494 Enhanced Senescence Phenotype In Human Medulloblastoma via Protein Phosphatase 2A Inhibition and Radiation Therapy

Abstract

INTRODUCTION:Medulloblastoma is the most common malignant pediatric brain tumor. Radiation(RT) is a part of standard care but causes significant neurocognitive impairment. Strategies to sensitize RT is critical to improving the quality of life and long-term survival of patients. Previous studies show that inhibition of protein-phosphatase 2A(PP2A)–a ubiquitous serine/threonine phosphatase–enhanced sensitivity to RT in multiple brain tumor xenografts. However, the mechanism behind enhanced radio-cytotoxicity is poorly understood. We hypothesize that PP2A inhibition combined with RT will increase cytoplasmic double-stranded DNA (dsDNA) and induce cGAS-cGAMP-STING signaling, promoting cellular senescence and thereby decreasing tumor growth.METHODS:D425-WT, a human group-3 medulloblastoma cell line, was used to generate a CRISPR sgPP2A knock out, D425-KO. We radiated WT and KO cells in vitro, quantified dsDNA in cytoplasmic extract using dsDNA Assay kit, and measured cGAMP levels in whole cell lysate using ELISA. Through FACS, we quantified the proportion of b-gal-expressing senescent cells. We measured protein expression of senescence markers (p21, p16, g-h2aX) by Western blot(WB). Finally, we injected D425 WT or KO cells orthotopically into cerebellum of nude NU/J mice followed by RT (10Gy) 3 days post-injection and monitored survival.RESULTS:In response to RT, D425-KO compared to WT showed significant increase in cytoplasmic dsDNA and intracellular cGAMP--a cyclic di-nucleotide STING agonist. There was also an increase in b-gal+ senescent cells and senescence markers expression in WB. In vivo, PP2AKO alone extended longevity, but PP2AKO with RT further prolonged survival compared to KO or RT alone.CONCLUSION:PP2A inhibition enhances RT induced cytosolic dsDNA damage and STING activation, promoting cellular senescence in vitro and sensitivity to RT in vivo.