Thomas R. Eykyn

Therapeutic target metabolism observed using hyperpolarized 15N choline

Choline is a precursor of cellular phospholipid metabolism that provides Magnetic Resonance (MR) and Positron Emission Tomography (PET) biomarkers for cancer detection and response assessment. Employing Dynamic Nuclear Polarization we show that the MR signal of 15N in choline can be enhanced by at least 4 orders of magnitude with a relaxation time of ca. 4 min, providing a method to observe the action of choline kinase, an important target for novel cancer therapeutics.

Model free approach to kinetic analysis of real-time hyperpolarized 13C magnetic resonance spectroscopy data.

Real-time detection of the rates of metabolic flux, or exchange rates of endogenous enzymatic reactions, is now feasible in biological systems using Dynamic Nuclear Polarization Magnetic Resonance. Derivation of reaction rate kinetics from this technique typically requires multi-compartmental modeling of dynamic data, and results are therefore model-dependent and prone to misinterpretation. We present a model-free formulism based on the ratio of total areas under the curve (AUC) of the injected and product metabolite, for example pyruvate and lactate. A theoretical framework to support this novel analysis approach is described, and demonstrates that the AUC ratio is proportional to the forward rate constant k. We show that the model-free approach strongly correlates with k for whole cell in vitro experiments across a range of cancer cell lines, and detects response in cells treated with the pan-class I PI3K inhibitor GDC-0941 with comparable or greater sensitivity. The same result is seen in vivo with tumor xenograft-bearing mice, in control tumors and following drug treatment with dichloroacetate. An important finding is that the area under the curve is independent of both the input function and of any other metabolic pathways arising from the injected metabolite. This model-free approach provides a robust and clinically relevant alternative to kinetic model-based rate measurements in the clinical translation of hyperpolarized 13C metabolic imaging in humans, where measurement of the input function can be problematic.

Dichloroacetate induces autophagy in colorectal cancer cells and tumours

Background:Dichloroacetate (DCA) has been found to have antitumour properties.Methods:We investigated the cellular and metabolic responses to DCA treatment and recovery in human colorectal (HT29, HCT116 WT and HCT116 Bax-ko), prostate carcinoma cells (PC3) and HT29 xenografts by flow cytometry, western blotting, electron microscopy, 1H and hyperpolarised 13C-magnetic resonance spectroscopy.Results:Increased expression of the autophagy markers LC3B II was observed following DCA treatment both in vitro and in vivo. We observed increased production of reactive oxygen species (ROS) and mTOR inhibition (decreased pS6 ribosomal protein and p4E-BP1 expression) as well as increased expression of MCT1 following DCA treatment. Steady-state lactate excretion and the apparent hyperpolarised [1-13C] pyruvate-to-lactate exchange rate (kPL) were decreased in DCA-treated cells, along with increased NAD+/NADH ratios and NAD+. Steady-state lactate excretion and kPL returned to, or exceeded, control levels in cells recovered from DCA treatment, accompanied by increased NAD+ and NADH. Reduced kPL with DCA treatment was found in HT29 tumour xenografts in vivo.Conclusions:DCA induces autophagy in cancer cells accompanied by ROS production and mTOR inhibition, reduced lactate excretion, reduced kPL and increased NAD+/NADH ratio. The observed cellular and metabolic changes recover on cessation of treatment.

Hyperpolarized (13)C magnetic resonance detection of carboxypeptidase G2 activity.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene‐directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance 13C magnetic resonance spectroscopy (MRS), we observed the CPG2‐mediated conversion of a novel hyperpolarized reporter probe 3,5‐difluorobenzoyl‐L‐glutamic acid (3,5‐DFBGlu) to 3,5‐difluorobenzoic acid (3,5‐DFBA) and L‐glutamic acid (L‐Glu) in vitro. Isotopic labeling of the relevant nuclei with 13C in 3,5‐DFBGlu or related substrates will yield a further factor of 100 increase in the signal‐to‐noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo. Magn Reson Med, 2009.

Measurement of compartment size in q-space experiments: Fourier transform of the second derivative

Restricted diffusion in compartmentalized systems can lead to spatial coherence phenomena being observed in q‐space plots from pulsed field gradient spin‐echo (PGSE) nuclear magnetic resonance (NMR) experiments. The underlying features observed in these plots contain information on the geometry of the compartments that is otherwise difficult to obtain. A numerical procedure is proposed that accentuates these coherence features: the data are weighted with a bell‐shaped window function, interpolated with a shifting cubic spline, and then the second derivative is taken prior to Fourier transformation. The window function provides apodization of the noisy data at high q values, while it and the second derivative are equivalent to applying a high‐pass filter to remove the zero‐ or low‐frequency components in the echo‐signal attenuation. Using a combination of theory, Monte Carlo simulations, and data from PGSE NMR experiments on human red blood cells, we demonstrate this to be a valuable processing tool for delineating the underlying coherence features. It should prove particularly useful where the coherence features are poorly defined or where more than one pattern is present in a q‐space plot. Magn Reson Med 52:907–912, 2004.

