ARTICLE ABSTRACTPurpose: The goal of this study is to optimize the activity of trabectedin for Ewing sarcoma by developing a molecularly targeted combination therapy.Experimental Design: We have recently shown that trabectedin interferes with the activity of EWS-FLI1 in Ewing sarcoma cells. In this report, we build on this work to develop a trabectedin-based combination therapy with improved EWS-FLI1 suppression that also targets the drug-associated DNA damage to Ewing sarcoma cells.Results: We demonstrate by siRNA experiments that EWS-FLI1 drives the expression of the Werner syndrome protein (WRN) in Ewing sarcoma cells. Because WRN-deficient cells are known to be hypersensitive to camptothecins, we utilize trabectedin to block EWS-FLI1 activity, suppress WRN expression, and selectively sensitize Ewing sarcoma cells to the DNA-damaging effects of SN38. We show that trabectedin and SN38 are synergistic, demonstrate an increase in DNA double-strand breaks, an accumulation of cells in S-phase and a low picomolar IC50. In addition, SN38 cooperates with trabectedin to augment the suppression of EWS-FLI1 downstream targets, leading to an improved therapeutic index in vivo. These effects translate into the marked regression of two Ewing sarcoma xenografts at a fraction of the dose of camptothecin used in other xenograft studies.Conclusions: These results provide the basis and rationale for translating this drug combination to the clinic. In addition, the study highlights an approach that utilizes a targeted agent to interfere with an oncogenic transcription factor and then exploits the resulting changes in gene expression to develop a molecularly targeted combination therapy. Clin Cancer Res; 20(5); 1190–203. ©2013 AACR.