Abstract Epithelioid sarcoma (EPS) is a rare, slow-growing yet highly metastatic mesenchymal neoplasm characterized by inactivation of SMARCB1. Although the EZH2 inhibitor Tazemetostat has been approved for EPS patients, clinical outcomes remain poor. In this study, we performed functional genomic studies to identify therapeutic vulnerabilities of EPS and to elucidate their underlying mechanisms.CRISPR screening using an sgRNA library targeting ∼2,700 druggable genes identified dependencies of EPS cells on; BAF complex subunits (SMARCA4, SMARCC2 and SMARCD1), integrin pathway genes (ITGAV, PTK2, PTPN11) and the cyclin-dependent kinases (CDK4 and CDK6). A clinically available CDK4/6 dual inhibitor Palbociclib effectively inhibited EPS cell proliferation in vitro and significantly delayed tumor growth in human EPS cell line-derived xenograft models. Moreover, the CDK4-selective inhibitor Atirmociclib showed profound cytotoxicity in cell lines with low CDK6 expression, suggesting the feasibility of tailoring CDK4/6-targeted therapy based on CDK6 expression levels.Re-expression of SMARCB1 using a Tet-ON conditional expression system induced growth arrest, accompanied by reduced RB phosphorylation and decreased cyclin A2 (CCNA2) expression. SMARCB1 re-expression also upregulated the CDK inhibitors, p21 and p27, indicating compromised cell cycle control. The strong dependency of EPS on p21/p27-CDK4/6 axis supports CDK4/6 inhibition as a therapeutic strategy. Our CRISPR screen and in vitro viability assays demonstrated that EPS cell lines are intrinsically resistant to Tazemetostat, with genetic and epigenetic silencing of p16 emerging as a potential mechanism underlying this resistance.Together, functional genomic screens define key therapeutic vulnerabilities in EPS, suggesting CDK4/6 inhibitors as a promising treatment option for EPS patients. We propose that expression levels of CDK6 and p16 may serve as predictive biomarkers for response to EZH2 and CDK4/6 inhibitors, thereby informing patient stratification and improving clinical outcomes. Citation Format: Jiyeon Park, Ryo Miyamoto, David G. Kirsch. Exploring actionable therapeutic targets associated with SMARCB1 deficiency in Epithelioid sarcoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 646.
Park et al. (Fri,) studied this question.