Search

Abstract Dimethyl fumarate is an ester from the Krebs cycle intermediate fumarate. This drug is approved and currently used for the treatment of psoriasis and multiple sclerosis, and its anti-angiogenic activity was reported some years ago. Due to the current clinical relevance of this compound and the recently manifested importance of endothelial cell metabolism on the angiogenic switch, we wanted to elucidate whether dimethyl fumarate has an effect on energetic metabolism of endothelial cells. Different experimental approximations were performed in endothelial cells, including proteomics, isotope tracing and metabolomics experimental approaches, in this work we studied the possible role of dimethyl fumarate in endothelial cell energetic metabolism. We demonstrate for the first time that dimethyl fumarate promotes glycolysis and diminishes cell respiration in endothelial cells, which could be a consequence of a down-regulation of serine and glycine synthesis through inhibition of PHGDH activity in these cells. Dimethyl fumarate alters the energetic metabolism of endothelial cells in vitro and in vivo through an unknown mechanism, which could be the cause or the consequence of its pharmacological activity. This new discovery on the targets of this compound could open a new field of study regarding the mechanism of action of dimethyl fumarate.

Summary: Fatty acid synthase (FASN) maintains de novo lipogenesis (DNL) to support rapid growth in most proliferating cancer cells. Lipogenic acetyl-coenzyme A (CoA) is primarily produced from carbohydrates but can arise from glutamine-dependent reductive carboxylation. Here, we show that reductive carboxylation also occurs in the absence of DNL. In FASN-deficient cells, reductive carboxylation is mainly catalyzed by isocitrate dehydrogenase-1 (IDH1), but IDH1-generated cytosolic citrate is not utilized for supplying DNL. Metabolic flux analysis (MFA) shows that FASN deficiency induces a net cytosol-to-mitochondria citrate flux through mitochondrial citrate transport protein (CTP). Previously, a similar pathway has been shown to mitigate detachment-induced oxidative stress in anchorage-independent tumor spheroids. We further report that tumor spheroids show reduced FASN activity and that FASN-deficient cells acquire resistance to oxidative stress in a CTP- and IDH1-dependent manner. Collectively, these data indicate that by inducing a cytosol-to-mitochondria citrate flux, anchorage-independent malignant cells can gain redox capacity by trading off FASN-supported rapid growth.

Malonyl-CoA-acyl carrier protein transacylase (MCAT) is an enzyme involved in mitochondrial fatty acid synthesis (mtFAS) and catalyzes the transfer of the malonyl moiety of malonyl-CoA to the mitochondrial acyl carrier protein (ACP). Previously, we showed that loss-of-function of mtFAS genes, including Mcat, is associated with severe loss of electron transport chain (ETC) complexes in mouse immortalized skeletal myoblasts (Nowinski et al., 2020). Here, we report a proband presenting with hypotonia, failure to thrive, nystagmus, and abnormal brain MRI findings. Using whole exome sequencing, we identified biallelic variants in MCAT. Protein levels for NDUFB8 and COXII, subunits of complex I and IV respectively, were markedly reduced in lymphoblasts and fibroblasts, as well as SDHB for complex II in fibroblasts. ETC enzyme activities were decreased in parallel. Re-expression of wild-type MCAT rescued the phenotype in patient fibroblasts. This is the first report of a patient with MCAT pathogenic variants and combined oxidative phosphorylation deficiency.

Mitochondrial electron transport chain (ETC) dysfunction due to mutations in the nuclear or mitochondrial genome is a common cause of metabolic disease in humans and displays striking tissue specificity depending on the affected gene. The mechanisms underlying tissue-specific phenotypes are not understood. Complex I (cI) is classically considered the entry point for electrons into the ETC, and in vitro experiments indicate that cI is required for basal respiration and maintenance of the NAD+/NADH ratio, an indicator of cellular redox status. This finding has largely not been tested in vivo. Here, we report that mitochondrial complex I is dispensable for homeostasis of the adult mouse liver; animals with hepatocyte-specific loss of cI function display no overt phenotypes or signs of liver damage, and maintain liver function, redox and oxygen status. Further analysis of cI-deficient livers did not reveal significant proteomic or metabolic changes, indicating little to no compensation is required in the setting of complex I loss. In contrast, complex IV (cIV) dysfunction in adult hepatocytes results in decreased liver function, impaired oxygen handling, steatosis, and liver damage, accompanied by significant metabolomic and proteomic perturbations. Our results support a model whereby complex I loss is tolerated in the mouse liver because hepatocytes use alternative electron donors to fuel the mitochondrial ETC.

Ling Cai et al. used transcriptomic profiling data of healthy lung, patient-derived small cell lung cancer cell lines, xenografts, and primary tumors to examine a link between neuroendocrine (NE) signatures and immune gene expression. Their findings suggest that cell-autonomous immune gene repression is a shared feature between healthy and tumor cells of NE lineage and may influence tumor-immune cell interaction and response to immunotherapy.

Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels.

Summary: Squamous cell carcinoma (SCC), a malignancy arising across multiple anatomical sites, is responsible for significant cancer mortality due to insufficient therapeutic options. Here, we identify exceptional glucose reliance among SCCs dictated by hyperactive GLUT1-mediated glucose influx. Mechanistically, squamous lineage transcription factors p63 and SOX2 transactivate the intronic enhancer cluster of SLC2A1. Elevated glucose influx fuels generation of NADPH and GSH, thereby heightening the anti-oxidative capacity in SCC tumors. Systemic glucose restriction by ketogenic diet and inhibiting renal glucose reabsorption with SGLT2 inhibitor precipitate intratumoral oxidative stress and tumor growth inhibition. Furthermore, reduction of blood glucose lowers blood insulin levels, which suppresses PI3K/AKT signaling in SCC cells. Clinically, we demonstrate a robust correlation between blood glucose concentration and worse survival among SCC patients. Collectively, this study identifies the exceptional glucose reliance of SCC and suggests its candidacy as a highly vulnerable cancer type to be targeted by systemic glucose restriction. : Hsieh et al. show that p63 and SOX2 cooperate to induce enhanced GLUT1 expression, driving a critical reliance on glucose, in squamous cell carcinomas. Systemic glucose restriction by ketogenic diet or SGLT2 inhibition attenuates squamous tumor progression by disrupting redox homeostasis and insulin/AKT pathways in vivo. Keywords: p63, SOX2, GLUT1, squamous cell carcinoma, glucose restriction, ketogenic diet, SGLT2

Summary: Autophagy is a cell survival process essential for the regulation of immune responses to infections. However, the role of T cell autophagy in anti-tumor immunity is less clear. Here, we demonstrate a cell-autonomous role for autophagy in the regulation of CD8+ T-cell-mediated control of tumors. Mice deficient for the essential autophagy genes Atg5, Atg14, or Atg16L1 display a dramatic impairment in the growth of syngeneic tumors. Moreover, T cells lacking Atg5 have a profound shift to an effector memory phenotype and produce greater amounts of interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α). Mechanistically, Atg5−/− CD8+ T cells exhibit enhanced glucose metabolism that results in alterations in histone methylation, increases in H3K4me3 density, and transcriptional upregulation of both metabolic and effector target genes. Nonetheless, glucose restriction is sufficient to suppress Atg5-dependent increases in effector function. Thus, autophagy-dependent changes in CD8+ T cell metabolism directly regulate anti-tumor immunity. : DeVorkin et al. show that loss of autophagy enhances CD8+ T-cell-mediated rejection of tumors. Mechanistically, suppression of autophagy shifts T cells to a glycolytic phenotype and causes a reduction in S-adenosylmethionine. As a consequence, autophagy-deficient T cells transcriptionally reprogram immune response genes to an effector memory state. Keywords: autophagy, CD8+ T cells, anti-tumor immunity, glycolysis, lactate, SAM, methylation

Summary: Inborn errors of metabolism (IEMs) link metabolic defects to human phenotypes. Modern genomics has accelerated IEM discovery, but assessing the impact of genomic variants is still challenging. Here, we integrate genomics and metabolomics to identify a cause of lactic acidosis and epilepsy. The proband is a compound heterozygote for variants in LIPT1, which encodes the lipoyltransferase required for 2-ketoacid dehydrogenase (2KDH) function. Metabolomics reveals abnormalities in lipids, amino acids, and 2-hydroxyglutarate consistent with loss of multiple 2KDHs. Homozygous knockin of a LIPT1 mutation reduces 2KDH lipoylation in utero and results in embryonic demise. In patient fibroblasts, defective 2KDH lipoylation and function are corrected by wild-type, but not mutant, LIPT1 alleles. Isotope tracing reveals that LIPT1 supports lipogenesis and balances oxidative and reductive glutamine metabolism. Altogether, the data extend the role of LIPT1 in metabolic regulation and demonstrate how integrating genomics and metabolomics can uncover broader aspects of IEM pathophysiology. : Ni et al. investigate human LIPT1 deficiency, which results in developmental delay, epilepsy, and broad metabolic abnormalities, including lactic acidosis, L- and D-2-hydroxyglutaric aciduria, defective lipogenesis, and an altered balance between oxidative and reductive glutamine metabolism. Keywords: inborn errors of metabolism, metabolomics, genomics, lactic acidosis, epilepsy,developmental delay, 2-ketoacid dehydrogenase, lipoylation, lipogenesis, fatty acid oxidation

KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and β-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and β-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis in vivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain.

