TET2 loss triggers ssDNA gap accumulation and heightened PARP inhibitor sensitivity.

Somatic mutations in the de novo methyltransferase DNMT3A and cytosine demethylase TET2 are common in age-related clonal hematopoiesis and hematopoietic neoplasms. TET2 mutations, unlike DNMT3A mutations, increase sensitivity to PARP inhibitors (PARPi). We find that TET2 loss, but not DNMT3A loss, alters DNA replication fork progression upon treatment with PARPi Olaparib, resulting in longer DNA replication tracts and increased DNA breaks. This reveals a new link between abnormal fork elongation and PARPi sensitivity in TET2-deficient cells. The elongated forks of TET2-deficient cells contain PRIMPOL-dependent single-stranded (ss)-DNA gaps. This phenotype is dependent on the catalytic activity of TET2 and is mitigated by TET1/3 depletion. Furthermore, TET2 loss causes an imbalance in cytosine methylation and an increase in 5hmU modifications. These modifications are processed by SMUG1, creating abasic sites, detectable via a modified DNA fiber method. A CRISPR screen identified the base excision repair nuclease APE1 as linked to increased PARPi sensitivity in TET2-deficient cells. We propose that abasic sites, skipped by PRIMPOL repriming, lead to ssDNA gaps that are converted into DNA breaks by APE1, increasing PARPi toxicity. Finally, we identify TET2 mutations as an unrecognized predictor of PARPi response in bone marrow samples from patients with myeloid malignancies.