Enhancement of methotrexate uptake in childhood ALL

描述(由申请人提供)：该项目的总体目标是通过选择性地调节白血病母细胞对甲氨蝶呤(MTX)的吸收和代谢来提高儿童急性淋巴细胞白血病(ALL)的治愈率，MTX是治愈这种疾病的关键药物。我们的方法是基于我们的独特发现，在临床前儿童ALL模型中，外源性5-氨基-4-咪唑-羧胺核苷(Z)增强了MTX的摄取、代谢和细胞毒力。由于Z已用于治疗心肌缺血的临床试验，我们的临床前研究可能很容易转化为儿科临床试验。正确使用Z作为增强剂的关键是对其机制的理解，这将通过五个具体目标来阐明：1)在儿童ALL细胞培养模型中表征Z对MTX摄取和代谢的增强作用。我们的结果表明，Z主要通过增强还原型叶酸载体(RFC)的活性来增强MTX对MTX多谷氨酸的积累和代谢，RFC也运输MTX。Z RFC对MTX摄取的增强作用以及其他因素在增强MTX聚谷氨酸积累中的作用必须得到充分的定义。2)确定二氢叶酸还原酶(DHFR)抑制在甲氨蝶呤摄取Z增强中的作用。3)确定增强MTX摄取是否需要特定的Z代谢。将通过使用特定核苷转运或代谢酶突变的细胞系和/或使用特定核苷转运或代谢酶突变的细胞系，以及通过直接定量放射性标记的Z的潜在代谢物来探索Z运输和代谢的需求。4)确定将Z信号转化为RFC活性增加的生化机制。由于不需要新的蛋白质合成(结果)，Z可以通过诱导翻译后修饰或变构来增加RFC的活性。这两类机制都将被探索。5)在儿童ALL的临床前活体模型中，通过测定MTX+Z联合用药的剂量、方案和治疗效果，确定Z增强的临床潜力。如果成功，这项研究可能会提供一种方法来提高甲氨蝶呤的有效性，甲氨蝶呤是治疗儿童急性淋巴细胞白血病的关键药物，从而提高这种疾病的治愈率。

DESCRIPTION (provided by applicant): The overall goal of this project is to increase cure of childhood acute lymphoblastic leukemia (ALL) by selectively modulating in leukemic blasts the uptake and metabolism of methotrexate (MTX), a drug key in curing this disease. Our approach is based on our unique finding that exogenous 5-amino-4-imidazole-carboxamide riboside (Z) potentiates uptake, metabolism, and cytotoxic potency of MTX in a preclinical childhood ALL model. Since Z has been used in clinical trials for treating cardiac ischemia, our preclinical studies may be readily translatable into a pediatric clinical trial. Critical to appropriate use of Z as a potentiator is an understanding of its mechanism, which will be elucidated through five Specific Aims: 1) Characterize Z potentiation of MTX uptake and metabolism in a cell culture model of childhood ALL. Our results show that Z potentiates MTX accumulation and metabolism to MTX polyglutamates primarily via a previously undiscovered enhancement in activity of the reduced folate carrier (RFC), which also transports MTX. Z potentiation of MTX uptake by the RFC and the role of other factors in potentiation of MTX polyglutamate accumulation must be fully defined. 2) Determine the role of dihydrofolate reductase (DHFR) inhibition in Z potentiation of MTX uptake. 3) Determine whether specific Z metabolism is required for potentiation of MTX uptake. A requirement for Z transport and metabolism will be explored by using inhibitors of, and/or using cell lines with mutations in, specific nucleoside transport or metabolizing enzymes and by using direct quantitation of potential metabolites of radiolabeled Z. 4) Determine the biochemical mechanisms that transduce the Z signal into increased RFC activity. Since new protein synthesis is not required (Results), Z could increase RFC activity by inducing a post-translational modification or allosterically. Both classes of mechanism will be explored. 5) Define the clinical potential of Z potentiation by determining the dose, schedule, and therapeutic efficacy of MTX+Z combinations in in vivo preclinical models of childhood ALL. If successful, this research could provide a way to increase the effectiveness of methotrexate, a key drug used in treating childhood acute lymphoblastic leukemia, and thus increase the cure rate in this disease.