Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

Abstract

Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction. Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction. Ewing sarcoma is a pediatric bone cancer that displays a characteristic small round blue cell morphology (1Ewing J. Classics in oncology. Diffuse endothelioma of bone. James Ewing. Proceedings of the New York Pathological Society, 1921.CA Cancer J. Clin. 1972; 22: 95-98Crossref PubMed Scopus (75) Google Scholar). An early investigation of the disease demonstrated that patient cells express an aberrant EWSR1/FLI1 fusion gene composed of the N-terminal coding region of the EWS RNA Binding Protein 1/Ewing Sarcoma Breakpoint Region 1 (EWSR1) gene and the C-terminal coding region of the FLI-1 Proto-Oncogene, ETS Transcription Factor (FLI1) gene (2Delattre O. Zucman J. Plougastel B. Desmaze C. Melot T. Peter M. Kovar H. Joubert I. de Jong P. Rouleau G. Aurias A. Thomas G. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours.Nature. 1992; 359: 162-165Crossref PubMed Scopus (1433) Google Scholar, 3Delattre O. Zucman J. Melot T. Garau X.S. Zucker J.M. Lenoir G.M. Ambros P.F. Sheer D. Turc-Carel C. Triche T.J. Aurias A. Thomas G. The Ewing family of tumors--a subgroup of small-round-cell tumors defined by specific chimeric transcripts.N. Engl. J. Med. 1994; 331: 294-299Crossref PubMed Scopus (867) Google Scholar). Subsequent studies demonstrated that 85% of Ewing sarcoma patients have tumors that express the EWSR1/FLI1 fusion gene, while the remaining patients have tumors that express fusion genes composed of EWSR1 and other ETS transcription factors (ETV1, ETV4, ERG, and FEV1) (4Jeon I.S. Davis J.N. Braun B.S. Sublett J.E. Roussel M.F. Denny C.T. Shapiro D.N. A variant Ewing's sarcoma translocation (7;22) fuses the EWS gene to the ETS gene ETV1.Oncogene. 1995; 10: 1229-1234PubMed Google Scholar, 5Kaneko Y. Yoshida K. Handa M. Toyoda Y. Nishihira H. Tanaka Y. Sasaki Y. Ishida S. Higashino F. Fujinaga K. 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In all cases, the EWSR1 fusion genes encode the transactivation domain from EWSR1 and the ETS DNA binding domain from the ETS transcription factor. The reported effects of EWSR1 fusion protein expression include: 1) altered epigenetic status due to aberrant recruitment of the chromatin remodeling complex, and 2) aberrant expression of target genes in Ewing sarcoma cells (9Jedlicka P. Ewing Sarcoma, an enigmatic malignancy of likely progenitor cell origin, driven by transcription factor oncogenic fusions.Int. J. Clin. Exp. Pathol. 2010; 3: 338-347PubMed Google Scholar, 10Lessnick S.L. Ladanyi M. Molecular pathogenesis of Ewing sarcoma: new therapeutic and transcriptional targets.Annu. Rev. Pathol. 2012; 7: 145-159Crossref PubMed Scopus (119) Google Scholar, 11Johnson K.M. Mahler N.R. Saund R.S. Theisen E.R. Taslim C. Callender N.W. Lessnick S.L. for the EWS domain of in binding required for Ewing sarcoma S. A. PubMed Scopus Google Scholar, C. M. A. P. S. C. Kovar H. of gene and by the oncogenic fusion protein 10: Full Text Full Text PDF PubMed Scopus Google Scholar, G. S. B. S. A. I. C. of by a Full Text Full Text PDF PubMed Scopus Google Scholar, E.R. We Ewing sarcoma and is the Rev. PubMed Scopus Google Scholar, H. K. transcription and in 2010; PubMed Scopus Google Scholar). In addition, gene expression from due to EWSR1/FLI1 fusion protein with RNA and with RNA Full Text Full Text PDF PubMed Scopus (119) Google Scholar, M. and fusion proteins RNA by Google Scholar, A. S. E.R. A. fusion protein is a that S. A. PubMed Scopus Google Scholar). Ewing sarcoma is to a of DNA J. Y. S. H. J. of the Ewing Sarcoma family of tumors Genet. 10: PubMed Scopus Google Scholar, F. D. M. A. S. M. S. T. G. of Ewing sarcoma an with of and PubMed Scopus Google Scholar, de F. A. S. M. A. M. S. M. B. gene in bone and PubMed Scopus Google Scholar). The of Ewing have and genes have as in these in have in of in in of and in in of patients J. Y. S. H. J. of the Ewing Sarcoma family of tumors Genet. 