Chromobox 4 (CBX4) Is a Novel Interactor of SS18::SSX in Synovial Sarcoma

Synovial sarcoma is an aggressive cancer generally affecting adolescents and young adults and is characterized by high rates of recurrence and metastasis. It is primarily driven by the fusion oncoprotein SS18::SSX, the product of a pathognomonic chromosomal translocation t(X;18), which facilitates widespread epigenetic dysregulation through interactions with complexes, such as the BRG1/BRM-associated factor complex and polycomb repressive complexes. Previous attempts to perform mass spectrometry (MS) of the SS18::SSX interactome have been limited by the lack of an antibody to detect the endogenous protein, hence relying on single-cell lines with exogenous tags. We previously used a monoclonal antibody, which specifically detects SS18::SSX containing the canonical fusion junction (seen in 95% of cases) and established its utility in several applications. Using that antibody, MS analysis revealed that the SS18::SSX protein undergoes alternative splicing of exon 8 in SS18. We next performed immunoprecipitation MS of SS18::SSX in 6 immortalized human synovial sarcoma cell lines and identified the canonical polycomb repressive complex member chromobox 4 (CBX4), as a novel interactor of the oncoprotein. Immunohistochemical staining of several epigenetic factors on a human synovial sarcoma tissue microarray showed an association of synovial sarcoma samples with higher CBX4 expression. Last, an analysis of CBX4 expression across 337 samples from 12 sarcoma subtypes, carcinomas, and normal tissue demonstrates higher expression in synovial sarcoma samples compared with other tissue types. These results highlight a crucial approach in identifying important partners of SS18::SSX in synovial sarcoma to establish new biological pathways that contribute to the disease.