Topoisomerase Targeting and Resistance in Leukemia

描述（由申请人提供）：本项目的总体目标是通过开发基于机制的拓扑异构酶靶向治疗来改善高危急性非淋巴细胞白血病患者的治疗。拓扑替康作为拓扑异构酶I靶向药物的最新发展，在白血病的治疗中具有活性，允许研究涉及拓扑异构酶I和拓扑异构酶II靶向药物的合理设计的组合方案。细胞培养模型表明拓扑替康耐药往往涉及药物外排，或拓扑异构酶I的下调或突变。新的数据还暗示泛素/蛋白酶体途径对喜树碱的耐药性。此外，细胞对喜树碱的耐药性通常伴随着拓扑异构酶II蛋白水平和催化活性的上调，导致对拓扑异构酶II靶向药物如依托泊苷的超敏反应。涉及从接受托泊替康治疗的患者获得的细胞的初步研究表明，这些临床前模型可能具有临床相关性。然而，关于白血病患者中治疗诱导的细胞拓扑异构酶水平和活性的改变知之甚少，并且在这些患者中有效的拓扑替康耐药机制尚不清楚。 在本项目的第一个目的中，将在从接受阿糖胞苷/拓扑替康诱导治疗的患者中获得的原始细胞中定量拓扑异构酶II蛋白水平和活性。将评价拓扑异构酶II峰改变的时间，以确定给予依托泊苷的最佳时间。随后，将确定该给药方案的依托泊苷最大耐受剂量。 第二个目的是确定拓扑替康耐药的临床机制，包括分析从患者中获得的原始细胞中的细胞内拓扑替康蓄积、BCRP表达、拓扑异构酶I突变以及拓扑异构酶I泛素化和降解。从第一个和第二个目标获得的数据将与第三个目标的临床结局相关。这些研究的结果应提供洞察拓扑替康耐药的临床机制，并允许合理开发拓扑异构酶靶向策略治疗白血病。

DESCRIPTION (provided by applicant): The overall goal of this project is to improve the treatment of patients with high-risk acute non-lymphocytic leukemia by developing mechanism-based topoisomerase-targeting therapies. The recent development of topotecan as a topoisomerase I-targeting drug that is active in the therapy of leukemia allows investigation of rationally designed combination regimens involving both topoisomerase I- and topoisomerase Il-targeting drugs. Cell culture models indicate that resistance to topotecan often involves drug efflux, or downregulation or mutation of topoisomerase I. New data also implicate ubiquitin/proteasome pathways in resistance to camptothecins. Moreover, cellular resistance to camptothecins is usually accompanied by upregulation of topoisomerase II protein levels and catalytic activity, resulting in hypersensitivity to topoisomerase Il-targeting drugs such as etoposide. Initial studies involving cells obtained from patients treated with topotecan suggest that these preclinical models may be clinically relevant. However, relatively little is known regarding treatment-induced alterations in cellular topoisomerase levels and activity in patients with leukemia, and the mechanisms of resistance to topotecan that are operative in these patients are unknown. In the first aim of this project, topoisomerase Il protein levels and activity will be quantified in blasts obtained from patients receiving cytosine arabinoside/topotecan induction therapy. The timing of peak topoisomerase Il alterations will be evaluated to determine an optimal time for administration of etoposide. Subsequently, the maximum-tolerated dose of etoposide will be identified for this schedule of drug administration. The second aim concerns identification of clinical mechanisms of resistance to topotecan, and includes analyses of intracellular topotecan accumulation, BCRP expression, topoisomerase I mutations, and topoisomerase I ubiquitylation and degradation in blasts obtained from patients. The data obtained from the first and second aims will be correlated with clinical outcomes in the third aim. The results of these studies should provide insight into clinical mechanisms of resistance to topotecan, and allow rational development of a topoisomerase-targeting strategy in the treatment of leukemia.