Abstract 2628: Mithramycin analogs with reduced toxicity for EWS-FLI1 targeting

Abstract

Abstract Introduction: EWS-FLI1 and related chromosomal translocations are prevalent in Ewing sarcoma and play a major role in modulating oncogenic transcription. Development of drugs that affect EWS-FLI1 oncoprotein function may lead to successful treatment for these patients. Mithramycin (MTM) was shown to inhibit transcriptional targets of EWS-FLI1, but it has a narrow therapeutic window attributed to its nonspecific toxicities. To overcome this, semisynthetic methods were developed to generate MTM analogs with unique pharmacologic properties. Mechanistic and pharmacologic studies are presented here. Methods: Studies were conducted using MTM and lead analogs (mithramycin-SK (MTM-SK), mithramycin-SA-tryptophan (MTM-SA-Trp), and mithramycin-SA-phenylalanine (MTM-SA-Phe)). EWS-FLI1 promoter occupancy was investigated using chromatin immunoprecipitation real-time PCR (ChIP-RTPCR). The effect of drug treatment on expression of genes controlled by EWS-FLI1 was evaluated by quantitative real-time PCR (qRT-PCR). The effect of treatment on cell cycle distribution was also compared among analogs. In vitro efficacy was evaluated by estimating GI50 parameters (72-hr). In addition, the maximum tolerated dose (MTD) and the effect of treatment on plasma total-calcium were used to assess relative toxicity in mice. Results: EWS-FLI1 promoter occupancy upstream from Nr0b1, Tgfbr2, and Rcor1 genes was evaluated in Ewing sarcoma cells (TC32) by ChIP-RTPCR. MTM and MTM-SA-Trp analog destabilized FLI1 binding to all three promoters and MTM-SA-Trp was shown to be the most destabilizing. Comparatively, MTM-SK appears to mostly stabilize FLI1. Additionally, qRTPCR showed that MTM and its analogs efficiently down-regulated mRNA expression in a dose-dependent manner (rank-order of efficiency: MTM-SA-Trp>MTM = MTM-SK). These data were in accord with the in vitro cytotoxicity data that show MTM-SA-Trp has relatively higher potency (lower GI50) among Ewing cell lines (n = 8) as compared to other analogs. Furthermore, the effect of drug treatment appears to lead to differences in cell-cycle progression. MTM and MTM-SK treated TC32 cells were primarily in G1/G2 phase, whereas MTM-SA-Trp treated cells showed increased S-phase accumulation. Compared to MTM, mice tolerated significantly higher single doses of the MTM analogs. Repeated dosing showed similar results except that MTM-SA-Trp was tolerated at lower doses. MTM treatment caused an acute drop in total-calcium, which also occurred with MTM-SA-Trp and MTM-SA-Phe analogs three days later within the two-week treatment. Comparatively, MTM-SK caused an increase in total-calcium. In all cases, total-calcium concentrations returned to baseline within two weeks following treatment. Conclusion: The work presented here demonstrates the ability to design more specific and less toxic analogs of MTM. Development of such analogs could lead to successful treatments of Ewing sarcoma. Citation Format: Joseph Eckenrode, Jamie Horn, Jhong-Min Chen, Jurgen Rohr, Markos Leggas. Mithramycin analogs with reduced toxicity for EWS-FLI1 targeting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2628. doi:10.1158/1538-7445.AM2015-2628