Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening

Grohar, Patrick J.; Woldemichael, Girma M.; Griffin, Laurie B.; Mendoza, Arnulfo; Chen, Qing Rong; Yeung, Choh; Currier, Duane; Davis, Sean; Khanna, Chand; Khan, Javed; McMahon, James B.; Helman, Lee J.

doi:10.1093/jnci/djr156

Cited by 178 publications

(207 citation statements)

References 67 publications

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“…[32][33][34][35][36][37] Recently, MTM was identified as the lead compound against the Friend leukemia virus integration 1 (EWS-FLI1) transcription factor. 38 EWS-FLI1 is believed to be responsible for malignant transformation and progression of Ewing sarcoma family tumors and was widely regarded as undruggable because it is a transcription factor. 38 A high-throughput screening of over 50,000 compounds identified MTM as the top candidate from the screen and greatly reduced and inhibited tumor volume in Ewing sarcoma family tumors in mouse xenograft models, and it is planned to reinstitute clinical trials for this application of MTM.…”

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confidence: 99%

Nanoparticulate formulations of mithramycin analogs for enhanced cytotoxicity

Scott

Rohr

Bae

2011

IJN

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Mithramycin (MTM), a natural product of soil bacteria from the Streptomyces genus, displays potent anticancer activity but has been limited clinically by severe side effects and toxicities. Engineering of the MTM biosynthetic pathway has produced the 3-side-chain-modified analogs MTM SK (SK) and MTM SDK (SDK), which have exhibited increased anticancer activity and improved therapeutic index. However, these analogs still suffer from low bioavailability, short plasma retention time, and low tumor accumulation. In an effort to aid with these shortcomings, two nanoparticulate formulations, poly(ethylene glycol)-poly(aspartate hydrazide) self-assembled and cross-linked micelles, were investigated with regard to the ability to load and pH dependently release the drugs. Micelles were successfully formed with both nanoparticulate formulations of each drug analog, with an average size of 8.36 ± 3.21 and 12.19 ± 2.77 nm for the SK and SDK micelles and 29.56 ± 4.67 nm and 30.48 ± 7.00 nm for the SK and SDK cross-linked micelles respectively. All of the drug-loaded formulations showed a pH-dependent release of the drugs, which was accelerated as pH decreased from 7.4 to 5.0. The micelles retained biological activity of SK and SDK entrapped in the micelles, suppressing human A549 lung cancer cells effectively.

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confidence: 99%

Nanoparticulate formulations of mithramycin analogs for enhanced cytotoxicity

Scott

Rohr

Bae

2011

IJN

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“…Cells and Culture Conditions-Growth and propagation conditions and characterization of TC32 cells have been described previously (11). EW8 and 5838 cells were obtained from Dr. Lee Helman of the Pediatric Oncology Branch, NCI, National Institutes of Health, whose laboratory performed authentication of the cell lines by short-tandem repeat genotyping.…”

Section: Methodsmentioning

confidence: 99%

“…Activity-A high throughput screen (HTS) developed using the Ewing sarcoma cell line TC32 stably transduced with a luciferase reporter construct that reports on EWS-FLI1 activity was used to screen over 60,000 natural products extracts (11). The top scoring extract in the primary screen was that of the African plant Phyllanthus engleri.…”

Section: High Throughput Screening Identifies Ea As An Inhibitor Of Ementioning

confidence: 99%

Englerin A Inhibits EWS-FLI1 DNA Binding in Ewing Sarcoma Cells

Caropreso

Darvishi

Turbyville

et al. 2016

Journal of Biological Chemistry

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High-throughput screening of extracts from plants, marine, and micro-organisms led to the identification of the extract from the plant Phyllanthus engleri as the most potent inhibitor of EWS-FLI1 induced luciferase reporter expression. Testing of compounds isolated from this extract in turn led to the identification of Englerin A (EA) as the active constituent of the extract. EA induced both necrosis and apoptosis in Ewing cells subsequent to a G2M accumulation of cells in the cell cycle. It also impacted clonogenic survival and anchorage-independent proliferation while also decreasing the proportion of chemotherapy-resistant cells identified by high ALDH activity. EA also caused a sustained increase in cytosolic calcium levels. EA appears to exert its effect on Ewing cells through a decrease in phosphorylation of EWS-FLI1 and its ability to bind DNA. This effect is mediated, at least in part, through a decrease in the levels of the calcium-dependent protein kinase PKC-␤I after a transient up-regulation.The hallmark that defines the Ewing sarcoma family of tumors is the presence of non-random chromosomal rearrangements between the Ewing sarcoma breakpoint region 1 gene (EWS) 2 located on chromosome 22q12 and genes of the E26 transformation-specific sequence (ETS) family of transcription factors. Reciprocal chromosomal translocations, t(11; 22) (q24;q12), that result in the fusion of the transactivational domain of EWS and the DNA binding domain of FLI1, an ETS transcription factor, generating the chimeric EWS-FLI1 fusion transcript underlie an overwhelming majority of cases of Ewing sarcoma (1). The disease arises in less than 3 per million people under the age of 20, with 90% of all cases being found in patients between 5 and 25 years of age (2). Prior to the advent of chemotherapy, up to 90% of patients succumbed to the disease when only surgery and/or radiation were used. Current standard treatment constitutes combination chemotherapy along with surgery and/or radiation. This approach has had a positive impact on event-free survival and overall survival. However, in 27% of patients diagnosed with localized disease, this treatment modality still fails to improve overall survival (3). Furthermore, 15-25% of patients present with metastases upon diagnosis and the cure rate among this group is below 20% (4). Overall the long-term cure rate in children and adolescents with Ewing sarcoma still remains under 60% (5). Various reports show the aberrant transcription factor EWS-FLI1 is a key contributor in the tumorigenesis and progression of Ewing sarcoma (6, 7). The discovery of molecules that modulate its activity are expected to not only provide a better understanding of the biology behind the disease but are also expected to lead to the development of chemotherapeutic agents targeting the disease. Herein we describe results from a mode-of-action study undertaken using a compound identified as an inhibitor from a plant extract in a primary high-throughput screen (HTS). Experimental ProceduresReagents-Synthetic EWS-...

