Translocations involving ETS-transcription factors, most commonly leading to the 
EWSR1-FLI1 fusion protein, are the hallmark of Ewing sarcoma. 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 TP53 
is commonly found to be wild-type, thus providing an attractive target for 
treatment. Here, we study TP53 wild-type (EW7, CADO-ES1 and TC32) and 
TP53-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 TP53 status. We show that simultaneous YK-4-279 treatment with Nutlin-3 to 
stabilize p53 resulted in an additive inhibition of TP53 wild-type Ewing sarcoma 
cell burden, whilst not affecting TP53-deleted Ewing sarcoma cells. Improved 
inhibition of proliferation and migration by combinatorial treatment was 
confirmed in vivo by 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.