American Association for Cancer Research
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FIGURE 5 from Piperacetazine Directly Binds to the PAX3::FOXO1 Fusion Protein and Inhibits Its Transcriptional Activity

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posted on 2023-10-06, 14:20 authored by Kay Nakazawa, Taryn Shaw, Young K. Song, Marilyn Kouassi-Brou, Anna Molotkova, Purushottam B. Tiwari, Hsien-Chao Chou, Xinyu Wen, Jun S. Wei, Emre Deniz, Jeffrey A. Toretsky, Charles Keller, Frederic G. Barr, Javed Khan, Aykut Üren

Piperacetazine inhibits anchorage-independent growth of FP-RMS cells. A, Average 48 hour IC50 values for indicated cell lines. Sample size indicated above each bar. Red bars: FP-RMS, blue bars: FN-RMS, black bars: non-RMS. U48484, U66788, U37125, and U57810 are cell lines from transgenic RMS mouse models. B, RH30 and RD colonies in soft agar with DMSO or 15 µmol/L piperacetazine treatment. The top row images are whole well images taken with Gelcount and the bottom row images are microscope images of the colonies at 40X magnification. C, Gelcount quantification of colonies in B (*, P < 0.0001; Student t test; ns, P > 0.05).


Children's Cancer Foundation (CCF)

HHS | NIH | National Cancer Institute (NCI)




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. On the basis of 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. RMS is a malignant soft-tissue tumor mainly affecting the pediatric population. A subgroup of RMS with worse prognosis harbors a unique chromosomal translocation creating an oncogenic fusion protein, PAX3::FOXO1. We identified piperacetazine as a direct inhibitor of PAX3::FOXO1, which may provide a scaffold for designing RMS-specific targeted therapy.