Abstract 1612: Identification of mithramycin analogs with improved targeting of the EWS/FLI1 transcription factor
Authors: Christy Osgood, Nichole Maloney, Christopher G. Kidd, Meti Gebregiorgis, Luz E. Nunez, Javier Gonzalez-Sabin, Lee J. Helman, Francisco Moris, Patrick J. Grohar
Journal: Cancer Research (2015)

Abstract Background: Ewing sarcoma is a bone a soft tissue sarcoma with a poor overall survival. This tumor absolutely depends on the continued expression of the EWS-FLI1 transcription factor for cell survival. We are therefore focused on developing small molecules that inhibit EWS-FLI1. We have previously completed a high throughput screen that identified mithramycin as an inhibitor of EWS-FLI1 and translated this compound to the clinic in a phase I-II trial. The success of this compound in the clinic has been challenged by drug associated liver toxicity that has necessitated dose reductions. Therefore the goal of this study is to identify less toxic and-or more potent mithramycin analogs. Methods: The less toxic analog, EC8042, was identified by evaluating animal toxicity data and serum pharmacokinetics in mice and rats. In order to identify a more potent compound, a panel of more than 20 mithramycin analogs was screened using an EWS-FLI1 reporter NR0B1 luciferase construct to identify EC8105. EWS-FLI1 suppression was confirmed using quantitative PCR and western blot analysis in vitro. The ability of the drug to block EWS-FLI1 binding to chromatin was evaluated by performing chromatin immunoprecipitation in the presence and absence of drug. The relative hepatotoxicity of the analogs was modeled in vitro by comparing doses that achieve suppression of EWS-FLI1 to toxic doses of the drug in HepG2 cells and confirmed in vivo in xenograft experiments. Finally, we tested the ability of both analogs to suppress tumor growth in xenograft models of Ewing sarcoma and confirmed suppression of EWS-FLI1 using immunofluorescence of formalin fixed tissue from these experiments. Results: EC8042 shows equivalent suppression of EWS-FLI1 activity but is substantially less toxic than the parent compound, allowing higher serum levels of drug in vivo in animal models. In contrast, EC8105 is approximately 8 times more potent than the parent compound and demonstrates improved suppression of the EWS-FLI1 gene signature. Both compounds work to block EWS-FLI1 binding to chromatin. More importantly, in contrast to mithramycin, both analogs suppress EWS-FLI1 activity at concentrations that are non-toxic to HepG2 cells. These effects translate into improved suppression of Ewing sarcoma xenograft growth with a corresponding increase in mouse survival and regression of several tumors in both cohorts. Conclusions: We have identified the mithramycin analogs EC8042 and EC8105 as EWS-FLI1 inhibitors. These compounds are less toxic and more potent than the parent compound and suppress EWS-FLI1 at concentrations that do not appear to cause liver toxicity. Together these results suggest that the clinical development of these analogs is warranted. Citation Format: Christy Osgood, Nichole Maloney, Christopher G. Kidd, Meti Gebregiorgis, Luz E. Nunez, Javier Gonzalez-Sabin, Lee J. Helman, Francisco Moris, Patrick J. Grohar. Identification of mithramycin analogs with improved targeting of the EWS/FLI1 transcription factor. [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 1612. doi:10.1158/1538-7445.AM2015-1612