Cellular and molecular basis of microRNA-29a Induced Acute Myeloid Leukemia

描述（由申请人提供）：microRNA-29 a诱导急性髓性白血病的细胞和分子基础。急性髓性白血病（AML）是由未成熟造血细胞中遗传和/或表观遗传变化的积累引起的。虽然白血病发生涉及多个步骤，包括起始、进展、转化和维持，但在人类疾病中研究这些过程存在许多实际挑战;然而，由于在各种体外和体内试验中相对容易地对细胞进行遗传修饰和检测细胞功能，因此充分表征的AML小鼠模型提供了研究其中每一个过程的机会。虽然AML起源于未成熟的造血细胞，但引发疾病或最终转化为白血病干细胞（LSC）的确切细胞尚不清楚。我们先前发现microRNA-29 a（miR-29 a）在人HSC和AML LSC中高度表达，并且miR-29 a在小鼠BM细胞中的异位表达足以诱导进展为AML的骨髓增殖性肿瘤（MPN）样疾病。我们表征了miR-29 a诱导的疾病，并表明自我更新的定向祖细胞在AML发展之前出现，并且miR-29 a诱导的AML含有可以基于特异性细胞表面标志物的表达前瞻性分离的LSC。虽然自我更新祖细胞的确切作用尚未确定，但它们的出现提出了一种有趣的可能性，即定向祖细胞异常获得自我更新可能是AML发病机制的早期事件。总之，这些数据表明miR-29 a诱导的AML模型是用于研究AML发病机制的稳健且实验上易于处理的模型。我们建议描述miR-29 a在AML发展过程中的作用，并阐明miR-29 a调节LSC和异常自我更新祖细胞自我更新的分子基础。我们将利用我们新开发的miR-29 a缺陷小鼠模型以及各种AML小鼠模型来确定miR-29 a是否是白血病发生所必需的。我们将鉴定在miR-29 a驱动的白血病发生过程中转化的细胞群，我们还将测试两个miR-29 a靶点Dnmt 3a和Smpd 3（在人AML中也反复突变的基因）对miR-29 a诱导的表型的贡献。为了鉴定可能与miR-29 a协同诱导白血病的潜在基因或突变，我们将对LSC和异常自我更新祖细胞进行RNA-Seq。最后，我们将利用我们在miR-29 a诱导的AML模型以及原代人类AML样本方面的经验，评估miR-29 a在连续移植环境中LSC功能中的作用，这是自我更新的金标准测定。最终，我们希望这些研究能够对miR-29 a在AML中的作用产生新的见解，定义miR-29 a在白血病前中间体中作用的分子机制（包括自我更新的分子基础），并确定miR-29 a或其下游介质是否可以作为AML的潜在治疗靶点。

DESCRIPTION (provided by applicant): Cellular and molecular basis of microRNA-29a induced acute myeloid leukemia. Acute myeloid leukemia (AML) arises from the accumulation of genetic and/or epigenetic changes in immature hematopoietic cells. While leukemogenesis involves multiple steps including initiation, progression, transformation, and maintenance, studying these processes in human disease presents many practical challenges; however, well-characterized mouse models of AML afford the opportunity to study each of them due to the relative ease of genetically modifying cells and testing cellular function in a variety of in vitroand in vivo assays. While AML arises from immature hematopoietic cells, the exact cell that initiates disease or ultimately transforms into the leukemia stem cell (LSC) is unclear. We previously showed that microRNA-29a (miR-29a) is highly expressed in human HSC and AML LSC, and that ectopic expression of miR-29a in mouse BM cells is sufficient to induce a myeloproliferative neoplasm (MPN)-like disease that progresses to AML. We characterized miR-29a induced disease and showed that self-renewing committed progenitors arise prior to the development of AML and that miR-29a induced AMLs contain LSCs that can be prospectively isolated based on the expression of specific cell surface markers. Although the precise role of the self-renewing progenitors has not been defined, their appearance raises the intriguing possibility that aberrant acquisition of self-renewal by committed progenitors may be an early event in AML pathogenesis. Overall, these data demonstrate that the miR-29a induced AML model is a robust and experimentally tractable model for studying AML pathogenesis. We propose to characterize miR-29a's roles during AML development and to elucidate the molecular basis of miR-29a's regulation of self-renewal in LSCs and aberrantly self-renewing progenitors. We will determine if miR-29a is required for leukemogenesis utilizing our newly developed miR-29a deficient mouse model as well as various mouse models of AML. We will identify the cell population/s that become transformed during miR- 29a driven leukemogenesis, and we will also test the contribution of two miR-29a targets, Dnmt3a and Smpd3 (genes also recurrently mutated in human AML) to miR-29a induced phenotypes. To identify potential genes or mutations that may cooperate with miR-29a to induce leukemia, we will perform RNA-Seq on LSCs and aberrantly self-renewing progenitors. Finally, we will leverage our experience working with the miR-29a induced AML model as well as with primary human AML samples to evaluate the role of miR-29a in LSC function in the serial transplantation setting, the gold-standard assay for self-renewal. Ultimately, we expect these studies to generate new insights into the role of miR-29a in AML, define the molecular mechanisms of miR-29a's actions in pre-leukemic intermediates (including the molecular basis for self-renewal), and determine whether miR-29a or its downstream mediators may serve as potential therapeutic targets in AML.