Matt Harlow1, Elissa Boguslawski2, Anderson Peck2, Zachary Madaj2, Maria J. G. Navarro3, Pablo Aviles3, Carlos Galmarini3, Patrick Grohar2. 1Vanderbilt University, Nashville, TN; 2Van Andel Research Institute, Grand Rapids, MI; 3PharmaMar, S.A., Madrid, Spain .
Disclosures :M. Harlow: None. E. Boguslawski: None. A. Peck: None. Z. Madaj: None. M.J.G. Navarro: ; PharmaMar. P. Aviles: ; PharmaMar. C. Galmarini: ; PharmaMar. P. Grohar: None.
Abstract
BACKGROUND:
Ewing sarcoma (ES) is a pediatric malignancy characterized by the EWS-FLI1 transcription factor. ES tumors require the continuous transcriptional activity of EWS-FLI1 for the oncogenic phenotype, regardless of fusion type, which makes EWS-FLI1 an ideal therapeutic target. To date, no EWS-FLI1 directed therapies have been developed. Interestingly, there have been reports of ES patients as exceptional responders in clinical trials, including a complete response in a refractory, metastatic patient that was administered the natural product trabectedin in a Phase I trial. We have since established trabectedin as an EWS-FLI1 inhibitor that acts through a re-localization mechanism at concentrations that are clinically achievable. Intriguingly, trabectedin failed in the Phase II setting after a change in schedule that lead to a decrease in reported Cmax. In this report, we unify the disparate clinical trial conclusions by examining the schedule-dependency of trabectedin in ES cells.
METHODS:
Here, we model a broad range of exposures to trabectedin and investigate the cell viability kinetics using live cell imaging analysis software and traditional MTS. In parallel, we present a novel assay to quantitatively measure the kinetics of drug clearance in vitro. In addition, we use qPCR and western blotting to examine EWS-FLI1 target gene expression changes in response to different exposures to trabectedin. Finally, we investigated ES cell lines containing variant EWS-FLI1 fusion types and identify an increased sensitivity of type III/IV ES cell lines to trabectedin treatment. We recapitulated these findings in vivo using ES xenograft models.
RESULTS:
Sensitivity of ES cells to trabectedin treatment is schedule-dependent. Here, we demonstrate that cells treated with trabectedin at equivalent exposures but different Cmax exhibit profound differences in cell viability. The differences in viability correlate with Cmax that are necessary to inhibit EWS-FLI1 activity in vitro. By measuring the kinetics of drug clearance, we show that the inhibitory effects on EWS-FLI1 are durable even after trabectedin has been washed out of the culture medium. In addition, we examine the sensitivity of variant EWS-FLI1 fusion proteins such as type III/IV fusions both in vitro and in vivo.
CONCLUSIONS:
In this report, we use clinical trial data to develop a more clinically relevant cell culture system for ES drug treatments. Importantly, we demonstrate that equivalent exposures do not lead to equivalent outcomes. We believe that this observation explains the discordant results of the Phase I and II clinical trials, and suggests that the Cmax achieved in serum dictates whether a patient will respond. In addition, we show that type III/IV EWS-FLI1 fusion proteins are particularly sensitive to the drug.