USP49 undergoes liquid-liquid phase separation and stabilizes RPA70 to induce radioresistance through homologous recombination repair in esophageal squamous cell carcinoma.

Radiotherapy is crucial in the comprehensive management of esophageal squamous cell carcinoma (ESCC). Nevertheless, its efficacy is compromised by the emergence of radioresistance, leading to poor prognoses. Accumulating research has underscored the critical role of deubiquitinases (DUBs) in modulating radiosensitivity. This study aimed to identify potential DUBs that mediate radioresistance in ESCC and to elucidate the underlying regulatory mechanisms. Our research has identified deubiquitinase USP49 as a critical regulator of radiosensitivity in ESCC both in vivo and in vitro. Elevated USP49 expression was related to a poorer prognosis in ESCC patients receiving chemoradiotherapy. Mechanistically, the ataxia telangiectasia mutated (ATM) promotes liquid-liquid phase separation (LLPS) of USP49 and its recruitment at DNA double-strand breaks (DSBs) upon ionizing radiation (IR) exposure. USP49 deubiquitinates and recruits RPA70 to DSBs along with Rad51, thereby promoting homologous recombination (HR) repair. Moreover, p53 further augments the transcription of USP49 upon IR exposure. Our findings clarify a previously unrecognized mechanism by which USP49 modulates HR and radioresistance, proposing that targeting USP49 could potentially enhance radiosensitivity and improve prognosis of ESCC patients.