The bone marrow extracellular matrix: scaffold of hematopoiesis

摘要 这项建议解决了了解原发性骨髓纤维化(PMF)疾病机制的迫切需要。 PMF是一种预后不佳的疾病。唯一有可能治愈的疗法，异基因干细胞 移植，是一种高风险的手术，死亡率至少为50%。骨髓纤维化的特征是 过度产生细胞外基质(ECM)，这是骨髓中的纤维，导致进行性 血细胞生产失败。一些基因突变，包括JAK2V617F，已经在 PMF。而携带基因突变的造血细胞被认为是异常增殖和分泌 刺激基质细胞过度产生细胞外基质的因素，抑制JAK2只能部分改善 疾病结果，以及专门针对ECM产生的补充疗法目前还没有 可用。对流行假说的依赖，其重点是基因突变，是 这一领域的进步。本项目建议从ECM的角度来解决这一问题。在基础上建设 我们发表的对老鼠和人类样本的初步研究，这是这一提议的中心假设 整合素(介导细胞与细胞外基质附着的黏附分子)的异常激活有助于 PMF细胞增殖情况。由于PMF的增殖是由ECM和整合素之间的相互作用引起的，因此 问题是，增殖是由ECM形成的异常还是由整合素的直接激活引发的。 因此，我将探索这种异常整合素激活背后的两种潜在机制，询问是否：(1) PMF细胞发育的血管壁龛产生异常高的细胞外基质，以及(2)赖氨酰氧化酶。 在PMF中上调的ECM酶(LOX)通过翻译后修饰激活PMF中的整合素。 这些研究将在最具代表性的人PMF小鼠模型上进行，转基因小鼠 JAK2V617F突变。整合素对PMF细胞增殖的影响将通过阻断来确定 整合素通过抗体发挥作用，并使用CRISPR/Cas9删除整合素基因。我将使用3D共焦 成像和高级计算分析，以确定细胞、ECM和 骨髓纤维化骨髓中的血管形成，通过详细的研究阐明细胞外基质的产生机制 维管壁龛的形态分析。翻译后修饰的蛋白质组学分析将 对整合素进行检测，以确定LOX氧化的特定赖氨酸残基，这对 针对整合素或LOX的治疗方法的发展。这是一个职业发展奖，而这个的PI 应用程序将得到具有该领域专业知识的调查人员的指导，以执行该项目。 该项目建立在恶性血液病PI以前的经验基础上。培训和专业知识 在此奖项期间获得的将为该公司的PI成功的独立职业生涯奠定基础 项目。

ABSTRACT This proposal addresses the urgent need to understand disease mechanisms in Primary Myelofibrosis (PMF). PMF is a disease with a dismal prognosis. The only potentially curative therapy, allogeneic stem cell transplantation, is a high-risk procedure, with a mortality rate of at least 50%. Myelofibrosis is characterized by excessive production of extracellular matrix (ECM), the fibers in bone marrow, which leads to a progressive failure in blood cell production. A number of gene mutations, including JAK2V617F, have been reported in PMF. While it is thought that hematopoietic cells carrying gene mutations abnormally proliferate and secrete factors that stimulate stromal cells to produce ECM excessively, inhibition of JAK2 only partially improves disease outcomes, and complementary therapies that specifically target ECM production are not currently available. Dependence on the prevalent hypothesis, with its focus on gene mutations, is a critical barrier to progress in the field. This project proposes instead to address the problem from the ECM angle. Building on our published and preliminary studies with mouse and human samples, the central hypothesis of this proposal is that abnormal activation of integrins (adhesive molecules that mediate cell attachment to ECM) facilitates proliferation of PMF cells. As PMF proliferation arises from interaction between ECM and integrins, the question is whether proliferation is triggered by an abnormality in ECM formation or by direct integrin activation. Thus, I will explore two potential mechanisms behind this abnormal integrin activation, asking whether: (1) abnormally high ECM production occurs in the vascular niche, where PMF cells develop, and (2) Lysyl oxidase (LOX), an ECM enzyme upregulated in PMF, activates integrins in PMF through post-translational modification. These studies will be performed on the most representative mouse model of human PMF, mice transgenic for the JAK2V617F mutation. Effects of integrins on cell proliferation in PMF will be determined by blocking integrin function with antibodies and by deleting integrin genes using CRISPR/Cas9. I will use 3D confocal imaging and advanced computational analysis to determine the spatial distribution of cells, ECM, and vasculature in myelofibrotic bone marrow, clarifying the mechanisms of ECM production by detailed morphological analysis of the vascular niche. Proteomic analysis of post-translational modifications will be performed on integrins to determine specific lysine residues oxidized by LOX, which has implications on the development of therapies targeting integrins or LOX. This is a Career Development Award, and the PI of this application will have the mentorship of investigators with expertise in the field for the execution of the project. The project builds on previous experience of the PI in malignant hematology. The training and the expertise acquired during this award will provide a foundation for a successful independent career for the PI of this project.