Measuring Asparagine Synthetase Expression in Leukemia

描述（由申请人提供）：l -天冬酰胺酶（L-Asp）是一种酶，催化氨基酸天冬酰胺水解为天冬氨酸和氨，导致血清中天冬酰胺的消耗。该药物是儿童急性淋巴细胞白血病（ALL）有效治疗的关键组成部分，强化L-Asp治疗已被证明可显著改善儿童ALL的预后。不幸的是，L-Asp的治疗指数很窄，对一些患者可能造成致命的严重副作用。一般认为，L-Asp的治疗效果是基于正常和恶性淋巴细胞中天冬酰胺合成酶（AS）的低内在表达水平，使它们无法合成高效的天冬酰胺，因此依赖于从血浆中输入天冬酰胺。ALL细胞系中AS的强迫过表达可产生对L-Asp的抗性，而AS表达升高被认为是人类ALL对L-Asp产生抗性的机制之一。最近的数据表明，ALL患者的基线和诱导AS mRNA表达存在显著差异，但AS水平、对L-Asp的治疗反应和预后之间的关系尚不清楚。提高对不同病例间AS表达差异的理解，测量原发性人类白血病中AS mRNA和蛋白的表达。这些方法将包括实时定量PCR来测量AS mRNA水平；Western blot、免疫组织化学和流式细胞术检测AS蛋白的表达；以及新的蛋白质组学方法，包括定量质谱分析。这些试剂和方法将利用l - asp敏感的Molt-4人ALL细胞系和l - asp抗性衍生物（Molt-4/R）进行开发和优化。这些方法和试剂将在从ALL患儿身上获得的标本中进行常规试验。该提案的长期目标是开发试剂和技术，可用于未来的研究，以确定基线和诱导AS表达与ALL儿童的预后之间的相关性，这些儿童随机接受标准治疗与强化L-Asp的标准治疗。更详细地了解AS在人类白血病中的表达作用，也将为新型人类AS在ALL中的潜在治疗效用提供重要信息。

DESCRIPTION (provided by applicant): L-Asparaginase (L-Asp) is an enzyme that catalyzes hydrolysis of the amino acid asparagine to aspartate and ammonia leading to depletion of asparagine from the serum. This agent is a key component of effective therapies for childhood acute lymphoblastic leukemia (ALL) and intensified L-Asp therapy has been demonstrated to lead to significant improvements in outcome of children with ALL. Unfortunately, L-Asp has a narrow therapeutic index and can cause significant side effects that are fatal in some patients. The therapeutic efficacy of L-Asp is generally believed to be based upon low intrinsic levels of asparagine synthetase (AS) expression in normal and malignant lymphocytes that render them incapable of synthesizing sui'ficient asparagine and therefore reliant on import of asparagine from the plasma. Forced over-expression of AS in ALL cell lines confers resistance to L-Asp, and elevated AS expression is postulated to be a mechanism of L-Asp resistance in human ALL. Recent data establish that there is significant variability in baseline and induced AS mRNA expression in ALL, but the relationship between AS levels, therapeutic response to L-Asp, and outcome are unknown. Improved understanding of how AS expression differs among cases measure AS mRNA and protein expression in primary human leukemias. These methodologies will include real time quantitative PCR to measure AS mRNA levels; Western blot, immunohistochemistry and flow cytometric measures of AS protein expression; and novel proteomic methods involving quantitative mass spectrometry. These reagents and methods will be developed and optimized using the L-Asp-sensitive Molt-4 human ALL cell line and an L-Aspresistant derivative (Molt-4/R). The methods and reagents will then be piloted for routine use in specimens obtained from children with ALL. The long-term goal of this proposal is to develop reagents and technologies that can be used in future studies to determine how baseline and induced AS expression correlates with outcome in children with ALL randomized to receive standard therapy versus standard therapy with intensified L-Asp. A more detailed understanding of the role of AS expression in human leukemia will also provide important information regarding the potential therapeutic utility of novel inhibitors of human AS in ALL.