Targeting glioblastoma multiforme cells with pharmacological ascorbate: Disrupting DNA damage response and mTOR cascades via extracellular HO.

Glioblastoma multiforme (GBM) is the most aggressive and lethal malignant brain tumor, with limited treatment options and poor patient prognosis. Novel therapeutic strategies are urgently needed to extend survival and improve quality of life for GBM patients. Our transcriptomic analysis revealed that GBM cells exhibit substantial upregulation of DNA damage response (DDR) pathways, identifying DDR as a promising therapeutic target. To leverage this vulnerability, we investigated pharmacological ascorbate (P-AscH; millimolar concentrations of vitamin C) as a potential treatment for GBM. Our findings demonstrated that P-AscH induces ROS-mediated cytotoxicity in both low- and high-grade glioma cells, primarily via extracellular HO production. This generated HO triggers oxidative DNA damage, PARP1 hyperactivation, and impairment of DDR signaling by targeting key regulators, including Chk1 and RPA2. Concurrently, extracellular HO following P-AscH exposure also disrupts the mTOR signaling pathway, a critical regulator of cell survival and DDR. Mechanistic studies showed that HO impairs both mTORC1 and mTORC2 pathways, as evidenced by decreased phosphorylation of mTORC1 substrates (S6K, S6 and 4EBP1) and the mTORC2 substrate Akt, along with reduced total protein levels of these key components. Furthermore, we demonstrated that P-AscH augments the cytotoxic effects of standard-of-care temozolomide and synergistically enhances the anticancer effects of mTOR inhibitors. These improved therapeutic responses were validated in a 3D-GBM spheroid model. Collectively, our findings suggest that P-AscH disrupts both DDR and mTOR signaling pathways, potentially sensitizing GBM cells to both existing therapies and investigational drugs. These results underscore the promise of P-AscH as an adjunctive treatment for GBM and other malignancies.