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Blood-supply boost in ischaemia The cellular oxygen-sensing enyzme PHD2 uses oxygen to degrade hypoxia-inducible factor 1α. Using a mouse model of limb ischaemia, Takeda et al . link oxygen sensing through PHD2 to macrophage phenotype and arteriogenesis. They show that myeloid-cell-specific PHD2 haplodeficiency results in improved arteriogenesis after ischaemia owing to hyperactivation of NFκB signalling in the macrophages and the promotion of an M2-like repair phenotype. This suggests that PHD2 inhibition might promote collateral vascularization in patients who are at risk of limb or heart ischaemia. PHD2 serves as an oxygen sensor that rescues blood supply by regulating vessel formation and shape in case of oxygen shortage 1 , 2 , 3 , 4 , 5 . However, it is unknown whether PHD2 can influence arteriogenesis. Here we studied the role of PHD2 in collateral artery growth by using hindlimb ischaemia as a model, a process that compensates for the lack of blood flow in case of major arterial occlusion 6 , 7 , 8 . We show that Phd2 (also known as Egln1 ) haplodeficient ( Phd2 +/− ) mice displayed preformed collateral arteries that preserved limb perfusion and prevented tissue necrosis in ischaemia. Improved arteriogenesis in Phd2 +/− mice was due to an expansion of tissue-resident, M2-like macrophages 9 , 10 and their increased release of arteriogenic factors, leading to enhanced smooth muscle cell (SMC) recruitment and growth. Both chronic and acute deletion of one Phd2 allele in macrophages was sufficient to skew their polarization towards a pro-arteriogenic phenotype. Mechanistically, collateral vessel preconditioning relied on the activation of canonical NF-κB pathway in Phd2 +/− macrophages. These results unravel how PHD2 regulates arteriogenesis and artery homeostasis by controlling a specific differentiation state in macrophages and suggest new treatment options for ischaemic disorders.

The nine identified human homologues of E . coli AlkB 2-oxoglutarate (2OG) and Fe(II)-dependent dioxygenase, ALKBH1-8 and FTO, display different substrate specificities and diverse biological functions. Here we discovered the combined overexpression of members of the ALKBH family in head and neck squamous cell carcinomas (HNSCC). We found direct correlation of ALKBH3 and FTO expression with primary HNSCC tumor size. We observed unidentified thus far cytoplasmic localization of ALKBH2 and 5 in HNSCC, suggesting abnormal role(s) of ALKBH proteins in cancer. Further, high expression of ALKBHs was observed not only in HNSCC, but also in several cancerous cell lines and silencing ALKBH expression in HeLa cancer cells resulted in dramatically decreased survival. Considering the discovered impact of high expression of ALKBH proteins on HNSCC development, we screened for ALKBH blockers among newly synthetized anthraquinone derivatives and demonstrated their potential to support standard anticancer therapy.

Collagen prolyl 4-hydroxylase (P4H) expression and collagen hydroxylation in cancer cells are necessary for breast cancer progression. Here, we show that P4H alpha 1 subunit (P4HA1) protein expression is induced in triple-negative breast cancer (TNBC) and HER2 positive breast cancer. By modulating alpha ketoglutarate (α-KG) and succinate levels P4HA1 expression reduces proline hydroxylation on hypoxia-inducible factor (HIF) 1α, enhancing its stability in cancer cells. Activation of the P4HA/HIF-1 axis enhances cancer cell stemness, accompanied by decreased oxidative phosphorylation and reactive oxygen species (ROS) levels. Inhibition of P4HA1 sensitizes TNBC to the chemotherapeutic agent docetaxel and doxorubicin in xenografts and patient-derived models. We also show that increased P4HA1 expression correlates with short relapse-free survival in TNBC patients who received chemotherapy. These results suggest that P4HA1 promotes chemoresistance by modulating HIF-1-dependent cancer cell stemness. Targeting collagen P4H is a promising strategy to inhibit tumor progression and sensitize TNBC to chemotherapeutic agents. Hyperactivation of HIF-1α is crucial in progression of triple-negative breast cancer, but how HIF-1α stability is maintained in a hypoxia-independent manner is unclear. Here, the authors show collagen prolyl-4-hydroylase 1 stabilises HIF-1α and is involved in chemoresistance in TNBC.

