Poster Presentation Australian Epigenetics Alliance Conference 2022

The Dopamine receptor D2 (DRD2) antagonist and Caseinolytic protease proteolytic subunit (ClpP) agonist ONC201 drives hypermethylation of Histone H3 lysine 27 in H3-K27-altered diffuse midline glioma. (#145)

Evangeline R Jackson 1 2 , Yolanda Colino Sanguino 3 4 , Ryan Duchatel 1 2 , Mika Persson 1 2 , Abdul Mannan 1 2 , Padraic S. Kearney 1 2 , Alicia M. Douglas 1 2 , Izac J. Findlay 1 2 , Dilana Staudt 1 2 , Zac Germon 1 2 , Sebastian M. Waszak 5 6 , Fatima Valdes Mora 3 4 , Matthew D. Dun 1 2
  1. Cancer Signalling Research Group, School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, Australia
  2. Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
  3. Cancer Epigenetics Biology and Therapeutics, Precision Medicine Theme, Children’s Cancer Institute, Sydney, NSW, Australia
  4. School of Children and Women Health, University of NSW, Sydney, NSW, Australia
  5. Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo, Norway
  6. Department of Neurology, , University of California, San Francisco, San Francisco, CA, USA

Diffuse midline gliomas (DMGs), including those diagnosed in the pons (diffuse intrinsic pontine glioma - DIPG), are paediatric CNS tumours recognised as the most lethal of all children’s cancers. Palliative radiotherapy remains the only approved treatment, with survival just 9-11 months. DIPG is an epigenetically driven cancer, where 80% of patients harbour recuring mutations in Histone 3 at lysine 27 (H3K27M). For the remaining DMG patients, they harbour overexpression of the EZH-inhibitory protein (EZHIP). Hence, DMGs are characterised by global hypomethylation of H3K27, causing an open-chromatin state and high plasticity. Thus, the use of drugs that remodel the DIPG epigenome could be an ideal therapeutic strategy. Here, we have interrogated the molecular mechanism of action of a promising brain-penetrant small molecule therapy, ONC201 in models of DIPG. In patients, ONC201 treatment shows preclinical and emerging efficacy in early-stage clinical trials (NCT03416530). However, many DMG patients do not respond, and for those who do, resistance develops. Regardless of sensitivity, we demonstrate that ONC201 elicits antagonism of the Dopamine receptor D2 (DRD2), and mitochondrial degradation through potent agonism of the mitochondrial protease ClpP. This ultimately causes proteolysis of the electron transport chain proteins and the critical mitochondrial tricarboxylic acid (TCA) cycle regulator, Isocitrate dehydrogenase 3 (IDH3A/B). The loss of key TCA proteins leads to the loss of α-ketoglutarate, an important metabolic cofactor for several chromatin-modifying enzymes, including histone demethylases. H3K27me3 ChIP-seq and Western blot data in ONC201 resistant cells showed that ONC201 promotes a global increase of H3K27me3 suggesting that this drug promotes epigenetic remodelling towards a repressive chromatin state. In addition, ChIP-seq showed an increase of H3K27me3 peaks at seeding genomic sites, however, H3K27me3- chromatin spreading was not observed. This suggests that resistance to ONC201 may be caused by the inability to fully reinstate chromatin silencing domains.