TM-10 * ALTERATIONS IN METABOLIC PATHWAYS IN GLIOBLASTOMA MULTIFORME CELLS IN RESPONSE TO HEXOKINASE-2 INHIBITION

Abstract

INTRODUCTION: GBMs are characterized by elevated glycolysis, however to date effective therapies targeting tumor glycolysis have not been identified. Also, the contribution of metabolic pathways other than glycolysis, such as glutamine and fatty acid oxidation, that are key in providing carbon to TCA cycle, have not been explored in GBMs. Aim: In this study we aim to decipher the adaptive mechanisms brought about by the inhibition of tumor glycolysis in GBMs. We have investigated the metabolic changes occurring in GBM cells after the knockdown of key glycolytic enzyme, hexokinase2 (HK2). METHODS AND RESULTS: We have generated doxycyclin-inducible HK2 knockdown GBM cells by transfecting shRNA in U87 and three Glioma Stem Cell lines with different baseline molecular characteristics We confirm specific knockdown of HK2, with no change in HK1 or HK3, using western blot. HK2 knockdown reduced cell number and proliferative capacity, in addition to decrease in lactate production and O2 consumption. HK2 loss significantly increased NADP:NADPH ratio. Metabolic profiling by LC/MS targeted metabolomics was conducted after exposing GBM cells to C13-labeled glucose media. LC/MS metabolomics demonstrate that decrease in HK2 significantly reduced the levels of metabolites belonging to glycolysis and Pentose Phosphate Pathway. These two pathways are known to be associated with NADPH and biomass production for rapidly proliferating tumor cells. In contrast, metabolites in TCA cycle increased in response to HK2 inhibition. CONCLUSION: Our data strongly suggests that GBM cells activate alternate metabolic pathways in response to inhibition of HK2 dependent glycolysis. These results have important clinical implications in designing combinatorial targeted therapy for blocking tumor metabolism in GBMs. Our ongoing work is focused on establishing the metabolites involved in glutamine catabolism and fatty acid oxidation, both in-vitro and in-vivo GBM models.