MKL in megakaryocytopoiesis

描述(申请人提供)：巨核细胞生成是指巨核细胞从双表型巨核细胞-红系前体细胞分化为巨核母细胞的过程，巨核细胞经过内减数分裂成为多倍体，然后成熟为成熟的巨核细胞，可以将血小板释放到血液循环中。这些阶段中的每一个阶段都是细胞产生功能血小板的关键，而血小板是止血的关键。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)中这一过程可能会如何出错，人们知之甚少。这一建议的重点是确定MKL1促进巨核细胞分化的机制(S)。MKL1首次被鉴定为参与t(1；22)易位，几乎只发生在新生儿AMKL中。为了了解t(1；22)易位编码的RBM15-MKL1融合蛋白促进白血病的机制，我们必须了解RBM15和MKL1在造血中的正常功能，这是最近才开始阐明的。我的实验室发现了几条关于MKL1正常功能的重要线索(S)：1)MKL1在巨核细胞分化过程中差异表达，在最成熟的多倍体巨核细胞中表达水平最高；2)野生型(WT)MKL1的过表达促进了人细胞系以及原代小鼠和人细胞的Mk分化，Mk数量增加，Mk倍体增加；3)MKL1基因敲除小鼠的巨核细胞分化受损，血小板数量减少，4)缺口刺激促进Mk分化，5)MKL和Notch对SRF和Notch反应启动子具有协同作用。这项建议的目的建立在这些观察的基础上，以阐明MKL1促进巨核细胞生成的机制。目的1用体内和体外方法研究MKL1与SRF和MKL2在巨核细胞生成中的功能相互作用。目的2验证MKL1和Noch协同作用促进巨核细胞分化的假说。目的3探讨MKL1促进巨核细胞多倍化的机制。这项拟议工作的临床相关性是明确的--这些研究将有助于阐明正常巨核细胞生成的机制，这是正常形成功能血小板的关键。此外，这些研究将为未来对AMKL中独特发现的RBM15-MKL融合蛋白的研究奠定基础，AMKL占儿童AML病例的10%。 与公共健康相关：我们血液中的血小板对预防出血至关重要。血小板是在骨髓中由称为巨核细胞的细胞形成的。拟议的工作重点是更好地了解巨核细胞生成过程，长期目标是为影响这些细胞的遗传和获得性疾病找到有效的治疗方法。急性巨核细胞白血病(AMKL)主要影响新生儿和出生一岁的儿童。当AMKL与1号染色体上的RBM15基因和22号染色体上的MKL1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，而且这种疾病几乎总是致命的。在这一新的应用中，我们建议建立在我们已发表的工作和初步数据的基础上，阐明MKL1在正常和恶性巨核细胞扩增、多倍化和成熟中的作用。具体地说，我们正在评估MKL1在巨核细胞分化中的作用，以及其作用机制。这些研究的结果将为MKL1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.)