Abstract

Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein1. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

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Change history

  • Corrected online 27 June 2018

    In this Letter, the sentence beginning "This work was funded...." in the Acknowledgements should have read "CPRIT (RP140105) to J.C.R." rather than "CPRIT (RP150445) to J.C.R." This error has been corrected online.

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Acknowledgements

We thank F. Chedin for the DRIP–seq protocol and genome-wide restriction enzyme sites file, and R. Crouch, J. Stark and Y. Shiio for plasmids. We are grateful to the UTH-SA/Cancer Center Sequencing core and the Histology & Immunohistochemistry Core at UTH-SA. This work was funded by the NIH (K22ES012264, 1R15ES019128, 1R01CA152063), a Voelcker Fund Young Investigator Award and CPRIT (RP150445) to A.J.R.B.; CPRIT (RP101491), a Translational Science Training Across Disciplines Scholarship (UTHSA) and an NCI postdoctoral training grant (T32CA148724) to A.G.; CPRIT (RP140105) to J.C.R.; NIH (P30CA054174) to Mays Cancer Center; NCI (R01CA204915) and Curing Kids Cancer to K.S.; NIH CTSA (1UL1RR025767-01, P30CA054174) and CPRIT (RP120685-C2) to Y.C.; NIH (1R01CA140394) to S.L.L. and NIH (1R01CA134605) to E.R.L.

Author information

Affiliations

  1. Department of Cell Systems and Anatomy, University of Texas Health at San Antonio, San Antonio, Texas 78229, USA

    • Aparna Gorthi
    • , July Carolina Romero
    • , Liesl A. Lawrence
    • , Xavier Bernard
    •  & Alexander J. R. Bishop
  2. Greehey Children’s Cancer Research Institute, University of Texas Health at San Antonio, San Antonio, Texas 78229, USA

    • Aparna Gorthi
    • , July Carolina Romero
    • , Eva Loranc
    • , Lin Cao
    • , Liesl A. Lawrence
    • , Elicia Goodale
    • , Xavier Bernard
    • , V. Pragathi Masamsetti
    • , Yidong Chen
    •  & Alexander J. R. Bishop
  3. Department of Pediatric Oncology, Dana-Farber Cancer Institute and Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA

    • Amanda Balboni Iniguez
    •  & Kimberly Stegmaier
  4. The Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    • Amanda Balboni Iniguez
    •  & Kimberly Stegmaier
  5. Departments of Oncology and Pediatrics, Georgetown University, Washington DC 20057, USA

    • Sydney Roston
    •  & Jeffrey A. Toretsky
  6. Departments of Pediatrics and Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA

    • Elizabeth R. Lawlor
  7. Center for Childhood Cancer and Blood Diseases, Nationwide Children’s Hospital, Columbus, Ohio 43205, USA

    • Stephen L. Lessnick
  8. Mays Cancer Center, University of Texas Health at San Antonio, Texas 78229, USA

    • Yidong Chen
    •  & Alexander J. R. Bishop
  9. Department of Epidemiology and Biostatistics, University of Texas Health at San Antonio, San Antonio, Texas 78229, USA

    • Yidong Chen

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Contributions

A.G. and A.J.R.B. conceived and designed the study and wrote the manuscript. A.G. conducted the majority of the research. J.C.R. contributed to replication of experiments for homologous recombination and BRCA1 localization. E.G. performed EU incorporation experiments. L.A.L. performed RNaseH rescue of replication stress and EU incorporation experiments. E.L., L.C., V.P.M. and X.B. provided technical support. A.B.I. and K.S. performed EWS–FLI1 knockdown for the phospho-RNAPII experiment. S.R. and J.A.T. provided recombinant EWS–FLI1 protein. Y.C. conducted bioinformatics analysis support. S.L.L. and E.R.L. provided reagents/insights.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Alexander J. R. Bishop.

Reviewer Information Nature thanks B. Braun and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

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  1. 1.

    Supplementary Figure 1

    This file contains full scans of the western blots presented in Figures 1e-g, 2e, 3i, 4a and Extended Data Figures 1b,c, 2a,b, 3a, 5a,d, 6d-g and 7a.

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    Damage-induced changes in gene expression between IMR90 and EwS cell lines. Supplementary Table 1.1 contains a list of genes that are at least two-fold altered upon etoposide treatment in IMR90 but not in EwS cell lines. The criterion for evaluation of EwS cells was as follows: genes that were upregulated at least 2-fold in IMR90 but less than 0-fold (no change or downregulated) in EwS and vice versa. Supplementary Table 1.2 contains a list of genes that are at least two-fold altered upon etoposide treatment in EwScell lines but not in IMR90. The criterion for evaluation is as follows: genes that were upregulated at least 2-fold in EwS but less than 0-fold (no change or downregulated) in IMR90 and vice versa. Supplementary Table 1.3 contains a list of genes that were similarly altered (minimum 2-fold change) by gene expression in response to damage between IMR90 and EwS.

  2. 2.

    Supplementary Table 2

    This table shows the top 5% hits in the Drosophila kc167 RNAi screens. Data was collected as percent survival upon damage induction (3 days) for each RNAi. The loci that mapped to NCBI gene IDs are listed

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https://doi.org/10.1038/nature25748

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