Analysis of E-selectin Ligands of Human Acute Leukemia Cells and their Biology in Leukemogenesis

摘要： 称为“唾液酸化刘易斯X”（sLeX; CD 15 s）的四糖是E- 选择素（CD 62 E），一种在血管内皮细胞上表达的Ca++依赖性凝集素。通过调查 我们的目标是揭示这种结构和潜在的糖基转移酶（GT）如何控制 其生物合成介导人类白血病发生。sLeX基序可以呈递在细胞表面上， 蛋白质（即，糖蛋白）和/或脂质（即，糖脂）支架，并且这些糖缀合物（称为“E-选择素 配体”）程序化对内皮细胞的抗剪切粘附。E-selectin通常不会在休息时显示。 血管内皮细胞，其表达由炎性细胞因子如TNF和IL-1诱导。怎么-- 然而，显著地，E-选择素在骨髓（BM）窦状血管上组成性表达， 已知在介导循环细胞向BM迁移中起关键作用，这是血细胞恢复的关键过程 造血干细胞移植（HSCT）后。除了它在招募造血干细胞/前体细胞方面的作用外， 骨髓微血管中E-选择素的表达是骨髓造血干细胞（HSPCs）向骨髓转化的重要机制之一。 在创造造血生长促进微环境中的重要作用，统称为“血管 壁龛”。我们实验室的研究表明，人HSPC表达多种E-选择素配体， 我们还观察到白血病原始细胞特征性地表达E-选择素配体。我们假设 E-选择素配体与人急性白血病细胞的结合可促进有效的BM转移， 使利基住宿，用于取代居民HSPC从其适当的生长微环境， 从而促进白血病细胞增殖。在这个建议中，使用E-选择素结合试验在静态和静态下， 和流体剪切条件，再加上流式细胞术和蛋白质印迹的补充技术，我们 将分析从急性白血病患者的血液和BM中分离的白血病细胞的E-选择素结合活性， 白血病我们将在不同类型的人leu中鉴定相关的携带sLeX的糖缀合物。 kemia细胞，测量这种糖缀合物参与E-选择素的能力，并确定如何表达 高尔基体GT的形状在不同的E-选择素配体之间产生sLeX修饰。这些信息将 与各种生物化学方法整合，包括糖基化和细胞表面的代谢抑制 糖工程化以定制修饰sLeX展示，以评估sLeX在特定的细胞上的呈现程度。 蛋白质和/或脂质支架允许E-选择素在来自人急性白血病的各种亚型的胚细胞之间结合。 白血病，以及相关E-选择素受体/配体相互作用在白血病细胞生物学中的影响。的 拟议的研究结果将是阐明白血病发生的糖生物学的关键，也应该有助于 通过选择性中断治疗急性白血病的新策略， sLeX表达和/或E-选择素配体介导的过程，以及潜在的治疗/预后分层 基于sLeX水平和/或急性白血病细胞上不同E-选择素配体的表达的模式。 相关性：急性白血病的发生是因为异常的白色细胞前体在特殊生长中增殖 骨髓内的区域。越来越多的证据表明，这些增长区是由显示器创建的， 血细胞表面的一种特殊的糖分子，就像尼龙搭扣一样将细胞锚在骨髓上 船舶.这项研究项目将研究这些糖的结构以及它们是如何形成的，预计 获得的信息将为更有效的急性白血病治疗铺平道路， 更精确地识别需要更强化治疗和/或更密切监测的患者。

ABSTRACT: The tetrasaccharide known as “sialylated Lewis X” (sLeX; CD15s) is the prototypical binding determinant for E- selectin (CD62E), a Ca++-dependent lectin expressed on vascular endothelial cells. Through investigations described herein, we aim to unveil how this structure, and the underlying glycosyltransferases (GTs) controlling its biosynthesis, mediate(s) human leukemogenesis. The sLeX motif can be presented on cell surfaces on protein (i.e., glycoprotein) and/or lipid (i.e., glycolipid) scaffolds, and these glycoconjugates (known as “E-selectin ligands”) program shear-resistant adhesion to endothelial cells. E-selectin is typically not displayed on resting vascular endothelial cells, and its expression is induced by inflammatory cytokines such as TNF and IL-1. How- ever, conspicuously, E-selectin is constitutively expressed on bone marrow (BM) sinusoidal vessels where it is known to play a key role in mediating migration of circulating cells to BM, a process critical to blood cell recovery following hematopoietic stem cell transplantation (HSCT). Beyond its role in recruiting hematopoietic stem/pro- genitor cells (HSPCs) to BM, it is well-known that E-selectin expression on marrow microvessels serves a fun- damental role in creation of hematopoietic growth-promoting microenvironments, collectively known as “vascular niches”. Studies from our laboratory have shown that human HSPCs express a variety of E-selectin ligands, and we have also observed that leukemic blasts characteristically express E-selectin ligands. We hypothesize that engagement of E-selectin ligands on human acute leukemia cells programs efficient BM metastasis and also enables niche lodgment, serving to displace resident HSPCs from their proper growth microenvironment and thereby promoting leukemic cell proliferation. In this proposal, using E-selectin binding assays under both static and fluid shear conditions, together with complementary techniques in flow cytometry and western blotting, we will analyze the E-selectin binding activity of leukemia cells isolated from blood and BM of patients with acute leukemias. We will identify the pertinent sLeX-bearing glycoconjugates among the different types of human leu- kemia cells, measure the ability of such glycoconjugates to engage E-selectin, and determine how expression of Golgi GTs shape creation of sLeX modifications among the different E-selectin ligands. This information will be integrated with various biochemical approaches including metabolic inhibition of glycosylation and cell surface glycoengineering to custom-modify sLeX display to assess the extent to which sLeX presentation on a specific protein and/or lipid scaffold licenses E-selectin binding among blasts from various subtypes of human acute leukemias, and the impact of the relevant E-selectin receptor/ligand interaction(s) in leukemia cell biology. The results of proposed studies will be key to elucidating the glycobiology of leukemogenesis, and should also pro- vide fundamental insights for establishing novel strategies to treat acute leukemias by selectively interrupting sLeX expression and/or E-selectin ligand-mediated processes, and for potential therapy/prognosis stratification schemas based on sLeX levels and/or the expression of distinct E-selectin ligands on acute leukemia cells. Relevance: Acute leukemia arises because abnormal white cell precursors proliferate within special growth zones within the bone marrow. There is increasing evidence that these growth zones are created by the display of particular sugar molecules on the surface of blood cells that act like Velcro to anchor the cells to marrow vessels. This research project will study these sugar structures and how they are made, with anticipation that information obtained will pave the way for more effective therapies for acute leukemias and may also allow for more precise identification of patients needing more intensive treatment and/or closer monitoring.