CK2 inhibitor CX-4945 targets EWS-FLI1 signaling network and shows therapeutic efficacy in metastatic mouse models of Ewing Sarcoma

Ewing sarcoma (ES) is an aggressive bone tumor that primarily affects children, adolescents, and young adults. EWS-FLI1 oncogenic fusion protein is indispensable for ES tumor survival and progression. Casein kinase II (CK2) is a serine/threonine kinase that plays an essential role in apoptosis, DNA damage repair, and the cell cycle. CK2 is highly expressed in ES and associated with metastatic disease and poor 5-year overall survival. Here, we show that CK2 inhibitor CX-4945 (silmitasertib) induced K48-specific ubiquitination and subsequent proteasomal degradation of EWS-FLI1. CK2 inhibition effectively altered fusion protein abundance and disrupted the ES oncogenic signaling, specifically repressing metastasis-associated gene programs. Phenotypically, CK2-depleted ES cells showed decreased migration and invasion in vitro. In the metastatic ES xenograft model, CX-4945 significantly suppressed tumor growth, reduced tumor burden in the lungs, and extended overall survival. CK2 genetic depletion phenocopied CX-4945 effects both in vitro and in vivo. Molecular analysis of treated tumors confirmed robust target engagement, characterized by significant decrease in CK2 substrate phosphorylation levels. CX-4945 showed synergistic cytotoxicity with Irinotecan, a commonly used chemotherapy for the treatment of relapsed ES. Our findings establish CK2 as a novel therapeutic target in ES and provide a mechanistic rationale for combining CK2 inhibitor with chemotherapy regimens. Given the established safety profile of CX-4945, these results support clinical testing of the CK2 inhibitor fusion for treatment of metastatic ES. A Phase 1/2 trial (NCT06541262) is currently evaluating CX-4945 in combination with chemotherapy for pediatric and young adults with relapsed or refractory solid tumors, including ES. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/677357v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@96e51aorg.highwire.dtl.DTLVardef@9b353eorg.highwire.dtl.DTLVardef@1c49da8org.highwire.dtl.DTLVardef@14abe06_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of Translational RelevanceOur study identifies Casein Kinase 2 (CK2) as a novel therapeutic target in Ewing Sarcoma (ES). We demonstrate that CK2 inhibition triggers K48-specific ubiquitination and subsequent proteasomal degradation of EWS-FLI1 oncoprotein. Additionally, CX-4945 simultaneously targets multiple oncogenic signaling pathways and EWS-FLI1 regulators, resulting in sustained suppression of proliferation and metastasis. In metastatic ES models, oral CX-4945 showed robust efficacy, significantly reducing tumor volume and lung metastasis while extending survival. These findings provide the mechanistic rationale for integrating CK2 inhibition into current chemotherapy regimens. The translational impact is immediate: CX-4945 has an established clinical development pathway, and its safety in combination with chemotherapy is currently being evaluated in an ongoing Phase 1 multicenter trial (NCT06541262), offering a novel targeted strategy for patients with metastatic Ewing Sarcoma.