Jeffrey A. Toretsky, MD

Developing a New Molecule to Treat Ewing's Sarcoma

Two Lombardi scientists have found a small molecule that could serve as the basis for a new treatment for Ewing's sarcoma. If further studies continue to show positive results, they believe the novel agent could be the first targeted therapy to treat the disease, which is a rare childhood cancer.

In the United States, about 500 patients are diagnosed with Ewing's sarcoma each year, and they are treated with a combination of five different chemotherapy drugs. Between 60-70 percent of patients survive over time, but many have long-term side effects that linger from the therapy.

Over 95 percent of patients with Ewing's sarcoma have the same mutation - called a translocation - where the end of chromosome 22 becomes fused to the end of chromosome 11. This very specific mutation fuses two normally unrelated proteins into one oncogenic protein called EWS-FLI1.

Jeffrey Toretsky, MD, led the Lombardi research to discover the potential new treatment. He has been at the forefront of Ewing's sarcoma research for a number of years. Working with Lombardi colleague Aykut Üren, MD, the Toretsky-Üren laboratory was the first to create a recombinant EWS-FLI fusion protein. This allowed them to study the activity of the oncogene in depth, and the team discovered an important binding partner called RNA helicase A (RHA).

Through a series of partner proteins including RHA, EWS-FLI1 initiates and sustains the cancer. In order to stop the continued proliferation of the tumors, Dr. Toretsky and his team knew they needed to disrupt the binding between the oncogenic protein and its partners. Focusing on RHA, they worked to identify the specific region on RHA that stuck to EWS-FLI1. This painstaking work took over ten years from idea to implementation. Throughout that time, The Children's Cancer Foundation of Baltimore, MD, supported Dr. Toretsky's research, believing in the promise of his approach.

In order to discover the binding patterns of EWS-FLI1, the laboratory must produce millions of copies of the RHA and EWS-FLI1 proteins. A generous gift from the Go4theGoal Foundation recently allowed Dr. Toretsky to purchase a new piece of equipment to purify the proteins. This ensures that his team is able to push forward with their research, including the testing of the new drug.

Finally, collaborating with Milton Brown, MD, PhD, Director of Georgetown's Drug Discovery Program, the team searched for a molecule that would keep the two proteins separated. In other words, they sought an agent that would stick to EWS-FLI1 in the very place that RHA bound to the fusion molecule.

Dr. Brown's laboratory is able to screen thousands of compounds for their potential drug-like properties in a matter of minutes instead of days or weeks. Using the known crystal structures of proteins, Dr. Brown and his team match the 3-dimensional structures of the target with a library of small molecules in silico, or on the computer. This way, they can conduct a very rapid assessment to find the best candidates for further testing in cells, which is a much more time consuming and expensive process.

In this case, Dr. Toretsky was able to use a library of small molecules loaned to Georgetown from the National Cancer Institute. The team of investigators screened 3,000 compounds for their potential to block the EWS-FLI1 interaction with RHA. Using a direct binding assay, the team found several molecules that bound to EWS-FLI1 and chose to pursue one of them, which seemed the most promising.

"We've taken this initial hit from the library and discovered the relationship between the small molecule and the EWS-FLI1 protein," said Dr. Brown. "This is very exciting because it provides us the opportunity to bridge the gap between basic science and pre-clinical studies."

The drug discovery team is now working to make the compound target the pocket more specifically in order to create a better candidate drug. While several stages of the research process remain before the drug can move into the clinic, this could be the first targeted therapy to be developed for Ewing's sarcoma.

Because of the relatively small number of patients diagnosed with the disease each year, there has been little research investment into new treatments. So-called "orphan" diseases such as Ewing's, garner little interest from pharmaceutical companies who most often conduct drug development research. Georgetown's Drug Discovery Program provides an academic solution, enabling basic laboratory research, like Dr. Toretsky's, to progress to pre-clinical stages in the drug discovery pipeline.

"Ewing's sarcoma is rare," said Dr. Toretsky, "but our work has the potential to improve treatment not only for patients with this disease but for others who have cancers with similar molecular characteristics."

This finding is also important for another reason - it demonstrates a proof of principle for a new method of designing drugs. The notion long accepted among scientists is that it is not possible to block protein-protein interactions given that the surface of these proteins is slippery, and much too flexible for a drug to bind to.

"These are wiggly proteins, yet this study shows that inhibiting protein-protein interactions with a small molecule is possible," said Dr. Toretsky.

This is unique because the small molecule developed by Drs. Toretsky and Brown has a different activity than other drugs that have been shown to work against fusion proteins. For example, Gleevac - which treats chronic myeloid leukemia formed by a chromosome translocation - blocks the function of a single protein, not the binding of two proteins. Dr. Toretsky is hopeful that this means that other fusion proteins, found in other sarcomas as well as diverse disorders, can be successfully treated as well.

The findings were presented at the 2008 annual meeting of the American Association for Cancer Research (AACR) in San Diego by Hayriye Verda Erkizan, PhD, a postdoctoral researcher in Dr. Toretsky's lab.

by Allison Whitney
(Published August 2008)