TIMELESS promotes glioma stemness and malignancy through JAK-STAT3 pathway.

Gliomas are the most common primary tumors of the central nervous system. Among them, glioblastoma (GBM), a World Health Organization (WHO) grade 4 glioma, is the most aggressive form and remains one of the most lethal human cancers. Its poor prognosis is largely attributable to rapid progression, frequent recurrence, and profound therapeutic resistance, all of which are closely associated with glioma stem cells (GSCs). Although several signaling pathways sustaining GSC properties have been identified, upstream regulators that coordinate these pathways remain incompletely understood. TIMELESS, originally identified as a component of the circadian clock, has recently been implicated in the regulation of brain function and is aberrantly overexpressed in multiple cancer types; however, its role in glioma malignancy has not been defined. Here, we demonstrate that TIMELESS mRNA expression increases with glioma grade and is associated with poor prognosis across multiple glioma cohorts. Genetic depletion of Timeless suppressed tumor aggressiveness and prolonged survival in an orthotopic mouse glioma model by attenuating GSC properties. Mechanistically, Timeless enhanced stemness of glioma by upregulating Janus kinases (JAK) expression and promoting phosphorylation of signal transducer and activator of transcription 3 (STAT3). These effects were conserved in human GBM cells, supporting the relevance of TIMELESS-mediated signaling across species. Together, our findings uncover an unexpected, clock-independent function of TIMELESS in sustaining glioma stemness and malignancy and highlight the TIMELESS-JAK-STAT3 axis as a non-canonical mechanism that operates beyond the traditional circadian clockwork in treatment-resistant glioma.