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

描述（由申请人提供）：microRNA-29a诱导急性髓系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的遗传和/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，表征良好的AML小鼠模型提供了研究它们的机会，因为基因修饰细胞和在各种体外和体内试验中测试细胞功能相对容易。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。我们之前的研究表明，microRNA-29a （miR-29a）在人HSC和AML LSC中高度表达，并且miR-29a在小鼠BM细胞中的异位表达足以诱导骨髓增生性肿瘤（MPN）样疾病进展为AML。我们表征了miR-29a诱导的疾病，并表明自我更新的承诺祖细胞在AML的发展之前出现，并且miR-29a诱导的AML含有LSCs，可以根据特定细胞表面标记物的表达进行前瞻性分离。虽然自我更新祖细胞的确切作用尚未明确，但它们的出现提出了一种有趣的可能性，即由承诺的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-29a诱导的AML模型是研究AML发病机制的一个稳健且实验可处理的模型。我们提出表征miR-29a在AML发展过程中的作用，并阐明miR-29a调控LSCs和异常自我更新祖细胞的分子基础。我们将利用我们新开发的miR-29a缺陷小鼠模型以及各种AML小鼠模型来确定miR-29a是否是白血病发生所必需的。我们将确定在miR-29a驱动的白血病发生过程中转化的细胞群/s，我们还将测试两个miR-29a靶点Dnmt3a和Smpd3（在人类AML中也经常突变的基因）对miR-29a诱导的表型的贡献。为了确定可能与miR-29a协同诱导白血病的潜在基因或突变，我们将对LSCs和异常自我更新的祖细胞进行RNA-Seq。最后，我们将利用我们在miR-29a诱导的AML模型以及原始人类AML样本上的工作经验来评估miR-29a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-29a在AML中的作用产生新的见解，定义miR-29a在白血病前期中间体中的作用的分子机制（包括自我更新的分子基础），并确定miR-29a或其下游介质是否可能作为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.