Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule Dependent Manner

Purpose: The successful clinical translation of compounds that target specific oncogenic transcription factors will require an understanding of the mechanism of target suppression to optimize the dose and schedule of administration. We have previously shown trabectedin reverses the gene signature of the EWS-FLI1 transcription factor. In this report, we establish the mechanism of suppression and use it to justify the re-evaluation of this drug in the clinic in Ewing sarcoma patients. Experimental Design: We demonstrate a novel epigenetic mechanism of trabectedin using biochemical fractionation and chromatin immunoprecipitation sequencing (CHIP-Seq). We link the effect to drug schedule and EWS-FLI1 downstream target expression using confocal microscopy, qPCR, western blot analysis and cell viability assays. Finally, we quantitate target suppression within the 3-dimensional architecture of the tumor in vivo using 18F-FLT imaging. Results: Trabectedin evicts the SWI/SNF chromatin remodeling complex from chromatin and redistributes EWS-FLI1 in the nucleus leading to a marked increase in H3K27me3 and H3K9me3 at EWS-FLI1 target genes. These effects only occur at high concentrations of trabectedin leading to suppression of EWS-FLI1 target genes and a loss of cell viability. In vivo, low dose irinotecan is required to improve the magnitude, penetrance and duration of target suppression in the 3-dimensional architecture of the tumor leading to differentiation of the Ewing sarcoma xenograft into benign mesenchymal tissue. Conclusions: These data provide the justification to evaluate trabectedin in the clinic on a short infusion schedule in combination with low dose irinotecan with 18F-FLT PET imaging in Ewing sarcoma patients.

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