Mutations in isocitrate dehydrogenase 1 (IDH1) characterize most adult low-grade gliomas. Mutant IDH1 catalyzes production of the oncometabolite 2-hydroxyglutarate (2-HG). We recently discovered that the IDH1 mutation also reprograms pyruvate metabolism in a 2-HG-dependent manner, and reprogramming of pyruvate metabolism is essential for cell proliferation in mutant IDH1 glioma cells.

Metabolic reprogramming is an important hallmark of cancer. Alterations in many metabolic pathways support the requirement for cellular building blocks that are essential for cancer cell proliferation. This metabolic reprogramming can be imaged using magnetic resonance spectroscopy (MRS). H MRS can inform on alterations in the steady-state levels of cellular metabolites, but the emergence of hyperpolarized C MRS has now also enabled imaging of metabolic fluxes in real-time, providing a new method for tumor detection and monitoring of therapeutic response. In the case of glioma, preclinical cell and animal studies have shown that the hyperpolarized C MRS metabolic imaging signature is specific to tumor type and can distinguish between mutant IDH1 glioma and primary glioblastoma. Here, we review these findings, first describing the main metabolic pathways that are altered in the different glioma subtypes, and then reporting on the use of hyperpolarized C MRS and MR spectroscopic imaging (MRSI) to probe these pathways. We show that the future translation of this hyperpolarized C MRS molecular metabolic imaging method to the clinic promises to improve the noninvasive detection, characterization, and response-monitoring of brain tumors resulting in improved patient diagnosis and clinical management.

Targeted therapeutic approaches are increasingly being implemented in the clinic, but early detection of response frequently presents a challenge as many new therapies lead to inhibition of tumor growth rather than tumor shrinkage. Development of novel non-invasive methods to monitor response to treatment is therefore needed. Magnetic resonance spectroscopy (MRS) and magnetic resonance spectroscopic imaging are non-invasive imaging methods that can be employed to monitor metabolism, and previous studies indicate that these methods can be useful for monitoring the metabolic consequences of treatment that are associated with early drug target modulation. However, single-metabolite biomarkers are often not specific to a particular therapy. Here we used an unbiased 1H MRS-based metabolomics approach to investigate the overall metabolic consequences of treatment with the phosphoinositide 3-kinase inhibitor LY294002 and the heat shock protein 90 inhibitor 17AAG in prostate and breast cancer cell lines. LY294002 treatment resulted in decreased intracellular lactate, alanine fumarate, phosphocholine and glutathione. Following 17AAG treatment, decreased intracellular lactate, alanine, fumarate and glutamine were also observed but phosphocholine accumulated in every case. Furthermore, citrate, which is typically observed in normal prostate tissue but not in tumors, increased following 17AAG treatment in prostate cells. This approach is likely to provide further information about the complex interactions between signaling and metabolic pathways. It also highlights the potential of MRS-based metabolomics to identify metabolic signatures that can specifically inform on molecular drug action.

The use of magnetic resonance spectroscopy (MRS) to investigate breast tumour biochemistry in vivo is reviewed. To this end, results obtained both from patients in vivo and from tumour extracts and model systems are discussed. An association has been observed between transformation and an increase in phosphomonoesters (PMEs) detected in the 31P MRS spectrum, as well as an increase in choline-containing metabolites detected in the 1H spectrum. A decrease in PME content after treatment is associated with response to treatment as assessed by tumour volume. Experiments in model systems aimed at understanding the underlying biochemical processes are presented, as well as data indicating the usefulness of MRS in monitoring the uptake and metabolism of some chemotherapeutic agents.

Alterations in cell metabolism are increasingly being recognized as a hallmark of cancer and are being exploited for the development of diagnostic tools and targeted therapeutics. Recently, ¹³C MRS-detectable hyperpolarized pyruvate to lactate conversion has been validated in models as a noninvasive imaging method for the detection of tumors and treatment response, and has successfully passed phase I clinical trials. To date, response to treatment has been associated with a decrease in hyperpolarized lactate production. In this study, we monitored the effect of treatment with the mitogen-activated protein kinase (MEK) inhibitor U0126 in prostate and breast cancer cells. Following treatment, we observed a 31% decrease in the flux of hyperpolarized ¹³C label in treated MCF-7 breast cancer cells relative to controls. In contrast, and unexpectedly, the flux increased to 167% in treated PC3 prostate cancer cells. To mechanistically explain these observations, we investigated treatment-induced changes in the different factors known to affect the pyruvate to lactate conversion. NADH (nicotinamide adenine dinucleotide, reduced form) levels remained unchanged, whereas lactate dehydrogenase expression and activity, as well as intracellular lactate, increased in both cell lines, providing an explanation for the elevated hyperpolarized lactate observed in PC3 cells. The expression of MCT1, which mediates pyruvate transport, decreased in treated MCF-7, but not PC3, cells. This identifies pyruvate transport as rate limiting in U0126-treated MCF-7 cells and explains the decrease in hyperpolarized lactate observed in these cells following treatment. Our findings highlight the complexity of interactions between MEK and metabolism, and the need for mechanistic validation before hyperpolarized ¹³C MRS can be used to monitor treatment-induced molecular responses.

