P09.04.A MECHANISTIC PATHWAY ANALYSIS OF PD-L1 UPREGULATION IN GLIOBLASTOMA CELLS FOLLOWING HYPOFRACTIONATED IRRADIATION

Abstract BACKGROUND Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults, characterized by high invasiveness and aggressive growth, resulting in a poor prognosis with a median overall survival rate of 14 to 17 months. Standard treatment schemes, including surgical resection, temozolomide and radiotherapy, have yielded only modest clinical benefit. While immunotherapeutic approaches have succeeded in various malignancies, their efficacy in GBM has been limited. The PD-1/PD-L1 pathway, crucial in immune regulation, has emerged as a promising therapeutic target. Through the PD-1/PD-L1 interaction, GBM cells evade immune surveillance by activating PD-1 receptors on immune cells, inhibiting T-cell responses and promoting tumor progression. GBM cells upregulate PD-L1 expression through receptors such as EGFR, IFNGR, and c-Met-R. Radiotherapy has also been shown to increase PD-L1 expression. Understanding the mechanisms driving PD-L1 expression is essential for overcoming therapeutic resistance and improving treatment strategies for GBM. This study seeks to identify the signaling pathways responsible for PD-L1 upregulation in GBM after irradiation. MATERIAL AND METHODS We investigated the impact of hypofractionated radiotherapy (2x5Gy) combined with various signaling pathway Inhibitors (Akt-Inhibitor VIII, c-Met-Inhibitor 1, Cobimetinib, Encorafenib, Erlotinib, m-TOR-Inhibitor 3, Vemurafenib and SC79) on the expression profile of the immune checkpoint molecules PD-L1/2 in the murine GBM cell line GL261-luc2 using flow cytometry. Cell viability and toxicity assays identified EC50 values. Combinations of the most effective inhibitors to decrease PD-L1 and PD-L2 expression were subsequently tested on human GBM cell lines (U118, U138, HROG-06). Additionally, RNA-sequencing was performed on irradiated human GBM cell lines. Based on the transcriptomic findings, further flow cytometric analyses were conducted to assess the effects of CXCR2 inhibition with Reparixin on PD-L1 regulation. Supernatants were collected to evaluate neuroinflammatory analytes. RESULTS Our findings indicate that the RAS/ERK and PI3K/Akt/mTOR signaling pathways contribute to the upregulation of irradiation-dependent expression of PD-L1. Inhibitors of these pathways show time- and dose-dependent toxicity and suppress cellular proliferation, requiring dose optimization in future studies. CONCLUSION Modulating these pathways post-irradiation may reduce GBM cell radioresistance, necessitating further investigation into potential molecular targets.