ADAR1 promotes Okazaki fragment maturation and is a target for breast cancer therapy

To initiate lagging strand DNA synthesis, the polymerase α-RNA primase complex synthesizes RNA primers, which are composed largely of adenine (A) and cytosine (C) residues that start each of the millions of Okazaki fragments required for eukaryotic chromosome replication. RNA primers are synthesized in an error prone manner. Adenosine-to-inosine (A-to-I) modifications are common RNA editing events performed by adenosine deaminase acting on RNA (ADAR1). A consequence of A-to-I editing is that I is interpreted as guanine (G), functional A-to-G substitutions and correction of A:C mismatches within pre-mRNAs, mRNAs, and noncoding RNAs. There are two ADAR1 isoforms, ADAR1p150 and ADAR1p110, which are predominantly localized to the cytoplasm and nucleus, respectively. By modifying cytoplasmic double-stranded viral RNA, ADAR1 p150 suppresses interferon signaling. The functions of nuclear ADAR1p110, however, have been understudied. We have found a novel role for ADAR1p110 in editing of RNA primers during DNA replication. ADAR1p110 is overexpressed in breast cancer, particularly triple negative breast cancer (TNBC), an aggressive subset characterized by elevated levels of replication stress. Our preliminary data suggests that ADAR1p110 localizes with primase at nascent DNA, and that ADAR1p110 A to I editing of nascent DNA suppresses DNA gap formation and stabilizes the replication fork. Consistently, loss of endonucleases FEN1 and DNA2, key enzymes in Okazaki fragment maturation, were synthetic lethal with ADAR1p110. Poly (ADP-ribose) polymerase (PARP) inhibitors induce the accumulation of excessive single stranded DNA gaps in the lagging strand of replicating DNA in cancer cells, resulting in cancer cell death. PARP inhibition is approved for BRCA1 and BRCA2 associated breast cancers. However, many patients have inherent or acquired resistance, and PARP inhibition is not an option for patients without BRCA1 or BRCA2 alterations. We found that genetic deletion of ADAR1 p110 or inhibition of ADAR1p110 dimerization with a novel ADAR1p110 specific small molecule inhibitor identified through virtual screening resulted in DNA gaps and tumor specific hypersensitivity to PARP inhibition and other replication gap targeted therapy. Further, we have found that the multifunctional protein, APEX1, may regulate this new ADAR1p110 role. We hypothesize that ADAR1p110 stabilizes replication forks by editing mis- matched RNA:DNA hybrids and suppressing replication gaps and is a target to overcome resistance to breast cancer therapy. Aim 1 will investigate ADAR1p110 RNA primer editing and Okazaki fragment processing. Aim 2 will examine the molecular function of APEX1 in ADAR1p110 regulation. Aim 3 will evaluate ADAR1p110 as a target for therapeutically resistant breast cancer. Project Number: 1R01CA299196-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Robert Mutter (+1 co-PI) | Institution: MAYO CLINIC ROCHESTER, ROCHESTER, MN | Award Amount: $367,731 | Activity Code: R01 | Study Section: Mechanisms of Cancer Therapeutics B Study Section[MCTB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11315561