Summary: Pathways underlying metabolic reprogramming in cancer remain incompletely understood. We identify the transmembrane serine protease TMPRSS11B as a gene that promotes transformation of immortalized human bronchial epithelial cells (HBECs). TMPRSS11B is upregulated in human lung squamous cell carcinomas (LSCCs), and high expression is associated with poor survival of non-small cell lung cancer patients. TMPRSS11B inhibition in human LSCCs reduces transformation and tumor growth. Given that TMPRSS11B harbors an extracellular (EC) protease domain, we hypothesized that catalysis of a membrane-bound substrate modulates tumor progression. Interrogation of a set of soluble receptors revealed that TMPRSS11B promotes solubilization of Basigin, an obligate chaperone of the lactate monocarboxylate transporter MCT4. Basigin release mediated by TMPRSS11B enhances lactate export and glycolytic metabolism, thereby promoting tumorigenesis. These findings establish an oncogenic role for TMPRSS11B and provide support for the development of therapies that target this enzyme at the surface of cancer cells. : Updegraff et al. show that transmembrane protease TMPRSS11B is upregulated in lung squamous cell carcinoma, where it interacts with MCT4 and its obligate chaperone Basigin. TMPRSS11B catalytic activity promotes Basigin solubilization, which enhances lactate export and glycolytic metabolism, thereby promoting tumorigenesis. Keywords: TMPRSS11B, lung squamous cell carcinoma, MCT4, Basigin, lactate export, lung cancer, transmembrane serine protease, glycolytic metabolism, transposon mutagenesis, CRISPR-mediated genome editing

KRAS mutation, which occurs in ∼95% of pancreatic ductal adenocarcinoma (PDA), has been shown to program tumor metabolism. MCT4 is highly upregulated in a subset of PDA with a glycolytic gene expression program and poor survival. Models with high levels of MCT4 preferentially employ glycolytic metabolism. Selectively in such “addicted” models, MCT4 attenuation compromised glycolytic flux with compensatory induction of oxidative phosphorylation and scavenging of metabolites by macropinocytosis and autophagy. In spite of these adaptations, MCT4 depletion induced cell death characterized by elevated reactive oxygen species and metabolic crisis. Cell death induced by MCT4-depletion was augmented by inhibition of compensatory pathways. In xenograft models, MCT4 had a significant impact on tumor metabolism and was required for rapid tumor growth. Together, these findings illustrate the metabolic diversity of PDA described by MCT4, delineate pathways through which this lactate transporter supports cancer growth, and demonstrate that PDA can be rationally targeted based on metabolic addictions.

Mammalian cells generate citrate by decarboxylating pyruvate in the mitochondria to supply the tricarboxylic acid (TCA) cycle. In contrast, hypoxia and other impairments of mitochondrial function induce an alternative pathway that produces citrate by reductively carboxylating α-ketoglutarate (AKG) via NADPH-dependent isocitrate dehydrogenase (IDH). It is unknown how cells generate reducing equivalents necessary to supply reductive carboxylation in the setting of mitochondrial impairment. Here, we identified shared metabolic features in cells using reductive carboxylation. Paradoxically, reductive carboxylation was accompanied by concomitant AKG oxidation in the TCA cycle. Inhibiting AKG oxidation decreased reducing equivalent availability and suppressed reductive carboxylation. Interrupting transfer of reducing equivalents from NADH to NADPH by nicotinamide nucleotide transhydrogenase increased NADH abundance and decreased NADPH abundance while suppressing reductive carboxylation. The data demonstrate that reductive carboxylation requires bidirectional AKG metabolism along oxidative and reductive pathways, with the oxidative pathway producing reducing equivalents used to operate IDH in reverse.

Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels. We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC) perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity. Additionally, we identified a mitochondrial adenylate kinase (AK4) that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.

Metformin is a biguanide widely prescribed to treat Type II diabetes that has gained interest as an antineoplastic agent. Recent work suggests that metformin directly antagonizes cancer cell growth through its actions on complex I of the mitochondrial electron transport chain (ETC). However, the mechanisms by which metformin arrests cancer cell proliferation remain poorly defined. Here we demonstrate that the metabolic checkpoint kinases AMP-activated protein kinase (AMPK) and LKB1 are not required for the antiproliferative effects of metformin. Rather, metformin inhibits cancer cell proliferation by suppressing mitochondrial-dependent biosynthetic activity. We show that in vitro metformin decreases the flow of glucose- and glutamine-derived metabolic intermediates into the Tricarboxylic Acid (TCA) cycle, leading to reduced citrate production and de novo lipid biosynthesis. Tumor cells lacking functional mitochondria maintain lipid biosynthesis in the presence of metformin via glutamine-dependent reductive carboxylation, and display reduced sensitivity to metformin-induced proliferative arrest. Our data indicate that metformin inhibits cancer cell proliferation by suppressing the production of mitochondrial-dependent metabolic intermediates required for cell growth, and that metabolic adaptations that bypass mitochondrial-dependent biosynthesis may provide a mechanism of tumor cell resistance to biguanide activity.