The major focus of my laboratory is to evaluate the role of EWS-FLI1 in the oncogenesis and maintenance of Ewing's Sarcoma Family of Tumors (ESFT). I seek novel therapies that can be implemented in the clinic to improve the survival of children with ESFT and reduce morbidity. A significant clinical challenge in ESFT is that patients often have a complete regression of their tumor following initial chemotherapy only to have a recurrence after chemotherapy ends. This suggests that a stem cell of ESFT survives the chemotherapy and allows for tumor re-growth. My laboratory is currently investigating three areas that address the problem of tumor re-growth by determining: (1) the role of insulin-like growth factor type 1 (IGF-I) in patients with ESFT, (2) the role of the protein tyrosine phosphatase L1 (PTPL1) as a significant target of the EWS-FLI1 transcription factor, and (3) the requirement of RNA helicase A (RHA) as a cofactor of EWS-FLI1 function. These projects have all been continuously funded, either by the Children’s Cancer Foundation or the NIH, since 1997 and 1999, respectively.

My research suggests that high IGF-I levels might decrease ESFT patient survival based upon a retrospective analysis of serum samples. I am prospectively evaluating this hypothesis by measuring IGF-I and IGFBP-3 levels, as part of a clinical trial in patients with ESFT. In addition, I found that blockade of phosphoinositide 3-OH kinase and Akt, downstream enzymes from IGF-IR that mediate cell survival, enhances the chemosensitivity of ESFT cells. These findings have led us develop and test Akt inhibitors to enhance apoptosis in ESFT.

My laboratory team discovered a novel role for phosphatases in Ewing’s sarcoma based upon observations from the IGF-IR studies conducted while I was a fellow at the NCI. The protein tyrosine phosphatase L1 is a direct transcriptional target of EWS-FLI1 and is highly expressed in ESFT tumors. Reduction of PTPL1 levels in ESFT cells decreases their anchorage-independent growth. This project is now funded by the NCI for the next five years. The three major aims of this project include: (1) to determine how PTPL1 modulates signaling in ESFT, (2) to develop small molecule inhibitors of PTPL1, and (3) to evaluate the levels of PTPL1 in patient tissues and the impact of PTPL1 upon patient survival.

An ideal cancer therapeutic target is a molecule that is unique to the cancer cell. EWS-FLI1 is a unique protein that is only found in ESFT cells. Reduction of EWS-FLI1 leads to the death of ESFT cells. Therapy directed towards EWS-FLI1 might eliminate a repopulating stem cell and render chemotherapy more effective towards the cure of ESFT patients. In order to take advantage of the uniqueness of EWS-FLI1, my lab identifies protein partners of EWS-FLI1. The interaction points between these protein partners and EWS-FLI1 have the potential to be unique in three-dimensional space. These unique interaction points could then be targets for EWS-FLI1 inactivation.

One key protein that interacts with EWS-FLI1 is RNA helicase A (RHA). RHA is an ubiquitously and highly expressed protein in 40 ESFT patient tumors. Thus far, immunoprecipitation experiments in ESFT cells confirm EWS-FLI1 and RHA in a complex. Addition of RHA to ESFT cells enhances EWS-FLI1 transcription and to EWS-FLI1 transformed fibroblasts enhances anchorage-independent growth.

Our laboratory utilizes recombinant EWS-FLI1 to directly screen small molecules as potential probes of EWS-FLI1 function. Small molecules that bind to EWS-FLI1 and inhibit EWS-FLI1 function will be evaluated as anti-tumor agents. Screening will occur in three ways. High throughput screening of compound libraries will be initially conducted using surface plasmon resonance. These screening assays will directly measure the dissociation of full-length EWS-FLI1 from full length RHA. Small molecules that cause disruption will be assessed in a dual promoter assay in ESFT cells. Small molecules will also be screened for inhibition of anchorage independent growth of ESFT cells.