Bioactive compound modulation of epigenetic regulator Sp1/NFkB/miR network in AML

AML 中表观遗传调节因子 Sp1/NFkB/miR 网络的生物活性复合调节

基本信息

批准号：
8207209
负责人：
Shujun Liu
金额：
$ 16.58万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8207209
关键词：
Aberrant DNA Methylation Acute Myelocytic Leukemia Address Adult Adult Acute Myeloblastic Leukemia Adverse effects Animal Model Antineoplastic Agents Apoptosis Applications Grants Area Attention Attenuated Binding Biological Blast Cell Blood Cells Bone Marrow Cancer cell line Cell Cycle Cell Line Cell Proliferation Cells Childhood Acute Myeloid Leukemia Clinic Clinical Clinical Research Clinical Trials Complex DNA DNA Methylation DNA Methyltransferase DNA Modification Methylases DNMT3a Decitabine Dependence Development Disease Disease Management Disease Outcome Disease remission Dose Drug Kinetics Drug resistance Effectiveness Epigenetic Process Evaluation Event Feedback Funding Genes Genetic Transcription Goals Health Hematopoietic Neoplasms Histone Deacetylation Hypermethylation Impairment In Vitro Individual Intervention Investigation Link Maintenance Malignant - descriptor Malignant Neoplasms Measures Mediating Medicinal Plants Medicine Methylation MicroRNAs Minority Modeling Modification Molecular Molecular Biology Mus Myelogenous Non-Malignant Normal Cell Nutrient Outcome Pancytopenia Pathway interactions Patients Pattern Pharmaceutical Preparations Pharmacodynamics Plant Extracts Plants Plasma Play Positioning Attribute Process Proliferating Property Protein Kinase Proteins Protocols documentation Regulatory Pathway Reporting Research Research Personnel Role Sampling Schedule Seeds Signal Pathway Signal Transduction Solid Neoplasm Specificity Staining method Stains Structure Testing Therapeutic Therapeutic Uses Toxic effect Trans-Activators Translating Translational Research Tumor Suppressor Genes United States Food and Drug Administration Volatile Oils Western Blotting Work base bioactive food component cancer cell cancer therapy clinical efficacy design early onset experience improved in vitro testing in vivo inhibitor/antagonist innovation interdisciplinary approach interest leukemia leukemogenesis liquid chromatography mass spectrometry mouse model new therapeutic target novel novel therapeutics pre-clinical preclinical study research study response therapeutic development therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Despite progress made in understanding the biological mechanisms of myeloid leukemogenesis, acute myeloid leukemia (AML) remains a deadly disease for most of the patients. It is well documented that silencing of tumor suppressor genes (TSGs) mediated by aberrant DNA methyltransferase (DNMT)-dependent DNA hypermethylation plays a critical pathogenic role in the development and progression of AML. Azanucleosides, i.e., decitabine, have been recently approved by FDA as hypomethylating agents and positive clinical outcome has been achieved for some patients. However, the clinical response is restricted to a minority of hematopoietic malignancies. Hence, further studies are urgently required to explore novel therapeutic strategies or agents to overcome the dismal outcome. Our long-term goals are to elucidate the regulatory mechanisms controlling DNA methylation thereby leukemogenesis as a prerequisite to the development of therapeutic protocols that can be used to attenuate the disease process. The objectives of this grant application are to explore novel DNA hypomethylation agents (DNMTi) with diverse structures through distinct mechanisms from decitabine. The specific hypothesis behind the proposed research is that bioactive food component Thymoquinone (TQ) may achieve higher efficacy of DNA hypomethylation through modulating methylation regulator Sp1/NFkB/miR network. That hypothesis is based on the following observations: 1) miR29b directly disrupts DNMT3a/3b and indirectly abolishes DNMT1 via impairment of its transactivator Sp1, thereby leading to DNA hypomethylation. 2) Sp1/NFkB complex suppresses miR29b expression and positively correlates to DNMT level in AML cell lines and patient samples, suggesting that DNA methylation is under control of a protein-miR network involving NFkB activity, Sp1/NFkB complex, DNMTs and miRs. 3) plant-derived drugs displayed efficiently anti-leukemic activities with huge therapeutic potentials, while the conventional medicine has lots of side effects, 4) TQ is a bioactive constituent and acts as anticancer agent, with minimal level of toxicity to normal cells, by significantly blocking NFkB signaling pathways. Based on these observations, the experimental focus of this proposal is on the hypomethylating effect of TQ in vitro and in vivo. The specific aims are designed to provide a comprehensive understanding of the mechanism(s) of TQ action(s) and to optimize the dose and schedule of administration of TQ effective against leukemic disease. The specific aims of this application are to: 1. Elucidate the mechanism of TQ antileukemic activity by the demonstration that TQ functions as DNA hypomethylation agent through Sp1/NFkB/miR29b network in AML cell lines and patient primary blasts. We will demonstrate that pharmacological modification of Sp1/NFkB/miR29b regulatory network by TQ induces DNA hypomethylation in vitro using i) Western blot and quantitative PCR (qPCR), ii) LC/MS/MS and iii) MTS and PI/AV staining. 2. Perform preclinical in vivo evaluation of the pharmacodynamic and pharmacokinetic activity of TQ in leukemic mice models. We will i) define the effective pharmacological dose and schedule of TQ administration that will modulate the misbalanced Sp1/NFkB/miR29b network thereby DNA hypomethylation in vivo using Western blot, qPCR, and LC/MS/MS, ii) determine the plasma and intracellular PK parameters of TQ and correlate these with PD and clinical efficacy endpoints, iii) determine the clinical efficacy measured by survival duration. This project will be carried out through an interdisciplinary approach by investigators with expertise in translational research (Liu) and PK/PD studies (Chan). If successful, the novel DNMTi can be applied to solid tumor or non-proliferating malignancies, this investigation may advance our understanding of epigenetic changes mediated by specific bioactive food component, the roles of Sp1/NFkB, miR and DNA methylation in leukemogenesis, the molecular pathways of TQ action and help us to adequately evaluate the specificity of individual nutrient.

