Oncometabolites d- and l-2-Hydroxyglutarate Inhibit the AlkB Family DNA Repair Enzymes under Physiological Conditions

Abstract

Cancer-associated mutations often lead to perturbed cellular energy metabolism and accumulation of potentially harmful oncometabolites. One example is the chiral molecule 2-hydroxyglutarate (2HG); its two stereoisomers (d- and l-2HG) have been found at abnormally high concentrations in tumors featuring anomalous metabolic pathways. 2HG has been demonstrated to competitively inhibit several α-ketoglutarate (αKG)- and non-heme iron-dependent dioxygenases, including some of the AlkB family DNA repair enzymes, such as ALKBH2 and ALKBH3. However, previous studies have only provided the IC₅₀ values of d-2HG on the enzymes, and the results have not been correlated to physiologically relevant concentrations of 2HG and αKG in cancer cells. In this work, we performed detailed kinetic analyses of DNA repair reactions catalyzed by ALKBH2, ALKBH3, and the bacterial AlkB in the presence of d- and l-2HG in both double- and single-stranded DNA contexts. We determined the kinetic parameters of inhibition, including k_cat, K_M, and K_i. We also correlated the relative concentrations of 2HG and αKG previously measured in tumor cells with the inhibitory effect of 2HG on the AlkB family enzymes. Both d- and l-2HG significantly inhibited the human DNA repair enzymes ALKBH2 and ALKBH3 at pathologically relevant concentrations (73-88% for d-2HG and 31-58% for l-2HG inhibition). This work provides a new perspective that the elevation of the d- or l-2HG concentration in cancer cells may contribute to an increased mutation rate by inhibiting the DNA repair performed by the AlkB family enzymes and thus exacerbate the genesis and progression of tumors.

Figure 1

a) Repair mechanism of the AlkB family enzymes on alkyl DNA lesions. Adduct m1A is used here as an example to show the steps of enzymatic catalysis. b) The generation of D- and L-2HG and mechanisms of inhibition to the AlkB family DNA repair enzymes.

Figure 2

HPLC analyses of the DNA repair reactions. a) Starting material 16mer oligonucleotide containing m1A at the lesion site in ss-DNA reaction. b) ss-Product 16mer oligonucleotide containing A at the “lesion site” in the ss-DNA reaction. c) ds-DNA reaction products of 16mer m1A with 23mer Tcp. The mixture containing ss-16mer m1A, ss-16mer A, ds-16mer m1A:23mer Tcp, and ds-16mer A:23mer Tcp. The latter two species were not fully separable by HPLC. d) ds-DNA reaction products of 16mer m1 A with 23mer Tcp and additional 23mer A, which is fully complementary to 23mer Tcp. The duplex of 23mer Tcp:23mer A was eluted as ds-DNA, thus releasing ss-16mer m1A and ss-16mer A for quantification.

Figure 3

Inhibition of ALKBH2 repair of ml A in ss- and ds-DNA by D- and L-2HG. a) Inhibition of D-2HG on ml A repair in ss-DNA. b) Inhibition of D-2HG on ml A repair in ds-DNA. c) Inhibition of D-2HG on ml A repair under D-2HG:αKG = 373:1 ratio conditions, d) Inhibition of L-2HG on ml A repair in ss-DNA. e) Inhibition of L-2HG on ml A repair in ds-DNA. f) Inhibition of L-2HG on ml A repair under L-2HG:αKG = 28:1 ratio conditions.

Figure 4

Addition of αKG reverses the inhibitory effect of 2-HG toward ALKBH2 repair of ml A. Different concentrations of αKG were added to a fixed concentration of 2HG (10 mM) to recover the repair of ml A by a) ALKBH2, b) ALKBH3 and c) AlkB.