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

文摘:

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.