Measuring Asparagine Synthetase Expression in Leukemia

说明(申请人提供)：L-天冬酰胺酶(L-天冬氨酸氨基转移酶)是一种催化氨基酸天冬氨酸天冬氨酸水解酶和氨导致天冬酰胺从血清中耗尽的酶。这种药物是儿童急性淋巴细胞白血病(ALL)有效治疗的关键成分，L-天冬氨酸强化疗法已被证明能显著改善儿童ALL的预后。不幸的是，L-天冬氨酸的治疗指数很窄，可能会引起严重的副作用，在一些患者中是致命的。一般认为L-天冬氨酸的疗效是基于正常和恶性淋巴细胞中天冬酰胺合成酶(AS)的低水平表达，使它们不能合成足够的天冬酰胺，因此依赖于从血浆中进口天冬酰胺。AS在所有细胞系中强制过表达使人ALL对L-天冬氨酸产生耐药，AS表达升高可能是人ALL对L-天冬氨酸耐药的机制之一。最近的数据表明，ALL的基线和诱导的AS基因表达存在显著的差异，但AS水平、对L-天冬氨酸的治疗反应和预后之间的关系尚不清楚。提高了对AS表达在不同病例之间的差异的理解，以衡量原发人类白血病的mRNA和蛋白质表达。这些方法将包括实时定量聚合酶链式反应来测量AS的mRNA水平；蛋白质印迹、免疫组织化学和流式细胞术的AS蛋白表达的测量；以及涉及定量质谱学的新的蛋白质组学方法。这些试剂和方法将使用L-天冬氨酸敏感的人ALL细胞系和L-天冬氨酸抗性衍生物(Molt-4/R)来开发和优化。然后，这些方法和试剂将用于从ALL儿童身上获得的标本的常规使用。这项建议的长期目标是开发可用于未来研究的试剂和技术，以确定基线和诱导AS表达与ALL儿童预后的相关性。更详细地了解AS在人类白血病中的表达作用也将为人类AS的新型抑制剂在ALL中的潜在治疗作用提供重要的信息。

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.