Piperacetazine directly binds to the PAX3::FOXO1 fusion protein and inhibits its transcriptional activity. Piperacetazine directly binds to the PAX3::FOXO1 fusion protein and inhibits its transcriptional activity

The tumor-specific chromosomal translocation product, PAX3::FOXO1, is an aberrant fusion protein that plays a key role for oncogenesis in the alveolar subtype of rhabdomyosarcoma (RMS). PAX3::FOXO1 represents a validated molecular target for alveolar RMS and successful inhibition of its oncogenic activity is likely to have significant clinical applications. Even though several PAX3::FOXO1 function-based screening studies have been successfully completed, a directly binding small molecule inhibitor of PAX3::FOXO1 has not been reported. Therefore, we screened small molecule libraries to identify compounds that were capable of directly binding to PAX3::FOXO1 protein using surface plasmon resonance technology. Compounds that directly bound to PAX3::FOXO1 were further evaluated in secondary transcriptional activation assays. We discovered that piperacetazine can directly bind to PAX3::FOXO1 protein and inhibit fusion protein-derived transcription in multiple alveolar RMS cell lines. Piperacetazine inhibited anchorage-independent growth of fusion positive alveolar RMS cells but not embryonal RMS cells. Based on our findings, piperacetazine is a molecular scaffold upon which derivatives could be developed as specific inhibitors of PAX3::FOXO1. These novel inhibitors could potentially be evaluated in future clinical trials for recurrent or metastatic alveolar RMS as novel targeted therapy options. Overall design: RH30 cells were treated with 30 µM of piperacetazine or DMSO for 24 hours before harvest for RNA using RNAeasy kits (Qiagen Cat# 74104, Hilden, Germany). RNA-Seq libraries were constructed using Illumina TruSeq stranded mRNA sample preparation kits and sequenced on a NextSeq500 sequencer using 2x75bp paired-end protocol (Illumina). Genes with >10 total reads in these samples were kept, and raw read counts were transformed to regularized logarithm values using DESeq2 package (RRID:SCR_015687) (35). The ranked list of genes for the comparison between piperacetazine-treated and DMSO control were sorted by log2 fold change. The Gene Set Enrichment Analysis (GSEA) was performed using command “gsea-cli.sh GSEAPreranked” and curated gene sets