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.

项目摘要/摘要 PARP1和PARP2是DNA损伤诱导的聚ADP-ribose（PAR）转移，被募集到并被DNA断裂激活。活跃的PARP1和2自身和组蛋白以促进DNA修复和染色质松弛。目前，用于PARP1和PARP2的双特异性抑制剂（PARPI）目前用于 BRCA1/2缺陷乳房，卵巢，胰腺和前列腺癌的治疗。但是，严重的贫血发生在约三分之一的患者中，导致降低剂量和过早终止帕普里治疗。 parpi 还会引起明显的克隆造血作用，这种疾病会增加髓样发育不全和急性的风险髓样白血病（MDS/AML）。这些造血毒性是出乎意料的，因为PARP1的完全损失这是导致> 80％的DNA损伤诱导的细胞中的Parylation的影响，对造血没有影响。到了解PARPI的这种潜在靶向毒性，我们用催化敲击产生了小鼠模型 PARP2中的非活性突变 - 多ADP-核糖基化（Parylation）缺陷（E534A，PARP2EA）或单ADP- 核糖基化（玛丽化）缺乏（H404A，PARP2HA）。与PARP2 - / - 小鼠的正常发育相反，表达pary不足PARP2（PARP2EA/EA）的小鼠在胚胎第16.5天（E16.5）死亡，严重死亡红细胞生成的贫血和特定阶段。 PARP2HA/HA小鼠可行，但显示脾脏肿大和慢性贫血。同时，我们的细胞生物学分析表明，临床使用的PARPI有效失速在激光诱导的DNA损伤位点上的PARP2，但不是PARP1。基于这些和其他观察，我们假设PARP2具有依赖于良好的结构函数，优先阻止了nick 在DNA复制过程中的连接，是PARP抑制剂诱导的红细胞生成缺陷和贫血的基础。特别是，我们建议将催化性无活性PARP2捕获在DNA断裂中，尤其是5'Pho-Nicks，它们可以防止其他修复因子（例如，连接1）进入DNA划痕。相应地，就像 PARP2EA/EA小鼠，lig1 - / - 小鼠也屈服于E16.5的致命性贫血。为了检验我们的假设，我们将目标1）研究使用活细胞在DNA损伤部位调节PARP2募集和动态的机制成像和散装生化测定；目标2）确定催化无活性PARP2对其他DNA修复因子的募集和功能，以及体内和体外的DNA复制；目标3）特征表达催化无效的小鼠模型-PARP2并研究TRP53，CHK2，两个的损失如何基因相关的克隆造血，调节PARP2EA/EA模型中的贫血。完成提案将表征PARP2的先前未识别的结构功能不活动的PARP2损害了DNA复制并有选择地废除红细胞生成，提供了策略通过减少PARP2停滞，以减轻PARP抑制剂的这种靶向毒性。