Abstract B150: Targeting EWS-FLI1 with mithramycin analogues for Ewing sarcoma treatment

Abstract

Abstract Ewing Sarcoma, a devastating malignancy affecting mainly pediatric and young adult populations, is characterized by the aberrant activity of the oncogenic EWS-FLI1 transcription. However, the development of treatments against EWS-FLI1 is lacking. Mithramycin analogues, exhibiting specificity towards EWS-FLI1, have been posited as a groundbreaking approach in drug discovery for the treatment of Ewing Sarcoma. A phase I/II trial and pharmacokinetic (PK) study in Ewing sarcoma patients demonstrated that mithramycin (MTM) had poor PK and dose-limiting hepatic and hematologic toxicities at subtherapeutic concentrations. Here we present a novel MTM analogue with significantly improved PK and a wider therapeutic window. MTMSA-Trp was prepared by chemical conversion of MTM to the MTMSA analogue and the subsequent conjugation of a tryptophan amino acid. The bulky amino acid substitution on the 3-side chain of the molecule appears to shift the ionization properties of the two hydroxy groups on the tricyclic core and to increase protein binding, both of which appear to lead to significantly improved pharmacokinetics. Further, based on crystallographic evaluation, the bulky hydrophobic substitution on the 3-side chain protrudes outside the DNA helix and interacts with bound FLI1. Luciferase reporter and FRET assays demonstrate 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. 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 (CD44, LOX). EWS-FLI1 modulation persists longer when cells are exposed to the 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 as compared to MTM. Initial efficacy studies in TC32 Ewing Sarcoma cells demonstrated tumor regression and improved survival at the maximum tolerated dose (MTD) and at 2/3 of the MTD on a daily x 5 intravenous bolus injection. Significantly, a more protracted dosing schedule which allowed for higher doses every third day for six doses led to an impressive reduction of tumors whose initial size was above 15 mm in one diameter (i.e., 1200-1600 mm3). Our studies with MTMSA-Trp demonstrate that improving the pharmacologic properties of mithramycin may lead to the development of an EWS-FLI1 inhibitor. Ongoing studies are evaluating the efficacy of MTMSA-Trp in additional Ewing models, and future work will focus on pre-IND studies. Citation Format: Markos Leggas, Kumar K Niloy, Rajesh Yetijaram, Jamie Horn, Yasuda Kazuto, Thomas Prisinzano, Jon S Thorson, Oleg Tsoikov, Jurgen Rohr. Targeting EWS-FLI1 with mithramycin analogues for Ewing sarcoma treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B150.