Abstract 2605: Mithramycin enhances the efficacy of etoposide in Ewing sarcoma cells and a mouse xenograft tumor model

Abstract

Abstract Ewing sarcoma (ES) tumors arise in bone or soft tissue of children, adolescents, and young adults. The fusion protein and transcription factor, EWS-FLI1 is over-expressed in >85% of ES tumors. High throughput screening with approximately 50,000 compounds identified Mithramycin (MIT) as an inhibitor of EWS-FLI1. A previous clinical trial to test the efficacy of MIT was not successful, presumably due to toxicity from the high dose required to inhibit EWS-FLI1. We hypothesize that the efficacy and safety of treatment can be enhanced if MIT is used along with standard chemotherapeutic agents. Accordingly, preclinical experiments were conducted to test combination treatment with chemotherapeutic agents used for the treatment (standard care) of ES. After screening several chemotherapeutic agents used in the standard treatment of ES, Etoposide (VP-16) was tested for further experiments. ES cells, CHLA10 and TC205 were treated in the presence of vehicle (Dimethyl sulfoxide), or optimized doses of MIT, VP-16 or MIT+VP-16. The effect of these treatments on cell growth inhibition was studied at 48-hour post-treatment using a luminescent (CellTiter-Glo®) cell viability assay kit. The effect of these treatments on programmed cell death was measured by evaluating the apoptotic cell population using Flow cytometry with Annexin V Apoptosis Detection Kit and the expression of a protein marker, Cleaved-Poly (ADP-ribose) polymerase (c-PARP), by Western blot analysis. Nude mice were injected subcutaneously with TC205 cells, treated with MIT, ETO or MIT+VP-16 and the tumor growth inhibition was assessed. As expected, all treatments showed a dose and time-dependent effect on ES cell growth inhibition; however, the combination treatment of MIT+VP-16 was significantly (p<0.01) more effective than individual agents. The cell growth inhibition in combination treatment was accompanied by an increase in apoptotic cells and c-PARP expression in both ES cell lines. The combination treatment significantly (p<0.01) decreased the tumor weight and volume when compared to control or the groups treated by either MIT or VP-16. The combination of MIT and VP-16 is effective when compared to individual treatments in the TC205 and CHLA10 cell lines and inhibited tumor growth in a mouse model. Among the two cell lines tested, CHLA10 cell line expresses EWS-FLI1 while mTC205 cell line does not express EWS-FLI1. These results demonstrate that the combination of MIT and VP-16 is inhibiting both cell lines, suggesting a novel mechanism of targeting the ES cells even in the absence of EWS-FLI1 expression. These results demonstrate that MIT in combination with the chemotherapeutic agent VP-16 increases therapeutic efficacy in a preclinical model for ES and demonstrates strong potential for translational application. Citation Format: Anish Ray, Umesh T. Sankpal, Lina Albeer, Abigail Hunter, Holly Lout, Kathryn Dunlap, W. Paul Bowman, Don Eslin, Riyaz M. Basha. Mithramycin enhances the efficacy of etoposide in Ewing sarcoma cells and a mouse xenograft tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2605.