Fumarate and Succinate Regulate Expression of Hypoxia-inducible Genes via TET Enzymes

Abstract

The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1-3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the K m value of Tets 1 and 2 for O 2 is 30 M, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe 2؉ and 2-oxoglutaratebinding residues increased the K m values for these factors 30 -80-fold and reduced the V max values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC 50 values ϳ400 -500 M. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIF␣ stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization.The 2-oxoglutarate-dependent dioxygenases (2-OGDDs) 2 comprise an enzyme family of about 70 members in humans (1, 2). These enzymes all share the same basic reaction mechanism, in which the substrate is hydroxylated by molecular oxygen in the presence of a divalent metal cofactor (most commonly Fe 2ϩ ) and the 2-oxoglutarate cosubstrate is decarboxylated to succinate and CO 2 (1). The substrates for 2-OGDDs vary from proteins to DNA, RNA, and fatty acids (1). Interestingly, a large number of 2-OGDDs act on the chromatin structure, most notably the ten-eleven-translocation 5-methylcytosine dioxygenases (TETs) and the Jumonji domain-containing histone demethylases (1-3). The stability of the ␣ subunit of the key regulator of the hypoxia response, the hypoxia-inducible factor (HIF), is also regulated by 2-OGDDs, namely the HIF-prolyl 4-hydroxylases (HIF-P4Hs), also known as PHDs and EglNs (1, 2, 4).The TET enzymes convert the 5-methylcytosine (5-mC) in DNA sequentially to 5-hydroxymethylcytosine (5-hmC), 5-formylcytocine, and 5-carboxylcytocine, leading to DNA demethylation (3, 5-7). 5-hmC is also likely to have its own epigenetic function beyond simply being a demethylating base (3). The highest levels of 5-hmC are found in stem cells of various origins and in neural tissues (6, 7). There are three human TET isoenzymes. TET1 is highly expressed in embryonic stem cells, whereas TETs 2 and 3 are required for normal hematopoiesis...

AMPK/α-Ketoglutarate Axis Dynamically Mediates DNA Demethylation in the Prdm16 Promoter and Brown Adipogenesis

“…Compared to progenitors, αKG concentration was vastly higher in BAT or differentiated brown adipocytes at day 3, while citrate content only slightly differed between progenitor cells and BAT (Figure 4A, 4B). Thus, αKG, due to its very low concentration (WT: 14.15 ± 2.2 µM at day 0; 33.49 ± 6.1 µM at day 3), likely is the rate limiting factor hindering TET-mediated DNA demethylation in progenitor cells considering αKG Km for TETs is around 50–60 µM (Laukka et al, 2016) (Figure 4A). We further compared metabolite contents between WT and Prkaa1 −/− cells and found that αKG was reduced due to Prkaa1 KO (Figure 4C), which was confirmed by chemical analysis (Figure 4D).…”

Cancer metabolism in space and time: Beyond the Warburg effect

“…Nev er the less, hy poxia stim u lates tu mor growth ad van tage also by de creas ing the ac tiv ity of TET (oxy gen de pen dent ten eleven translo ca tion en zymes) demethy lases, lead ing to re duced tran scrip tion of on co sup pres sor genes [43,44]. In deed, se vere hy poxia (0.5% O ) de creased the ac tiv ity of TET en zymes in sev eral mouse and hu man cell lines [44].…”

Mitochondrial dysfunction in cancer: Potential roles of ATF5 and the mitochondrial UPR

“…For example, mutations in the TCA cycle gene encoding isocitrate dehydrogenase (IDH) cause an over-production of the onco-metabolite 2-hydroxyglutarate, which contributes to glioma formation and leukemogensis [24, 25]. And, succinate dehydrogenase (SDH) or fumarate hydratase (FH) mutations also cause accumulation of the TCA cycle metabolic intermediates, succinate and fumarate, that activate hypoxia-inducible factor 1 alpha (HIF-1α), which promotes cancer cell growth and survival [26–29]. Moreover, mutations in genes encoding complex III components of electron transport chain impair apoptosis, thus contributing to tumor progression [30].…”