From the beginning of the project to the end of the reporting period, we have made substantial progress toward understanding the molecular mechanisms of pediatric sarcomas and establishing new experimental systems for their study.
In Aim 1 we uncovered how the SS18::SSX fusion protein, the oncogenic driver of synovial sarcoma, interacts with the chromatin. A domain-specific CRISPR screen revealed that the SSX C-terminal tail is essential for targeting the fusion protein to H2AK119ub1-marked chromatin regions deposited by PRC1.1. This SSX domain not only localizes SS18::SSX to these regions but also stabilizes PRC1.1 activity, reinforcing aberrant transcription by the oncofusion. Importantly, the same SSX-mediated mechanism applies to other SSX-containing fusions, pointing to a broader oncogenic principle (Benabdallah et al., Nat. Struct. Mol. Biol., 2023). We also demonstrated that other variant PRC1 complexes (vPRC1s) play roles in synovial sarcoma. However, PRC1.1 (PCGF1) is the main complex that deposits H2AK119ub1thereby sustaining SS18::SSX-driven transcription. These findings have important implications when designing strategies to treat this disease by impairing oncofusion activity.
In Aim2, we investigated the role of PRC1.1 deregulation in BCOR-driven sarcomas, where BCOR alterations are frequent. By engineering mesenchymal stem cell models expressing BCOR fusions, we observed activation of disease-specific gene signatures matching those in patient tumors from the INFORM registry. Mechanistic studies revealed that BCOR fusions preserve PRC1.1 integrity but reprogram Polycomb-controlled regions by recruiting transcriptional co-activators. Validation in patient-derived BCOR::CCNB3 cells confirmed these observations. This work established a new paradigm whereby BCOR fusions hijack co-activators to override Polycomb repression, providing a conceptual framework for future therapeutic approaches targeting these dependencies.
Im Aim3, we established a new strategy to model sarcoma in mice. To overcome the scarcity of robust preclinical models, we developed the EPO-GEMM platform, a flexible system for electroporation-based sarcoma modeling in immunocompetent mice. By combining transposon vectors, CRISPR/Cas9 tools, and in vivo muscle electroporation, we successfully established more than 10 genetically defined sarcoma models, including fusion-driven subtypes. These models recapitulate the histological and transcriptomic features of their human counterparts and allow tumor induction at neonatal or later postnatal stages. We further generated syngeneic allograft models (SAMs) to facilitate standardized preclinical testing. As proof-of-concept, an ETV6::NTRK3-driven model confirmed sensitivity to NTRK inhibition in vivo (Imle et al., Nat. Commun., 2025).
The results of this project have been disseminated through high-impact publications (Nat. Struct. Mol. Biol., 2023; Nat. Commun., 2025; additional manuscripts in preparation and under review) and presentations at international scientific conferences. The newly developed sarcoma models are available through the ITTC-P4 preclinical platform, ensuring broad accessibility for the research community.
