Exploiting a Novel DNA Repair Defect in Ewing Sarcoma - Project Summary Ewing sarcoma (EwS) is an aggressive subtype of bone and soft tissue cancers in adolescents and young adults. EwS is recognized as a radiosensitive and chemosensitive cancer. The current 5-year overall survival for patients with localized disease is 65% to 75%. While the localized disease can be cured by multimodality treatment in 70% of patients, those with metastatic disease have a poor prognosis with a 5-year overall survival (OS) of less than 30%. Relapsed Ewing sarcoma has an extremely poor prognosis with a five-year overall survival after relapse of only 10%. Recent studies have identified abnormalities in DNA Repair (DDR) and Homologous Recombination (HR) in Ewing sarcoma cells. However, the mechanism by which EWS-FLI1 influences DDR pathways is still largely unknown, and other more profound DNA repair defects, beyond the HRD phenotype, may exist. Importantly, our laboratory has surprising new data demonstrating that EwS has an intrinsic defect in Microhomology-Mediated End- Joining (MMEJ). Previous studies have suggested that EWSR1 is an RNA binding protein with splicing activity. Our new data demonstrate that loss of EWSR1 results in exon skipping of the POLQ mRNA, generating a premature termination codon and a profound reduction of POLQ protein expression and MMEJ activity.As a result, EWSR1 knockout cells showed similar phenotypes to POLQ deficient cells, including hypersensitivity to topoisomerase inhibitors and irradiation, as well as synthetic lethality with BRCA1/2 loss. Our new data also show that EWSR1 mutations can disrupt other aspects of MMEJ. For instance, the fusion protein EWS-FLI1, perhaps through its ability to form condensates, inhibits the recruitment of POLQ to the DSB loci, resulting in a severe impairment of MMEJ. Our data therefore indicate that EwS is the only known human cancer that has this unique DDR defect – impaired MMEJ- activity.We believe this unique DDR defect could be exploited for therapeutic benefit by inhibiting other DNA repair pathways. Based on our preliminary data, we propose the following three aims: Aim 1 is to investigate the molecular mechanisms by which the MMEJ pathway is suppressed in EwS. Aim 2 is to assess the function of the MMEJ pathway in EwS primary tumor tissue from patients.Aim 3 is to exploit the MMEJ defect for therapeutic benefit in patients with EwS.
