH3T3 mouse fibroblast cells. Cancer Res 2008;68:9231–8. 3 (FGFR3) signaling active in chondrocytes and multiple myeloma cells. 25. Sun M, Wang G, Paciga JE, Feldman RI, Yuan ZQ, Ma XL, et al. AKT1/ J Biol Chem 2010;285:20644–53. PKBalpha kinase is frequently elevated in human cancers and its consti- 43. Webster MK, D'Avis PY, Robertson SC, Donoghue DJ. Profound ligand- tutive activation is required for oncogenic transformation in NIH3T3 cells. independent kinase activation of fibroblast growth factor receptor 3 by the Am J Pathol 2001;159:431–7. activation loop mutation responsible for a lethal skeletal dysplasia, tha- 26. Petti LM, Irusta PM, DiMaio D. Oncogenic activation of the PDGF beta natophoric dysplasia type II. Mol Cell Biol 1996;16:4081–7. receptor by the transmembrane domain of p185neu. Oncogene 1998;16: 44. Wu YM, Su F, Kalyana-Sundaram S, Khazanov N, Ateeq B, Cao X, et al. 843–51. Identification of targetable FGFR gene fusions in diverse cancers. 27. Roll JD, Reuther GW. ALK-activating homologous mutations in LTK induce Cancer Discov 2013;3:636–47. cellular transformation. PLoS One 2012;7:e31733. 45. Gutierrez-Caballero C, Burgess SG, Bayliss R, Royle SJ. TACC3-ch-TOG 28. Kawai H, Matsushita H, Suzuki R, Sheng Y, Lu J, Matsuzawa H, et al. track the growing tips of microtubules independently of clathrin and Functional analysis of the SEPT9-ABL1 chimeric fusion gene derived from Aurora-A phosphorylation. Biol Open 2015;4:170–9. T-prolymphocytic leukemia. Leuk Res 2014;38:1451–9. 46. Singh D, Chan JM, Zoppoli P, Niola F, Sullivan R, Castano A, et al. 29. Tao W, Leng X, Chakraborty SN, Ma H, Arlinghaus RB. c-Abl activates janus Transforming fusions of FGFR and TACC genes in human glioblastoma. kinase 2 in normal hematopoietic cells. J Biol Chem 2014;289:21463–72. Science 2012;337:1231–5. 30. Laneuville P, Heisterkamp N, Groffen J. Expression of the chronic mye- 47. Acquaviva J, He S, Zhang C, Jimenez JP, Nagai M, Sang J, et al. FGFR3 logenous leukemia-associated p210bcr/abl oncoprotein in a murine IL-3 translocations in bladder cancer: differential sensitivity to HSP90 inhibi- dependent myeloid cell line. Oncogene 1991;6:275–82. tion based on drug metabolism. Mol Cancer Res 2014;12:1042–54.

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Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

Katelyn N. Nelson, April N. Meyer, Asma Siari, et al.

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