2. Involvement of EWS-FLI1 in Transcription, Epigenetic Reprogramming, and Alternative Splicing in EwS
2.1. EWS-FLI1 in Transcription and Epigenetic Reprogramming
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
[10][11][12][13][14][15][16][17][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
[18][19][20][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
[10][11][18][20][29,30,37,39]. The EWS-FLI1 protein complex includes RNA polymerase II
[21][22][23,40], the core subunit hsRBP7 (human RNA polymerase II)
[23][24][41,42], E2F3
[25][26][43,44], EWSR1
[27][45], CBP/p300
[28][46], WDR5, ASH2, MLL
[11][30], and the BAF complex (mammalian SWI/SNF complex)
[10][29][29,47]. The threshold of GGAA motifs optimal for maximal expression is 20–26
[30][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
[31][32][49,50]. This specific coupling results in the activation of many genes (
Figure 1), such as NKX2.2
[33][51], NROB1
[34][35][36][52,53,54], IGF1R
[37][55], BCL11B
[38][56], EZH2
[17][36], VRK1
[11][30], GLI1
[39][57], PTPL1
[40][58], PPPR1A
[41][59], ERG2
[42][60], GSTM4
[43][61], PAX7
[44][62], CHM1
[45][63], REST
[46][64], PHF19
[13][32], STEAP1
[47][48][65,66], SLFN11(Schlafen 11)
[49][67], HDAC3
[50][68], TNC
[51][69], APCDD1
[31][49], IL1RAP
[52][53][70,71], MYC
[54][72], and PRC1 (protein regulator of cytokinesis 1)
[55][73].
Figure 1. The EWS-FLI1 protein complex drives the specific transcription profile of EwS. EWS-FLI1 recruits E2F3, hsRBP7, BAF, RING1B, RHA, P300, and MEIS1, among others, to GGAA repeats and further activates CHM1, EZH2, PAX7, NKX2.2, NROB1, and STEAP1, among others. EWS-FLI1 functions as a protein complex with ERG, BAF, RHA, DDX5, and U1C to drive alternative splicing. Among the genes, the purple ones, such as CHM1, could serve as TCR-based immunotherapy targets; the red ones, such as NKX2-2, serve as diagnostic markers in clinic diagnosis. EWS/FLI1 recruits LSD1 and unknown transcription factors (?) to repress TGFBR1 and IGFBP3, which still needs further research.
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
[56][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
[57][75], such as STEAP1
[58][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
[59][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
[49][60][67,78]. STEAP1 and IL1RAP are vital for the redox homeostasis of EwS
[47][52][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
[61][79] and PHLDA1
[35][53] to drive oncogenic transformation
[12][62][31,80]. The nucleosome remodeling and deacetylase (NuRD) complex is a typical ATP-dependent chromatin remodeling complex
[63][81] that plays a critical role in transcription and determines differentiation and development
[64][82]. EWS-FLI1 recruits the NuRD-LSD1 complex to repress LOX and TGFBR2
[62][65][80,83]. It also affects the transcriptional activation of AP-1
[14][33] and MRTFB
[66][84] and binds to the promotor of FOXO1 to repress its expression, thereby increasing tumor growth
[67][85]. EWS-FLI1 promotes the phosphorylation of cyclin-dependent kinase-2 and AKT to inhibit the activity of FOXO1, thus rewiring transcriptional repression
[67][85]. EWS-FLI1 is also involved in the regulation of microRNAs (miRNAs)
[68][86]. It downregulates miRNA-145 to initiate mesenchymal stem-cell reprogramming toward EwS stem cells
[69][87] and represses miR-708, which induces the overexpression of EYA3 and contributes to the chemoresistance to etoposide and doxorubicin
[70][88].
The histone methyltransferase EZH2 exhibits silencing activity via methylation of H3K27
[71][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
[17][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
[72][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
[73][74][75][91,92,93], interacts with EWS-FLI1 based on mass spectrometry
[76][94]. XPO5 is highly expressed in various cancers including EwS. Furthermore, the phosphorylation of XPO5 alters the nucleus and cytoplasm shift
[77][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
[78][79][80][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
[81][99]. However, PCAF expression is lower in EwS tissues, which is a common feature of cancer.