Key Points Active DNA demethylation in mammals is achieved through TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution of oxidized 5mC or thymine DNA glycosylase (TDG)-mediated excision of 5fC and 5caC coupled with base excision repair. Active DNA demethylation is regulated at various levels, including substrate and cofactor availability, post-transcriptional and post-translational regulation of TET and TDG, and genomic localization of the demethylation machinery. Studies of tissue distribution, genomic distribution and the dynamics of oxidized 5mC provide insights into the mechanism and function of active DNA demethylation as well as the potential roles of oxidized 5mC. Active DNA demethylation and oxidized 5mC are involved in pre-implantation embryo development, primordial germ cell development, pluripotency and differentiation, as well as neuronal functions. In certain biological contexts, such as in pre-implantation embryos, the biological meaning of TET-mediated oxidation is not fully understood. In some other biological contexts, such as neurons, the extent of active DNA demethylation and its function require further study. TET may function in a catalytic-activity-independent manner. Further analysis is needed to distinguish the functions of the TET proteins themselves from the function of active DNA demethylation. Emerging evidence suggests an interplay between TET, active DNA demethylation and genomic instability and the DNA damage response. A key mode of regulating DNA methylation is through active demethylation driven by TET-mediated oxidation of 5-methylcytosine (5mC). This Review discusses our latest understanding of the mechanisms and regulation of active DNA demethylation, and the roles of active demethylation (and the oxidized 5mC intermediates) in gene regulation, genome stability, development and disease. In mammals, DNA methylation in the form of 5-methylcytosine (5mC) can be actively reversed to unmodified cytosine (C) through TET dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution or thymine DNA glycosylase (TDG)-dependent base excision repair. In the past few years, biochemical and structural studies have revealed mechanistic insights into how TET and TDG mediate active DNA demethylation. Additionally, many regulatory mechanisms of this process have been identified. Technological advances in mapping and tracing the oxidized forms of 5mC allow further dissection of their functions. Furthermore, the biological functions of active DNA demethylation in various biological contexts have also been revealed. In this Review, we summarize the recent advances and highlight key unanswered questions.

Background Indoleamine 2,3- dioxygenase 1 (IDO1) is an immunosuppressive enzyme that has been correlated with shorter disease-specific survival in patients with urothelial carcinoma (UC). IDO1 may counteract the antitumor effects of immune checkpoint inhibitors. Epacadostat is a potent and highly selective inhibitor of IDO1. In the phase I/II ECHO-202/KEYNOTE-037 study, epacadostat plus pembrolizumab resulted in a preliminary objective response rate (ORR) of 35% in a cohort of patients with advanced UC. Methods ECHO-307/KEYNOTE-672 was a double-blinded, randomized, phase III study. Eligible adults had confirmed locally advanced/unresectable or metastatic UC of the urinary tract and were ineligible to receive cisplatin-based chemotherapy. Participants were randomly assigned (1:1) to receive epacadostat (100 mg twice daily) plus pembrolizumab (200 mg every 3 weeks) or placebo plus pembrolizumab for up to 35 pembrolizumab infusions. The primary endpoint was investigator-assessed ORR per Response Evaluation Criteria in Solid Tumors (version 1.1). Results A total of 93 patients were randomized (epacadostat plus pembrolizumab, n  = 44; placebo plus pembrolizumab, n  = 49). Enrollment was stopped early due to emerging data from the phase III ECHO-301/KEYNOTE-252 study. The median duration of follow-up was 64 days in both arms. Based on all available data at cutoff, ORR (unconfirmed) was 31.8% (95% CI, 22.46–55.24%) for epacadostat plus pembrolizumab and 24.5% (95% CI, 15.33–43.67%) for placebo plus pembrolizumab. Circulating kynurenine levels numerically increased from C1D1 to C2D1 in the placebo-plus-pembrolizumab arm and decreased in the epacadostat-plus-pembrolizumab arm. Epacadostat-plus-pembrolizumab combination treatment was well tolerated with a safety profile similar to the placebo arm. Treatment discontinuations due to treatment-related adverse events were more frequent with epacadostat (11.6% vs. 4.1%). Conclusions Treatment with epacadostat plus pembrolizumab resulted in a similar ORR and safety profile as placebo plus pembrolizumab in cisplatin-ineligible patients with previously untreated locally advanced/unresectable or metastatic UC. At a dose of 100 mg twice daily, epacadostat did not appear to completely normalize circulating kynurenine levels when administered with pembrolizumab. Larger studies with longer follow-up and possibly testing higher doses of epacadostat, potentially in different therapy settings, may be warranted. Trial registration ClinicalTrials.gov identifier: NCT03361865, retrospectively registered December 5, 2017.

