Mithramycin analogues trap the EWS-FLI1 transcriptional complex, evict ETV6, and disable oncogenic condensate function in Ewing sarcoma

Background : Ewing sarcoma is an aggressive pediatric cancer driven by the EWS-FLI1 fusion oncoprotein, which assembles into nuclear transcriptional condensates essential for oncogenic gene regulation. MTMSA-Trp is a synthetic mithramycin (MTM) analogue with improved pharmacokinetics and in vivo efficacy in Ewing sarcoma. MTMSA-Trp binds to the minor groove of DNA and interacts with the major groove-bound EWS-FLI1, but the mechanistic details of these interactions are not well understood. Methods and Results : Using luciferase reporter assays, we show that MTMSA-Trp selectively inhibits EWS-FLI1-dependent transcription at nanomolar potency, with weaker effects on Sp1-driven transcription. MTMSA-Trp activity is attenuated in EWS-FLI1 knockdown cells, indicating functional dependence. Western blotting confirmed MTMSA-Trp-mediated suppression of EWS-FLI1-regulated targets in multiple Ewing sarcoma cell lines, while non-Ewing lines exhibited minimal response. qRT-PCR analyses revealed that MTMSA-Trp downregulates EWS-FLI1 mRNA, while paradoxically stabilizing its protein and shifting downstream transcriptional programs consistent with EWS-FLI1 antagonism. Biophysical assays demonstrated increased thermal and proteolytic stability of EWS-FLI1 upon MTMSA-Trp treatment, suggesting drug-induced stabilization of the EWS-FLI1 complex. Protein stability assays further showed that MTMSA-Trp prolongs the half-life of EWS-FLI1 in an EWS-FLI1-dependent manner, whereas ETV6, though functionally linked, was not stabilized but essentially evicted from the nucleus. Subcellular fractionation revealed that MTMSA-Trp increases nuclear retention of EWS-FLI1 while dynamically redistributing ETV6 in an EWS-FLI1-dependent manner. These effects extended to chromatin-associated partners of EWS-FLI1 condensates, including BAF155, BAF60a, and ARID1a. Immunofluorescence confirmed that MTMSA-Trp preserved ARID1a-containing nuclear condensates, protecting them from degradation by cycloheximide, and did so only in the presence of EWS-FLI1. At the transcriptional level, MTMSA-Trp downregulated CDK7 and hyperphosphorylated RNA Pol II CTD accompanied by accelerated RPB1 degradation. These effects were abolished when EWS-FLI1 was silenced. Conclusions : Our results show that MTMSA-Trp binds to and stabilizes the EWS-FLI1 transcriptional complex, maintains its associated condensates, and alters gene expression by disrupting RNA Pol II activity. These findings reveal a previously overlooked mechanism of action for MTM analogues in their interaction with the transcriptional complex, ETV6, and phase-separated oncogenic complexes. They further support the potential of these compounds as a therapeutic approach for Ewing sarcoma.