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）是第一个“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）治疗留下了一群对这些药物表现出先天抗性的细胞。这些是静止的CML干细胞（HSC），代表活跃的癌症储存库。因此，很明显，只有能够安全有效地靶向这些造血干细胞而不损害正常造血干细胞的药物才有可能导致疾病的根除。我们发表的初步数据表明，可能的机制涉及激酶（如BCR-ABL1和Jak2）和磷酸酶（如PP2A）之间的异常平衡，这可能取决于特定microrna （miRs）表达的改变。事实上，我们发现miRs的表达改变可能靶向Jak2-hnRNP A1-SET/PP2A-2-catenin HSC通路，这对于静止CML HSC的生存和自我更新至关重要，但以BCR-ABL1表达而不是激酶依赖的方式运作。a)静止CML hsc中BCR-ABL1蛋白升高，而mRNA和Jak2的表达/活性未升高；b) miR水平在白血病中异常，在干细胞和祖细胞中发生变化；c)最近发现的RNA诱骗活性不仅限于miR-328，其他mir也可能相互作用并干扰hnRNP功能；该假说认为，静止的Ph+ hsc表现出miR表达失调，这取决于BCR-ABL1的表达，而不是激酶活性，这有助于增强白血病hsc的生存和自我更新。基于这些考虑，本提案的总体目标是了解miR表达改变对维持静止CML hsc储库的要求，并通过鉴定和整合miRs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，该分析以规范和/或诱饵的方式，通过直接干扰BCR-ABL1-Jak2-hnRNP A1-SET-2-catenin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hsc富集的CD34+/CD38-(CD90+) BM细胞，独特的scl - ta - bcr /ABL小鼠模型和合成miRs或拮抗剂来治疗移植了GFP+/Luc+ CML hsc的NSG小鼠，以便：1)识别CML hsc中失调的miRs，这些miRs通过其规范和/或hnRNP A1诱变活性干扰BCR-ABL1-Jak2途径；2)评估miR水平的调节是否会损害CML造血干细胞的体外和体内存活和自我更新；3)利用2-O-MePS miRs和拮抗剂确定miR表达调节在CML根除中的治疗作用。我们相信，这项工作的成功完成将提高我们对白血病HSC生物学的认识，并且基于我们在过去几年的发现，可以肯定地预测，在这一领域将会有新的观察，其中一些将揭示新的治疗干预策略。因此，这项工作对基础和转化癌症研究具有很强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-2-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-2-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.