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Projects

Grohar Laboratory

Project Description

The goal of our laboratory is to develop and clinically translate novel targeted therapies for pediatric sarcomas to improve patient outcomes. We employ a bench to bedside and back again approach to identify, characterize and develop new compounds and drug combinations for Ewing sarcoma.

Projects_EWS-FLI1_KoemanThe EWS-FLI1 Transcription Factor

To date, our lab has primarily focused on targeting the EWS-FLI1 transcription factor for Ewing sarcoma. EWS-FLI1 is an oncogenic transcription factor formed by the t(11;22)(q24;12) chromosomal translocation that leads to the fusion of the EWSR1 gene to FLI1 (figure 1). The resulting dysregulated transcription factor alters the expression of more than 500 genes to drive tumorigenesis and progression. Importantly, a number of independent studies have shown that silencing of EWS-FLI1 is incompatible with Ewing sarcoma cell survival. Therefore, the goal of this work is to develop a small molecule therapy that blocks EWS-FLI1 activity.

Studies of Trabectedin

Trabectedin (ET-743; Ecteinascidin 743; Yondelis) is a natural product originally isolated from the sea squirt Ectenascidia turbinata. This compound has been found to be active in translocation-positive sarcomas. In addition, early preclinical work suggests a particular sensitivity of Ewing sarcoma cells to the drug. In order to tie these two together, we showed that the drug blocks the activity of EWS-FLI1.

To further develop the drug, we showed that the combination of trabectedin and irinotecan are synergistic. The mechanism of this synergy is (figure 3):

  • EWS-FLI1 drives expression of the WRN gene. Cells deficient in WRN are hypersensitive to irinotecan because WRN helps resolve stalled replication forks.
  • Irinotecan feeds back to augment the trabectedin-mediated blockade of EWS-FLI1.

We translated this compound back to the clinic in collaboration with European groups (the drug is approved in Europe). We have reported a case of a patient with multiply recurrent/relapsed refractory Ewing sarcoma who achieved a sustained response to the combination of irinotecan and trabectedin.*

Future efforts are focused on developing second and third generation trabectedin analogs as EWS-FLI1 inhibitors in collaboration with PharmaMar.

Studies of Mithramycin

In order to identify other EWS-FLI1 inhibitors, we have utilized a high-throughput screening approach. We developed a cell-based assay and screened more than 50,000 compounds in collaboration with NCI at Frederick to identify mithramycin as an inhibitor of EWS-FLI1. We showed that the drug inhibits EWS-FLI1 and suppresses key targets such as NR0B1 and suppresses the gene signature of EWS-FLI1 that has been generated by other labs. We also showed activity in our xenograft model of Ewing sarcoma and suppression of NR0B1 in vivo (figure 4).

Again we translated the compound to the clinic in collaboration with the National Cancer Institute. However, the trial is currently on hold.

Future work is focused on characterizing the mechanism of suppression, identifying second-generation mithramycin analogs with improved target suppression, and developing pharmacodynamic markers of EWS-FLI1 suppression so that the suppression of targets can be followed in patients (figure 5).

*Tancredi R, Zambelli A, De Prada GA, Fregoni V, Pavesi L, Riccardi A, Burdach S, Grohar PJ, D’Incalci M. 2015. Targeting the EWS-FLI1 transcription factor in Ewing sarcoma. Cancer Chemother Pharmacol 75(6):1317–1320