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Poly ADP-ribose polymerase PARP1 and PARP2-catalyzed ADP-ribose polymerization (PARylation) is involved in a variety of physiological processes, especially in the process of DNA damage repair. There is a synergistic effect between two different DNA damage repairs, mediated by PARP1/2 and BRCT1/2, which together ensure the integrity of the tissue cell genome. Based on the concept of synthetic lethality, PARP inhibitors have been widely used in clinical practice to target the treatment of BRCA1/2 mutated cancers, which can specifically kill BRCA1/2 mutated cancer cells without affecting normal cells. Currently, the FDA has approved four PARP inhibitors for the treatment of BRCA1/2 mutated cancers. While they have shown great potential in treatment, their use has recently been limited due to unexplained serious hematologic side effects, including anemia and drug-related leukemia. In 2022, two of these PARP inhibitors were withdrawn from the FDA for maintenance therapy because they cause severe anemia and increase the risk of treatment-associated myeloid leukemia.
The four PARP inhibitors currently used in clinical practice are dual inhibitors of PARP1 and PARP2. Not only were they able to inhibit the enzymatic activity of both PARP1 and PARP2, but they were also able to trap PARP1 and PARP2 at sites of DNA damage. Numerous studies have shown that retention of PARP1 is critical for the efficacy of PARP inhibitors in cancer treatment. Knockdown of PAPR1 greatly reduces the sensitivity of cancer cells to PARP inhibitors. However, knockdown of PARP2 had no effect. There is still a lack of effective research on the physiological consequences of PARP2 inactivation.
2024年10月8日，来自美国的哥伦比亚大学查珊团队在Molecular Cell期刊上发表了题为Inactive Parp2 causes Tp53-dependent lethal anemia by blocking replication-associated nick ligation in erythroblasts的研究论文，文章表明失去活性的PARP2可能是引起PARP抑制剂相关血液学副作用的因子。
To investigate the physiological effects of PAPR2 inactivation, the researchers introduced a loss-of-activity mutation (PARP2-E534A) in situ on mouse chromosome PARP2 by homology recombination. Inactivation of E534A not only effectively inhibits Parp2 activity, but also retains Parp2 at DNA damage sites. It is a good simulation of Parp2 under the action of PARP inhibitors. The results of the study showed that Parp2 inactivation led to the lethality of mouse embryos. Further analysis of embryonic development showed that the embryos of Parp2EA/EA were able to develop normally to about E13.5. However, embryos at this stage exhibit significant erythropoiesis disorders – compared to wild-type embryos, Parp2EA/EA embryos lack blood color and are smaller and more miserable white fetal liver (the organ that produces red blood cells during the embryonic stage). By way of comparison, mice with complete Parp2 deletion (Parp2-/-) were able to be born normally, and the embryos of E13.5 did not have significant erythrocyte defects. Inactivating PARP2, rather than its deletion, has been shown to be a key factor in the development of fatal anemia in mice.
Studies have also shown that inactivated PARP2 interferes with DNA replication, triggers genomic instability, and leads to cell cycle arrest and apoptosis through Tp53 and Chk2-dependent pathways. Knockdown of Tp53 or Chk2 reverses these phenotypes and rescues embryos.
Interestingly, the team's previous study, published in PNAS in July 2023, noted that inactivated PARP1 also led to the death of mouse embryos. However, the embryo disappeared completely before E9.5, well before the time of embryonic erythropoiesis (E11.5). Inactivating PARP2 specifically impedes erythropogenesis. This is related to the different DNA substrate binding activities of PARP1 and PARP2: PARP1 can bind to a variety of DNA structures through its N-terminal 3 zinc finger structure and WGR domain, including single-stranded DNA break (SSB), double-stranded DNA break (DSB), etc.; However, the N-terminus of PARP2 contains only one DNA-binding domain, the WGR domain. PARP2 is only able to bind to and be activated by the 5'-phosphorylated DNA crack (5'p-Nick). In normally replicating cells, the large amount of 5'p-Nick produced during the maturation of the Okazaki fragment of the DNA lagging strand needs to be ligated into intact DNA by the DNA ligase LIG1. In vitro 5'p-Nick DNA ligation assays showed that PARP2 with loss of activity inhibited LIG1-mediated ligation. In vivo super-resolution fluorescence imaging has also shown that inactivated PARP2 prevents LIG1 from binding to replicating DNA. Interestingly, mice with LIG1 deletion also exhibited a similar embryonic lethal anemia phenotype, but their phenotype was slightly weaker than that of Parp2EA/EA. Further studies have shown that Parp2-EA can inhibit not only LIG1-mediated junction, but also LIG3-mediated junction. LIG3 has also been shown to be able to participate in the joining of Okazaki fragments in the absence of LIG1. "Inactivated PARP2 interferes with key DNA break ligations mediated by LIG1 and LIG3, causing the replication fork to collapse," the researchers stated. "This collapse is particularly devastating in erythrocyte blasts with ultra-fast replication fork velocities." By measuring the PARylation levels of different tissue cells, it was found that although DNA damage usually preferentially activates PARP1, replication stress predominantly activates PARP2, revealing a previously unknown replication-specific role of PARP2 in erythropoiesis.
In summary, inactivated PARP2 selectively binds and stays at 5'p Nick in an allosteric manner, interfering with Nick junction mediated by LIG1 and LIG3, including the maturation of the Okazaki fragment. In erythroblasts that rely on fast replication of Okazaki fragments in a timely manner, the presence of inactivated PARP2 triggers replication fork collapse, DNA strand breaks, and ultimately Tp53- and Chk2-dependent checkpoint activation, apoptosis, and fatal anemia. Patients receiving clinical maintenance therapy with PARP inhibitors may have an increased risk of leukemia due to long-term hematopoietic pressure due to increased acquired Tp53 and Chk2 mutations (Fig. 1). This study provides a mechanistic explanation for the severe hematologic toxicity associated with current PARP inhibitors, and reveals the functional differences between PARP1 and PARP2 in DNA repair and replication, providing a new idea for further optimizing the therapeutic window of PARP inhibitors and other DNA damage response inhibitors.
模式图（Credit: Molecular Cell）
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https://www.cell.com/molecular-cell/abstract/S1097-2765(24)00776-7
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