The symbiotic interactions between cancer stem cells and the tumor microenvironment (TME) are critical for tumor progression. However, the molecular mechanism underlying this symbiosis in glioblastoma (GBM) remains enigmatic. Here, we show that circadian locomotor output cycles kaput (CLOCK) and its heterodimeric partner brain and muscle ARNT-like 1 (BMAL1) in glioma stem cells (GSC) drive immunosuppression in GBM. Integrated analyses of the data from transcriptome profiling, single-cell RNA sequencing, and TCGA datasets, coupled with functional studies, identified legumain (LGMN) as a direct transcriptional target of the CLOCK–BMAL1 complex in GSCs. Moreover, CLOCK-directed olfactomedin-like 3 (OLFML3) upregulates LGMN in GSCs via hypoxia-inducible factor 1-alpha (HIF1α) signaling. Consequently, LGMN promotes microglial infiltration into the GBM TME via upregulating CD162 and polarizes infiltrating microglia toward an immune-suppressive phenotype. In GBM mouse models, inhibition of the CLOCK–OLFML3–HIF1α–LGMN–CD162 axis reduces intratumoral immune-suppressive microglia, increases CD8+ T-cell infiltration, activation, and cytotoxicity, and synergizes with anti–programmed cell death protein 1 (anti–PD-1 therapy). In human GBM, the CLOCK-regulated LGMN signaling correlates positively with microglial abundance and poor prognosis. Together, these findings uncover the CLOCK–OLFML3–HIF1α–LGMN axis as a molecular switch that controls microglial biology and immunosuppression, thus revealing potential new therapeutic targets for patients with GBM.

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