描述（由申请人提供）：尽管在了解髓系白血病发生的生物学机制方面取得了进展，但急性髓系白血病（AML）对于大多数患者来说仍然是一种致命的疾病。有充分证据表明，异常 DNA 甲基转移酶 (DNMT) 依赖性 DNA 高甲基化介导的肿瘤抑制基因 (TSG) 沉默在 AML 的发生和进展中发挥着关键的致病作用。氮杂核苷，即地西他滨，最近已被 FDA 批准作为低甲基化药物，并且已经在一些患者中取得了积极的临床结果。然而，临床反应仅限于少数造血系统恶性肿瘤。因此，迫切需要进一步的研究来探索新的治疗策略或药物来克服令人沮丧的结果。我们的长期目标是阐明控制 DNA 甲基化从而控制白血病发生的调节机制，这是开发可用于减轻疾病过程的治疗方案的先决条件。本次拨款申请的目的是通过与地西他滨不同的机制探索具有不同结构的新型 DNA 低甲基化剂 (DNMTi)。这项研究背后的具体假设是，生物活性食品成分百里醌 (TQ) 可能通过调节甲基化调节因子 Sp1/NFkB/miR 网络来实现更高的 DNA 低甲基化功效。该假设基于以下观察结果：1) miR29b 直接破坏 DNMT3a/3b，并通过损害其反式激活蛋白 Sp1 间接废除 DNMT1，从而导致 DNA 低甲基化。 2) Sp1/NFkB 复合物抑制 miR29b 表达，并与 AML 细胞系和患者样本中的 DNMT 水平呈正相关，表明 DNA 甲基化受到涉及 NFkB 活性、Sp1/NFkB 复合物、DNMT 和 miR 的蛋白质-miR 网络的控制。 3) 植物源药物显示出高效的抗白血病活性，具有巨大的治疗潜力，而传统药物有很多副作用。 4) TQ 是一种生物活性成分，可作为抗癌剂，对正常细胞的毒性极小。显着阻断 NFkB 信号通路。基于这些观察，本提案的实验重点是TQ在体外和体内的低甲基化作用。具体目标旨在提供对 TQ 作用机制的全面了解，并优化有效对抗白血病的 TQ 剂量和给药方案。本申请的具体目的是： 1. 通过证明 TQ 在 AML 细胞系和患者原代母细胞中通过 Sp1/NFkB/miR29b 网络发挥 DNA 低甲基化剂的作用，阐明 TQ 抗白血病活性的机制。我们将使用 i) 蛋白质印迹和定量 PCR (qPCR)、ii) LC/MS/MS 和 iii) MTS 和 PI/AV 染色证明 TQ 对 Sp1/NFkB/miR29b 调控网络的药理学修饰可在体外诱导 DNA 低甲基化。 2. 对TQ在白血病小鼠模型中的药效学和药代动力学活性进行临床前体内评价。我们将 i) 确定 TQ 给药的有效药理学剂量和时间表，以调节不平衡的 Sp1/NFkB/miR29b 网络，从而使用蛋白质印迹、qPCR 和 LC/MS/MS 来调节体内 DNA 低甲基化，ii) 确定血浆和细胞内TQ 的 PK 参数并将这些参数与 PD 和临床疗效终点相关联，iii) 确定通过生存持续时间衡量的临床疗效。该项目将由具有转化研究 (Liu) 和 PK/PD 研究 (Chan) 专业知识的研究人员通过跨学科方法进行。如果成功，新型 DNMTi 可应用于实体瘤或非增殖性恶性肿瘤，这项研究可能会增进我们对特定生物活性食物成分介导的表观遗传变化、Sp1/NFkB、miR 和 DNA 甲基化在白血病发生中的作用、分子TQ 作用途径并帮助我们充分评估单个营养素的特异性。