This review is focused on describing the use of magnetic resonance (MR) spectroscopy for metabolic imaging of brain tumors. We will first review the MR metabolic imaging findings generated from preclinical models, focusing primarily on in vivo studies, and will then describe the use of metabolic imaging in the clinical setting. We will address relatively well-established (1) H MRS approaches, as well as (31) P MRS, (13) C MRS and emerging hyperpolarized (13) C MRS methodologies, and will describe the use of metabolic imaging for understanding the basic biology of glioma as well as for improving the characterization and monitoring of brain tumors in the clinic.

Metabolic reprogramming is increasingly being viewed as a hallmark of cancer. Accordingly, metabolic readouts can serve as biomarkers of response to therapy. The goal of this study was to investigate some of the MRS-detectable metabolic consequences of mitogen-activated protein kinase kinase (MEK) inhibition. We investigated PC3 prostate cancer, MCF-7 breast cancer and A375 melanoma cells, and determined that, consistent with previous studies, MRS-detectable levels of phosphocholine decreased significantly in all cell lines (to 63%, 50% and 18% of the control, respectively) following MEK inhibition with U0126. This effect was mediated by a decrease in the expression of choline kinase α, the enzyme that catalyzes the phosphorylation of choline. In contrast, the impact of MEK inhibition on glycolysis was cell line dependent. A375 cells, which express mutant BRAF, demonstrated significant decreases in glucose uptake (to 36% of control) and lactate production (to 42% of control) in line with positron emission tomography data. In contrast, in PC3 and MCF-7 cells, increases in glucose uptake (to 198% and 192% of control, respectively) and lactate production (to 177% and 212% of control, respectively) were observed, in line with a previous hyperpolarized (13) C MRS study. This effect is probably mediated by the activation of the phosphoinositide 3-kinase pathway and AMP-activated protein kinase. Our findings demonstrate the value of translatable non-invasive MRS methods for the provision of information on cellular metabolism as an indication of the activation of potential feedback loops following MEK inhibition.

Abstract Introduction 17-allyamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is currently in clinical trials in breast cancer. However, 17-AAG treatment often results in inhibition of tumor growth rather than shrinkage, making detection of response a challenge. Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) are noninvasive imaging methods than can be used to monitor metabolic biomarkers of drug-target modulation. This study set out to examine the MRS-detectable metabolic consequences of Hsp90 inhibition in a breast cancer model. Methods MCF-7 breast cancer cells were investigated, and MRS studies were performed both on live cells and on cell extracts. 31P and 1H MRS were used to determine total cellular metabolite concentrations and 13C MRS was used to probe the metabolism of [1,2-13C]-choline. To explain the MRS metabolic findings, microarray and RT-PCR were used to analyze gene expression, and in vitro activity assays were performed to determine changes in enzymatic activity following 17-AAG treatment. Results Treatment of MCF-7 cells with 17-AAG for 48 hours caused a significant increase in intracellular levels of choline (to 266 ± 18% of control, P = 0.05) and phosphocholine (PC; to 181 ± 10% of control, P = 0.001) associated with an increase in expression of choline transporter SLC44A1 and an elevation in the de novo synthesis of PC. We also detected an increase in intracellular levels of glycerophosphocholine (GPC; to 176 ± 38% of control, P = 0.03) associated with an increase in PLA2 expression and activity. Conclusions This study determined that in the MCF-7 breast cancer model inhibition of Hsp90 by 17-AAG results in a significant MRS-detectable increase in choline, PC and GPC, which is likely due to an increase in choline transport into the cell and phospholipase activation. 1H MRSI can be used in the clinical setting to detect levels of total choline-containing metabolite (t-Cho, composed of intracellular choline, PC and GPC). As Hsp90 inhibitors enter routine clinical use, t-Cho could thus provide an easily detectable, noninvasive metabolic biomarker of Hsp90 inhibition in breast cancer patients.

Proinflammatory mononuclear phagocytes (MPs) play a crucial role in the progression of multiple sclerosis (MS) and other neurodegenerative diseases. Despite advances in neuroimaging, there are currently limited available methods enabling noninvasive detection of MPs in vivo. Interestingly, upon activation and subsequent differentiation toward a proinflammatory phenotype MPs undergo metabolic reprogramming that results in increased glycolysis and production of lactate. Hyperpolarized (HP) ¹³C magnetic resonance spectroscopic imaging (MRSI) is a clinically translatable imaging method that allows noninvasive monitoring of metabolic pathways in real time. This method has proven highly useful to monitor the Warburg effect in cancer, through MR detection of increased HP [1-¹³C]pyruvate-to-lactate conversion. However, to date, this method has never been applied to the study of neuroinflammation. Here, we questioned the potential of ¹³C MRSI of HP [1-¹³C]pyruvate to monitor the presence of neuroinflammatory lesions in vivo in the cuprizone mouse model of MS. First, we demonstrated that ¹³C MRSI could detect a significant increase in HP [1-¹³C]pyruvate-to-lactate conversion, which was associated with a high density of proinflammatory MPs. We further demonstrated that the increase in HP [1-¹³C]lactate was likely mediated by pyruvate dehydrogenase kinase 1 up-regulation in activated MPs, resulting in regional pyruvate dehydrogenase inhibition. Altogether, our results demonstrate a potential for ¹³C MRSI of HP [1-¹³C]pyruvate as a neuroimaging method for assessment of inflammatory lesions. This approach could prove useful not only in MS but also in other neurological diseases presenting inflammatory components.

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 (13)C magnetic resonance spectroscopic imaging (MRSI) and (1)H 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 (13)C MRSI, and (1)H MRS to monitor the effects of treatment. In our study, (1)H 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.