Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma.
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ABSTRACT:
SUBMITTER: Grohar PJ
PROVIDER: S-EPMC4755295 | biostudies-literature | 2016 Jan
REPOSITORIES: biostudies-literature
Publications
Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma.
Grohar Patrick J PJ Kim Suntae S Rangel Rivera Guillermo O GO Sen Nirmalya N Haddock Sara S Harlow Matt L ML Maloney Nichole K NK Zhu Jack J O'Neill Maura M Jones Tamara L TL Huppi Konrad K Grandin Magdalena M Gehlhaus Kristen K Klumpp-Thomas Carleen A CA Buehler Eugen E Helman Lee J LJ Martin Scott E SE Caplen Natasha J NJ
Cell reports 20160114 3
Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the ...[more]
Similar Datasets
Project description:Chromosomal translocations between the EWS gene and members of the ETS gene family are characteristic molecular features of the Ewing sarcoma. The most common translocation t(11;22)(q24;q12) fuses the EWS gene to FLI1, and is present in 85-90% of Ewing sarcomas. In the present study, a specifically designed multiplex long-range PCR assay was applied to amplify genomic EWS-FLI1 fusion sites from as little as 100 ng template DNA. Characterization of the EWS-FLI1 fusion sites of 42 pediatric and young adult Ewing sarcoma patients and seven cell lines revealed a clustering in the 5' region of the EWS-breakpoint cluster region (BCR), in contrast to random distribution of breakpoints in the FLI1-BCR. No association of breakpoints with various recombination-inducing sequence motifs was identified. The occurrence of small deletions and duplications at the genomic junction is characteristic of involvement of the non-homologous end-joining (NHEJ) repair system, similar to findings at chromosomal breakpoints in pediatric leukemia and lymphoma.
| S-EPMC3575406 | biostudies-literature
Project description:Ewing sarcoma is the second most common pediatric bone and soft tissue tumor presenting with an aggressive behavior and prevalence to metastasize. The diagnostic translocation t(22;11)(q24;12) leads to expression of the chimeric oncoprotein EWS-FLI1 which is uniquely expressed in all tumor cells and maintains their survival. Constant EWS-FLI1 protein turnover is regulated by the ubiquitin proteasome system. Here, we now identified ubiquitin specific protease 19 (USP19) as a regulator of EWS-FLI1 stability using an siRNA based screening approach. Depletion of USP19 resulted in diminished EWS-FLI1 protein levels and, vice versa, upregulation of active USP19 stabilized the fusion protein. Importantly, stabilization appears to be specific for the fusion protein as it could not be observed neither for EWSR1 nor for FLI1 wild type proteins even though USP19 binds to the N-terminal EWS region to regulate deubiquitination of both EWS-FLI1 and EWSR1. Further, stable shUSP19 depletion resulted in decreased cell growth and diminished colony forming capacity in vitro, and significantly delayed tumor growth in vivo. Our findings not only provide novel insights into the importance of the N-terminal EWSR1 domain for regulation of fusion protein stability, but also indicate that inhibition of deubiquitinating enzyme(s) might constitute a novel therapeutic strategy in treatment of Ewing sarcoma.
