Pia Eriksson

Hyperpolarized [1-13C] glutamate: a metabolic imaging biomarker of IDH1 mutational status in glioma

Mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade glioma and secondary glioblastoma, represent an early pathogenic event, and are associated with epigenetically driven modulations of metabolism. Of particular interest is the recently uncovered relationship between the IDH1 mutation and decreased activity of the branched-chain amino acid transaminase 1 (BCAT1) enzyme. Noninvasive imaging methods that can assess BCAT1 activity could therefore improve detection of mutant IDH1 tumors and aid in developing and monitoring new targeted therapies. BCAT1 catalyzes the transamination of branched-chain amino acids while converting α-ketoglutarate (α-KG) to glutamate. Our goal was to use (13)C magnetic resonance spectroscopy to probe the conversion of hyperpolarized [1-(13)C] α-KG to hyperpolarized [1-(13)C] glutamate as a readout of BCAT1 activity. We investigated two isogenic glioblastoma lines that differed only in their IDH1 status and performed experiments in live cells and in vivo in rat orthotopic tumors. Following injection of hyperpolarized [1-(13)C] α-KG, hyperpolarized [1-(13)C] glutamate production was detected both in cells and in vivo, and the level of hyperpolarized [1-(13)C] glutamate was significantly lower in mutant IDH1 cells and tumors compared with their IDH1-wild-type counterparts. Importantly however, in our cells the observed drop in hyperpolarized [1-(13)C] glutamate was likely mediated not only by a drop in BCAT1 activity, but also by reductions in aspartate transaminase and glutamate dehydrogenase activities, suggesting additional metabolic reprogramming at least in our model. Hyperpolarized [1-(13)C] glutamate could thus inform on multiple mutant IDH1-associated metabolic events that mediate reduced glutamate production.

IDH1 Mutation Induces Reprogramming of Pyruvate Metabolism.

Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized (13)C-magnetic resonance spectroscopy ((13)C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-(13)C-pyruvate to 5-(13)C-glutamate, relative to cells expressing wild-type IDH1. (13)C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-(13)C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications.

Metabolic Reprogramming in Mutant IDH1 Glioma Cells

Background Mutations in isocitrate dehydrogenase (IDH) 1 have been reported in over 70% of low-grade gliomas and secondary glioblastomas. IDH1 is the enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate while mutant IDH1 catalyzes the conversion of α-ketoglutarate into 2-hydroxyglutarate. These mutations are associated with the accumulation of 2-hydroxyglutarate within the tumor and are believed to be one of the earliest events in the development of low-grade gliomas. The goal of this work was to determine whether the IDH1 mutation leads to additional magnetic resonance spectroscopy (MRS)–detectable changes in the cellular metabolome. Methods Two genetically engineered cell models were investigated, a U87-based model and an E6/E7/hTERT immortalized normal human astrocyte (NHA)-based model. For both models, wild-type IDH1 cells were generated by transduction with a lentiviral vector coding for the wild-type IDH1 gene while mutant IDH1 cells were generated by transduction with a lentiviral vector coding for the R132H IDH1 mutant gene. Metabolites were extracted from the cells using the dual-phase extraction method and analyzed by 1H-MRS. Principal Component Analysis was used to analyze the MRS data. Results Principal Component Analysis clearly discriminated between wild-type and mutant IDH1 cells. Analysis of the loading plots revealed significant metabolic changes associated with the IDH1 mutation. Specifically, a significant drop in the concentration of glutamate, lactate and phosphocholine as well as the expected elevation in 2-hydroxyglutarate were observed in mutant IDH1 cells when compared to their wild-type counterparts. Conclusion The IDH1 mutation leads to several, potentially translatable MRS-detectable metabolic changes beyond the production of 2-hydroxyglutarate.

HDAC Inhibition Induces Increased Choline Uptake and Elevated Phosphocholine Levels in MCF7 Breast Cancer Cells

