Petros A. Tyrakis

S -2-hydroxyglutarate regulates CD8 + T-lymphocyte fate

R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8+ T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8+ T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8+ T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic–epigenetic axis, to immune fate and function.

A Late-Stage Intermediate in Salinomycin Biosynthesis Is Revealed by Specific Mutation in the Biosynthetic Gene Cluster

Salinomycin (1) from Streptomyces albus DSM 41398 is an antibiotic polyether ionophore with a complex tricyclic bispiroacetal core structure that selectively binds K ions and transports them across cell membranes, thus leading to cell death. The therapeutic use of salinomycin is limited by its toxicity, but it is widely used in animal husbandry as a coccidiostat. Salinomycin has attracted strong renewed interest owing to its potent and selective activity against cancer stem cells and cancer cell lines. Engineering the biosynthetic pathway to salinomycin offers an attractive route to novel and potentially useful analogues of this complex molecule. We have cloned and analysed the salinomycin gene cluster from S. albus DSM 41398, and found that the polyketide chain is synthesised on an assembly line of nine polyketide synthase (PKS) multienzymes. We have also initiated targeted deletion of the genes that control oxidative cyclisation so as to probe the mechanism of polyether ring formation. One such mutant produces a novel polyketide diene whose structure provides the first evidence for the likely order of key steps in the biosynthesis. Polyether ionophores make up a particularly numerous subclass of complex polyketide antibiotics. They are synthesised on canonical modular PKS assembly-line multienzymes, in which each module houses fatty acid synthase-like enzyme domains, and there is colinearity between the order (and enzyme domain content) of successive modules and the chemistry of the product. The antibiotic polyethers adopt a characteristic conformation in which multiple oxygen atoms provide ligands for a centrally held specific cation, whereas the external surface is exclusively nonpolar. A general model for the biosynthesis of polyethers has been proposed in which the PKS produces a linear polyketide chain containing two or more trans or E double bonds. Stereoselective epoxidation of these double bonds leads to a polyepoxide, whose ring opening (in a series of controlled SN2-like reactions) generates the characteristic rings of the polyether. This model is strongly supported by the results of extensive genetic studies on the cloned and characterised biosynthetic gene clusters for monensin A and several other polyethers. Comparison of these gene clusters has revealed the presence of a conserved set of genes that are a hallmark of polyether biosynthesis: the monC family b] that encode a flavin-linked epoxidase, and the monB family c] that encode novel epoxide hydrolase/cyclase enzymes. It also appears that all stages of the biosynthesis take place while the intermediates are tethered either to a discrete acyl carrier protein (ACP) or to an ACP domain within the PKS. 11] However, important aspects of the oxidative cyclisation process remain undefined, especially the molecular basis for the exquisite stereospecificity and stereoselectivity of the process. Salinomycin, and related polyether metabolites produced by S. albus DSM 41398, possess an unusual and particularly complex tricyclic

An HIF-1α/VEGF-A Axis in Cytotoxic T Cells Regulates Tumor Progression

Summary Cytotoxic T cells infiltrating tumors are thought to utilize HIF transcription factors during adaptation to the hypoxic tumor microenvironment. Deletion analyses of the two key HIF isoforms found that HIF-1α, but not HIF-2α, was essential for the effector state in CD8+ T cells. Furthermore, loss of HIF-1α in CD8+ T cells reduced tumor infiltration and tumor cell killing, and altered tumor vascularization. Deletion of VEGF-A, an HIF target gene, in CD8+ T cells accelerated tumorigenesis while also altering vascularization. Analyses of human breast cancer showed inverse correlations between VEGF-A expression and CD8+ T cell infiltration, and a link between T cell infiltration and vascularization. These data demonstrate that the HIF-1α/VEGF-A axis is an essential aspect of tumor immunity.

Mycobacterium tuberculosis Dihydrofolate Reductase Reveals Two Conformational States and a Possible Low Affinity Mechanism to Antifolate Drugs.

Inhibition of the biosynthesis of tetrahydrofolate (THF) has long been a focus in the treatment of both cancer and infectious diseases. Dihydrofolate reductase (DHFR), which catalyzes the last step, is one of the most thoroughly explored targets of this pathway, but there are no DHFR inhibitors used for tuberculosis treatment. Here, we report a structural, site-directed mutagenesis and calorimetric analysis of Mycobacterium tuberculosis DHFR (MtDHFR) in complex with classical DHFR inhibitors. Our study provides insights into the weak inhibition of MtDHFR by trimethoprim and other antifolate drugs, such as pyrimethamine and cycloguanil. The construction of the mutant Y100F, together with calorimetric studies, gives insights into low affinity of MtDHFR for classical DHFR inhibitors. Finally, the structures of MtDHFR in complex with pyrimethamine and cycloguanil define important interactions in the active site and provide clues to the more effective design of antibiotics targeted against MtDHFR.

