Abstract 5716: Using copper chelating agents to simultaneously target epigenetic mechanisms and receptor tyrosine kinase (RTK) signaling in diffuse intrinsic pontine glioma (DIPG)
Authors: Michniewicz Filip , Saletta Federica , Watkinson Tayla , Mercatelli Daniele , Giorgi Federico M. , Bell Jessica , Tsoli Maria , Ziegler David , Vittorio Orazio 
Published: June 2022

Abstract
DIPG is an incurable and inoperable pediatric brain cancer in the ventral pons characterized by its complex mutational profile. Epigenetic mutations to H3K27M are seen in up to 81% of patients, resulting in the global loss of the repressive H3K27 trimethylation marker, increasing chromatin accessibility and deregulating gene expression. However, these epigenetic changes are often partnered by secondary mutations, with activating mutations and copy number gains observed in a wide variety of RTK genes and their associated signaling pathways. The combination of these two mutational paradigms results in the broad alteration of cellular and metabolic mechanisms, complicating the search for effective targeted therapeutic strategies. Copper, a metal ion essential for normal cellular function is known to be highly expressed in the pons, and influence ERK and PI3K/AKT signaling. Importantly, excess copper accumulation has been implicated in the pathogenesis of multiple neurological diseases and cancers. Copper chelation therapy is a clinically approved strategy for pediatric patients with Wilson’s Disease, known to improve their neurological symptoms, and is under investigation for use in a number of cancers. Therefore, we hypothesized copper chelation may be an effective therapeutic strategy for DIPG. Cytotoxicity assays using copper chelator tetraethylenepentamine (TEPA) demonstrated millimolar efficacy and synergized with mTOR inhibitor everolimus in a panel of DIPG cell lines, with H3-K27M mutant cell lines found to be more sensitive. Western blots were used to identify copper integrated pathways, with DIPG cell lines stimulated with sub-lethal copper concentrations demonstrating increased phosphorylated expression of ERK1/2, ERK5, AKT and p70SK61. TEPA reduced the phosphorylation of these markers compared to controls. Importantly, epigenetic mechanisms were sensitive to copper, as both copper stimulation and TEPA modulated H3K27 trimethylation, and TEPA decreased pro-expression H3K27 acetylation. In further blots examining epigenetic regulators, TEPA reduced the expression of EZH2, DNMT3B and DNMT1. RNA-sequencing analysis revealed TEPA downregulated genes involved in S-adenosylmethionine production metabolism, DNA regulation, cell cycle and mTORC signaling and specifically, EZH2, DNMT3B and DNMT1 expression. In vivo investigations demonstrated TEPA’s ability to improve survival in an orthotopic xenograft model, and via xenogen luminescence imaging complete tumor regression in 25% of treated mice. This study indicates copper directly impacts epigenetic and RTK mechanisms, and that their targeting through copper chelation agents represents a potential therapeutic strategy for DIPG, both as a single agent and in combination.
Citation Format: Filip Michniewicz, Federica Saletta, Tayla Watkinson, Daniele Mercatelli, Federico M. Giorgi, Jessica Bell, Maria Tsoli, David Ziegler, Orazio Vittorio. Using copper chelating agents to simultaneously target epigenetic mechanisms and receptor tyrosine kinase (RTK) signaling in diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5716.