Abstract A039: Novel microRNA-mediated G2/M vulnerabilities in glioblastoma revealed by a CRISPR/Cas9 screen

Abstract Glioblastoma (GBM) is the most common and aggressive adult brain tumor, known for its resistance to standard treatments primarily due to the presence of therapy-resistant glioblastoma stem cells (GSCs). Despite the critical role that GSCs play in GBM progression, there are currently no clinical therapies specifically targeting these cells. While some microRNAs (miRNAs) have been identified to be essential for the survival and differentiation of GSCs, the roles of most miRNAs in these cells remain poorly understood. To address this knowledge gap, we conducted a microRNA essentiality screen in patient-derived GSC-enriched cultures using our newly designed miRNA-focused CRISPR/Cas9 library. By systematically testing the essentiality of nearly all known miRNAs, we identified 23 miRNAs that are crucial for the survival of U3013MG GSCs. Validation experiments confirmed that 5 of these miRNA candidates are essential for the growth of 4 GSC models and 2 differentiated GBM models. Essentiality was also validated in a 3D growth context using a neurosphere assay, where we observed a significant reduction in tumor-initiating potential in U3013MG following miRNA knockout. Ongoing experiments have demonstrated similar phenotypes using orthogonal miRNA inhibition methods, including miRNA sponges, which will be reported soon. We will also examine whether inhibiting miRNAs in combination with temozolomide, the standard chemotherapy treatment, sensitizes GBM cells to chemotherapy. Notably, overexpression of some candidate miRNA increased the growth and tumor-initiating potential of GBM cells suggesting that they demonstrate reciprocal oncogenic properties upon knockout and overexpression. Overall, our results support the hypothesis that a subset of miRNA contribute to GBM cell viability and tumor progression, indicating their potential as therapeutic targets in GBM. Ultimately, our aim is to identify and exploit new miRNA vulnerabilities in GSCs to develop effective therapies for GBM. Our current research is focused on uncovering the mechanisms through which essential miRNAs contribute to the survival of GBM, with the goal of evaluating their therapeutic potential. Our experiments have demonstrated that each of our candidate miRNA knockouts consistently induced a G2/M cell cycle arrest across all tested cell lines. This G2/M arrest was associated with increased cell death and mitotic abnormalities in GBM cells. Collectively, these findings suggest that miRNA candidates are crucial for GBM growth by regulating specific G2/M-associated vulnerabilities. We are currently conducting further experiments to explore this vulnerability in greater depth. Specifically, our next objective is to identify and characterize the molecular targets and signaling pathways affected by these miRNAs. Overall, we identified 5 miRNAs that are essential for the GBM survival by inducing G2/M arrest followed by cell death and mitotic abnormalities. Our research showcases 5 novel and promising miRNA candidates as potential therapeutic strategies for GBM. Citation Format: Iulia A. Grigore, Athulram Rajagopal, Jonathan Tak-Sum Chow, Yumeng Zhou, Sukhleen Randhawa, Byeong Yeop Lee, Leonardo Salmena. Novel microRNA-mediated G2/M vulnerabilities in glioblastoma revealed by a CRISPR/Cas9 screen abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (6Suppl): Abstract nr A039.