2.2. EWS-FLI1 in Alternative Splicing
Pre-mRNA splicing is critical for gene expression, and most protein-encoding transcripts are alternatively spliced to provide diverse functions
[82][83][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
[21][23], thus demonstrating the negative property of this chimeric protein
[84][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
[85][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
[86][104]. Work by Selvanathan
[76][94] demonstrates that EWS-FLI1 is involved in the alternative splicing of CLK1, CASP3, PPFIBP1, and TERT, which potentially regulate the oncogenesis of EwS.
3. The Regulation of EWS-FLI1
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
[13][15][87][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
[88][106]. MiR-145 and let-7 repress EWS-FLI1 by targeting its mRNA
[69][89][90][91][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
[90][108]. At the post-transcriptional level, EWS-FLI1 degradation is proteasome dependent, and the protein has a half-life of 2–4 h
[92][110]. This process can be protected by the action of ubiquitin-specific protease 19 (USP19) at the N-terminus
[93][111] and accelerated by tripartite-motif-containing 8 (TRIM8) at K334
[94][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
[95][113]. The inhibitor of chromosomal maintenance 1 (CRM1 and XPO1), KPT-330
[96][114], and IFN-γ
[97][115] suppress expression of EWS-FLI1 at the protein level. FOXM1, a downstream factor of EWS-FLI1, upregulates its expression
[98][116]. Cytosine arabinoside (ARA-C) downregulates EWS-FLI1 at the protein level and inhibits tumor growth
[99][117]; however, it shows hematologic toxicity and minimal activity in patients
[100][118].
STAG2 (stromal antigen 2) is a core subunit of the cohesion complex and is frequently mutated in multiple cancers
[101][119] including EwS
[102][103][10,120]. Mutation of STAG2 in EwS is associated with poor outcomes by improving metastasis
[102][10]. Mechanically, in addition to the disruption of PRC2-mediated regulation of gene expression in EwS
[104][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
[104][105][121,122].
Unlike STAG2, there was no evidence showing mutations of the ETS transcription factor ETV6 in EwS
[106][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
[107][108][124,125]. Upon inactivating ETV6, EWS-FLI1 overtakes and activates these cis-elements to promote mesenchymal differentiation by upregulating the expression of SOX11
[108][125].
4. CAR-T Therapy
Unlike TCR-based T-cell therapy, which is limited to specific HLA restriction and deficient HLA expression in EwS
[109][205] because of the presence of myeloid-derived suppressor cells, F2 fibrocytes, and M2-like macrophages in the microenvironment
[110][190], CAR-engineered T-cell therapy can target specific cell-surface antigens in tumors, independent of HLA. VEGFR2 is a potential target for CAR-T-cell therapy in EwS
[111][206]. In addition to TCR-T-cells, CAR-T-cells
[112][207] can target STEAP1, which is involved in the malignant phenotype of EwS
[47][65].
CAR-T targeting GPR64, ROR1, and IGF1R, which are highly expressed in EwS
[113][114][137,208], leads to a selective killing of EwS in vivo
[115][209]. LINGO1, which is highly expressed in EwS
[116][210], is a direct target of EWS-FLI1
(Supplementary Figure S4B). EZH2 inhibition by GSK-126 induces GD2 surface expression in EwS
[117][211], and the combination of CAR-T therapy targeting GD2 and EZH2 inhibitors have synthetic cytotoxic in the treatment of EwS; this kind of combination provides new options for the clinical application. IL1RAP, a direct target of EWS-FLI1, is highly expressed in EwS, but minimally expressed in normal tissues, which makes it a promising surface target for EwS
[52][70] and a potential candidate for advanced CAR-T therapy. ICAM-1 can promote tumor cell/T-cell interaction and T-cell activation, and the knockdown of EWS-FLI1 upregulates ICAM-1 expression and leads to the upregulation of PD-L1 and PD-L2, both proteins that inhibit the activity of T-cells
[97][115]. Blocking PD-1 with a checkpoint inhibitor could increase the T-cell-mediated killing of EwS cells with low expression of EWS-FLI1.