Fumarate Hydratase Loss Causes Combined Respiratory Chain Defects

Summary Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid (TCA) cycle mutated in hereditary and sporadic cancers. Despite recent advances in understanding its role in tumorigenesis, the effects of FH loss on mitochondrial metabolism are still unclear. Here, we used mouse and human cell lines to assess mitochondrial function of FH-deficient cells. We found that human and mouse FH-deficient cells exhibit decreased respiration, accompanied by a varying degree of dysfunction of respiratory chain (RC) complex I and II. Moreover, we show that fumarate induces succination of key components of the iron-sulfur cluster biogenesis family of proteins, leading to defects in the biogenesis of iron-sulfur clusters that affect complex I function. We also demonstrate that suppression of complex II activity is caused by product inhibition due to fumarate accumulation. Overall, our work provides evidence that the loss of a single TCA cycle enzyme is sufficient to cause combined RC activity dysfunction.

The Factor Inhibiting HIF Asparaginyl Hydroxylase Regulates Oxidative Metabolism and Accelerates Metabolic Adaptation to Hypoxia

Summary Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia.

Modelling pulmonary microthrombosis coupled to metastasis: distinct effects of thrombogenesis on tumorigenesis

ABSTRACT Thrombosis can cause localized ischemia and tissue hypoxia, and both of these are linked to cancer metastasis. Vascular micro-occlusion can occur as a result of arrest of circulating tumour cells in small capillaries, giving rise to microthrombotic events that affect flow, creating localized hypoxic regions. To better understand the association between metastasis and thrombotic events, we generated an experimental strategy whereby we modelled the effect of microvascular occlusion in metastatic efficiency by using inert microbeads to obstruct lung microvasculature before, during and after intravenous tumour cell injection. We found that controlled induction of a specific number of these microthrombotic insults in the lungs caused an increase in expression of the hypoxia-inducible transcription factors (HIFs), a pro-angiogenic and pro-tumorigenic environment, as well as an increase in myeloid cell infiltration. Induction of pulmonary microthrombosis prior to introduction of tumour cells to the lungs had no effect on tumorigenic success, but thrombosis at the time of tumour cell seeding increased number and size of tumours in the lung, and this effect was strikingly more pronounced when the micro-occlusion occurred on the day following introduction of tumour cells. The tumorigenic effect of microbead treatment was seen even when thrombosis was induced five days after tumour cell injection. We also found positive correlations between thrombotic factors and expression of HIF2α in human tumours. The model system described here demonstrates the importance of thrombotic insult in metastatic success and can be used to improve understanding of thrombosis-associated tumorigenesis and its treatment. Summary: Induction of pulmonary microthrombosis by three distinct methods enhances HIF-a expression and tumour formation; increases in tumorigenesis that are induced by these thrombotic insults occur in a time- and mode-dependent manner.

Abstract A19: Pulmonary microthrombosis enhances tumorigenesis via myeloid hypoxia-inducible factors

Thrombosis causes local blood flow restriction and tissue hypoxia, and both of these are associated with tumor cell metastasis. To better understand the regulation of thrombosis-induced metastasis, we created a model incorporating elements of both processes. Controlled induction of pulmonary microthrombosis caused an increase in expression of first hypoxia-inducible factor (HIF)1α, and subsequently HIF2α. Induction of thrombosis before the introduction of tumor cells to venous circulation had no effect on pulmonary tumor number or size; but thrombosis at the time of tumor cell seeding increased number and size of tumors in the lung. Thrombosis on the day after seeding of tumor cells caused an even greater increase in tumor number and size, and this effect persisted until even when thrombosis was induced five days after the introduction of tumor cells into the blood. Experiments on myeloid HIF1α or HIF2α knockout mice demonstrated that loss of either HIF1α or HIF2α eliminated the advantage given to pulmonary tumorigenesis by thrombotic insult. In primary human tumours, markers of thrombosis were positively correlated with expression of the 2 HIFα isoforms. These data demonstrate the importance of microthrombosis in a novel model of metastasis and the essential role of the myeloid cell-specific HIFα response in mediating this process. Citation Format: Colin E. Evans, Asis Palazon, Jingwei Sim, Petros A. Tyrakis, Par-Ola Bendahl, Mattias Belting, Helene Rundqvist, Cristina Branco, Randall S. Johnson. Pulmonary microthrombosis enhances tumorigenesis via myeloid hypoxia-inducible factors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A19.