| S-EPMC6353870 | biostudies-other
Project description:PURPOSE:In a preclinical drug screen, mithramycin was identified as a potent inhibitor of the Ewing sarcoma EWS-FLI1 transcription factor. We conducted a phase I/II trial to determine the dose-limiting toxicities (DLT), maximum tolerated dose (MTD), and pharmacokinetics (PK) of mithramycin in children with refractory solid tumors, and the activity in children and adults with refractory Ewing sarcoma. PATIENTS AND METHODS:Mithramycin was administered intravenously over 6 h once daily for 7 days for 28 day cycles. Adult patients (phase II) initially received mithramycin at the previously determined recommended dose of 25 µg/kg/dose. The planned starting dose for children (phase I) was 17.5 µg/kg/dose. Plasma samples were obtained for mithramycin PK analysis. RESULTS:The first two adult patients experienced reversible grade 4 alanine aminotransferase (ALT)/aspartate aminotransferase (AST) elevation exceeding the MTD. Subsequent adult patients received mithramycin at 17.5 µg/kg/dose, and children at 13 µg/kg/dose with dexamethasone pretreatment. None of the four subsequent adult and two pediatric patients experienced cycle 1 DLT. No clinical responses were observed. The average maximal mithramycin plasma concentration in four patients was 17.8 ± 4.6 ng/mL. This is substantially below the sustained mithramycin concentrations ?50 nmol/L required to suppress EWS-FLI1 transcriptional activity in preclinical studies. Due to inability to safely achieve the desired mithramycin exposure, the trial was closed to enrollment. CONCLUSIONS:Hepatotoxicity precluded the administration of a mithramycin at a dose required to inhibit EWS-FLI1. Evaluation of mithramycin in patients selected for decreased susceptibility to elevated transaminases may allow for improved drug exposure.
| S-EPMC6413860 | biostudies-literature
Project description:Core regulatory circuitry (CRC)-dependent transcriptional network is critical for developmental tumors in children and adolescents carrying few gene mutations. However, whether and how CRC contributes to transcription regulation in Ewing sarcoma is unknown. Here, we identify and functionally validate a CRC 'trio' constituted by three transcription factors (TFs): KLF15, TCF4 and NKX2-2, in Ewing sarcoma cells. Epigenomic analyses demonstrate that EWS-FLI1, the primary fusion driver for this cancer, directly establishes super-enhancers of each of these three TFs to activate their transcription. In turn, KLF15, TCF4 and NKX2-2 co-bind to their own and each other's super-enhancers and promoters, forming an inter-connected auto-regulatory loop. Functionally, CRC factors contribute significantly to cell proliferation of Ewing sarcoma both in vitro and in vivo. Mechanistically, CRC factors exhibit prominent capacity of co-regulating the epigenome in cooperation with EWS-FLI1, occupying 77.2% of promoters and 55.6% of enhancers genome-wide. Downstream, CRC TFs coordinately regulate gene expression networks in Ewing sarcoma, controlling important signaling pathways for cancer, such as lipid metabolism pathway, PI3K/AKT and MAPK signaling pathways. Together, molecular characterization of the oncogenic CRC model advances our understanding of the biology of Ewing sarcoma. Moreover, CRC-downstream genes and signaling pathways may contain potential therapeutic targets for this malignancy.
| S-EPMC7672457 | biostudies-literature
Project description:Ewing sarcomas are driven by EWS-ETS fusions, most commonly EWS-FLI1, which promotes widespread metabolic reprogramming, including activation of serine biosynthesis. We previously reported that serine biosynthesis is also activated in Ewing sarcoma by the scaffolding protein menin through as yet undefined mechanisms. Here, we investigated whether EWS-FLI1 and/or menin orchestrate serine biosynthesis via modulation of ATF4, a stress-response gene that acts as a master transcriptional regulator of serine biosynthesis in other tumors. Our results show that in Ewing sarcoma, ATF4 levels are high and that ATF4 modulates transcription of core serine synthesis pathway (SSP) genes. Inhibition of either EWS-FLI1 or menin leads to loss of ATF4, and this is associated with diminished expression of SSP transcripts and proteins. We identified and validated an EWS-FLI1 binding site at the ATF4 promoter, indicating that the fusion can directly activate ATF4 transcription. In contrast, our results suggest that menin-dependent regulation of ATF4 is mediated by transcriptional and post-transcriptional mechanisms. Importantly, our data also reveal that the downregulation of SSP genes that occurs in the context of EWS-FLI1 or menin loss is indicative of broader inhibition of ATF4-dependent transcription. Moreover, we find that menin inhibition similarly leads to loss of ATF4 and the ATF4-dependent transcriptional signature in MLL-rearranged B-cell acute lymphoblastic leukemia, extending our findings to another cancer in which menin serves an oncogenic role. IMPLICATIONS: These studies provide new insights into metabolic reprogramming in Ewing sarcoma and also uncover a previously undescribed role for menin in the regulation of ATF4.
