Role of microRNAs in the regulation of CML stem cell self renewal and survival

描述（由申请人提供）：慢性髓性白血病（CML）是第一种“临床治愈”的干细胞来源的造血肿瘤;然而，酪氨酸激酶抑制剂（TKI）治疗留下了对这些药物显示先天耐药性的细胞库。这些是静止的CML干细胞（HSC），代表了活跃的癌症储库。因此，很明显，只有能够安全有效地靶向这些HSC而不损害正常HSC的药物才有可能根除疾病。我们发表的初步数据表明，一个可能的机制涉及激酶（即BCR-ABL 1和Jak 2）和磷酸酶（即PP 2A）之间的异常平衡，这可能取决于特定microRNA（miR）的表达改变。事实上，我们发现miR的表达改变，其可能靶向Jak 2-hnRNP A1-SET/PP 2A-β-连环蛋白HSC通路，该通路对于静止期CML HSC的存活和自我更新是必需的，但以BCR-ABL 1表达依赖性方式而非激酶依赖性方式起作用。鉴于a）BCR-ABL 1蛋白而非mRNA和Jak 2表达/活性在静止期CML HSC中升高; B）miR水平在白血病中异常，并且在干细胞与祖细胞中发生变化;以及c）最近发现的RNA诱饵活性不限于miR-328，而是其他miR可能与hnRNP功能相互作用并干扰hnRNP功能;推动这一提议的假设是，静止的Ph+ HSC表现出失调的miR表达，其依赖于BCR-ABL 1表达而不是激酶活性，并有助于增强白血病HSC的存活和自我更新。基于这些考虑，本提案的总体目标是理解改变的miR表达对于维持CML HSC的静止储库的需求，并通过miR的鉴定和整合的体外和体内分析来确定miR表达的药理学恢复的治疗相关性，其以典型和/或诱饵方式，通过直接干扰BCR-ABL 1-Jak 2-hnRNP A1-SET-β-连环蛋白通路影响CML HSC存活和自我更新。因此，为了实现这些目标，我们将依赖于使用来自CML患者的高度HSC富集的CD 34 +/CD 38-（CD 90+）BM细胞、独特的SCL-tTA-BCR/ABL小鼠模型和合成的miR或miR来治疗移植有GFP+/Luc+ CML HSC的NSG小鼠，以便：1）识别CML HSC中失调的miR，这些miR通过其典型和/或hnRNP A1诱饵活性干扰BCR-ABL 1-Jak 2途径; 2）评估miR水平的调节是否损害CML HSC的体外和体内存活和自我更新;和3）通过使用2-O-MePS miR和miR-100 miR确定miR表达的调节在CML根除中的治疗作用。我们有信心，成功完成拟议的工作将推进我们的知识白血病HSC生物学，并根据我们在过去几年中所取得的发现，它是安全的预测，新的观察将在这一领域，其中一些将揭示新的策略，治疗干预。因此，这项工作对基础和转化癌症研究的重要性和高度相关性。

DESCRIPTION (provided by applicant): Chronic myeloid leukemia (CML) is the first "clinically cured" stem cell-derived hematopoietic neoplasm; however, tyrosine kinase inhibitor (TKI) therapy leaves behind a pool of cells showing innate resistance to these drugs. These are quiescent CML stem cells (HSC) that represent an active cancer reservoir. Thus, it becomes clear that only drugs that can safely and efficiently target these HSCs without harming the normal ones have the potential to lead to disease eradication. Our published and preliminary data suggest that a likely mechanism involve an aberrant balance between kinases (i.e. BCR-ABL1 and Jak2) and phosphatases (i.e. PP2A) and this may depend on altered expression of specific microRNAs (miRs). In fact, we found altered expression of miRs that may target the Jak2-hnRNP A1-SET/PP2A-ß-catenin HSC pathway, which is essential for survival and self-renewal of quiescent CML HSCs but operates in a BCR-ABL1 expression- but not kinase- dependent manner. Given that a) BCR-ABL1 protein but not mRNA and Jak2 expression/activity are elevated in quiescent CML HSCs; b) miR levels are aberrant in leukemias and change in stem vs. progenitor cells; and c) the recently discovered RNA decoy activity is not limited to miR-328 but other miRs likely interact and interfere with hnRNP function; the hypothesis driving this proposal is that quiescent Ph+ HSCs display a dysregulated miR expression which depends on BCR-ABL1 expression but not kinase activity, and contributes to enhanced survival and self-renewal of leukemic HSCs. Based on these considerations, the overall objective of this proposal is to understand the requirement of altered miR expression for the maintenance of the quiescent reservoir of CML HSCs and determine the therapeutic relevance of pharmacologic restoration of miR expression through the identification and integrated in vitro and in vivo analysis of the miRs, which in a canonical and/or decoy manner, affect CML HSC survival and self-renewal through direct interference with the BCR-ABL1-Jak2-hnRNP A1-SET-ß-catenin pathway. Thus, to accomplish these goals we will rely on the use of highly HSC-enriched CD34+/CD38-(CD90+) BM cells form CML patients, the unique SCL-tTA-BCR/ABL mouse model and synthetic miRs or antagomiR to treat NSG mice transplanted with GFP+/Luc+ CML HSCs in order to: 1) Identify miRs dysregulated in CML HSCs that interfere with the BCR-ABL1-Jak2 pathway through their canonical and/or hnRNP A1 decoy activities; 2) Assess whether modulation of miR levels impairs in vitro and in vivo survival and self-renewal of CML HSCs; and 3) Determine the therapeutic role of modulation of miR expression in eradication of CML by using 2-O-MePS miRs and antagomiRs. We are confident that the successful completion of the proposed work will advance our knowledge of leukemic HSC biology and, based on the discoveries we made in the past few years, it is safe to predict that new observations will be made in this field and that some of them will reveal new strategies for therapeutic intervention. Hence, the strong importance and high relevance of this work for basic and translational cancer research.