Ewing sarcoma is a small blue round cell tumour occurring in the bone and soft tissue of young children. The causative genetic defect underlying the development of this tumour is a translocation fusing an Ews gene to an Ets transcription factor family gene, most frequently resulting in the fusion genes Ews-Fli1 (~85% of all cases) or Ews-Erg (~10% of all cases). Despite knowledge of this driving molecular event, an effective therapeutic strategy is lacking. To test potential treatment regimes, we established a novel Ewing sarcoma zebrafish engraftment model allowing time-effective, dynamic quantification of Ewing sarcoma progression and tumour burden in vivo, applicable for screening of single and combined compounds. In Ewing sarcoma, the tumour-suppressor gene p53 is commonly found to be wild-type, thus providing an attractive target for treatment. Here, we study p53-wild-type (EW7, CADO-ES1 and TC32) and p53-deleted (SK-N-MC) Ewing sarcoma cell lines to investigate the potentiating effect of p53 reactivation by Nutlin-3 on treatment with YK-4-279 to block transcriptional activity of EWSR1-FLI1 protein. Blocking EWSR1-FLI1 transcriptional activity reduced Ewing sarcoma tumour cell burden irrespective of p53 status. We show that simultaneous YK-4-279 treatment with Nutlin-3 to stabilise p53 resulted in an additive inhibition of p53-wild-type Ewing sarcoma cell burden, whilst not affecting p53-deleted Ewing sarcoma cells. Improved inhibition of proliferation and migration by combinatorial treatment was confirmed in vivo with zebrafish engraftments. Mechanistically, both compounds together additively induced apoptosis of tumour cells in vivo by engaging distinct pathways. We propose reactivation of the p53 pathway in combination with complementary targeted therapy by EWSR1-FLI1 transcriptional activity disruption as a valuable strategy against p53-wild-type Ewing sarcoma. In addition, a high-throughput engraft model in blastula-stage embryos is developed for the rapid screening of novel anticancer compounds. This HT-model proved to be suitable to screen the inhibitory effects of multiple compounds on various Ewings sarcoma lines.
Disclosure: The study was funded by Stichting Kinderen Kankervrij, Project 30677.