Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant infiltration of tumor-associated macrophages (TAMs). TAMs have been reported to drive resistance to gemcitabine, a frontline ...chemotherapy in PDA, though the mechanism of this resistance remains unclear. Profiling metabolite exchange, we demonstrate that macrophages programmed by PDA cells release a spectrum of pyrimidine species. These include deoxycytidine, which inhibits gemcitabine through molecular competition at the level of drug uptake and metabolism. Accordingly, genetic or pharmacological depletion of TAMs in murine models of PDA sensitizes these tumors to gemcitabine. Consistent with this, patients with low macrophage burden demonstrate superior response to gemcitabine treatment. Together, these findings provide insights into the role of macrophages in pancreatic cancer therapy and have potential to inform the design of future treatments. Additionally, we report that pyrimidine release is a general function of alternatively activated macrophage cells, suggesting an unknown physiological role of pyrimidine exchange by immune cells.
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•Macrophages polarized by pancreatic cancer cells release pyrimidine nucleosides•Pyrimidine release is a property of alternatively activated macrophage metabolism•Deoxycytidine from macrophages inhibits gemcitabine treatment of cancer cells•Targeting macrophages enhances gemcitabine treatment of pancreatic cancer
Macrophages are present in high abundance in pancreatic ductal adenocarcinoma. Halbrook et al. identify that alternatively activated macrophages release a spectrum of pyrimidine nucleosides that are consumed by pancreatic cancer cells. Among these, deoxycytidine can directly compete with gemcitabine, hindering its efficiency as a chemotherapy.
Metabolic reprogramming is observed across all cancer types. Indeed, the success of many classic chemotherapies stems from their targeting of cancer metabolism. Contemporary research in this area has ...refined our understanding of tumor-specific metabolic mechanisms and has revealed strategies for exploiting these vulnerabilities selectively. Based on this growing understanding, new small-molecule tools and drugs have been developed to study and target tumor metabolism. Here, we highlight allosteric modulation of metabolic enzymes as an attractive mechanism of action for small molecules that target metabolic enzymes. We then discuss the mechanistic insights garnered from their application in cancer studies and highlight the achievements of this approach in targeting cancer metabolism. Finally, we discuss technological advances in drug discovery for allosteric modulators of enzyme activity.
Abnormal epigenetic patterns correlate with effector T cell malfunction in tumours
, but the cause of this link is unknown. Here we show that tumour cells disrupt methionine metabolism in CD8
T ...cells, thereby lowering intracellular levels of methionine and the methyl donor S-adenosylmethionine (SAM) and resulting in loss of dimethylation at lysine 79 of histone H3 (H3K79me2). Loss of H3K79me2 led to low expression of STAT5 and impaired T cell immunity. Mechanistically, tumour cells avidly consumed methionine and outcompeted T cells for methionine by expressing high levels of the methionine transporter SLC43A2. Genetic and biochemical inhibition of tumour SLC43A2 restored H3K79me2 in T cells, thereby boosting spontaneous and checkpoint-induced tumour immunity. Moreover, methionine supplementation improved the expression of H3K79me2 and STAT5 in T cells, and this was accompanied by increased T cell immunity in tumour-bearing mice and patients with colon cancer. Clinically, tumour SLC43A2 correlated negatively with T cell histone methylation and functional gene signatures. Our results identify a mechanistic connection between methionine metabolism, histone patterns, and T cell immunity in the tumour microenvironment. Thus, cancer methionine consumption is an immune evasion mechanism, and targeting cancer methionine signalling may provide an immunotherapeutic approach.
Targeted tandem mass spectrometry (LC-MS/MS) has been extremely useful for profiling small molecules extracted from biological sources, such as cells, bodily fluids and tissues. Here, we present a ...protocol for analysing incorporation of the non-radioactive stable isotopes carbon-13 (
C) and nitrogen-15 (
N) into polar metabolites in central carbon metabolism and related pathways. Our platform utilizes selected reaction monitoring (SRM) with polarity switching and amide hydrophilic interaction liquid chromatography (HILIC) to capture transitions for carbon and nitrogen incorporation into selected metabolites using a hybrid triple quadrupole (QQQ) mass spectrometer. This protocol represents an extension of a previously published protocol for targeted metabolomics of unlabeled species and has been used extensively in tracing the metabolism of nutrients such as
C-labeled glucose,
C-glutamine and
N-glutamine in a variety of biological settings (e.g., cell culture experiments and in vivo mouse labelling via i.p. injection). SRM signals are integrated to produce an array of peak areas for each labelling form that serve as the output for further analysis. The processed data are then used to obtain the degree and distribution of labelling of the targeted molecules (termed fluxomics). Each method can be customized on the basis of known unlabeled Q1/Q3 SRM transitions and adjusted to account for the corresponding
C or
N incorporation. The entire procedure takes ~6-7 h for a single sample from experimental labelling and metabolite extraction to peak integration.
