Advancements in Pulsed Stable Isotope-Resolved Metabolomics.

The understanding of biochemical processes of metabolism is gained through the measurement of the concentration of intermediates and the rate of metabolite conversion. However, the measurement of metabolite concentrations does not give a full representation of this dynamic system. To understand the kinetics of metabolism, the system must be described and quantified in terms of metabolite flow as a function of time. In order to measure the metabolite flow, or more precisely the metabolic flux through a biological system, substrates of the cell are labelled with stable isotopes. The usage of these substrates by the cell leads to the incorporation of the isotopes into downstream intermediates.The most important metabolic pathways are encompassed in the central carbon metabolism (CCM). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), the central carbon metabolism "is the most basic aspect of life". It includes all metabolites and enzymatic reactions within: glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), amino acids and nucleotide metabolic pathways. Some molecules are at the crossroad of metabolic pathways, interconnecting diverse metabolic and therefore functional outcomes. Labelling these nodal metabolites and analysing their isotopic composition allows the precise determination of the metabolic flow within the biochemical networks that they are in.Application of stable isotope labelled substrates allows the measurement of metabolic flux through a biochemical pathway. The rapid turnover of metabolites in pathways requires pulse-feeding cells with a labelled substrate. This method allows for the determination of different cell states. For example, the action of a drug from immediate impact until the compensatory response of the metabolic system (cell, organs, organisms). Pulsed labelling is an elegant way to analyse the action of small molecules and drugs and enables the analysis of regulatory metabolic processes in short time scales.

Inhibiting phosphoglycerate dehydrogenase counteracts chemotherapeutic efficacy against MYCN-amplified neuroblastoma.

Here we sought metabolic alterations specifically associated with MYCN amplification as nodes to indirectly target the MYCN oncogene. Liquid chromatography-mass spectrometry-based proteomics identified seven proteins consistently correlated with MYCN in proteomes from 49 neuroblastoma biopsies and 13 cell lines. Among these was phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in de novo serine synthesis. MYCN associated with two regions in the PHGDH promoter, supporting transcriptional PHGDH regulation by MYCN. Pulsed stable isotope-resolved metabolomics utilizing 13 C-glucose labeling demonstrated higher de novo serine synthesis in MYCN-amplified cells compared to cells with diploid MYCN. An independence of MYCN-amplified cells from exogenous serine and glycine was demonstrated by serine and glycine starvation, which attenuated nucleotide pools and proliferation only in cells with diploid MYCN but did not diminish these endpoints in MYCN-amplified cells. Proliferation was attenuated in MYCN-amplified cells by CRISPR/Cas9-mediated PHGDH knockout or treatment with PHGDH small molecule inhibitors without affecting cell viability. PHGDH inhibitors administered as single-agent therapy to NOG mice harboring patient-derived MYCN-amplified neuroblastoma xenografts slowed tumor growth. However, combining a PHGDH inhibitor with the standard-of-care chemotherapy drug, cisplatin, revealed antagonism of chemotherapy efficacy in vivo. Emergence of chemotherapy resistance was confirmed in the genetic PHGDH knockout model in vitro. Altogether, PHGDH knockout or inhibition by small molecules consistently slows proliferation, but stops short of killing the cells, which then establish resistance to classical chemotherapy. Although PHGDH inhibition with small molecules has produced encouraging results in other preclinical cancer models, this approach has limited attractiveness for patients with neuroblastoma.

L-Glyceraldehyde Inhibits Neuroblastoma Cell Growth via a Multi-Modal Mechanism on Metabolism and Signaling.

Glyceraldehyde (GA) is a three-carbon monosaccharide that can be present in cells as a by-product of fructose metabolism. Bruno Mendel and Otto Warburg showed that the application of GA to cancer cells inhibits glycolysis and their growth. However, the molecular mechanism by which this occurred was not clarified. We describe a novel multi-modal mechanism by which the L-isomer of GA (L-GA) inhibits neuroblastoma cell growth. L-GA induces significant changes in the metabolic profile, promotes oxidative stress and hinders nucleotide biosynthesis. GC-MS and 13C-labeling was employed to measure the flow of carbon through glycolytic intermediates under L-GA treatment. It was found that L-GA is a potent inhibitor of glycolysis due to its proposed targeting of NAD(H)-dependent reactions. This results in growth inhibition, apoptosis and a redox crisis in neuroblastoma cells. It was confirmed that the redox mechanisms were modulated via L-GA by proteomic analysis. Analysis of nucleotide pools in L-GA-treated cells depicted a previously unreported observation, in which nucleotide biosynthesis is significantly inhibited. The inhibitory action of L-GA was partially relieved with the co-application of the antioxidant N-acetyl-cysteine. We present novel evidence for a simple sugar that inhibits cancer cell proliferation via dysregulating its fragile homeostatic environment.

Proteomic profiling reveals ACSS2 facilitating metabolic support in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by genomic aberrations in oncogenes, cytogenetic abnormalities, and an aberrant epigenetic landscape. Nearly 50% of AML cases will relapse with current treatment. A major source of therapy resistance is the interaction of mesenchymal stroma with leukemic cells resulting in therapeutic protection. We aimed to determine pro-survival/anti-apoptotic protein networks involved in the stroma protection of leukemic cells. Proteomic profiling of cultured primary AML (n = 14) with Hs5 stroma cell line uncovered an up-regulation of energy-favorable metabolic proteins. Next, we modulated stroma-induced drug resistance with an epigenetic drug library, resulting in reduced apoptosis with histone deacetylase inhibitor (HDACi) treatment versus other epigenetic modifying compounds. Quantitative phosphoproteomic probing of this effect further revealed a metabolic-enriched phosphoproteome including significant up-regulation of acetyl-coenzyme A synthetase (ACSS2, S30) in leukemia-stroma HDACi treated cocultures compared with untreated monocultures. Validating these findings, we show ACSS2 substrate, acetate, promotes leukemic proliferation, ACSS2 knockout in leukemia cells inhibits leukemic proliferation and ACSS2 knockout in the stroma impairs leukemic metabolic fitness. Finally, we identify ACSS1/ACSS2-high expression AML subtype correlating with poor overall survival. Collectively, this study uncovers the leukemia-stroma phosphoproteome emphasizing a role for ACSS2 in mediating AML growth and drug resistance.

