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Radioresistance is one of the main causes of cancer treatment failure, which leads to relapse and inferior survival outcome of cancer patients. Liquid-liquid phase separation (LLPS) of proteins is known to be involved in various biological processes, whereas its role in the regulation of radiosensitivity remains largely unknown. In this study, we characterized NONO, an RNA/DNA binding protein with LLPS capacity, as an essential regulator of tumor radioresistance. In vitro assay showed that NONO involved in DNA repair via non-homologous end joining (NHEJ) manner. NONO knockout significantly reduced DNA damage repair and sensitized tumor cells to irradiation in vitro and in vivo. NONO overexpression was correlated with an inferior survival outcome in cancer patients. Mechanically, NONO was associated with nuclear EGFR (nEGFR). Both irradiation and EGF treatment induced nEGFR accumulation, thereby increased the association between NONO and nEGFR. However, NONO was not a substrate of EGFR kinase. Furthermore, NONO promoted DNA damage-induced DNA-PK phosphorylation at T2609 by enhancing the interaction between EGFR and DNA-PK. Importantly, NONO protein formed high concentration LLPS droplets in vitro, and recruited EGFR and DNA-PK. Disruption of NONO droplets with LLPS inhibitor significantly reduced the interaction between EGFR and DNA-PK, and suppressed DNA damage-induced phosphorylation of T2609-DNA-PK. Taken together, LLPS of NONO recruits nuclear EGFR and DNA-PK and enhances their interaction, further increases DNA damage-activated pT2609-DNA-PK and promotes NHEJ-mediated DNA repair, finally leads to tumor radioresistance. NONO phase separation-mediated radioresistance may serve as a novel molecular target to sensitize tumor cell to radiotherapy., Competing Interests: None., (AJCR Copyright © 2021.)

Siglec15 is a recently characterized immunosuppressive transmembrane protein, which expresses in various types of solid tumors and promotes cancer development. Several studies reported that Siglec15 is a prognostic biomarker of cancer patients, and targeting Siglec15 may be a promising strategy for cancer therapy. However, the regulation of Siglec15 function remains unclear. Here we show that the immunosuppression activity of Siglec15 is largely modulated by N-glycosylation. Through mass spectrum and site mutation analysis, we identified that Siglec15 was extensively glycosylated at N172 (N173 for mouse) in cancer cells. Meanwhile, Siglec15 N172Q had a similar molecular weight with PNGase-F-treated Siglec15, suggesting N172 as the only one glycosylation residue. In xenograft model, glycosylation deficiency of Siglec15 reduced tumor growth in C57BL/6 mice, but had no impact in nude mice, indicating the requirement of N-glycosylation for immunosuppressive function of Siglec15. Furthermore, colorectal cancer patients with high Siglec15 expression had a poor response to neoadjuvant chemo-radiotherapy and short survival time. Interestingly, removal of N-glycosylation enhances the detection of Siglec15, which may be employed in the prediction of immunotherapy response. Together, our results disclose a pivotal role of glycosylated Siglec15 in tumor immune escape, which may be a therapeutic target for cancer immunotherapy., Competing Interests: None., (AJCR Copyright © 2021.)