The product of the Delta gene, acting as ligand, and that of the Notch gene, acting as receptor, are key components in a lateral-inhibition signalling pathway that regulates the detailed patterning of many different tissues in Drosophila. During neurogenesis in particular, neural precursors, by expressing Delta, inhibit neighbouring Notch-expressing cells from becoming committed to a neural fate. Vertebrates are known to have several Notch genes, but their functions are unclear and their ligands hitherto unidentified. Here we identify and describe a chick Delta homologue, C-Delta-1. We show that C-Delta-1 is expressed in prospective neurons during neurogenesis, as new cells are being born and their fates decided. Our data from the chick, combined with parallel evidence from Xenopus, suggest that both the Delta/Notch signalling mechanism and its role in neurogenesis have been conserved in vertebrates.

SummaryThe Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Mechanistic analysis revealed that fumarate modifies cysteine residues within the Kelch-like ECH-associated protein 1 (KEAP1), abrogating its ability to repress the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway, suggesting a role for Nrf2 dysregulation in FH-associated cysts and tumors.

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid cycle (TCA cycle) that catalyses the hydration of fumarate into malate. Germline mutations of FH are responsible for hereditary leiomyomatosis and renal-cell cancer (HLRCC). It has previously been demonstrated that the absence of FH leads to the accumulation of fumarate, which activates hypoxia-inducible factors (HIFs) at normal oxygen tensions. However, so far no mechanism that explains the ability of cells to survive without a functional TCA cycle has been provided. Here we use newly characterized genetically modified kidney mouse cells in which Fh1 has been deleted, and apply a newly developed computer model of the metabolism of these cells to predict and experimentally validate a linear metabolic pathway beginning with glutamine uptake and ending with bilirubin excretion from Fh1-deficient cells. This pathway, which involves the biosynthesis and degradation of haem, enables Fh1-deficient cells to use the accumulated TCA cycle metabolites and permits partial mitochondrial NADH production. We predicted and confirmed that targeting this pathway would render Fh1-deficient cells non-viable, while sparing wild-type Fh1-containing cells. This work goes beyond identifying a metabolic pathway that is induced in Fh1-deficient cells to demonstrate that inhibition of haem oxygenation is synthetically lethal when combined with Fh1 deficiency, providing a new potential target for treating HLRCC patients.

Nascent retinal neurons, by expressing Dl1, deliver lateral inhibition to neighbouring progenitors; this signal is essential to prevent progenitors from entering the neuronal differentiation pathway. Lateral inhibition serves the key function of maintaining a balanced mixture of dividing progenitors and differentiating progeny. We propose that the same mechanism operates throughout the vertebrate CNS, enabling large numbers of neurons to be produced sequentially and adopt different characters in response to a variety of signals. A similar mechanism of lateral inhibition, mediated by Delta and Notch proteins, may regulate stem-cell function in other tissues.

Germline mutations in the FH gene encoding the Krebs cycle enzyme fumarate hydratase predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. FH-deficient cells and tissues accumulate high levels of fumarate, which may act as an oncometabolite and contribute to tumourigenesis. A recently proposed role for fumarate in the covalent modification of cysteine residues to S-(2-succinyl) cysteine (2SC) (termed protein succination) prompted us to assess 2SC levels in our existing models of HLRCC. Herein, using a previously characterized antibody against 2SC, we show that genetic ablation of FH causes high levels of protein succination. We next hypothesized that immunohistochemistry for 2SC would serve as a metabolic biomarker for the in situ detection of FH-deficient tissues. Robust detection of 2SC was observed in Fh1 (murine FH)-deficient renal cysts and in a retrospective series of HLRCC tumours (n = 16) with established FH mutations. Importantly, 2SC was undetectable in normal tissues (n = 200) and tumour types not associated with HLRCC (n = 1342). In a prospective evaluation of cases referred for genetic testing for HLRCC, the presence of 2SC-modified proteins (2SCP) correctly predicted genetic alterations in FH in every case. In two series of unselected type II papillary renal cancer (PRCC), prospectively analysed by 2SCP staining followed by genetic analysis, the biomarker accurately identified previously unsuspected FH mutations (2/33 and 1/36). The investigation of whether metabolites in other tumour types produce protein modification signature(s) that can be assayed using similar strategies will be of interest in future studies of cancer.

SummaryThe gene encoding the Krebs cycle enzyme fumarate hydratase (FH) is mutated in hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity causes accumulation of intracellular fumarate, which can directly modify cysteine residues to form 2-succinocysteine through succination. We undertook a proteomic-based screen in cells and renal cysts from Fh1 (murine FH)-deficient mice and identified 94 protein succination targets. Notably, we identified the succination of three cysteine residues in mitochondrial Aconitase2 (ACO2) crucial for iron-sulfur cluster binding. We show that fumarate exerts a dose-dependent inhibition of ACO2 activity, which correlates with increased succination as determined by mass spectrometry, possibly by interfering with iron chelation. Importantly, we show that aconitase activity is impaired in FH-deficient cells. Our data provide evidence that succination, resulting from FH deficiency, targets and potentially alters the function of multiple proteins and may contribute to the dysregulated metabolism observed in HLRCC.

The drive to understand how altered cellular metabolism and cancer are linked has caused a paradigm shift in the focus of cancer research. The discovery of a mutated metabolic enzyme, isocitrate dehydrogenase 1, that leads to accumulation of the oncometabolite 2-hydroxyglutarate, provided significant direct evidence that dysfunctional metabolism plays an important role in oncogenesis. Striking parallels exist with the Krebs cycle enzyme fumarate hydratase (FH), a tumor suppressor, whose mutation is associated with the development of leiomyomata, renal cysts, and tumors. Loss of FH enzymatic activity results in accumulation of intracellular fumarate which has been proposed to act as a competitive inhibitor of 2-oxoglutarate-dependent oxygenases including the hypoxia-inducible factor (HIF) hydroxylases, thus activating oncogenic HIF pathways. Interestingly, our studies have questioned the role of HIF and have highlighted other candidate mechanisms, in particular the non-enzymatic modification of cysteine residues (succination) that could lead to disruption or loss of protein functions, dysfunctional cell metabolism and cell signaling. Here, we discuss the evidence for proposing fumarate as an onco-metabolite.

SummaryIsocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.