10: PubMed Scopus Google Scholar, F. D. M. A. S. M. S. T. G. of Ewing sarcoma an with of and PubMed Scopus Google Scholar). While the in these genes to an in disease the incidence of the of an mechanism is the high incidence of aneuploidy in Ewing the of chromosome in of the of chromosome in of and the of of chromosome in of patients F. D. M. A. S. M. S. T. G. of Ewing sarcoma an with of and PubMed Scopus Google Scholar, J. J. F. C. J. A. G. A. A. P. and associated with DNA and in Ewing's PubMed Scopus Google Scholar). the cells a high incidence of aneuploidy, it is whether aneuploidy is in the early of and promotes the of the it is a of and during is the of chromosome caused by the of of mitotic chromosome mitotic to the of the the the of by in A. the midzone in the at the 7: PubMed Scopus Google Scholar, J.M. D. J. S. T. D. and in human Scopus Google Scholar). We previously demonstrated that EWSR1/FLI1 impairs the recruitment of the mitotic Aurora B kinase to the (the of and midzone proteins located between segregating chromosomes during H. K. C. B. M. Ewing sarcoma EWS protein midzone by Aurora B kinase to the PubMed Scopus Google Scholar). is that localization of Aurora B to the midzone to due to in the chromosome and the induction of aneuploidy J. B. The kinase to the mitotic at metaphase and is required for 10: Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. A. A is required for cell and of factors in PubMed Google Scholar, J.M. A. an protein kinase associated with chromosomes and is required for and in PubMed Scopus Google Scholar). mitotic dysfunction by EWSR1/FLI1 a critical in the induction of Ewing To the induction of aneuploidy by EWSR1/FLI1, we generated Tet-on EWSR1/FLI1 inducible cell lines using the P. K.M. J. M. J.E. J.E. G.M. human PubMed Scopus Google Scholar). cell lines to the of the cell and the expression of EWSR1/FLI1. we that EWSR1/FLI1 expression to Aurora and to the induction of aneuploidy 1 cell induction of EWSR1/FLI1 that it is an early in disease Furthermore, amino acid residue Thr 79 of EWSR1/FLI1 is essential for 1) colocalization of the fusion protein with Aurora B during 2) localization of Aurora B during and the induction of aneuploidy. substitution of amino acid of EWSR1/FLI1 with alanine the of the cells to induce these phenotypes, expression of EWSR1/FLI1-T79D the phosphomimetic substitution of for Thr retained the high activity as wild-type EWSR1/FLI1. suggest that phosphorylation of Thr 79 of EWSR1/FLI1 is critical for induction of aneuploidy, likely by the localization of the fusion protein on the leading to mitotic a mechanism to for the high incidence of aneuploidy in Ewing sarcoma and to the of the pathogenesis of this To the mechanism of mitotic dysfunction Aurora B we by the mutant of EWSR1/FLI1 that the to induce aberrant localization of Aurora B. mutant to the amino acid of the fusion protein that for induction of midzone is an for protein the critical mutant as a in to the that the of EWSR1/FLI1 is required to induce mitotic we a to the critical of the fusion protein M. Ewing sarcoma fusion protein EWSR1/FLI1 with EWSR1 leading to mitotic in and human cell PubMed Scopus Google Scholar). We residue Thr 79 of EWSR1/FLI1, is located in the of the from EWSR1 it is a for Thr 79 is cells expressing EWSR1/FLI1 with a S. Davis P. of Ewing's sarcoma protein and in to DNA J. PubMed Scopus Google Scholar). Thr 79 is a for the expression of of DNA Binding D.N. G. M. M. Kovar H. is in the transcriptional activity of in Ewing's PubMed Scopus Google Scholar). We generated DNA with EWSR1/FLI1 and with amino acid at residue 79 in EWSR1/FLI1. The substitution a Thr to substitution a Thr to substitution phosphomimetic these DNA we stable Tet-on