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TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma.

ABSTRACT: Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.

SUBMITTER: Seong BKA

PROVIDER: S-EPMC8443273 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The role of FLI-1-EWS, a fusion gene reciprocal to EWS-FLI-1, in Ewing sarcoma.

Project description:Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation involving EWS and an Ets family transcription factor, most commonly FLI-1. The EWS-FLI-1 fusion oncogene is widely believed to play a central role in Ewing sarcoma. The EWS-FLI-1 gene product regulates the expression of a number of genes important for cancer progression, can transform mouse cells such as NIH3T3 and C3H10T1/2, and is necessary for proliferation and tumorigenicity of Ewing sarcoma cells, suggesting that EWS-FLI-1 is the causative oncogene. However, a variety of evidence also suggest that EWS-FLI-1 alone cannot fully explain the Ewing sarcomagenesis. Here we report that FLI-1-EWS, a fusion gene reciprocal to EWS-FLI-1, is frequently expressed in Ewing sarcoma. We present evidence suggesting that endogenous FLI-1-EWS is required for Ewing sarcoma growth and that FLI-1-EWS cooperates with EWS-FLI-1 in human mesenchymal stem cells, putative cells of origin of Ewing sarcoma, through abrogation of the proliferation arrest induced by EWS- FLI-1.

| S-EPMC4701224 | biostudies-literature

Ewing sarcoma cells secrete EWS/Fli-1 fusion mRNA via microvesicles.

Project description:Tumours defined as Ewing sarcoma (ES) constitute a group of highly malignant neoplasms that most often affect children and young adults in the first 2 decades of life. The EWS/Fli-1 fusion gene, a product of the translocation t(11;22) (q24; 12), is detected in 95% of ES patients. Recently, it was validated that cells emit a heterogeneous mixture of vesicular, organelle-like structures (microvesicles, MVs) into their surroundings including blood and body fluids, and that these MVs contain a selected set of tumor-related proteins and high levels of mRNAs and miRNAs. In this present study, we detected the Ewing sarcoma-specific EWS/Fli-1 mRNA in MVs from the culture medium of ES cell lines carrying t(11;22) (q24; 12). Also, we detected this fusion gene in approximately 40% of the blood samples from mice inoculated with xenografts of TC135 or A673 cells. These findings indicate the EWS/Fli-1 mRNA in MVs might be a new non-invasive diagnostic marker for specific cases of Ewing sarcoma.

| S-EPMC3790721 | biostudies-literature

Mechanism and relevance of EWS/FLI-mediated transcriptional repression in Ewing sarcoma.

Project description:Ewing sarcoma provides an important model for transcription-factor-mediated oncogenic transformation because of its reliance on the ETS-type fusion oncoprotein EWS/FLI. EWS/FLI functions as a transcriptional activator and transcriptional activation is required for its oncogenic activity. Here, we demonstrate that a previously less-well characterized transcriptional repressive function of the EWS/FLI fusion is also required for the transformed phenotype of Ewing sarcoma. Through comparison of EWS/FLI transcriptional profiling and genome-wide localization data, we define the complement of EWS/FLI direct downregulated target genes. We demonstrate that LOX is a previously undescribed EWS/FLI-repressed target that inhibits the transformed phenotype of Ewing sarcoma cells. Mechanistic studies demonstrate that the NuRD co-repressor complex interacts with EWS/FLI, and that its associated histone deacetylase and LSD1 activities contribute to the repressive function. Taken together, these data reveal a previously unknown molecular function for EWS/FLI, demonstrate a more highly coordinated oncogenic transcriptional hierarchy mediated by EWS/FLI than previously suspected, and implicate a new paradigm for therapeutic intervention aimed at controlling NuRD activity in Ewing sarcoma tumors.

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EWS/FLI is a Master Regulator of Metabolic Reprogramming in Ewing Sarcoma.

Project description:Ewing sarcoma is a bone malignancy driven by a translocation event resulting in the fusion protein EWS/FLI1 (EF). EF functions as an aberrant and oncogenic transcription factor that misregulates the expression of thousands of genes. Previous work has focused principally on determining important transcriptional targets of EF, as well as characterizing important regulatory partnerships in EF-dependent transcriptional programs. Less is known, however, about EF-dependent metabolic changes or their role in Ewing sarcoma biology. Therefore, the metabolic effects of silencing EF in Ewing sarcoma cells were determined. Metabolomic analyses revealed distinct separation of metabolic profiles in EF-knockdown versus control-knockdown cells. Mitochondrial stress tests demonstrated that knockdown of EF increased respiratory as well as glycolytic functions. Enzymes and metabolites in several metabolic pathways were altered, including de novo serine synthesis and elements of one-carbon metabolism. Furthermore, phosphoglycerate dehydrogenase (PHGDH) was found to be highly expressed in Ewing sarcoma and correlated with worse patient survival. PHGDH knockdown or pharmacologic inhibition in vitro caused impaired proliferation and cell death. Interestingly, PHGDH modulation also led to elevated histone expression and methylation. These studies demonstrate that the translocation-derived fusion protein EF is a master regulator of metabolic reprogramming in Ewing sarcoma, diverting metabolites toward biosynthesis. As such, these data suggest that the metabolic aberrations induced by EF are important contributors to the oncogenic biology of these tumors.Implications: This previously unexplored role of EWS/FLI1-driven metabolic changes expands the understanding of Ewing sarcoma biology, and has potential to significantly inform development of therapeutic strategies. Mol Cancer Res; 15(11); 1517-30. ©2017 AACR.

| S-EPMC5668171 | biostudies-literature

EWS/FLI utilizes NKX2-2 to repress mesenchymal features of Ewing sarcoma.

