Disruption of Microhomology-mediated End-joining in Ewing Sarcoma

  • bioRxiv. 2025 Sep 18:2025.05.06.651696. doi: 10.1101/2025.05.06.651696.
Shuhei Asada  1 ,  Guangli Zhu  1  2 ,  Jithma Prasad Abeykoon  1  3 ,  Yutaro Tanaka  4  5 ,  Huy Nguyen  1  6 ,  Yuna Hirohashi  1 ,  Divya R Iyer  1 ,  Nicholas William Ashton  1 ,  Sirisha Mukkavalli  1 ,  Martha Velazquez  1 ,  Lige Jiang  1 ,  Miles Del Busso  3 ,  Judith Jebastin Thangaiah  7 ,  Steven I Robinson  8 ,  Kalindi Parmar  1  6 ,  Eliezer M Van Allen  5  9  10  11 ,  Riaz Gillani  4  5  9  12 ,  Geoffrey I Shapiro  10 ,  Alan D D'Andrea  1  6  13
Affiliations
  • 1. Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
  • 2. Department of Radiation Oncology, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
  • 3. Division of Hematology, Department of Internal Medicine, Mayo Clinic, MN, USA.
  • 4. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
  • 5. Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
  • 6. Center for DNA Damage and Repair, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
  • 7. Department of Pathology, Mayo Clinic, MN, USA.
  • 8. Department of Oncology, Mayo Clinic, MN, USA.
  • 9. Harvard Medical School, Boston, MA, USA.
  • 10. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
  • 11. Center for Cancer Genomics, Dana-Farber Cancer Institute, Boston, MA, USA.
  • 12. Boston Children's Hospital, Boston, MA, USA.
  • 13. Lead contact.
PMID: 40568108 DOI: 10.1101/2025.05.06.651696
Abstract

Ewing sarcoma (EwS) is a group of bone and soft tissue cancers in children and young adults. Since EwS cells have pronounced sensitivity to radiation and chemotherapy-induced DNA damage, the role of the oncoprotein, EWS-FLI1, in DNA repair is likely. Here, we demonstrate that EWS-FLI1 causes a defect in microhomology-mediated end-joining (MMEJ) repair. EWSR1 is a splicing factor that promotes the faithful splicing of the POLQ pre-mRNA, required for the expression of POLΘ, a critical protein in the MMEJ pathway. Expression of EWS-FLI1, or loss of EWSR1, causes exon 25 skipping of the POLQ transcript, decreased POLΘ expression, impaired MMEJ, and cellular sensitivity to inhibitors of the Fanconi Anemia (FA), NHEJ, or HR pathways, through the mechanism of synthetic lethality. Knockdown of EWS-FLI1 expression restores POLΘ mitotic foci and increases MMEJ activity. Inhibitors of the FA, NHEJ, or HR therefore may provide a targeted therapy for patients with EwS.

Keywords
Alternative end-joining; CDK12; DNA double-strand breaks; DNA-PKc; Ewing’s Sarcoma; TMEJ; the Fanconi Anemia pathway.
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