Targeting EWS-FLI1 with Small Molecule Inhibitors


Ewing’s Sarcoma is a rare disease affecting bones and adjacent soft tissues that can occur in any part of the human body. This report describes the progress made with a grant awarded by the Liddy Shriver Sarcoma Initiative and the Amschwand Sarcoma Cancer Foundation. This grant enabled the continuation of the project until support was gained by both an NIH R01 mechanism and the discovery and subsequent publication of the first small molecule targeted to EWS-FLI1.1

Transcription factors are responsible for controlling all the genes in a cell and work together in large, multi-protein complexes to switch genes on and off. Ewing’s Sarcoma contains a transcription factor that is produced because of the t(11;22) chromosomal translocation that is specific to this disease. This transcription factor is called EWS-FLI1. EWS-FLI1 is thus a perfect target because it is only present in Ewing’s Sarcoma and not in normal cells. When EWS-FLI1 is removed from Ewing’s Sarcoma cells growing in Petri dishes, the cells will die. Though EWS-FLI1 is a perfect target because of this uniqueness, there are particular challenges to developing a treatment that can attack this perfect target.

EWS-FLI1 is a protein and, to reiterate, its purpose in cells is to be a transcription factor. Since transcription factors need to work together, one way to turn off an individual factor is to disrupt, or take apart, the group of proteins. In order to create a treatment to switch off EWS-FLI1, EWS-FLI1 needs to be disrupted from other transcription factors. Therefore, it was necessary to identify a critical partner that EWS-FLI1 required for its oncogenic function.2

Disrupting Transcription Factor EWS-FLI1 Leads to New Medicines

There are many technical reasons why scientists have been discouraged from directly creating therapies to disrupt transcription factors such as EWS-FLI1. These technical reasons include aspects of biochemistry that challenged synthesis and purification of EWS-FLI1. Proteins fold into three-dimensional shapes in order to work; however, some proteins like EWS-FLI1 are disordered and lack a stable three-dimensional structure. EWS-FLI1 is a combination of two proteins, half EWS and half FLI1. Investigators have identified some contributions of these two halves, while full-length EWS-FLI1 has been shown to switch on and off certain genes. However, the structural contributions and, specifically, key protein interactions are still incompletely understood.

New medicines that disrupt general transcription factors could affect normal cells; however, in Ewing’s Sarcoma, the EWS-FLI1 provides a very specific target. Our previously published research showed that the three-dimensional shape of EWS-FLI1 allows it to work together with another transcription factor called RNA Helicase A (RHA). A group of proteins that includes EWS-FLI1 and RHA are responsible for forming Ewing’s Sarcoma. We constructed a mimic of the portion of RHA that adheres to EWS-FLI1, called peptide E9R. Peptides are simply small units that link together to form a complete protein. E9R can prevent EWS-FLI1 from working with RHA (Figure, top), and thus E9R inhibits EWS-FLI1 from making tumors. Peptides, however, have properties that make their translation into medicine problematic. We therefore chose to move our research into identifying small molecules that might act like the peptide. Small molecules are the basis of most current medicines.

Figure 1

Figure: Turning Off EWS-FLI1 Turns Off Ewing’s Sarcoma Tumor Growth.

We used the same method to purify EWS-FLI1 and successfully identify RHA as a partner that binds to EWS-FLI1. This validated that a peptide could keep the two proteins apart, useful information to screen for small molecules that could bind to EWS-FLI1. Screening identified a series of structures that were evaluated by medicinal chemists who are part of our research team. One small molecule looked promising to these chemists and we showed that this promising molecule did indeed reduce the growth of Ewing’s Sarcoma cells. The chemists then made over 20 modifications to our initial molecule for improvement. These modified molecules were tested for their ability to block EWS-FLI1 from binding to RHA as well as their potency in cell growth assays (Figure, bottom). These small molecules will form the basis for medicines that could potentially have a specific target and could therefore be the "magic bullet" for Ewing’s Sarcoma.

Many leukemias and other sarcomas have closely related, critical protein transcription factors. Pioneering work with a model, such as Ewing’s Sarcoma, may lead directly to new medicines for these cancers, many of which are lethal. Alternatively, work in Ewing’s Sarcoma may provide approaches that can be repeated for many different types of cancer. Discoveries and therapies for Ewing’s Sarcoma would therefore have benefits to many additional patients with a wide range of cancers.

