Imaging brain glucose metabolism in vivo reveals propionate as a major anaplerotic substrate in pyruvate dehydrogenase deficiency.

A vexing problem in mitochondrial medicine is our limited capacity to evaluate the extent of brain disease in vivo. This limitation has hindered our understanding of the mechanisms that underlie the imaging phenotype in the brain of patients with mitochondrial diseases and our capacity to identify new biomarkers and therapeutic targets. Using comprehensive imaging, we analyzed the metabolic network that drives the brain structural and metabolic features of a mouse model of pyruvate dehydrogenase deficiency (PDHD). As the disease progressed in this animal, in vivo brain glucose uptake and glycolysis increased. Propionate served as a major anaplerotic substrate, predominantly metabolized by glial cells. A combination of propionate and a ketogenic diet extended lifespan, improved neuropathology, and ameliorated motor deficits in these animals. Together, intermediary metabolism is quite distinct in the PDHD brain-it plays a key role in the imaging phenotype, and it may uncover new treatments for this condition.

An enzyme-coupled microplate assay for activity and inhibition of hmdUMP hydrolysis by DNPH1.

2'-Deoxynucleoside 5'-monophosphate N-glycosidase 1 (DNPH1) hydrolyzes the epigenetically modified nucleotide 5-hydroxymethyl 2'-deoxyuridine 5'-monophosphate (hmdUMP) derived from DNA metabolism. Published assays of DNPH1 activity are low throughput, use high concentrations of DNPH1, and have not incorporated or characterized reactivity with the natural substrate. We describe the enzymatic synthesis of hmdUMP from commercially available materials and define its steady-state kinetics with DNPH1 using a sensitive, two-pathway enzyme coupled assay. This continuous absorbance-based assay works in 96-well plate format using nearly 500-fold less DNPH1 than previous methods. With a Z prime value of 0.92, the assay is suitable for high-throughput assays, screening of DNPH1 inhibitors, or characterization of other deoxynucleotide monophosphate hydrolases.

Hyperpolarized [5-13C,4,4-2H2,5-15N]-L-glutamine provides a means of annotating in vivo metabolic utilization of glutamine.

Glutamine is consumed by rapidly proliferating cells and can provide the carbon and nitrogen required for growth through various metabolic pathways. However, delineating the metabolic fate of glutamine is challenging to interrogate in vivo. Hyperpolarized magnetic resonance, by providing high transient nuclear magnetic resonance signals, provides an approach to measure fast biochemical processes in vivo. Aminohydrolysis of glutamine at carbon-5 plays an important role in providing nitrogen and carbon for multiple pathways. Here, we provide a synthetic strategy for isotope-enriched forms of glutamine that prolongs glutamine-C5 relaxation times and thereby reveals in vivo reactions involving carbon-5. We investigate multiple enrichment states, finding [5-13C,4,4-2H2,5-15N]-L-glutamine to be optimal for hyperpolarized measurement of glutamine conversion to glutamate in vivo. Leveraging this compound, we explore pancreatic cancer glutamine metabolism in vivo. Taken together, this work provides a means for studying glutamine metabolic flux in vivo and demonstrates on-target effects of metabolic enzyme inhibitors.

Ketohexokinase-mediated fructose metabolism is lost in hepatocellular carcinoma and can be leveraged for metabolic imaging.

The ability to break down fructose is dependent on ketohexokinase (KHK) that phosphorylates fructose to fructose-1-phosphate (F1P). We show that KHK expression is tightly controlled and limited to a small number of organs and is down-regulated in liver and intestinal cancer cells. Loss of fructose metabolism is also apparent in hepatocellular adenoma and carcinoma (HCC) patient samples. KHK overexpression in liver cancer cells results in decreased fructose flux through glycolysis. We then developed a strategy to detect this metabolic switch in vivo using hyperpolarized magnetic resonance spectroscopy. Uniformly deuterating [2-13C]-fructose and dissolving in D2O increased its spin-lattice relaxation time (T1) fivefold, enabling detection of F1P and its loss in models of HCC. In summary, we posit that in the liver, fructolysis to F1P is lost in the development of cancer and can be used as a biomarker of tissue function in the clinic using metabolic imaging.