cell lines by EWSR1/FLI1, EWSR1/FLI1-T79A, and EWSR1/FLI1-T79D with an the safe-harbor AAVS1 locus of DLD-1 cells using CRISPR/Cas9 technology P. K.M. J. M. J.E. J.E. G.M. human PubMed Scopus Google Scholar, D. Y. P. I. and DNA in using a locus in the human 2010; PubMed Scopus Google Scholar). The DLD-1 cancer cell it express EWSR1/FLI1 and it a it as a to proteins of for to induce aneuploidy J.M. D. J. S. T. D. and in human Scopus Google Scholar, C. B. in 1997; PubMed Scopus Google Scholar). The inducible to the activity of EWSR1/FLI1 in the induction of aneuploidy a cell of EWSR1/FLI1, EWSR1/FLI1-T79A, and EWSR1/FLI1-T79D in cells by using an and by using the We the by the EWSR1/FLI1 and The cells at by using the and by for the of EWSR1/FLI1 and using for In the EWSR1/FLI1 expressing cells displayed in the chromosomes during prophase and metaphase and in the in cells to express the in of the cells on the chromosomes in the during prophase and metaphase anaphase, and the and the on the chromosomes Together, these suggest that residue of EWSR1/FLI1 is critical for localization to the chromosomes during early mitosis. To this the localization of EWSR1/FLI1 and using a cell lines with using the and for The cells and as and chromosome and all of the to using that DLD-1 cells express levels of the EWSR1/FLI1 and with the using the in the chromosome in cells expressing EWSR1/FLI1, and is in this for the of the for the EWSR1/FLI1 and in the chromosome from that the of the that of EWSR1/FLI1 as that the expression of the is that of EWSR1/FLI1 the localization of on the chromosome that of EWSR1/FLI1. the that the localization of the EWSR1/FLI1 on the chromosome is by the of the the residue Thr 79 is required for EWSR1/FLI1 to to chromosomes during mitosis. This is the first that EWSR1/FLI1 is to the chromosome in a Thr 79 To address whether the localization of EWSR1/FLI1 on the chromosome with the localization of Aurora B, EWSR1/FLI1 and DLD-1 cell lines by the in the cells to using and and B and of the cells and with for and Aurora B using the of We a of in in in EWSR1/FLI1 expressing cells compared with cells expressing The for Aurora B and using the cells expressing EWSR1/FLI1 a of localization cells expressing The that residue Thr 79 is essential for the colocalization between EWSR1/FLI1 and Aurora B. To whether the stable lines of on midzone localization of Aurora B using expression of EWSR1/FLI1, the stable cell lines with by using an B and an to the transgene M. Ewing sarcoma fusion protein EWSR1/FLI1 with EWSR1 leading to mitotic in and human cell PubMed Scopus Google Scholar). The localization of Aurora B by the of cells Aurora B at the with using Ewing sarcoma cells and the DLD-1 cells that to express EWSR1/FLI1 displayed a greater incidence of aberrant Aurora B localization cells H. K. C. B. M. Ewing sarcoma EWS protein midzone by Aurora B kinase to the PubMed Scopus Google Scholar, M. Ewing sarcoma fusion protein EWSR1/FLI1 with EWSR1 leading to mitotic in and human cell PubMed Scopus Google Scholar). We cells in of In cells expressing EWSR1/FLI1, the of cells aberrant Aurora B localization compared with that in noninduced cells we the of the residue substitution on midzone The expression the of Aurora B the midline The expressing cells a in incidence of Aurora B compared with that in noninduced cells suggest that amino acid Thr 79 of EWSR1/FLI1 is critical to induce of Aurora B at the midzone during anaphase. We whether EWSR1/FLI1 induces aneuploidy and whether Thr 79 is in this induction. the of DLD-1 cell is and the induction of EWSR1/FLI1 by the stable DLD-1 cell lines with for to whether the EWSR1/FLI1 induces aneuploidy cell P. of the of human to by Google Scholar). The to a metaphase chromosome by of the chromosomes with The of chromosomes in the cells of The of EWSR1/FLI1 expressing cells aberrant of the in displayed of in the of