Using Copper Chelating Agents To Target Receptor Tyrosine Kinase Signalling In Diffuse Intrinsic Pontine Glioma (Dipg)

Abstract DIPG is a universally fatal pediatric brain cancer. Receptor tyrosine kinase (RTK) pathway alterations are among the defining characteristics in many patients. Copper is a transition metal essential for cellular signaling, known to impact PI3K/AKT and MAPK/ERK pathways. Copper chelating agents are clinically approved for use in children with Wilson’s Disease, documented to reduce brain copper levels and are cited as potential cancer therapeutics. Due to copper’s wide cellular integration, we propose that targeting copper in DIPG through use of copper chelators is a viable therapeutic strategy and are strong candidates for combination therapy. Cytotoxicity assays performed in a panel of DIPG cell lines using copper chelator tetraethylenepentamine (TEPA) demonstrated a millimolar range of efficacy. To identify copper integrated pathways, western blots were performed on DIPG cell lines dosed with sub-lethal copper concentrations, which increased phosphorylated expression of AKT, ERK1/2, ERK5 and STAT3. Conversely, western blots performed after TEPA treatment demonstrated reduced phosphorylated expression of all these proteins compared to controls. Western blots investigating TEPA in combination with Everolimus and Trametinib demonstrated synergistic targeting of these proteins. Our results indicate that adding copper in the culture media initiated two RTK-mediated downstream signal transductions, including AKT and ERK and additionally STAT signaling. The use of copper chelator TEPA affected copper homeostasis and reduced DIPG cell proliferation. Our study proposes copper plays an important role in RTK-mediated signaling promoting DIPG proliferation. This implies that reducing copper with clinically available chelation agents can represent a potential anti-cancer treatment for DIPG.