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.
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