Mithramycin Alters EWS::FLI1 DNA Binding and RNA Polymerase II Processivity to Inhibit Nascent Transcription [CUT&Tag]
Although many DNA binding natural products exert their effects through non-specific mechanisms, a therapeutic opportunity exists for a subset of these compounds that alter the expression or activity of specific driver oncogenes in specific cell contexts. In this study, we integrate CUT&Tag with Global Run-On Sequencing (CUT, Tag, and GRO) to show that the minor groove binding compound, mithramycin (MMA), inhibits the Ewing sarcoma oncogenic driver, the EWS::FLI1 transcription factor. MMA causes either an increase or decrease in EWS::FLI1 binding to chromatin at downstream target response elements to poison nascent transcription. The reversal of EWS::FLI1 activity is limited by non-specific effects of the drug on RNAPII processivity but can be optimized by continuous administration at low concentration to cause more precise reversal of the oncogenic transcriptome and striking Ewing sarcoma xenograft regressions. The activity in vivo is further improved with a less-toxic second-generation analog, AIT-102. Overall design: CUT&Tag data using FLI1 antibody, H3K27ac antibody or IgG control following incubation with PBS solvent control or mithramycin at either 20 nmol/L for 72 hours or 100 nmol/L for 18 hours.