| S-EPMC8462528 | biostudies-literature
Project description:Ewing sarcoma (EwS) is the second most common bone cancer in children and adolescents with a high metastatic potential. EwS development is driven by a specific chromosomal translocation resulting in the generation of a chimeric EWS-ETS transcription factor, most frequently EWS-FLI1.Nicotinamide adenine dinucleotide (NAD) is a key metabolite of energy metabolism involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability. This study describes targeting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD synthesis, by FK866 in EwS cells. Here we report that blocking NAMPT leads to exhaustive NAD depletion in EwS cells, followed by a metabolic collapse and cell death. Using conditional EWS-FLI1 knockdown by doxycycline-inducible shRNA revealed that EWS-FLI1 depletion significantly reduces the sensitivity of EwS cells to NAMPT inhibition. Consistent with this finding, a comparison of 7 EwS cell lines of different genotypes with 5 Non-EwS cell lines and mesenchymal stem cells revealed significantly higher FK866 sensitivity of EWS-ETS positive EwS cells, with IC50 values mostly below 1nM.Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of EwS cells and suggest NAMPT inhibition as a potential new treatment approach for Ewing sarcoma.
| S-EPMC5421879 | biostudies-literature
Project description:ET-743 (trabectedin; Yondelis) is approved in Europe for the treatment of soft tissue sarcomas. Emerging phase 1 and 2 clinical data have shown high response rates in myxoid liposarcoma in part owing to the inhibition of the FUS-CHOP transcription factor. In this report, we show that modulation of specific oncogenic transcription factors by ET-743 may extend to other tumor types. We demonstrate that, among a panel of pediatric sarcomas, Ewing sarcoma family of tumors (ESFTs) cell lines bearing the EWS-FLI1 transcription factor are the most sensitive to treatment with ET-743 compared with osteosarcoma, rhabdomyosarcoma, and synovial sarcoma. We show that ET-743 reverses a gene signature of induced downstream targets of EWS-FLI1 in two different ESFT cell lines (P = .001). In addition, ET-743 directly suppresses the promoter activity of a known EWS-FLI1 downstream target NR0B1 luciferase reporter construct without changing the activity of a constitutively active control in ESFT cells. Furthermore, the effect is specific to EWS-FLI1, as forced expression of EWS-FLI1 in a cell type that normally lacks this fusion protein, HT1080 cells, induces the same NR0B1 promoter, but this activation is completely blocked by ET-743 treatment. Finally, we used gene set enrichment analysis to confirm that other mechanisms of ET-743 are active in ESFT cells. These results suggest a particular role for ET-743 in the treatment of translocation-positive tumors. In addition, the modulation of EWS-FLI1 makes it a novel targeting agent for ESFT and suggests that further development of this compound for the treatment of ESFT is warranted.
| S-EPMC3033593 | biostudies-literature
Project description:Chromosomal translocation results in development of an Ewing sarcoma breakpoint region 1-Friend leukemia integration 1 (EWS-FLI1) fusion oncogene in the majority of Ewing sarcoma. The persistent dependence of the tumor for this oncoprotein points to EWS-FLI1 as an ideal drug target. Although EWS-FLI1 transcriptional targets and binding partners are evaluated, the mechanisms regulating EWS-FLI1 protein stability remain elusive. Speckle-type POZ protein (SPOP) and OTU domain-containing protein 7A (OTUD7A) are identified as the bona fide E3 ligase and deubiquitinase, respectively, that control EWS-FLI1 protein turnover in Ewing sarcoma. Casein kinase 1-mediated phosphorylation of the VTSSS degron in the FLI1 domain enhances SPOP activity to degrade EWS-FLI1. Opposing this process, OTUD7A deubiquitinates and stabilizes EWS-FLI1. Depletion of OTUD7A in Ewing sarcoma cell lines reduces EWS-FLI1 protein abundance and impedes Ewing sarcoma growth in vitro and in mice. Performing an artificial-intelligence-based virtual drug screen of a 4-million small molecule library, 7Ai is identified as a potential OTUD7A catalytic inhibitor. 7Ai reduces EWS-FLI1 protein levels and decreases Ewing sarcoma growth in vitro and in a xenograft mouse model. This study supports the therapeutic targeting of OTUD7A as a novel strategy for Ewing sarcoma bearing EWS-FLI1 and related fusions, and may also be applicable to other cancers dependent on aberrant FLI1 expression.
