Small Molecules

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

Jeffrey A. Toretsky, M.D.

Associate Professor

Department of Oncology and Pediatrics

Lombardi Comprehensive Cancer Center

Georgetown University

Washington, DC

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 (Refs. 1 and 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 (Ref. 3). In addition, small molecule allosteric inhibitors have been developed that block the interaction of Runx1 and CBFbeta in leukemia (Ref. 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).

The Ewing's Sarcoma Family of Tumors

An excellent review article by R. Lor Randall, MD, "Ewing's Sarcoma Family of Tumors (ESFT)" appears in ESUN, Vol. 1, N. 3, 2004. Click here to access it.

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) (Refs. 5 and 6) or small interfering RNAs (siRNA) (Refs. 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.

Figure 1: Turning Off EWS-FLI1 Turns Off ESFT Tumor Growth. Different strategies have been used to turn off EWS-FLI1 (see Legend).

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 (Ref. 10). RHA expression enhanced EWS-FLI1 mediated anchorage-independent colony formation, while an inactivating mutation of RHA prevented colony formation (Ref. 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.

Summary

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 (Ref. 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.

Acknowledgement

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

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