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

• Published in: Cell death discovery 2024-08, Vol.10 (1), p.351-20, Article 351
• Main Authors: Cassandri, Matteo, Porrazzo, Antonella, Pomella, Silvia, Noce, Beatrice, Zwergel, Clemens, Aiello, Francesca Antonella, Vulcano, Francesca, Milazzo, Luisa, Camero, Simona, Pajalunga, Deborah, Spada, Massimo, Manzi, Valeria, Gravina, Giovanni Luca, Codenotti, Silvia, Piccione, Michela, Tomaciello, Miriam, Signore, Michele, Barillari, Giovanni, Marchese, Cinzia, Fanzani, Alessandro, De Angelis, Biagio, Quintarelli, Concetta, Vakoc, Christopher R., Chen, Eleanor Y., Megiorni, Francesca, Locatelli, Franco, Valente, Sergio, Mai, Antonello, Rota, Rossella, Marampon, Francesco
• Format: Article
• Language: English
• Subjects: 631/337/1427/2122; 631/67/1798; 631/67/2332; Apoptosis; Biochemistry; Biomedical and Life Sciences; Caspase; Cell activation; Cell Biology; Cell Cycle Analysis; Children; Clinical trials; CRISPR; DNA damage; FOXO1 protein; Histone deacetylase; Histone H2A; Isoforms; Life Sciences; Pax3 protein; Phosphorylation; Poly(ADP-ribose) polymerase; Protein kinase C; Rad51 protein; Radiation therapy; Radioresistance; Rhabdomyosarcoma; siRNA; Soft tissue sarcoma; Solid tumors; Stem Cells; Tumors
• ISSN: 2058-7716; 2058-7716
• DOI: 10.1038/s41420-024-02115-y

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.