Team of Chemists, Biologists, Oncologists, and even Mathematicians to Make New Medicines

The construction of a multidisciplinary group of scientists specializing in different fields is a vital element of our project. We constantly interact as a team including: chemists to create the small-molecules; in addition, the chemists can identify aspects of the small molecules that would affect their ability to become viable medications. Biologists on our team will test small molecules in the laboratory so that only the most promising candidates become medicines. One key representative on our team is the mathematicians. These experts provide us with algorithms used to compare current molecules and also help predict which molecules might be most effective to improve drug potency and specificity. Most importantly, our team includes oncologists who are intimately familiar with the needs of patients and the challenges of administering new medicines. Each stage of the development of the new medicines needs specialized support and is critical to effectively creating cutting-edge treatments.

Is the Small Molecule Ready to be Tested in Ewing's Sarcoma Patients?

While we have published the structure of our first compound and have shown data suggesting our quest will be successful, there are hurdles that remain. Our current efforts are focused on optimizing our current lead compound and exploring other chemical structures that may work in a similar fashion. Once a group of compounds shows exceptional promise, by medicinal chemistry standards, we will test the small molecules for unexpected toxicities prior to advancing to Human Phase One trials.

The recently published Small molecule selected to disrupt oncogenic protein EWS-FLI1 interaction with RNA Helicase A inhibits Ewing's Sarcoma (Nature Medicine, July 2009) demonstrates a novel therapeutic opportunity based on the direct targeting of EWS-FLI1. This publication begins with the demonstration that RNA Helicase A (RHA) is a required partner of EWS-FLI1. The work advances from this genetic experiment through a series of additional proofs that the interaction of EWS-FLI1 and RHA is critical for EWS-FLI1 oncogenesis. These proofs include a series of experiments with a peptide that disrupts RHA from EWS-FLI1 using: an in vitro assay, exogenous peptide administration and peptide expression. 

An NIH compound library was then screened for small molecules that bind to EWS-FLI1. One of these compounds was shown to disrupt EWS-FLI1 from RHA, both to further validate the target and serve as a chemical scaffold. Using a multidisciplinary team that included synthetic organic chemists, a chemical modification generated a lead compound that was more potent in both disrupting EWS-FLI1 from RHA and in ESFT cytotoxicity. The small molecule, YK-4-279 was shown to be relatively specific for Ewing’s Tumors and inhibited murine xenograft growth. Additional homologous transcription factors are being pursued in other sarcomas and carcinomas for therapeutic advancement. Current drug discovery pipelines are running dry with kinase inhibitors and are looking for novel druggable targets. This combination of novel biology and therapeutics is presented in Nature Medicine paper.


This work demonstrates important principles in the creation of small molecules that disrupt protein-protein interactions. Our current compound, YK-4-279, may be the scaffold for the EWS-FLI1 "magic bullet" or it may simply prove that such a protein can be targeted. Future small molecule experiments will also reveal additional details of how EWS-FLI1 works to cause and maintain Ewing’s Sarcoma growth. This forward momentum caused by using small molecules with the potential to become therapeutics and to inform biology has potential as a new generation of cancer therapy.

By Jeffrey A. Toretsky, MD
Associate Professor at the Department of Oncology and Pediatrics
Lombardi Comprehensive Cancer Center
Georgetown University
Washington, DC


1.  Erkizan HV, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg JS, Yuan L, Abaan OD, Chou TH, Dakshanamurthy S, Brown ML, Uren A, Toretsky JA. A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing's sarcoma. Nat Med, 15(7):750-6, 2009.

2.  Uren A, Toretsky JA. Ewing's Sarcoma Oncoprotein EWS-FLI1: the Perfect Target without a Therapeutic Agent. Future Onc, 1(4):521-8, 2005.

Small Molecules That Can Inhibit EWS-FLI1 May Represent Novel and Specific Therapy for ESFT Patients

Small Molecules Can Disrupt Protein-Protein Interactions

The use of small molecules to prevent or alter protein-protein interactions is thought to be challenging, albeit surmountable.1,2 There are many important anti-cancer small molecules that interfere with protein interactions in widespread use today. The vinca alkaloids represent a class of small molecule containing extremely effective anti-cancer agents that exert their effects by binding to β-tubulin and inhibiting its polymerization.3 In addition, small molecule allosteric inhibitors have been developed that block the interaction of Runx1 and CBFbeta in leukemia.4 Given the significant challenges of systemic oligonucleotide delivery in patients, and the very successful transport of small molecule pharmacologic agents, we are directing our drug discovery efforts towards identification of small molecule protein-protein interaction inhibitors (SMPPII).