Synthesis of α,ß-unsaturated epoxy ketones utilizing a bifunctional sulfonium/phosphonium ylide.

Herein, a new protocol for rapid synthesis of α,ß-unsaturated epoxy ketones utilizing a bifunctional sulfonium/phosphonium ylide is described. This approach comprises two sequential chemoselective reactions between sulfonium and phosphonium ylides and two distinct aldehydes, which allows for the rapid construction of a variety of unsymmetric α,ß-unsaturated epoxy ketones. This methodology allows the rapid construction of the core reactive functionality of a family of lipid peroxidation products, the epoxyketooctadecenoic acids, but can be further broadly utilized as a useful synthon for the synthesis of natural products, particularly those derived from oxidized fatty acids. Accordingly, a protocol utilizing this approach to synthesize the epoxyketooctadecenoic acid family of molecules is described.

Multi-sample measurement of hyperpolarized pyruvate-to-lactate flux in melanoma cells.

Hyperpolarized [1-13 C] pyruvate can be used to examine the metabolic state of cancer cells, highlighting a key metabolic characteristic of cancer: the upregulated metabolic flux to lactate, even in the presence of oxygen (Warburg effect). Thus, the rate constant of 13 C exchange of pyruvate to lactate, kPL , can serve as a metabolic biomarker of cancer presence, aggressiveness and therapy response. Established in vitro hyperpolarized experiments dissolve the probe for each cell sample independently, an inefficient process that consumes excessive time and resources. Expanding on our previous development of a microcoil with greatly increased detection sensitivity (103 -fold) compared with traditional in vitro methods, we present a novel microcoil equipped with a 10-µL vertical reservoir and an experimental protocol utilizing deuterated dissolution buffer to measure metabolic flux in multiple mass-limited cell suspension samples using a single dissolution. This method increases efficiency and potentially reduces the methodological variability associated with hyperpolarized experiments. This technique was used to measure pyruvate-to-lactate flux in melanoma cells to assess BRAF-inhibition treatment response. There was a significant reduction of kPL in BRAFV600E cells following 24 and 48 hours of treatment with 2 µM vemurafenib (P ≤ .05). This agrees with significant changes observed in the pool sizes of extracellular lactate (P ≤ .05) and glucose (P ≤ .001) following 6 and 48 hours of treatment, respectively, and a significant reduction in cell proliferation following 72 hours of treatment (P ≤ .01). BRAF inhibition had no significant effect on the metabolic flux of BRAFWT cells. These data demonstrate a 6-8-fold increase in efficiency for the measurement of kPL in cell suspension samples compared with traditional hyperpolarized in vitro methods.

Dynamic volumetric hyperpolarized 13 C imaging with multi-echo EPI.

PURPOSE: To generate dynamic, volumetric maps of hyperpolarized [1-13 C]pyruvate and its metabolic products in vivo. METHODS: Maps of chemical species were generated with iterative least squares (IDEAL) reconstruction from multiecho echo-planar imaging (EPI) of phantoms of thermally polarized 13 C-labeled chemicals and mice injected with hyperpolarized [1-13 C]pyruvate on a preclinical 3T scanner. The quality of the IDEAL decomposition of single-shot and multishot phantom images was evaluated using quantitative results from a simple pulse-and-acquire sequence as the gold standard. Time course and area-under-the-curve plots were created to analyze the distribution of metabolites in vivo. RESULTS: Improved separation of chemical species by IDEAL, evaluated by the amount of residual signal measured for chemicals not present in the phantoms, was observed as the number of EPI shots was increased from one to four. Dynamic three-dimensional metabolite maps of [1-13 C]pyruvate,[1-13 C]pyruvatehydrate, [1-13 C]lactate, [1-13 C]bicarbonate, and [1-13 C]alanine generated by IDEAL from interleaved multishot multiecho EPI of live mice were used to construct time course and area-under-the-curve graphs for the heart, kidneys, and liver, which showed good agreement with previously published results. CONCLUSIONS: IDEAL decomposition of multishot multiecho 13C EPI images is a simple, yet robust method for generating high-quality dynamic volumetric maps of hyperpolarized [1-13 C]pyruvate and its products in vivo and has potential applications for the assessment of multiorgan metabolic phenomena.

