Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate

Ten-eleven translocation enzymes (TETs) are Fe(II)/2-oxoglutarate (2OG) oxygenases that catalyze the sequential oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in eukaryotic DNA. Despite their roles in epigenetic regulation, there is a lack of reported TET inhibitors. The extent to which 2OG oxygenase inhibitors, including clinically used inhibitors and oncometabolites, modulate DNA modifications via TETs has been unclear. Here, we report studies on human TET1–3 inhibition by a set of 2OG oxygenase-focused inhibitors, employing both enzyme-based and cellular assays. Most inhibitors manifested similar potencies for TET1–3 and caused increases in cellular 5hmC levels. (R)-2-Hydroxyglutarate, an oncometabolite elevated in isocitrate dehydrogenase mutant cancer cells, showed different degrees of inhibition, with TET1 being less potently inhibited than TET3 and TET2, potentially reflecting the proposed role of TET2 mutations in tumorigenesis. The results highlight the tractability of TETs as drug targets and provide starting points for selective inhibitor design.


Table of Contents Page
Table S1.Kinetic parameters for inhibition of recombinant TETs by IOX1 3 and 2HGs 22,23.S17 Table S2.Inhibition data for R-2HG and S-2HG with isolated 2OG dependent dioxygenases.S18 Table S3.Summary of cellular inhibition data for R-2HG and S-2HG with 2OG dependent dioxygenases.S19   The turnover to 5hm C 2 product was calculated using an internal standard curve (Figure S2B) carried out on each independent assay plate.

Figure S2 .
Figure S2.AlphaScreen assay for measuring activity of TET enzymes.(A) Schematic representation of the AlphaScreen assay.The streptavidin conjugated donor beads bind biotinylated DNA which, on TET catalysed oxidation of 5m C to 5hm C, can bind to a 5hm C selective antibody.The product is captured by Protein A conjugated acceptor beads.When the beads are excited (680 nm), the donor beads release singlet molecular oxygen that is converted to chemiluminescence by proximate acceptor beads (520-620 nm).(B) Standard curves for AlphaScreen for ssDNA containing 5m C 1 (green), 5hm C 2 (orange) or 5hm C 2 / 5m C 1 mixture (total concentration of DNA maintained to 10 nM, plotted as a function of 5hm C concentration) (blue).A standard curve for the 5hm C 2 / 5m C 1 mixture was used as an internal for calculating the activities of the TETs.Data are plotted as the mean ± StDev (n > 7).

Figure S9 .
Figure S9.Quantitative analysis of global 5hm C levels of stably transfected TET1CD expressing U2OS cells using high-content immunofluorescence imaging.FLAG-TET1CD wild-type (WT) or catalytically inactive mutant (MUT) stable U2OS cells were treated with or without Dox for 24 h, fixed and stained with DAPI (nuclear), anti-FLAG and anti-5hm C antibodies.The global 5hm C levels were measured as the mean ± s.e.m (n > 3000 cells).(A) An increase in 5hm C levels correlates with TET1CD wild-type expression, but not with mutant.(B) An increase in cellular FLAG staining correlating with the expression of FLAG-TET1CD (WT) and FLAG-TET1CD (MUT) were observed when the cells were treated with Dox.Statistical analysis was carried out using the unpaired t-test with Welsch's correction (**** = P < 0.001, ns = P > 0.05).(C) Exemplar dose-response curves of compound treatment generated using the immunofluorescence assay.Dox-inducible wild-type or mutant TET1CD expressing stable U2OS cells with or without Dox were treated with DMSO, JIB-04 14 or DMOG 30 (1% (v/v) DMSO final conc.) for 24 h.The 5hm C levels of 1% (v/v) DMSO treated controls of TET1CD-WT (dottedline, black) and TET1CD-MUT (dashed-line, grey) with and without Dox are indicated as additional grid lines on the y-axis.Note 5hmC levels for MUT induced and uninduced cells are the same.Data are plotted as mean ± s.e.m (n > 3000 cells).TET1CD-WT data were fitted using non-linear regression (four parameters) with top and bottom constraints as Dox-induced and uninduced DMSO treated WT cells, respectively.In some cases, the WT uninduced cells had slightly higher 5hm C levels than induced mutant expressing cells, in part due to basal level of leaky TET1 expression (FigureS7).FLAG-TET1CD-WT and FLAG-TET1CD-MUT U2OS cells were treated at the same time, on different 96-well plates.Images acquired using Operetta CLS High-Content Analysis (PerkinElmer).

Figure S10 .S14Figure S11 .
Figure S10.Structures of prodrugs used in cellular assays in this study.

Table S3 . Summary of cellular inhibition data for R-2HG and S-2HG with 2OG dependent dioxygenases.
Reported inhibition data is referenced.Experimental screening method on cell line with: a: by immunofluorescence; b: western blot; or c: by dot blot and LC-MS/MS.