P02.07.B TARGETING GLIOBLASTOMA MITOCHONDRIAL METABOLISM WITH S-GBOXIN INDUCES CYTOTOXICITY UNDER CONDITIONS OF THE TUMOR MICROENVIRONMENT

Abstract BACKGROUND Glioblastoma (GB) is the most common primary malignant brain tumor in adults with a dismal prognosis. Targeted therapy approaches that exploit cancer-specific traits have yielded disappointing results in clinical trials. A novel compound targeting oxidative phosphorylation in GB via inhibition of complex V of the respiratory chain is Gboxin. S-Gboxin, a pharmacokinetically optimized version with improved in vivo stability, was efficient in a mouse GB model but has not been tested under conditions of tumor microenvironment, including hypoxia and nutrient deprivation. MATERIAL AND METHODS Cytotoxicity and cell death under S-Gboxin treatment were analyzed in human GB cell lines and primary tumor cell cultures, as well as immortalized and primary human astrocytes. Additionally, culture conditions were modulated to induce nutrient deprivation, hypoxia, and dependency on mitochondrial fuels. As further readouts, oxygen consumption and effects of migration were measured. To analyze co-targets for a potential synergistic effect with S-Gboxin, we employed pharmacological and genetic models of inhibition of the integrated stress response (ISR) and of AMP-activated protein kinase (AMPK). RESULTS S-Gboxin cytotoxicity was detectable starting at concentrations of approximately 4 µM and nutrient replete conditions after 24 h in human GB cells. Nutrient starvation and mitochondrial fuels enhanced cell death. Hypoxia did not diminish the effects of S-Gboxin. Oxygen consumption was reduced under S-Gboxin. Additionally, S-Gboxin treatment led to elevated lactate production and increased glucose consumption. ATF4 as a marker of the ISR was induced in a dose dependent manner in ATF4 reporter cells, but its inhibition did not mediate resistance to S-Gboxin. In contrast, AMPK inhibition, in combination with S-Gboxin, dramatically enhanced cell death. A similar effect of increased cell death was detected under incubation with galactose, whose metabolism relies on mitochondria. CONCLUSION Nutrient deprivation and mitochondrial fuels, as well as AMPK inhibition, enhanced S-Gboxin dependent cell death. Hypoxia did not reduce S-Gboxin efficacy. While the ISR was induced by S-Gboxin in a dose dependent manner, this was not relevant for therapy resistance. In summary, S-Gboxin is a promising compound for targeting cancer-specific metabolic pathways in GB cells, especially in combination with AMPK-inhibition.