Small Molecule Inhibition of PAX3-FOXO1 through AKT Activation Suppresses Malignant Phenotypes of Alveolar Rhabdomyosarcoma

Abstract

Alveolar rhabdomyosarcoma (ARMS) comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of ARMS is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. ARMS cells are dependent to the oncogenic activity of PAX3-FOXO1 and its expression status in ARMS tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small-molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/Endoplasmic Reticulum Ca2+-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in ARMS cells demonstrated that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteosomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of ARMS cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an ARMS xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against ARMS.

PLK1 Phosphorylates PAX3-FOXO1, the Inhibition of Which Triggers Regression of Alveolar Rhabdomyosarcoma

“…Currently, we aim to mechanistically understand these different levels of regulation required for the oncogenic function. We, as well as others, were able to demonstrate that the transcriptional activity of PAX3-FOXO1 depends on its phosphorylation state providing novel therapeutic opportunities (23)(24)(25).…”

p38α MAPK disables KMT1A-mediated repression of myogenic differentiation program

“…Reporter luciferase assay was performed as described [37]. Luciferase activity was determined using the luciferase assay system (Promega) and normalized with protein concentration determined by DC protein assay reagent (Bio-Rad).…”

Intergenically Spliced Chimeric RNAs in Cancer

“…For instance, many small inhibitors of PAX3-FOXO1 chimeric proteins can suppress malignant phenotypes of ARMS and may be attractive therapeutic strategies in this subset of patients [58, 59]. However, since the fusion occurs during skeletal muscle differentiation, potential side effects on muscle development in a pediatric population should be considered.…”