Rewired metabolism is a hallmark of pancreatic ductal adenocarcinomas (PDA). Previously, we demonstrated that PDA cells enhance glycosylation precursor biogenesis through the hexosamine biosynthetic ...pathway (HBP) via activation of the rate limiting enzyme, glutamine-fructose 6-phosphate amidotransferase 1 (GFAT1). Here, we genetically ablated GFAT1 in human PDA cell lines, which completely blocked proliferation in vitro and led to cell death. In contrast, GFAT1 knockout did not preclude the growth of human tumor xenografts in mice, suggesting that cancer cells can maintain fidelity of glycosylation precursor pools by scavenging nutrients from the tumor microenvironment. We found that hyaluronic acid (HA), an abundant carbohydrate polymer in pancreatic tumors composed of repeating
-acetyl-glucosamine (GlcNAc) and glucuronic acid sugars, can bypass GFAT1 to refuel the HBP via the GlcNAc salvage pathway. Together, these data show HA can serve as a nutrient fueling PDA metabolism beyond its previously appreciated structural and signaling roles.
Introduction
We previously developed a tandem mass spectrometry-based label-free targeted metabolomics analysis framework coupled to two distinct chromatographic methods, reversed-phase liquid ...chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC), with dynamic multiple reaction monitoring (dMRM) for simultaneous detection of over 200 metabolites to study core metabolic pathways.
Objectives
We aim to analyze a large-scale heterogeneous data compendium generated from our LC–MS/MS platform with both RPLC and HILIC methods to systematically assess measurement quality in biological replicate groups and to investigate metabolite abundance changes and patterns across different biological conditions.
Methods
Our metabolomics framework was applied in a wide range of experimental systems including cancer cell lines, tumors, extracellular media, primary cells, immune cells, organoids, organs (e.g. pancreata), tissues, and sera from human and mice. We also developed computational and statistical analysis pipelines, which include hierarchical clustering, replicate-group CV analysis, correlation analysis, and case–control paired analysis.
Results
We generated a compendium of 42 heterogeneous deidentified datasets with 635 samples using both RPLC and HILIC methods. There exist metabolite signatures that correspond to various phenotypes of the heterogeneous datasets, involved in several metabolic pathways. The RPLC method shows overall better reproducibility than the HILIC method for most metabolites including polar amino acids. Correlation analysis reveals high confidence metabolites irrespective of experimental systems such as methionine, phenylalanine, and taurine. We also identify homocystine, reduced glutathione, and phosphoenolpyruvic acid as highly dynamic metabolites across all case–control paired samples.
Conclusions
Our study is expected to serve as a resource and a reference point for a systematic analysis of label-free LC–MS/MS targeted metabolomics data in both RPLC and HILIC methods with dMRM.
Self-renewing embryonic stem cells (ESCs) respond to environmental cues by exiting pluripotency or entering a quiescent state. The molecular basis underlying this fate choice remains unclear. Here, ...we show that histone acetyltransferase MOF plays a critical role in this process through directly activating fatty acid oxidation (FAO) in the ground-state ESCs. We further show that the ground-state ESCs particularly rely on elevated FAO for oxidative phosphorylation (OXPHOS) and energy production. Mof deletion or FAO inhibition induces bona fide quiescent ground-state ESCs with an intact core pluripotency network and transcriptome signatures akin to the diapaused epiblasts in vivo. Mechanistically, MOF/FAO inhibition acts through reducing mitochondrial respiration (i.e., OXPHOS), which in turn triggers reversible pluripotent quiescence specifically in the ground-state ESCs. The inhibition of FAO/OXPHOS also induces quiescence in naive human ESCs. Our study suggests a general function of the MOF/FAO/OXPHOS axis in regulating cell fate determination in stem cells.
