Research ArticleMOLECULAR BIOLOGY

Degradation of 5hmC-marked stalled replication forks by APE1 causes genomic instability

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Science Signaling  18 Aug 2020:
Vol. 13, Issue 645, eaba8091
DOI: 10.1126/scisignal.aba8091

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TET2 and 5hmC mark the forks for APE1

Inhibitors of the enzyme PARP, commonly used to treat various tumors, block the repair and restart of stalled replication forks, which eventually kills cancer cells. Kharat et al. found that the DNA demethylase TET2—which is associated with epigenetic control of transcription—was critical for sensitivity to PARP inhibitors by promoting repair-mediated degradation of stalled forks. The TET2 product 5-hydroxymethylcytosine (5hmC) at stalled replication forks served as a recruitment marker for the base excision repair–associated endonuclease APE1. Without TET2, stalled replication forks were not degraded but were stabilized, thereby not only reducing PARP inhibitor sensitivity but also contributing to genomic instability. The findings reveal a function for DNA methylation and demethylation dynamics in genome integrity mechanisms and suggest that PARP inhibitors may not be effective in TET2-deficient tumors.

Abstract

Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.21 mouse mammary tumor cells. We found that resistance to multiple PARPi emerged with reduced expression of TET2 (ten-eleven translocation), which promotes DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC) and other products. TET2 knockdown in BRCA2-deficient cells protected stalled replication forks (RFs). Increasing 5hmC abundance induced the degradation of stalled RFs in KB2P1.21 and human cancer cells by recruiting the base excision repair–associated apurinic/apyrimidinic endonuclease APE1, independent of the BRCA2 status. TET2 loss did not affect the recruitment of the repair protein RAD51 to sites of double-strand breaks (DSBs) or the abundance of proteins associated with RF integrity. The loss of TET2, of its product 5hmC, and of APE1 recruitment to stalled RFs promoted resistance to the chemotherapeutic cisplatin. Our findings reveal a previously unknown role for the epigenetic mark 5hmC in maintaining the integrity of stalled RFs and a potential resistance mechanism to PARPi and cisplatin.

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