Abstract Number: LB-177
Presentation Time: Monday, Apr 18, 2016, 1:00 PM - 5:00 PM
Author Block:
Matt Harlow1, Matthew Easton2, Maria Jose Guillén Navarro3, Lisa Turner2, Galen Hostetter2, Carlos Galmarini3, Pablo Aviles3, Patrick Grohar2. 1Vanderbilt University, Nashville, TN; 2Van Andel Research Institute, Grand Rapids, MI; 3Pharmamar S.A., Madrid, Spain .
Abstract Body: BACKGROUND:
Ewing sarcoma is a bone and soft tissue sarcoma characterized by fusion of the wild-type EWSR1 and FLI1 genes, which creates a constitutively activated transcription factor, EWS/FLI1. Ewing sarcoma cells are dependent on the continued activity of EWS/FLI1 to block differentiation and promote proliferation. For these reasons, EWS/FLI1 represents an ideal drug target. With this in mind, we have shown that the small molecule trabectedin inhibits EWS/FLI1 activity and synergizes with irinotecan in vivo. In addition, we showed that the gene expression response of Ewing sarcoma cells to trabectedin treatment is identical to keratinocytes exposed to UV light. Interestingly, wild-type EWSR1 accumulates in the nucleolus in response to UV light damage. Therefore, we hypothesize that the response and sensitivity of Ewing sarcoma cells to trabectedin is due to the re-localization of EWS/FLI1 in the nucleus.
METHODS:
Here, we utilize confocal microscopy and chromatin immunoprecipitation to characterize the localization of EWS/FLI1 and EWS/FLI1 deletion mutants in response to trabectedin. Because trabectedin has a narrow therapeutic index, we characterize the effect of PM01183, a second-generation structural analog with an improved pharmacokinetic profile, on EWS/FLI1 localization. In addition, we use qPCR and western blotting to determine the kinetics of EWS/FLI1 inhibition in response to this class of compounds. We demonstrate the effects of PM01183 treatment on the EWS/FLI1 gene signature genome-wide. Finally, we translate these findings in vivo using two Ewing sarcoma xenograft models.
RESULTS:
Treatment of Ewing sarcoma cells with Trabectedin and PM01183 causes EWS/FLI1 to accumulate in the nucleolus. This effect is accompanied by a loss of binding at target sequences and a reversal in expression of the gene signature of EWS/FLI1. We found the effect on EWS/FLI1 to be sustained, even after removal of the drug from the cell culture medium. We extended these findings in vivo to demonstrate the efficacy of a previously characterized combination therapy. Interestingly, treatment of Ewing sarcoma xenografts with the combination therapy caused the tumor to be replaced by benign adipocytes.
CONCLUSIONS:
We characterize a novel method of targeting an oncogenic fusion protein by leveraging a property of the wild-type protein to inactivate the oncogenic transcription factor. We demonstrate that wild-type EWSR1 protein’s propensity to accumulate in the nucleolus upon UV light damage is maintained within the oncogenic EWS/FLI1 protein with trabectedin or PM01183 treatment. We believe that this accounts for the heightened sensitivity of Ewing sarcoma cells to this class of compounds and has numerous clinical implications.