Glutamine synthetase (GS) catalyzes de novo synthesis of glutamine that facilitates cancer cell growth. In the liver, GS functions next to the urea cycle to remove ammonia waste. As a dysregulated urea cycle is implicated in cancer development, the impact of GS’s ammonia clearance function has not been explored in cancer. Here, we show that oncogenic activation of β-catenin (encoded by CTNNB1) led to a decreased urea cycle and elevated ammonia waste burden. While β-catenin induced the expression of GS, which is thought to be cancer promoting, surprisingly, genetic ablation of hepatic GS accelerated the onset of liver tumors in several mouse models that involved β-catenin activation. Mechanistically, GS ablation exacerbated hyperammonemia and facilitated the production of glutamate-derived nonessential amino acids, which subsequently stimulated mechanistic target of rapamycin complex 1 (mTORC1). Pharmacological and genetic inhibition of mTORC1 and glutamic transaminases suppressed tumorigenesis facilitated by GS ablation. While patients with hepatocellular carcinoma, especially those with CTNNB1 mutations, have an overall defective urea cycle and increased expression of GS, there exists a subset of patients with low GS expression that is associated with mTORC1 hyperactivation. Therefore, GS-mediated ammonia clearance serves as a tumor-suppressing mechanism in livers that harbor β-catenin activation mutations and a compromised urea cycle.

Cancer immunotherapy, exemplified by the remarkable clinical benefits of the immune checkpoint blockade and chimeric antigen receptor T-cell therapy, is revolutionizing cancer therapy. They induce long-term tumor regression and overall survival benefit in many types of cancer. With the advances in our knowledge about the tumor immune microenvironment, remarkable progress has been made in the development of small-molecule drugs for immunotherapy. Small molecules targeting PRR-associated pathways, immune checkpoints, oncogenic signaling, metabolic pathways, cytokine/chemokine signaling, and immune-related kinases have been extensively investigated. Monotherapy of small-molecule immunotherapeutic drugs and their combinations with other antitumor modalities are under active clinical investigations to overcome immune tolerance and circumvent immune checkpoint inhibitor resistance. Here, we review the latest development of small-molecule agents for cancer immunotherapy by targeting defined pathways and highlighting their progress in recent clinical investigations.

Fasting results in major metabolic changes including a switch from glycogenolysis to gluconeogenesis to maintain glucose homeostasis. However, the relationship between the length of fasting and the relative contribution of gluconeogenic substrates remains unclear. We investigated the relative contribution of glycogen, lactate, and glycerol in glucose production of male C57BL/6 J-albino mice after 6, 12, and 18 h of fasting. We used non-perturbative infusions of 13C3 lactate, 13C3 glycerol, and 13C6 glucose combined with liquid chromatography mass spectrometry and metabolic flux analysis to study the contribution of substrates in gluconeogenesis (GNG). During infusion studies, both lactate and glycerol significantly label about 60% and 30-50% glucose carbon, respectively, but glucose labels much more lactate (∼90%) than glycerol carbon (∼10%). Our analyses indicate that lactate, but not glycerol is largely recycled during all fasting periods such that lactate is the largest direct contributor to GNG via the Cori cycle but a minor source of new glucose carbon (overall contribution). In contrast, glycerol is not only a significant direct contributor to GNG but also the largest overall contributor to GNG regardless of fasting length. Prolonged fasting decreases both the whole body turnover rate of glucose and lactate but increases that of glycerol, indicating that the usage of glycerol in GNG become more significant with longer fasting. Collectively, these findings suggest that glycerol is the dominant overall contributor of net glucose carbon in GNG during both short and prolonged fasting.

Metabolic flux analysis (MFA) is an increasingly important tool to study metabolism quantitatively. Unlike the concentrations of metabolites, the fluxes, which are the rates at which intracellular metabolites interconvert, are not directly measurable. MFA uses stable isotope labeled tracers to reveal information related to the fluxes. The conceptual idea of MFA is that in tracer experiments the isotope labeling patterns of intracellular metabolites are determined by the fluxes, therefore by measuring the labeling patterns we can infer the fluxes in the network. In this review, we will discuss the basic concept of MFA using a simplified upper glycolysis network as an example. We will show how the fluxes are reflected in the isotope labeling patterns. The central idea we wish to deliver is that under metabolic and isotopic steady-state the labeling pattern of a metabolite is the flux-weighted average of the substrates’ labeling patterns. As a result, MFA can tell the relative contributions of converging metabolic pathways only when these pathways make substrates in different labeling patterns for the shared product. This is the fundamental principle guiding the design of isotope labeling experiment for MFA including tracer selection. In addition, we will also discuss the basic biochemical assumptions of MFA, and we will show the flux-solving procedure and result evaluation. Finally, we will highlight the link between isotopically stationary and nonstationary flux analysis.

