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Dual targeting of EWS-FLI1 activity and the associated DNA damage response with trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth.

ABSTRACT: PURPOSE: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.

SUBMITTER: Grohar PJ

PROVIDER: S-EPMC5510643 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Dual targeting of EWS-FLI1 activity and the associated DNA damage response with trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth.

Grohar Patrick J PJ Segars Laure E LE Yeung Choh C Pommier Yves Y D'Incalci Maurizio M Mendoza Arnulfo A Helman Lee J LJ

Clinical cancer research : an official journal of the American Association for Cancer Research 20131125 5

<h4>Purpose</h4>The goal of this study is to optimize the activity of trabectedin for Ewing sarcoma by developing a molecularly targeted combination therapy.<h4>Experimental design</h4>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.<h4>Results</h ...[more]

PMID: 24277455

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Project description:PurposePropagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity.Experimental designExisting tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays.ResultsWe found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis.ConclusionsEwing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13. See related commentary by Weiss and Bailey, p. 4360.

Dataset Information

Dual targeting of EWS-FLI1 activity and the associated DNA damage response with trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth.

Publications

Dual targeting of EWS-FLI1 activity and the associated DNA damage response with trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth.

Similar Datasets

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