Hikari A.I. Yoshihara

Magnetic Resonance of 2-Hydroxyglutarate in IDH1-Mutated Low-Grade Gliomas

2-Hydroxyglutarate can be detected ex vivo in biopsy tissue from IDH1-mutant low-grade gliomas with proton high-resolution magic angle spinning NMR spectroscopy. Gliomas Make the Grade Tumors of the central nervous systems can be classified, or “graded,” on a scale of I to IV, according to their capacity to proliferate and invade surrounding tissue (with I being benign). Although determining the grade of brain tumor malignancy is important for doctors to predict survival and prescribe treatment, it cannot sufficiently explain the variation in clinical outcome. Some have attempted to classify brain tumors on the basis of acquired mutations, which has provided further insight into the characteristic diversity observed in survival. For example, one mutation in the gene isocitrate dehydrogenase (IDH) has demonstrated prolonged life expectancy for patients with low-grade brain tumors. Now, Elkhaled, Jalbert, and colleagues have shown that accumulation of a metabolite resulting from this mutation can be detected using magnetic resonance imaging techniques. Under normal conditions, the IDH enzyme converts the metabolite isocitrate to α-ketoglutarate. When IDH is mutated, its enzyme product further converts α-ketoglutarate to an otherwise scarce metabolite, 2-hydroxyglutarate (2HG). Because improved patient outcome has been associated with IDH mutations, the accumulation of 2HG might therefore be able to predict favorable genotypes. Elkhaled, Jalbert, and colleagues used an imaging method based on proton high-resolution magic angle spinning (1H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy to determine whether the presence of 2HG was detectable. A total of 104 tissue (biopsy) samples from 52 patients with recurrent grade II gliomas (some of which had converted to grades III or IV) were evaluated for the presence of 2HG and the IDH1 mutation. 2HG proved to be detectable by spectroscopic analysis and showed approximately 86% concordance with the status of IDH1 mutation, as determined by antibody staining and genetic sequencing. Furthermore, 2HG abundance was shown to be similar across all brain tumor grades when normalized by cellularity, suggesting that its relative production per cell remains the same even after lesions have converted to higher histologic grades. This finding bears considerable significance for clinical evaluation of the malignancy grade and extent of tumor lesions. Finally, 2HG levels were determined to negatively correlate with normal vascularity. Given the current hypotheses in the literature regarding the influence of 2HG on vascular endothelial growth factor (VEGF), this result may be of interest for designing anti-angiogenic strategies for treating tumors with IDH mutations. This ex vivo study—along with its in vivo companion by Andronesi et al.—shows that the use of magnetic resonance imaging technology, already routine in the clinic, could significantly improve the management of brain tumors. Recent studies have indicated that a significant survival advantage is conferred to patients with gliomas whose lesions harbor mutations in the genes isocitrate dehydrogenase 1 and 2 (IDH1/2). IDH1/2 mutations result in aberrant enzymatic production of the potential oncometabolite d-2-hydroxyglutarate (2HG). Here, we report on the ex vivo detection of 2HG in IDH1-mutated tissue samples from patients with recurrent low-grade gliomas using the nuclear magnetic resonance technique of proton high-resolution magic angle spinning spectroscopy. Relative 2HG levels from pathologically confirmed mutant IDH1 tissues correlated with levels of other ex vivo metabolites and histopathology parameters associated with increases in mitotic activity, relative tumor content, and cellularity. Ex vivo spectroscopic measurements of choline-containing species and in vivo magnetic resonance measurements of diffusion parameters were also correlated with 2HG levels. These data provide extensive characterization of mutant IDH1 lesions while confirming the potential diagnostic value of 2HG as a surrogate marker of patient survival. Such information may augment the ability of clinicians to monitor therapeutic response and provide criteria for stratifying patients to specific treatment regimens.

Non-invasive in vivo assessment of IDH1 mutational status in glioma

Gain-of-function mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade gliomas and secondary glioblastoma. They lead to intracellular accumulation of the oncometabolite 2-hydroxyglutarate, represent an early pathogenic event and are considered a therapeutic target. Here we show, in this proof-of-concept study, that [1-(13)C] α-ketoglutarate can serve as a metabolic imaging agent for non-invasive, real-time, in vivo monitoring of mutant IDH1 activity, and can inform on IDH1 status. Using (13)C magnetic resonance spectroscopy in combination with dissolution dynamic nuclear polarization, the metabolic fate of hyperpolarized [1-(13)C] α-ketoglutarate is studied in isogenic glioblastoma cells that differ only in their IDH1 status. In lysates and tumours that express wild-type IDH1, only hyperpolarized [1-(13)C] α-ketoglutarate can be detected. In contrast, in cells that express mutant IDH1, hyperpolarized [1-(13)C] 2-hydroxyglutarate is also observed, both in cell lysates and in vivo in orthotopic tumours.