Gluconeogenesis (GNG) is de novo production of glucose from endogenous carbon sources. Although it is a commonly studied pathway, particularly in disease, there is a lack of consensus about substrate preference. Moreover, primary hepatocytes are the current gold standard for in vitro liver studies, but no direct comparison of substrate preference at physiological fasting concentrations has been performed. We show that mouse primary hepatocytes prefer glycerol to pyruvate/lactate in glucose production assays and ¹³C isotope tracing studies at the high concentrations commonly used in the literature, as well as at more relevant fasting, physiological concentrations. In addition, when glycerol, pyruvate/lactate, and glutamine are all present, glycerol is responsible for over 75% of all glucose carbons labeled. We also found that glycerol can induce a rate-limiting enzyme of GNG, glucose-6-phosphatase. Lastly, we suggest that glycerol is a better substrate than pyruvate to test in vivo production of glucose in fasting mice. In conclusion, glycerol is the major carbon source for GNG in vitro and in vivo and should be compared with other substrates when studying GNG in the context of metabolic disease states.

STUDY QUESTION: Are single nucleotide variants (SNVs) in Aurora kinases B and C (AURKB, AURKC) associated with risk of aneuploid conception?SUMMARY ANSWER: Two SNVs were found in patients with extreme aneuploid concepti rates with respect to their age; one variant, AURKC p.I79V, is benign, while another, AURKB p.L39P, is a potential gain-of-function mutant with increased efficiency in promoting chromosome alignment.WHAT IS KNOWN ALREADY: Maternal age does not always predict aneuploidy risk, and rare gene variants can be drivers of disease. The AURKB and AURKC regulate chromosome segregation, and are associated with reproductive impairments in mouse and human.STUDY DESIGN, SIZE, DURATION: An extreme phenotype sample selection scheme was performed for variant discovery. Ninety-six DNA samples were from young patients with higher than average embryonic aneuploidy rates and an additional 96 DNA samples were from older patients with lower than average aneuploidy rates.PARTICIPANTS/MATERIALS, SETTING, METHODS: Using the192 DNA samples, the coding regions of AURKB and AURKC were sequenced using next generation sequencing. To assess biological significance, we expressed complementary RNA encoding the human variants in mouse oocytes. Assays such as determining subcellular localization and assessing catalytic activity were performed to determine alterations in protein function during meiosis.MAIN RESULTS AND THE ROLE OF CHANCE: Ten SNVs were identified using three independent variant-calling methods. Two of the SNVs (AURKB p.L39P and AURKC p.I79V) were non-synonymous and identified by at least two variant-identification methods. The variant encoding AURKC p.I79V, identified in a young woman with a higher than average rate of aneuploid embryos, showed wild-type localization pattern and catalytic activity. On the other hand, the variant encoding AURKB p.L39P, identified in an older woman with lower than average rates of aneuploid embryos, increased the protein’s ability to regulate alignment of chromosomes at the metaphase plate. These experiments were repeated three independent times using 2–3 mice for each trial.LARGE SCALE DATA: N/A.LIMITATIONS, REASONS FOR CAUTION: Biological significance of the human variants was assessed in an in vitro mouse oocyte model where the variants are over-expressed. Therefore, the human protein may not function identically to the mouse homolog, or the same in mouse oocytes as in human oocytes. Furthermore, supraphysiological expression levels may not accurately reflect endogenous activity. Moreover, the evaluated variants were identified in one patient each, and no trial linking the SNV to pregnancy outcomes was conducted. Finally, the patient aneuploidy rates were established by performing comprehensive chromosome screening in blastocysts, and because of the link between female gamete aneuploidy giving rise to aneuploid embryos, we evaluate the role of the variants in Meiosis I. However, it is possible that the chromosome segregation mistake arose during Meiosis II or in mitosis in the preimplantation embryo. Their implications in human female meiosis and aneuploidy risk remain to be determined.WIDER IMPLICATIONS OF THE FINDINGS: The data provide evidence that gene variants exist in reproductively younger or advanced aged women that are predictive of the risk of producing aneuploid concepti in humans. Furthermore, a single amino acid in the N-terminus of AURKB is a gain-of-function mutant that could be protective of euploidy.STUDY FUNDING/COMPETING INTERESTS: This work was supported by a Research Grant from the American Society of Reproductive Medicine and support from the Charles and Johanna Busch Memorial Fund at Rutgers, the State University of NJ to K.S. and the Foundation for Embryonic Competence, Inc to N.T. The authors declare no conflicts of interest.