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Locus-selective targeting of the oncogenic fusion EWSR1-FLI1 in Ewing Sarcoma by CRISPR/dCas9 silencers

ABSTRACT: Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle for clinical applications. Here, we utilize precision molecular targeting and delivery strategies based on CRISPR/dCas9 systems adapted for epigenetic repression (dCas9-KRAB) to silence oncogenic drivers with high selectivity. As proof-of-principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing Sarcoma (EWS)-an aggressive childhood malignancy. We describe the development of a programmable non-viral polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for selective EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in EWS-related patient-derived xenografts (PDXs) of EWS. We show that silencing of EWSR1–FLI1 is accompanied by potent anti-tumor effects. To our knowledge, this represents the first effective non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which could be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE334757 | GEO | 2026/09/03

REPOSITORIES: GEO

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Locus-selective targeting of the oncogenic fusion EWSR1-FLI1 in Ewing Sarcoma by CRISPR/dCas9 silencers [DNA methylation]

Project description:Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle for clinical applications. Here, we utilize precision molecular targeting and delivery strategies based on CRISPR/dCas9 systems adapted for epigenetic repression (dCas9-KRAB) to silence oncogenic drivers with high selectivity. As proof-of-principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing Sarcoma (EWS)-an aggressive childhood malignancy. We describe the development of a programmable non-viral polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for selective EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in EWS-related patient-derived xenografts (PDXs) of EWS. We show that silencing of EWSR1–FLI1 is accompanied by potent anti-tumor effects. To our knowledge, this represents the first effective non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which could be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.

2026-09-03 | GSE334759 | GEO

KDM6A and B-mediated transcriptional activation of EWSR1-FLI1 targets coordinates demethylase dependent and independent mechanisms in Ewing sarcoma

Project description:Ewing Sarcoma (EwS) is a highly aggressive tumor arising in bones and soft tissues driven by the fusion oncoprotein EWSR1::FLI1. This aberrant transcription factor binds to GGAA microsatellites, causing epigenetic reprogramming through the formation of active neo-enhancers in a permissive cellular context. Inhibition of the oncogene remains challenging, and current efforts seek to exploit emergent epigenetic treatments targeting EWSR1::FLI1 cofactors. In EwS, neo-enhancers are characterized by strong enrichment of the active H3K27ac mark and concomitant lack of the repressive H3K27me3 mark. These regions are typically decorated with high levels of H3K27me3 prior to EWSR1::FLI1-activation. Here, upon expression of EWSR1::FLI1 in human pediatric mesenchymal stem cells, considered the putative cell of origin of EwS, we unraveled the genome-wide redistribution of H3K27me3. Stemming from these results, we elucidated the contribution of the H3K27me3 demethylases KDM6A/UTX and KDM6B/JMJD3 in the transcriptional activity of EWSR1::FLI1 induced enhancers. KDM6A had a demethylase-independent role in recruiting BRG1 at EWSR1::FLI1-primed enhancers containing single GGAA motifs, which was critical for EwS tumor growth. Conversely, KDM6B demethylated H3K27me3 at specific EWSR1::FLI1-active enhancers, co-localizing with BRG1 at GGAA repeats. Loss of KDM6B impaired the growth of EwS tumor xenografts. These results highlight KDM6 demethylases as EWSR1::FLI1 functional partners with potential as targets for treating EwS.

2025-08-18 | GSE211743 | GEO

Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

Project description:Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). The LC domain in EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and can self-assemble through a process known as phase separation. The ability of EWSR1 and related RNA-binding proteins to assemble into ribonucleoprotein granules in cells has been intensely studied but the role of phase separation in EWS-FLI1 activity is less understood. We investigated the overlapping functions of EWSR1 and EWS-FLI1 in controlling gene expression and tumorigenic cell growth in Ewing sarcoma, and our results suggested that these proteins function closely together. We then studied the nature of interactions among EWS-FLI1, EWSR1, and RNA Pol II. We observed EWSR1 and RNA Pol II to be present in protein granules in cells. We then identified protein granules in cells associated with the fusion protein, EWS-FLI1. The tyrosine residues in the LC domain are required for the abilities of EWS-FLI1 to bind its partners, EWSR1 and RNA Pol II, and to incorporate into protein granules. These data suggest that interactions among EWS-FLI1, RNA Pol II, and EWSR1 in Ewing sarcoma can occur in the context of a molecular scaffold found within protein granules in the cell.

2020-07-23 | GSE154944 | GEO

Ewing sarcoma immunopeptidomics

Project description:Ewing Sarcoma (EwS) is a rare pediatric malignancy characterized by a unique t(11:22) (q24;q12) translocation resulting in the EWSR1::FLI1 fusion. Recent reports indicate the EWSR1::FLI1 fusion onco-protein drives aberrant expression of numerous transcripts, including Lipoxygenase Homology Domains 1 (LOX¬HD1). Given its highly restricted protein expression pattern and role in EwS tumorigenesis and metastasis, LOXHD1 may serve as a novel immunotherapeutic target in this malignancy. LOXHD1 immunogenic epitopes restricted to HLA-A*02:01 allowed for the isolation of high avidity TCRs. LOXHD1-specific TCR engineered CD8+ T cells confer cytotoxic activity against a panel of HLA-A*02:01+ EwS tumor cell lines and adoptive transfer leads to tumor eradication in a mouse xenograft model of EwS. This study nominates LOXHD1 as an onco-fusion regulated / non-mutated EwS tumor associated antigen with expression limited to inner hair cells of the cochlea, adult testis, and EwS

