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.

Deregulated signal transduction and energy metabolism are hallmarks of cancer and both play a fundamental role in tumorigenesis. While it is increasingly recognised that signalling and metabolism are highly interconnected, the underpinning mechanisms of their co-regulation are still largely unknown. Here we designed and acquired proteomics, phosphoproteomics, and metabolomics experiments in fumarate hydratase (FH) deficient cells and developed a computational modelling approach to identify putative regulatory phosphorylation-sites of metabolic enzymes. We identified previously reported functionally relevant phosphosites and potentially novel regulatory residues in enzymes of the central carbon metabolism. In particular, we showed that pyruvate dehydrogenase (PDHA1) enzymatic activity is inhibited by increased phosphorylation in FH-deficient cells, restricting carbon entry from glucose to the tricarboxylic acid cycle. Moreover, we confirmed PDHA1 phosphorylation in human FH-deficient tumours. Our work provides a novel approach to investigate how post-translational modifications of enzymes regulate metabolism and could have important implications for understanding the metabolic transformation of FH-deficient cancers with potential clinical applications.

Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

Mutations of the tricarboxylic acid cycle enzyme fumarate hydratase cause hereditary leiomyomatosis and renal cell cancer. Fumarate hydratase-deficient renal cancers are highly aggressive and metastasize even when small, leading to a very poor clinical outcome. Fumarate, a small molecule metabolite that accumulates in fumarate hydratase-deficient cells, plays a key role in cell transformation, making it a bona fide oncometabolite. Fumarate has been shown to inhibit α-ketoglutarate-dependent dioxygenases that are involved in DNA and histone demethylation. However, the link between fumarate accumulation, epigenetic changes, and tumorigenesis is unclear. Here we show that loss of fumarate hydratase and the subsequent accumulation of fumarate in mouse and human cells elicits an epithelial-to-mesenchymal-transition (EMT), a phenotypic switch associated with cancer initiation, invasion, and metastasis. We demonstrate that fumarate inhibits Tet-mediated demethylation of a regulatory region of the antimetastatic miRNA cluster mir-200ba429, leading to the expression of EMT-related transcription factors and enhanced migratory properties. These epigenetic and phenotypic changes are recapitulated by the incubation of fumarate hydratase-proficient cells with cell-permeable fumarate. Loss of fumarate hydratase is associated with suppression of miR-200 and the EMT signature in renal cancer and is associated with poor clinical outcome. These results imply that loss of fumarate hydratase and fumarate accumulation contribute to the aggressive features of fumarate hydratase-deficient tumours.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a cancer syndrome caused by inactivating germline mutations in fumarate hydratase (FH) and subsequent accumulation of fumarate. Fumarate accumulation leads to profound epigenetic changes and the activation of an anti-oxidant response via nuclear translocation of the transcription factor NRF2. The extent to which chromatin remodeling shapes this anti-oxidant response is currently unknown. Here, we explored the effects of FH loss on the chromatin landscape to identify transcription factor networks involved in the remodeled chromatin landscape of FH-deficient cells. We identify FOXA2 as a key transcription factor that regulates anti-oxidant response genes and subsequent metabolic rewiring cooperating without direct interaction with the anti-oxidant regulator NRF2. The identification of FOXA2 as an anti-oxidant regulator provides additional insights into the molecular mechanisms behind cell responses to fumarate accumulation and potentially provides further avenues for therapeutic intervention for HLRCC.