TMIC-95. Hypoxic Tumor Microenvironment Promotes Hippo Pathway Transcriptional Activity to Enrich Glioma Stem Cells in the Peri-necrotic Niche of Glioblastoma

Abstract Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis, marked by extensive necrosis and enrichment of tumor-associated macrophages (TAMs) and glioma stem cells (GSCs) in peri-necrotic regions. Analysis of Ivy-GAP and single-cell RNA-seq data revealed elevated Hippo pathway transcriptional activity in the hypoxic peri-necrotic niche despite low expression of canonical stemness factors like SOX2, OLIG2, and FOXM1. We hypothesize that Hippo activation supports GSC maintenance through hypoxia and TAM-derived cytokines. In patient-derived GBM neurospheres, 1% hypoxia increased nuclear YAP1/TAZ and CYR61 expression. Zyxin (Zyx), a potential upstream regulator, was upregulated and phosphorylated under hypoxia. AKT, previously known to phosphorylate Zyx and be regulated by hypoxia, was also activated, suggesting hypoxia promotes Zyx phosphorylation and YAP/TAZ nuclear translocation via AKT. AKT inhibition with Capivasertib reduced Zyx phosphorylation and Hippo transcription in hypoxia. Conditioned media from primary macrophages activated Hippo signaling in GBM cells. NicheNetR identified TGF-β1 as a hypoxia-specific TAM-derived cytokine promoting malignant gene programs. Exogenous TGF-β1 increased CYR61, while TGFBR2 expression is elevated under hypoxia. Blocking TGFBR2 with inhibitor LY2109761 abolished TGF-β1-induced Hippo activation. Hypoxia and TGF-β1 synergistically enhanced GSC maintenance in limiting dilution assays. In vivo, pharmacologic inhibition of YAP/TAZ with Verteporfin or genetic TAZ knockdown in the RCAS/tv-a mouse model reduced tumor burden and prolonged survival. Flow cytometry confirmed decreased CD133+/CD44+ GSCs in TAZ-deficient tumors. These results demonstrate hypoxia and TGF-β1 signaling to activate YAP/TAZ-driven Hippo signaling, promoting GSC maintenance in the peri-necrotic GBM niche.