19 Autophagy related metals in IDH1 mutant glioma: Chloroquine and TMZ as a potential novel strategy to treat IDH1 mt gliomas

Abstract

Glioblastoma is considered among the most aggressive cancers, dismal prognosis and overall survival is only 14 months, 80% of primary low grade gliomas and seccondary GBMs that progress from low grade to grade II or III WHO classification have isocitrate dehydrogenase, (IDH1) or IDH2, mutations [1]. IDH1 mutant glioma is characterized by impaired glycolysis activity resulting in an abnormal production of 2-hydroxyglutarate (2-HG) [2] resulting in an undifferentiated phenotype with permanent hyper-methylation status and enhanced proliferation and invasion[3]. Interestingly, the IDH1 mutant phenotype of U87MG glioma cells shows resistance to autophagy induced cell death even in starving and low oxygen conditions [4]. Recent evidence has demonstrated increased autophagy activity on IDH1 mt cytotoxic activity when cells are exposed to autophagy inhibitors such as chloroquine [5]. To this date it remains elusive whether increased autophagy phenotype is the result of IDH1 mutation or if it either represents a secondary co-existing condition in the setting of the IDH1 mutation. Autophagy is also a mechanism of detoxification induced by chronic heavy metal exposure in both normal and cancer cells. Thus, we hypothesized that autophagy activity in IDH1 mt glioma is partially induced by chronic heavy metal exposure, leading to increased cell survival and abnormal DNA repair. Our approach included characterization and quantification of metal content on IDH1 mt glioma cell lines and tissues, in addition to correlation analyses of the celullar metallome with autophagy markers, ROS and DNA repair of IDH1 mt glioma cells allowed us to explore targets responsible for cell survival and DNA repair response. Furthermore, we evaluated the potential therapeutic value of Chloroquine (CQ) and bafylomicin for IDH1 mutant gliomas targeting autophagy pathway in combination with TMZ and radiation. We demonstrated that 2-HG induces autophagy activity via LC3B activation, and autophagy inhibiton by beclin gene silencing results in a reduction of 2-HG leading to cell starvation and apoptosis. Remarkably, we observed a positive correlation on at least six different metals with autophagy induced LC3B and beclin1 expression that significantly differed between the mutant and the wt genotype in glioma cell lines. ROS and DNA repair were also positively associated with at least 6 different metals and only seen in the IDH1 mt cell lines, then suggesting a possible explanation for the increase on autophagy, analysis of both LC3B and beclin 1 expression demonstrated a positive correlation with Mo98, Fe54, and Zn 66 on IDH1 mt cell lines and a positively correlation with Mo98 and V concentrations in relation to H2AX expression. Co, SeO Mo, V and Mg were positively correlated to ROS expression. TMZ and CQ induced autophagy pathway activation as measured by LC3B, Beclin, Atg expression. Silencing beclin in IDH1 mutant glioma cell lines induced apoptosis and reduction on 2-HG production after treatment with TMZ and radiation. Overall the results contained in this study 1) identify cellular metal content in relationship to mechanisms leading to increased autophagy on IDH1 mt glioma cells. 2) evaluate the combination of CQ and TMZ to potentially target and inhibit autophagy as a mechanism downstream the 2-HG production in IDH1 mt glioma cells.