FET fusion oncoproteins in sarcoma tumorigenesis - lnteractions with the SWI/SNF chromatin remodeling complex and epigenetic effects

Abstract

The FET family of fusion oncoproteins containing the N-terminal of either FUS, EWSR1 or TAF15 together with a transcription factor partner is the characteristic oncogenic driver of more than 20 types of sarcoma and leukemia, including myxoid liposarcoma and Ewing sarcoma. The FET oncoproteins are thought to function as aberrant transcription factors, however, the exact oncogenic mechanism remains to be elucidated. The aim of this PhD project was to study the effects of the FET oncoproteins, especially on the SWI/SNF chromatin remodeling complex and downstream epigenetic effects. We therefore developed a comprehensive proteomic workflow, where subcellular fractionation by nuclear extraction and enrichment using immunoprecipitation was employed prior to western blot or mass spectrometry analysis to study nuclear interactions. First, we evaluated the contribution of each part of the fusion oncoprotein. The transcription factor partner DDIT3 bound specific genomic loci via its dimerization partners and repressed a set of target genes. The FET N terminal domain in both normal and oncogenic FET proteins interacted with the SWI/SNF complex, however, in a dysregulated way for FET oncoproteins. Further analysis showed that FET oncoproteins interact with all three main subtypes of the SWI/SNF complex and that the core of the FET oncoprotein-bound SWI/SNF complexes remain intact. However, a minor effect on the complex composition cannot be excluded. We then showed that FET oncoproteins interact with the transcriptional coactivator BRD4, via the SWI/SNF complex. Importantly, further analysis revealed that FET oncoproteins, SWI/SNF components and BRD4 co-localize on chromatin and interact with Mediator and RNA polymerase II components thus providing a direct link to mechanisms in chromatin remodeling and transcriptional regulation. Downstream analyses indicated that FET oncoproteins affect the repressive histone mark H3K27me3 and disturb the important transcriptional balance between SWI/SNF and polycomb repressive complexes. Furthermore, the FET oncoprotein FUS-DDIT3 interacted with pSTAT3, a transcription factor activated by the JAK-STAT signaling pathway, indicating a role of this pathway in FET sarcoma. In conclusion, dysregulation of SWI/SNF and downstream epigenetic processes provide a unifying oncogenic mechanism for tumors caused by FET fusion oncoproteins thus providing an opportunity for development of new targeted therapies.