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调节白血病原始细胞中甲氨蝶呤（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是治疗这种疾病的关键药物。我们的方法是基于我们的独特发现，即外源性5-氨基-4-咪唑-甲酰胺核苷（Z）在临床前儿童ALL模型中增强MTX的摄取、代谢和细胞毒性效力。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。适当使用Z作为增效剂的关键是理解其机制，这将通过五个具体目标来阐明：1）表征儿童ALL细胞培养模型中MTX摄取和代谢的Z增效作用。我们的研究结果表明，Z增强MTX的积累和代谢，MTX聚谷氨酸盐主要是通过以前未发现的增强活性的还原叶酸载体（RFC），这也运输MTX。RFC对MTX摄取的Z增强作用以及其他因素在MTX聚谷氨酸积累增强中的作用必须完全明确。2)确定二氢叶酸还原酶（DHFR）抑制在MTX摄取的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。将通过使用特异性核苷转运或代谢酶的抑制剂和/或使用特异性核苷转运或代谢酶突变的细胞系以及通过使用放射性标记Z的潜在代谢物的直接定量来探索Z转运和代谢的要求。4)确定将Z信号转化为增加的RFC活性的生化机制。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活性。这两类机制都将被探讨。5)通过确定MTX+Z组合在儿童ALL的体内临床前模型中的剂量、时间表和疗效，确定Z增强的临床潜力。如果成功，这项研究可以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.