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bioRxiv · 10.1101/2023.05.15.540760

H2A.Z histone variants facilitate HDACi-dependent removal of H3.3K27M mutant protein in paediatric high-grade glioma cells

Leszczynska, K. B.·Pereira de Freitas, A.·Jayaprakash, C.·Dzwigonska, M.·Wojnicki, K.·Gielniewski, B.·Szadkowska, P.·Kaza, B.·Ciolkowski, M. K.·Trubicka, J.·Grajkowska, W.·Kaminska, B.·Mieczkowski, J.
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Abstract

Diffuse intrinsic pontine gliomas (DIPG) are deadly paediatric brain tumours, non-resectable due to brainstem localisation and diffusive growth. Patients with DIPG have a dismal prognosis of 9-12 months of survival with no effective therapy. Over 80% of DIPGs harbour a mutation in histone 3 (H3.3 or H3.1) resulting in a lysine to methionine substitution (H3K27M). H3K27M causes global epigenetic alterations (a loss of H3K27 trimethylation and an increase in H3K27 acetylation) resulting in aberrant gene expression. To date, no therapeutic strategy exists to suppress the levels of oncogenic H3K27M. We show that pan-HDAC inhibitors (HDACi) lead to the temporary but significant reduction in the H3.3K27M protein (up to 80%) in multiple glioma cell lines expressing the H3.3K27M histone variant, without changes in the H3F3A mRNA expression. The H3.3K27M occupancy at the chromatin is greatly reduced upon HDACi (SB939) treatment, as shown by ChIPseq analysis. H3.3K27M loss is most striking at SB939-upregulated genes suggesting the role in repression of these genes. In addition, genes previously reported as H3K27M-dependent become downregulated in response to SB939 treatment. We discover that the SB939-mediated loss of H3.3K27M is partially blocked by a lysosomal inhibitor, chloroquine. Moreover, the loss of H3.3K27M is facilitated by co-occurrence of H2A.Z, as evidenced by the knock-down of H2A.Z histone isoforms. ChIPseq analysis confirms the occupancy of H3.3K27M and H2A.Z at the same SB939-inducible genes. Altogether, we provide new insight into disease-specific mechanism of HDAC inhibition and demonstrate pharmacological modulation of the oncogenic H3.3K27M protein levels. These findings open a new possibility to directly target the H3.3K27M oncohistone, which may be exploited in future therapies.

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Leszczynska, K. B., Pereira de Freitas, A., Jayaprakash, C., Dzwigonska, M., Wojnicki, K., Gielniewski, B., Szadkowska, P., Kaza, B., Ciolkowski, M. K., Trubicka, J., Grajkowska, W., Kaminska, B., Mieczkowski, J.. 2023-05-24. H2A.Z histone variants facilitate HDACi-dependent removal of H3.3K27M mutant protein in paediatric high-grade glioma cells. https://doi.org/10.1101/2023.05.15.540760

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