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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.

Table S1, PCR primers used in gene signature panel. Table S2, Evidence for selection of each gene as an EWS-FLI1 target. S3A Mean fold change in expression of NR0B1 as a function of GAPDH (2Î"Î"CT) following treatment with MMA or EC-8105. Data is the average of 3 independent experiments as measured by qPCR. S3B Mean fold change in expression of EWS-FLI1-induced targets as a function of GAPDH (2Î"Î"CT) for treatment with an siRNA targeted at the breakpoint of EWS/FLI1 for 24 h. Data is the average of 3 independent experiments as measured by qPCR S3C Mean fold change in expression of EWS-FLI1-suppressed targets as a function of GAPDH (2Î"Î"CT) for treatment with an siRNA targeted at the breakpoint of EWS-FLI1for 24 h. Data is the average of 3 independent experiments as measured by qPCR S4A: Structures and NSC numbers of mithramycin analogs S4A: Structures and NSC numbers of mithramycin analogs (cont'd). S4C. Allometric scaling estimates for dosing as a function of weight of an organism for mithramycin (MMA) and EC-8042. (see text for reference). S5A Pearson correlation analysis showing similar responses of the PPTP panel of cell lines for EC-8105 and EC-8042 relative to mithramycin. S5B IC50 values for cell lines in PPTP. Columns are all cell lines tested, exclusion of rhabdoid tumor (minus rhabdoid) and exclusion of rhabdoid tumor and acute lymphoblastic leukemia cell lines (minus rhabdoid and ALL). The statistics are shown in boxes at the bottom. S5C Graph of IC50 as a function of p53 status. Cell lines with wild-type p53 (P53 wt) were more sensitive to treatment with mithramycin (MMA) and EC-8105 than lines with mutant p53 (P53 Mut) across the entire PPTP panel of cell lines independent of histology. Note there was no difference with the non-DNA damaging analogy, EC-8042. S6 Prediction plot showing mean tumor volume as a function of days of treatment with EC-8105 (1.0 mg/kg IP, M/W/F schedule, 8 doses). Thick, dotted lines represent mean tumor growth and the individual lines in the back represent the individual tumor growth by cohort. S7 Weight of each mouse in cohort of EC-8105 treated mice (black lines) treated on a 1.5 mg/kg/dose IV Q3D X 8 doses schedule relative to weight change of control mice (grey line). S8 Survival curves for control mice (black) relative to EC-8042-treated mice (grey) treated at 24 mg/kg dose X 8 either intravenously (S8A) or intraperitoneal (S8B). Arrows indicate end of treatment. S9. Response of mice treated with 24 mg/kg/dose EC-8042 IP M, W, F X8 (S9A). Regression of tumors for every mouse mouse in the cohort. Each line represents an individual mouse. Asterisks (*) highlight treatment days. S9B Mean tumor volume (+/- SEM) of every mouse in the control cohort (3177 mm3 +/- 308.2) relative to the EC-8042-treated cohort (422.5 mm3 +/- 48) on day 11 of treatment (P < 0.0001). S10: Suppression of NR0B1 staining in cells treated with 1.5 mg/kg EC-8105 IV, 48 hours after treatment. Three representative sections are shown stained for NR0B1 (red) or DAPI (blue) since the tumors were large and showed a range of staining.

Background: Ewing sarcoma (ES) is a primary bone tumor of adolescence with poor prognosis. ES is characterized by the presence of EWS-FLI1 which acts as an oncogenic transcription factor to promote and maintain a tumorigenic phenotype. All ES cells express EWS-FLI1, however, other actionable mutations are exceedingly rare. We previously identified mithramycin (MMA) as a potent and specific EWS-FLI1 inhibitor. MMA effectively decreases ES tumor cell viability and tumor volume in vivo by reversing the EWS-FLI1 transcriptional signature. Structural and biochemical data suggest that sub-micromolar MMA increases the stability of the DNA binding domain of EWS-FLI1 at GGAA repeats. These data suggest that MMA may prevent EWS-FLI1 from successfully promoting Pol II transcriptional elongation. Methods: We used ChIP-qPCR to detect EWS-FLI1 binding at the promoter of well characterized targets. We used a Pol II processivity assay to measure the efficiency of transcriptional elongation at EWS-FLI1 targets. We use RT-qPCR, western blotting, and RNAseq to confirm that MMA sensitizes ES cells to elongation inhibitors. Finally, we use GROseq to identify the changes to Pol II dynamics at EWS-FLI1 targets following MMA treatment. Results: Our ChIP-qPCR data demonstrate an increased fraction of EWS-FLI1 DNA binding at the promoters of target genes with GGAA microsatellite regions. MMA significantly inhibits the processivity of Pol II at EWS-FLI1 targets when added both into a run-on reaction or following ES cell pretreatment. Combining MMA with elongation inhibitors induces strong synergy as measured by Bliss-Independence, renders multiple ES cell lines unviable by inhibiting the effects of EWS-FLI1 at the mRNA and protein level, and is effective in vivo. MMA biases GROseq reads toward the 5’ end of genes, a result consistent with a promoter trapping mechanism. We are currently correlating GROseq data to global EWS-FLI1 DNA binding using ChIPseq. Conclusions: We characterized a mechanism of MMA-induced EWS-FLI1 inhibition that involves increased stabilization of the fusion protein at target promoter regions. This mechanism is supported by changes to EWS-FLI1 DNA-binding, EWS-FLI1 target transcriptional processivity, and a reversal of the EWS-FLI1 transcriptional signature by combining MMA with downstream elongation inhibition. We hope that this work will inform future endeavors to develop a targeted therapy for ES that inhibits the main driver of disease, EWS-FLI1. Citation Format: Guillermo Flores, Susan Kitchen-Goosen, Brandon Oswald, Elissa Boguslawski, Marie Adams, Ian Beddows, Zachary Madaj, Patrick Grohar. Mithramycin impedes EWS-FLI1 driven transcription by preventing target promoter clearance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2882.