Histone deacetylase (HDAC) inhibitors have emerged as effective antineoplastic agents in the clinic. Studies from our lab and others have reported that magnetic resonance spectroscopy (MRS)-detectable phosphocholine (PC) is elevated following SAHA treatment, providing a potential noninvasive biomarker of response. Typically, elevated PC is associated with cancer while a decrease in PC accompanies response to antineoplastic treatment. The goal of this study was therefore to elucidate the underlying biochemical mechanism by which HDAC inhibition leads to elevated PC. We investigated the effect of SAHA on MCF-7 breast cancer cells using 13C MRS to monitor [1,2-13C] choline uptake and phosphorylation to PC. We found that PC synthesis was significantly higher in treated cells, representing 154±19% of control. This was within standard deviation of the increase in total PC levels detected by 31P MRS (129±7% of control). Furthermore, cellular choline kinase activity was elevated (177±31%), while cytidylyltransferase activity was unchanged. Expression of the intermediate-affinity choline transporter SLC44A1 and choline kinase α increased (144% and 161%, respectively) relative to control, as determined by mRNA microarray analysis with protein-level confirmation by Western blotting. Taken together, our findings indicate that the increase in PC levels following SAHA treatment results from its elevated synthesis. Additionally, the concentration of glycerophosphocholine (GPC) increased significantly with treatment to 210±45%. This is likely due to the upregulated expression of several phospholipase A2 (PLA2) isoforms, resulting in increased PLA2 activity (162±18%) in SAHA-treated cells. Importantly, the levels of total choline (tCho)-containing metabolites, comprised of choline, PC and GPC, are readily detectable clinically using 1H MRS. Our findings thus provide an important step in validating clinically translatable non-invasive imaging methods for follow-up diagnostics of HDAC inhibitor treatment.

Glioma Cells with the IDH1 Mutation Modulate Metabolic Fractional Flux through Pyruvate Carboxylase

Background Over 70% of low-grade gliomas carry a heterozygous R132H mutation in the gene coding for isocitrate dehydrogenase 1 (IDH1). This confers the enzyme with the novel ability to convert α-ketoglutarate to 2-hydroxyglutarate, ultimately leading to tumorigenesis. The major source of 2-hydroxyglutarate production is glutamine, which, in cancer, is also a source for tricarboxylic acid cycle (TCA) anaplerosis. An alternate source of anaplerosis is pyruvate flux via pyruvate carboxylase (PC), which is a common pathway in normal astrocytes. The goal of this study was to determine whether PC serves as a source of TCA anaplerosis in IDH1 mutant cells wherein glutamine is used for 2-hydroxyglutarate production. Methods Immortalized normal human astrocytes engineered to express heterozygous mutant IDH1 or wild-type IDH1 were investigated. Flux of pyruvate via PC and via pyruvate dehydrogenase (PDH) was determined by using magnetic resonance spectroscopy to probe the labeling of [2-13C]glucose-derived 13C-labeled glutamate and glutamine. Activity assays, RT-PCR and western blotting were used to probe the expression and activity of relevant enzymes. The Cancer Genome Atlas (TCGA) data was analyzed to assess the expression of enzymes in human glioma samples. Results Compared to wild-type cells, mutant IDH1 cells significantly increased fractional flux through PC. This was associated with a significant increase in PC activity and expression. Concurrently, PDH activity significantly decreased, likely mediated by significantly increased inhibitory PDH phosphorylation by PDH kinase 3. Consistent with the observation in cells, analysis of TCGA data indicated a significant increase in PC expression in mutant IDH-expressing human glioma samples compared to wild-type IDH. Conclusions Our findings suggest that changes in PC and PDH may be an important part of cellular adaptation to the IDH1 mutation and may serve as potential therapeutic targets.

MR Studies of Glioblastoma Models Treated with Dual PI3K/mTOR Inhibitor and Temozolomide:Metabolic Changes Are Associated with Enhanced Survival.

The current standard of care for glioblastoma (GBM) is surgical resection, radiotherapy, and treatment with temozolomide (TMZ). However, resistance to current therapies and recurrence are common. To improve survival, agents that target the PI3K signaling pathway, which is activated in approximately 88% of GBM, are currently in clinical trials. A challenge with such therapies is that tumor shrinkage is not always observed. New imaging methods are therefore needed to monitor response to therapy and predict survival. The goal of this study was to determine whether hyperpolarized 13C magnetic resonance spectroscopic imaging (MRSI) and 1H magnetic resonance spectroscopy (MRS) can be used to monitor response to the second-generation dual PI3K/mTOR inhibitor voxtalisib (XL765, SAR245409), alone or in combination with TMZ. We investigated GS-2 and U87-MG GBM orthotopic tumors in mice, and used MRI, hyperpolarized 13C MRSI, and 1H MRS to monitor the effects of treatment. In our study, 1H MRS could not predict tumor response to therapy. However, in both our models, we observed a significantly lower hyperpolarized lactate-to-pyruvate ratio in animals treated with voxtalisib, TMZ, or combination therapy, when compared with controls. This metabolic alteration was observed prior to MRI-detectable changes in tumor size, was consistent with drug action, and was associated with enhanced animal survival. Our findings confirm the potential translational value of the hyperpolarized lactate-to-pyruvate ratio as a biomarker for noninvasively assessing the effects of emerging therapies for patients with GBM. Mol Cancer Ther; 15(5); 1113–22. ©2016 AACR.

