Two Krebs cycle genes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), are mutated in a subset of human cancers, leading to accumulation of their substrates, fumarate and succinate, respectively. Here we demonstrate that fumarate and succinate are competitive inhibitors of multiple a-ketoglutarate (a-KG)-dependent dioxygenases, including histone demethylases, prolyl hydroxylases, collagen prolyl-4-hydroxylases, and the TET (ten-eleven translocation) family of 5-methlycytosine (5mC) hydroxylases. Knockdown of FH and SDH results in elevated intracellular levels of fumarate and succinate, respectively, which act as competitors of a-KG to broadly inhibit the activity of a-KG-dependent dioxygenases. In addition, ectopic expression of tumor-derived FH and SDH mutants inhibits histone demethylation and hydroxylation of 5mC. Our study suggests that tumor-derived FH and SDH mutations accumulate fumarate and succinate, leading to enzymatic inhibition of multiple a-KG-dependent dioxygenases and consequent alterations of genome-wide histone and DNA methylation. These epigenetic alterations associated with mutations of FH and SDH likely contribute to tumorigenesis.[Keywords: FH; SDH; metabolites; a-KG-dependent dioxygenases; DNA methylation; histone methylation] Supplemental material is available for this article. Received March 7, 2012; revised version accepted May 9, 2012. Several lines of evidence, including the recent identification of mutations affecting isocitrate dehydrogenase (IDH), fumarate hydratase (FH), and succinate dehydrogenase (SDH), have demonstrated that mutations in certain metabolic enzymes may play a causal role in tumorigenesis. The NADP + -dependent IDH genes IDH1 and IDH2 are frequently mutated in >75% of glioma (Parsons et al. 2008),

SUMMARY Protein acetylation has emerged as a major mechanism in regulating cellular metabolism. Whereas most glycolytic steps are reversible, the reaction catalyzed by pyruvate kinase is irreversible and the reverse reaction requires phosphoenolpyruvate carboxykinase (PEPCK1) to commit for gluconeogenesis. Here we show that acetylation regulates the stability of the gluconeogenic rate limiting enzyme PEPCK1, thereby modulating cellular response to glucose. High glucose destabilizes PEPCK1 by stimulating its acetylation. PEPCK1 is acetylated by the P300 acetyltransferase and this acetylation stimulates the interaction between PEPCK1 and UBR5, a HECT domain containing E3 ubiquitin ligase, therefore promoting PEPCK1 ubiquitinylation and degradation. Conversely, SIRT2 deacetylates and stabilizes PEPCK1. These observations represent an example that acetylation targets a metabolic enzyme to a specific E3 ligase in response to metabolic condition changes. Given that increased levels of PEPCK is linked with type II diabetes, this study also identifies potential therapeutic targets for diabetes.

The TET2 DNA dioxygenase regulates cell identity and suppresses tumorigenesis by modulating DNA methylation and expression of a large number of genes. How TET2, like most other chromatin modifying enzymes, is recruited to specific genomic sites is unknown. Here we report that WT1, a sequence-specific transcription factor, is mutated in a mutually exclusive manner with TET2, IDH1 and IDH2 in acute myeloid leukemia (AML). WT1 physically interacts with and recruits TET2 to its target genes to activate their expression. The interaction between WT1 and TET2 is disrupted by multiple AML-derived TET2 mutations. TET2 suppresses leukemia cell proliferation and colony formation in a manner dependent on WT1. These results provide a mechanism for targeting TET2 to specific DNA sequence in the genome. Our results also provide an explanation for the mutual exclusivity of WT1 and TET2 mutations in AML and suggest an IDH1/2-TET2-WT1 pathway in suppressing AML.

Hyperphosphorylation of signal transducer and activator of transcription 3 (STAT3) has been found in various types of human cancers, including head and neck cancer (HNC). Although smoking is critical in the development and progression of HNC, how tobacco components activate STAT3 is unclear. We demonstrated that exposure of HNC cell lines to a tobacco extract induced a rapid Y705 phosphorylation of STAT3 and a rapid increase in the SUMO protease SENP3 that depended on a simultaneous increase in reactive oxygen species. We identified that SUMOylation at the lysine 451 site facilitated STAT3 binding to the phosphatase TC45 through an SUMO-interacting motif of TC45. SENP3 could thus enhance STAT3 phosphorylation by de-conjugating the SUMO2/3 modification of STAT3. Knocking-down of SENP3 greatly impaired basal and induced STAT3 phosphorylation by tobacco extract or interleukin 6. A correlation between SENP3 protein levels and STAT3 Y705 phosphorylation levels in human laryngeal carcinoma specimens was found, which was more significant in the specimens derived from the smoker patients and with poor clinicopathological parameters. Our data identified SUMOylation as a previously undescribed post-translational modification of STAT3 and SENP3 as a critical positive modulator of tobacco- or cytokine-induced STAT3 activation. These findings provide novel insights into the hyperphosphorylation of STAT3 in development of HNC.