Figure 2.
The combination of mithramycin and PHA-767491 reverses the activity of EWS-FLI1. A and B, 100 nM mithramycin for 18 hours blocks the expression of the EWS-FLI1 induced targets EZH2 and NR0B1 while inducing the expression of the repressed target PHLDA. Lower concentration (20 nM for 18 hours) of mithramycin had a minimal impact on expression of EWS-FLI1 induced (NR0B1, EZH2) or repressed targets (PHLDA1) unless combined with 2 μM PHA-767491. Data represents fold change (2ΔΔCT) in expression relative to GAPDH as measured by RT-qPCR in TC32 (n=6), TC252 (n=6), and TC71 (n=3) treated with either media (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C) for 18 hours. Each biological replicate had 3 or 4 technical replicates. * = P < 0.05, ** = P < 0.01, *** = P <0.001, **** = P < 0.0001, error bars show standard deviation. C, EWS-FLI1 downstream target proteins are suppressed with high dose mithramycin or the combination of mithramycin and PHA-767491. Immunoblot showing expression of the EWS-FLI1 downstream targets (NR0B1, EZH2) relative to loading control (GAPDH) following 18-hour exposure to medium (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C). Immunoblots shown are representative of three independent experiments per cell line. D and E, Reversal of the EWS-FLI1 gene signature requires either high dose mithramycin or combination treatment as demonstrated by RNA sequencing following treatment with medium (M), solvent (S), or 100 nM mithramycin (100 MMA), 20 nM mithramycin (20), 2 μM PHA-767491 (PHA), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (Combo) for 12 hours in TC32 (n=3) and TC252 (n=3) cell lines.