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. 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