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“…In addition, this study has shown once more how important it is to identify additional therapeutic options for patients with GIST that make use of these tumors' specifi c molecular makeup aside from KIT/ PDGFRA mutations. Especially targeting the transcriptional machinery-either globally or with the aim of reducing the expression levels of specifi c genes (such as KIT and ETV1 )-has recently emerged as an important new angle for the treatment of GIST and other oncogene-driven malignancies (8)(9)(10). For example, inhibitors of the 26S proteasome, such as bortezomib (Velcade), have been shown to inhibit ongoing gene transcription in GIST, leading to a dramatic loss of KIT protein expression and subsequent apoptosis ( 8 ).…”

Section: ;Rosa26mentioning

confidence: 99%

“…For example, inhibitors of the 26S proteasome, such as bortezomib (Velcade), have been shown to inhibit ongoing gene transcription in GIST, leading to a dramatic loss of KIT protein expression and subsequent apoptosis ( 8 ). The SP1 inhibitor mithramycin A, a "classical" transcriptional inhibitor, has recently gained attention again for being the top hit in a 50,000 compound screen against the EWS-FLI1 fusion oncogene in Ewing family sarcoma ( 9 ). It was also identifi ed in a smaller library screen as being effective for GISTs, with its main mechanism of action lying in the reduction of KIT ( 10 ).…”

Section: ;Rosa26mentioning

confidence: 99%

Targeting ETV1 in Gastrointestinal Stromal Tumors: Tripping the Circuit Breaker in GIST?

Duensing

2015

Cancer Discovery

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Summary: Activating mutations in the KIT or PDGFRA receptor tyrosine kinase genes are the key oncogenic drivers in the majority of gastrointestinal stromal tumors (GIST), but novel results now show that aberrant kinase signaling is potentiated by a positive feedback circuit that involves the ETS transcription factor ETV1. Targeting ETV1 can disrupt this circuit and represents a promising new therapeutic approach for the treatment of GISTs.See related article by Ran and colleagues, p. 304 (5).Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract and one of the most common subtypes of sarcomas. Because of their unique molecular properties, GISTs spearheaded the use of targeted therapies in solid tumors, a concept that eventually led to a paradigm shift in oncologic therapy. GISTs were the fi rst solid tumor entity that could successfully be treated with small-molecule inhibitors, specifi cally tyrosine kinase inhibitors such as imatinib mesylate (Gleevec; ref. 1 ). This notion came shortly after the discovery that an oncogenic mutation in the KIT (75%-85%) or PDGFRA (platelet-derived growth factor receptor alpha; 5%-7%) gene is the tumor-initiating event in the vast majority of GISTs and leads to constitutive activation of the encoded receptor tyrosine kinase ( 2, 3 ). Major responses are seen after fi rst-line treatment with the KIT/PDGFRA inhibitor imatinib, and approximately 85% of patients with metastatic and/or inoperable GIST benefi t from this therapy. However, complete tumor remissions are rare, and about 50% of patients experience disease recurrence within 2 years of treatment. There are several reasons for this, including the emergence of secondary mutations in KIT/PDGFRA that confer drug resistance, entry into a state of cellular quiescence, and the potential existence of GIST stem/progenitor cells that express low levels of KIT and are intrinsically imatinib resistant. Given these reasons, it is unlikely that GISTs can be cured with imatinib alone, and additional therapeutic approaches are urgently needed.GISTs are thought to arise from a specialized cell type in the bowel wall, the interstitial cells of Cajal (ICC), or a common progenitor. These cells are present throughout the entire digestive tract, where they serve as pacemaker cells to coordinate peristalsis. Interestingly, there are several subsets of ICCs that are located in distinct microscopical locations within the gut, and only certain types seem to give rise to GIST. In an earlier study, Chi and colleagues ( 4 ) discovered that the ETV1 transcription factor is a master regulator not only of the ICC population giving rise to GIST, but also of an ICC-GIST-specifi c transcriptional network. ETV1 is highly expressed in GISTs (but not in other sarcomas) and is required for the growth and proliferation of GIST cells.A new study by the same group ( 5 ), published in this issue of Cancer Discovery , adds an important new spin to their previous observations. The latest data not only provide in vi...

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Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening

Abstract: Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.

Cited by 178 publications

References 67 publications

Nanoparticulate formulations of mithramycin analogs for enhanced cytotoxicity

Nanoparticulate formulations of mithramycin analogs for enhanced cytotoxicity

Englerin A Inhibits EWS-FLI1 DNA Binding in Ewing Sarcoma Cells

Targeting ETV1 in Gastrointestinal Stromal Tumors: Tripping the Circuit Breaker in GIST?

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