Abstract

Ewing sarcoma (ES) is a primary malignant bone tumor that predominantly affects children, adolescents, and young adults. First described by James Ewing in the early 20th century, ES arises from a chromosomal translocation involving a member of the FET protein family, most commonly {EWSR1} (EWS RNA-binding protein 1), and a member of the ETS transcription factor family. In approximately 85% of cases, the translocation {t(11;22)(q24;q12)} fuses {EWSR1} with {FLI1}, generating the oncogenic protein {EWSR1–FLI1} {1522903}. This aberrant transcription factor reshapes the epigenetic landscape, driving malignant transformation by simultaneously activating oncogenes and repressing tumor suppressors {29945296}. Although {EWSR1–FLI1} is the defining molecular lesion of ES, tumor cells display remarkable heterogeneity in its expression and activity {28135250}, giving rise to diverse phenotypic states that underlie therapy resistance and disease progression {32049009}. Understanding the molecular circuitry orchestrated by {EWSR1–FLI1} is therefore crucial, as the identification of novel therapeutic and prognostic targets remains a major unmet need in this aggressive disease.

Content

Introduction
Ewing sarcoma encompasses a group of highly aggressive tumors that most frequently originate in bone, though in some cases they arise in the surrounding soft tissues ¹. The femur, tibia, pelvis, and ribs are the most common primary sites, whereas vertebral involvement is less frequent. Although the disease can occur at any age, its incidence peaks in children and adolescents, in whom it represents the second most common primary malignant bone tumor ². ES is not generally considered a hereditary cancer, yet rare susceptibility loci have been reported, suggesting that multifactorial familial transmission may occur in exceptional cases ³. Despite recent progress in the molecular understanding of this neoplasm, survival outcomes remain disappointing, with cure rates of 70–80% in patients with localized, standard-risk disease, but only about 30% for those presenting with metastasis ⁴. The aggressive nature of ES is largely due to frequent relapses, therapy resistance, and a high metastatic potential. Genetically, ES is characterized by a very low mutational burden ⁵, and its oncogenesis is instead driven by recurrent chromosomal translocations that give rise to fusion genes. In about 85% of cases, the translocation fuses EWSR1 on chromosome 22 with FLI1 on chromosome 11, producing the EWSR1::FLI1 oncoprotein ⁶. In approximately 10% of cases, EWSR1 fuses with ERG, generating EWSR1::ERG ⁷, while the remaining cases involve other ETS family members. The type of translocation does not appear to significantly influence survival ⁸. The EWSR1::FLI1 fusion protein acts as an aberrant transcription factor and chromatin regulator, promoting widespread reprogramming of gene expression and thereby transforming precursor cells into malignant ones ⁹.
The FET and ETS Protein Families
The oncogenic potential of ES is rooted in the biology of the FET and ETS families. The FET family includes FUS (chromosome 16), TAF15 (chromosome 17), and EWSR1 (chromosome 22) ¹⁰. These proteins share characteristic domains that regulate transcription, post-transcriptional processing, and DNA damage repair ^10,11. Their structure is characterized by an intrinsically disordered prion-like N-terminal domain rich in SYGQ repeats, a central RNA recognition motif (RRM), three arginine–glycine–glycine (RGG) domains, and a zinc finger domain. The ETS family, to which FLI1 belongs, is composed of 26 transcription factors defined by a conserved winged helix-turn-helix DNA-binding domain that recognizes purine-rich motifs (GGA[A/T]). The FLI1 protein contains several functional regions: the FLI1-specific (FLS) domain with an N-terminal activation sequence, a high-affinity DNA-binding ETS domain, and a C-terminal activation domain ^12,13. ETS proteins regulate a wide spectrum of cellular processes, including proliferation, differentiation, migration, and survival ¹⁴.
The EWSR1::FLI1 Fusion Protein
The chromosomal translocation between EWSR1 and FLI1 produces multiple transcripts depending on the breakpoint, the most frequent being the so-called “type I” fusion, which joins exons 1–7 of EWSR1 with exons 6–10 of FLI1 ⁶. The resulting chimeric protein integrates the low-complexity SYGQ transactivation domain of EWSR1 with the FLS, ETS, and C-terminal activation domains of FLI1 ¹⁵. Functionally, this hybrid structure confers the ability to bind DNA at both canonical ETS motifs and extended GGAA repeats ¹⁶. Importantly, the protein can convert GGAA microsatellites into active regulatory elements, with transcriptional strength depending on the number of repeats, optimally between 18 and 26 ¹⁷. Genes associated with multiple GGAA repeats are typically activated, whereas those with fewer repeats are often repressed ¹⁸. This gain-of-function property allows EWSR1::FLI1 to regulate a broader gene repertoire than wild-type FLI1.

Figure 1
The figure illustrates the molecular pathway that leads to the formation of EWSR/FLI1 fusion protein. The cromosomal translocation produces the fusion gene EWSR-FLI1, with a specific combination of domains: N-terminal domain of EWSR1 SYGQ characterized by low complexity and intrinsically disordered, and the FLS, ETS and PCYT1A domains from FLI1. Resulting from this specific domain combination the fusion protein binds DNA using the ETS 3’ domain of FLI1 and induces gene expression through the TAD amino-terminal EWSR1 domain.

