Oxygen regulation of TET enzymes.

Hypoxia has a significant impact on many physiological and pathological processes. Over the recent years, its role in modulation of epigenetic remodelling has also become clearer. In cancer, low oxygen environments and aberrant epigenomes often go hand in hand, and changes in DNA methylation are now commonly recognised as potential outcome indicators. TET (ten-eleven translocation) family enzymes are alpha-ketoglutarate-, iron- and oxygen-dependent DNA demethylases and are key players in these processes. Although TETs have historically been considered tumour suppressors, recent studies suggest that their functions in cancer might not be straightforward. Recently, inhibition of TETs has been reported to have positive impact in cancer immunotherapy and vaccination studies. This underlines the current interest in developing targeted pharmaceutical inhibitors of these enzymes. Here, we will survey the complexity of TET roles in cancer, and its hypoxic modulation, as well as highlight the potential of these enzymes as therapeutic targets

Replication stress and chromatin context link ATM activation to a role in DNA replication

ATM-mediated signaling in response to DNA damage is a barrier to tumorigenesis. Here we asked whether replication stress could also contribute to ATM signaling. We demonstrate that, in the absence of DNA damage, ATM responds to replication stress in a hypoxia-induced heterochromatin-like context. In certain hypoxic conditions, replication stress occurs in the absence of detectable DNA damage. Hypoxia also induces H3K9me3, a histone modification associated with gene repression and heterochromatin. Hypoxia-induced replication stress together with increased H3K9me3 leads to ATM activation. Importantly, ATM prevents the accumulation of DNA damage in hypoxia. Most significantly, we describe a stress-specific role for ATM in maintaining DNA replication rates in a background of increased H3K9me3. Furthermore, the ATM-mediated response to oncogene-induced replication stress is enhanced in hypoxic conditions. Together, these data indicate that hypoxia plays a critical role in the activation of the DNA damage response, therefore contributing to this barrier to tumorigenesis

Hypoxia-induced SETX links replication stress with the unfolded protein response.

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways

Lactate exposure shapes the metabolic and transcriptomic profile of CD8+ T cells

IntroductionCD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in local tissues can result in significant exposure of cytotoxic T cells to the lactate metabolite. Lactate has been known to act as an immunosuppressor, at least in part due to its association with tissue acidosis.MethodsTo dissect the role of the lactate anion, independently of pH, we performed phenotypical and metabolic assays, high-throughput RNA sequencing, and mass spectrometry, on primary cultures of murine or human CD8+ T cells exposed to high doses of pH-neutral sodium lactate.ResultsThe lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and can displace glucose as a carbon source. Activation in the presence of sodium lactate significantly alters the CD8+ T cell transcriptome, including the expression key effector differentiation markers such as granzyme B and interferon-gamma.DiscussionOur studies reveal novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells

RRM2B: An oxygen-requiring protein with a role in hypoxia

How tumor cells adapt and survive under hypoxia significantly impacts patient prognosis. We recently demonstrated that the oxygen-requiring ribonucleotide reductase (RNR) enzyme, which provides cells with deoxyribonucleotides, responds to limited oxygen availability by switching small subunits from RRM2 to RRM2B. This property of RNR is essential for hypoxic cell viability and therefore contributes to the most aggressive and therapy-resistant fraction of tumors

Vitamin B6 Metabolism Determines T Cell Anti-Tumor Responses.

Funder: Knut och Alice Wallenbergs StiftelseFunder: VetenskapsrådetFunder: BarncancerfondenFunder: CancerfondenTargeting T cell metabolism is an established method of immunomodulation. Following activation, T cells engage distinct metabolic programs leading to the uptake and processing of nutrients that determine cell proliferation and differentiation. Redirection of T cell fate by modulation of these metabolic programs has been shown to boost or suppress immune responses in vitro and in vivo. Using publicly available T cell transcriptomic and proteomic datasets we identified vitamin B6-dependent transaminases as key metabolic enzymes driving T cell activation and differentiation. Inhibition of vitamin B6 metabolism using the pyridoxal 5'-phosphate (PLP) inhibitor, aminoxyacetic acid (AOA), suppresses CD8+ T cell proliferation and effector differentiation in a dose-dependent manner. We show that pyridoxal phosphate phosphatase (PDXP), a negative regulator of intracellular vitamin B6 levels, is under the control of the hypoxia-inducible transcription factor (HIF1), a central driver of T cell metabolism. Furthermore, by adoptive transfer of CD8 T cells into a C57BL/6 mouse melanoma model, we demonstrate the requirement for vitamin B6-dependent enzyme activity in mediating effective anti-tumor responses. Our findings show that vitamin B6 metabolism is required for CD8+ T cell proliferation and effector differentiation in vitro and in vivo. Targeting vitamin B6 metabolism may therefore serve as an immunodulatory strategy to improve anti-tumor immunotherapy

Modified Hypoxia-Inducible Factor Expression in CD8+ T Cells Increases Antitumor Efficacy.