cells that displayed aberrant of chromosomes between the expressing cells and the cells The of cells in the EWSR1/FLI1 the and the chromosomes In contrast, the of cells expressing EWSR1/FLI1 chromosome suggest that EWSR1/FLI1 induces aneuploidy and that the Thr 79 residue of EWSR1/FLI1 a critical in this The of the cell that residue Thr 79 of EWSR1/FLI1 a critical in the of Aurora B during metaphase and and in the induction of aneuploidy. Thr is to we to whether the phosphorylation of amino acid Thr 79 of EWSR1/FLI1 the of Aurora B during and the induction of aneuploidy. To address this, we the phosphomimetic EWSR1/FLI1-T79D mutant a Thr to substitution at residue 79, and compared activity with that of EWSR1/FLI1. The localization of EWSR1/FLI1 and EWSR1/FLI1-T79D during mitosis. The cells using and for by using by the cells with at mitotic to EWSR1/FLI1, of the expressing cells displayed on the chromosomes during all mitotic We the localization of EWSR1/FLI1-T79D in and chromosome using the in the in and D. with the by EWSR1/FLI1 and EWSR1/FLI1-T79D in the chromosome in cells of the for the EWSR1/FLI1 and EWSR1/FLI1-T79D in the chromosome that the for EWSR1/FLI1-T79D that of EWSR1/FLI1 as the expression of the and EWSR1/FLI1-T79D EWSR1/FLI1 and cell the localization of on the chromosome that of EWSR1/FLI1. Together, these suggest that the phosphorylation of the residue Thr 79 of EWSR1/FLI1 promotes localization to chromosomes during mitosis. We whether EWSR1/FLI1-T79D and Aurora B on metaphase the in the stable cell lines with and The EWSR1/FLI1 and EWSR1/FLI1-T79D expressing DLD-1 cell lines to using and and B and by the colocalization as using the of In to the in with a in the between EWSR1/FLI1 and EWSR1/FLI1-T79D of colocalization between EWSR1/FLI1-T79D and Aurora B compared with that of EWSR1/FLI1 and Aurora B. The that the phosphorylation of residue Thr 79 of EWSR1/FLI1 is essential for the colocalization of EWSR1/FLI1 and Aurora B. the to Aurora B to the we the of phosphorylation of EWSR1/FLI1 residue on Aurora B localization using stable cell lines with the phosphomimetic mutant cells expressing EWSR1/FLI1 and EWSR1/FLI1-T79D to using and B expressing EWSR1/FLI1 EWSR1/FLI1-T79D displayed a greater incidence of aberrant Aurora B localization cells H. K. C. B. M. Ewing sarcoma EWS protein midzone by Aurora B kinase to the PubMed Scopus Google Scholar, M. Ewing sarcoma fusion protein EWSR1/FLI1 with EWSR1 leading to mitotic in and human cell PubMed Scopus Google Scholar). We the localization of Aurora B at the midzone in cells in the of cells that displayed aberrant Aurora B localization in cells that express EWSR1/FLI1 that in noninduced cells expressing the phosphomimetic EWSR1/FLI1-T79D displayed a in incidence of Aurora B compared with that in noninduced cells between the incidence of aberrant Aurora B localization in cells and cells suggest that phosphorylation of the Thr 79 residue of EWSR1/FLI1 is critical for the of Aurora B at the midzone during anaphase. To address whether phosphorylation of EWSR1/FLI1 at Thr 79 is a critical required for the induction of aneuploidy in cells expressing EWSR1/FLI1, chromosome on cells expressing EWSR1/FLI1 and EWSR1/FLI1-T79D with the in cells expressing EWSR1/FLI1 a incidence of aberrant of chromosomes cells that express the fusion cells expressing EWSR1/FLI1-T79D displayed a incidence of aberrant of chromosomes compared with cells and in the incidence of cells containing aberrant chromosome between cells expressing EWSR1/FLI1 and cells expressing the phosphomimetic mutant EWSR1/FLI1-T79D The of chromosomes cell in cells expressing EWSR1/FLI1 and cells expressing EWSR1/FLI1-T79D in suggest that EWSR1/FLI1 induces aneuploidy the Thr 79 residue of EWSR1/FLI1 is Together, these studies that phosphorylation of Thr 79 of EWSR1/FLI1 promotes localization to mitotic chromosomes during prophase and