Regulation of EWSR1–FLI1 Expression
The expression of EWSR1::FLI1 is tightly controlled at multiple levels. Transcriptionally, the fusion gene is driven by the constitutively active EWSR1 promoter ⁴, with histone modifications such as H3K4me3, H3K9ac, and H3K27ac facilitating transcriptional initiation ¹⁹. The transcription factor SP1 contributes to promoter activity ²⁰, while pre-mRNA splicing involves SF3B1 and HNRNPH1 ^21,22, and stabilization of the mature transcript is mediated by LIN28B ²². At the post-translational level, protein stability is regulated by ubiquitination. Degradation is promoted by the E3 ligases TRIM8 and SPOP, whereas deubiquitinases such as USP19 and OTUD7A counteract proteasomal degradation, thereby prolonging protein half-life ^23,24.
Mechanisms of Action of EWSR1::FLI1
The pathogenic effects of EWSR1::FLI1 arise from its ability to reprogram transcriptional networks. The fusion protein simultaneously induces oncogenes such as MYC ²⁵ and GLI1 ²⁶, while repressing tumor suppressors including FOXO1 ²⁷ and IER3 ²⁸. These effects are mediated by the synergy between its ETS DNA-binding domain and the intrinsically disordered prion-like SYGQ domain, which enables recruitment of multiple chromatin regulators ^18,29. Among its key partners are SWI/SNF ³⁰, histone acetyltransferase EP300 ¹⁸, BRD1 ³¹, RNApolII, STAG2 ³², KDM1A with or without NuRD ³³, BRD4 ³⁴, and KDM3A ³⁵. At the chromatin level, EWSR1–FLI1 binding to GGAA repeats creates de novo enhancers marked by H3K4me1 and H3K27ac, while binding to canonical ETS motifs can lead to repression, frequently involving histone deacetylases (HDAC9) ³⁶ or LSD1–NuRD complexes ^33,37. These activities result in profound remodeling of 3D chromatin structure, including enhancer–promoter looping that activates oncogenic transcription ^38,39. The outcome of EWSR1::FLI1 action is also influenced by cooperating and antagonistic transcription factors. MEIS1 and RUNX3 can enhance its transactivation potential ^40,41, while HOXD13 counteracts its repressive functions ⁴².
Heterogeneity of Ewing Sarcoma
Despite being driven by a single defining translocation, ES is a heterogeneous disease ⁴³. Tumor cells vary in their levels of EWSR1::FLI1 expression and transcriptional activity, resulting in distinct phenotypic states ⁴⁴. Cells with high expression of the fusion protein exhibit increased proliferation, oxidative phosphorylation, and tight cell–cell adhesion, whereas cells with low expression display enhanced migratory capacity, extracellular matrix interactions, and a hypoxia-adapted phenotype, which are associated with metastatic potential ^24,45. This phenotypic plasticity arises from dynamic modulation of EWSR1::FLI1 activity by chromatin remodelers, transcriptional cofactors, and external cues such as Wnt/β-catenin signaling, the YAP/TAZ/TEAD axis, receptor tyrosine kinases (RTK), hypoxia, growth factors, and immunosuppressive T cells in the bone marrow ⁴⁶.

Figure 2. 
EWSR1::FLI1 by binding GGAA promoters and enhancers interacts with histone aceryltransferase EP300, MEIS and KMT2A leading to histone modifications such as H3K27ac H3K4me3. These modifications can make chromatin more accessible to RNApolII, transcription factors and regulators proteins.


Figure 3. 
EWSR1::FLI1 by binding to sites with one or a few repetitions GGAA displace the ETS transcription factors and chromatin remodelers associated with them (which normally determine open chromatin) inactivating the potentiators and leading to reduced gene transcription. Associated with the repressor activity of EWSR1::FLI1 have been identified proteins that determine chromatin changes associated with repression such as histone deacetylases (HDAC), KDM1A with NuRD.


Conclusions
EWSR1::FLI1 is the central oncogenic driver of Ewing sarcoma, functioning as an aberrant transcription factor and chromatin remodeler that reprograms gene expression and underlies malignant transformation. While advances in understanding its molecular mechanisms have identified new therapeutic opportunities, the protein remains a challenging target due to its extensive interactome. Nevertheless, the cofactors and pathways modulated by EWSR1::FLI1 provide promising alternative targets for intervention. Crucially, the heterogeneity and plasticity of ES must be taken into account, as therapeutic strategies aimed solely at inhibiting EWSR1::FLI1 may fail to eliminate subpopulations with EWSR1::FLI1 “low” activity or may inadvertently promote their expansion. Deeper mechanistic insights into how EWSR1::FLI1 governs tumor biology are therefore essential for the development of more effective therapies capable of improving patient survival and quality of life.