Abstract 2900: Musashi-1 regulated oncogenic switch enhances therapy resistance to DNA damage in glioblastoma

Abstract Glioblastoma (GBM) is notoriously resistant to treatment, largely due to its inherent enhancement of DNA repair mechanisms. Standard therapies, which include ionizing radiation and alkylating agents, primarily exert their effects by damaging DNA. Therefore, targeting the DNA damage response (DDR) has emerged as a promising strategy to sensitize tumor cells to these treatments. The RNA-binding protein Musashi-1 (MSI1) is known to contribute to the aggressive nature of GBM, yet its specific role in DNA damage-mediated therapy resistance remains largely unexplored. This study employed three GBM cell lines, an orthotopic GBM model in immunodeficient mice, and tissue samples from 35 GBM patients. We developed a specialized antibody to detect phosphorylation at the MSI1-S347 site in both cells and tissues. Site-directed mutagenesis and transcriptome sequencing were used to investigate the effects of MSI1 phosphorylation on GBM. The comet and colony formation assays were conducted to assess therapy resistance in GBM cells following treatment with carmustine (BCNU) or X-ray irradiation. Flow cytometry, immunofluorescence, and immunohistochemistry were utilized to identify cancer stem cell markers in GBM. A luminescence reporter system was created to pinpoint the binding site of non-phosphorylated MSI1 and to establish a visual drug screening platform targeting MSI1-S347. We identified a new phosphorylation site at serine-347 (S347) on the C-terminal of MSI1, which transcriptome analysis linked to the processing of DNA double-strand breaks in GBM. Patient tissue samples showed significantly higher levels of phosphorylated MSI1-S347 compared to normal brain tissues, regardless of tumor location or GBM subtype. Phosphorylation at MSI1-S347 enhanced BCNU resistance by promoting DNA repair mechanisms, and it also increased resistance to X-ray irradiation by preserving colony-forming ability. Additionally, higher levels of S347 phosphorylation were associated with reduced survival rates in GBM animal models. Mechanistically, we observed a positive correlation between phosphorylated S347 and the cancer stem cell marker CD133 expression. Further analysis revealed changes in binding interactions between MSI1 and p21 mRNA depending on the S347 phosphorylation state. To address this, we developed a luminescence reporter system to visually screen drugs that can modulate MSI1-S347 phosphorylation. This platform measures S347 phosphorylation levels in vivo and serves as a drug screening tool targeting GBM therapy resistance. This study uncovers a novel mechanism by which MSI1 enhances DNA damage repair, contributing to therapy resistance in GBM. Understanding the DNA damage response can guide clinical strategies to inhibit the MSI1 binding switch and enhance treatment efficacy. Targeting the MSI1 binding switch could offer a more effective approach to overcoming GBM therapy resistance. Citation Format: Xian Liu, Tsoi Pok Kong, Man Ding Wong, Liang Ting Lin. Musashi-1 regulated oncogenic switch enhances therapy resistance to DNA damage in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2900.