Oxidation of thymine by TET2 dioxygenase carrying leukemia-derived neomorphic mutations

TET enzymes oxidize 5-methylcytosine (mC) in DNA, contributing to the regulation of gene transcription and genome functions. Deficiency of TET2 promotes the development of clonal hematopoiesis and various blood cancers, yet the mechanism by which TET2 mutations facilitate tumorigenesis remains to be explored. Here, we report that a subset of TET2 mutations identified in leukemia patients alter the substrate specificity of TET2 from acting on mC to thymine. This neomorphic activity results from substitutions at key residues involved in the interactions with the mC base, including Asn1387 and His1904. Recombinant human TET2 proteins harboring the mutation of these residues can catalyze the oxidation of thymine to 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU). Exogenous expression of the TET2 Asn1387Thr (N1387T) mutant in transfected cells leads to hmU accumulation, with the highest level in cells lacking SMUG1-initiated base excision repair pathway. Endogenous knock-in of N1300T, the murine equivalent of N1387T, in mouse embryonic stem cells induces hmU production, causing DNA lesions and transcriptional activation of DNA damage response genes. These findings suggest that certain patient-derived TET2 mutations acquire unexpected gain-of-functions beyond an impairment in mC oxidation, and thymine oxidation by the neomorphic mutants may impose risks to genome stability, presumably contributing to malignant transformation.