EWS-FLI1 is an aberrant transcription factor that drives cellular transformation by rewiring the epigenome to induce a large number of oncogenes. The N-terminus of EWSR1 contains a prion-like domain, characterized by an intrinsically disordered structure and low complexity. This domain has phase transition properties and manipulates multiple proteins involved in epigenome reprogramming and epigenetic alterations [29,30,31,32,33,34,35,36]. In addition to the canonical ETS-binding sites, EWS-FLI1 binds to DNA sequences at the GGAA/T core motif [37,38,39] via a conserved ETS domain. It regulates multiple proteins through its prion-like domain to tumor-specific enhancers and promotors, recruiting acetyltransferases and establishing de novo enhancers by generating H3K27ac, thus opening the chromosomal architecture, which contributes to the activation of target genes [29,30,37,39]. The EWS-FLI1 protein complex includes RNA polymerase II [23,40], the core subunit hsRBP7 (human RNA polymerase II) [41,42], E2F3 [43,44], EWSR1 [45], CBP/p300 [46], WDR5, ASH2, MLL [30], and the BAF complex (mammalian SWI/SNF complex) [29,47]. The threshold of GGAA motifs optimal for maximal expression is 20–26 [48], which differs from that in wild-type FLI1. Super-enhancer-associated MEIS1 and RING1B also contribute to the chromatin reprogramming through co-localization with EWS-FLI1 at the active enhancers to drive the malignancy of EwS [49,50]. This specific coupling results in the activation of many genes (Figure 1), such as NKX2.2 [51], NROB1 [52,53,54], IGF1R [55], BCL11B [56], EZH2 [36], VRK1 [30], GLI1 [57], PTPL1 [58], PPPR1A [59], ERG2 [60], GSTM4 [61], PAX7 [62], CHM1 [63], REST [64], PHF19 [32], STEAP1 [65,66], SLFN11(Schlafen 11) [67], HDAC3 [68], TNC [69], APCDD1 [49], IL1RAP [70,71], MYC [72], and PRC1 (protein regulator of cytokinesis 1) [73].

Among the direct targets of EWS-FLI1, NKX2-2 mediates oncogenic transformation via transcriptional repression and is necessary and sufficient for the oncogenic phenotype of EwS [74]. Further work demonstrates that NKX2-2, KLF15, and TCF4 occupy similar super-enhancers and promoters, forming an inter-connected auto-regulatory loop and occupying 77.2% of promoters and 55.6% of enhancers shared with EWS-FLI1 [75], such as STEAP1 [76]; this kind of coordinate regulation drives the proliferation of EwS. NROB1 directly interacts with EWS-FLI1 to modulate multiple gene expressions and mediate the oncogenic phenotype of EwS [77]. SLFN11 is a putative DNA/RNA helicase that recruits to the stressed replication fork and irreversibly triggers replication block and cell death. Overexpression of SLFN11 is associated with resistance to topoisomerase I inhibitors and poly (ADP-ribose) polymerase (PARP) inhibitor combinations [67,78]. STEAP1 and IL1RAP are vital for the redox homeostasis of EwS [65,70]. APCDD1, PHF19, GSTM4, and PTPL1 are genes that are involved in the proliferation of EwS.

EWS-FLI1 is also involved in transcriptional repression of tumor suppressors such as IGFBP3 [79] and PHLDA1 [53] to drive oncogenic transformation [31,80]. The nucleosome remodeling and deacetylase (NuRD) complex is a typical ATP-dependent chromatin remodeling complex [81] that plays a critical role in transcription and determines differentiation and development [82]. EWS-FLI1 recruits the NuRD-LSD1 complex to repress LOX and TGFBR2 [80,83]. It also affects the transcriptional activation of AP-1 [33] and MRTFB [84] and binds to the promotor of FOXO1 to repress its expression, thereby increasing tumor growth [85]. EWS-FLI1 promotes the phosphorylation of cyclin-dependent kinase-2 and AKT to inhibit the activity of FOXO1, thus rewiring transcriptional repression [85]. EWS-FLI1 is also involved in the regulation of microRNAs (miRNAs) [86]. It downregulates miRNA-145 to initiate mesenchymal stem-cell reprogramming toward EwS stem cells [87] and represses miR-708, which induces the overexpression of EYA3 and contributes to the chemoresistance to etoposide and doxorubicin [88].

The histone methyltransferase EZH2 exhibits silencing activity via methylation of H3K27 [89]. In EwS, EWS-FLI1 upregulates EZH2 expression by interacting with the EZH2 promoter, thereby promoting tumor growth/metastasis and blocking endothelial/neuro-ectodermal differentiation [36].

