Abstract 3244: EWS-FLI1-driven upregulation of UGT3A2 unlocks drug sensitivity pathway in Ewing sarcoma
Authors: Olaoye Olasunkanmi , Harrington Colleen , Ross Kenneth , Seong Bo Kyung A. , de Waal Luk , Qi Jun , Roth Jennifer , Stegmaier Kimberly 
Published: March 2024

Ewing sarcoma (ES), an aggressive pediatric bone tumor, presents a clinical challenge, particularly for metastatic or relapsed disease. The hallmark genetic alteration involves the fusion of the FET RNA-binding protein, EWSR1, with an ETS transcription factor, typically FLI1, resulting in the EWS-FLI1 fusion oncoprotein. EWS-FLI1 orchestrates a unique epigenetic program, leading to the aberrant expression of genes crucial for cell-cycle regulation, DNA damage response, and cell migration.

Targeting the EWS-FLI1 fusion oncoprotein is a daunting task, which has prompted us to pursue other strategies in parallel. Here, we present an innovative approach harnessing the power of phenotypic drug screens to uncover amenable pathways for targeted medicines in ES. Leveraging data from the Broad Institute’s repurposing dataset encompassing 4,518 drug molecules screened across 578 adherent cancer cell lines using the high-throughput multiplexed screening platform known as PRISM, we identified compounds with selective efficacy against ES.

The top-ranking compound identified for selective ES toxicity in the screen was R112, an ATP-competitive inhibitor of Spleen Tyrosine Kinase (SYK). In low-throughput evaluation, R112 induced selective ES cell death when compared to other sarcomas. However, the reported target of R112, SYK, is a dependency in ES based on the Cancer Dependency Map (DepMap) resource.

Subsequent validation of compounds identified in the PRISM screen as selectively toxic in ES revealed that while majority of them induced selective ES cell death, their reported targets were diverse and not linked to known dependencies in ES. Notably, one of the emerging compounds, luteolin, a natural compound known for its antioxidative properties, stood out and was selected for further studies alongside R112.

Comprehensive genome-wide CRISPR-rescue screens identified UGT3A2, encoding a UDP glycosyltransferase, along with its solute carrier, SLC35B4, as pivotal genes that when deleted rescued both R112 and luteolin-induced cytotoxicity in ES cells. UGT3A2, a direct target gene of EWS-FLI1, demonstrated hyperexpression in EWS-FLI1-positive ES cells and no expression in most cancer and normal cells. While ES cells do not depend on UGT3A2 for survival, this abnormal elevation of UGT3A2 expression emerged as a key determinant driving their hypersensitivity to a subset of additional small molecules identified from the PRISM screen. Despite divergent mechanisms and direct targets, these drugs shared a common phenoxy motif, which UGT3A2 glycosylated.

This specific EWS-FLI1-induced UGT3A2 upregulation, thus, unveils a novel biomarker for drug sensitivity,paving the way for precision medicine strategies tailored for Ewing sarcoma.

Beyond Ewing sarcoma, this work holds promise in reshaping therapeutic paradigms and offering new avenues for discovering safe and enhanced treatments across a spectrum of cancers.

Citation Format: Olasunkanmi Olaoye, Colleen Harrington, Kenneth Ross, Bo Kyung A. Seong, Luk de Waal, Jun Qi, Jennifer Roth, Kimberly Stegmaier. EWS-FLI1-driven upregulation of UGT3A2 unlocks drug sensitivity pathway in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3244.