Abstract 4360: Development of a novel mithramycin analogue with improved pharmacokinetics and therapeutic window for targeted inhibition of EWS-FLI1 in Ewing sarcoma

Background: Ewing Sarcoma (EWS) is characterized by aberrant activity of the oncogenic EWS-FLI1 transcription. Mithramycin (MTM) has been identified to be a specific inhibitor of transcription mediated by the EWS-FLI1. A phase I/II trial and PK study in Ewing sarcoma patients demonstrated that MTM had poor PK and dose-limiting hepatic and hematologic toxicities at subtherapeutic concentrations. Here, we are presenting a novel MTM analogue with significantly improved PK and a wider therapeutic window for targeted inhibition of. Results and Discussion: MTMSA-Trp was prepared by chemical conversion of MTM to the MTMSA analogue and the subsequent conjugation of a tryptophan amino acid. This substitution appears to shift the ionization properties of the two hydroxy groups on the tricyclic core and to increase protein binding, which significantly improves PK. The cytotoxicity of MTMSA-Trp is in the order of low nM across several cell lines expressing EWS-ETS fusions, and the compound disrupts the expression of positively regulated proteins (NR0B1, ID2) and induces expression of negatively regulated ones (PHLDA1, LOX). MTMSA-Trp mediates apoptosis during which upstream caspases proteolytically processes for Caspase-7 activation. This effector caspase drives the cleavage of downstream substrates PARP and high secondary DNA damage (gH2A.X). Luciferase reporter assay demonstrates a dose-dependent effect of MTMSA-Trp on the attenuation of EWS-FLI1 transcription and DNA binding, and based on CETSA evidence, there is physical interaction between MTMSA-Trp and EWS-FLI1. MTMSA-Trp was found to modify the prototypical activity of EWS-FLI1 by disrupting R-loops. Evaluation of MTMSA-Trp effect on EWS-FLI1 mediated transcriptional activity by RNA Seq highlighted 287 genes among overlapping DEGs. ATAC seq analysis revealed that TEAD, ETS, CEBP, and NFKB TF families have divergent TF binding profiles between Ewing and non-Ewing cells treated with MTMSA-Trp. EWS-FLI1 modulation persists longer when cells are exposed to GI90 concentration for a short time compared to the GI50 for longer times, suggesting that daily dosing or continuous exposure is not required. MTMSA-Trp has improved PK in athymic nu/nu mice with a greater than 10-fold reduction in clearance and significantly reduced partition in the liver compared to MTM. Initial efficacy studies in TC32 EWS cells demonstrated tumor regression and improved survival at the maximum tolerated dose (MTD) and at of the MTD on a daily x 5 intravenous bolus injection. Significantly, a more protracted dosing schedule, which allowed higher doses every 3 days for 6 doses, led to an impressive reduction of tumors whose initial size was 1200-1600 mm3. Conclusions: MTMSA-Trp demonstrate that improving the pharmacologic properties of mithramycin may lead to the development of an EWS-FLI1 inhibitor. Citation Format: Rajesh Yetirajam, Srijan Acharya, Kumar Kuldeep Niloy, Yasuda Kazuto, Jamie Horn, Thomas Prisinzano, Jon S. Thorson, Oleg Tsodikov, Jurgen Rohr, Markos Leggas. Development of a novel mithramycin analogue with improved pharmacokinetics and therapeutic window for targeted inhibition of EWS-FLI1 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4360.