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TET2- and TET3- dependence of vitamin C-induced epigenomic alterations in acute myeloid leukemia Wong, Jasper


Neomorphic mutations in isocitrate dehydrogenase 1/2 (IDH1/2) and inactivating mutations in ten eleven translocation dioxygenase 2 (TET2) are frequent and mutually exclusive events in de novo acute myeloid leukemia (AML). IDH1/2 mutations drive epigenomic dysfunction through production of the oncometabolite R-2-hydroxyglutarate (R-2HG), which inhibits the ability of TET2 to oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and causes DNA hypermethylation. Vitamin C (vitC) induces epigenetic reprogramming and cellular differentiation through TET re-activation in a murine leukemia model with mutant IDH1ᴿ¹³²ᴴ. Although TET mutations in AML occur exclusively in the TET2 gene, TET2 and TET3 are expressed at similar levels in de novo AML blasts. The role of TET3 in the regulation of myeloid activation and its role in AML is currently unknown. Based on the functional redundancy among TET enzymes, I hypothesize that the vitC-induced re-establishment of methylation homeostasis and differentiation in IDH1 mutant cells is also mediated through TET3. To delineate the contributions of TET2 and TET3 to vitC-induced reprogramming, we inactivated TET2 (TET2KO) and TET3 (TET3KO) individually or in combination (DKO) in HOXA9/IDH1ᴿ¹³²ᴴ (R132H) cells using CRISPR/Cas9. After 15 and 72 hours of vitC treatment, we sequenced total RNA and 5hmC/5mC-immunoprecipitated DNA. We showed that IDH1ᴿ¹³²ᴴ inhibition of TET is incomplete and that loss of either TET2 or TET3 down-regulates a set of 136 genes related to myeloid activation. A subset of the TET2/3-dependent genes can be rescued with vitC activation of the remaining TET, from which we identified PU.1, CEBPE, and RUNX1 as putative transcription factors. We identified a hypermethylation phenotype at enhancers in the context of the IDH1ᴿ¹³²ᴴ, which can be reversed through vitC treatment in a TET2- and TET3- dependent mechanism. Pathway analyses of nearby genes and genes up-regulated by vitC suggest a common myeloid differentiation pathway that both TET2 and TET3 can activate. To verify this, TET2KO and TET3KO but not DKO cells showed reduced proliferation and increased levels of myeloid differentiation markers from vitC treatment. These findings support my hypothesis and suggest a model in which vitC activates both TET2 and TET3 to reprogram the enhancer landscape of IDH1ᴿ¹³²ᴴ leukemic cells to drive differentiation.

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