SummaryReprogrammed cellular metabolism is a common characteristic observed in various cancers1,2. However, whether metabolic changes directly regulate cancer development and progression remains poorly understood. Here we show that BCAT1, a cytosolic aminotransferase for the branched-chain amino acids (BCAAs), is aberrantly activated and functionally required for chronic myeloid leukemia (CML). BCAT1 is up-regulated during CML progression and promotes BCAA production in leukemia cells by aminating the branched-chain keto acids. Blocking BCAT1 expression or enzymatic activity induces cellular differentiation and impairs the propagation of blast crisis CML (BC-CML) both in vitro and in vivo. Stable isotope tracer experiments combined with NMR-based metabolic analysis demonstrate the intracellular production of BCAAs by BCAT1. Direct supplementation with BCAAs ameliorates the defects caused by BCAT1 knockdown, indicating that BCAT1 exerts its oncogenic function via BCAA production in BC-CML cells. Importantly, BCAT1 expression not only is activated in human BC-CML and de novo acute myeloid leukemia but also predicts disease outcome in patients. As an upstream regulator of BCAT1 expression, we identified Musashi2 (MSI2), an oncogenic RNA binding protein that is required for BC-CML. MSI2 is physically associated with the BCAT1 transcript and positively regulates its protein expression in leukemia. Taken together, this work reveals that altered BCAA metabolism activated through the MSI2-BCAT1 axis drives cancer progression in myeloid leukemia.

Recently, the enhanced resolution and sensitivity offered by chemoselective isotope tags have enabled new and enhanced methods for detecting hundreds of quantifiable metabolites in biofluids using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry. However, the inability to effectively detect the same metabolites using both complementary analytical techniques has hindered the correlation of data derived from the two powerful platforms and thereby the maximization of their combined strengths for applications such as biomarker discovery of the identification of unknown metabolites. With the goal of alleviating this bottleneck, we describe a smart isotope tag, 15N-cholamine, which possesses two important properties: an NMR sensitive isotope, and a permanent charge for MS sensitivity. Using this tag, we demonstrate the detection of carboxyl group containing metabolites in both human serum and urine. By combining the individual strengths of the 15N label and permanent charge, the smart isotope tag facilitates effective detection of the carboxyl-containing metabolome by both analytical methods. This study demonstrates a unique approach to exploit the combined strength of MS and NMR in the field of metabolomics.

Dense time-series metabolomics data are essential for unraveling the underlying dynamic properties of metabolism. Here we extend high-resolution-magic angle spinning (HR-MAS) to enable continuous in vivo monitoring of metabolism by NMR (CIVM-NMR) and provide analysis tools for these data. First, we reproduced a result in human chronic lymphoid leukemia cells by using isotope-edited CIVM-NMR to rapidly and unambiguously demonstrate unidirectional flux in branched-chain amino acid metabolism. We then collected untargeted CIVM-NMR datasets for Neurospora crassa , a classic multicellular model organism, and uncovered dynamics between central carbon metabolism, amino acid metabolism, energy storage molecules, and lipid and cell wall precursors. Virtually no sample preparation was required to yield a dynamic metabolic fingerprint over hours to days at ~4-min temporal resolution with little noise. CIVM-NMR is simple and readily adapted to different types of cells and microorganisms, offering an experimental complement to kinetic models of metabolism for diverse biological systems.

In addition to fatty acids, glucose and lactate are important myocardial substrates under physiological and stress conditions. They are metabolized to pyruvate that enters mitochondria via the mitochondrial pyruvate carrier (MPC) for citric acid cycle (CAC) metabolism. Here, we show that MPC-mediated mitochondrial pyruvate utilization is essential for the partitioning of glucose-derived cytosolic metabolic intermediates, which modulate myocardial stress adaptation. Mice with cardiomyocyte-restricted deletion of subunit 1 of MPC (cMPC1 −/− ) developed age-dependent pathologic cardiac hypertrophy, transitioning to a dilated cardiomyopathy and premature death. Hypertrophied hearts accumulated lactate, pyruvate, and glycogen and displayed increased protein O-GlcNacylation (O-GlcNAc), which was prevented by increasing availability of non-glucose substrates in vivo by ketogenic (KD) or high-fat (HFD) diets, which reversed the structural, metabolic and functional remodeling of non-stressed cMPC1 −/− hearts. While concurrent short-term KD did not rescue cMPC1 −/− hearts from rapid decompensation and early mortality following pressure overload, 3-week of KD prior to TAC was sufficient to rescue this phenotype. Together, our results highlight the centrality of pyruvate metabolism to myocardial metabolism and function.

