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 the second most common bone cancer that develops in adolescents and young adults. Majority of Ewing sarcoma patients (~85%) carry an EWSR1-FLI1 fusion gene derived from t(11;22)(q24;q12) chromosomal translocation. The formation of EWSR1-FLI1 leads to the loss of one wild type EWSR1 allele, however, how loss of the gene contributes to the pathogenesis of Ewing sarcoma remains unclear. Our previous study revealed that the EWSR1 is required for a faithful mitosis. We demonstrated that the mutant ewsa (a homologue of human EWSR1) zebrafish induces mitotic defects, aneuploidy, and promotes tumorigenesis (with tp53 mutation). Consistently, Ewing sarcoma patients display high incidence of aneuploidy (e.g. approximately 47% of the patients display trisomy 8). For these reasons, we aim to elucidate the molecular function of EWSR1 in faithful chromosome segregation and in maintenance of chromosome stability. To accomplish our goal, we employed the Auxin-Inducible Degron (AID) system because it allows us to conditionally degrade EWSR1 proteins in a cell line. Both EWSR1 alleles were tagged with mini-AID using CRISPR/Cas9 system in a stable DLD-1 cell that expresses a plant E3 ligase TIR1. The western blot verified the efficient degradation of EWSR1 when the cells were treated with Auxin (AUX+) for 24hrs. To determine whether EWSR1 maintains chromosomal stability, the cells were synchronized to pro-metaphase using thymidine/nocodazole protocol, and were treated with AUX for 24hrs (underwent one cell cycle) concurrently. The EWSR1 knockdown (AUX+) cells displayed significantly higher incidence of lagging chromosomes compared to the control (AUX-) cells. Furthermore, immunocytochemistry using the antibody against the key mitotic regulator, Aurora B, revealed that the EWSR1 knockdown (AUX+) cells displayed significantly less incidence of Aurora B localization at inner centromere, but higher incidence of localization on kinetochore compared to the control (AUX-) cells. Consistently, when the cells that were treated with AUX for 48hrs (underwent two cell cycles), it displayed significantly higher incidence of aberrant numbers of chromosome compared to the control (AUX-) cells. In general, impaired chromosome segregation leads to mitotic delay, accompanied by the activation of mitotic checkpoint. However, the EWSR1 knockdown (AUX+) cells did not undergo any arrest in mitosis. Therefore, our data suggest that the EWSR1 knockdown (AUX+) cells not only induces aneuploidy by inducing lagging chromosome, but also it may override mitotic checkpoint. Currently, we are in a process of elucidating the molecular mechanism of EWSR1 knockdown dependent mitigation of mitotic arrest. Successful completion of the study may shed light on how/whether haploinsufficiency of EWSR1 contributes to the molecular pathogenesis of Ewing sarcoma. Citation Format: Haeyoung Kim, Mizuki Azuma. Function of Ewing sarcoma EWSR1 protein in the maintenance of chromosome stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2299.

EWSR1 (Ewing sarcoma breakpoint region 1) was originally identified as a part of an aberrant EWSR1/FLI1 fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the EWSR1/FLI1 fusion gene in the tumor genome, the cell loses one wild type EWSR1 allele. Our previous study demonstrated that the loss of ewsr1a (homologue of human EWSR1) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the tp53 mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using an Auxin Inducible Degron (AID) system. When both EWSR1 genes of DLD-1 cell were tagged with mini-AID at its 5′-end using a CRISPR/Cas9 system, treatment of the (AID-EWSR1/AID-EWSR1) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the EWSR1 knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at Kinetochore proximal centromere compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of EWSR1-mCherry and EWSR1:R565A-mCherry (a mutant that has low affinity for Aurora B) in the (AID-EWSR1/AID-EWSR1) DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 prevents the induction of lagging chromosomes, and of aneuploidy through the interaction with Aurora B.

EWSR1 (Ewing sarcoma breakpoint region 1) was originally identified as a part of an aberrant EWSR1/FLI1 fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the EWSR1/FLI1 fusion gene in the tumor genome, the cell loses one wild type EWSR1 allele. Our previous study demonstrated that the loss of ewsr1a (homologue of human EWSR1) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the tp53 mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using Auxin Inducible Degron (AID) system. When both EWSR1 genes of DLD-1 cell were tagged with mini-AID using CRISPR/Cas9 system, treatment of the (AID-EWSR1/AID-EWSR1) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the EWSR1 knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at kinetochores compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not arrest at mitosis, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of EWSR1-mCherry and EWSR1:R565A-mCherry (a mutant that has low affinity for Aurora B) in the (AID-EWSR1/AID-EWSR1) DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 is essential to prevent aneuploidy through interaction with Aurora B, most likely by regulating the localization of Aurora B at centromere.