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Identification of Mithramycin Analogues with Improved Targeting of the EWS-FLI1 Transcription Factor.

ABSTRACT: PURPOSE:The goal of this study was to identify second-generation mithramycin analogues that better target the EWS-FLI1 transcription factor for Ewing sarcoma. We previously established mithramycin as an EWS-FLI1 inhibitor, but the compound's toxicity prevented its use at effective concentrations in patients. EXPERIMENTAL DESIGN:We screened a panel of mithralogs to establish their ability to inhibit EWS-FLI1 in Ewing sarcoma. We compared the IC50 with the MTD established in mice to determine the relationship between efficacy and toxicity. We confirmed the suppression of EWS-FLI1 at the promoter, mRNA, gene signature, and protein levels. We established an improved therapeutic window by using time-lapse microscopy to model the effects on cellular proliferation in Ewing sarcoma cells relative to HepG2 control cells. Finally, we established an improved therapeutic window using a xenograft model of Ewing sarcoma. RESULTS:EC-8105 was found to be the most potent analogue and was able to suppress EWS-FLI1 activity at concentrations nontoxic to other cell types. EC-8042 was substantially less toxic than mithramycin in multiple species but maintained suppression of EWS-FLI1 at similar concentrations. Both compounds markedly suppressed Ewing sarcoma xenograft growth and inhibited EWS-FLI1 in vivo CONCLUSIONS:These results provide a basis for the continued development of EC-8042 and EC-8105 as EWS-FLI1 inhibitors for the clinic. Clin Cancer Res; 22(16); 4105-18. ©2016 AACR.

SUBMITTER: Osgood CL

PROVIDER: S-EPMC4987166 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Identification of Mithramycin Analogues with Improved Targeting of the EWS-FLI1 Transcription Factor.

Osgood Christy L CL Maloney Nichole N Kidd Christopher G CG Kitchen-Goosen Susan S Segars Laura L Gebregiorgis Meti M Woldemichael Girma M GM He Min M Sankar Savita S Lessnick Stephen L SL Kang Min M Smith Malcolm M Turner Lisa L Madaj Zachary B ZB Winn Mary E ME Núñez Luz-Elena LE González-Sabín Javier J Helman Lee J LJ Morís Francisco F Grohar Patrick J PJ

Clinical cancer research : an official journal of the American Association for Cancer Research 20160315 16

<h4>Purpose</h4>The goal of this study was to identify second-generation mithramycin analogues that better target the EWS-FLI1 transcription factor for Ewing sarcoma. We previously established mithramycin as an EWS-FLI1 inhibitor, but the compound's toxicity prevented its use at effective concentrations in patients.<h4>Experimental design</h4>We screened a panel of mithralogs to establish their ability to inhibit EWS-FLI1 in Ewing sarcoma. We compared the IC50 with the MTD established in mice to ...[more]

PMID: 26979396

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Project description:BackgroundChromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable."MethodsWe developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided.ResultsMithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm(3), difference = 2319 mm(3), 95% CI = 1766 to 2872 mm(3), P < .001).ConclusionMithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.

Dataset Information

Identification of Mithramycin Analogues with Improved Targeting of the EWS-FLI1 Transcription Factor.

Publications

Identification of Mithramycin Analogues with Improved Targeting of the EWS-FLI1 Transcription Factor.

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