Ewing’s Sarcoma Family of Tumors Contain a Unique Fusion Protein: EWS-FLI1

The Ewing’s sarcoma family of tumors (ESFT) are characterized by a translocation, occurring in 95% of tumors, between the central exons of the EWS gene (Ewing Sarcoma) located on chromosome 22 to the central exons of an ets family gene; either FLI1 (Friend Leukemia Insertion) located on chromosome 11, t(11;22), or ERG located on chromosome 21, t(21;22).

Figure 1

Figure: Turning Off EWS-FLI1 Turns Off Ewing’s Sarcoma Tumor Growth.

The EWS-FLI1 fusion transcript encodes a 55 kDa protein (electrophoretic motility of approximately 68 kD) with two primary domains. Reduced expression levels of EWS-FLI1 using either antisense oligodeoxynucleotides (ODN)5,6 or small interfering RNAs (siRNA)7-9 cause decreased proliferation of ESFT cell lines and regression of tumors in nude mice (Figure 1, TOP). Unfortunately, the pharmacological use of siRNA and antisense ODN in humans has been less than satisfactory.

Legend for Figure 1

  1. Antisense and small interfering RNAs (siRNA) both use oligodeoxynucleotide to bind to mRNA decreasing production of EWS-FLI1. If there is no EWS-FLI1, there is no complex and tumors stop growing. The challenge with this approach is making stable antisense or siRNA and getting these agents to tumors in humans.
  2. Small peptides (chain of amino-acids) can stick to EWS-FLI1 in a very specific way and prevent RNA Helicase A (RHA) from attaching. When RHA does not attach to EWS-FLI1, ESFT cells stop growing. The challenge with this approach is making stable peptides and getting these agents to tumors in humans.
  3. Small molecules (pharmaceuticals are mostly small molecules) that specifically stick to parts of EWS-FLI1 can prevent it from interacting with RHA. The challenge is to find the specific small molecules. Small molecules can be given to patients by oral or intravenous routes and are relatively easier to formulate at this time.

EWS-FLI1 Binds RNA Helicase A In Vitro and In Vivo

We hypothesized that protein-protein interactions of EWS-FLI1 may contribute to its oncogenic potential; therefore, we sought novel proteins that directly interact with and functionally modulate EWS-FLI1. We identified RHA Helicase A (RHA), using phage display, as a binding partner of EWS-FLI1. Immunoprecipitation, ELISA, and chromatin immunoprecipitation assays using fragments of RHA in ESFT cell lysate or recombinant EWS-FLI1 confirmed specificity and identified similar binding regions in support of the phage-display discovery data.10 RHA expression enhanced EWS-FLI1 mediated anchorage-independent colony formation, while an inactivating mutation of RHA prevented colony formation.10 A peptide might block the interaction of RHA with EWS-FLI1 to decrease tumor growth (Figure 1, MIDDLE). Depending on the length and stability of the appropriate peptide, this approach may lead to new therapeutics specifically directed against ESFT.

Our Experimental Plan

Therapies directed towards the inactivation of EWS-FLI1 might reduce the toxic effects of therapy and address the significant problem of recurrent disease for Ewing’s Sarcoma patients. Our approach is to develop SMPPII that disrupt EWS-FLI1 from RNA Helicase A (RHA) to progress towards a new therapeutic agent (Figure 1, BOTTOM). We will accomplish our objective by developing small molecule protein-protein interaction inhibitors to function as lead therapeutic compounds. We obtained a collection of 3,000 compounds from the National Cancer Institute and tested these for the ability to bind to EWS-FLI1. A series of compounds did bind to EWS-FLI1 and are going to be further investigated in these investigations supported by the Liddy Shriver Sarcoma Initiative and the Amschwand Sarcoma Cancer Foundation. We will identify the chemical characteristics that allow for the best binding to EWS-FLI1 and chemically modify the lead compounds to enhance this binding. The growth effects of compounds upon ESFT cells and non-tumor controls will also be tested. Promising compounds will advance into ESFT animal model studies.


