STEM-05. HYPOXIA AND ACIDOSIS PROMOTE YAP/TAZ ACTIVATION, POTENTIALLY SUPPORTING GLIOMA STEM CELL MAINTENANCE IN THE PERI-NECROTIC ZONES

Abstract

Abstract Glioblastoma (GBM) is a highly malignant brain tumor characterized by rapid growth and poor clinical outcomes. The development of necrosis is associated with massive tumor microenvironment (TME) reshaping and rapid progression characterized by accumulation of tumor-associated macrophages (TAMs) and glioma stem cells (GSCs) in peri-necrotic zones. Using Ivy-GAP and single-cell RNA seq data, we found that many canonical stem-cell associated transcription factors (SOX2, OLIG2, FOXM1) were not upregulated in the hypoxic peri-necrotic zone, yet Hippo transcriptional activation was consistently noted under these conditions. We hypothesized that Hippo activation promotes GSC stemness and may be due to direct effects of hypoxic; the acidic conditions associated with necrosis; or by cytokines secreted by adjacent TAMs. Using patient-derived GBM neurosphere cultures, we exposed glioma cells to 1% hypoxia and found that nuclear expression of YAP1 and TAZ, as well as Hippo pathway readout CYR61, were upregulated compared to normoxia (21%) at 48 hrs, indicating Hippo activation. Exposing cells to an acidic environment (pH 6.3) also caused Hippo transcriptional activation independent of hypoxia. Using single-cell RNA-seq data, we uncovered potential hypoxia-regulated upstream genes that could be responsible for nuclear YAP/TAZ transportation under hypoxia, including Zyxin, WBP2, and PP2A. We also found that culturing GBM cells with media conditioned by human primary macrophages activated Hippo transcription. Using human cytokine arrays, we identified cytokines that showed increased levels in TAM-conditioned media, including EGF, CXCL8, TGF-β, and CXCL5. Treating GBM cells with either hypoxia or a pharmacological activator of YAP/TAZ led to the upregulation of proteins in the inhibitor of differentiation (ID) family (ID1, ID2), which could represent a downstream mechanism related to stem-like behavior following Hippo activation. Our data support the conclusion that hypoxia and TAM cytokines directly stimulate YAP/TAZ activation to potentially promote GSC maintenance in the GBM peri-necrotic niche.