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Abstract

Synovial sarcoma is an aggressive soft tissue cancer that primarily affects adolescents and young adults and is characterised by a chromosomal translocation, t(X:18), that results in the expression of the fusion oncoprotein SS18::SSX. Proteomic studies have established that SS18::SSX interacts with key epigenetic regulators and complexes to induce widespread epigenetic dysregulation that drives and maintains sarcoma cells. However, these studies have been limited to one or two cell lines with exogenously-tagged SS18::SSX to detect the fusion oncoprotein. The current study establishes the use of a new commercial SS18::SSX antibody to detect the fusion oncoprotein specifically in tumour tissue samples expressing SS18::SSX in its most common canonical form, found in 95% of clinical synovial sarcoma cases. Furthermore, this tool can also demonstrate spatial interaction between SS18::SSX and its interactors in cell lines and formalin-fixed paraffin embedded primary samples. Mass spectrometry investigation of endogenous SS18::SSX proves that it is expressed in two isoforms that are generated through alternative splicing of exon 8 in SS18. Using an antibody directed against endogenous SS18, mass spectrometry analysis of the interactome of six synovial sarcoma human cell lines reveals enrichment of RNA processing proteins. These include FUS, EWSR1 and TAF15 (FET) proteins which are involved in other translocation-associated sarcomas and also have epigenetic functions, as well as splicing factors. These interactions are validated using the new SS18::SSX antibody-based techniques established earlier. Probing the SS18::SSX interactome reveals a novel interactor, CBX4, which is a member of the canonical PRC1 complex and also has a dual role as an E3 SUMO ligase. CBX4 is shown to be more highly expressed in synovial sarcoma primary tissue as compared to other epigenetic proteins. Analysis of CBX4 expression across multiple sarcoma samples on tissue microarray slides reveals higher expression in synovial sarcomas. Ultimately, these tools represent important methodologies to uncover and validate interactors of SS18::SSX that influence and inform on the biology of SS18::SSX in synovial sarcoma.