Our recent studies on the treatment of twenty-four patients with end stage acute myeloid leukemia and myeloid blast crisis of granulocytic leukemia with tiazofurin revealed that in this poor prognosis group of patients five attained complete seen in four others. The effect lasted for 3 to 10 months with intermittent drug treatment. We have demonstrated that only 50% of the patients respond; therefore, we need a predictive test to spare those patients who might not benefit. Therefore, the first Specific Aim of the proposal is to test the hypothesis that an in vitro test to predict the sensitivity of leukemic cells of patients to tiazofurin can be developed on the basis of biochemical targets. To test this hypothesis, we will elucidate the relationship between the predictive test, which would be based on the levels of TAD (thiazole-4-carboxamide adenine dinucleotide, active metabolite of tiazofurin) and the associated decrease in the GTP pools (target of tiazofurin action); and tiazofurin metabolizing enzyme activities (tiazofurin phosphorylation, NAD pyrophosphorylase and TAD phosphodiesterase). To achieve this goal the project plans to separate and enrich leukemic cells from leukemic patients and their counter parts (from healthy volunteers) and incubate in vitro with radiolabeled tiazofurin. The second Specific Aim of this project is to test the hypothesis that the regulation of tiazofurin metabolism and the extent of conversion of tiazofurin to TAD is correlated with hematologic response in refractory leukemia. To test this hypothesis blood samples will be obtained from patients undergoing tiazofurin therapy and the concentration of TAD in leukemic cells will be correlated with GTP levels and hematologic response. The activities of tiazofurin metabolizing enzymes will be determined in duplicate cell samples prepared for the predictive test. The third Specific Aim of this 5-year program is to test the hypotheses that the emergence of tiazofurin resistant leukemic cells is regulated by tiazofurin metabolism. to test this hypothesis we will elucidate the regulation of TAD by monitoring the extent of TAD synthesis by tiazofurin metabolizing enzymes, and the targets, IMP dehydrogenase activity and GTP pools in leukemic cells obtained from patients sensitive and clinically refractory to tiazofurin. This program should yield novel conceptual approaches to the treatment of human leukemia.
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