**10. Vitamin C as a powerful modulator of TET2 activity**

Decreased TET expression and loss of 5hmC have been observed in a wide variety of solid tumors, as well as in many hematological malignancies, including acute myeloid leukemias, myelodysplastic syndromes, and clonal hematopoiesis [114].

Recent experimental studies suggest that pharmacological dose of Vitamin C may represent a potentially important strategy in leukemia therapy through a stimulatory effect on TET2 activation and restoration in leukemic cells. Vitamin C is a co-factor of TET2 enzyme and is capable of interacting with the catalytic domain of TET2, enhancing the enzymatic oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) [115]. This epigenetic modulation elicited by Vitamin C is able to improve the generation of pluripotent stem cells [116] and to induce a blastocyst-like state in mouse embryonic stem cells [117].

Two recent studies explored the possible epigenetic effects of Vitamin C on leukemia models, mediated by activation and restoration of TET2 activity in leukemic cells. In the first one, authors used a murine model of IDH1-dependent acute myeloid leukemia [118], and 2-phosphate l-ascorbic acid (Asc 2-P). Asc 2-P, unlike native Vitamin C, remains oxidatively stable under standard cell culture conditions [119], and possesses the same modulatory effects of Vitamin C, but, unlike Vitamin C, it does not induce cytotoxic effects of through stimulation of H2 O2 production. Asc 2-P added to the cells in culture is stable and releases Vitamin C by plasma membrane alkaline phosphatase hydrolysis [120]. Therefore, Asc 2-P allows a better characterization of the epigenetic activity of Vitamin C, without the "disturbing" H<sup>2</sup> O2 -mediated cytotoxic effects of the native molecule. Asc 2-P treatment of the IDH1 AML-mutant mice induced an increase of 5hmC levels, a reduction of leukemic proliferation and an increase in expression of genes involved in leukocyte differentiation [118]. The stimulatory effect of Vitamin C on myeloid differentiation is mediated though the restoration of a normal expression and function of transcription factors, such as PU.1 and RUNX1, required for normal myeloid differentiation.

A second study provided clear evidence that in various leukemia models, Vitamin C treatment induces the restoration of TET2 function, blocking aberrant self-renewal and leukemia progression. Treatment with Vitamin C, mimics TET2 restoration, driving DNA hypomethylation and, by enhancing 5hmC formation, suppresses leukemic colony formation and leukemic progression of primary human leukemia patient-derived xenografts (PDXs). Interestingly, TET2-mediated DNA oxidation induced by Vitamin C-treated leukemic cells, greatly enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to target TET-deficient leukemic cells. These observations suggest that future clinical trials could incorporate high-dose Vitamin C as an adjuvant to standard chemotherapy/demethylating therapy, particularly in TET2-deficient neoplasms [121].
