Targeting microRNAs to eradicate leukemia stem cells

白血病干细胞(LSC)包括急性或慢性白血病中已获得 “干细胞”的特性包括承受无限自我更新、维持异常克隆的能力。 在暴露于化疗或其他生物应激源时，从而实现造血和静止 导致对抗白血病治疗产生抗药性。目前可用的细胞周期依赖性化疗和 其他分子靶向剂无法消除这些LSCs。因此，新的有效治疗方法 废除LSC是一种未得到满足的需求。MicroRNAs(MiRNAs)是一种短的非编码RNA，调节 多个靶蛋白，从而控制广泛的细胞程序。在符合以下条件的miRNA中 白血病中miR-126-3p(miR-126)高表达与LSC基因表达相关 签名和糟糕的结果。此外，在正常情况下，较高水平的miR-126控制静止 造血干细胞(HSC)和LSC，但在抑制miR-126活性的同时增加HSC的造血量 输出，它会使LSC筋疲力尽。这一提议的中心假设是miR-126对于 LSC的动态平衡并介导LSC的治疗耐药，因此代表了一种有前景的LSC导向的 治疗靶点。本应用程序的主要目标是了解miR-126的表达方式 并开发一种有效的治疗方法来抑制LSC中的miR-126，而 少做正常的造血。作为一项原则证明，我们将重点放在急性髓系的miR-126靶点上。 (AML)和慢性髓系白血病(CML)，但类似的原理可以扩展到其他类型的 白血病。我们提出了以下具体目标(SA)：SA1。剖析和攻克分子 一种新发现的SPRED1/miR-126自身调节基因介导的药物耐药机制 在LSC中循环。我们将测试依赖于酪氨酸激酶(TK)的SPRED1/miR-126自动调节环是 在AML和CML中起作用，介导依赖miR-126的酪氨酸激酶耐药机制 抑制剂(TKI)。我们将1)评估SPRED1/miR-126自动调节环在不同亚型中的活性 2)定义TK依赖的SPRED1磷酸化位点；3)建立白血病小鼠模型，解剖 SPRED1/miR-126自动调节环与异常激活的Tk的相互作用。SA2.为了定义miR的作用- 126在维持骨髓微环境中的LSC生态位方面。我们将从基因上发展 在LSC和内皮细胞(EC)中构建有条件miR-126缺失的AML和CML模型。我们会 确定1)LSC生产的miR-126的贡献；2)miR-126在欧共体的贡献 3)miR-126缺失是否能增强治疗介导的LSC消除。SA3.至 开发和优化一种针对LSC和LSC利基中的miR-126的合成抑制剂。我们会 进行PK和PD分析以及临床前研究，以确定AntimiR-126的有效剂量/时间表 与CpG-寡脱氧核苷酸(ODN)偶联，实现最佳靶向细胞递送。

Leukemia stem cells (LSC) comprise subpopulations of cells in acute or chronic leukemia that have acquired “stem cell” properties including the ability to endure unlimited self-renewal, maintain aberrant clonal hematopoiesis and achieve quiescence upon exposure to chemotherapy or other bio-stressors thereby conferring resistance to antileukemia treatments. Currently available cell-cycle-dependent chemotherapy and other molecular targeting agents are unable to eliminate these LSCs. Thus new effective treatments to abrogate LSC are an unmet need. MicroRNAs (miRNAs) are short non-coding RNAs that regulate levels of multiple target proteins, thereby controlling a wide array of cellular programs. Among miRNAs that are deregulated in leukemia, higher expression of miR-126-3p (miR-126) is associated with LSC-gene expression signatures and poor outcome. Furthermore, higher levels of miR-126 controls quiescence both in normal hematopoietic stem cells (HSC) and LSC, but while attenuated miR-126 activity increases HSC hematopoietic output, it drives LSC to exhaustion. The central hypothesis of this proposal is that miR-126 is critical for the homeostasis of LSC and mediates LSC therapy resistance, thus represents a promising LSC-directed therapeutic target. The major objective of this application is to understand how miR-126 expression is aberrantly regulated in LSC and to develop an effective therapeutic approach to inhibit miR-126 in LSC, while sparing normal hematopoiesis. As a proof-of-principle, we will focus on targeting miR-126 in acute myeloid (AML) and chronic myeloid leukemia (CML), but similar principles could be expanded to other types of leukemia. We propose the following specific aims (SA): SA1. To dissect and overcome the molecular mechanisms of therapy resistance mediated by a newly discovered SPRED1/miR-126 autoregulatory loop in LSC. We will test that a tyrosine kinase (TK)-dependent SPRED1/miR-126 autoregulatory loop is operative in AML and CML, which mediate miR-126-dependent mechanisms of resistance to tyrosine kinase inhibitors (TKI). We will 1) assess the activity of SPRED1/miR-126 autoregulatory loop in distinct subtypes of AML; 2) define TK-dependent SPRED1 phosphorylation sites; 3) create leukemia mouse models to dissect the interplay of SPRED1/miR-126 autoregulatory loop with aberrantly active TK. SA2. To define the role of miR- 126 in maintaining a LSC niche within the bone marrow microenvironment. We will develop genetically engineered AML and CML models with conditional miR-126 deletion in LSC and endothelial cells (EC). We will determine 1) the contribution of miR-126 produced by LSC; 2) the contribution of miR-126 in the EC compartment; 3) whether deletion of miR-126 could enhance treatment-mediated elimination of LSC. SA3. To develop and optimize a synthetic inhibitor that targets miR-126 in LSC and the LSC niche. We will perform PK and PD analyses and preclinical studies to define the active dose/schedule of an antimiR-126 conjugated with CpG-oligodeoxynucleotide (ODN) for optimal targeted cell delivery.