Targeting MicroRNAs to Eradicate Leukemia Stem Cells

项目总结 白血病干细胞(LSCs)位于急性髓系白血病(AML)细胞层次结构的顶端。这个 LSCs的静止部分提供了一个自我更新细胞的储存库，维持白血病的生长，防止克隆 因此，消除LSCs是任何抗白血病治疗的“圣杯”。 在以前的研究中，我们表明miR-126对于维持LSCs的静止亚组分是必要的， 防止克隆耗尽。我们演示了SPRED1/miR-126如何在LSC和BM中进行自动调节循环 内皮细胞(ECs)聚集以增加LSCs中miR-126的水平，保护它们并支持白血病的生长。 我们表明，高miR-126水平是由于这两种LSC自主机制，导致增强 来自内皮细胞的外源miR-126提供的内源性生产和非自主机制。 为了去除LSCs和ECs中的miR-126，我们设计了一种新型的寡脱氧核苷酸反义miR-126抑制剂，称为 米尔斯滕。我们的数据显示，miRisten对miR-126的药理学剥夺显著降低了LSC 内源性miR-126的产生和减少内皮细胞miR-126的外源性供应。最终结果是 是miR-126的显著减少，破坏了LSCs的动态平衡和活性，如 一系列的移植实验。此外，我们现在有证据表明miR-126增强了线粒体 LSCs的代谢(即氧化磷酸化)和线粒体动力学(即线粒体融合) 通过SPRED1/ERK/p-bcl2/NRF2信号转导途径。因此，miR-126通过miRisten治疗而耗尽 显著下调bcl2，扰乱线粒体新陈代谢，导致反应性水平升高 氧物种与LSCs的凋亡。此外，miRisten通过上调LSC线粒体的表达来破坏LSC线粒体的功能 动力蛋白相关蛋白1(Drp1)，诱导线粒体分裂，降低线粒体膜 有丝分裂标记蛋白的潜在和诱导表达。由于以线粒体为中心的新陈代谢 LSCs的主要代谢能量来源，我们建议剖析miRisten如何利用线粒体 代谢易损性作为消除LSCs的新作用机制。此外，在进行了 研究性新药应用(IND)--支持药代动力学、药效学和毒理学研究； 我们将迅速将miRisten从替补席转移到旁边，这是美国首个人类1期临床试验 复发/难治(r/r)AML患者。这一提议的中心假设是miRisten针对miR- LSC的126依赖代谢易感性，将为消除LSC提供一种新的治疗方法 在AML中。我们提出了以下具体目标(SA)：SA#1：确定miRisten诱导的机制 腰椎干细胞中的线粒体代谢脆弱性。SA#2：进行药代动力学、药效学、疗效 以及miRisten的毒理学研究，为人体研究的剂量和时间表选择提供信息。SA#3：品行 MiRisten在r/r急性髓系白血病患者中的首个人类1期试验。该项目将把新发现转化为 通过进行临床前研究，最终实现了对miRisten的首次人体试验。

PROJECT SUMMARY Leukemia stem cells (LSCs) are at the apex of the acute myeloid leukemia (AML) cellular hierarchy. The quiescent fraction of LSCs provides a reservoir of self-renewing cells that sustain leukemia growth, prevent clonal exhaustion, and are treatment resistant; thus, eliminating LSCs is the `holy grail' of any anti-leukemia treatment. In previous studies, we showed that miR-126 is necessary to maintain a quiescent subfraction of LSCs that prevent clonal exhaustion. We demonstrated how SPRED1/miR-126 autoregulatory loop in LSCs and in BM endothelial cells (ECs) converge to increase miR-126 levels in LSCs, protect them and support leukemia growth. We showed that high miR-126 levels are due to both LSC autonomous mechanisms, resulting in enhanced endogenous production, and non-autonomous mechanisms, through exogenous miR-126 supply from ECs. To deplete miR-126 in LSCs and ECs, we designed a novel oligodeoxynucleotide anti-miR-126 inhibitor, called miRisten. Our data show that pharmacological miR-126 deprivation by miRisten significantly decreases LSC endogenous production of miR-126 and decreases the exogenous supply of endothelial miR-126. The net result is a significant decrease of miR-126 that damages the homeostasis and activity of LSCs, as demonstrated in serial transplant experiments. In addition, we now have evidence that miR-126 enhances mitochondrial metabolism (i.e., oxidative phosphorylation) and mitochondrial dynamics (i.e., mitochondrial fusion) in LSCs through SPRED1/ERK/p-BCL-2/NRF2 signaling. Accordingly, depletion of miR-126 by miRisten treatment significantly downregulates BCL-2 and disrupts mitochondrial metabolism, leading to increased levels of reactive oxygen species and apoptosis of LSCs. In addition, miRisten disrupts LSC mitochondrial function by upregulating the dynamin related protein 1 (DRP1), inducing mitochondrial fission, decreasing mitochondrial membrane potential, and inducing expression of mitophagy marker proteins. Since mitochondria-centered metabolism is the main metabolic energetic source for LSCs, we propose to dissect how miRisten exploits the mitochondrial metabolic vulnerability as a novel mechanism of action to eliminate LSCs. Furthermore, after conducting Investigational New Drug application (IND)-enabling pharmacokinetic, pharmacodynamic and toxicology studies, we will rapidly translate miRisten from bench to beside with a first-in-human phase 1 clinical trial of miRisten in patients with relapsed/refractory (r/r) AML. The central hypothesis of this proposal is that miRisten targets miR- 126-depended metabolic vulnerability of LSCs and will provide a novel therapeutic approach for LSC elimination in AML. We propose the following Specific Aims (SAs): SA#1: Determine the mechanisms of miRisten-induced mitochondrial metabolic vulnerability in LSCs. SA#2: Conduct pharmacokinetic, pharmacodynamic, efficacy and toxicology studies of miRisten to inform dose and schedule selection for human studies. SA#3: Conduct a first-in-human phase 1 trial of miRisten in patients with r/r AML. This project will translate novel discoveries on miR-126 into the clinic, by conducting preclincal studies that culminate in a first-in-human trial of miRisten.