Neuro-Oncology Advances 3(1), 1–13, 2021 | https://doi.org/10.1093/noajnl/vdab096 | Advance Access date 05 July 2021 1 © The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology. Lily Keane † , Mathilde Cheray † , Dalel Saidi, Caoimhe Kirby, Lara Friess, Patricia Gonzalez-Rodriguez, Maren Elisabeth Gerdes, Kathleen Grabert, Barry W. McColl, and Bertrand Joseph Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden (L.K., M.C., D.S., L.F., P.G.-R., M.E.G., K.G., B.J.); UK Dementia Research Institute, Center for Discovery Brain Sciences, University of Edinburgh, Edinburgh, UK (C.K., B.W.M.) Corresponding Author: Bertrand Joseph, PhD, Nobels väg 13, Institute of Environmental Medicine, Karolinska Institutet, 17177 Stockholm, Sweden (bertrand.joseph@ki.se). † These authors are co-first authors. Abstract Background. Diffuse intrinsic pontine gliomas (DIPG), within diffuse midline gliomas are aggressive pediatric brain tumors characterized by histone H3-K27M mutation. Small-molecule inhibitors for the EZH2-H3K27 histone methyltransferase have shown promise in preclinical animal models of DIPG, despite having little effect on DIPG cells in vitro. Therefore, we hypothesized that the effect of EZH2 inhibition could be mediated through targeting of this histone modifying enzyme in tumor-associated microglia. Methods. Primary DIPG tissues, and cocultures between microglia and patient-derived DIPG or -pediatric high- grade glioma (pHGG) cell lines, were used to establish the H3-K27M status of each cell type. Antisense RNA strategies were used to target EZH2 gene expression in both microglia and glioma cells. Microglia anti-tumoral properties were assessed by gene expression profle, tumor cell invasion capacity, microglial phagocytic activity, and associated tumor cell death. Results. In primary DIPG tissues, microglia do not carry the H3-K27M mutation, otherwise characteristic of the cancer cells. Activation of a microglial tumor-supportive phenotype by pHGG, independently of their H3-K27M status, is asso- ciated with a transient H3K27me3 downregulation. Repression of EZH2 in DIPG cells has no impact on tumor cell sur- vival or their ability to activate microglia. However, repression of EZH2 in microglia induces an anti-tumor phenotype resulting in decreased cancer cell invasion capability, increased microglial phagocytosis, and tumor-related cell death. Conclusions. These results indicate that microglia, beyond the tumor cells, contribute to the observed response of DIPG to EZH2 inhibition. Results highlight the potential importance of microglia as a new therapeutic avenue in DIPG. Key Points • Methylation of H3K27 is important for DIPG-induced activation of microglia. • EZH2 inhibition in microglia leads to robust anti-tumoral effects in pediatric HGG. • Microglia may represent a novel therapeutic avenue in the treatment of DIPG. Pediatric diffuse midline gliomas are a group of diseases that affect the brainstem, spinal cord, and thalamus. One subtype of this group, pediatric diffuse midline gliomas H3-K27M mutant, formerly known as diffuse intrinsic pontine glioma (DIPG), is an incredibly aggressive pediatric brain tumor which originates in the pons. 1 DIPG strikes at the heart of childhood, with a median Inhibition of microglial EZH2 leads to anti-tumoral effects in pediatric diffuse midline gliomas This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/noa/article/3/1/vdab096/6315204 by guest on 03 September 2021