Satellite record reveals 1960s acceleration of Totten Ice Shelf in East Antarctica.

Wilkes Land and Totten Glacier (TG) in East Antarctica (EA) have been losing ice mass significantly since 1989. There is a lack of knowledge of long-term mass balance in the region which hinders the estimation of its contribution to global sea level rise. Here we show that this acceleration trend in TG has occurred since the 1960s. We reconstruct ice flow velocity fields of 1963-1989 in TG from the first-generation satellite images of ARGON and Landsat-1&4, and build a five decade-long record of ice dynamics. We find a persistent long-term ice discharge rate of 68 ± 1 Gt/y and an acceleration of 0.17 ± 0.02 Gt/y2 from 1963 to 2018, making TG the greatest contributor to global sea level rise in EA. We attribute the long-term acceleration near grounding line from 1963 to 2018 to basal melting likely induced by warm modified Circumpolar Deep Water. The speed up in shelf front during 1973-1989 was caused by a large calving front retreat. As the current trend continues, intensified monitoring in the TG region is recommended in the next decades.

Investigating the role of GLUL as a survival factor in cellular adaptation to glutamine depletion via targeted stable isotope resolved metabolomics.

Cellular glutamine synthesis is thought to be an important resistance factor in protecting cells from nutrient deprivation and may also contribute to drug resistance. The application of ?targeted stable isotope resolved metabolomics" allowed to directly measure the activity of glutamine synthetase in the cell. With the help of this method, the fate of glutamine derived nitrogen within the biochemical network of the cells was traced. The application of stable isotope labelled substrates and analyses of isotope enrichment in metabolic intermediates allows the determination of metabolic activity and flux in biological systems. In our study we used stable isotope labelled substrates of glutamine synthetase to demonstrate its role in the starvation response of cancer cells. We applied 13C labelled glutamate and 15N labelled ammonium and determined the enrichment of both isotopes in glutamine and nucleotide species. Our results show that the metabolic compensatory pathways to overcome glutamine depletion depend on the ability to synthesise glutamine via glutamine synthetase. We demonstrate that the application of dual-isotope tracing can be used to address specific reactions within the biochemical network directly. Our study highlights the potential of concurrent isotope tracing methods in medical research.

Analysing central metabolism in ultra-high resolution: At the crossroads of carbon and nitrogen.

BACKGROUND: Cancer cell metabolism can be characterised by adaptive metabolic alterations, which support abnormal proliferative cell growth with high energetic demand. De novo nucleotide biosynthesis is essential for providing nucleotides for RNA and DNA synthesis, and drugs targeting this biosynthetic pathway have proven to be effective anticancer therapeutics. Nevertheless, cancers are often able to circumvent chemotherapeutic interventions and become therapy resistant. Our understanding of the changing metabolic profile of the cancer cell and the mode of action of therapeutics is dependent on technological advances in biochemical analysis. SCOPE OF REVIEW: This review begins with information about carbon- and nitrogen-donating pathways to build purine and pyrimidine moieties in the course of nucleotide biosynthesis. We discuss the application of stable isotope resolved metabolomics to investigate the dynamics of cancer cell metabolism and outline the benefits of high-resolution accurate mass spectrometry, which enables multiple tracer studies. CONCLUSION: With the technological advances in mass spectrometry that allow for the analysis of the metabolome in high resolution, the application of stable isotope resolved metabolomics has become an important technique in the investigation of biological processes. The literature in the area of isotope labelling is dominated by 13C tracer studies. Metabolic pathways have to be considered as complex interconnected networks and should be investigated as such. Moving forward to simultaneous tracing of different stable isotopes will help elucidate the interplay between carbon and nitrogen flow and the dynamics of de novo nucleotide biosynthesis within the cell.

Next-Generation Sequencing-Assisted DNA-Based Digital PCR for a Personalized Approach to the Detection and Quantification of Residual Disease in Chronic Myeloid Leukemia Patients.

Recent studies indicate that 40% of chronic myeloid leukemia patients who achieve sustained undetectable BCR-ABL1 transcripts on tyrosine kinase inhibitor therapy remain disease-free after drug discontinuation. In contrast, 60% experience return of detectable disease and have to restart treatment, thus highlighting the need for an improved method of identifying patients with the lowest likelihood of relapse. Here we describe the validation of a personalized DNA-based digital PCR (dPCR) approach for quantifying very low levels of residual disease, which involves the rapid identification of t(9;22) fusion junctions using targeted next-generation sequencing coupled with the use of a dPCR platform. t(9;22) genomic breakpoints were successfully mapped in samples from 32 of 32 patients with early stage disease. Disease quantification by DNA-based dPCR was performed using the Fluidigm BioMark platform on 46 follow-up samples from 6 of the 32 patients, including 36 samples that were in deep molecular remission. dPCR detected persistent disease in 81% of molecular-remission samples, outperforming both RT-dPCR (25%) and DNA-based quantitative PCR (19%). We conclude that dPCR for BCR-ABL1 DNA is the most sensitive available method of residual-disease detection in chronic myeloid leukemia and may prove useful in the management of tyrosine kinase inhibitor withdrawal.