Supplementary Figures from Identification of Mithramycin Analogues with Improved Targeting of the EWS-FLI1 Transcription Factor

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.