1333 THY1 Upregulates Glioma Stem Cell Phenotype and Induces Treatment Resistance in Glioblastoma

INTRODUCTION: Glioblastoma(GBM) is a lethal intracranial neoplasm that inevitably recurs despite treatment with DNA damaging therapies, radiation(RT) and temozolomide(TMZ). Recurrence is likely driven by inherently resistant glioma stem-like cells(GSCs), which have baseline upregulation of DNA damage response(DDR) pathways due to elevated DNA replication stress(RS). Elevated DDR confers resistance to RT/TMZ however factors mediating elevated RS and therefore upregulated DDR in GSC are poorly defined. METHODS: THY1 was overexpressed or silenced in patient-derived neurosphere and organoid models utilizing inducible shRNA systems. Models were irradiated and collected at various timepoints. Alterations in activation and completion of DNA repair were evaluated using yH2AX immunofluorescence and ATM/ATR activation via western blot. In parallel, GBM tissue from over 20 patients underwent single cell RNA sequencing and spatial transcriptomics to assess pathways associated with THY1. RESULTS: Spatial transcriptomics and single cell RNA sequencing data demonstrate significant upregulation of GSC pathways in high-THY1 cells, and constitutive upregulation of DDR pathways in high-THY1 cells compared to low-THY1 cells(p<2e-16), especially homologous recombination. In high-THY1 in vitro models, DNA repair activation is upregulated at baseline. Following irradiation, high-THY1 models mediate more rapid repair of DNA, with peak repair 2 hours after radiation. CONCLUSIONS: These findings demonstrate that GSCs exhibit elevated levels of DNA replication stress, and high baseline DDR pathway activation which allows for extremely rapid DNA repair. Here we demonstrate that THY1 mediates treatment resistance as result of constitutively upregulated DNA repair. Elucidation of the mechanisms mediating the GSC phenotype in high-THY1 cells is underway to better characterize the resulting DNA replication stress and upregulation of DNA repair, and identify a targetable pathway.