Detection of cancer in cervical tissue biopsies using mobile lipid resonances measured with diffusion‐weighted 1H magnetic resonance spectroscopy

The purpose of this study was to implement a diffusion‐weighted sequence for visualisation of mobile lipid resonances (MLR) using high resolution magic angle spinning (HR‐MAS) 1H MRS and to evaluate its use in establishing differences between tissues from patients with cervical carcinoma that contain cancer from those that do not. A stimulated echo sequence with bipolar gradients was modified to allow T1 and T2 measurements and optimised by recording signal loss in HR‐MAS spectra as a function of gradient strength in model lipids and tissues. Diffusion coefficients, T1 and apparent T2 relaxation times were measured in model lipid systems. MLR profiles were characterised in relation to T1 and apparent T2 relaxation in human cervical cancer tissue samples. Diffusion‐weighted (DW) spectra of cervical biopsies were quantified and peak areas analysed using linear discriminant analysis (LDA).

MEK1/2 Inhibition Decreases Lactate in BRAF-Driven Human Cancer Cells

The RAS/BRAF/MEK/ERK signaling pathway is a central driver in cancer with many BRAF and MEK inhibitors being evaluated in clinical trials. Identifying noninvasive biomarkers of early pharmacodynamic responses is important for development of these targeted drugs. As increased aerobic glycolysis is often observed in cancer, we hypothesized that MEK1/2 (MAP2K1/MAP2K2) inhibitors may reduce lactate levels as detected by magnetic resonance spectroscopy (MRS), as a metabolic biomarker for the pharmacodynamic response. MRS was used to monitor intracellular and extracellular levels of lactate in human cancer cells in vitro and in melanoma tumors ex vivo. In addition, we used (1)H MRS and a fluorescent glucose analog to evaluate the effect of MEK inhibition on glucose uptake. MEK1/2 signaling inhibition reduced extracellular lactate levels in BRAF-dependent cells but not BRAF-independent cells. The reduction in extracellular lactate in BRAF-driven melanoma cells was time-dependent and associated with reduced expression of hexokinase-II driven by c-Myc depletion. Taken together, these results reveal how MEK1/2 inhibition affects cancer cell metabolism in the context of BRAF oncogene addiction. Furthermore, they offer a preclinical proof-of-concept for the use of MRS to measure lactate as a noninvasive metabolic biomarker for pharmacodynamic response to MEK1/2 inhibition in BRAF-driven cancers.

Cardiac Hypoxia Imaging: Second-Generation Analogues of 64Cu-ATSM

Myocardial hypoxia is an attractive target for diagnostic and prognostic imaging, but current approaches are insufficiently sensitive for clinical use. The PET tracer copper(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) has promise, but its selectivity and sensitivity could be improved by structural modification. We have therefore evaluated a range of 64Cu-ATSM analogs for imaging hypoxic myocardium. Methods: Isolated rat hearts (n = 5/group) were perfused with normoxic buffer for 30 min and then hypoxic buffer for 45 min within a custom-built triple-γ-detector system to quantify radiotracer infusion, hypoxia-dependent cardiac uptake, and washout. A 1-MBq bolus of each candidate tracer (and 18F-fluoromisonidazole for comparative purposes) was injected into the arterial line during normoxia, and during early and late hypoxia, and their hypoxia selectivity and pharmacokinetics were evaluated. The in vivo pharmacokinetics of promising candidates in healthy rats were then assessed by PET imaging and biodistribution. Results: All tested analogs exhibited hypoxia sensitivity within 5 min. Complexes less lipophilic than 64Cu-ATSM provided significant gains in hypoxic-to-normoxic contrast (14:1 for 64Cu-2,3-butanedione bis(thiosemicarbazone) (ATS), 17:1 for 64Cu-2,3-pentanedione bis(thiosemicarbazone) (CTS), 8:1 for 64Cu-ATSM, P < 0.05). Hypoxic first-pass uptake was 78.2% ± 7.2% for 64Cu-ATS and 70.7% ± 14.5% for 64Cu-CTS, compared with 63.9% ± 11.7% for 64Cu-ATSM. Cardiac retention of 18F-fluoromisonidazole increased from 0.44% ± 0.17% during normoxia to 2.24% ± 0.08% during hypoxia. In vivo, normoxic cardiac retention of 64Cu-CTS was significantly lower than that of 64Cu-ATSM and 64Cu-ATS (0.13% ± 0.02% vs. 0.25% ± 0.04% and 0.24% ± 0.03% injected dose, P < 0.05), with retention of all 3 tracers falling to less than 0.7% injected dose within 6 min. 64Cu-CTS also exhibited lower uptake in liver and lung. Conclusion: 64Cu-ATS and 64Cu-CTS exhibit better cardiac hypoxia selectivity and imaging characteristics than the current lead hypoxia tracers, 64Cu-ATSM and 18F-fluoromisonidazole.

Normalized one-dimensional NOE measurements in isotopically labeled macromolecules using two-way cross-polarization

A novel one-dimensional NOE experiment is presented where a selected proton is excited by two-way heteronuclear cross- polarization between protons and nitrogen-15 or carbon-13. The utility of the method is demonstrated for a sample of 15N labeled human ubiquitin. Inter- and intra-residue NOEs are clearly observed in a very time-effective manner. The signal intensities can be easily normalized.

Lactate and Choline Metabolites Detected In Vitro by Nuclear Magnetic Resonance Spectroscopy Are Potential Metabolic Biomarkers for PI3K Inhibition in Pediatric Glioblastoma

The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline (tCho) and lactate levels (p<0.02) as detected by phosphorus (31P)- and proton (1H)-NMR. Similar changes were also detected using the pan–Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p<0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNA-damaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential non-invasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma.