HDAC3 genetic and pharmacologic inhibition radiosensitizes fusion positive rhabdomyosarcoma by promoting DNA double-strand breaks

radiotherapy (RT) plays a critical role in the management of rhabdomyosarcoma (RMS), the prevalent soft tissue sarcoma in childhood. the high risk PAX3-FOXO1 fusion-positive subtype (FP-RMS) is often resistant to RT. we have recently demonstrated that inhibition of class-I histone deacetylases (HDACs) radiosensitizes FP-RMS both in vitro and in vivo. however, HDAC inhibitors exhibited limited success on solid tumors in human clinical trials, at least in part due to the presence of off-target effects. hence, identifying specific HDAC isoforms that can be targeted to radiosensitize FP-RMS is imperative. we, here, found that only HDAC3 silencing, among all class-I HDACs screened by siRNA, radiosensitizes FP-RMS cells by inhibiting colony formation. thus, we dissected the effects of HDAC3 depletion using CRISPR/Cas9-dependent HDAC3 knock-out (KO) in FP-RMS cells, which resulted in endoplasmatic reticulum stress activation, ERK inactivation, PARP1- and caspase-dependent apoptosis and reduced stemness when combined with irradiation compared to single treatments. HDAC3 loss-of-function increased DNA damage in irradiated cells augmenting H2AX phosphorylation and DNA double-strand breaks (DSBs) and counteracting irradiation-dependent activation of ATM and DNA-Pkcs as well as Rad51 protein induction. moreover, HDAC3 depletion hampers FP-RMS tumor growth in vivo and maximally inhibits the growth of irradiated tumors compared to single approaches. we, then, developed a new HDAC3 inhibitor, MC4448, which showed specific cell anti-tumor effects and mirrors the radiosensitizing effects of HDAC3 depletion in vitro synergizing with ERKs inhibition. overall, our findings dissect the pro-survival role of HDAC3 in FP-RMS and suggest HDAC3 genetic or pharmacologic inhibition as a new promising strategy to overcome radioresistance in this tumor.