Arginine deprivation, either by nutritional starvation or exposure to ADI-PEG20, induces adaptive transcriptional upregulation of
ASS1 and ASL in glioblastoma multiforme ex vivo cultures and cell lines. This adaptive transcriptional upregulation is blocked by
neoplasia-specific CpG island methylation in either gene, causing arginine auxotrophy and cell death. In cells with methylated
ASS1 or ASL CpG islands, 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 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 occur in both of the two key
genes of arginine biosynthesis in human cancer and confer sensitivity to therapeutic arginine deprivation. We demonstrate that
methylation status of the CpG islands, rather than expression levels per se of the genes, predicts sensitivity to arginine
deprivation. 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.