Abstract 1612: Identification of mithramycin analogs with improved targeting of the EWS/FLI1 transcription factor

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

18F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma

“…5B ). Interestingly, EC-8042 showed the greatest degree of suppression of 18 F-FLT activity and the most marked changes in staining, which correlates with the most profound response to treatment that we recently reported 26 . In contrast, the staining was more variable with EC-8105 and mithramycin, consistent with the relative activity of these drugs by the intraperitoneal route ( Supplementary Fig.…”

“…In addition, we showed that the second-generation mithramycin analogs EC-8042 and EC-8105 also suppressed expression of those three molecules in a highly significant manner ( Fig. 2B ; statistics in Supplementary Table 3a ) 26 . In order to exclude nonspecific cytotoxicity as a cause of the suppression, we also showed that the chemotherapeutic agent 5FU did not suppress expression of TK1, despite a known sensitivity of Ewing sarcoma cells to the cytotoxic effects of the drug 27 .…”

BET bromodomain inhibitors suppress EWS-FLI1-dependent transcription and the IGF1 autocrine mechanism in Ewing sarcoma

“…Certain chemotherapy drugs, such as cytarabine, mithramycin and trabectedin, have been shown to impair the transcriptional activity of EWS-FLI1 [28, 29, 48, 49]. However, the therapeutic indexes of these chemo drugs are limited by their toxicity, which may be overcome by further modifications of their chemical structures [50]. In addition, a first-in-class compound selectively targeting the interaction between EWS-FLI1 and RNA helicase A (YK-4-279) has been developed to modulate EWS-FLI1 activities [51].…”

Inhibition of SP1 by the mithramycin analog EC-8042 efficiently targets tumor initiating cells in sarcoma

“…EC-8042 altered the expression of cell cycle related genes resulting in cell cycle arrest and apoptosis in breast cancer cell lines [ 21 ]. In Ewing sarcoma, EC-8042 was substantially less toxic than mithramycin but maintained suppression of EWS-FLI1 at similar concentrations as MTM, and markedly suppressed Ewing sarcoma xenograft growth [ 22 ]. More importantly, EC-8042 shows a potent in vitro and in vivo antitumor activity against several tumors types similar to other analogues but is 10-fold less toxic in vivo than MTM [ 17 ], enabling it as the lead candidate in the quest for mithralogs with improved therapeutic window potentially useful in the clinical setting.…”