Grohar Laboratory Research Overview

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We reported the synergistic combination of trabectedin and irinotecan and related the synergy to EWS-FLI1. Trabectedin exposure suppresses EWS-FLI1 to block expression of the WRN helicase and sensitize the cells to the DNA-damaging effects of irinotecan. Irinotecan feeds back to amplify and sustain EWS-FLI1 suppression. Both effects require EWS-FLI1 which is only found in Ewing sarcoma cells.

We found that trabectedin causes a redistribution of EWS-FLI1 within the nucleus to the nucleolus, likely accounting for the suppression of activity. Importantly, we identified lurbinectedin as a second-generation compound with an improved toxicity profile that also causes this phenotype.

Clinical anecdotal responses to trabectedin/irinotecan have been described.

We have determined that this class of compounds triggers an epigenetic switch leading to an increase and redistribution of histone post-translational modifications associated with gene silencing, H3K27me3 and H3K9me3. Importantly, these effects are schedule dependent and require a threshold concentration. This provides the justification for our clinical study of trabectedin and the basis for the use of lurbinectedin in the clinic in this disease type.

We continue to delve into the mechanism of EWS-FLI1 suppression of this class of compounds as a means to understand therapeutic response and resistance and an opportunity to discover new drug combinations. In addition, we are exploring the activity in related tumor types.

Our initial efforts at compound screening were completed in collaboration with the National Cancer Institute at Frederick. In this effort, we screened more than 50,000 compounds and identified mithramycin as an inhibitor of the EWS-FLI1 transcription factor.

We subsequently screened a series of analogs of mithramycin to identify both a more potent and less toxic second-generation analog, EC8105 and EC8042.

Additional cell line screens suggested that an aggressive pediatric soft tissue sarcoma called rhabdoid tumor was particularly sensitive to this class of compounds. We characterized the mechanism of hypersensitivity of rhabdoid tumor and demonstrated inhibition of the defining molecular lesion of the tumor, the SWI/SNF chromatin remodeling complex. Importantly, SWI/SNF has been identified by other groups as the complex associated with EWS-FLI1. Therefore, heightened activity in these two cell types may be related to this activity.

Our lab collaborated with groups at the National Cancer Institute in our initial efforts with genetic screens. We identified RNA processing as a therapeutic vulnerability in subsets of Ewing sarcoma tumors. We now have the ability to perform genome-wide screens in our lab and collaborate with investigators at the University of Pennsylvania High-Throughput Screening Core.

Improving outcomes in high-risk Ewing sarcoma will likely require combination therapies. Matrix drug screening is one approach to identify these combinations. We have used this approach to identify the combination of mithramycin and CDK9 inhibitors that may both improve activity and limit the toxicity of mithramycin.

The Grohar Lab team have ongoing projects and unpublished data in all of these discovery areas. We continue to use screening approaches and novel chemical probes to delineate mechanisms of chemotherapeutic efficacy in Ewing sarcoma and related tumors.

A major focus of our translational studies is understanding the relationships between drug exposure and target suppression both in vitro and in vivo. In addition, we have worked to develop methods to quantitate target suppression in tissue.

A number of years ago, we recognized the need for a pharmacodynamic marker of EWS-FLI1 suppression. We found that 18F-FLT PET reflects EWS-FLI1 activity because the fusion protein drives expression of the proteins responsible for tracer activity. It is notable that this effect is not related to perturbation of the cell cycle.

Correlative biology that supports clinical translation guides our future studies. We are collaborating with investigators at Penn and the NCI to evaluate this tracer in patients in the clinic. In addition, we are using the tracer in our preclinical studies to examine the contribution of tumor architecture to EWS-FLI1 suppression. Finally, we have expanded our translational efforts trying to better understand mechanisms of drug sensitivity and resistance using patient tissue collected in our study.

The fundamental goal of our work is to improve outcomes for the patients we serve. This makes the clinical translation of our therapies the most important step in the process. We collaborate with expert clinical trialists to develop clinical trials rooted in our preclinical work. The goal of these studies is to test these agents in a rigorous fashion and at the same time gain insight into the fundamental biology of the disease. SARC037 is the realization of this approach. This study was developed in collaboration with national experts in sarcoma and provides the basis for future clinical studies of this compound and analogs such as lurbinectedin.

Importantly this study also features excellent key collaborations with translational scientists locally at CHOP/Penn and across the country to allow us to perform 18F-FLT PET Imaging, single cell and bulk transcriptional analysis, model building, translocation typing and mutational profiling of individual patients.

We have a number of projects in various stages of clinical translation. All of our lab studies are performed with the clinic as the goal with careful consideration of translational principles at a very early stage in project development.