| S-EPMC8292909 | biostudies-literature
Project description:EWS-FLI1 constitutes an oncogenic transcription factor that plays key roles in Ewing sarcoma development and maintenance. We have recently succeeded in generating an ex vivo mouse model for Ewing sarcoma by introducing EWS-FLI1 into embryonic osteochondrogenic progenitors. The model well recapitulates the biological characteristics, small round cell morphology, and gene expression profiles of human Ewing sarcoma. Here, we clarified the global DNA binding properties of EWS-FLI1 in mouse Ewing sarcoma. GGAA microsatellites were found to serve as binding sites of EWS-FLI1 albeit with less frequency than that in human Ewing sarcoma; moreover, genomic distribution was not conserved between human and mouse. Nevertheless, EWS-FLI1 binding sites within GGAA microsatellites were frequently associated with the histone H3K27Ac enhancer mark, suggesting that EWS-FLI1 could affect global gene expression by binding its target sites. In particular, the Fox transcription factor binding motif was frequently observed within EWS-FLI1 peaks and Foxq1 was identified as the cooperative partner that interacts with the EWS portion of EWS-FLI1. Trib1 and Nrg1 were demonstrated as target genes that are co-regulated by EWS-FLI1 and Foxq1, and are important for cell proliferation and survival of Ewing sarcoma. Collectively, our findings present novel aspects of EWS-FLI1 function as well as the importance of GGAA microsatellites.
| S-EPMC6125457 | biostudies-literature
Project description:Transcription factors have long been deemed 'undruggable' targets for therapeutics. Enhanced recognition of protein biochemistry as well as the need to have more targeted approaches to treat cancer has rendered transcription factors approachable for therapeutic development. Since transcription factors lack enzymatic domains, the specific targeting of these proteins has unique challenges. One challenge is the hydrophobic microenvironment that affects small molecules gaining access to block protein interactions. The most attractive transcription factors to target are those formed from tumor specific chromosomal translocations that are validated oncogenic driver proteins. EWS-FLI1 is a fusion protein that results from the pathognomonic translocation of Ewing sarcoma (ES). Our past work created the small molecule YK-4-279 that blocks EWS-FLI1 from interacting with RNA Helicase A (RHA). To fulfill long-standing promise in the field by creating a clinically useful drug, steps are required to allow for in vivo administration. These investigations identify the need for continuous presence of the small molecule protein-protein inhibitor for a period of days. We describe the pharmacokinetics of YK-4-279 and its individual enantiomers. In vivo studies confirm prior in vitro experiments showing (S)-YK-4-279 as the EWS-FLI1 specific enantiomer demonstrating both induction of apoptosis and reduction of EWS-FLI1 regulated caveolin-1 protein. We have created the first rat xenograft model of ES, treated with (S)-YK-4-279 dosing based upon PK modeling leading to a sustained complete response in 2 of 6 ES tumors. Combining laboratory studies, pharmacokinetic measurements, and modeling has allowed us to create a paradigm that can be optimized for in vivo systems using both in vitro data and pharmacokinetic simulations. Thus, (S)-YK-4-279 as a small molecule drug is ready for continued development towards a first-in-human, first-in-class, clinical trial.
| S-EPMC3964211 | biostudies-literature