The biguanide metformin that inhibits mitochondrial complex I activity is now in over 200 oncology clinical trials. In this proposal we will determine how to enhance cancer cell sensitivity towards metformin therapy. There are two principal modes of cellular energy production – glycolysis and mitochondrial oxidation of glycose and fatty acids. Metformin inhibits mitochondrial energy production. Therefore, when switched to a low glucose medium (that reduces glycolysis), cancer cells become sensitized to metformin. Physiological glucose concentration in tissues is however significantly lower compared to that used in most cell culture studies and glucose concentration is even lower in tumor tissues. At this physiological range of glucose, cancer cells maintain high glycolytic rate and genes that regulate cancer cell glycolysis may resist biguanide action by upregulating compensatory glycolysis when mitochondria is inhibited. Therefore, identification and inhibition of such genes may enhance cancer cell liability towards biguanides. Despite promise in other cancers, our data shows that glioma stem cells (GSCs) are resistant to biguanides at physiological glucose. We discovered that the cellular energy sensor AMP kinase (AMPK) that augments glycolysis during stress in cardiac and skeletal muscle is co-opted by GSCs for optimal glycolysis. We propose to test the mechanisms by which AMPK regulates glycolysis in GSCs in vitro and in vivo. Through dose escalation pharmacological studies in mice we will determine maximum tolerated metformin dose in brain tumor-bearing mice, quantitate metformin plasma levels, and metformin concentrations attained in normal brain and tumor tissue. We will test if genetic inhibition of AMPK in GSCs reduces glycolysis, suppress proliferation and tumor growth and improves metformin sensitivity.