Ewing sarcoma is an aggressive bone cancer of children and young adults defined by the presence of a chromosomal translocation: t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the amino-terminal domain of EWS to the carboxyl-terminus of FLI. The EWS portion is an intrinsically disordered transcriptional regulatory domain, while the FLI portion contains an ETS DNA-binding domain and two flanking regions of unknown function. Early studies using non-Ewing sarcoma models provided conflicting information on the roles of each domain of FLI in EWS/FLI oncogenic function. We therefore sought to define the specific contributions of each FLI domain to EWS/FLI activity in a well-validated Ewing sarcoma model and, in doing so, to better understand Ewing sarcoma development mediated by the fusion protein. We analyzed a series of engineered EWS/FLI mutants with alterations in the FLI portion using a variety of assays. Fluorescence anisotropy, CUT&RUN, and ATAC-sequencing experiments revealed that the isolated ETS domain is sufficient to maintain the normal DNA-binding and chromatin accessibility function of EWS/FLI. In contrast, RNA-sequencing and soft agar colony formation assays revealed that the ETS domain alone was insufficient for transcriptional regulatory and oncogenic transformation functions of the fusion protein. We found that an additional alpha-helix immediately downstream of the ETS domain is required for full transcriptional regulation and EWS/FLI-mediated oncogenesis. These data demonstrate a previously unknown role for FLI in transcriptional regulation that is distinct from its DNA-binding activity. This activity is critical for the cancer-causing function of EWS/FLI and may lead to novel therapeutic approaches.

Ewing sarcoma is a prototypical fusion transcription factor-associated pediatric cancer that expresses EWS/FLI or a highly related FET/ETS chimera. EWS/FLI dysregulates transcription to induce and maintain sarcomagenesis, but the mechanisms utilized are not fully understood. We therefore sought to define the global effects of EWS/FLI on chromatin conformation and transcription in Ewing sarcoma cells using a well-validated ‘knock-down/rescue’ model of EWS/FLI function in combination with next generation sequencing assays to evaluate how the chromatin landscape changes with loss, and recovery, of EWS/FLI expression. We found that EWS/FLI (and EWS/ERG) genomic localization is largely conserved across multiple patient-derived Ewing sarcoma cell lines. This EWS/FLI binding signature is associated with establishment of topologically-associated domain (TAD) boundaries, compartment activation, enhancer-promoter looping that involve both intra- and inter-TAD interactions, and gene activation. In addition, EWS/FLI co-localizes with the loop-extrusion factor cohesin to promote chromatin loops and TAD boundaries. Importantly, local chromatin features provide the basis for transcriptional heterogeneity in regulation of direct EWS/FLI target genes across different Ewing sarcoma cell lines. These data demonstrate a key role of EWS/FLI in mediating genome-wide changes in chromatin configuration and support the notion that fusion transcription factors serve as master regulators of three-dimensional reprogramming of chromatin.

Tenofovir (TFV) is an antiviral drug approved for treating Human Immunodeficiency Virus (HIV) and Hepatitis B. TFV is administered orally as the prodrug tenofovir disoproxil fumarate (TDF) which then is deesterified to the active drug TFV. TFV induces nephrotoxicity characterized by renal failure and Fanconi Syndrome. The mechanism of this toxicity remains unknown due to limited experimental models. This study investigated the cellular mechanism of cytotoxicity using a human renal proximal tubular epithelial cell line (HK-2). HK-2 cells were grown for 48 h followed by 24 to 72 h exposure to 0–28.8 μM TFV or vehicle, phosphate buffered saline (PBS). MTT (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and Trypan blue indicated that TFV diminished cell viability at 24–72 h. TFV decreased ATP levels at 72 h when compared to vehicle, reflecting mitochondrial dysfunction. TFV increased the oxidative stress biomarkers of protein carbonylation and 4-hydroxynonenol (4-HNE) adduct formation. Tumor necrosis factor alpha (TNFα) was released into the media following exposure to 14.5 and 28.8 μM TFV. Caspase 3 and 9 cleavage was induced by TFV compared to vehicle at 72 h. These studies show that HK-2 cells are a sensitive model for TFV cytotoxicity and suggest that mitochondrial stress and apoptosis occur in HK-2 cells treated with TFV.

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 Ewing sarcoma patient.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. ImplicationsIdentification 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.

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.

Objective: Ewing sarcoma (ES) is the second most common pediatric bone cancer. To address a blatant lack of advancement in treatment, it is of the utmost importance to understand the underlying biology driving disease and through these developments, we hope to better outcomes through bench-to-bedside projects. ES is driven by a chromosomal translocation, which produces a fusion oncoprotein known as EWS/FLI1. Though it is known that FLI1 contains a DNA-binding domain that is essential for protein function and disease formation, very little further research has been completed to understand precisely how it contributes to ES etiology and how we can disrupt these functions. Our goal is to complete structure-function mapping that allows an in-depth analysis of FLI1 domain contributions that ultimately may elicit new targets for treatment development. Methods: EWS/FLI1 cDNA was used to create several different versions of EWS/FLI1, including a full-length protein and two EWS/FLI1 proteins with truncated FLI1 domains. A ES cellular model was used to test functionality of these constructs. A variety of molecular biology and new sequencing techniques were employed to establish structure-function relationships of the FLI1 domain of EWS/FLI1, including luciferase reporter assays, soft agar assays, RNA-sequencing, CUT&RUN-sequencing, and ATAC-sequencing. Results: In vitro transcriptional activation assays revealed EWS/FLI1 containing only the DNA-binding domain of FLI1 (EWS/FLI1DBD) was sufficient to drive transcription, but this result did not translate to the ability to oncogenically transform ES cells in a soft agar assay. We used various sequencing techniques to understand the process that allows EWS/FLI1 to actually drive transformation. CUT&RUN-sequencing revealed that EWS/FLI1 binds various places throughout the genome and, surprisingly, that EWS/FLI1DBD was able to bind all the same places. RNA-sequencing showed that EWS/FLI1 was able to regulate gene expression at the majority of bound regions, but EWS/FLI1DBD starkly lacked this ability. These results caused us to question what role EWS/FLI1 has as a pioneer transcription factor and what happens between DNA-binding and gene regulation. Through ATAC-sequencing results, we expect to see that EWS/FLI1 has the ability to open closed chromatin regions, but that EWS/FLI1DBD will lack this ability. It is likely that EWS/FLI1 recruits various additional co-factors to open chromatin and through these constructs, we hope to identify more of these important proteins. Conclusions: We have revealed FLI1 has a novel role in EWS/FLI1 activity that goes beyond DNA-binding, as well as isolated the essential regions of FLI1 necessary for activity. Ultimately, we hope that these novel properties or interactions will provide new ways to target ES cells that can benefit patient outcomes. Citation Format: Megann A. Boone, Jesse C. Crow, Julia Selich-Anderson, Cenny Taslim, Emily R. Theisen, Stephen L. Lessnick. Structure-function mapping reveal FLI1 contributes more than the DNA-binding properties to drive Ewing sarcoma biology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1305.

<p>Supp Figure 4 depicts the overlap analysis of EWS/ETS and FUS/ETS proteins at both the genomic localization and transcriptional regulatory levels.</p>

<p>Supp Figure 2 shows knockdown of endogenous EWS/FLI and expression of alternative FET/ETS fusions in the A673 Ewing sarcoma cell line.</p>