Tumor metabolism is reprogrammed to meet the demands of proliferating cancer cells. In particular, cancer cells upregulate synthesis of the membrane phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) in order to allow for rapid membrane turnover. Nonetheless, we show here that, in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas, which produce the oncometabolite 2-hydroxyglutarate (2-HG), PtdCho and PtdE biosynthesis is downregulated and results in lower levels of both phospholipids when compared with wild-type IDH1 cells. 2-HG inhibited collagen-4-prolyl hydroxylase activity, leading to accumulation of misfolded procollagen-IV in the endoplasmic reticulum (ER) of both genetically engineered and patient-derived IDHmut glioma models. The resulting ER stress triggered increased expression of FAM134b, which mediated autophagic degradation of the ER (ER-phagy) and a reduction in the ER area. Because the ER is the site of phospholipid synthesis, ER-phagy led to reduced PtdCho and PtdE biosynthesis. Inhibition of ER-phagy via pharmacological or molecular approaches restored phospholipid biosynthesis in IDHmut glioma cells, triggered apoptotic cell death, inhibited tumor growth, and prolonged the survival of orthotopic IDHmut glioma-bearing mice, pointing to a potential therapeutic opportunity. Glioma patient biopsies also exhibited increased ER-phagy and downregulation of PtdCho and PtdE levels in IDHmut samples compared with wild-type, clinically validating our observations. Collectively, this study provides detailed and clinically relevant insights into the functional link between oncometabolite-driven ER-phagy and phospholipid biosynthesis in IDHmut gliomas.Significance: Downregulation of phospholipid biosynthesis via ER-phagy is essential for proliferation and clonogenicity of mutant IDH1 gliomas, a finding with immediate therapeutic implications. Cancer Res; 78(9); 2290-304. ©2018 AACR.

Approximately 80% of low-grade glioma (LGGs) harbor mutant isocitrate dehydrogenase 1/2 (IDH1/2) driver mutations leading to accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). Thus, inhibition of mutant IDH is considered a potential therapeutic target. Several mutant IDH inhibitors are currently in clinical trials, including AG-881 and BAY-1436032. However, to date, early detection of response remains a challenge. In this study we used high resolution ¹H magnetic resonance spectroscopy (¹H-MRS) to identify early noninvasive MR (Magnetic Resonance)-detectable metabolic biomarkers of response to mutant IDH inhibition. In vivo ¹H-MRS was performed on mice orthotopically-implanted with either genetically engineered (U87IDHmut) or patient-derived (BT257 and SF10417) mutant IDH1 cells. Treatment with either AG-881 or BAY-1436032 induced a significant reduction in 2-HG. Moreover, both inhibitors led to a significant early and sustained increase in glutamate and the sum of glutamate and glutamine (GLX) in all three models. A transient early increase in N-acetylaspartate (NAA) was also observed. Importantly, all models demonstrated enhanced animal survival following both treatments and the metabolic alterations were observed prior to any detectable differences in tumor volume between control and treated tumors. Our study therefore identifies potential translatable early metabolic biomarkers of drug delivery, mutant IDH inhibition and glioma response to treatment with emerging clinically relevant therapies.

Background

The alternative lengthening of telomeres (ALT) pathway is essential for tumor proliferation in astrocytomas. The goal of this study was to identify metabolic alterations linked to the ALT pathway that can be exploited for noninvasive magnetic resonance spectroscopy (MRS)-based imaging of astrocytomas in vivo.

Methods

Genetic and pharmacological methods were used to dissect the association between the ALT pathway and glucose metabolism in genetically engineered and patient-derived astrocytoma models. 2H-MRS was used for noninvasive imaging of ALT-linked modulation of glycolytic flux in mice bearing orthotopic astrocytomas in vivo.

Results

The ALT pathway was associated with higher activity of the rate-limiting glycolytic enzyme phosphofructokinase-1 and concomitantly elevated flux of glucose to lactate in astrocytoma cells. Silencing the ALT pathway or treating with the poly(ADP-ribose) polymerase inhibitor niraparib that induces telomeric fusion in ALT-dependent astrocytoma cells abrogated glycolytic flux. Importantly, this metabolic reprogramming could be non-invasively visualized by 2H-MRS. Lactate production from [6,6'-2H]-glucose was higher in ALT-dependent astrocytoma tumors relative to the normal brain in vivo. Furthermore, treatment of orthotopic astrocytoma-bearing mice with niraparib reduced lactate production from [6,6'-2H]-glucose at early timepoints when alterations in tumor volume could not be detected by anatomical imaging, pointing to the ability of [6,6'-2H]-glucose to report on pseudoprogression in vivo.

Conclusions

We have mechanistically linked the ALT pathway to elevated glycolytic flux and demonstrated the ability of [6,6'-2H]-glucose to non-invasively assess tumor burden and response to therapy in astrocytomas. Our findings point to a novel, clinically translatable method for metabolic imaging of astrocytoma patients.

Background

Magnetic resonance spectroscopy (MRS) studies have identified elevated levels of the phospholipid precursor phosphocholine (PC) and phosphoethanolamine (PE) as metabolic hallmarks of cancer. Unusually, however, PC and PE levels are reduced in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas that produce the oncometabolite 2-hydroxyglutarate (2-HG) relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was to determine the molecular mechanism underlying this unusual metabolic reprogramming in IDHmut gliomas.

Methods

Steady-state PC and PE were quantified using ³¹P-MRS. To quantify de novo PC and PE synthesis, we used ¹³C-MRS and measured flux to ¹³C-PC and ¹³C-PE in cells incubated with [1,2-¹³C]-choline and [1,2-¹³C]-ethanolamine. The activities of choline kinase (CK) and ethanolamine kinase (EK), the enzymes responsible for PC and PE synthesis, were quantified using ³¹P-MR-based assays. To interrogate the role of 2-HG, we examined IDHwt cells incubated with 2-HG and, conversely, IDHmut cells treated with the IDHmut inhibitor AGI-5198. To examine the role of hypoxia-inducible factor 1-α (HIF-1α), we silenced HIF-1α using RNA interference. To confirm our findings in vivo and in the clinic, we studied IDHwt and IDHmut orthotopic tumor xenografts and glioma patient biopsies.

Results

De novo synthesis of PC and PE was reduced in IDHmut cells relative to IDHwt. Concomitantly, CK activity and EK activity were reduced in IDHmut cells. Pharmacological manipulation of 2-HG levels established that 2-HG was responsible for reduced CK activity, EK activity, PC and PE. 2-HG has previously been reported to stabilize levels of HIF-1α, a known regulator of CK activity. Silencing HIF-1α in IDHmut cells restored CK activity, EK activity, PC and PE to IDHwt levels. Our findings were recapitulated in IDHmut orthotopic tumor xenografts and, most importantly, in IDHmut patient biopsies, validating our findings in vivo and in the clinic.

Conclusions

This study identifies, to our knowledge for the first time, a direct role for 2-HG in the downregulation of CK and EK activity, and thereby, PC and PE synthesis in IDHmut gliomas. These results highlight the unusual reprogramming of phospholipid metabolism in IDHmut gliomas and have implications for the identification of MRS-detectable metabolic biomarkers associated with 2-HG status.