# A collaborative approach to analyze and target the EWS-FLI1 transcription factor in patients with Ewing sarcoma

> **NIH NIH U01** · SARC · 2020 · $494,871

## Abstract

Project Abstract
It has been known for more than 25 years that Ewing sarcoma cells are absolutely dependent on the EWS-
FLI1 transcription factor for cell survival. EWS-FLI1 is a dysregulated transcription factor formed by the
t(11;22)(q24;q12) chromosomal translocation. This fusion oncogene alters the expression of over 500 genes
to establish a transcriptional program responsible for tumorigenesis and progression. Despite this known
dependence, the clinical realization of an EWS-FLI1 inhibitor has not been achieved. We have previously
shown that trabectedin interferes with EWS-FLI1 activity. We showed that the drug blocks expression of EWS-
FLI1 downstream targets, including the very specific target gene NR0B1. We demonstrated reversal of
expression at the promoter, mRNA, and protein levels both in vitro and in vivo in xenograft models of the
disease. In addition, we showed that the drug reverses the gene signature of EWS-FLI1 using gene expression
profiling and Gene Set Enrichment Analysis (GSEA). We subsequently determined the mechanism of EWS-
FLI1 suppression. Treatment of Ewing sarcoma cells with trabectedin redistributes EWS-FLI1 within the
nucleus to the nucleolus. Importantly, this redistribution of EWS-FLI1 is extremely concentration dependent,
requiring relatively high, but clinically achievable concentrations of the drug. In addition, irinotecan can both
amplify and sustain the trabectedin mediated block of EWS-FLI1 activity. Therefore, the goal of this study is to
use the combination of trabectedin and irinotecan to achieve the therapeutic suppression of EWS-FLI1. In
order to accomplish this, in aim 1, we will establish the optimal dose and schedule of administration of
trabectedin in combination with low dose irinotecan. We will use a traditional 3 + 3 phase I design and
administer trabectedin as a 1-hour infusion in order to achieve the highest serum concentration of drug
possible. We will then perform a limited dose escalation of irinotecan to both amplify and sustain suppression
of EWS-FLI1. In aim 2, we will determine if we can use 18F-FLT as a biomarker of EWS-FLI1 suppression. We
have demonstrated that silencing of EWS-FLI1 suppresses expression of the proteins responsible for 18F-FLT
activity, ENT1, ENT2 and TK1. This results in a loss of PET avidity of Ewing sarcoma cells with EWS-FLI1
suppression in preclinical models of the disease. In this study, we will determine if these effects translate to
patients. Finally, in aim 3, we will do a series of correlative studies to characterize the EWS-FLI1
transcriptome for the first time in patients. We will perform both single cell and bulk tumor RNA sequencing to
identify the putative EWS-FLI1 targets for the first time in the clinic. We will organize the gene signatures into
transcriptional networks, compare the results to preclinical data and establish a series of Patient Derived
Xenografts (PDXs). This will provide insight into mechanisms of tumorigenesis and drug resistance. In
...

## Key facts

- **NIH application ID:** 9954027
- **Project number:** 5U01CA236220-02
- **Recipient organization:** SARC
- **Principal Investigator:** Patrick J Grohar
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $494,871
- **Award type:** 5
- **Project period:** 2019-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9954027

## Citation

> US National Institutes of Health, RePORTER application 9954027, A collaborative approach to analyze and target the EWS-FLI1 transcription factor in patients with Ewing sarcoma (5U01CA236220-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9954027. Licensed CC0.

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