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.

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.

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Mutations in the gene encoding the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer in affected individuals. FH-associated neoplasia is characterized by defective mitochondrial function and by upregulation of transcriptional pathways mediated by hypoxia-inducible factor (HIF), although whether and by what means these processes are linked has been disputed. We analysed the HIF pathway in Fh1-/- mouse embryonic fibroblasts (MEFs), in FH-defective neoplastic tissues and in Fh1-/- MEFs re-expressing either wild-type or an extra-mitochondrial restricted form of FH. These experiments demonstrated that upregulation of HIF-1alpha occurs as a direct consequence of FH inactivation. Fh1-/- cells accumulated intracellular fumarate and manifested severe impairment of HIF prolyl but not asparaginyl hydroxylation which was corrected by provision of exogenous 2-oxoglutarate (2-OG). Re-expression of the extra-mitochondrial form of FH in Fh1-/- cells was sufficient to reduce intracellular fumarate and to correct dysregulation of the HIF pathway completely, even in cells that remained profoundly defective in mitochondrial energy metabolism. The findings indicate that upregulation of HIF-1alpha arises from competitive inhibition of the 2-OG-dependent HIF hydroxylases by fumarate and not from disruption of mitochondrial energy metabolism.

Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia.

Circulating tumor cells (CTCs) hold great potential to answer key questions of how non-small cell lung cancer (NSCLC) evolves and develops resistance upon anti-PD-1/PD-L1 treatment. Currently, their clinical utility in NSCLC is compromised by a low detection rate with the established, Food and Drug Administration (FDA)-approved, EpCAM-based CellSearch® System. We tested an epitope-independent method (ParsortixTM system) and utilized it to assess PD-L1 expression of CTCs from NSCLC patients. We prospectively collected 127 samples, 97 of which were analyzed with the epitope-independent system in comparison to the CellSearch system. CTCs were determined by immunocytochemistry as intact, nucleated, CD45−, pankeratins (K)+ cells. PD-L1 status of CTCs was evaluated from 89 samples. With the epitope-independent system, ≥1 CTC per blood sample was detected in 59 samples (61%) compared to 31 samples (32%) with the EpCAM-based system. Upon PD-L1 staining, 47% of patients harbored only PD-L1+CTCs, 47% had PD-L1+ and PD-L1−CTCs, and only 7% displayed exclusively PD-L1−CTCs. The percentage of PD-L1+CTCs did not correlate with the percentage of PD-L1+ in biopsies determined by immunohistochemistry (p = 0.179). Upon disease progression, all patients showed an increase in PD-L1+CTCs, while no change or a decrease in PD-L1+CTCs was observed in responding patients (n = 11; p = 0.001). Our data show a considerable heterogeneity in the PD-L1 status of CTCs from NSCLC patients. An increase of PD-L1+CTCs holds potential to predict resistance to PD-1/PD-L1 inhibitors.

SummaryThe identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.