Abstract A103: ET-743 and irinotecan cooperate to inhibit the EWS/FLI1 transcription factor and the associated DNA damage response.
Authors: Segars Laura E. , Mendoza Arnulfo , Helman Lee J. , Grohar Patrick J. 
Published: June 2012

Background: ET-743 (Trabectedin, Yondelis™) is a natural product recently identified as a particularly active agent for Ewing's sarcoma cells in vitro. ET-743 interferes with the activity of the oncogenic transcription factor EWS/FLI1 in Ewing's sarcoma cells. In addition ET-743 induces DNA damage and poisons DNA damage repair pathways. In this report, we hypothesized that the EWS-FLI1 transcriptional program may mediate aspects of the DNA damage response. Therefore, interference with EWS/FLI1 activity by ET-743 may confer additional sensitivity to specific cytotoxic agents.

Methods: DNA repair pathways and EWS/FLI1 targets were evaluated by microarray expression, western blot analysis and siRNA interference. To optimize the activity of ET-743 in Ewing's sarcoma several combination therapies were investigated in vitro. The combination of ET-743 and Irinotecan was further evaluated in vivo with two ESFT xenograft models.

Results: ESFT cells displayed increased sensitivity to the combination of ET-743 and the topoisomerase I inhibitor, SN-38. This result was unique to topoisomerase I inhibitors and further restricted to ESFT cell lines. Preliminary xenograft results suggest the combination is also effective in vivo. This sensitivity can be accounted for by both the impaired DNA damage response and greater transcriptional inhibition of EWS/FLI1. Previous reports have established that cells deficient in WRN, a critical protein in the NHEJ DNA repair pathway, are hypersensitive to topoisomerase I inhibitors. Microarray expression and western blot analysis revealed that ET-743 suppresses WRN expression. Further siRNA data showed that the EWS/FLI1 transcription factor drives the expression of WRN. These results suggest that WRN suppression by ET-743 is occurring through an EWS/FLI1 dependent mechanism. Additionally Irinotecan augments the transcriptional inhibition of EWS/FLI1, as evident by suppression of downstream targets.

Conclusion: We have proposed a mechanism for the clinically relevant combination treatment of ET-743 and Irinotecan by interfering with the DNA damage response and augmenting the transcriptional interference of EWS/FLI1. By linking the EWS-FLI1 transcriptional program to the DNA damage response in ESFT cells, we expose an inherent weakness to topoisomerase I inhibitors thus creating a cell type specific sensitivity to the drug. In addition, we found Irinotecan cooperates with the ET-743 mediated suppression of EWS/FLI1 targets. These two effects increase the sensitivity of ESFT cells to this drug combination. Finally, the report provides a novel method of oncogenic transcription factor targeting that involves direct suppression of downstream targets and exploitation of the resulting changes in gene expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A103.