ESFT patients require improved therapy to increase survival and reduce treatment-related morbidity. The chromosomal translocation, t(11;22) leading to the unique and critical fusion protein EWS-FLI1 is a perfect cancer target. Since many other sarcomas share similar translocation variants,11 our objectives have potential for application in many other cancer types. We will approach this using small molecule protein-protein interaction inhibitors (SMPPII). The development of successful SMPPII will help resolve the molecular mechanisms of sarcomagenesis. Future projects will further develop and test lead compounds in animal models of ESFT. The goal is ultimately to develop less toxic and more successful therapy for ESFT patients.


Thank you to Ms. Gabriela Perazza for manuscript and figure design assistance.

By Jeffrey A. Toretsky, MD
Associate Professor at the Department of Oncology and Pediatrics
Lombardi Comprehensive Cancer Center
Georgetown University
Washington, DC


1. Sillerud LO, Larson RS. Design and structure of peptide and peptidomimetic antagonists of protein-protein interaction. Curr Protein Pept Sci 2005;6(2):151-69.

2. Pagliaro L, Felding J, Audouze K, et al. Emerging classes of protein-protein interaction inhibitors and new tools for their development. Curr Opin Chem Biol 2004;8(4):442-9.

3. Gadek TR, Nicholas JB. Small molecule antagonists of proteins. Biochem Pharmacol 2003;65(1):1-8.

4. Gorczynski MJ, Grembecka J, Zhou Y, et al. Allosteric inhibition of the protein-protein interaction between the leukemia-associated proteins Runx1 and CBFbeta. Chemistry & biology 2007;14(10):1186-97.

5. Toretsky JA, Connell Y, Neckers L, Bhat NK. Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides. J Neurooncol 1997;31(1-2):9-16.

6. Tanaka K, Iwakuma T, Harimaya K, Sato H, Iwamoto Y. EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J Clin Invest 1997;99(2):239-47.

7. Ouchida M, Ohno T, Fujimura Y, Rao VN, Reddy ES. Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts. Oncogene 1995;11(6):1049-54.

8. Maksimenko A, Malvy C, Lambert G, et al. Oligonucleotides targeted against a junction oncogene are made efficient by nanotechnologies. Pharm Res 2003;20(10):1565-7.

9. Kovar H, Aryee DN, Jug G, et al. EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells in vitro. Cell Growth Differ 1996;7(4):429-37.

10. Toretsky JA, Erkizan V, Levenson A, et al. Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res 2006;66(11):5574-81.

11. Helman LJ, Meltzer P. Mechanisms of sarcoma development. Nat Rev Cancer 2003;3(9):685-94.

Grant Funding

The Liddy Shriver Sarcoma Initiative and the Amschwand Sarcoma Cancer Foundation co-funded this $50,000 grant in February 2008. The study was made possible, in part, by a generous gift from the Arlo and Susan Ellison family to the Liddy Shriver Sarcoma Initiative. Its findings were covered in the following press release by Georgetown University:

Discovery Highlights New Direction For Drug Discovery

Researchers did what others thought was not possible by finding a small molecule to stop "slippery" protein from binding to another, causing Ewing’s Sarcoma

Washington, DC – In a discovery that rebuffs conventional scientific thinking, researchers at the Lombardi Comprehensive Cancer Center at Georgetown University Medical Center (GUMC) have discovered a novel way to block the activity of the fusion protein responsible for Ewing’s sarcoma, a rare cancer found in children and young adults.

In the paper published online July 5 in Nature Medicine, they report discovering and successfully testing a small molecule that keeps the fusion protein from sticking to another protein that is critical for tumor formation. The researchers say this interaction is unique – and is especially surprising since the Ewing’s sarcoma fusion protein is extremely flexible, which allows it to change shape constantly.

"Most targeted small molecule cancer drugs inhibit the intrinsic activity of a single protein, but our agent stops two proteins from interacting. This has never been shown before with a cancer-causing fusion protein and represents a potentially novel medical therapy in the future," says the study’s lead investigator, Jeffrey Toretsky, MD, a pediatric oncology physician and researcher at GUMC’s Lombardi Comprehensive Cancer Center.

The study could provide a model upon which to design treatment for other disorders caused by the interaction between two proteins, and may be especially useful in cancers caused by translocations of genes, such as sarcomas and leukemias, the researchers say. Agents in use now that work against fusion proteins inhibit a single protein to stop intrinsic enzymatic activity; one example is Gleevec, used for chronic myelogenous leukemia (CML). The Ewing’s sarcoma fusion protein, known as EWS-FLI1, lacks enzymatic activity, "and this difference is why our work is significant," Toretsky says.

