# Mithramycin alters EWS::FLI1 DNA binding and RNA polymerase II processivity to inhibit nascent transcription

## TL;DR

Mithramycin disrupts a key cancer-causing protein in Ewing sarcoma, and a newer version of the drug shows promise for treatment.

## Contribution

Mithramycin's mechanism of inhibiting EWS::FLI1 and its optimized use with a less-toxic analog in Ewing sarcoma is newly revealed.

## Key findings

- Mithramycin alters EWS::FLI1 DNA binding and RNA polymerase II processivity.
- Continuous low-dose MMA reverses the oncogenic transcriptome in Ewing sarcoma.
- The analog AIT-102 improves in vivo activity with reduced toxicity.

## Abstract

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.

This study shows that mithramycin suppresses the EWS::FLI1 transcription factor in Ewing sarcoma using a hybrid CUT, Tag & GRO assay, revealing mechanism, need for continuous dosing, and supporting clinical use of next-gen analog AIT-102.