(P 362) IDENTIFICATION OF A NOVEL INTERACTOR OF ENDOGENOUS SS18-SSX THROUGH MASS SPECTOMETRY-BASED ANALYSIS IN SYNOVIAL SARCOMA CELLS

Objective: The role of the fusion oncoprotein SS18-SSX in synovial sarcoma has been studied extensively to understand how one mutant protein is able to cause and sustain oncogenesis. Experiments using mass spectrometry to analyse interactors of SS18-SSX have mostly relied on cell line models engineered to tag the protein for pulldown experiments, resulting in discovery of its association with a diverse set of proteins including HDAC, ATF2, TLE1, KDM2B and the mammalian SWI/SNF (complex). The lack of an antibody specific to SS18-SSX has limited these studies to epitope-tagged single cell-line models.
In the current study, immunoprecipitation of the native SS18-SSX protein was performed in multiple synovial sarcoma cell lines with subsequent analyses of the interacting proteins through tandem mass spectrometry. The study aims to identify novel interactors of the synovial sarcoma fusion oncoprotein in endogenous cellular backgrounds.

Methods: Whole cell lysates were collected from six synovial sarcoma cell lines which have previously been shown to express the fusion oncoprotein using a commercially available SS18-SSX fusion specific antibody. An osteosarcoma cell line was included as a negative control. Dynabeads were used to perform immunoprecipitation (IP) with the SS18-SSX antibody and a control rabbit IgG antibody, in triplicates. Samples were digested with trypsin and processed for tandem mass tag labelling of peptides and subsequent analysis on an Orbitrap Fusion Tribrid MS platform. Peptides were analysed using Proteome Discoverer Software, and reporter abundance was averaged across triplicate runs.

Results: Protein abundance was compared in each cell line and log2 fold change values calculated for proteins enriched in the SS18-SSX IP group over the control IgG group. Comparison of the top hits in all synovial sarcoma samples identified a canonical PRC1 complex member, CBX4, as a positive hit in 4 out of 6 cell lines. As part of the canonical PRC1 complex, CBX4 utilises its chromodomain to recognise the epigenetic H3K27me3 mark on histones to mediate transcriptional repression. Furthermore, CBX4 also functions in a dual role as an E3 SUMO ligase within the SUMOylation pathway, whose other members (SUMO2, UBC9) have been shown to be enriched in synovial sarcoma lineages (DepMap). Co-IP of SS18-SSX followed by western blot detection showed CBX4 to be present in all 6 cell lines and immunofluorescence imaging confirmed the co-localisation of SS18-SSX with CBX4 in the nucleus.

Conclusion: Using the fusion-specific antibody for immunoprecipitation of the endogenous SS18-SSX protein helps to detect common interactors amongst different synovial sarcoma cell lines, and uncover new pathways and roles for the protein in sarcomagenesis. We uncover a novel interaction of SS18-SSX with CBX4, a canonical PRC1 member involved in transcriptional repression. This interaction presents itself as a potential druggable target in synovial sarcoma with the use of existing molecules that can inhibit CBX4 function.