Cysteine Depletion-induced Ferroptosis as a Therapeutic Vulnerability in Glioblastoma Temozolomide Resistance

Glioblastoma is the most malignant and commonly diagnosed primary brain tumor in adults with a dismal median overall survival of 14 to 16 months. In 2005 the Stupp regimen changed clinical care with the discovery that the chemotherapeutic drug temozolomide (TMZ), with the addition of surgery and radiation, could extend patient survival. However, new therapeutic avenues have remained stagnant and with no second-line therapeutic options showing significant improvement in recurrent GBM tumors, resistance to TMZ is uniformly fatal. To this end we sought to better understand the molecular mechanisms of TMZ-resistant disease to provide patients a potential second-line therapeutic option with a focus on cysteine depletion-induced ferroptosis in TMZ-resistant GBM. Ferroptosis is an iron-dependent form of cell death which has recently gained attention as an attractive avenue to eradicate otherwise drug resistant cancer cells. Our preliminary data support the role of a previously unidentified metabolic enzyme in the induction of cysteine depletion-induced ferroptosis. Our findings strongly suggest that the y-glutamyl enzyme (y-glutamylcyclotransferase; GGCT) recycles cysteine and prevents glutathione (GSH) production – the main goal of the y-glutamyl pathway. We further show that TMZ-resistant cells have changes consistent with a sensitivity to ferroptosis induction such as an increase in reactive oxygen species (ROS), cysteine uptake and the cysteine/glutamate antiporter, xCT, as well as mislocalized perinuclear mitochondria. Finally, we show that the neuroprotective agent, ebselen which has already been shown to cross the blood brain barrier – a major hurdle for GBM treatment, specifically targets TMZ-resistant GBM cells in vitro. Based on these results, we propose to further investigate the role of cysteine recycling via GGCT and its potential therapeutic potential as a treatment vulnerability in patient-derived xenograft (PDX) TMZ-resistant GBM orthotopic pre-clinical animal models. Lastly, we seek to establish prognostic biomarkers of this aberrant cysteine recycling through a metabolic byproduct of GGCTs enzymatic activity in clinical glioma specimens. Overall, this proposal will give insight into a new avenue of ferroptosis induction in drug resistant GBM, and potentially pave the way for cysteine deprivation-induced ferroptosis in other drug resistant cancers.