3-hydroxykynurenine is a ROS-inducing cytotoxic tryptophan metabolite that disrupts the TCA cycle.

Tryptophan is an essential amino acid that is extensively characterized as a regulator of cellular function through its metabolism by indoleamine 2,3-deoxygenase (IDO) into the kynurenine pathway. However, despite decades of research on tryptophan metabolism, the metabolic regulatory roles of it and its metabolites are not well understood. To address this, we performed an activity metabolomics screen of tryptophan and most of its known metabolites in cell culture. We discovered that treatment of human colon cancer cells (HCT116) with 3-hydroxykynurenine (3-HK), a metabolite of kynurenine, potently disrupted TCA cycle function. Citrate and aconitate levels were increased, while isocitrate and all downstream TCA metabolites were decreased, suggesting decreased aconitase function. We hypothesized that 3HK or one of its metabolites increased reactive oxygen species (ROS) and inhibited aconitase activity. Accordingly, we observed almost complete depletion of reduced glutathione and a decrease in total glutathione levels. We observed a dose-dependent decrease in cell viability after 48 hours of 3HK treatment. These data suggest that raising the intracellular levels of 3HK could be sufficient to induce ROS-mediated apoptosis. We modulated the intracellular levels of 3HK by combined induction of IDO and knockdown of kynureninase (KYNU) in HCT116 cells. Cell viability decreased significantly after 48 hours of KYNU knockdown compared to controls, which was accompanied by increased ROS production and Annexin V staining revealing apoptosis. Finally, we identify xanthommatin production from 3-HK as a candidate radical-producing, cytotoxic mechanism. Our work indicates that KYNU may be a target for disrupting tryptophan metabolism. Interestingly, many cancers exhibit overexpression of IDO, providing a cancer-specific metabolic vulnerability that could be exploited by KYNU inhibition.

Isolated Effects of Plasma Freezing versus Thawing on Metabolite Stability.

Freezing and thawing plasma samples is known to perturb metabolite stability. However, no study has systematically tested how different freezing and thawing methods affect plasma metabolite levels. The objective of this study was to isolate the effects of freezing from thawing on mouse plasma metabolite levels, by comparing a matrix of freezing and thawing conditions through 10 freeze-thaw cycles. We tested freezing with liquid nitrogen (LN2), at -80 °C, or at -20 °C, and thawing quickly in room temperature water or slowly on ice. Plasma samples were extracted and the relative abundance of 87 metabolites was obtained via liquid chromatography-mass spectrometry (LC-MS). Observed changes in metabolite abundance by treatment group correlated with the amount of time it took for samples to freeze or thaw. Thus, snap-freezing with LN2 and quick-thawing with water led to minimal changes in metabolite levels. Conversely, samples frozen at -20 °C exhibited the most changes in metabolite levels, likely because freezing required about 4 h, versus freezing instantaneously in LN2. Overall, our results show that plasma samples subjected to up to 10 cycles of LN2 snap-freezing with room temperature water quick-thawing exhibit remarkable metabolomic stability.

Malic Enzyme 1 Absence in Synovial Sarcoma Shifts Antioxidant System Dependence and Increases Sensitivity to Ferroptosis Induction with ACXT-3102.

PURPOSE: To investigate the metabolism of synovial sarcoma (SS) and elucidate the effect of malic enzyme 1 absence on SS redox homeostasis. EXPERIMENTAL DESIGN: ME1 expression was measured in SS clinical samples, SS cell lines, and tumors from an SS mouse model. The effect of ME1 absence on glucose metabolism was evaluated utilizing Seahorse assays, metabolomics, and C13 tracings. The impact of ME1 absence on SS redox homeostasis was evaluated by metabolomics, cell death assays with inhibitors of antioxidant systems, and measurements of intracellular reactive oxygen species (ROS). The susceptibility of ME1-null SS to ferroptosis induction was interrogated in vitro and in vivo. RESULTS: ME1 absence in SS was confirmed in clinical samples, SS cell lines, and an SS tumor model. Investigation of SS glucose metabolism revealed that ME1-null cells exhibit higher rates of glycolysis and higher flux of glucose into the pentose phosphate pathway (PPP), which is necessary to produce NADPH. Evaluation of cellular redox homeostasis demonstrated that ME1 absence shifts dependence from the glutathione system to the thioredoxin system. Concomitantly, ME1 absence drives the accumulation of ROS and labile iron. ROS and iron accumulation enhances the susceptibility of ME1-null cells to ferroptosis induction with inhibitors of xCT (erastin and ACXT-3102). In vivo xenograft models of ME1-null SS demonstrate significantly increased tumor response to ACXT-3102 compared with ME1-expressing controls. CONCLUSIONS: These findings demonstrate the translational potential of targeting redox homeostasis in ME1-null cancers and establish the preclinical rationale for a phase I trial of ACXT-3102 in SS patients. See related commentary by Subbiah and Gan, p. 3408.

Mitochondrial Pyruvate Carrier Function in Health and Disease across the Lifespan.

As a nodal mediator of pyruvate metabolism, the mitochondrial pyruvate carrier (MPC) plays a pivotal role in many physiological and pathological processes across the human lifespan, from embryonic development to aging-associated neurodegeneration. Emerging research highlights the importance of the MPC in diverse conditions, such as immune cell activation, cancer cell stemness, and dopamine production in Parkinson's disease models. Whether MPC function ameliorates or contributes to disease is highly specific to tissue and cell type. Cell- and tissue-specific differences in MPC content and activity suggest that MPC function is tightly regulated as a mechanism of metabolic, cellular, and organismal control. Accordingly, recent studies on cancer and diabetes have identified protein-protein interactions, post-translational processes, and transcriptional factors that modulate MPC function. This growing body of literature demonstrates that the MPC and other mitochondrial carriers comprise a versatile and dynamic network undergirding the metabolism of health and disease.

Disrupting Mitochondrial Pyruvate Uptake Directs Glutamine into the TCA Cycle away from Glutathione Synthesis and Impairs Hepatocellular Tumorigenesis.

Hepatocellular carcinoma (HCC) is a devastating cancer increasingly caused by non-alcoholic fatty liver disease (NAFLD). Disrupting the liver Mitochondrial Pyruvate Carrier (MPC) in mice attenuates NAFLD. Thus, we considered whether liver MPC disruption also prevents HCC. Here, we use the N-nitrosodiethylamine plus carbon tetrachloride model of HCC development to test how liver-specific MPC knock out affects hepatocellular tumorigenesis. Our data show that liver MPC ablation markedly decreases tumorigenesis and that MPC-deficient tumors transcriptomically downregulate glutathione metabolism. We observe that MPC disruption and glutathione depletion in cultured hepatomas are synthetically lethal. Stable isotope tracing shows that hepatocyte MPC disruption reroutes glutamine from glutathione synthesis into the tricarboxylic acid (TCA) cycle. These results support a model where inducing metabolic competition for glutamine by MPC disruption impairs hepatocellular tumorigenesis by limiting glutathione synthesis. These findings raise the possibility that combining MPC disruption and glutathione stress may be therapeutically useful in HCC and additional cancers.