Limited Environmental Serine and Glycine Confer Brain Metastasis Sensitivity to PHGDH Inhibition.

A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacologic inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggest that PHGDH inhibitors may be useful in the treatment of brain metastasis. SIGNIFICANCE: Using proteomics, metabolomics, and multiple brain metastasis models, we demonstrate that the nutrient-limited environment of the brain potentiates brain metastasis susceptibility to serine synthesis inhibition. These findings underscore the importance of studying cancer metabolism in physiologically relevant contexts, and provide a rationale for using PHGDH inhibitors to treat brain metastasis.This article is highlighted in the In This Issue feature, p. 1241.

Hyperpolarized [6-13C,15N3]-Arginine as a Probe for in Vivo Arginase Activity.

Alterations in arginase enzyme expression are linked with various diseases and have been shown to support disease progression, thus motivating the development of an imaging probe for this enzymatic target. 13C-enriched arginine can be used as a hyperpolarized (HP) magnetic resonance (MR) probe for arginase flux since the arginine carbon-6 resonance (157 ppm) is converted to urea (163 ppm) following arginase-catalyzed hydrolysis. However, scalar relaxation from adjacent 14N-nuclei shortens cabon-6 T 1 and T 2 times, yielding poor spectral properties. To address these limitations, we report the synthesis of [6-13C,15N3]-arginine and demonstrate that 15N-enrichment increases carbon-6 relaxation times, thereby improving signal-to-noise ratio and spectral resolution. By overcoming these limitations with this novel isotope-labeling scheme, we were able to perform in vitro and in vivo arginase activity measurements with HP MR. We present HP [6-13C,15N3]-arginine as a noninvasive arginase imaging agent for preclinical studies, with the potential for future clinical diagnostic use.

Hyperpolarized MRI Visualizes Warburg Effects and Predicts Treatment Response to mTOR Inhibitors in Patient-Derived ccRCC Xenograft Models.

The ever-changing tumor microenvironment constantly challenges individual cancer cells to balance supply and demand, presenting tumor vulnerabilities and therapeutic opportunities. Everolimus and temsirolimus are inhibitors of mTOR (mTORi) approved for treating metastatic renal cell carcinoma (mRCC). However, treatment outcome varies greatly among patients. Accordingly, administration of mTORi in mRCC is diminishing, which could potentially result in missing timely delivery of effective treatment for select patients. Here, we implemented a clinically applicable, integrated platform encompassing a single dose of [1-13C] pyruvate to visualize the in vivo effect of mTORi on the conversion of pyruvate to lactate using hyperpolarized MRI. A striking difference that predicts treatment benefit was demonstrated using two preclinical models derived from patients with clear cell RCC (ccRCC) who exhibited primary resistance to VEGFRi and quickly succumbed to their diseases within 6 months after the diagnosis of metastasis without receiving mTORi. Our findings suggest that hyperpolarized MRI could be further developed to personalize kidney cancer treatment. SIGNIFICANCE: These findings demonstrate hyperpolarized [1-13C]pyruvate MRI as a tool for accurately assessing the clinical success of mTOR inhibition in patients with ccRCC.

A non-synthetic approach to extending the lifetime of hyperpolarized molecules using D2O solvation.

Although dissolution dynamic nuclear polarization is a robust technique to significantly increase magnetic resonance signal, the short T1 relaxation time of most 13C-nuclei limits the timescale of hyperpolarized experiments. To address this issue, we have characterized a non-synthetic approach to extend the hyperpolarized lifetime of 13C-nuclei in close proximity to solvent-exchangeable protons. Protons exhibit stronger dipolar relaxation than deuterium, so dissolving these compounds in D2O to exchange labile protons with solvating deuterons results in longer-lived hyperpolarization of the 13C-nucleus 2-bonds away. 13C T1 and T2 times were longer in D2O versus H2O for all molecules in this study. This phenomenon can be utilized to improve hyperpolarized signal-to-noise ratio as a function of longer T1, and enhanced spectral and imaging resolution via longer T2.

