Abstract 5664: Identifying novel vulnerabilities in clear cell sarcoma through functional and genomic screening

Abstract Introduction: Clear Cell Sarcoma (CCSA) is a rare soft tissue sarcoma characterized by specific gene fusions EWSR1::ATF1 or EWSR1::CREB1, leading to the expression of an oncogenic fusion protein. Metastasized CCSA is resistant to conventional chemotherapy, targeted therapy or immunotherapy. This study aims to uncover new therapeutic strategies for CCSA by utilizing a whole-genome CRISPR/Cas9 knock-out screen combined with functional drug screening. Methods: We performed a genome-wide knock-out screen in the CCSA cell line model KAS using the pooled Brunello sgRNA library (Addgene # 73178, ∼4 guides/gene, 19'114 genes). In parallel, three CCSA patient samples and multiple CCSA cell lines (SU-CCS-1, DTC-1, KAS) were treated with a library of 84 solid tumor pan-cancer drugs, and a custom drug library tailored to the hits of the CRISPR screen (70 compounds). Genes identified as critical vulnerabilities in CCSA were validated through single-gene knockouts, and promising therapeutic targets were further mechanistically interrogated and evaluated in a CCSA xenograft model. Results: Results revealed 1'552 significantly depleted genes (log fold change <-1 and FDR < 0.01) after 21 days, with enriched pathways in proteasome activity, mismatch repair, DNA replication, and, mTORC1 signaling, revealing critical vulnerabilities. Notable highly ranked depleted genes include the transcription factor and CCSA marker SOX10, as well as MDM2, MCL1, CHEK1, HSPA5, PSMA5 and mTOR. Through integration of genetic knock-out and pharmacological inhibition data identified in our dual screening, we were able to identify several overlapping vulnerabilities. Initial validation of the therapeutic potential of PI3K/mTOR inhibition in CCSA cell models in vitro revealed an induction of apoptosis upon treatment. We then corroborated those findings in a CCSA xenograft mouse model in vivo. Treatment with the two combined PI3K/mTOR inhibitors omipalisib and copanlisib both resulted in significantly improved survival (P = 0.027 and P = 0.036, respectively) of tumor bearing mice, and reduced tumor growth, each compared to vehicle treatment. Conclusion: These findings offer new insights into actionable vulnerabilities in CCSA, with PI3K/mTOR inhibitors showing potential for therapeutic development. Future studies will investigate synergistic combinations to further optimize treatment efficacy and validate further vulnerabilities identified by our dual screening approach. Citation Format: Laura Leuenberger, Larissa Isenegger, Yanjiang Chen, Miles Burri, Sarah Kollar, Lara Planas-Paz, Peter Karl Bode, Agnieszka Wozniak, Sebastian Bauer, Patrick Schöffski, Chantal Pauli, Christian Britschgi. Identifying novel vulnerabilities in clear cell sarcoma through functional and genomic screening [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 5664.