Abstract 6698: Inducible miRNA therapeutics: A multi-targeted strategy for treating diffuse midline gliomas

Abstract Diffuse Midline Gliomas (DMGs) are highly aggressive gliomas that account for ∼20% of pediatric central nervous system tumors. The H3K27M-altered subset is particularly lethal, with 100% tumor recurrence and no effective long-term treatments. Thus, a desperate need to develop new therapeutic avenues remains. RNA therapies, a new class of therapeutics that include microRNA (miRNA) therapies, utilize RNA mimetics to target and treat cancer and other diseases. Compared to single-target small molecule inhibitors, miRNAs represent potent future tools capable of simultaneously targeting multiple aberrant signaling pathways in DMG to increase therapeutic efficacy and prevent tumor resistance and recurrence. To identify targetable, aberrant gene expression in H3K27M-mutated DMG, we performed RNA sequencing of nascent, newly synthesized RNA in the human DMG cell line SU-DIPG-XIII and its isogenic pair SU-DIPG-XIII-H3K27M-KO, where oncohistone expression is prevented by CRISPR gene editing. We compared transcriptional profiles in isogenic cells to render a list of potential disease/tumor-driving genes, expressed solely in H3K27M DMGs, that can be targeted. Computational analysis using miRNet identified miRNAs hsa-miR-124-3p, hsa-miR-27a-3p, and hsa-miR-146a-5p as promising candidate RNA mimetics to develop as new therapeutic options for DMG H3K27M-altered. These miRNAs are predicted to each target 300-400 genes upregulated specifically in H3K27M DMGs, highlighting the powerful capability of this new class of RNA therapeutics. Notably, preliminary proliferation assays in SU-DIPG-XIII cells showed that DMG-specific miRNAs significantly reduced tumor cell growth, highlighting their therapeutic potential. To evaluate efficacy and cellular consequences of identified miRNAs in a panel of human and murine DMG H3K27M-altered cells, we have employed an inducible lentiviral system that allows for precise on/off gene regulation with miRNA mimetics. This system enables controlled manipulation of gene expression, providing a powerful tool to study the functional consequences of miRNAs in various cellular contexts. Furthermore, the panel of DMGs with inducible miRNA will be utilized for in vivo testing in relevant orthotopic models and adapted to the PiggyBac intrauterine electroporation DMG model. Our research will pioneer the use of microRNA mimetics as the first proof-of-concept study of using DMG-specific miRNAs to control aberrant gene expression. While this strategy demonstrates significant promise, we acknowledge that advancements in RNA delivery methods will be essential for translating this approach into a clinical reality in the future. In summary, our findings will provide proof-of-concept for use of a new class of RNA therapeutics for the development of innovative future therapies for DMG. Citation Format: Riley Bigger, Niloofar Khairkhah, Rhea Sridhara, Hernando Lopez-Bertoni, Stefanie Galban. Inducible miRNA therapeutics: A multi-targeted strategy for treating diffuse midline gliomas [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 6698.