MiR-34a inhibits the proliferation and increases the sensitivity of EwS to doxorubicin and vincristine and is a strong predictor of a favorable prognosis in EwS [90]. However, the exact mechanism underlying its downregulation remains elusive. Exportin 5 (XPO5), which mediates the nuclear export of pre-miRNAs and short hairpin RNAs [91,92,93], interacts with EWS-FLI1 based on mass spectrometry [94]. XPO5 is highly expressed in various cancers including EwS. Furthermore, the phosphorylation of XPO5 alters the nucleus and cytoplasm shift [95]. Investigating XPO5 and its relationship with EWS-FLI1 may offer new insights into the therapy of EwS. Post-translational modifications of EWS-FLI1 modulate its transcriptional activity. Phosphorylation and O-GlcNAcylation of the N-terminus of EWSR1 [96,97,98], as well as acetylation of the C-terminal FLI1 domain by PCAF (KAT2B, lysine acetyltransferase 2B), enhance the transcriptional activity of EWS-FLI1 [99]. However, PCAF expression is lower in EwS tissues, which is a common feature of cancer.

Pre-mRNA splicing is critical for gene expression, and most protein-encoding transcripts are alternatively spliced to provide diverse functions [100,101]. The N-terminus of EWSR1 interacts with the hyperphosphorylated RNA polymerase II and recruits serine-arginine (SR) through its C-terminus. After chromosome translocation, the C-terminus of wild-type EWSR1 is replaced by FLI1, which hinders the recruitment of SR-splicing factors and interferes with mRNA splicing [23], thus demonstrating the negative property of this chimeric protein [102]. This leads to comprehensive alternative splicing of numerous genes. Meanwhile, EWS-FLI1 interacts with the splicing components (snRNP) U1C and SF1 to modulate pre-mRNA splicing [103]. It also recruits the BAF complex to drive the alternative splicing of ARID1A and the preferential splicing of ARID1A-L, which is necessary for tumor growth [104]. Work by Selvanathan [94] demonstrates that EWS-FLI1 is involved in the alternative splicing of CLK1, CASP3, PPFIBP1, and TERT, which potentially regulate the oncogenesis of EwS.

Transcription and post-transcriptional modifications are involved in the regulation of expression and activity of EWS-FLI1. Although the transcriptional regulation of EWS-FLI1 remains elusive, the BRD4 inhibitor JQ1 suppresses this activity [32,34,105]. HDAC6 deacetylates specificity protein 1 (SP1), thereby inhibiting the recruitment of the SP1/P300 complex to the promoters of EWSR1 and EWS-FLI1 and downregulating EWS-FLI1 [106]. MiR-145 and let-7 repress EWS-FLI1 by targeting its mRNA [87,107,108,109] and inhibit the proliferation of EwS. The RNA-binding protein LIN28B interacts with EWS-FLI1 transcripts to maintain the stability and ensure the expression of EWS-FLI1 to enhance the tumorigenicity of the self-renewal of EwS [108]. At the post-transcriptional level, EWS-FLI1 degradation is proteasome dependent, and the protein has a half-life of 2–4 h [110]. This process can be protected by the action of ubiquitin-specific protease 19 (USP19) at the N-terminus [111] and accelerated by tripartite-motif-containing 8 (TRIM8) at K334 [112]; however, USP19 is expressed at low levels in EwS. Casein kinase 1 (CK1)-mediated phosphorylation of the VTSSS degron in the FLI1 domain activates speckle-type POZ protein (SPOP) activity, which degrades EWS-FLI1. In contrast, OTU-domain-containing protein 7A (OTUD7A) participates in the deubiquitination of the C-terminus and stabilizes EWS-FLI1 [113]. The inhibitor of chromosomal maintenance 1 (CRM1 and XPO1), KPT-330 [114], and IFN-γ [115] suppress expression of EWS-FLI1 at the protein level. FOXM1, a downstream factor of EWS-FLI1, upregulates its expression [116]. Cytosine arabinoside (ARA-C) downregulates EWS-FLI1 at the protein level and inhibits tumor growth [117]; however, it shows hematologic toxicity and minimal activity in patients [118].

STAG2 (stromal antigen 2) is a core subunit of the cohesion complex and is frequently mutated in multiple cancers [119] including EwS [10,120]. Mutation of STAG2 in EwS is associated with poor outcomes by improving metastasis [10]. Mechanically, in addition to the disruption of PRC2-mediated regulation of gene expression in EwS [121], the inactivation of STAG2 strongly altered CTCF-anchored loop extrusion and decreases promotor-enhancer interactions. As a result, the cis-mediated EWS-FLI1 activity at GGAA microsatellite neo-enhancers is downregulated and the cells are enhanced in their migration and invasion properties [121,122].

Unlike STAG2, there was no evidence showing mutations of the ETS transcription factor ETV6 in EwS [123]. ETV6 does not change the expression of EWS-FLI1 but co-occupy loci genome wide at the short consecutive GGAA repeats and constrains the transcriptional activity of EWS-FLI1 [124,125]. Upon inactivating ETV6, EWS-FLI1 overtakes and activates these cis-elements to promote mesenchymal differentiation by upregulating the expression of SOX11 [125].

4. CAR-T Therapy