Abstract

TET2 mutations (TET2MT) are ubiquitous in myeloid neoplasia, but are most frequent in MDS and MDS/MPN. TET2 is an iron-catalyzed dioxygenase critical in active DNA demethylation that progressively oxidizes 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5CaC). As 5hmC residues are not recognized by DNMT3A, their methylation pattern is not copied onto the daughter DNA strand, leading to net DNA demethylation: TET2 can also therefore be considered a demethylase.

Ascorbic acid (AA) is required as a prosthetic group of TET-mediated cytosine demethylation. AA may increase the residual function/throughput of intact TET2 in cases with heterozygous TET2MT and restore hydroxymethylation, thus reversing the expected epigenetic phenotype. We hypothesized that in heterozygous TET2MT neoplasms, AA alone or in combination may constitute a rational targeted therapy to restore TET2 function.

We first assessed the effects of AA on TET2 in cell-free experiments. We combined purified wild-type TET2 (TET2WT) with a synthetic poly mC substrate and 0-4 mM AA, and measured levels of 5mC, 5hmC, 5fC, and 5CaC via 2D-UPLC-MS/MS profiling. AA increased the ratio of 5hmC, 5CaC, and 5fC/5mC in a dose-dependent fashion (peak effect 4.5 to 9). To model heterozygous TET2MT seen in myeloid neoplasia, we combined TET2WT with a catalytically-dead TET2S1898F in a 1:1 ratio. AA again increased the ratio of 5hmC, 5fC, and 5CaC to 5mC (peak effect 3 to 6, p<.0001); AA had no effect on TET2S1898F alone.

To determine if AA TET2 upregulation could be reproduced in human cells, K562 and MOLM-13 AML lines were treated for 24 hours with 0-1000 uM of AA before subjecting them to 2D-UPLC-MS/MS analysis. AA increased 5hmC levels in K562 cells by 1.8-fold (500 uM, p=.04) and 1.7-fold (1000 uM, p=.15), and increased 5fC levels by 2.1-fold (500 uM, p <0.0001) and 3.1-fold (1000 uM, p=.035). Similarly, AA increased 5hmC levels in MOLM13 cells by 1.8-fold (500 uM, p=.01) and 2-fold (1000 uM, p<.0001), and increased 5fC levels by 3.6-fold (500 uM, p<.0001; 1000 uM, p=.01).

We then studied the effect of TET2 loss on leukemogenesis in dual Tet2+/- ; Gulo -/- knockout mice unable to produce endogenous AA, making them ideal to determine the effect of exogenous AA on in vivo TET2 function. Bone marrow samples isolated from Tet2+/- ; Gulo -/- mice had a 1.7-fold reduction in 5hmC (p<.0001), a 2-fold reduction in 5fC (p=.0009), and a 3.4-fold reduction in 5CaC (p<.0001). Treatment of Tet2+/- ; Gulo -/- mice with 3.3 g/L ascorbic acid (in the drinking water for 6 months) partially rescued these DNA oxidation products in their bone marrow cells, with significantly increased levels of 5fC (p = .02) and 5CaC (p =.04) compared to untreated Tet2+/- ; Gulo -/- mice. Compared to TET2WT mice, Tet2+/- ; Gulo -/- mice had only a 1.2-fold reduction in 5hmC (p = 0.2), a 1.15-fold reduction in 5fC (p = .004), and a 1.7-fold reduction in 5CaC (p =.3).

Similar to the TET2 knockout mice results, 8/8 of TET2MT patient samples profiled had reduced levels of at least one TET2-dependent DNA oxidation product. Intriguingly, 24-hour treatment with 250 uM AA treatment had no significant effect on any TET2 MT bone marrow samples; in TET2 WT samples, 250 uM AA increased 5fC levels by 2-fold (p =.014) and 5CaC levels by 3-fold (p =.0025), though it nonsignificantly reduced 5hmC levels by 80% (p =.069).

In our study, AA significantly increased the levels of TET2-dependent DNA oxidation products in purified TET2, Tet2+/- ; Gulo -/- knockout mice, TET2WT human cell lines, and TET2WT patient samples. Therapeutic AA may therefore play a role in treating myeloid neoplasia with IDH1 or IDH2 mutations, which have structurally-intact but functionally hypoactive TET2 from loss of the IDH-catalyzed TET2 substrate alpha-ketoglutarate. Given that AA was unable to restore TET2 activity in samples from TET2MT patients, AA may not be beneficial in TET2MT myeloid neoplasia.

Our study also tells a cautionary tale about using 5hmC levels alone to estimate TET2 activity. 5hmC levels were normal in 3/8 (37.5%) of patients with pathogenic TET2MT; simultaneous measurement of multiple TET2-dependent products is therefore important to accurately assess in-vivo TET2 activity and determine who might benefit from therapeutic AA intervention.

Disclosures

Greenberg: Acetylon Pharmaceuticals Inc.: Patents & Royalties: Treatment of Protein Degradation Disorders; Oisin Biotechnologies: Consultancy. Makishima: Yasuda Medical Foundation: Research Funding. Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski: Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy.

Author notes

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Asterisk with author names denotes non-ASH members.