The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct and separable from its DNA-binding function in Ewing sarcoma

Abstract

Background: Ewing sarcoma is an aggressive bone cancer in children and young adults that contains a pathognomonic chromosomal translocation, t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the low-complexity amino-terminal portion of EWS to the carboxyl-terminus of FLI. The FLI portion contains a central ETS DNA-binding domain and adjacent amino- and carboxyl-regions. Early studies using non-Ewing sarcoma cellular models provided conflicting information on the role of these adjacent regions in the oncogenic function of EWS/FLI. We therefore sought to define the specific contributions of each FLI region to EWS/FLI activity in an appropriate Ewing model, and in doing so, to better understand Ewing sarcoma development mediated by the fusion protein. Methods: We used a knock-down/rescue system to replace endogenous EWS/FLI expression with mutant forms of the protein in Ewing sarcoma cells and tested these for oncogenic transformation using soft-agar colony forming assays. These data were complemented by DNA-binding assays using fluorescence anisotropy, genomic localization assays using CUT&RUN, transcriptional regulation studies using luciferase reporter assays and RNA-sequencing, as well as chromatin accessibility assays using ATAC-sequencing. Results: We found that the DNA-binding domain and short flanking regions of FLI were required for oncogenic transformation, gene expression, genomic localization and chromatin accessibility when fused to the amino-terminal EWS-portion from EWS/FLI, but that the remaining regions of FLI were dispensable for these functions. Removal of a carboxyl-terminal alpha-helix from the short flanking regions of the DNA-binding domain of FLI created a hypomorphic EWS/FLI that retained normal DNA binding, genomic localization, and chromatin accessibility, but had significantly restricted transcriptional activity and a near total loss of oncogenic transformational capacity. Conclusions: The DNA-binding domain and carboxyl-terminal short flanking region of FLI are the only portions of FLI required for EWS/FLI-mediated oncogenic transformation in a Ewing sarcoma cellular context. In addition to the well-defined DNA-binding function of FLI, this additional alpha-helix immediately downstream of the DNA-binding domain contributes a previously-undescribed function in gene regulation and oncogenic transformation. Understanding the function of this critical region could provide new therapeutic opportunities to target EWS/FLI in Ewing sarcoma.

One oncogene, several vulnerabilities: EWS/FLI targeted therapies for Ewing sarcoma

“…EWS/FLI is a powerful oncogenic driver that, through a series of complex mechanisms involving several binding partners, binds both high affinity GGAA sequences and GGAA repeat regions on DNA to establish de novo enhancers that drive tumor formation and progression [21] , [22] , [23] , [24] , [25] , [26] . Indeed, genes induced by EWS/FLI drive proliferation, DNA damage response genes and alter the cell cycle, while repressed genes maintain a block in proliferation and inhibit cell communication [27] , [28] , [29] , [30] , [31] , [32] .…”