with localization of Aurora B at the midzone and induction of aneuploidy The of a amino acid at residue 79 of EWSR1/FLI1 from Thr to to the induction of the In contrast, the substitution of amino acid 79 of EWSR1/FLI1 from Thr to the induction of the Furthermore, suggest these a the localization of EWSR1/FLI1 to the chromosomes during prophase and metaphase in the midzone and to the induction of aneuploidy the first for an between the localization of EWSR1/FLI1 on mitotic chromosomes during early localization of Aurora B at the and induction of aneuploidy. Furthermore, we that the phosphorylation of Thr 79 of EWSR1/FLI1 is critical to this of the that the phosphorylation of 79, activity and mitotic is essential for the pathogenesis of Ewing A demonstrated that DNA promotes the phosphorylation of Thr 79 of EWSR1/FLI1 S. Davis P. of Ewing's sarcoma protein and in to DNA J. PubMed Scopus Google Scholar). to the in it is essential to whether the kinase phosphorylation during mitosis. In addition, phosphorylation and as Thr This is a mechanism for the of the fusion demonstrated that of (the that from in cells in increased levels of of EWSR1 as as aberrant localization of EWSR1 during metaphase C. M. M. C. expression promotes mitotic and 15: PubMed Scopus Google Scholar). EWSR1 and EWSR1/FLI1 and EWSR1/FLI1 the as the of Thr it is that EWSR1/FLI1 activity is by at Thr 79 D.N. G. M. M. Kovar H. is in the transcriptional activity of in Ewing's PubMed Scopus Google Scholar, K. the localization of EWS with an in in PubMed Scopus Google Scholar). demonstration of the of in the induction of aneuploidy, the and EWSR1/FLI1-T79D that we an for the proteins to and phosphorylation at the Thr and the of the cells expressing proteins altered by the it is that and phosphorylation and and that the phosphorylation of EWSR1/FLI1 is the critical for the induction of mitotic phosphorylation of EWSR1/FLI1 is in the induction of aneuploidy and these protein is to whether EWSR1 a in this is studies have that EWSR1/FLI1 with and the of EWSR1 M. Ewing sarcoma fusion protein EWSR1/FLI1 with EWSR1 leading to mitotic in and human cell PubMed Scopus Google Scholar, C. D.N. Kovar H. and of and oncogenic fusion 22: PubMed Scopus Google Scholar, A. S. E.R. K. Lessnick S.L. Y. transcription to and in Ewing PubMed Scopus Google Scholar). studies demonstrated that EWSR1 a in the localization of Aurora B to the midzone between the M. Ewing sarcoma fusion protein EWSR1/FLI1 with EWSR1 leading to mitotic in and human cell PubMed Scopus Google Scholar). In addition, EWSR1 in chromosome and in H. T. J. M. of Ewing sarcoma EWS promotes by in PubMed Scopus Google Scholar). To the mechanism of Aurora B it is to the between Aurora B, and EWSR1/FLI1. is that EWSR1/FLI1, EWSR1 and Aurora B a that between EWSR1/FLI1 and Aurora B with of B. In addition, it is essential to whether these on the the critical of amino acid in the of EWSR1/FLI1, the amino acid and EWSR1/FLI1-T79D of the pathogenesis of the to chromosomes during early we this the to with EWSR1 Aurora B. with EWSR1 Aurora B, of to indicating that localization is by an unknown it is that the EWSR1/FLI1-T79D an activity compared with Furthermore, while to the of EWSR1/FLI1 in it is to address whether the of EWSR1/FLI1 in transcription and the induction of aneuploidy of Aurora B in the cell of for Ewing in an F. K. A. B. P. Delattre O. cell of Ewing Full Text Full Text PDF PubMed Scopus Google Scholar, D. P. S. J.M. K. I. expression a Ewing's sarcoma in human PubMed Scopus Google Scholar). studies required to the mechanism for the of aneuploidy of specific chromosomes the high incidence of of chromosome in Ewing sarcoma cells. that phosphorylation of Thr 79 of EWSR1/FLI1 promotes the induction of aneuploidy, and the cells of cells expressing EWSR1/FLI1 EWSR1/FLI1-T79D displayed a of chromosome is that is for specific chromosomes in the