Modulation of TET2 Activity By Ascorbic Acid and Factors Affecting Lysine Acetylation
Authors: Guan Yihong , Greenberg Edward F , Hasipek Metis , Hirsch Cassandra M. , Graham Amy C , Radivoyevitch Tomas , Chen Shi , Liu Xiaochen , Visconte Valeria , You Dewen , Negoro Eiju , Przychodzen Bartlomiej P. , Makishima Hideki , Nazha Aziz , Gackowski Daniel , Zarakowska Ewelina , Mukherji Mridul , Sekeres Mikkael A. , Xu Mingjiang , Ólinski Ryszard , Maciejewski Jaroslaw P. , Jha Babal K. 
Published: October 2019

Abstract
Loss-of-function TET2 mutations (TET2mt) strongly associate with myeloid neoplasia. TET2 is an α-ketoglutarate (αKG) and Fe2+-dependent DNA-dioxygenase that hydroxylates 5-methylcytosine-DNA (5mC), producing 5-hydroxymethylcytosine-DNA (5hmC). Ascorbic acid (AA) activates TET2 and other dioxygenases via several direct and indirect mechanisms, including recharging Fe3+ to Fe2+. Recent studies, chiefly in murine models, indicated that increased AA availability may alleviate the decreased of dioxygenase activity in cells affected by hypomorphic mutations via up-modulation of the remaining functional activity, particularly in heterozygous TET2mt, but it may also act via increased compensatory activity of other TET enzymes. Beyond these observations, the mechanisms of AA action in TET2mt bone marrow cells are not well understood. Yet to be identified are factors that modulate the effects of AA.
We performed in silico binding prediction validated by surface plasmon resonance assay coupled with confirmatory in vitro and in vivo assays. In cell free system we demonstrated the AA increases the production of 5mC-DNA oxidation products (TDOP, 5hmC/5fmC/5caC) in a mixture of recombinant TET2wt:TET2S1898F 1:1 mimicking heterozygous TET2mt neoplasia. Using cell-free system with recombinant TET2 protein and the aforementioned biophysicochemical analyses, we further demonstrated that AA binds to the TET2 catalytic domain adjacent to Fe2+/aKG binding site and recycle Fe2+ back into the catalysis.
In vitro cultures demonstrated that AA increases TDOP and slows proliferation in Tet2+/- and Tet2-/- mouse bone marrow progenitor cells. A similar but less pronounced effect was also observed in shRNA TET2KD MOLM13 and K562 cells in which AA restored the original proliferation rate of these cells which was increased by TET2KD. Finally, using cultures of healthy human marrows (retrovirally transduced with TET2 or scrambled shRNA), we showed that AA decreased the proliferation rate of these cells similar to ectopic overexpression of TET2. When primary MDS bone marrow samples with various TET2mt (n=4) were studied, the effects of AA were less consistent. Consistent with the observation, in SIG-M5 cell line characterized by natural TET2 mutations, AA only modestly increased 5hmC 2-fold. While, in high TET2 expression CMK cell line, AA increased 5hmC 6-fold. In vivo, high-dose oral AA increased TDOP, decreased spleen size and myeloproliferation, and prolonged survival in sublethally-irradiated Tet2+/-Gulo-/- and Tet2+/- mice. However the AA treatment has only a modest effect on the overall survival of these mice.
Recent reports indicated that TET2 activity is down regulated by HDAC1/2 by deacetylation at the N-terminus (Zhang et al., Molecular Cell 65, 323-335, 2017) and up regulated by Sirtuin 1 (Class III HDAC) via lysine deacetylation in the catalytic domain (Sun et al., abstract, Blood 2016 128:1053). In search of factors that modulate the effects of TET, experiments were performed to determine if acetylation also modulates the activity of TET in leukemia cells. Inhibition of class I and II but not class III HDACs by trichostatin A (TSA) increased the 5hmC level 1.5-fold. However, when combined with AA, TSA amplifies the 5hmC level nearly 4-fold. Acetylation of lysine in the catalytic site inactivates TET via structural perturbations. Therefore, activation of class III HDACs should activate TET2 and inhibition should inhibit it. Indeed, consistent with this presumption, in leukemia cells, we observed that activation of sirtuin, a class III HDAC, by small molecule SRT1720, significantly increased 5hmC while the inhibitors 3-TYP and sirtnol reduced 5hmC. Context-dependent TET2 acetylation/deacetylation significantly affects its AA-mediated activation, and thus MDS/MPN clones with dysregulation of histone acetyltransferases and/or histone deacetylases may be less responsive to AA alone.
In sum, we demonstrate the mechanism and context of AA-mediated TET activation which may guide novel treatment strategies for TET2-deficient neoplasia. Our results show that TET2 activity can be modulated by various mechanisms including acetylation/deacetylation interactions with the effects of AA.

Disclosures
Nazha: MEI: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: 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, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.