As one of the deadliest malignancies, gastric cancer (GC) is often accompanied by a low 5-year survival following initial diagnosis, which accounts for a substantial proportion of cancer-related deaths each year worldwide. Altered epigenetic modifications of cancer oncogenes and tumor suppressor genes emerge as novel mechanisms have been implicated the pathogenesis of GC. In the current study, we aim to elucidate whether histone deacetylase 3 (HDAC3) exerts oncogenic role in GC, and investigate the possible mechanism. Initially, we collected 64 paired cancerous and noncancerous tissues surgically resected from GC patients. Positive expression of HDAC3, FTO, and MYC in the tissues was measured using Immunohistochemistry. Meanwhile, GC cell line BGC-823/AGS was selected and treated with lentivirus vectors for alteration of HDAC3, FTO, or FOXA2 expressions, followed by detection on mRNA and protein levels of HDAC3, FOXA2, FTO, and MYC using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot assays. The results demonstrated that the expressions of HDAC3, FTO and MYC were upregulated, while FOXA2 expression was downregulated in GC tissues and cells. After that, the cell viability, migration, and invasion of GC cells were assessed by CCK-8 and Transwell assays, revealing that HDAC3 accelerated GC cell viability, migration and invasion by degrading FOXA2. Subsequently, the binding relationship among HDAC3, FOXA2, FTO, and MYC was assessed by assays of immunoprecipitation, dual-luciferase reporter gene, and chromatin immunoprecipitation assay. Methylation of m6A mRNA in GC cells was detected via gene-specific m6A qPCR and dot-blot assays. The transcription factor FOXA2 was found to bind to the FTO gene promoter and decreased its expression, while FTO stabilized MYC mRNA by reducing m6A methylation of MYC in GC cells. In addition, HDAC3 was observed to maintain the FTO/m6A/MYC signaling and regulated GC progression, which was also supported by in vivo animal study data of GC cell tumorigenesis in nude mice. These key observations uncover the tumor-initiating activities of HDAC3 in GC through its regulation on FOXA2-mediated FTO/m6A/MYC axis, highlighting the potential of therapeutically targeting epigenetic modifications to combat GC.

Tryptophan (Trp) is an essential amino acid, whose metabolism is a key gatekeeper of intestinal homeostasis. Yet, its systemic effects, particularly on atherosclerosis, remain unknown. Here we show that high-fat diet (HFD) increases the activity of intestinal indoleamine 2, 3-dioxygenase 1 (IDO), which shifts Trp metabolism from the production of microbiota-derived indole metabolites towards kynurenine production. Under HFD, the specific deletion of IDO in intestinal epithelial cells leads to intestinal inflammation, impaired intestinal barrier, augmented lesional T lymphocytes and atherosclerosis. This is associated with an increase in serotonin production and a decrease in indole metabolites, thus hijacking Trp for the serotonin pathway. Inhibition of intestinal serotonin production or supplementation with indole derivatives alleviates plaque inflammation and atherosclerosis. In summary, we uncover a pivotal role of intestinal IDO in the fine-tuning of Trp metabolism with systemic effects on atherosclerosis, paving the way for new therapeutic strategies to relieve gut-associated inflammatory diseases.

The ( R )-enantiomer of 2-hydroxyglutarate, which is produced when IDH is mutated in human tumours, is shown to stimulate the activity of the EGLN prolyl 4-hydroxylases, leading to diminished levels of HIF and enhanced human astrocyte proliferation. Cancer induction by isocitrate dehydrogenase mutation Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 have been identified in gliomas, the most common form of brain tumour, and in other cancers including leukaemias. The mutated enzymes produce 2-hydroxyglutarate (2HG), which is a potential oncometabolite. Three papers in this issue of Nature examine the mechanisms through which IDH mutations promote cancers. Lu et al . show that 2HG-producing IDH mutants can prevent the histone demethylation that is required for progenitor cells to differentiate, potentially contributing to tumour-cell accumulation. Turcan et al . show that IDH1 mutation in primary human astrocytes induces DNA hypermethylation and reshapes the methylome to resemble that of the CIMP phenotype, a common feature of gliomas and other solid tumours. Koivunen et al . show that the ( R )-enantiomer of 2HG (but not the ( S )-enantiomer) can stimulate the activity of the EGLN prolyl 4-hydroxylases, leading to diminished levels of hypoxia-inducible factor (HIF), which in turn can enhance cell proliferation. These papers establish a framework for understanding gliomagenesis and highlight the interplay between genomic and epigenomic changes in human cancers. The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation 1 . Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2, which catalyse the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the ( R )-enantiomer of 2-hydroxyglutarate (( R )-2HG) 2 , 3 . Here we show that ( R )-2HG, but not ( S )-2HG, stimulates EGLN activity, leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the ( R )-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy.

Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-K mO₂) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein–coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.

Based on its effects on both tumour cell intrinsic malignant properties as well as anti-tumour immune responses, tryptophan catabolism has emerged as an important metabolic regulator of cancer progression. Three enzymes, indoleamine-2,3-dioxygenase 1 and 2 (IDO1/2) and tryptophan-2,3-dioxygenase (TDO2), catalyse the first step of the degradation of the essential amino acid tryptophan (Trp) to kynurenine (Kyn). The notion of inhibiting IDO1 using small-molecule inhibitors elicited high hopes of a positive impact in the field of immuno-oncology, by restoring anti-tumour immune responses and synergising with other immunotherapies such as immune checkpoint inhibition. However, clinical trials with IDO1 inhibitors have yielded disappointing results, hence raising many questions. This review will discuss strategies to target Trp-degrading enzymes and possible down-stream consequences of their inhibition. We aim to provide comprehensive background information on Trp catabolic enzymes as targets in immuno-oncology and their current state of development. Details of the clinical trials with IDO1 inhibitors, including patient stratification, possible effects of the inhibitors themselves, effects of pre-treatments and the therapies the inhibitors were combined with, are discussed and mechanisms proposed that might have compensated for IDO1 inhibition. Finally, alternative approaches are suggested to circumvent these problems.