Transcriptional silencing disrupts two levels of arginine biosynthesis in glioblastoma multiforme (GBM): a novel, targeted therapeutic strategy for GBM

Presented at BNOS June 2012

N Syed, F Roncaroli, K Janczar, P Singh, K O’Neil, C Lo Nigro, L Lattanzio, HM Coley, E Hatzimichael, J Bomalaski, P Szlosarek, T Crook.

British Neuro-oncology Society:  Abstract submission 2012

Changes in the structure and expression of enzymes involved in key metabolic pathways (such as isocitrate dehydrogenase) have been identified in glial brain tumours. To seek novel candidate brain tumour suppressors, we have performed pharmacological methylation reversal.

Methylation reversal was performed in GBM cell lines using 5’ azacytidine (5’AZA), coupled with micro-array analysis. Gene expression was analysed using qPCR and western blotting. Methylation was analysed using methylation specific PCR and quantitative pyrosequencing.

Arginine deprivation, using ADI-PEG20 induces adaptive transcriptional up-regulation of ASS1 and ASL in glioblastoma multiforme which is blocked by neoplasia-specific CpG island methylation, causing arginine auxotrophy and cell death. ADI-PEG20 initially induces a protective autophagic response, but abrogation of this by chloroquine accelerates and potentiates cytotoxicity. Concomitant methylation in the CpG islands of both ASS1 and ASL, observed in a subset of cases, confers hypersensitivity to the cytotoxic effects of ADI-PEG20. Cancer stem cells positive for CD133 and methylation in the ASL CpG island retain sensitivity to ADI-PEG20. Our results show for the first time that epigenetic changes in the two key genes of arginine biosynthesis occur in human cancer and confer sensitivity to therapeutic autophagy due to arginine deprivation. Moreover, we demonstrate that methylation status of the CpG islands, rather than expression levels per se of the genes, predicts sensitivity to autophagy.

Our results suggest a novel therapeutic strategy for this invariably fatal central nervous system neoplasm for which we have identified robust biomarkers and which overcomes the limitations to conventional chemotherapy imposed by the blood:brain barrier.