TMIC-48. HYPOXIC AND PERI-NECROTIC ZONES IN GLIOBLASTOMA CAUSE HIPPO-OFF SIGNALING, POTENTIALLY SUPPORTING GLIOMA STEM CELLS

Abstract

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor with pre-necrotic and post-necrotic growth phases. The development of necrosis is associated with extensive tumor micro-environmental (TME) rearrangement and rapid biologic advancement. Compared to pre-necrotic GBMs, post-necrotic GBMs are heterogenous and contain multi-focal regions of necrosis surrounded by pseudopalisading cells and characterized by severe hypoxia. Hypoxic, peri-necrotic zones are highly enriched for glioma stem cells (GSCs) and tumor associated macrophages (TAMs), the latter accounting for 30-40% total tumor cellularity. Using Ivy-GAP data, we performed gene set enrichment analysis (GSEA) for stem-cell associated markers, comparing peri-necrotic to non-necrotic regions. We also used TCGA/CGGA data to identify correlations between hypoxia markers and classic stem-cell associated markers (SOX2, SOX9, nestin, STAT3, prominin). None of these showed enrichment in the peri-necrotic zone or association with hypoxia markers. However, we uncovered strong associations of Hippo-off signaling with peri-necrotic regions, signifying activation of YAP/TAZ transcriptional activation. We therefore hypothesized that Hippo-off (YAP/TAZ transcriptional activation) in hypoxic peri-necrotic zones promotes GSC self-renewing division. To support this, we performed GSEA for Hippo pathway readout genes and found peri-necrotic enrichment of Hippo regulated genes CYR61, BIRC2, SERPINE1 and AREG. Using patient-derived GBM neurosphere cultures, we exposed glioma cells to 1% hypoxia and found that p-YAP1 and p-TAZ protein levels were downregulated compared to normoxia (21%) at 24 hrs, correlating with Hippo-on. Hippo pathway gene targets were also upregulated, indicating activation of YAP/TAZ transcription. Using Ivy-GAP, TCGA and CGGA data, we also found peri-necrotic/hypoxic enrichment of two cytokines, IL-1β and IL-6, known to trigger YAP/TAZ activation. Using cytokine multiplex analysis, we observed an upregulation in both IL-1β and IL-6 secretion from hypoxic monocytes and microglia, raising the possibility that these cytokines directly stimulate Hippo-off and promote GSC self-renewing division in the GBM peri-necrotic niche.