TM-11 METABOLOMIC ANALYSIS OF GLIOBLASTOMA MULTIFORME UPON ARGININE DEPRIVATION TREATMENT

INTRODUCTION: Treatment of glioblastoma (GBM) remains challenging, and metabolic targeting opens novel treatment approaches. Arginine is a semi-essential amino-acid that plays a central role in various proliferative cellular reactions. Argininosuccinate synthetase-1 (ASS1) is the rate-limiting enzyme of de novo arginine synthesis, and methylation specific silencing of ASS1 exerts an auxotrophic dependency for extracellular arginine in GBM, amongst other cancers. We have identified that ASS1-silenced GBM cell lines targeted by arginine-deprivation therapy through pegylated arginine deiminase (ADI-PEG20) resulted in arrested proliferation as well as autophagy, highlighting the key role of arginine in GBM cell proliferation. Consequences of arginine deprivation by ADI-PEG20 were analysed with an untargeted metabolomics approach, with the aim of identifying novel metabolic targets for therapy in ASS1 positive and negative cells. METHODS: ASS1 positive and negative GBM cell lines were exposed to arginine deprivation by ADI-PEG20, and samples for 1H NMR and GC/TOF-MS metabolic analysis were prepared, and the data was processed according to established protocols. This included data analysis via principal component analysis (PCA) using SIMCA-P software, and further multi- and uni-variate studies were performed in MATLAB as well as Graphpad Prism. RESULTS: 1H NMR and GC/TOF-MS analysis, via PCA, revealed separation in the metabolic profiles between ADI-PEG20 treated and non-treated GBM cell lines, particularly striking in ASS1-deficient cell lines. OPLS-DA analysis of 1H NMR data highlighted discriminant metabolites including intermediates of the arginine metabolism, such as decreases in creatine and choline levels, not previously linked to arginine deprivation. Functional investigations demonstrated that creatine was able to promote proliferation in GBM cell lines. CONCLUSIONS: We have identified differentially altered metabolic pathways in GBM as a result of arginine deprivation that are involved in proliferation, including creatine. Changes observed in GBM lines showed potential to use metabolic profiles for monitoring treatment response, and open new avenues for therapies.