Role of Sialic Acid Modification in ALL Survival and Drug Resistance

描述(申请人提供)：急性淋巴细胞性白血病(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，并在ALL细胞接受化疗时提供抗凋亡信号。在之前的支持期间，该项目在了解所有细胞同时受到药物治疗和基质支持的挑战时如何反应方面取得了重大进展。通过唾液酸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，在小鼠和人类ALL细胞上，唾液酸9-O-乙酰化水平都异常高，随着它们产生耐药性，这种水平一直显著增加。令人惊讶的是，当9-O-乙酰化通过激活从唾液酸中分解R9乙酰基的特定酯酶而被去除时，在移植小鼠的保护性骨髓微环境中增殖的人类ALL细胞被根除。我们假设所有细胞都产生9-O-乙酰化、唾液酸修饰的糖蛋白和糖脂，作为一种保护自发性和药物诱导的细胞凋亡的机制。为了解决这一问题，目标1将通过在与保护性基质共培养的人Pre-B ALL和小鼠野生型和GD3-/-Pre-B ALL细胞中表达可诱导的9-O-乙酰酯酶，来研究Pre-B ALL细胞中由于9-O-乙酰化从SIAs中去除而激活的细胞内途径。目的通过比较不同9-O-乙酰化唾液酸水平的前B细胞在体外和MOU模型中的药物敏感性和耐药性，确定SIAs的9-O-乙酰化水平增加是如何促进耐药发展的。GD3的具体作用将在st8sia1-/-Pre-B ALL细胞中进行检测。目标3将确定SIAS的乙酰化如何将唾液酸糖蛋白或GD3的反应性改变为有利于所有细胞存活的构象。GD3氧化状态对其促凋亡活性的影响将在缺乏内源性GD3的st8sia1-/-Pre-B ALL细胞中进行评估。我们还将使用专门检测这种修饰的Antennarius凝集素来表征在人类和小鼠ALL细胞中9-O-乙酰化的关键唾液酸糖蛋白的亚细胞定位、药物治疗的诱导动力学和耐药性的发展。免疫亲和纯化和质谱学将用于鉴定被9-O-乙酰唾液酸化修饰的核心蛋白，这些核心蛋白在所有细胞在体外基质支持下产生耐药性时上调。9-O-唾液酸化并没有在所有的耐药性中被检测，但我们的数据表明它与它们的生长和生存极其相关。这些创新研究将为所有细胞耐受药物治疗的机制提供新的见解，并导致治疗这种癌症和其他类型癌症的新靶点和方法。

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.