DISCOVERY OF SMALL MOLECULAR ACTIVATORS OF NR4A ORPHAN NUCLEAR RECEPTORS

描述(由申请人提供)：这项提案的总体目标是通过激活NR4A亚类孤儿核受体来寻找治疗急性髓系白血病(AML)的新型抗白血病药物。急性髓系白血病主要是髓系细胞发育的正常转录控制的腐败疾病，导致未成熟母细胞的聚集和具有长期自我更新特性的转化的白血病启动细胞(LIC)群体的出现，能够维持白血病的扩张。NR4A1和NR4A3是AML的有效肿瘤抑制基因。它们在所有人类急性髓系白血病LIC中都是沉默的，与患者的细胞遗传学无关。由于造血干细胞(HSC)动态平衡的破坏和转化为抗辐射的LIC的出现，小鼠的缺失导致出生后极快的AML。此外，急性挽救人类AML细胞中的NR4A1或NR4A3会抑制它们的增殖，并对区分所有原发人类LIC与正常HSC的基因特征子集进行重新编程，而不考虑细胞遗传学。我们假设，NR4A沉默是AML发展过程中的一个必要步骤，而针对其重新激活的策略可能在AML的治疗中具有普遍的治疗益处。通过将我们基于NR4A靶标的基因组数据与电子化学基因组学筛选相结合，我们采用了一种新的整合策略，成功地识别了在AML细胞中重新激活NR4A的小分子，并可用于揭示NR4A沉默的机制。我们在这项建议中的具体目标是：1)通过分析NR4A在人AML细胞中重新激活的细胞和分子后果，进一步验证NR4A作为治疗人AML的新的治疗靶点；2)通过分析NR4A小分子激活剂在AML细胞系、AML患者原代细胞和人类AML动物模型中消除LICs的有效性，以及通过比较它们对正常人HSC的影响来检测它们对AML的选择性，以及3)揭示NR4A在AML细胞中沉默和重新激活的机制。为此，我们将结合小分子激活剂的信息通路分析和NR4A基因染色质图谱的分子分析来识别AML中NR4A沉默的标志和介体，并揭示参与其重新激活的信号通路。鉴于NR4A在AML患者中的广泛沉默以及它们在引起AML方面的充分失活，我们相信这些研究在新药发现方面的成功结果将很快导致新的临床试验，这将对AML患者的治疗产生重大影响。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to identify novel antileukemic drugs for treatment of acute myeloid leukemias (AMLs) via activation of the NR4A subclass of orphan nuclear receptors. AMLs are primarily diseases of corruption of normal transcriptional control of myeloid cell development leading to accumulation of immature blasts and emergence of a transformed leukemic initiating cell (LIC) population with long term self renewal properties capable of sustaining leukemic expansion. NR4A1 and NR4A3 are potent tumor suppressors of AML. They are silenced in all human AML LICs irrespective of patient cytogenetics. Deletions in mice lead to extremely rapid postnatal AML due to disruption of hematopoietic stem cell (HSC) homeostasis and the emergence of a transformed radio-resistant LIC. Further, acute rescue of NR4A1 or NR4A3 in human AML cells inhibits their proliferation and reprograms a subset of gene signatures that distinguish all primary human LICs from normal HSCs regardless of cytogenetics. We hypothesize that NR4A silencing is an obligate step in AML development and that strategies directed toward their reactivation may be of general therapeutic benefit in treatment of AMLs. By intersection of our NR4A target based genomics data with in silico chemical genomics screening, we have employed a novel integrative strategy to successfully identify small molecules that reactivate NR4As in AML cells and that can be used to disclose mechanisms of NR4A silencing. Our specific aims in this proposal are: 1) to further validate NR4As as novel therapeutic targets for treatment of human AMLs by analysis of the cellular and molecular consequences of NR4A reactivation in human AML cells, 2) to test the anti-leukemic properties of small molecule activators of NR4As in human AML cells by analysis of their efficacy in elimination of LICs using AML cell lines, primary cells from AML patients and animal models of human AML and by interrogation of their AML selectivity by comparison of their effects on normal human HSCs, and 3) to disclose mechanisms of silencing and reactivation of NR4As in AML cells. In this aim, we will combine informatics pathway analysis of small molecule activators with molecular analysis of the chromatin landscape of the NR4A genes to identify marks and mediators of NR4A silencing in AML and to disclose signaling pathways involved in their reactivation. Given the widespread silencing of NR4As in AML patients and the sufficiency of their inactivation in causing AML, we believe that a successful outcome from these studies in terms of new drug discovery will quickly result in new clinical trials that will have major impact on treatment of AML patients.