Transformation by the (R)-enantiomer of 2-hydroxyglutarate linked to EGLN activation

Abstract

The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2, which catalyse the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG, but not (S)-2HG, stimulates EGLN activity, leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy.

Figure 1. Oncogenic Properties of IDH1 R132H

a,b, Anti-HA immunoblot (a) and LC-MS analysis (b) of immortalized human astrocytes (passage 4) infected with retroviruses encoding HA-tagged versions of the indicated IDH1 variants. c,d, In vitro proliferation under standard culture conditions (c) or in soft agar (d)(passage 23). Bars in (d) = 0.5 mm. e, Number of macroscopic soft agar colonies in (d) (*P<0.01, **P<0.005). Error bars, s.d.; n=3.

Fig. 2. R-2HG can serve as an EglN cosubstrate

a, b, In vitro prolyl 4-hydroxylation assays conducted with recombinant EglNs (a) and collagen P4H-I (b) in the presence of the indicated amounts of 2-OG or 2HG. L-[2,3,4,5-³H]proline-labeled HIF1α oxygen-dependent degradation domain (ODDD) (a) and [¹⁴C]proline-labeled protocollagen (b) were used as substrates. Enzymes were produced in insect cells using baculoviruses and affinity-purified. Error bars, s.d.; n = 3-4. c, d, K_m and V_max values for R-2HG for EglN family members. K_m and V_max values for 2-OG are included for comparison. e, LC-MS analysis of succinate, 2-OG and R-2HG from enzymatic reactions with EglN1, HIF1α oxygen-dependent degradation domain (ODDD) polypeptide and either 5 mM R-2HG (red) or 80 μM 2-OG (black) as cofactors. Numbers next to each peak indicate elution times (plain font) and peak areas (bold font). No peaks above background were detected in samples in which 2-OG and R-2HG were both omitted (data not shown). f, Model of R-2HG (green) and S-2HG (cyan) bound to the active site of EglN1. N-oxalylglycine (magneta) bound in the original structure is shown for comparison. The active site water molecule, which has been shown to be the O₂ binding site , is shown in red and the peptide substrate in light blue. Hydrogen bonds are indicated by dash lines.

Fig 3. HIF activity is diminished in IDH mutant cells

a-d, Immunoblot analysis of immortalized human astrocytes (passage 10) (a,b), oligodendroglioma cells (c) and HCT116 colorectal cells (d) expressing the indicated IDH1 variants grown under 21% (N) or 7.5% (H) oxygen for 24 hours prior to lysis or under 21% oxygen in presence of DFO (D). In (b) cells grown under 21% oxygen were treated with increasing amounts of DMOG or DFO for 16 hours prior to lysis. e, Quantitative real-time PCR analysis of cells in (d) under normoxic conditions. Error bars, s.d. n=3. f, Heat map depicting expression of HIF target genes (blue = lower expression and red = higher expression) in proneural tumors clustered based on IDH status (red = mutant and green = wild-type in horizontal bar atop matrix).

Fig 4. Decreased HIF activity contributes to transformation by mutant IDH

a,c, Soft agar colony formation by human astrocytes after stable infection with lentiviruses encoding the indicated HIF1α shRNAs or scrambled (Scr) shRNA (a) or lentiviruses encoding EglN1, EglN1 (P317R), or venus fluorescent protein (c). Bars = 0.5 mm. b,d, Number of macroscopic soft agar colonies in (a) and (c), respectively. (*P<0.05, **P<0.005). Error bars, s.d. n=3. e,f, Proliferation (e) and immunoblot analysis (f) of human astrocytes expressing wild-type or R132H IDH1 and an shRNA against EglN1 (10578) (or scrambled control). g,h, Proliferation of human astrocytes expressing IDH1 R132H and shRNAs against HIF1α (0819), EglN1 (10578), or both. Scr= scrambled shRNA.