Adoptive transfer of antitumor cytotoxic T cells is an emerging form of cancer immunotherapy. A key challenge to expanding the utility of adoptive cell therapies is how to enhance the survival and function of the transferred T cells. Immune-cell survival requires adaptation to different microenvironments and particularly to the hypoxic milieu of solid tumors. The hypoxia-inducible factor (HIF) transcription factors are an essential aspect of this adaptation. In this study, we undertook experiments to define structural determinants of HIF that potentiate antitumor efficacy in cytotoxic T cells. We first created retroviral vectors to deliver ectopic expression of HIF1α and HIF2α in mouse CD8+ T cells, together or individually and with or without sensitivity to the oxygen-dependent HIFα inhibitors Von Hippel-Lindau and factor-inhibiting HIF (FIH). HIF2α, but not HIF1α, drove broad transcriptional changes in CD8+ T cells, resulting in increased cytotoxic differentiation and cytolytic function against tumor targets. A specific mutation replacing the hydroxyl group-acceptor site for FIH in HIF2α gave rise to the most effective antitumor T cells after adoptive transfer in vivo In addition, codelivering an FIH-insensitive form of HIF2α with an anti-CD19 chimeric antigen receptor greatly enhanced cytolytic function of human CD8+ T cells against lymphoma cells both in vitro and in a xenograft adoptive transfer model. These experiments point to a means to increase the antitumor efficacy of therapeutic CD8+ T cells via ectopic expression of the HIF transcription factor.See related Spotlight on p. 364

Lactate exposure shapes the metabolic and transcriptomic profile of CD8+ T cells.

Funder: Wellcome TrustFunder: Cancer Research UKFunder: Medical Research CouncilINTRODUCTION: CD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in local tissues can result in significant exposure of cytotoxic T cells to the lactate metabolite. Lactate has been known to act as an immunosuppressor, at least in part due to its association with tissue acidosis. METHODS: To dissect the role of the lactate anion, independently of pH, we performed phenotypical and metabolic assays, high-throughput RNA sequencing, and mass spectrometry, on primary cultures of murine or human CD8+ T cells exposed to high doses of pH-neutral sodium lactate. RESULTS: The lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and can displace glucose as a carbon source. Activation in the presence of sodium lactate significantly alters the CD8+ T cell transcriptome, including the expression key effector differentiation markers such as granzyme B and interferon-gamma. DISCUSSION: Our studies reveal novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells

The Debaryomyces hansenii carboxylate transporters Jen1 homologues are functional in Saccharomyces cerevisiae

We have functionally characterized the four Saccharomyces cerevisiae (Sc) Jen1 homologues of Debaryomyces hansenii (Dh) by heterologous expression in S. cerevisiae. Debaryomyces hansenii cells display mediated transport for the uptake of lactate, acetate, succinate and malate. DHJEN genes expression was detected by RT-PCR in all carbon sources assayed, namely lactate, succinate, citrate, glycerol and glucose. The heterologous expression in the S. cerevisiae W303-1A jen1 Lambda ady2 Lambda strain demonstrated that the D. hansenii JEN genes encode four carboxylate transporters. DH27 gene encodes an acetate transporter (K-m 0.94 +/- 0.17 mM; V-max 0.43 +/- 0.03 nmol s(-1) mg(-1)), DH17 encodes a malate transporter (K-m 0.27 +/- 0.04 mM; V-max 0.11 +/- 0.01 nmol s(-1) mg(-1)) and both DH18 and DH24 encode succinate transporters with the following kinetic parameters, respectively, K-m 0.31 +/- 0.06 mM; V-max 0.83 +/- 0.04 nmol s(-1) mg(-1)and K-m 0.16 +/- 0.02 mM; V-max 0.19 +/- 0.02 nmol s(-1) mg(-1). Surprisingly, no lactate transporter was found, although D. hansenii presents a mediated transport for this acid. This work advanced the current knowledge on yeast carboxylate transporters by characterizing four new plasma membrane transporters in D. hansenii.This work was supported by FCT I. P. through the strategic funding UID/BIA/04050/2013 and the project PTDC/BIA-MIC/5184/2014. DR and ISS acknowledge FCT for the SFRH/BD/96166/2013 and SFRH/BPD/101016/2014 grants.info:eu-repo/semantics/publishedVersio

Lactate exposure shapes the metabolic and transcriptomic profile of CD8+ T cells.

Peer reviewed: TrueINTRODUCTION: CD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in local tissues can result in significant exposure of cytotoxic T cells to the lactate metabolite. Lactate has been known to act as an immunosuppressor, at least in part due to its association with tissue acidosis. METHODS: To dissect the role of the lactate anion, independently of pH, we performed phenotypical and metabolic assays, high-throughput RNA sequencing, and mass spectrometry, on primary cultures of murine or human CD8+ T cells exposed to high doses of pH-neutral sodium lactate. RESULTS: The lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and can displace glucose as a carbon source. Activation in the presence of sodium lactate significantly alters the CD8+ T cell transcriptome, including the expression key effector differentiation markers such as granzyme B and interferon-gamma. DISCUSSION: Our studies reveal novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells