Notch1 O-GlcNAcylation drives tumor stemness and mechanoadaptation to a stiff microenvironment and promotes chordoma recurrence.

Chordomas are rare malignant osseous neoplasms with a striking rate of recurrence. Primary chordomas typically originate from embryonic notochord remnants, whereas recurrent chordomas usually stem from tumor cells infiltrating bone or cartilage after surgery. Clinically, the recurrent chordomas exhibit a stiffer extracellular microenvironment (ECM) than primary tumors. Intriguingly, this study identified cytoskeleton rearrangement, stress fiber reorganization, enhanced stemness, and Notch signaling activation in recurrent chordoma tissues or cell lines surviving stiff substrates, indicating the critical roles of mechanical remodeling and tumor stemness in stiffness resistance. We propose a potentially novel recurrence model where tumor cells experience mechanoadaptive organization, which enables them to resist stiff microenvironment-induced cell death. O-GlcNAcylation of Notch1 intracellular domain (NICD1) is central to this process. Mechanistically, the stiff ECM-driven ligand-independent phosphorylation of EPHA2 sequentially activated LYN kinase and subsequently triggered O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) activity by phosphorylating Y989 and Y418, critical residues for OGT glycosyltransferase activity; this induced NICD1 O-GlcNAcylation at T2063, T2090, and S2162, specifically promoting transcription of mechanical and stemness-related genes. MIR31 deletion upregulated LYN, enhancing stiffness perception and promoting O-GlcNAc addition to NICD1, finally resulting in mechanoadaptation- and tumor stemness-driven recurrence. Consequently, MIR31 deletion is a potential biomarker for recurrence and patient stratification in Notch- or OGT-targeted therapies.