Abstract 6733: Targeting Sp1 in Ewing sarcoma: a multi-approach method for the utilization of mithramycin

Abstract

Abstract Purpose: Ewing Sarcoma (ES) is a bone and soft tissue cancer affecting young adults and children. Localized ES presents with a 5-year survival rate of 70%, while metastatic cases range from 15-30%. Our laboratory investigates combination treatments using less toxic agents to induce sensitization to chemotherapy in ES. The anti-cancer activity of an antineoplastic antibiotic, Mithramycin (Mit), against ES cells has been shown. Mit inhibits Specificity protein 1 (Sp1) a marker associated with aggressive cancer cell growth and resistance to chemo/radiation therapies. However, its mechanistic effects on other oncogenic proteins have yet to be elucidated in ES. The purpose of this study is to evaluate the effectiveness of Mit and combination with other chemotherapeutic agents, Etoposide and Vincristine, to inhibit ES cell growth and assess the effect on cancer related proteins regulated by Sp1. Future studies will expand upon Mithramycin's mechanism of action in Ewing Sarcoma utilizing RNA sequencing and computational methods. Methods: Anti-proliferative activity of Mit and/or Vincristine and Etoposide against ES cell lines, TC205 and CHLA10, was evaluated using CellTiterGlo kit. Dose curves and IC50 values were determined by Sigma-Plot software. The expression of Sp1 and survivin was determined by Western blot analysis. Cell lines were obtained from Children’s Oncology Group. The specific type of drug effect of the combination treatments were determined by analyzing the combination index obtained via Calcusyn software. Nude mice were injected with TC-205 cells and treated over two weeks with either Mit (1mg/kg per week) and/or Etoposide (5mg/kg per week) and tumor volume was compared. Protein models were obtained from RCSB PDB and homology tests were performed in the Swiss-model workspace. Results: Mit significantly decreased ES cell line viability and tumor volume in nude mice. Mit showed the ability to reduce the expression of Sp1 and offered differing effects on survivin expression, indicative of anti-apoptotic mechanisms being implemented in the ES cell lines. IC50 values of both chemotherapeutics and Mit were decreased by nearly 50% when used in combination and this effect was mirrored in further decreases in Sp1 expression. Synergistic drug responses were shown in the combination of Mit with both Vincristine and Etoposide. Sp1 and survivin protein models were established and homology verification using Ramachandran plots and QMEAN Z-scores indicated quality protein models for further computational studies. Conclusions: Mit may effectively sensitize ES cells and improve the response of chemotherapy while lowering necessary effective dosages. Studies to understand the mechanism of action of Mit on Sp1, survivin, and other proteins involved in Ewing Sarcomagenesis are underway. This study is partially supported by a grant from the Cancer Prevention and Research Institute of Texas (Award#: RP210046) Citation Format: Christoffer Lambring, Riyaz Basha, Umesh Sankpal. Targeting Sp1 in Ewing sarcoma: a multi-approach method for the utilization of mithramycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6733.