Background: Diffuse Intrinsic Pontine Gliomas (DIPG) are incurable paediatric brain cancers driven by histone mutation H3K27M, globally reducing H3K27 trimethylation altering the epigenome and deregulating gene expression. This alteration is often partnered with mutations to kinase signalling, complicating targeted and combination therapeutic approaches, which often develop resistance. Copper is an ion essential for normal brain development which is highly concentrated in the pons. However, excess copper has been linked to both neurological and cancer pathologies. Interestingly, the H3-H4 dimer is a copper reductase, and copper is established to stimulate kinase signalling. Copper chelators are clinically approved agents used to treat paediatric Wilson’s Disease patients, improving neurological symptoms and are under clinical investigation for several cancers. We hypothesised that targeting copper through chelation therapy in DIPG may effectively target two key mechanisms of DIPG pathology.
Aims: Investigate links between copper, epigenetics and kinase signalling in DIPG and determine therapeutic potential.
Methods: Transcriptomic interrogation of patient datasets, in vitro investigations in a panel of DIPG cell lines and an in vivo investigation using an orthotopic xenograft model of DIPG (SU-DIPGVI-Luc) was performed. DIPG cells were treated with copper chelator tetraethylenepentamine-pentachloride (TEPA). Apoptotic caspase 3/7 assay was performed using IncuCyte device and software. Transcriptomic (RNA-Seq), proteomic and metabolomic (Mass Spectrometry) investigations, western blots and flow cytometry determined efficacy and mechanism. In vivo investigation consisted of survival study with concurrent luminescence imaging, histology and copper assay.
Results: High copper chaperone expression was found to be inversely proportional to epigenetic regulator transcriptomic expression in patient data. Apoptotic assays determined H3K27M cell lines were more sensitive to copper chelation than H3-WT cell lines. Transcriptomic analysis revealed significant downregulation of G2/M cell cycle gene expression, corroborated by proteomic and western blot downregulation of NIMA and PLK kinase signalling, essential for M-phase. Epigenetic regulators EZH2, SETDB1, DNMT1 and DNMT3B were also downregulated in transcriptomic and proteomic investigations, with dysregulation of DIPG epigenome observed. Metabolomic investigations revealed ATP, GTP and S-adenosylmethionine (SAM) downregulation. Flow cytometry cell cycle analysis further showed an increase in the sub-G1 fraction. TEPA increased survival in orthotopic PDX model with luminescence imaging revealing complete tumour regression in 25% of treated mice alongside reductions in copper accumulation.
Conclusions: Data indicates copper chelation interrupts M-phase chromatin packaging, with DNA fragmenting and cells subsequently undergoing apoptosis. This alongside efficacy in vivo suggest copper chelation represents a suitable therapeutic option for DIPG patients warranting clinical investigation.