CLINICAL PHENOTYPE TARGETED ANTIFOLATE CHEMOTHERAPY

人类白血病的药物敏感性和临床结果非常高， 这取决于它们的细胞来源。这项建议旨在 定义关于药物代谢的生化表型， 解释了抗叶酸剂表现出的谱系特异性反应， 人类白血病经典和新型抗叶酸剂的聚谷氨酰化 酶Folylpolyglutamate合成酶（FPGS）是它们生长所必需的。 药理活性，导致细胞内滞留时间延长 和增加的细胞毒性。 拟议的研究将测试 假设人类白血病对抗叶酸剂的反应依赖于 FPGS的表达。我们将研究生物化学和 据报道，临床观察的分子基础是， 体内白血病母细胞凋亡后， 接触这些药物。这些研究将确定底物的作用 对FPGS的亲和力和对关键叶酸代谢酶的抑制，以及 非多聚谷氨酰化抗叶酸剂和天然叶酸剂的作用。 对正常造血祖细胞在暴露于 抗叶酸剂将确定FPGS在药物选择性中的潜在作用。 此外，在正常人中，DNA甲基化和FPGS mRNA表达的变化 将研究骨髓细胞和白血病母细胞。评价 这些结果的临床意义，FPGS和多聚谷氨酰化 将在抗叶酸剂的临床样品中测定相关参数 敏感和耐药的白血病。 FPGS的临床意义 抗叶酸反应也将通过生长因子诱导的 耐药髓系白血病母细胞暴露于 这些试剂，并且在转染诱导型表达系统后， 将编码hFPGS转化为“酶缺陷型”抗性白血病表型。 总的来说，这一建议应提供一个明确的答案：1。如何 FPGS在抗叶酸剂的临床反应中是否重要？2.是什么 新型抗叶酸剂对白血病母细胞FPGS表达的影响， 这些在敏感表型、耐药表型和正常骨方面是否有差异 骨髓祖细胞？3. FPGS的谱系特异性表达是 生化表型预测因子重要临床决定因素 抗叶酸肿瘤反应？

The drug sensitivity and clinical outcome of human leukemias is highly dependent on their cell lineage of origin. This proposal will aim to define biochemical phenotypes with respect to drug metabolism that account for the lineage-specific response to antifolates exhibited by human leukemias. Polyglutamylation of classical and novel antifolates by the enzyme Folylpolyglutamate synthetase (FPGS) is essential to their pharmacological activity, resulting in prolonged intracellular retention and increased cytotoxicity. The proposed studies will test the hypothesis that the response of human leukemias to antifolates depend upon the expression of FPGS. We will investigate the biochemical and molecular basis for the reported clinical observation that a lineage- specific increase in FPGS activity occurs after in vivo leukemic blasts' exposure to these drugs. These studies will define the role of substrate affinity for FPGS and inhibition of key folate-metabolizing enzymes, and the effects of non-polyglutamylatable antifolates and natural folates. Similar studies with normal hematopoietic progenitors after exposure to antifolates will define the potential role of FPGS in drug selectivity. Further, changes in DNA methylation and FPGS mRNA expression in normal bone marrow cells and leukemic blasts will be investigated. To evaluate the clinical significance of these results, FPGS and polyglutamylation related parameters will be determined in clinical samples from antifolate sensitive and resistant leukemias. The clinical relevance of FPGS in antifolate response will also be tested by growth factor-induced upregulation of FPGS in resistant myeloid leukemic blasts' exposed to these agents, and after transfection of an inducible expression system encoding hFPGS to an "enzyme deficient" resistant leukemic phenotype. Overall, this proposal should provide a definitive answer to: 1. How important is FPGS in the clinical response to antifolates? 2. What are the effects of novel antifolates on leukemic blasts' FPGS expression, and do these differ in sensitive vs resistant phenotypes vs normal bone marrow progenitors? 3. Is the lineage-specific expression of FPGS an important clinical determinant of a biochemical phenotype predictor of antifolate tumor response?