Abstract 6455: Development of a H3K27M inducible and reversible model of diffuse midline glioma (DMG) to characterize tumor microenvironment and epigenetic memory

Diffuse Midline Gliomas (DMGs) with a lysine to methionine mutation in Histone 3 are lethal pediatric brain tumors. Despite ongoing efforts to develop new therapies, it remains unclear whether targeting this oncohistone or downstream epigenetic pathway activation are sufficient to halt and eradicate tumors. Here, we introduce a H3K27M inducible and reversible mouse model for DMG that allows precise control of oncohistone expression. This model enables us to study the effects of H3K27M activation, inactivation and re-activation on tumor growth and recurrence and additionally, characterize changes in the microenvironmental and epigenetic memory. We employed the in utero electroporation-based (IUE) delivery method to express the PiggyBac-based expression vectors mutant PDGFRA and dominant-negative TP53 and the newly designed tetracycline inducible H3K27M, along with the transposon. Upon tetracycline induced H3K27M activation, DMG tumors were detected and dissected to generate a new DMG cell line (PPiK27M), which can also be used for intracranial implantations in immunocompetent mice. In addition, we used two patient-derived DMG cell models wherein H3K27M was CRISPR edited (H3K27M-KO) to reintroduce the inducible and reversible H3K27M construct to provide an orthotopic model closely recapitulating human disease. We validated inducibility and reversibility of H3K27M by intracellular flow cytometry and western blotting in the murine and human cell models. We detected characteristic morphological changes and repression of astrocytic markers upon H3K27M re-expression in KO cells. In conclusion, our results indicate that the new inducible and reversible H3K27M IUE model and cells faithfully recapitulate DMG characteristics. We are currently performing single-cell RNA sequencing (scRNA-seq) of tumors to track cellular and molecular alterations in the surrounding tumor immune microenvironment. Additionally, we are characterizing the epigenome to understand whether transcriptional changes persist after inactivation of the oncohistone. Our study provides new tools to mechanistically decipher the role of the oncohistone, and potentially, identify new therapeutic targets for DMG. Citation Format: Niloofar Khairkhah, Sienna L. Galban, Megan Faunce, Rodrigo T. Cartaxo, Carl Koschmann, Yue Zhao, Stefanie Galban. Development of a H3K27M inducible and reversible model of diffuse midline glioma (DMG) to characterize tumor microenvironment and epigenetic memory [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 6455.