FIGURE 4 from Piperacetazine Directly Binds to the PAX3::FOXO1 Fusion Protein and Inhibits Its Transcriptional Activity

Abstract

<p>Piperacetazine alters the expression of PAX3::FOXO1 target genes. <b>A,</b> Protein expression of PAX3::FOXO1 target genes was evaluated by Western blot analysis in RH30 cells treated with 10 µmol/L piperacetazine for 6 days. <b>B,</b> RNA-seq was performed in fusion-positive RH30 cells treated with 30 µmol/L piperacetazine for 24 hours. The ranked gene expression list was compared with existing lists of genes using GSEA. Gene set descriptions: Top left: genes upregulated during human skeletal muscle myoblast differentiation. Top right: Hallmark genes during myogenesis. Bottom left: Genes downregulated in SAOS-2 (osteosarcoma) cells upon expression of PAX3::FOXO1. Bottom right: Genes downregulated in fusion-positive versus fusion-negative RMS cell lines. NES = normalized enrichment score, FDR = false discovery rate). <b>C,</b> Piperacetazine does not cause PAX3::FOXO1 to shift its intracellular localization. RH30 cells were treated with 10 µmol/L piperacetazine for 24 hours prior to cellular fractionation, which were analyzed via Western blot analysis. Lamin A/C and alpha-tubulin were used as positive controls for nuclear and cytoplasmic fractions, respectively. <b>D,</b> Piperacetazine does not alter PAX3::FOXO1 protein levels or phosphorylation of Ser256, as evaluated by Western blot analysis. RH30 cells were treated with 10 µmol/L piperacetazine or vehicle for 3 days. <b>E,</b> PAX3::FOXO1 protein was immobilized on a CM5 chip, and double-stranded PAX3::FOXO1 oligonucleotide (100 nmol/L), piperacetazine (10 µmol/L), or a combination of the two were injected over the chip surface. Piperacetazine did not inhibit DNA binding to PAX3::FOXO1.</p>