Therapeutic efficacy of CK2 inhibitor in patient-derived lung metastatic model of Ewing sarcoma

Introduction: Metastatic Ewing sarcoma (ES), a highly aggressive bone cancer, has an extremely poor 5-year overall survival rate of <25%. EWS-FLI oncogenic fusion protein drives tumor progression in nearly all cases. Casein kinase II (CK2) is a serine/threonine kinase that is essential for normal cellular processes and is overactive in cancer. CK2 high-expressing ES patients have a high relapse rate and worse 5-year overall survival. CX-4945 (silmitasertib) is an orally bioavailable, CK2-selective small molecule inhibitor in clinical trial for the treatment of pediatric cancers. Methods: Patient-derived xenograft (PDX) cell lines derived from relapsed refractory metastatic EWS-FLI1 fusion-positive ES (n=15) were treated with CX-4945 and analyzed. We generated metastatic models of EWS-FLI fusion-positive ES following subcutaneous implant in athymic nude mice. After 4 weeks of treatment with CX-4945, mice were analyzed for the presence of lung and bone metastasis using histology, immunohistochemistry (IHC), and pathology review. We scored the lung metastasis burden. The number and area of tumor cell foci within the lung tissue were noted. Tumor cells and inflammatory cells in the extra-parenchymal space and tumor cells circulating within the blood vessels were confirmed using IHC. Using flow cytometry with a human CD99 antibody, we confirmed tumor cells in the lung tissue. Tumor size and survival (time to reach endpoint) were noted. We then assess the effect of CX-4945 treatment on the occurrence and severity of lung metastasis. Results: Four weeks of treatment with CX-4945 alone showed slow tumor progression and significantly prolonged survival in two xenograft models. More importantly, treated mice showed significantly less tumor burden in lungs and bone marrow. Conclusions: These results support further preclinical characterization and mechanistic investigation of CX-4945 for treating ES. A phase 1 clinical trial is evaluating the safety of CX-4945 in pediatric solid tumors, including ES (NCT06541262). Patient derived lung metastasis model of Ewing sarcoma development and assessment for effect of treatment is feasible and essential to determine clinically meaningful therapeutic effect of anti-cancer agent.