Abstract 6916: CK2 inhibitor shows anti-tumor activity in Ewing sarcoma preclinical models

Abstract Introduction: Ewing sarcoma (EWS) is a highly aggressive bone cancer. EWS-FLI chromosomal fusion is detected in more than 85% of cases and is indispensable for EWS tumor survival and growth. Casein kinase II (CK2) is a serine/threonine kinase encoded by the CSNK2A1 gene that is essential for normal cellular processes and is overexpressed in EWS. Studies report that EWS protein is phosphorylated by CK2 at Ser325 and forced expression of CK2 in 293T cells increased EWS-FL1 protein. Here, we report that CK2 inhibition negatively affects EWS-FLI protein abundance, suppresses its downstream target GLI1, and shows anti-tumor activity in EWS xenografts. CX-4945 (silmitasertib) is a selective small molecule inhibitor of CK2 currently in clinical trial. Methods: EWS cell lines (n=6) were treated with serial dilution of CX-4945 for up to 48 hours, and cell viability, apoptosis, migration, and invasion potential were analyzed. We measured the mRNA and protein levels of known CK2 and EWS-FLI1 targets. RNA sequencing and gene expression analysis were done to evaluate gene expression changes following treatment with CX-4945. A673 cells were transduced with a clone of GFP+ lentiviral particles expressing CK2a-specific shRNA, and control shRNA, and phenotype was assessed using the above methods. A673 cell line-derived xenograft and patient-derived xenograft of EWS were treated with CX-4945 at a dose of 75mg/kg oral gavage twice daily for 28 days or vehicle. Tumor size and survival was measured. Results: CX-4945 at inhibitory concentrations of 2-8μM, increased apoptosis, decreased migration, invasion in all cell lines. We confirmed that CX-4945 treatment decreased EWS-FLI protein level, expression of EWS-FLI1 targets (GLI1, RNF2), known CK2 substrates (pAKT s129). Pharmacological and genetic inhibition of CK2 resulted in decreased cell viability, increased apoptosis, and decreased migration and invasion in vitro. Single-drug treatment with CX-4945 achieved 50-80% tumor inhibition and significantly prolonged survival in treated mice after 4 weeks of therapy. Following in vivo treatment with CX-4945, no significant organ toxicity was noted in tumor-bearing mice when compared to vehicle-treated mice. CX-4945 shows synergistic cytotoxic activity with Temozolomide and Irinotecan. Conclusions: CSNK2A1 expression is high in EWS and correlates with poor overall survival. CK2 inhibition decreases EWS-FLI protein abundance and suppresses its downstream target GLI1. CX-4945 shows excellent anti-tumor activity and target inhibition in EWS xenografts. CX-4945 works synergistically with cytotoxic agents like Temozolomide and Irinotecan. These results support further pre-clinical characterization and clinical development of CX-4945 for treating EWS in combination with cytotoxic therapy. A Phase I clinical trial is evaluating the safety and tolerability of CX-4945 in pediatric solid tumors, including EWS (NCT06541262). Citation Format: Muhammad Danial, Rajesh Rajaiah, Marudhu Pandiyan Shanmugam, Sholler Chloe, Jeremey Hengst, Abhinav Beeravally Nagulapally, Yasin Uzun, Giselle Saulnier Sholler, Chandrika Gowda behura. CK2 inhibitor shows anti-tumor activity in Ewing sarcoma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6916.