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•MOF blocks acquisition of the quiescent state in ground-state ESCs•MOF directly activates the fatty acid oxidation (FAO) pathway•MOF/FAO axis provides the carbon source for fueling mitochondrial respiration•FAO/OXPHOS axis-mediated blocking of quiescence is conserved in naive human ESCs
Khoa et al. find that ground-state embryonic stem cells (ESCs) readily use fatty acid oxidation (FAO) as the carbon source for mitochondrial oxidative phosphorylation (OXPHOS), which is directly regulated by histone acetyltransferase MOF. ESCs lacking the MOF/FAO/OXPHOS axis reside in a reversible pluripotent quiescence, mimicking embryonic diapause in mammals.
Pancreatic cancer cells are characterized by deregulated metabolic programs that facilitate growth and resistance to oxidative stress. Among these programs, pancreatic cancers preferentially utilize ...a metabolic pathway through the enzyme aspartate aminotransferase 1 also known as glutamate oxaloacetate transaminase 1 (GOT1) to support cellular redox homeostasis. As such, small molecule inhibitors that target GOT1 could serve as starting points for the development of new therapies for pancreatic cancer. We ran a high-throughput screen for inhibitors of GOT1 and identified a small molecule, iGOT1-01, with in vitro GOT1 inhibitor activity. Application in pancreatic cancer cells revealed metabolic and growth inhibitory activity reflecting a promiscuous inhibitory profile. We then performed an in silico docking analysis to study inhibitor–GOT1 interactions with iGOT1-01 analogues that possess improved solubility and potency properties. These results suggested that the GOT1 inhibitor competed for binding to the pyridoxal 5-phosphate (PLP) cofactor site of GOT1. To analyze how the GOT1 inhibitor bound to GOT1, a series of GOT1 mutant enzymes that abolished PLP binding were generated. Application of the mutants in X-ray crystallography and thermal shift assays again suggested but were unable to formally conclude that the GOT1 inhibitor bound to the PLP site. Mutational studies revealed the relationship between PLP binding and the thermal stability of GOT1 while highlighting the essential nature of several residues for GOT1 catalytic activity. Insight into the mode of action of GOT1 inhibitors may provide leads to the development of drugs that target redox balance in pancreatic cancer.
This paper investigates energy harvesting using nonlinear energy sink. First a novel apparatus is described in detail outlining how the essential nonlinearity and energy harvesting are achieved. Then ...the system modeling is addressed, including the equations of motion for the mechanical system and the electromechanical system, and a formula for the transduction factor. The experimental identification is conducted to determine several key parameters and relationships. Using the established models, a computer simulation is carried out to investigate the apparatus׳s performance under transient responses in terms of vibration absorption and energy harvesting. Finally experiments are conducted to validate the simulation results. It is shown that the system performs well, being capable of energy localization as well as broad band vibration absorption. The system is also shown to be capable of harvesting energy.
Known for his visual style as well as for his experimentation in virtually every genre of narrative cinema, award-winning director Sidney J. Furie also has the distinction of having made Canada's ...first ever feature-length fictional film in English,A Dangerous Age(1957). With a body of work that includesThe Ipcress File(1965),Lady Sings the Blues(1972), andThe Entity(1982), he has collaborated with major stars such as Marlon Brando, Frank Sinatra, Robert Redford, and Michael Caine, and his films have inspired some of Hollywood's most celebrated directors, including Stanley Kubrick and Quentin Tarantino.
In this first biography of the prolific filmmaker, author Daniel Kremer offers a comprehensive look at the director's unique career. Furie pioneered techniques such as improvisation in large-scale film productions, and sometimes shot his films in sequence to develop the characters from the ground up and improve the performers' in-the-moment spontaneity. Not only has Stanley Kubrick acknowledged that Furie'sThe Boys in Company C(1978) informed and influencedFull Metal Jacket(1987), but Martin Scorsese has said that he considersThe Entityto be one of the scariest horror films of all time. However, Furie was often later criticized for accepting lowbrow work, and as a result, little serious study has been devoted to the director.
Meticulously researched and enhanced by Kremer's close relationship with the filmmaker, this definitive biography captures the highs and lows of an exceptional but underexamined career, taking readers behind the scenes with a director who was often ahead of his time.
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