STEM-07. UNVEILING THE ROLE OF HYPOXIA AND TAM-SECRETED TGF-Β1 IN PROMOTING GLIOMA STEM CELL MAINTENANCE VIA YAP/TAZ ACTIVATION IN GLIOBLASTOMA PERI-NECROTIC NICHE

Abstract Glioblastoma (GBM) is a highly malignant brain tumor with rapid growth and poor outcomes. In the tumor microenvironment (TME), necrosis triggers significant reshaping and rapid progression, marked by tumor-associated macrophages (TAMs) and glioma stem cells (GSCs) accumulation in peri-necrotic zones. Analysis of Ivy-GAP and single-cell RNA-Seq data revealed consistent Hippo pathway activation in the hypoxic peri-necrotic zone, despite canonical stem-cell transcription factors such as SOX2, OLIG2, and FOXM1 not being upregulated. We hypothesize that Hippo transcription activation may promote GSC stemness via direct effects of hypoxia and/or TAM-secreted cytokines. Exposing patient-derived GBM neurospheres to 1% hypoxia increased nuclear YAP1 and TAZ expression, along with CYR61, a Hippo pathway readout, compared to normoxia at 48 hours. Single-cell RNA-Seq identified potential hypoxia-regulated genes upstream of YAP/TAZ, including Zyxin, upregulated in 1% hypoxia-treated cultures, possibly facilitating nuclear YAP/TAZ transport. Culturing GBM cells with primary macrophage-conditioned media activated Hippo transcription, with TGF-β1 identified as a hypoxia-specific driver of malignant cell gene expression with single-cell RNA-Seq analysis using NichenetR. Treating GBM cells with TGF-β1 increased CYR61 expression, signifying Hippo transcription activation. Furthermore, we showed that hypoxia and TGF-β1 synergistically maintained GSCs with extreme limiting dilution assays. In the immunocompetent RCAS/tv-a mouse model, Verteporfin, a YAP/TAZ inhibitor, significantly reduced tumor size and prolonged mouse lifespan. TAZ knockdown similarly extended lifespan and reduced tumor growth, with flow cytometry showing a decrease in CD133/CD44 positive GSC populations in knockdown mice. These findings suggest the conclusion that hypoxia and TAM-secreted TGF-β1 directly stimulate YAP/TAZ activation and potentially promote GSC stemness in the GBM peri-necrotic niche.