The non-catalytic function of PARP2 in DNA repair and cancer therapy

PARP2在DNA修复和癌症治疗中的非催化功能

基本信息

批准号：
10540084
负责人：
Shan Zha
金额：
$ 59.71万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10540084
关键词：
ADP Ribose Transferases ADP ribosylation Acute Myelocytic Leukemia Address Adult Affect Affinity Anemia Animals BRCA deficient BRCA1 gene BRCA2 gene Base Excision Repairs Binding Binding Proteins Biochemical Biochemistry Biological Assay Bone Marrow CHEK2 gene Cancer Patient Cells Cellular biology Charge Chromatin Chronic Cisplatin Clinical Clonal Expansion DNA DNA Binding DNA Damage DNA Ligases DNA Repair DNA Repair Gene DNA biosynthesis DNA lesion Defect Development Dose Dysplasia Embryo Erythropoiesis Flow Cytometry Fluorescence Genes Genetic Epistasis Goals Hematopoiesis Hematopoietic Histones Hypersensitivity Impairment In Vitro Knock-in Knockout Mice Lasers Ligase Ligation Link Malignant neoplasm of ovary Malignant neoplasm of pancreas Malignant neoplasm of prostate Mediating Modeling Molecular Biology Mus Mutation Myelogenous Okazaki fragments Patients Photobleaching Physical condensation Poly Adenosine Diphosphate Ribose Proteins Relaxation Risk Role S phase Site Specificity Splenomegaly TP53 gene Testing Toxic effect Withdrawal XRCC1 gene base brca gene cancer cell cancer therapy chemotherapy effective therapy in vivo inhibitor live cell imaging malignant breast neoplasm mouse genetics mouse model novel premature prevent recruit repaired

项目摘要

PROJECT SUMMARY/ABSTRACT PARP1 and PARP2 are DNA damage-induced poly-ADP-ribose (PAR) transferases, which are recruited to and activated by DNA breaks. Active PARP1&2 PARylate themselves and histones to promote DNA repair and chromatin relaxation. Dual-specificity inhibitors (PARPi) for PARP1 and PARP2 are currently used for the treatment of BRCA1/2-deficient breast, ovarian, pancreatic, and prostate cancers. However, severe anemia occurs in ~ one-third of patients, leading to dose reduction and premature termination of PAPRi therapy. PARPi also cause significant clonal hematopoiesis, a condition that increases the risk for myeloid dysplasia and acute- myeloid leukemia (MDS/AML). These hematopoietic toxicities are unexpected since the complete loss of Parp1 that is responsible for >80% of DNA damage-induced PARylation in cells, has no impact on hematopoiesis. To understand this potential on-target toxicity of PARPi, we generated mouse models with knockin catalytically inactive mutations in Parp2 – Poly-ADP-Ribosylation (PARylation) deficient (E534A, Parp2EA) or Mono-ADP- Ribosylation (MARylation) deficient (H404A, Parp2HA). In contrast to the normal development of Parp2-/- mice, mice expressing PARylation deficient PARP2 (Parp2EA/EA) died at embryonic day 16.5 (E16.5) with severe anemia and stage-specific blocks in erythropoiesis. The Parp2HA/HA mice are viable but display splenomegaly and chronic anemia. Meanwhile, our cell biology analyses suggest that clinically used PARPi effectively stalls PARP2, but not PARP1, on laser-induced DNA damage sites. Based on these and other observations, we hypothesize that PARP2 has PARylation-dependent structural functions that preferentially block nick ligation during DNA replication, underlie the PARP inhibitors-induced erythropoiesis defects and anemia. Specifically, we propose that catalytically inactive PARP2 were trapped on DNA breaks, especially 5’pho-nicks, where they prevent other repair factors (e.g., Ligase1) from accessing the DNA nicks. Correspondingly, like the Parp2EA/EA mice, Lig1-/- mice also succumbed to lethal anemia at E16.5. To test our hypothesis, we will Aim 1) investigate the mechanism that regulates PARP2 recruitment and dynamics at DNA damage sites using live-cell imaging and bulk biochemical assays; Aim 2) determine the impacts of catalytically inactive PARP2 on the recruitment and function of other DNA repair factors, and DNA replication in vivo and in vitro; Aim 3) characterize the mouse models expressing catalytically inactive - Parp2 and investigate how the loss of Trp53, CHK2, two genes associated clonal hematopoiesis, modulates the anemia in Parp2EA/EA models. The completion of the proposal will characterize the previously unrecognized structural functions of PARP2, address how catalytically inactive PARP2 compromises DNA replication and selectively abrogates erythropoiesis, providing the strategy to mitigate this on-targeted toxicity of PARP inhibitors by reducing PARP2 stalling.

项目概要/摘要 PARP1 和 PARP2 是 DNA 损伤诱导的聚 ADP 核糖 (PAR) 转移酶，它们被招募活性 PARP1&2 自身和组蛋白 PARylate 并被 DNA 断裂激活，以促进 DNA 修复。 PARP1 和 PARP2 的双特异性抑制剂 (PARPi) 目前用于治疗 BRCA1/2 缺陷型乳腺癌、卵巢癌、胰腺癌和前列腺癌，但可治疗严重贫血。大约三分之一的患者会发生这种情况，导致 PAPRi 治疗剂量减少和提前终止。还会引起显着的克隆性造血，这种情况会增加骨髓发育不良和急性白血病的风险由于 Parp1 完全丧失，这些造血毒性是出乎意料的。它负责细胞中超过 80% 的 DNA 损伤诱导的 PARylation，对造血没有影响。为了了解 PARPi 的这种潜在的靶向毒性，我们通过催化敲入生成了小鼠模型 Parp2 中的失活突变 – 聚 ADP 核糖基化 (PARylation) 缺陷（E534A、Parp2EA）或单 ADP- 核糖基化（MARylation）缺陷（H404A、Parp2HA）与 Parp2-/- 小鼠的正常发育相反，表达 PARylation 缺陷的 PARP2 (Parp2EA/EA) 的小鼠在胚胎第 16.5 天 (E16.5) 死亡，患有严重的 Parp2HA/HA 小鼠可存活，但出现脾肿大。同时，我们的细胞生物学分析表明，临床使用的 PARPi 可以有效地阻止贫血。基于这些和其他观察，我们对激光诱导的 DNA 损伤位点进行了 PARP2（而非 PARP1）。坚持认为 PARP2 具有 PARylation 依赖性结构功能，可优先阻断切口 DNA 复制期间的连接是 PARP 抑制剂诱导的红细胞生成缺陷和贫血的基础。具体来说，我们建议催化失活的 PARP2 被困在 DNA 断裂处，尤其是 5'pho 缺口，它们相应地阻止其他修复因子（例如连接酶 1）访问 DNA 缺口。 Parp2EA/EA 小鼠、Lig1-/- 小鼠也在 E16.5 时死于致命性贫血。为了检验我们的假设，我们将目标 1)。使用活细胞研究调节 DNA 损伤位点 PARP2 募集和动态的机制成像和批量生化检测；目标 2) 确定催化失活的 PARP2 对其他 DNA 修复因子的募集和功能，以及 DNA 体内和体外复制的特征；表达催化失活的小鼠模型 - Parp2 并研究 Trp53、CHK2、两个与克隆造血相关的基因，调节 Parp2EA/EA 模型中的贫血。该提案将描述 PARP2 以前未被认识的结构功能，解决如何催化失活的 PARP2 会损害 DNA 复制并选择性地消除红细胞生成，从而提供策略通过减少 PARP2 停滞来减轻 PARP 抑制剂的这种靶向毒性。