2025-05-06 | PXD046803 | Pride

Therapeutic Targeting Ewing Sarcoma through Inhibition of the FACT Complex (RNA-Seq II)

Project description:EWS/ETS fusion transcription factors, most commonly EWSR1-FLI1, drive initiation and progression of Ewing sarcoma (EwS), a highly aggressive childhood cancer of bone and soft tissues. Even though direct targeting EWSR1-FLI1 is a formidable challenge, epigenetic or transcriptional modulators have been recently proved to be promising therapeutic targets for indirectly disrupting its expression and/or function. Here, we performed transcriptome and functional genomics dataset analyses, and combined with small molecule screening of EwS lines to identify novel epigenetic/transcriptional-targeted therapeutic strategies. SSRP1 and SUPT16H, two subunits of the Facilitates Chromatin Transcription (FACT) complex, are both found to be EWSR1-FLI1-induced essential oncogenes in EwS. The FACT-targeted drug CBL0137 exhibits potent therapeutic efficacy against multiple EwS preclinical models in vitro and in vivo. Mechanistically, the FACT complex and EWS-FLI1 form oncogenic positive feedback loop via mutual transcriptional regulation and activation, and cooperatively promote cell cycle/DNA replication process and IGF1R-PI3K-AKT-mTOR pathway to drive EwS oncogenesis. The FACT inhibitor drug CBL0137 effectively targets the EWSR1-FLI1-FACT circuit, resulting in transcriptional disruption of EWS-FLI1, SSRP1 and their downstream effector oncogenic signatures. Our study illustrates a crucial role of the FACT complex in facilitating the expression and function of EWSR1-FLI1 and demonstrates FACT inhibition as a novel therapeutic strategy against EwS via indirect targeting the oncogenic fusion TF, providing preclinical support for adding EwS to CBL0137’s future clinical trials.

2022-12-31 | GSE195804 | GEO

Therapeutic Targeting Ewing Sarcoma through Inhibition of the FACT Complex (RNA-Seq I)

2022-12-31 | GSE195803 | GEO

Therapeutic Targeting Ewing Sarcoma through Inhibition of the FACT Complex (ChIP-Seq)

2022-12-31 | GSE195802 | GEO

New Cell lines Expanding the Diversity of Ewing Sarcoma Models – WXS Data

Project description:Ewings sarcoma (EwS) is a highly aggressive, malignant, solid tumor of childhood and adolescence. Most EwS develop in bone, while it originates from connective, adipose or muscle tissue less frequently. We report the establishment of new human EwS cell lines, all of which carry a t(11;22) (q24;q12) translocation generating the oncogenic transcription factor EWSR1::FLI1. Sequencing of the chimeric mRNAs indicated genomic DNA breakpoints localized in intron 8 of the EWSR1 gene and 3 different introns of translocation partner gene FLI1. While three EwS cell lines carry the EWSR1::FLI1 fusions of ex7/ex6 (type I) or ex7/ex5 (type II), the EWSR1::FLI1 fusion variant ex7/ex7 (type IV) is described for the first time in a continuous EwS model. The cell lines presented genomic, epigenomic and transcriptomic stability over a period of six months, though some variations in the chromosomal aberrations were observed in one cell line. The transcriptional variation within the novel EwS cell line panel might have been influenced by the EWSR1::FLI1 fusion type and TP53 mutations. Furthermore, the TP53 mutational status seemed to have the biggest impact on drug sensitivity profiles. The new EwS models presented here may help to identify small molecule inhibitors that act directly on EWSR1::FLI1 fusion proteins or uncover other genetic vulnerabilities of the altered epigenome and transcriptome in EwS, which would contribute to a better understanding of Ewing sarcoma tumorigenesis.

2025-08-22 | GSE289064 | GEO

New Cell lines Expanding the Diversity of Ewing Sarcoma Models – WGS Data

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New Cell lines Expanding the Diversity of Ewing Sarcoma Models - methylation

Project description:Ewings sarcoma (EwS) is a highly aggressive, malignant, solid tumor of childhood and adolescence. Most EwS develop in bone, while it originates from connective, adipose or muscle tissue less frequently. We report the establishment of new human EwS cell lines, all of which carry a t(11;22) (q24;q12) translocation generating the oncogenic transcription factor EWSR1::FLI1. Sequencing of the chimeric mRNAs indicated genomic DNA breakpoints localized in intron 8 of the EWSR1 gene and 3 different introns of translocation partner gene FLI1. While three EwS cell lines carry the EWSR1::FLI1 fusions of ex7/ex6 (type I) or ex7/ex5 (type II), the EWSR1::FLI1 fusion variant ex7/ex7 (type IV) is described for the first time in a continuous EwS model. The cell lines presented genomic, epigenomic and transcriptomic stability over a period of six months, though some variations in the chromosomal aberrations were observed in one cell line. Transcriptome and epigenome were stable over time and there was only little variation between the novel EwS cell lines. The TP53 mutational status seemed to have the biggest impact on drug sensitivity profiles. The new EwS models presented here may help to identify small molecule inhibitors that act directly on EWSR1::FLI1 fusion proteins or uncover other genetic vulnerabilities of the altered epigenome and transcriptome in EwS, which would contribute to a better understanding of Ewing sarcoma tumorigenesis.

2025-08-22 | GSE289061 | GEO

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