Abstract 5817: Oxaliplatin and ATR inhibitors show strong synergy in Ewing sarcoma cells
Authors: Sorensen Kathleen , Jess Jennifer , Goosen Susan , Flores Guillermo , Madaj Zach , Bogulaslawski Elissa , Grohar Patrick J. 
Published: August 2018

Abstract
Background: Ewing sarcoma (ES) is the second most common childhood bone tumor that is in need of new molecularly targeted agents. This tumor is driven by the aberrant activity of the EWS-FLI1 transcription factor that both drives proliferation and blocks differentiation. We, and others, have focused on developing methods to directly target EWS-FLI1. A complementary approach, employed in this study, is to target inherent sensitivities to specific agents created by the many gene expression changes induced by the fusion protein.
Experimental Design: It is known that EWS-FLI1 alters the expression of multiple gene involved in the DNA damage response. Therefore, an initial unbiased drug matrix screen was conducted to determine synergistic combinations between DNA damage response inhibitors and twelve different chemotherapeutic agents. The most synergistic combinations were analyzed by GI50 shifts to determine the sensitivity of ES cells to the chemotherapeutic agent and DNA damage response inhibitor alone and in combination and linked to mechanism via western blot analysis. Finally, gene transfer studies are used to link the effects to EWS-FLI1.
Results: Ewing sarcoma cells show increased sensitivity to an ATR inhibitor (ATRi) relative to control cell lines. In addition, we found marked synergy with platinum agents that was superior to synergy the other 11 chemotherapeutic agents evaluated. This sensitivity was observed with three different platinum agents and shifted the IC50 by over an order of magnitude in five different ES cell lines. In addition, although oxaliplatin showed a lower magnitude of shift in IC50, the synergy could be linked to EWS-FLI1. Oxaliplatin was antagonistic to ATRi in HT1080 cells that lack EWS-FLI1 but showed the opposite effect when these cells were forced to express EWS-FLI1 and the two agents showed synergy. Work continues to understand the mechanism of this heightened sensitivity of oxaliplatin in combination with ATRi as it relates to EWS-FLI1 and to evaluate both combinations in vivo in animal models of the disease.
Conclusion. In conclusion, ES cells are sensitive to ATRi with a striking hypersensitivity when used in combination with platinum agents. Specifically, ES cells are more sensitive to a combination therapy with cisplatin and ATRi. Yet, experiments with oxaliplatin indicate that this platinum agent in combination with ATRi affects cells transfected with EWS/FLI1 more than cells lacking this key transcription factor. Therefore, it is likely that the combination of oxaliplatin and ATRi would show a broad therapeutic window as the two agents may be antagonistic in normal cells that do not express EWS-FLI1 while synergize in Ewing sarcoma cells.
Citation Format: Kathleen Sorensen, Jennifer Jess, Susan Goosen, Guillermo Flores, Zach Madaj, Elissa Bogulaslawski, Patrick J. Grohar. Oxaliplatin and ATR inhibitors show strong synergy in Ewing sarcoma cells [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 5817.