SLFN5 mediates liquid-to-solid phase transition of NOTCH1 to suppress stemness and radioresistance of non-small cell lung cancer

Background: Radiotherapy remains one of the principal therapeutic modalities for non-small cell lung cancer (NSCLC), yet its therapeutic efficacy is frequently compromised by tumor recurrence, metastasis, and the development of radioresistance. Accumulating evidence identifies cancer stem cells (CSCs) as a critical driver of radioresistance. Therefore, elucidating the underlying molecular mechanisms and developing therapeutic targets to enhance radiosensitivity are of critical importance. Methods: To identify key mediators of adaptive radioresistance in NSCLC, we established radioresistant NSCLC cell lines through repeated cycles of irradiation and subsequently performed RNA-seq analysis comparing them with their parental counterparts. The effects of SLFN5 on radiosensitivity were evaluated by flow cytometry and colony formation assays, and further validated in vivo using a nude mouse xenograft tumor model. Cancer stem-like properties were assessed by RT-PCR, Western blot, flow cytometry, sphere formation assay, and in vivo limiting dilution tumorigenesis assays. Subsequently, we employed mass spectrometry, co-immunoprecipitation, and proximity ligation assay to identify the interaction between SLFN5 and NOTCH1. Furthermore, phase separation assay, fluorescence recovery after photobleaching, CUT&Tag-seq, and ATAC-seq were used to investigate the role of NOTCH1 phase separation in promoting stemness and radioresistance, and the regulatory effect of SLFN5 on this process. Finally, the methylation status of the SLFN5 promoter was analyzed using bisulfite sequencing PCR. Results: Our results demonstrate that SLFN5 was significantly downregulated in radioresistant NSCLC cell lines. Overexpressing SLFN5 effectively suppressed cancer stemness and epithelial-mesenchymal transition, thereby enhancing radiosensitivity both in vitro and in vivo. Conversely, knockdown of SLFN5 had significantly opposite effects. Mechanistically, liquid-liquid phase separation property is critical for NOTCH1 mediated stemness and radioresistance. Notably, SLFN5 interacts with NOTCH1 and induces the liquid-to-solid phase transition of NOTCH1, thereby impairing NOTCH1 signaling and the subsequent stemness and radioresistance. Finally, we identified DNMT3A and DNMT3B as the epigenetic regulators responsible for promoter hypermethylation and consequent silencing of SLFN5. Conclusion: Our study reveals that DNA methylation downregulated SLFN5 resulting in radioresistance of NSCLC via liberating NOTCH1 from gel-like phase to liquid droplet to potentiating stemness of cancer cells. This research provides a potential therapeutic strategy to overcome radioresistance in NSCLC.