The DegraBase: A Database of Proteolysis in Healthy and Apoptotic Human Cells

Proteolysis is a critical post-translational modification for regulation of cellular processes. Our lab has previously developed a technique for specifically labeling unmodified protein N termini, the α-aminome, using the engineered enzyme, subtiligase. Here we present a database, called the DegraBase (http://wellslab.ucsf.edu/degrabase/), which compiles 8090 unique N termini from 3206 proteins directly identified in subtiligase-based positive enrichment mass spectrometry experiments in healthy and apoptotic human cell lines. We include both previously published and unpublished data in our analysis, resulting in a total of 2144 unique α-amines identified in healthy cells, and 6990 in cells undergoing apoptosis. The N termini derive from three general categories of proteolysis with respect to cleavage location and functional role: translational N-terminal methionine processing (∼10% of total proteolysis), sites close to the translational N terminus that likely represent removal of transit or signal peptides (∼25% of total), and finally, other endoproteolytic cuts (∼65% of total). Induction of apoptosis causes relatively little change in the first two proteolytic categories, but dramatic changes are seen in endoproteolysis. For example, we observed 1706 putative apoptotic caspase cuts, more than double the total annotated sites in the CASBAH and MEROPS databases. In the endoproteolysis category, there are a total of nearly 3000 noncaspase nontryptic cleavages that are not currently reported in the MEROPS database. These studies significantly increase the annotation for all categories of proteolysis in human cells and allow public access for investigators to explore interesting proteolytic events in healthy and apoptotic human cells.

Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

The recent development of hyperpolarized (13)C magnetic resonance spectroscopic imaging provides a novel method for in vivo metabolic imaging with potential applications for detection of cancer and response to treatment. Chemotherapy-induced apoptosis was shown to decrease the flux of hyperpolarized (13)C label from pyruvate to lactate due to depletion of NADH, the coenzyme of lactate dehydrogenase. In contrast, we show here that in PC-3MM2 tumors, inhibition of platelet-derived growth factor receptor with imatinib reduces the conversion of hyperpolarized pyruvate to lactate by lowering the expression of lactate dehydrogenase itself. This was accompanied by reduced expression of vascular endothelial growth factor and glutaminase, and is likely mediated by reduced expression of their transcriptional factors hypoxia-inducible factor-1 and c-Myc. Our results indicate that hyperpolarized (13)C MRSI could potentially detect the molecular effect of various cell signaling inhibitors, thus providing a radiation-free method to predict tumor response.

Investigating tumor perfusion and metabolism using multiple hyperpolarized 13C compounds: HP001, pyruvate and urea

The metabolically inactive hyperpolarized agents HP001 (bis-1,1-(hydroxymethyl)-[1-(13)C]cyclopropane-d(8)) and urea enable a new type of perfusion magnetic resonance imaging based on a direct signal source that is background-free. The addition of perfusion information to metabolic information obtained by spectroscopic imaging of hyperpolarized [1-(13)C]pyruvate would be of great value in exploring the relationship between perfusion and metabolism in cancer. In preclinical normal murine and cancer model studies, we performed both dynamic multislice imaging of the specialized hyperpolarized perfusion compound HP001 (T(1)=95 s ex vivo, 32 s in vivo at 3 T) using a pulse sequence with balanced steady-state free precession and ramped flip angle over time for efficient utilization of the hyperpolarized magnetization and three-dimensional echo-planar spectroscopic imaging of urea copolarized with [1-(13)C]pyruvate, with compressed sensing for resolution enhancement. For the dynamic data, peak signal maps and blood flow maps derived from perfusion modeling were generated. The spatial heterogeneity of perfusion was increased 2.9-fold in tumor tissues (P=.05), and slower washout was observed in the dynamic data. The results of separate dynamic HP001 imaging and copolarized pyruvate/urea imaging were compared. A strong and significant correlation (R=0.73, P=.02) detected between the urea and HP001 data confirmed the value of copolarizing urea with pyruvate for simultaneous assessment of perfusion and metabolism.