Hyperpolarized 13C MR imaging detects no lactate production in mutant IDH1 gliomas: Implications for diagnosis and response monitoring

Metabolic imaging of brain tumors using 13C Magnetic Resonance Spectroscopy (MRS) of hyperpolarized [1-13C] pyruvate is a promising neuroimaging strategy which, after a decade of preclinical success in glioblastoma (GBM) models, is now entering clinical trials in multiple centers. Typically, the presence of GBM has been associated with elevated hyperpolarized [1-13C] lactate produced from [1-13C] pyruvate, and response to therapy has been associated with a drop in hyperpolarized [1-13C] lactate. However, to date, lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1) mutation, which, in addition to initiating tumor development, also induces metabolic reprogramming. In particular, mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA) and monocarboxylate transporters 1 and 4 (MCT1, MCT4), three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of 13C MRS of hyperpolarized [1-13C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA, MCT1 and MCT4, and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-13C] lactate compared to GBM, consistent with their metabolic reprogramming. Furthermore, hyperpolarized [1-13C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ) in mutant IDH1 tumors, in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas, which, when combined with other clinically available imaging methods, could be used to detect the presence of the IDH1 mutation in vivo.

GLUT3 upregulation promotes metabolic reprogramming associated with antiangiogenic therapy resistance

Clinical trials revealed limited response duration of glioblastomas to VEGF-neutralizing antibody bevacizumab. Thriving in the devascularized microenvironment occurring after antiangiogenic therapy requires tumor cell adaptation to decreased glucose, with 50% less glucose identified in bevacizumab-treated xenografts. Compared with bevacizumab-responsive xenograft cells, resistant cells exhibited increased glucose uptake, glycolysis, 13C NMR pyruvate to lactate conversion, and survival in low glucose. Glucose transporter 3 (GLUT3) was upregulated in bevacizumab-resistant versus sensitive xenografts and patient specimens in a HIF-1α-dependent manner. Resistant versus sensitive cell mitochondria in oxidative phosphorylation-selective conditions produced less ATP. Despite unchanged mitochondrial numbers, normoxic resistant cells had lower mitochondrial membrane potential than sensitive cells, confirming poorer mitochondrial health, but avoided the mitochondrial dysfunction of hypoxic sensitive cells. Thin-layer chromatography revealed increased triglycerides in bevacizumab-resistant versus sensitive xenografts, a change driven by mitochondrial stress. A glycogen synthase kinase-3β inhibitor suppressing GLUT3 transcription caused greater cell death in bevacizumab-resistant than -responsive cells. Overexpressing GLUT3 in tumor cells recapitulated bevacizumab-resistant cell features: survival and proliferation in low glucose, increased glycolysis, impaired oxidative phosphorylation, and rapid in vivo proliferation only slowed by bevacizumab to that of untreated bevacizumab-responsive tumors. Targeting GLUT3 or the increased glycolysis reliance in resistant tumors could unlock the potential of antiangiogenic treatments.

Mutant IDH1 expression is associated with down-regulation of monocarboxylate transporters

Mutations in isocitrate dehydrogenase 1 (IDH1) are characteristic of low-grade gliomas. We recently showed that mutant IDH1 cells reprogram cellular metabolism by down-regulating pyruvate dehydrogenase (PDH) activity. Reduced pyruvate metabolism via PDH could lead to increased pyruvate conversion to lactate. The goal of this study was therefore to investigate the impact of the IDH1 mutation on the pyruvate-to-lactate flux. We used 13C magnetic resonance spectroscopy and compared the conversion of hyperpolarized [1-13C]-pyruvate to [1-13C]-lactate in immortalized normal human astrocytes expressing mutant or wild-type IDH1 (NHAIDHmut and NHAIDHwt). Our results indicate that hyperpolarized lactate production is reduced in NHAIDHmut cells compared to NHAIDHwt. This reduction was associated with lower expression of the monocarboxylate transporters MCT1 and MCT4 in NHAIDHmut cells. Furthermore, hyperpolarized lactate production was comparable in lysates of NHAIDHmut and NHAIDHwt cells, wherein MCTs do not impact hyperpolarized pyruvate delivery and lactate production. Collectively, our findings indicated that lower MCT expression was a key contributor to lower hyperpolarized lactate production in NHAIDHmut cells. The SLC16A3 (MCT4) promoter but not SLC16A1 (MCT1) promoter was hypermethylated in NHAIDHmut cells, pointing to possibly different mechanisms mediating reduced MCT expression. Finally analysis of low-grade glioma patient biopsy data from The Cancer Genome Atlas revealed that MCT1 and MCT4 expression was significantly reduced in mutant IDH1 tumors compared to wild-type. Taken together, our study shows that reduced MCT expression is part of the metabolic reprogramming of mutant IDH1 gliomas. This finding could impact treatment and has important implications for metabolic imaging of mutant IDH1 gliomas.