Abstract 1632: Suppression of EWS-FLI1 transcription using a combination therapy of mithramycin and cyclin-dependent kinase 9 inhibition
Authors: Guillermo Flores, Joel Everett, Brandon Oswald, Natasha Caplen, Lee Helman, Zachary Madaj, Patrick Grohar
Journal: Cancer Research (2018)

Abstract Background: Ewing sarcoma (ES) is a pediatric soft tissue tumor with a poor prognosis. ES is dependent on the presence and activity of the oncogenic transcription factor, EWS-FLI1, that dysregulates gene expression at hundreds of targets. We have previously identified mithramycin (MMA) as a potent inhibitor of EWS-FLI1 driven transcription. However, only sub-therapeutic concentrations have been achieved in pediatric patients. To maximize inhibition, we conducted an siRNA screen to identify gene targets that potentiated the effects of MMA in ES. Knock-down of several transcriptionally related genes significantly sensitized ES cells to the effects of MMA. We next developed a drug matrix screening platform to identify commercially available transcriptional inhibitors that also potentiated the effects of MMA on EWS-FLI1 driven transcription and ES cell viability. PHA-767491, a cyclin-dependent kinase 9 inhibitor (CDK9i), reverses the effect on EWS-FLI1 on several targets at both the mRNA and protein level across multiple ES cell lines when combined with MMA. Methods: We utilize matrix drug screening to identify that PHA-767491 synergizes with MMA in terms of a reduction in cell viability as measured by MTS and cell growth as measured by time-lapse microscopy. We use RT-qPCR to measure changes in gene expression across multiple well characterized targets of EWS-FLI1 driven transcription. We use western blot analysis to measure changes at the protein level for these targets as well. We then use an orthotopic xenograft mouse model to measure changes in tumor size after administration of our MMA-CDK9i combination therapy. Results: Our combination of MMA and PHA-767491 displays strong synergy as measured by Bliss-Independence. Multiple ES cell lines become unviable, with minimal effect on non-ES cells, and this effect is stable after removal of the compounds. This synergy is recapitulated as a reversal of EWS-FLI1 driven transcription across multiple targets at both the mRNA and protein levels. We are currently evaluating the combination therapy in our animal model comparing MMA-CDK9i to control or either agent alone. Importantly our MMA-CDK9i combination uses drug concentrations that are clinically achievable. Conclusions: We describe an MMA-CDK9i combination that displays excellent activity against EWS-FLI1 driven transcription. We confirmed this using multiple independent assays in both in vitro and in vivo models. We complete this work with the hope that it can eventually be translated to patients. Citation Format: Guillermo Flores, Joel Everett, Brandon Oswald, Natasha Caplen, Lee Helman, Zachary Madaj, Patrick Grohar. Suppression of EWS-FLI1 transcription using a combination therapy of mithramycin and cyclin-dependent kinase 9 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1632.