Myxoid Liposarcoma: Models and Mechanisms of Sarcomagenesis and Response to Radiation Therapy
Myxoid liposarcoma (MLPS) is a malignant soft tissue sarcoma characterized by a pathognomonic t(12;16)(q13;p11) translocation that produces a fusion oncoprotein, FUS-CHOP. This cancer is remarkably sensitive to radiotherapy and exhibits a unique pattern of extrapulmonary metastasis. However, the mechanism for its radiosensitivity is unknown. In order to further understand the biological mechanisms underlying MLPS response to radiotherapy we studied the fusion oncoprotein FUS-CHOP and linked its role in sarcomagenesis to the radiosensitivity phenotype.
Here we investigate a molecular mechanism of radiosensitization that couples ionizing radiation to inhibition of translocation-driven sarcomagenesis in myxoid liposarcoma. We performed co-immunoprecipitation (co-IP) to identify proteins interacting with FUS-CHOP. Incucyte assays measured cell proliferation after knockdown of interacting proteins fusion-negative and fusion-positive primary murine sarcoma cell lines from a novel FUS-CHOP genetically engineered mouse model (GEMM). ChIP-seq/CUT&RUN mapped genome-wide binding sites of FUS-CHOP and identified DNA-binding motifs for the fusion oncoprotein. Co-IP of irradiated human MLPS cell lines were performed to evaluate post-translational modification of FUS-CHOP after irradiation, and to investigate regulation of protein-protein interactions by these modifications.
We detected functionally important interactions between FUS-CHOP and multiple chromatin remodeling complexes via co-IP including a new interaction with SNF2H, the ATPase subunit of the imitation switch (ISWI) complex. Using knockdown systems, we demonstrated that these interacting chromatin remodelers are functionally important for proliferation specifically in FUS-CHOP-driven, but not Kras-driven murine sarcoma cells. ChIP-seq and CUT&RUN profiling of human MLPS cell lines identified DNA-binding motifs and genomic loci targeted by FUS-CHOP, which co-localized with SNF2H and H3K27ac marks of active chromatin. We further hypothesized that post-translational modification of the FUS-CHOP PrLD may regulate the protein-protein interactions between FUS-CHOP and chromatin remodelers. Using irradiated human MLPS cell lines, we show that FUS-CHOP is a target of phosphorylation by the DNA damage response kinases DNA-PK and ATM after irradiation. Finally, we show that phosphorylation of the PrLD of FUS-CHOP diminishes protein-protein interactions with chromatin remodeling complexes and the ability for FUS-CHOP to transform NIH-3T3 cells.
We also report the generation and characterization of a spatially and temporally restricted mouse model of sarcoma driven by FUS-CHOP. Using different Cre-drivers in the adipocyte lineage, we initiated in vivo tumorigenesis by expressing FUS-CHOP in Prrx1+ mesenchymal progenitor cells. In contrast, expression of FUS-CHOP in more differentiated cells does not form tumors in vivo, and early expression of the oncoprotein during embryogenesis is lethal. We also employ in vivo electroporation and CRISPR technology to rapidly generate spatially and temporally restricted mouse models and cell lines of high grade FUS-CHOP-driven sarcomas for preclinical studies.
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