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

描述(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，但对大多数患者来说，急性髓系白血病(AML)仍然是一种致命的疾病。研究表明，依赖于DNA甲基转移酶(DNMT)的DNA高甲基化介导的肿瘤抑制基因(TSG)沉默在AML的发生发展中起重要作用。氮杂核苷，即地西他滨，最近已被FDA批准为去甲基化药物，并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血液系统恶性肿瘤。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甲基化的调控机制，从而作为开发可用于减轻疾病过程的治疗方案的先决条件。这项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低甲基化药物(DNMTi)。这项研究背后的具体假设是，生物活性食品成分胸腺醌(TQ)可能通过调节甲基化调节因子Sp1/NFkB/miR网络实现更高的DNA去甲基化效率。这一假说基于以下观察：1)miR29b直接破坏DNMT3a/3b，并通过损害其反式激活因子Sp1间接取消DNMT1，从而导致DNA低甲基化。2)Sp1/NFkB复合体抑制miR29b的表达，并与DNMT水平呈正相关，提示DNA甲基化受蛋白质-miR网络的控制，涉及NFkB活性、Sp1/NFkB复合体、Dnmts和miRs。3)植物源性药物显示出有效的抗白血病活性，具有巨大的治疗潜力，而传统药物副作用较大；4)TQ是一种生物活性成分，具有抗癌作用，对正常细胞的毒性最小，显著阻断了NFkB信号通路。基于这些观察，这项建议的实验重点是TQ在体外和体内的低甲基化作用。具体目的是为了全面了解TQ的作用机制(S)(S)，并优化有效治疗白血病的TQ的剂量和给药方案。本研究的具体目的是：1.通过研究TQ通过Sp1/NFkB/miR29b网络在AML细胞和患者原代细胞中发挥DNA去甲基化作用，阐明TQ的抗白血病作用机制。我们将使用1)Western印迹和定量聚合酶链式反应(QPCR)，2)LC/MS/MS和3)MTS和PI/AV染色，在体外证明TQ对Sp1/NFkB/miR29b调控网络的药理学修饰可以诱导DNA低甲基化。2.对TQ在白血病小鼠模型中的药效学和药动学活性进行临床前体内评价。我们将i)确定TQ的有效药理剂量和给药方案，以调节失衡的Sp1/NFkB/miR29b网络，从而通过Western印迹、qPCR和LC/MS/MS确定体内DNA低甲基化；ii)测定TQ的血浆和细胞内PK参数，并将这些参数与PD和临床疗效终点相关联；iii)以生存期衡量临床疗效。该项目将由在翻译研究(刘)和PK/PD研究(Chan)方面有专长的调查人员通过跨学科方法进行。如果成功，新的DNMTI可以应用于实体瘤或非增殖性恶性肿瘤，这一研究将有助于我们理解特定生物活性食物成分介导的表观遗传学变化，Sp1/NFkB，miR和DNA甲基化在白血病发生中的作用，TQ作用的分子途径，并帮助我们充分评估个体营养的特异性。

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.