In the United States, about 500 patients annually are diagnosed with the cancer, and they are treated with a combination of five different chemotherapy drugs. Between 60-70 percent of patients survive over time, but with side effects from the treatment. Few additional treatment options are available for patients whose cancer progresses, Toretsky says.

Ewing’s sarcoma is caused by the exchange of DNA between two chromosomes, a process known as a translocation. The new EWS-FLI1 gene is created when the EWS gene on chromosome 22 fuses to the FLI1 gene on chromosome 11, and its product is the fusion protein responsible for cancer formation. It is a so-called disordered protein, which means it does not have a rigid structure. A number of cancer-causing proteins are disordered.

In their 15-year search for a new treatment for Ewing’s sarcoma, Toretsky and his colleagues were the first to make a recombinant EWS-FLI1 fusion protein. They used it to discover that the fusion protein stuck to another protein, RNA helicase A (RHA), a molecule that forms protein complexes in order to control gene transcription. "We believe that when RHA binds to EWS-FLI1, the combination becomes more powerful at turning genes on and off," says the study’s first author, Hayriye Verda Erkizan, PhD, a postdoctoral researcher in Toretsky’s lab.

Then, from a library of 3,000 small molecules loaned to Georgetown from the National Cancer Institute, the researchers searched for a small molecule that would bind on to EWS-FLI1. They found one, and further discovered the same molecule, NSC635437, could stop EWS-FLI1’s fusion protein from sticking to RHA.

This was a wonderful discovery, Erkizan says, because the notion long accepted among scientists is that it is not possible to block protein-protein interactions given that the surface of many of these proteins are slippery - much too flexible for a drug to bind to.

They tested the agent in laboratory cell culture, and with the help of GUMC’s Drug Discovery Program, the researchers designed a stronger derivative compound they called YK-4-279. In this study, they tested YK-4-279 in two different animal models of Ewing’s sarcoma and found that the agent significantly inhibited the growth of tumors. There was an 80% reduction in the growth of treated tumors compared to untreated tumors.

Toretsky says that while the agent needs to be "optimized," these results serve as a proof of principle that inhibiting protein-protein interaction can work as a novel therapeutic that will target only cancer cells.

"We may be able to use this strategy to attack proteins we thought to be impervious to manipulation," he says.

Liddy Shriver Sarcoma InitiativeThe study was funded by grants from the National Institutes of Health, Children’s Cancer Foundation of Baltimore, MD, Go4theGoal Foundation, Dani’s Foundation of Denver, the Liddy Shriver Sarcoma Initiative, the Amschwand Sarcoma Cancer Foundation, the Burroughs-Wellcome Clinical Scientist Award in Translational Research, and the GUMC Drug Discovery Program.

Toretsky and co-authors Milton L. Brown, Aykut Üren and Yali Kong are inventors on a patent application that has been filed by Georgetown University related to the technology described in this paper. The other authors report no related financial interests.

About Georgetown University Medical Center 
Georgetown University Medical Center is an internationally recognized academic medical center with a three-part mission of research, teaching and patient care (through Georgetown’s affiliation with MedStar Health). GUMC’s mission is carried out with a strong emphasis on public service and a dedication to the Catholic, Jesuit principle of cura personalis -- or "care of the whole person." The Medical Center includes the School of Medicine and the School of Nursing and Health Studies, both nationally ranked, the world-renowned Lombardi Comprehensive Cancer Center and the Biomedical Graduate Research Organization (BGRO), home to 60 percent of the university’s sponsored research funding.

  • Figure: Turning Off EWS-FLI1 Turns Off Ewing’s Sarcoma Tumor Growth.
    Different strategies have been used to switch off EWS-FLI1, leading to reduction of Ewing’s Sarcoma growth. These include: (Top) Small peptides (chain of amino acids) can stick to EWS-FLI1 in a very specific way and prevent RNA Helicase A (RHA) from attaching. When RHA does not attach to EWS-FLI1, Ewing’s Sarcoma cells stop growing. The challenges with this approach are assembling stable peptides and transporting these agents to tumors in humans. (Bottom) Small molecules (pharmaceuticals are mostly small molecules) that specifically stick to parts of EWS-FLI1 can prevent it from interacting with RHA. The challenge is to find the specific small molecules. Small molecules can be given to patients by oral or intravenous routes and are relatively easier to formulate at this time.