Identification of a Novel FUS/ETV4 Fusion and Comparative Analysis with Other Ewing Sarcoma Fusion Proteins

Abstract

Ewing sarcoma is a pediatric bone cancer defined by a chromosomal translocation fusing one of the FET family members to an ETS transcription factor. There have been seven reported chromosomal translocations, with the most recent reported over a decade ago. We now report a novel FET/ETS translocation involving FUS and ETV4 detected in a patient with Ewing sarcoma. Here, we characterized FUS/ETV4 by performing genomic localization and transcriptional regulatory studies on numerous FET/ETS fusions in a Ewing sarcoma cellular model. Through this comparative analysis, we demonstrate significant similarities across these fusions, and in doing so, validate FUS/ETV4 as a bona fide Ewing sarcoma translocation. This study presents the first genomic comparison of Ewing sarcoma-associated translocations and reveals that the FET/ETS fusions share highly similar, but not identical, genomic localization and transcriptional regulation patterns. These data strengthen the notion that FET/ETS fusions are key drivers of, and thus pathognomonic for, Ewing sarcoma. IMPLICATIONS: Identification and initial characterization of the novel Ewing sarcoma fusion, FUS/ETV4, expands the family of Ewing fusions and extends the diagnostic possibilities for this aggressive tumor of adolescents and young adults.

Graphical abstract

Figure 1.

Neonatal patient presenting with Ewing sarcoma tumor. A, Coronal MRI scan revealed a left posterior mediastinal mass. B, Hematoxylin and eosin staining of patient tumor biopsy revealed sheets of undifferentiated, mitotically active, small round blue cells with dispersed chromatin and minimal amphophilic cytoplasm (50 μm scale bar depicted on image). C, CD99 immunochemistry reveals diffuse membranous expression (50 μm scale bar depicted on image). D, NKX2–2 IHC shows diffuse strong nuclear immunoreactivity (50 μm scale bar depicted on image).

Figure 2.

EWS/ETV4 and FUS/ETV4 DNA-binding and transcriptional-profile overlap reveals similar biological functions. A, Protein schematic of 3xFLAG-tagged (3F) EWS/ETV4 and FUS/ETV4 constructs. EWS is represented in light grey, FUS in dark grey, and ETV4 in light blue. Exons included in each fusion are noted. B, Venn diagram overlap analysis performed on CUT&Tag-detected genomic localization data for EWS/ETV4 and FUS/ETV4 expressed in A673 knockdown/rescue cells, as compared with control cells (Control: iEF + Empty Vector; EWS/ETV4: iEF + EWS/ETV4; FUS/ETV4: iEF + FUS/ETV4; N = 2 biological replicates). The number of peaks uniquely bound by each construct or those that are similarly bound are indicated in the figure. Significance of overlap: P < 2.2 × 10^–16. C, Venn-diagram analysis of RNA-seq results depicting significantly regulated genes for EWS/ETV4 and FUS/ETV4-expressing A673 knockdown/rescue cells, as compared with iEF + Empty Vector control cells (N = 2 biological replicates). Number of regulated genes for each construct is indicated in the figure. Significance of overlap: P < 2.2 × 10^–16.

Figure 3.

Comparison of FET/ETS fusions demonstrate similar biological function for both genomic localization and transcriptional-regulatory capacities. A, Protein schematic of cDNA constructs, including EWS/ERG, FUS/ERG, EWS/FEV, and FUS/FEV. EWS is depicted in light grey, FUS in dark grey, ERG in teal, and FEV in indigo. Exons included in each fusion are noted. B, Venn-diagram overlap analysis of CUT&Tag genomic localization data for the corresponding fusion protein listed expressed in A673 knockdown/rescue cells (iEF + Construct), as compared with control cells (iEF + Empty Vector; N = 2 biological replicates). Number of bound regions for each construct depicted in figure. Significance of overlap: P < 2.2 × 10^–16. C, Venn-diagram overlap analysis of RNA-seq expression data for genes called as significantly regulated by the corresponding construct expressed in A673 knockdown/rescue cells, as compared with control cells (iEF + Empty Vector; N = 2 biological replicates). Number of significantly regulated genes by each fusion listed in figure. Significance of overlap: P < 2.2 × 10^–16. D, Venn-diagram overlap analysis of CUT&Tag genomic localization–binding data of FET/ETS fusions in A673 knockdown/rescue cells (N = 2 biological replicates, left). All DNA-bound regions are called as significant for the corresponding fusion as compared with control cells (iEF + Empty Vector). Significance of overlap: P < 2.2 × 10^–16. Venn-diagram analysis of significantly regulated genes by corresponding FET/ETS fusion, as compared with control cells (iEF + Empty Vector), determined using RNA-seq (N = 2 biological replicates, right). Significance of overlap: P < 2.2 × 10^–16.