Role of Sialic Acid Modification in ALL Survival and Drug Resistance

描述（申请人提供）：急性淋巴细胞白血病（ALL）的治疗失败和复发是由骨髓基质细胞促进的，骨髓基质细胞在稳态条件下提供生长刺激，当ALL细胞接受化疗时提供抗凋亡信号。在之前的支持期间，该项目在了解ALL细胞同时受到药物治疗和基质支持时的反应方面取得了重大进展。通过唾液化修饰细胞表面分子，调节免疫细胞与微环境的相互作用，调节信号转导强度。我们的初步研究表明，小鼠和人类ALL细胞中唾液酸9- o -乙酰化水平异常高，随着它们产生耐药性，这一水平持续而显著地增加。引人注目的是，在移植小鼠的保护性骨髓微环境中增殖的人ALL细胞，通过激活一种特定的酯酶（该酯酶可从唾液酸中切割R9乙酰基）来去除9- o乙酰化，从而被根除。我们假设ALL细胞产生9- o乙酰化、唾液酸修饰的糖蛋白和糖脂，作为防止自发和药物诱导的细胞凋亡的机制。为了解决这一问题，Aim 1将通过在人类b前ALL和小鼠野生型和GD3-/- b前ALL细胞与保护性基质共培养中表达可诱导的9- o-乙酰酯酶，研究b前ALL细胞中由于Sias的9- o-乙酰化去除而在死亡期间激活的细胞内通路。目的2将通过比较体外和小鼠模型中具有不同9- o -乙酰化唾液酸水平的b前ALL细胞的药物敏感性和耐药性，确定Sias的9- o -乙酰化水平的增加如何促进耐药性的发展。我们将在st8sia1-/- pre-B ALL细胞中检测GD3的特异性贡献。目的3将确定Sias的乙酰化如何将唾液糖蛋白或GD3的反应性改变为有利于ALL细胞存活的构象。我们将在缺乏内源性GD3的st8sia1-/- pre-B ALL细胞中评估GD3氧化状态对其促凋亡活性的影响。我们还将描述在人和小鼠ALL细胞中被9- o乙酰化的关键唾液糖蛋白的亚细胞定位、药物治疗诱导动力学和耐药性的发展，使用特异性检测这种修饰的C. antennarius凝集素。免疫亲和纯化和质谱将用于鉴定经9- o -乙酰唾液化修饰的核心蛋白，并在体外基质支持下ALL细胞产生耐药性时上调核心蛋白。9- o -唾液酰化尚未在ALL耐药中得到检验，但我们的数据显示，它与ALL的生长和生存极其相关。这些创新的研究将对ALL细胞耐受药物治疗的机制产生新的见解，并导致新的靶点和方法来治疗这种和其他类型的癌症。

DESCRIPTION (provided by applicant): Treatment failure and relapse in acute lymphoblastic leukemia (ALL) is promoted by bone marrow stromal cells that provide growth stimulation under steady-state conditions and anti-apoptotic signals when the ALL cells are treated with chemotherapy. In the previous period of support, this project has made significant advances in understanding how ALL cells respond when they are simultaneously challenged by drug treatment and provided with stromal support. Modification of cell surface molecules through sialylation regulates interactions of immune cells with the microenvironment and modulates signal transduction strength. Our preliminary studies show abnormally high levels of sialic acid 9-O-acetylation on both mouse and human ALL cells, which is consistently and significantly increased as they develop drug resistance. Strikingly, human ALL cells proliferating in the protective bone marrow microenvironment of transplanted mice were eradicated when 9-O-acetylation was removed through activation of a specific esterase that cleaves the R9 acetyl group from sialic acid. We hypothesize that ALL cells generate 9-O-acetylated, sialic acid-modified glycoproteins and glycolipids as a mechanism of protection against spontaneous and drug- induced apoptosis. To address this, Aim 1 will investigate intracellular pathways activated during death due to 9-O-acetylation removal from Sias in pre-B ALL cells, by expression of inducible 9-O-acetyl esterases in human pre-B ALL and mouse wild type and GD3-/- pre-B ALL cells in co-culture with protective stroma. Aim 2 will determine how increased 9-O-acetylation levels of Sias contribute to drug resistance development by comparing drug sensitivity and resistance in pre-B ALL cells with different 9-O-acetylated sialic acid levels in vitro and in mous models. The specific contribution of GD3 will be examined in st8sia1-/- pre-B ALL cells. Aim 3 will determine how acetylation of Sias changes the reactivity of sialoglycoproteins or of GD3 to a conformation favorable for ALL cell survival. The effect of the oxidative state of GD3 on its pro-apoptotic activity will be evaluated in st8sia1-/- pre-B ALL cells that lack endogenous GD3. We will also characterize critical sialoglycoproteins that are 9-O-acetylated in human and mouse ALL cells for subcellular location, induction kinetics upon drug treatment and development of drug resistance using the C. antennarius lectin that specifically detects this modification. Immunoaffinity purification and mass spectrometry will be used to identify the core proteins that are modified by 9-O-acetyl sialylation and are upregulated as ALL cells develop drug resistance in the presence of stromal support in vitro. 9-O-sialylation has not been examined in drug resistance in ALL but our data show it is extremely relevant to their growth and survival. These innovative studies will yield new insight into the mechanisms through which ALL cells withstand drug treatment and lead to novel targets and approaches to treat this and other types of cancer.