EWSR1/FLI1-dependent induction of aneuploidy, during cell cells that chromosomes to and during Ewing sarcoma that we have demonstrated the of phosphorylation of in the induction of aneuploidy, studies the mechanism leading to in chromosome essential to human cells the cells of for Ewing sarcoma to the of specific chromosome and F. K. A. B. P. Delattre O. cell of Ewing Full Text Full Text PDF PubMed Scopus Google Scholar, H. Toyoda M. K. H. K. Y. M. H. T. Y. H. T. Y. T. to of human cells by chimeric with PubMed Scopus Google Scholar). of in the high of due to this it is to the incidence of aneuploidy of specific of the cell by the cell the is a cell of of Ewing it to the of these the using to the induction of aneuploidy in a specific chromosome and the of during Ewing sarcoma This a for the of EWSR1 fusion proteins Aurora B and induce aneuploidy. the and EWSR1/FLI1-T79D mutant as as and fused to other genes to the of phosphorylation in disease of express fusion genes that have of EWSR1 Thr 79, this the to a mechanism for the pathogenesis all EWSR1 fusion of the kinase that the activity of Thr 79 of EWSR1/FLI1 fusion protein and associated with chromosome to the of new therapeutic and from the the using the the and genes by from and using the that a with and the gene from the using the that a with and Y. The by to 2012; PubMed Scopus Google A new generated in the by the with and an containing the DNA of EWSR1/FLI1, EWSR1/FLI1-T79A, EWSR1/FLI1-T79D and the and of the T. T. Y. protein in human cells by with 15: Full Text Full Text PDF PubMed Scopus Google Scholar). The in on the H. A. M. Y. the and localization of proteins on mitotic PubMed Google Scholar). The of of using the CRISPR/Cas9 P. K.M. J. M. J.E. J.E. G.M. human PubMed Scopus Google Scholar). DLD-1 cells in and with the and RNA the AAVS1 locus AAVS1 in using the P. K.M. J. M. J.E. J.E. G.M. human PubMed Scopus Google Scholar, D. Y. P. I. and DNA in using a locus in the human 2010; PubMed Scopus Google Scholar). The cells for in at in containing The cells for with and in the cells and the expression of the transgene by and from EWSR1/FLI1 and the from and the In addition, from EWSR1/FLI1-T79D and the DLD-1 cell lines in with at with The expression of EWSR1/FLI1, EWSR1/FLI1-T79A, and EWSR1/FLI1-T79D in the DLD-1 cells by with for cells in this The cells in using for by with The with containing and the cells for The to the cell for to the the cell The cells in using an cell with K. M. midzone recruitment of is essential for Full Text Full Text PDF PubMed Scopus Google Scholar). the cells with for from the by with the with containing and the cells for the cells with of for and the mitotic cells by a mitotic by with The mitotic cells for by in on and the cells to the the cell by for to the The cells for and to as with H. K. C. B. M. Ewing sarcoma EWS protein midzone by Aurora B kinase to the PubMed Scopus Google Scholar). The B and H. K. C. B. M. Ewing sarcoma EWS protein midzone by Aurora B kinase to the PubMed Scopus Google Scholar). cells with at with a using The of and at using The of and at using in and as previously C. H. M. Ewing sarcoma protein of of in 10: PubMed Scopus Google Scholar). to for at and the as the The in a and to for at The from this to the and the from this as the from and cells to using a of of as by with a of and The cell from and stable cells to using a of by with a of The of the with a of and a of of using the and cells with the cells at The cells for with and and with of at for of acid to the cell while the using a The cells at for in 1 of acid and for on this the cells in of acid and this cell The DNA on the with The of chromosomes at with a using is as for The defined at by by in the The that have of with the of this H. P. and H. K. H. for Y. A. and for C. S. and for and M. A. and This by the of and Cancer

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