StagX1: An Isoquinolinone Compound Targeting Carboxylesterase 1 (CES1) Positive Cells as a Potential Therapeutic Agent for Ewing Sarcoma
Introduction
Ewing sarcoma (EWS) is a highly aggressive tumor and the second most common bone or soft tissue tumor in children and young adults. It is characterized by chromosomal translocations involving a member of the FET gene family and an ETS transcription factor, such as EWSR1–FLI1 (occurring in approximately 85% of cases), as well as EWSR1-ERG and other less frequent fusions (approximately 10-15% of cases). The resulting fusion proteins are tumor-specific transcription factors that reprogram the transcriptome and epigenetics of the genome. In order to find new therapeutic agents for EWS, we identified a compound called StagX1 through high-throughput screening. Here we report the mechanism of StagX1 that inhibits the growth of some Ewing sarcoma (EWS) cell lines.
Methods
We used HPLC-MS to identify the metabolites of StagX1. Following the StagX1 treatment, we measured cell viability using CellTiter Blue assay, and determined the expression of carboxylesterases (CESs) using immunoblotting and RNA-seq.
Results
Our findings revealed that StagX1 is a carboxyl ester with limited stability in liver microsomes and mouse plasma and gets hydrolyzed to form an acid product (StagX1-acid). Inhibitors of CESs prevented StagX1 from being converted to StagX1-acid, thereby eliminating the effect of StagX1 on cell growth inhibition. RNA-seq and immunoblotting assays indicated that StagX1-sensitive cell lines had significant overexpression of CES1, suggesting that CES1 is responsible for the hydrolysis of StagX1. In vitro enzymatic assays confirmed that CES1 is the enzyme responsible for hydrolyzing StagX1. StagX1 was stable in tissue culture medium and was gradually converted to StagX1-acid when added to cells. Both StagX1 and StagX1-acid were found inside cells within 15 minutes, suggesting that StagX1 is a prodrug that is hydrolyzed into StagX1-acid by CES1 inside cells. Although StagX1-acid is stable, it cannot inhibit cell growth, likely because of its hydrophilic nature that prevents it from entering cells. However, StagX1-acid liposome could inhibit the cells that are not sensitive to StagX1.
Conclusion
Our study provides evidence that StagX1 is a prodrug that is hydrolyzed by CES1 to form StagX1-acid. Upon treatment with StagX1, it enters cells and gets converted into StagX1-acid in cells that express CES1. This conversion triggers apoptosis, ultimately leading to the inhibition of cell growth. These findings have important implications for the development of more effective treatments for EWS and highlight the potential of targeting CES1-positive EWS tumors as a therapeutic strategy for this aggressive cancer.