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

摘要： 被称为“唾液酸化的Lewis X”(SLeX；CD15s)的四糖是E-的典型结合决定簇。 选择素(CD62E)是一种表达于血管内皮细胞的钙依赖凝集素。通过调查 在这里，我们的目标是揭示这种结构和潜在的糖基转移酶(GTS)是如何控制的 其生物合成，介导(S)人白血病的发生。SLeX基序可以呈现在细胞表面的 蛋白质(即，糖蛋白)和/或脂类(即，糖脂)支架，以及这些糖共轭化合物(称为“E-选择素 配体“)程序与内皮细胞的抗剪切粘附性。休息时通常不显示E-选择素 血管内皮细胞，其表达由炎性细胞因子如肿瘤坏死因子和白介素1诱导。怎么- 值得注意的是，E-选择素在其所在的骨髓(BM)窦血管上有结构性地表达 已知在调节循环细胞向骨髓迁移的过程中发挥关键作用，这是血细胞恢复的关键过程 在造血干细胞移植(HSCT)后。除了在招募造血干细胞/前体方面的作用外- 骨髓微血管上E-选择素的表达在骨髓细胞对骨髓间充质干细胞(HSPC)的分化中起重要作用。 在创造促进造血生长的微环境中的基础作用，统称为“血管 利基市场“。我们实验室的研究表明，人类HSPC表达多种E-选择素配体，并且 我们还观察到白血病细胞特有地表达E-选择素配体。我们假设 E-选择素配体在人急性白血病细胞上的结合可促进有效的骨髓转移 支持利基寄宿，使居住的HSPC脱离其适当的生长微环境，并 从而促进白血病细胞增殖。在本方案中，使用E-选择素结合分析在两种静态条件下 和流体剪切条件，再加上流式细胞术和免疫印迹的补充技术，我们 将分析急性白血病患者血液和骨髓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类白血病中鉴定相关的含SLeX的糖偶联物。 白血病细胞，测量这种糖结合物结合E-选择素的能力，并确定如何表达 高尔基GTS的形状在不同的E-选择素配体之间产生SLeX修饰。此信息将 与各种生化方法相结合，包括代谢抑制糖基化和细胞表面 自定义修改SLeX显示以评估SLeX在特定环境中的演示程度 蛋白质和/或脂质支架允许E-选择素在人类急性白血病不同亚型的原始细胞中结合 白血病，以及相关的E-选择素受体/配体相互作用(S)在白血病细胞生物学中的影响。这个 拟议的研究结果将是阐明白血病发生的糖生物学的关键，也应该有利于 通过选择性阻断来建立治疗急性白血病的新策略的基本见解 SLeX表达和/或E-选择素配体介导的过程和潜在治疗/预后分层 基于SLeX水平和/或急性白血病细胞上不同E-选择素配体表达的模式。 相关性：急性白血病的发生是因为异常的白细胞前体在特殊的生长过程中增殖 骨髓内的区域。越来越多的证据表明，这些生长带是由显示器造成的 血细胞表面的特殊糖分子，它们像尼龙搭扣一样将细胞固定在骨髓上 船只。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 所获得的信息将为更有效地治疗急性白血病铺平道路，并可能允许 更准确地识别需要更密集治疗和/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.