Purpose The goal of this study was to identify second-generation mithramycin analogs 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 to the maximum tolerated dose 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 analog 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.

There is a great need to develop novel approaches to target oncogenic transcription factors with small molecules. Ewing sarcoma is emblematic of this need, as it depends on the continued activity of the EWS-FLI1 transcription factor to maintain the malignant phenotype. We have previously shown that the small molecule trabectedin interferes with EWS-FLI1. Here we report important mechanistic advances and a second-generation inhibitor to provide insight into the therapeutic targeting of EWS-FLI1. We discovered that trabectedin functionally inactivated EWS-FLI1 by redistributing the protein within the nucleus to the nucleolus. This effect was rooted in the wild-type functions of the EWSR1, compromising the N-terminal half of the chimeric oncoprotein, which is known to be similarly redistributed within the nucleus in the presence of UV light damage. A second-generation trabectedin analog lurbinectedin (PM01183) caused the same nuclear redistribution of EWS-FLI1, leading to a loss of activity at the promoter, mRNA, and protein levels of expression. Tumor xenograft studies confirmed this effect and it was increased in combination with irinotecan, leading to tumor regression and replacement of Ewing sarcoma cells with benign fat cells. The net result of combined lurbinectedin and irinotecan treatment was a complete reversal of EWS-FLI1 activity and elimination of established tumors in 30-70% of mice after only 11 days of therapy. Our results illustrate the preclinical safety and efficacy of a disease-specific therapy targeting the central oncogenic driver in Ewing sarcoma.

Summary Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss-of-function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1 driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.

A seminatural, factorial‐design experiment was used to quantify dynamics of the pathogen Mycoplasma agassizii and upper respiratory tract disease in the Mojave desert tortoise (Gopherus agassizii) over 2 years. Groups of initially healthy animals were separated into serologically positive (seropositive), seronegative, and artificially infected groups and paired into 23 pens. We found no evidence of long‐term immune protection to M. agassizii or of immunological memory. Initially seronegative, healthy tortoises experienced an equal amount of disease when paired with other seronegative groups as when paired with seropositive and artificially infected groups—suggesting that recrudescence is as significant as transmission in introducing disease in individuals in this host–pathogen system. Artificially infected groups of tortoises showed reduced levels of morbidity when paired with initially seronegative animals—suggesting either a dilution effect or a strong effect of pathogen load in this system. Physiological dynamics within the host appear to be instrumental in producing morbidity, recrudescence, and infectiousness, and thus of population‐level dynamics. We suggest new avenues for studying diseases in long‐lived ectothermic vertebrates and a shift in modeling such diseases.

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