Poster Presentation NSW State Cancer Conference 2023

Targeting Post-Translational Protein Modifications as a Novel Therapeutic Treatment Strategy in Diffuse Intrinsic Pontine Gliomas (#145)

Arjayeeta Samadder 1 2 , Holly Holliday 1 2 , Jean B Bertoldo 1 2 , Elisha Hayden 1 2 , Benjamin S Rayner 1 2 , David S Ziegler 1 2 3
  1. Children's Cancer Institute, UNSW, Kensington, NSW, Australia
  2. School of Clinical Medicine, UNSW Medicine and Health, Sydney, NSW, Australia
  3. Kids Cancer Centre, Sydney Children’s Hospital, Sydney, NSW, Australia

Introduction: Diffuse intrinsic pontine glioma (DIPG) is a rare, aggressive, and incurable form of paediatric high-grade glioma, originating in the brain stem of children between ~5-11 years of age with a long-term survival of <1% [1]. Due to the sensitive location, surgical removal is impossible, chemotherapy is ineffective and radiation therapy is palliative. Over 80% of DIPGs are driven by the mutation of the lysine 27 residue at the amino terminal of the histone H3.1 or H3.3, inducing global K27 hypomethylation and resulting in epigenetic reprogramming of the genome that affects gene expression [1]. Citrullination, the conversion of protein arginine residues to citrulline, catalysed by the peptidylarginine deiminase (PAD) family of enzymes, is an irreversible post-translational modification affecting protein function and potentially driving tumour progression [2]. Preliminary studies from our laboratory have shown that PAD inhibition modifies the DIPG epigenome, activating tumour suppressor pathways, and causing DIPG cell apoptosis.

Objective: The aim of this project is to investigate the therapeutic efficacy and mechanism of targeting PAD-mediated citrullination in DIPG.

Methods and Results: We aim to study the effects of PAD activity in DIPG through pharmacological PAD inhibition with Cl-amidine [3], GSK484 [4] and a novel inhibitor, JBD-1. Quantitative PCR and western blotting following PAD inhibition showed a decrease in citrullination of histone H3 at positions R2, 8 and 17 , along with activation of antioxidant defence pathways leading to reactive oxygen species (ROS) production,  initiation of the unfolded protein response and DIPG cell toxicity in a dose dependent manner.  We aim to perform epigenetic profiling (CUT&RUN) and RNA-seq to study the genes associated with histone citrullination in DIPG, and mass spectrometry to investigate the effects of PAD inhibitors on protein citrullination and signalling pathways involved in tumour metabolism.

Conclusions: Protein citrullination potentially plays a critical role in DIPG tumourigenesis. Studying the underlying mechanism involved in PAD activity and citrullination may provide a novel therapeutic strategy for children with DIPG.

 

  1. [1] Chung, C. et al. (2020) “Integrated Metabolic and Epigenomic Reprograming by H3K27M Mutations in Diffuse Intrinsic Pontine Gliomas.” Cancer Cell, 38(3)
  2. [2] Falcão, A.M. et al. (2019) “PAD2-Mediated Citrullination Contributes to Efficient Oligodendrocyte Differentiation and Myelination.” Cell Reports, 27(4)
  3. [3] Uysal-Onganer, P. et al. (2021) “Peptidylarginine Deiminase Inhibitor Application, Using Cl-Amidine, PAD2, PAD3 and PAD4 Isozyme-Specific Inhibitors in Pancreatic Cancer Cells, Reveals Roles for PAD2 and PAD3 in Cancer Invasion and Modulation of Extracellular Vesicle Signatures.” International Journal of Molecular Sciences, 22(3), p. 1396. DOI: 10.3390/ijms22031396.
  4. [4] Chen, H. et al. (2021) “Inhibition of PAD4 Enhances Radiosensitivity and Inhibits Aggressive Phenotypes of Nasopharyngeal Carcinoma Cells.” Cellular & Molecular Biology Letters, 26(1). DOI: 10.1186/s11658-021-00251-2.