Abstract Diffuse Intrinsic Pontine Glioma is one of the worst pediatric brain tumors regarding prognosis due notably to intrinsic cell resistance to radio and chemotherapy. One of the main characteristics of DIPG cells is the presence of a mono-allelic mutation on the lysine 27 of histone H3 (H3K27M). This mutation inhibits the trimethylation of this lysine that leads to strong modifications of gene expression. Until now, even though this mutation seems to be a driver event in tumorigenesis, its role in cell resistance to treatment has not been deciphered, due to a lack of relevant cellular models. This way, in order to evaluate the role of the mutation on resistance to treatment, we first induced the mutation in three H3K27-unmutated pediatric glioma cell lines. In parallel, using the CRISPR/Cas9 technology, we are establishing DIPG cellular models in which the mutation will be reversed. By gene trapping approach, we aim to restore an H3F3Awt/wt genotype. After validation, these models would result in original tools to study the impact of H3K27M mutation in DIPG cells resistance to treatment. For the model of induction, the transfected cell lines exhibit the mutation accompanied by a loss of H3K27me3 mark and H3.3 overexpression. For now, we showed an increased cell growth due to the mutation in two cell lines, under normoxia as well as under hypoxia. On contrary there was no impact on resistance to chemotherapy or ionizing radiation. In the third cell line, we didn't observe any impact on cell growth, but an increase of cell radioresistance. Concerning the mutation reversion, our preliminary results show homologous recombination at the right locus in the genome, and some clones present a loss of the mutation confirmed by sequencing. After the removal of resistance cassette by action of a recombinase protein, we will be able to evaluate the biological effects of mutation reversion. To sum up, these different models would allow us to decipher cellular and molecular mechanism induced by the H3.3K27M mutation in DIPG cells including resistance to treatment, and thus, to possibly identify putative therapeutic targets. Citation Format: Quentin Bailleul, Mélanie Arcicasa, Audrey Hochart, Andria Rakotomalala, Marie Castets, Eddy Pasquier, Pierre-Olivier Angrand, Eric Adriaenssens, Xuefen Le Bourhis, Pierre Leblond, Samuel Meignan. Impact of H3.3K27M mutation on diffuse intrinsic pontine glioma's resistance to treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5005.