Abstract 525: A comprehensive approach: Copper chelation therapy modulates epigenetic, kinase signaling and metabolic pathways in diffuse midline gliomas (DMG)
Authors: Michniewicz Filip , Cazzoli Riccardo , Bell Jessica , Saletta Federica , Poursani Ensieh , Rouaen Jourdin R. , Shai-Hee Tyler , Pham Vu , Jue Toni Rose , Biery Matthew , Bramberger Liesl , Lim Terry , Khan Aaminah , Giorgi Federico M. , Mercatelli Daniele , Mayoh Chelsea , Cirillo Giuseppe , Macmillan Alexander , Whan Renee , Barlow Christopher T. , Tsoli Maria , Vitanza Nicholas , Berry Scott , Faridi Pouya , Ziegler David , Vittorio Orazio 
Published: March 2024

Abstract
Diffuse Midline Gliomas (DMG) are incurable pediatric brain tumors driven by histone mutation H3K27M, globally reducing H3K27 trimethylation altering the epigenome and deregulating gene expression. These mutations often partner with and are strongly integrated into kinase signaling and metabolic alterations, driving aggressive tumor growth with many mechanisms which can compensate for one another and therefore evade traditional single agent therapies. Copper over-accumulation has been linked to epigenetic, kinase and metabolic dysregulation in various cancers and neurological disorders. Copper chelators are clinically approved for use in pediatric Wilson’s Disease patients, improve neurological symptoms and are under clinical investigation for several cancers. We propose copper chelators may simultaneously target these mechanisms in DMG. We performed transcriptomic interrogation of patient datasets, in vitro investigations in a panel of DMG cell lines and an in vivo investigation using an orthotopic xenograft model of DIPG (SU-DIPGVI-Luc). Copper chelator tetraethylenepentamine-pentachloride (TEPA) was used to treat DMG cells. Transcriptomic (RNA-Seq), proteomic, phospho-proteomic and metabolomic (Mass Spectrometry) investigations, western blots and immunofluorescent microscopy determined efficacy and mechanism. In vivo investigation consisted of survival study with concurrent luminescence imaging, histology, and copper assay. We demonstrate copper chaperone transcript expression is increased in DMG patient samples compared to non-DMG samples. In vitro, apoptosis and growth assays demonstrated H3K27M DMG were more sensitive to TEPA than H3-WT DMG. In H3K27M DMG, TEPA downregulated ATP, GTP and SAM metabolism which corresponded with downregulated kinase signaling and altered H3K27 trimethylation and DNA methylation patterns around cell cycle genes. Affected genes included those regulating centrosome segregation and ATP synthesis. Further transcriptomic and proteomic investigations highlighted TEPA downregulated G2-M phase kinase pathways, with immunofluorescence demonstrating TEPA reduced expression of mitotic marker p-H3S10 and epigenetic methylators EZH2 and DNMT1. In vivo, TEPA reduced brain copper accumulation, eliminated tumors and increased survival in an orthotopic patient-derived xenograft DMG model. Collectively, our data indicates TEPA induced multi-modal targeting of M-phase cell cycle progression through epigenetic, kinase signaling and metabolic mechanisms. Efficacy was observed both in vitro and in vivo, providing a promising basis for future investigation of copper chelators in DMG. We propose copper-chelation is a viable therapeutic strategy for DMG patients warranting clinical investigation.
Citation Format: Filip Michniewicz, Riccardo Cazzoli, Jessica Bell, Federica Saletta, Ensieh Poursani, Jourdin R. Rouaen, Tyler Shai-Hee, Vu Pham, Toni Rose Jue, Matthew Biery, Liesl Bramberger, Terry Lim, Aaminah Khan, Federico M. Giorgi, Daniele Mercatelli, Chelsea Mayoh, Giuseppe Cirillo, Alexander Macmillan, Renee Whan, Christopher T. Barlow, Maria Tsoli, Nicholas Vitanza, Scott Berry, Pouya Faridi, David Ziegler, Orazio Vittorio. A comprehensive approach: Copper chelation therapy modulates epigenetic, kinase signaling and metabolic pathways in diffuse midline gliomas (DMG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 525.