Kevin M. Brindle

A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations

A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are “silent,” that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing “metabolic snapshots,” can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY—an abbreviation for functional analysis by co-responses in yeast.

Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy.

Measurements of early tumor responses to therapy have been shown, in some cases, to predict treatment outcome. We show in lymphoma-bearing mice injected intravenously with hyperpolarized [1-13C]pyruvate that the lactate dehydrogenase–catalyzed flux of 13C label between the carboxyl groups of pyruvate and lactate in the tumor can be measured using 13C magnetic resonance spectroscopy and spectroscopic imaging, and that this flux is inhibited within 24 h of chemotherapy. The reduction in the measured flux after drug treatment and the induction of tumor cell death can be explained by loss of the coenzyme NAD(H) and decreases in concentrations of lactate and enzyme in the tumors. The technique could provide a new way to assess tumor responses to treatment in the clinic.

Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate.

As alterations in tissue pH underlie many pathological processes, the capability to image tissue pH in the clinic could offer new ways of detecting disease and response to treatment. Dynamic nuclear polarization is an emerging technique for substantially increasing the sensitivity of magnetic resonance imaging experiments. Here we show that tissue pH can be imaged in vivo from the ratio of the signal intensities of hyperpolarized bicarbonate (H13CO3-) and 13CO2 following intravenous injection of hyperpolarized H13CO3-. The technique was demonstrated in a mouse tumour model, which showed that the average tumour interstitial pH was significantly lower than the surrounding tissue. Given that bicarbonate is an endogenous molecule that can be infused in relatively high concentrations into patients, we propose that this technique could be used clinically to image pathological processes that are associated with alterations in tissue pH, such as cancer, ischaemia and inflammation.

Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent

The C2 domain of synaptotagmin I, which binds to anionic phospholipids in cell membranes, was shown to bind to the plasma membrane of apoptotic cells by both flow cytometry and confocal microscopy. Conjugation of the protein to superparamagnetic iron oxide nanoparticles allowed detection of this binding using magnetic resonance imaging. Detection of apoptotic cells, using this novel contrast agent, was demonstrated both in vitro, with isolated apoptotic tumor cells, and in vivo, in a tumor treated with chemotherapeutic drugs.

New approaches for imaging tumour responses to treatment

Tumour responses to treatment are still largely assessed from imaging measurements of reductions in tumour size. However, this can take several weeks to become manifest and in some cases may not occur at all, despite a positive response to treatment. There has been considerable interest, therefore, in non-invasive techniques for imaging tissue function that can give early evidence of response. These can be used in clinical trials of new drugs to give an early indication of drug efficacy, and subsequently in the clinic to select the most effective therapy at an early stage of treatment.

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin‐dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone‐dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the Pkd1 gene

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation in the kidney, liver, and pancreas and is associated often with cardiovascular abnormalities such as hypertension, mitral valve prolapse, and intracranial aneurysms. It is caused by mutations in PKD1 or PKD2, encoding polycystin-1 and -2, which together form a cell surface nonselective cation ion channel. Pkd2−/− mice have cysts in the kidney and pancreas and defects in cardiac septation, whereas Pkd1del34 −/− and Pkd1L −/− mice have cysts but no cardiac abnormalities, although vascular fragility was reported in the latter. Here we describe mice carrying a targeted mutation in Pkd1 (Pkd1del17–21βgeo), which defines its expression pattern by using a lacZ reporter gene and may identify novel functions for polycystin-1. Although Pkd1del17–21βgeo +/− adult mice develop renal and hepatic cysts, Pkd1del17–21βgeo −/− embryos die at embryonic days 13.5–14.5 from a primary cardiovascular defect that includes double outflow right ventricle, disorganized myocardium, and abnormal atrio-ventricular septation. Skeletal development is also severely compromised. These abnormalities correlate with the major sites of Pkd1 expression. During nephrogenesis, Pkd1 is expressed in maturing tubular epithelial cells from embryonic day 15.5. This expression coincides with the onset of cyst formation in Pkd1del34 −/−, Pkd1L −/−, and Pkd2−/− mice, supporting the hypothesis that polycystin-1 and polycystin-2 interact in vivo and that their failure to do so leads to abnormalities in tubule morphology and function.