Project description:In Ewing sarcoma, NKX2-2 is a critical activated target of the oncogenic transcription factor EWS/FLI that is required for transformation. However, its biological function in this malignancy is unknown. Here we provide evidence that NKX2-2 mediates the EWS/FLI-controlled block of mesenchymal features. Transcriptome-wide RNA sequencing revealed that NKX2-2 represses cell adhesion and extracellular matrix organization genes. NKX2-2-depleted cells form more focal adhesions and organized actin stress fibers, and spread over a wider area-hallmarks of mesenchymally derived cells. Furthermore, NKX2-2 represses the actin-stabilizing protein zyxin, suggesting that these morphological changes are attributable to zyxin de-repression. In addition, NKX2-2-knockdown cells display marked increases in migration and substrate adhesion. However, only part of the EWS/FLI phenotype is NKX2-2-dependent; consequently, NKX2-2 is insufficient to rescue EWS/FLI repression of mesenchymalization. Strikingly, we found that EWS/FLI-and NKX22-repressed genes are activated by ZEB2, which was previously shown to block Ewing sarcoma epithelialization. Together, these data support an emerging theme wherein Ewing sarcoma cells highly express transcription factors that maintain an undifferentiated state. Importantly, co-opting epithelial and mesenchymal traits by Ewing sarcoma cells may explain how the primary tumor grows rapidly while also "passively" metastasizing, without the need for transitions toward differentiated states, as in carcinomas.

| S-EPMC4426950 | biostudies-literature

EWS/FLI Confers Tumor Cell Synthetic Lethality to CDK12 Inhibition in Ewing Sarcoma.

Project description:Many cancer types are driven by oncogenic transcription factors that have been difficult to drug. Transcriptional inhibitors, however, may offer inroads into targeting these cancers. Through chemical genomics screening, we identified that Ewing sarcoma is a disease with preferential sensitivity to THZ1, a covalent small-molecule CDK7/12/13 inhibitor. The selective CDK12/13 inhibitor, THZ531, impairs DNA damage repair in an EWS/FLI-dependent manner, supporting a synthetic lethal relationship between response to THZ1/THZ531 and EWS/FLI expression. The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing sarcoma, including a PDX, in vivo without hematopoietic toxicity.

| S-EPMC5846483 | biostudies-literature

RNA-seq profiling of Ewing sarcoma cells after TRIM8 knockout and EWS/FLI over-expression

Project description:Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif- containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared to >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion-oncoprotein specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF driven cancers.

2021-06-30 | GSE150244 | GEO

Role for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing sarcoma anchorage independent growth.

Project description:Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro. These sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. We now demonstrate the critical role of an EWS/FLI-bound GGAA-microsatellite in regulation of the NR0B1 gene as well as for Ewing sarcoma proliferation and anchorage-independent growth. Clinically, genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal "sweet-spot" GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this remains unknown. Our biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion), demonstrate a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the microsatellite was increased to the sweet-spot length. In contrast, a fully functional EWS/FLI mutant (Mut9, which retains approximately half of the EWS portion of the fusion) showed low affinity for smaller GGAA-microsatellites but instead significantly increased its affinity at sweet-spot microsatellite lengths. Single-gene ChIP and genome-wide ChIP-sequencing (ChIP-seq) and RNA-seq studies extended these findings to the in vivo setting. Together, these data demonstrate the critical requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unexpected role for the EWS portion of the EWS/FLI fusion in binding to sweet-spot GGAA-microsatellites.

| S-EPMC5603999 | biostudies-literature

Human A673 Ewing sarcoma secretome - effect of EWS-FLI-1 silencing

Project description:EWS-FLI-1 was silenced by an shRNA in A673 Ewing sarcoma cells and the resulting alterations in the secretome was analyzed by GeLC-MS/MS approach (six gel slices for each sample, luciferase shRNA-expressing cell secretome as control)

2021-07-06 | PXD016052 | Pride

BCL11B is up-regulated by EWS/FLI and contributes to the transformed phenotype in Ewing sarcoma.

Project description:The EWS/FLI translocation product is the causative oncogene in Ewing sarcoma and acts as an aberrant transcription factor. EWS/FLI dysregulates gene expression during tumorigenesis by abnormally activating or repressing genes. The expression levels of thousands of genes are affected in Ewing sarcoma, however, it is unknown which of these genes contribute to the transformed phenotype. Here we characterize BCL11B as an up-regulated EWS/FLI target that is necessary for the maintenance of transformation in patient derived Ewing sarcoma cells lines. BCL11B, a zinc finger transcription factor, acts as a transcriptional repressor in Ewing's sarcoma and contributes to the EWS/FLI repressed gene signature. BCL11B repressive activity is mediated by the NuRD co-repressor complex. We further demonstrate that re-expression of SPRY1, a repressed target of BCL11B, limits the transformation capacity of Ewing sarcoma cells. These data define a new pathway downstream of EWS/FLI required for oncogenic maintenance in Ewing sarcoma.

| S-EPMC3601955 | biostudies-literature

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