Abstract 6732: Identifying novel combination therapies for Ewing sarcoma
Authors: De Los Santos Maria Ana Isabel C. , Blandin Anne-Florence , Aguilar Alejandra E. , Hall Matthew , Shen Min , Zhang Ya-Qin , Crompton Brian D. 
Published: April 2023

Abstract
Ewing sarcoma is the second most common pediatric bone cancer in children and young adults. The standard of care therapy consists of chemotherapy, surgery, and radiation. Despite therapeutic advances, metastatic and relapsed Ewing sarcoma have poor outcomes. This is partially because transcription factors, such as the Ewing sarcoma oncoprotein EWS-FLI1, are difficult targets for the development of small molecules. Novel synergistic combination treatments are needed, and drug repurposing efforts can fast track potential therapies into clinical trials. To identify synergistic anti-Ewing combinations of targeted cancer agents, we conducted an initial library screen of 26 tyrosine kinase inhibitors, cell cycle inhibitors, and chemotherapies on two Ewing cell lines. While the screening results revealed multiple synergistic combinations of tyrosine kinase inhibitors and cell cycle inhibitors, we focused our follow-up validation studies on combinations that met at least one of the following criteria: 1) agents that exhibited additivity when combined with VEGFR inhibitors, a class of agents already shown to having clinical efficacy in Ewing sarcoma and 2) combinations involving approved FDA drugs that could be rapidly translated into early phase trials for patients with Ewing sarcoma. Efficacy of selected synergistic combinations were then tested in vitro on a larger collection of Ewing cell lines. Based on these results, two combinations, one with a VEGFR inhibitor and one combination of FDA approved agents, was tested in an aggressive Ewing sarcoma xenograft mouse model for efficacy and impact on survival. We demonstrated that the combination of ribociclib, a CDK 4/6 inhibitor, and regorafenib, a VEGFR inhibitor, exhibited additivity and synergy in Ewing cell lines. Xenografted mice treated with this combination had significantly impaired tumor proliferation in a subcutaneous tumor model and prolonged survival (median 35 days) compared to mice treated with vehicle (median 28 days) or single-agent therapy. Similarly, cell lines treated with copanlisib, a PI3K inhibitor, and ribociclib, two FDA-approved drugs, synergistically inhibited cell line viability in vitro and impaired xenograft proliferation in vivo. This combination also significantly improved survival (median 48 days) in a mouse xenograft compared to treatment with vehicle (median 27 days) or either drug alone. This work demonstrates that CDK 4/6 inhibitors show synergistic activity with both VEGFR inhibitors and PI3K inhibitors in Ewing Sarcoma by impairing the growth of cultured Ewing cells and improving the survival of mice with Ewing sarcoma. We believe that pre-clinical validation of novel approved drug combinations in Ewing sarcoma will accelerate the development of early phase trials for patients with relapsed refractory disease, a group of patients greatly in need of new therapeutic opportunities.
Citation Format: Maria Ana Isabel C. De Los Santos, Anne-Florence Blandin, Alejandra E. Aguilar, Matthew Hall, Min Shen, Ya-Qin Zhang, Brian D. Crompton. Identifying novel combination therapies for Ewing sarcoma [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 6732.