Targeted AKT Inhibition in Prostate Cancer Cells and Spheroids Reduces Aerobic Glycolysis and Generation of Hyperpolarized [1-13C] Lactate.

The PI3K/AKT/mTOR (PAM) signaling pathway is frequently mutated in prostate cancer. Specific AKT inhibitors are now in advanced clinical trials, and this study investigates the effect of MK2206, a non-ATP-competitive inhibitor, on the cellular metabolism of prostate cancer cells. We observed a reduction in cell motility and aerobic glycolysis in prostate cancer cells with treatment. These changes were not accompanied by a reduction in the ratio of high-energy phosphates or a change in total protein levels of enzymes and transporters involved in glycolysis. However, a decreased ratio of NAD+/NADH was observed, motivating the use of hyperpolarized magnetic resonance spectroscopy (HP-MRS) to detect treatment response. Spectroscopic experiments were performed on tumor spheroids, 3D structures that self-organize in the presence of an extracellular matrix. Treated spheroids showed decreased lactate production with on-target inhibition confirmed using IHC, demonstrating that HP-MRS can be used to probe treatment response in prostate cancer spheroids and can provide a biomarker for treatment response. Mol Cancer Res; 16(3); 453-60. ©2018 AACR.

Real-time quantitative analysis of metabolic flux in live cells using a hyperpolarized micromagnetic resonance spectrometer.

Metabolic reprogramming is widely considered a hallmark of cancer, and understanding metabolic dynamics described by the conversion rates or "fluxes" of metabolites can shed light onto biological processes of tumorigenesis and response to therapy. For real-time analysis of metabolic flux in intact cells or organisms, magnetic resonance (MR) spectroscopy and imaging methods have been developed in conjunction with hyperpolarization of nuclear spins. These approaches enable noninvasive monitoring of tumor progression and treatment efficacy and are being tested in multiple clinical trials. However, because of their limited sensitivity, these methods require a larger number of cells, on the order of 107, which is impractical for analyzing scant target cells or mass-limited samples. We present a new technology platform, a hyperpolarized micromagnetic resonance spectrometer (HMRS), that achieves real-time, 103-fold more sensitive metabolic analysis on live cells. This platform enables quantification of the metabolic flux in a wide range of cell types, including leukemia stem cells, without significant changes in viability, which allows downstream molecular analyses in tandem. It also enables rapid assessment of metabolic changes by a given drug, which may direct therapeutic choices in patients. We further advanced this platform for high-throughput analysis of hyperpolarized molecules by integrating a three-dimensionally printed microfluidic system. The HMRS platform holds promise as a sensitive method for studying metabolic dynamics in mass-limited samples, including primary cancer cells, providing novel therapeutic targets and an enhanced understanding of cellular metabolism.

Sampling Hyperpolarized Molecules Utilizing a 1 Tesla Permanent Magnetic Field.

Hyperpolarized magnetic resonance spectroscopy (HP MRS) using dynamic nuclear polarization (DNP) is a technique that has greatly enhanced the sensitivity of detecting (13)C nuclei. However, the HP MRS polarization decays in the liquid state according to the spin-lattice relaxation time (T1) of the nucleus. Sampling of the signal also destroys polarization, resulting in a limited temporal ability to observe biologically interesting reactions. In this study, we demonstrate that sampling hyperpolarized signals using a permanent magnet at 1 Tesla (1T) is a simple and cost-effective method to increase T1s without sacrificing signal-to-noise. Biologically-relevant information may be obtained with a permanent magnet using enzyme solutions and in whole cells. Of significance, our findings indicate that changes in pyruvate metabolism can also be quantified in a xenograft model at this field strength.