We describe here the agreed upon first development steps and priority objectives of a community engagement effort to address current challenges in quality assurance (QA) and quality control (QC) in untargeted metabolomic studies. This has included (1) a QA and QC questionnaire responded to by the metabolomics community in 2015 which recommended education of the metabolomics community, development of appropriate standard reference materials and providing incentives for laboratories to apply QA and QC; (2) a 2-day ‘Think Tank on Quality Assurance and Quality Control for Untargeted Metabolomic Studies’ held at the National Cancer Institute’s Shady Grove Campus and (3) establishment of the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) to drive forward developments in a coordinated manner.

The Metabolomics Quality Assurance and Quality Control Consortium (mQACC) evolved from the recognized need for a community-wide consensus on improving and systematizing quality assurance (QA) and quality control (QC) practices for untargeted metabolomics. As an initial step, members of the consortium and several non-members who used liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics were asked to voluntarily participate in a collaborative research project and took part by providing the QA and QC practices utilized in their laboratories, via a six-page questionnaire composed of over 120 questions and comment fields. All contributors to this project are authors. Responses were then analyzed to identify common and divergent QA and QC practices among the contributing laboratories. For QA, many laboratories reported documenting maintenance, calibration and tuning (82%); having established data storage and archival processes (71%); depositing data in public repositories (55%); having standard operating procedures (SOPs) in place for all laboratory processes (68%) and training staff on laboratory processes (55%). For QC, universal practices included using system suitability procedures (100%) and using a robust system of identification (Metabolomics Standards Initiative level 1 identification standards) for at least some of the detected compounds. Most laboratories used QC samples (>86%); used internal standards (91%); used a designated analytical acquisition template with randomized experimental samples (91%); and manually reviewed peak integration following data acquisition (86%). A minority of laboratories included technical replicates of experimental samples in their workflows (36%). Due to the recruitment method for participants and its voluntary nature, although the 23 contributors were researchers with diverse and international backgrounds from academia, industry and government, most being current members of mQACC, they are not necessarily representative of the worldwide pool of practitioners. The findings presented here, in addition to other data gathering efforts within mQACC, will be used to guide discussions for recommendations of best practices within the community and to establish internationally agreed upon reporting standards.

Breast cancer, a heterogeneous disease with variable pathophysiology and biology, is classified into four major subtypes. While hormonal- and antibody-targeted therapies are effective in the patients with luminal and HER-2 subtypes, the patients with triple-negative breast cancer (TNBC) subtype do not benefit from these therapies. The incidence rates of TNBC subtype are higher in African-American women, and the evidence indicates that these women have worse prognosis compared to women of European descent. The reasons for this disparity remain unclear but are often attributed to TNBC biology. In this study, we performed metabolic analysis of breast tissues to identify how TNBC differs from luminal A breast cancer (LABC) subtypes within the African-American and Caucasian breast cancer patients, respectively. We used High-Resolution Magic Angle Spinning (HR-MAS) 1H Nuclear magnetic resonance (NMR) to perform the metabolomic analysis of breast cancer and adjacent normal tissues (total n=82 samples). TNBC and LABC subtypes in African American women exhibited different metabolic profiles. Metabolic profiles of these subtypes were also distinct from those revealed in Caucasian women. TNBC in African-American women expressed higher levels of glutathione, choline, and glutamine as well as profound metabolic alterations characterized by decreased mitochondrial respiration and increased glycolysis concomitant with decreased levels of ATP. TNBC in Caucasian women was associated with increased pyrimidine synthesis. These metabolic alterations could potentially be exploited as novel treatment targets for TNBC.

NMR spectroscopy is a powerful analytical tool for both qualitative and quantitative analysis. However, accurate quantitative analysis in complex fluids such as human blood plasma is challenging, and analysis using one-dimensional NMR is limited by signal overlap. It is impractical to use heteronuclear experiments involving natural abundance 13C on a routine basis due to low sensitivity, despite their improved resolution. Focusing on circumventing such bottlenecks, this study demonstrates the utility of a combination of isotope tagged NMR experiments to analyze metabolites in human blood plasma. 1H-15N HSQC and 1H-13C HSQC experiments on the isotope tagged samples combined with the conventional 1H one-dimensional and 1H-1H TOCSY experiments provide quantitative information on a large number of metabolites in plasma. The methods were first tested on a mixture of 28 synthetic analogues of metabolites commonly present in human blood; twenty-seven metabolites in a standard NIST (National Institute of Standards and Technology) human blood plasma were then identified and quantified with an average coefficient of variation of 2.4 % for 17 metabolites and 5.6% when all the metabolites were considered. Carboxylic acids and amines represent a majority of the metabolites in body fluids and their analysis by isotope tagging enables a significant enhancement of the metabolic pool for biomarker discovery applications. Improved sensitivity and resolution of NMR experiments imparted by 15N and 13C isotope tagging is attractive for both the enhancement of the detectable metabolic pool and accurate analysis of plasma metabolites. The approach can be easily extended to many additional metabolites in almost any biological mixture.