In situ proximity ligation assays in the study of FET fusion oncoprotein interactions in sarcoma cells

Abstract

Cancer is a major global health challenge, and deeper understanding of molecular mechanisms driving tumor development is essential for the development of targeted therapies. FET sarcoma is a rare malignant cancer type driven by FET fusion oncoproteins, such as FUS::DDIT3 in myxoid liposarcoma. These fusion oncoproteins alter gene expression through aberrant interactions with chromatin-regulating proteins, including SWI/SNF chromatin remodeling complex and transcriptional co-activator BRD4. Understanding these interactions at the nanoscale is essential for investigating the mechanisms causing oncogenic transcription and for identifying novel therapies. The aim of this work was to employ and optimize an in situ proximity ligation assay (PLA) protocol for detection of proximity events indicative of protein protein interactions involving the FET fusion oncoprotein FUS::DDIT3 in sarcoma cells. In situ PLA combines antibody-based recognition with DNA amplification of oligonucleotide-probes, enabling highly sensitive visualization of proximity events within 40 nm and providing single molecule sensitivity. In situ PLA was employed to study interactions between FUS::DDIT3, SWI/SNF chromatin remodeling complex subunit BRG1, and the transcriptional co-activator BRD4 in fixed cancer cells. Experiments were conducted using a myxoid liposarcoma cell line endogenously expressing the fusion oncoprotein FUS::DDIT3. Additionally, the effects of a combination drug treatment consisting of the BRD4 inhibitor AZD5153 and the histone deacetylase inhibitor Panobinostat were studied. Confocal imaging demonstrated successful detection and quantification of PLA signals, indicating close proximity between BRG1 and FUS::DDIT3, BRD4 and BRG1, and BRD4 and FUS::DDIT3 in myxoid liposarcoma cells. Moreover, preliminary visual observations of PLA signals in confocal z-stack images indicated that these interactions were disturbed when treated with the drug combination. This suggests that at least part of the drug mechanism, targeting the oncogenic role of FET oncoproteins, is via disruption of essential protein interactions. Additional image acquisition, data analysis, and PLA signal quantification is needed in future experiments in order to draw any conclusions regarding the efficiency of the drug treatment.