Outcome-related metabolomic patterns from 1H/31P NMR after mild hypothermia treatments of oxygen–glucose deprivation in a neonatal brain slice model of asphyxia

Human clinical trials using 72 hours of mild hypothermia (32°C–34°C) after neonatal asphyxia have found substantially improved neurologic outcomes. As temperature changes differently modulate numerous metabolite fluxes and concentrations, we hypothesized that 1H/31P nuclear magnetic resonance (NMR) spectroscopy of intracellular metabolites can distinguish different insults, treatments, and recovery stages. Three groups of superfused neonatal rat brain slices underwent 45 minutes oxygen–glucose deprivation (OGD) and then were: treated for 3 hours with mild hypothermia (32°C) that began with OGD, or similarly treated with hypothermia after a 15-minute delay, or not treated (normothermic control group, 37°C). Hypothermia was followed by 3 hours of normothermic recovery. Slices collected at different predetermined times were processed, respectively, for 14.1 Tesla NMR analysis, enzyme-linked immunosorbent assay (ELISA) cell-death quantification, and superoxide production. Forty-nine NMR-observable metabolites underwent a multivariate analysis. Separated clustering in scores plots was found for treatment and outcome groups. Final ATP (adenosine triphosphate) levels, severely decreased at normothermia, were restored equally by immediate and delayed hypothermia. Cell death was decreased by immediate hypothermia, but was equally substantially greater with normothermia and delayed hypothermia. Potentially important biomarkers in the 1H spectra included PCr-1H (phosphocreatine in the 1H spectrum), ATP-1H (adenosine triphosphate in the 1H spectrum), and ADP-1H (adenosine diphosphate in the 1H spectrum). The findings suggest a potential role for metabolomic monitoring during therapeutic hypothermia.

Characterization of metabolites in infiltrating gliomas using ex vivo 1H high-resolution magic angle spinning spectroscopy

Gliomas are routinely graded according to histopathological criteria established by the World Health Organization. Although this classification can be used to understand some of the variance in the clinical outcome of patients, there is still substantial heterogeneity within and between lesions of the same grade. This study evaluated image‐guided tissue samples acquired from a large cohort of patients presenting with either new or recurrent gliomas of grades II–IV using ex vivo proton high‐resolution magic angle spinning spectroscopy. The quantification of metabolite levels revealed several discrete profiles associated with primary glioma subtypes, as well as secondary subtypes that had undergone transformation to a higher grade at the time of recurrence. Statistical modeling further demonstrated that these metabolomic profiles could be differentially classified with respect to pathological grading and inter‐grade conversions. Importantly, the myo‐inositol to total choline index allowed for a separation of recurrent low‐grade gliomas on different pathological trajectories, the heightened ratio of phosphocholine to glycerophosphocholine uniformly characterized several forms of glioblastoma multiforme, and the onco‐metabolite D‐2‐hydroxyglutarate was shown to help distinguish secondary from primary grade IV glioma, as well as grade II and III from grade IV glioma. These data provide evidence that metabolite levels are of interest in the assessment of both intra‐grade and intra‐lesional malignancy. Such information could be used to enhance the diagnostic specificity of in vivo spectroscopy and to aid in the selection of the most appropriate therapy for individual patients.

Use of hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate to probe the effects of the anticancer agent dichloroacetate on mitochondrial metabolism in vivo in the normal rat☆

Development of hyperpolarized technology utilizing dynamic nuclear polarization has enabled the measurement of (13)C metabolism in vivo at very high signal-to-noise ratio (SNR). In vivo mitochondrial metabolism can, in principle, be monitored with pyruvate, which is catalyzed to acetyl-CoA via pyruvate dehydrogenase (PDH). The purpose of this work was to determine whether the compound sodium dichloroacetate (DCA) could aid the study of mitochondrial metabolism with hyperpolarized pyruvate. DCA stimulates PDH by inhibiting its inhibitor, pyruvate dehydrogenase kinase. In this work, hyperpolarized [1-(13)C]pyruvate and [2-(13)C]pyruvate were used to probe mitochondrial metabolism in normal rats. Increased conversion to bicarbonate (+181±69%, P=.025) was measured when [1-(13)C]pyruvate was injected after DCA administration, and increased glutamate (+74±23%, P=.004), acetoacetate (+504±281%, P=.009) and acetylcarnitine (+377±157%, P=.003) were detected when [2-(13)C]pyruvate was used.

A designed antagonist of the thyroid hormone receptor

We synthesized an analogue of the thyromimetic GC-1 bearing the same hydrophobic appendage as the estrogen receptor antagonist ICI-164,384. While having reduced affinity for the thyroid hormone receptors compared to GC-1, it behaves in a manner consistent with a competitive antagonist in a transactivation assay.

Improved synthesis of the iodine-free thyromimetic GC-1.

Synthesis of the TRbeta-selective thyromimetic GC-1 has been improved using methoxymethyl (MOM) and triisopropylsilyl (TiPS) substituents as phenolic protecting groups. The new synthetic route is adaptable to analogue design.