Clinical Proton MR Spectroscopy in Central Nervous System Disorders

A large body of published work shows that proton (hydrogen 1 [(1)H]) magnetic resonance (MR) spectroscopy has evolved from a research tool into a clinical neuroimaging modality. Herein, the authors present a summary of brain disorders in which MR spectroscopy has an impact on patient management, together with a critical consideration of common data acquisition and processing procedures. The article documents the impact of (1)H MR spectroscopy in the clinical evaluation of disorders of the central nervous system. The clinical usefulness of (1)H MR spectroscopy has been established for brain neoplasms, neonatal and pediatric disorders (hypoxia-ischemia, inherited metabolic diseases, and traumatic brain injury), demyelinating disorders, and infectious brain lesions. The growing list of disorders for which (1)H MR spectroscopy may contribute to patient management extends to neurodegenerative diseases, epilepsy, and stroke. To facilitate expanded clinical acceptance and standardization of MR spectroscopy methodology, guidelines are provided for data acquisition and analysis, quality assessment, and interpretation. Finally, the authors offer recommendations to expedite the use of robust MR spectroscopy methodology in the clinical setting, including incorporation of technical advances on clinical units.

Production of hyperpolarized [1,4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors.

Dynamic nuclear polarization of 13C-labeled cell substrates has been shown to massively increase their sensitivity to detection in NMR experiments. The sensitivity gain is sufficiently large that if these polarized molecules are injected intravenously, their spatial distribution and subsequent conversion into other cell metabolites can be imaged. We have used this method to image the conversion of fumarate to malate in a murine lymphoma tumor in vivo after i.v. injection of hyperpolarized [1,4-13C2]fumarate. In isolated lymphoma cells, the rate of labeled malate production was unaffected by coadministration of succinate, which competes with fumarate for transport into the cell. There was, however, a correlation with the percentage of cells that had lost plasma membrane integrity, suggesting that the production of labeled malate from fumarate is a sensitive marker of cellular necrosis. Twenty-four hours after treating implanted lymphoma tumors with etoposide, at which point there were significant levels of tumor cell necrosis, there was a 2.4-fold increase in hyperpolarized [1,4-13C2]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized 13C-labeled malate from [1,4-13C2]fumarate appears to be a sensitive marker of tumor cell death in vivo and could be used to detect the early response of tumors to treatment. Given that fumarate is an endogenous molecule, this technique has the potential to be used clinically.

Tumor imaging using hyperpolarized 13C magnetic resonance spectroscopy.

Dynamic nuclear polarization is an emerging technique for increasing the sensitivity of magnetic resonance imaging and spectroscopy, particularly for low‐γ nuclei. The technique has been applied recently to a number of 13C‐labeled cell metabolites in biological systems: the increase in signal‐to‐noise allows the spatial distribution of an injected molecule to be imaged as well as its metabolic product or products. This review highlights the most significant molecules investigated to date in preclinical cancer models, either in terms of their demonstrated metabolism in vivo or the biological processes that they can probe. In particular, label exchange between hyperpolarized 13C‐labeled pyruvate and lactate, catalyzed by lactate dehydrogenase, has been shown to have a number of potential applications. Finally, techniques to image these molecules are also discussed as well as methods that may extend the lifetime of the hyperpolarized signal. Hyperpolarized magnetic resonance imaging and magnetic resonance spectroscopic imaging have shown great promise for the imaging of cancer in preclinical work, both for diagnosis and for monitoring therapy response. If the challenges in translating this technique to human imaging can be overcome, then it has the potential to significantly alter the management of cancer patients. Magn Reson Med, 2011.