MKL in megakaryocytopoiesis

描述（由申请方提供）：巨核细胞生成是真核细胞从双表型巨核细胞-红细胞前体分化为巨核母细胞的过程，巨核母细胞经历胞内有丝分裂成为多倍体，随后经历成熟成为成熟巨核细胞，其可将血小板释放到循环中。这些阶段中的每一个对于细胞产生功能血小板至关重要，这对于止血至关重要。尽管我们对造血的理解有了很大的进步，但关于巨核细胞生成是如何调节的，以及这在影响血小板形成的巨核细胞疾病和急性巨核细胞白血病（AMKL）中是如何出错的，我们知之甚少。该建议的重点是确定MKL 1促进巨核细胞分化的机制。MKL 1首先通过其参与t（1;22）易位而被鉴定，该易位几乎仅发生在新生儿的AMKL中。为了理解由t（1;22）易位编码的RBM 15-MKL 1融合蛋白促进白血病的机制，我们必须理解RBM 15和MKL 1在造血中的正常功能，这些功能最近才开始阐明。我的实验室已经发现了关于MKL 1正常功能的几个重要线索：1）MKL 1在巨核细胞分化期间差异表达，在最成熟的多倍体巨核细胞（Mk）中具有最高水平，2）野生型（WT）MKL 1的过表达促进人细胞系以及原代鼠和人细胞的Mk分化，其中Mk数量增加，Mk倍性增加，3）MKL 1敲除小鼠具有受损的巨核细胞分化和减少的血小板数量，4）notch刺激促进Mk分化和5）MKL和notch协同作用于SRF和notch应答启动子。本建议的目的是建立在这些观察，以阐明MKL 1促进巨核细胞生成的机制。目的1：通过体内和体外实验研究MKL 1、SRF和MKL 2在巨核细胞生成中的相互作用。目的2是检验MKL 1和notch协同作用促进巨核细胞分化的假设。目的3探讨MKL 1促进巨核细胞多倍体化的机制。这项工作的临床意义是明确的-这些研究将有助于阐明正常巨核细胞生成的机制，这是功能性血小板正常形成的关键。此外，这些研究将为未来研究AMKL中独特的RBM 15-MKL融合蛋白奠定基础，AMKL占儿童AML病例的10%。 公共卫生相关性：我们血液中的血小板对预防出血至关重要。血小板在骨髓中由称为巨核细胞的细胞形成。建议的工作重点是更好地了解巨核细胞生成的过程，长期目标是为影响这些细胞的遗传和获得性疾病提供有效的治疗方法。急性巨核细胞白血病（AMKL）主要影响新生儿和一岁以内的儿童。当AMKL与1号染色体上的RBM 15基因和22号染色体上的MKL 1基因之间的t（1;22）染色体翻译相关时，几乎没有有效的治疗选择，并且该疾病几乎总是致命的。在这个新的应用中，我们建议建立在我们已发表的工作和初步数据阐明MKL 1在正常和恶性巨核细胞扩增，多倍化和成熟中的作用。具体而言，我们正在评估MKL 1在巨核细胞分化中的作用及其作用机制。这些研究的结果将为MKL 1在正常和恶性细胞中的作用提供新的见解。

DESCRIPTION (provided by applicant): Megakaryocytopoiesis is the process by which egakaryocytes differentiate from biphenotypic megakaryocyte-erythroid precursors, to megakaryoblasts, which undergo endomitosis to become polyploid, and subsequently undergo maturation to mature megakaryocytes that can release platelets into the circulation. Each of these stages is critical for the cells to produce function platelets, which are critical for hemostasis. Despite great advances in our understanding of hematopoiesis, relatively little is known regarding how megakaryocytopoiesis is regulated, and how this may go awry in diseases of megakaryocytes affecting platelet formation and in acute megakaryoblastic leukemia (AMKL). This proposal is focused on determining the mechanism(s) by which MKL1 promotes megakaryocytic differentiation. MKL1 was first identified by its involvement in the t(1;22) translocation, which occurs almost exclusively in AMKL of newborns. To understand the mechanism by which the RBM15-MKL1 fusion protein encoded by the t(1;22) translocation promotes leukemia, we must understand the normal functions of RBM15 and MKL1 in hematopoiesis, which have only recently begun to be elucidated. My laboratory has discovered several important clues regarding the normal function(s) of MKL1: 1) MKL1 is differentially expressing during megakaryocytic differentiation with the highest levels in the most mature polyploid megakaryocytes (Mks), 2) overexpression of wildtype (WT) MKL1 promotes Mk differentiation of human cell lines as well as primary murine and human cells with increased numbers of Mk and increased ploidy of Mk, 3) MKL1 knockout mice have impaired megakaryocytic differentiation and decreased platelet numbers, 4) notch stimulation promotes Mk differentiation and 5) MKL and notch act synergistically on both SRF and notch responsive promoters. The aims of this proposal build upon these observations to elucidate the mechanisms by which MKL1 promotes megakaryocytopoiesis. Aim 1 is to determine the functional interactions of MKL1 with SRF and MKL2 in megakaryocytopoiesis using in vivo and in vitro approaches. Aim 2 is to test hypothesis that MKL1 and notch act synergistically to promote megakaryocytic differentiation. Aim 3 is to assess the mechanism by which MKL1 promotes polyploidization of megakaryocytes. The clinical relevance of the proposed work is clear - these studies will help to elucidate the mechanisms underlying normal megakaryocytopoiesis, which is key for normal formation of functional platelets. In addition, these studies will form the basis for future studies on the RBM15-MKL fusion protein found uniquely in AMKL, which accounts for 10% of AML cases in children. PUBLIC HEALTH RELEVANCE: The platelets in our blood are critical for prevention f bleeding. Platelets are formed in the bone marrow from cells called megakaryocytes. The work proposed is focused on a obtaining a better understanding of the process of megakaryocytopoiesis with the long-term goals of deriving effective therapies for genetic and acquired diseases affecting these cells. Acute megakaryoblastic leukemia (AMKL) affects primarily newborn infants and children during the first year of life. When AMKL is associated with a t(1;22) chromosomal translation between the RBM15 gene on chromosome 1 and the MKL1 gene on chromosome 22, there are few effective treatment options, and the disease is nearly always fatal. In this new application, we propose to build on our published work and preliminary data elucidating the role of MKL1 in normal and malignant megakaryocyte expansion, polyploidization and maturation. Specifically, we are assessing the role of MKL1 in megakaryocyte differentiation, and the mechanisms underlying its action. The results of these studies will provide new insights into the role of MKL1 in normal as well as malignant cells.)