Molecular Correlates of Methotrexate in Childhood ALL

描述（由申请人提供）：该项目是首席研究员、基础科学家和内科科学家长期合作的结果，旨在确定儿童急性淋巴细胞白血病（ALL）治疗反应的决定因素。重点是甲氨蝶呤（MTX）和6-巯基嘌呤（6-MP），这两种抗代谢物仍然是ALL现代治疗方案的基石，以及与这些药物的有效临床使用有关的关键未解问题。虽然在治疗儿童ALL方面取得了重大进展，但这在一定程度上反映了包括近50%预后良好的患者的亚组。然而，在治疗其他病人方面的成功却落后了。我们之前证明：(a)在ALL淋巴细胞中，人类减少叶酸载体（hRFC）， MTX和四氢叶酸的膜运输系统的表达范围非常广（88倍），并且通常与MTX运输水平不成比例(b)在预后良好的ALL表型中，包括高二倍体和t(12;21)；(e) hRFC mRNA剪接形式改变，hRFC编码序列中的基因序列变异，以及hRFC启动子中的高频序列缺失。研究旨在探讨(d)亚甲基四氢叶酸还原酶（MTHFR）基因中C677T单核苷酸高频多态性和(e)多药耐药蛋白（MRPs）家族对儿科all化疗反应和耐药的贡献。在这项应用中，我们建议使用ALL原代标本和移植到NOD-SCID小鼠体内的ALL异种移植物来系统地建立：(i) hRFC表达模式与MTX转运和敏感性之间的关系；（ii）通过体外和体内实验，在hRFC- b主要启动子的原始标本中，导致hRFC广泛表达的分子机制；（iii）通过对ALL和非ALL标本的高通量筛选以及转染细胞的功能分析，hRFC和MTHFR基因和转录序列变异作为MTX反应和耐药性的决定因素的作用；（iv） MRPs在ALL和转染细胞中作为MTX和6-MP积累和敏感性的决定因素的作用。我们的研究结果应该通过确定最有可能对抗代谢物治疗有反应的患者和/或可能经历过度毒性的患者来促进儿童ALL的合理化疗。我们的研究也应该导致新的ALL治疗方法，并与抗叶酸药物治疗其他恶性和非恶性疾病相关。从hRFC在哺乳动物细胞中叶酸积累中的关键作用以及叶酸缺乏对染色体不稳定和恶性转化、心血管疾病和胎儿异常的潜在贡献来看，我们的研究结果应该对人类健康和疾病具有深远的意义。

DESCRIPTION (provided by applicant): This project is the result of a long-term collaboration between the principal investigator, a basic scientist, and physician scientists and is designed to identify determinants of treatment response in childhood acute lymphoblastic leukemia (ALL). The focus is on methotrexate (MTX) and 6-mercaptopurine (6-MP), antimetabolites that continue to be cornerstones of modern treatment protocols for ALL, and on critical unanswered questions relating to the effective clinical use of these agents. While major progress has been made in treating childhood ALL, this, in part, reflects subgroups comprising nearly 50% of patients with uniquely good prognoses. However, success in the treatment of other patients has lagged behind. We previously demonstrated: (a) a extraordinarily wide range (88-fold) of expression of the human reduced folate carrier (hRFC), the membrane transport system for MTX and tetrahydrofolates, in ALL lymphoblasts, and often disproportionate to levels of MTX transport (b) elevated hRFC expression in good prognosis ALL phenotypes, including hyperdiploidy and t(12;21); and (e) altered hRFC mRNA splice forms, gene sequence variants in the hRFC coding sequence, and a high frequency sequence deletion in a hRFC promoter. Studies were initiated to explore the contributions of (d) the high frequency C677T single nucleotide polymorphism in the methylene tetrahydrofolate reductase (MTHFR) gene and (e) the family of multidrug resistance proteins (MRPs) to chemotherapy response and resistance in pediatric ALLs. In this application, we propose to use primary ALL specimens and ALL xenografts engrafted in NOD-SCID mice to systematically establish: (i) the relationships between patterns of hRFC expression and MTX transport and sensitivities; (ii) the molecular mechanisms that result in the wide range of hRFC expression in primary specimens from the major hRFC-B promoter by in vitro and in vivo assays; (iii) the roles of gene and transcript sequence variants for hRFC and MTHFR as determinants of MTX response and resistance by high-throughput screening of ALL and non-ALL specimens and functional analysis in transfected cells; and (iv) the roles of the MRPs as determinants of MTX and 6-MP accumulations and sensitivities in ALL and transfected cells. Our results should foster the rational chemotherapy of childhood ALL by identifying patients most likely to respond to antimetabolite treatments, and/or who are likely to experience excessive toxicities. Our studies should also lead to new ALL therapies and be relevant to other malignant and non-malignant diseases treated with antifolate drugs. From the key role of hRFC in folate accumulations in mammalian cells and the potential contributions of folate deficiencies to chromosomal instability and malignant transformation, cardiovascular disease, and fetal abnormalities, our results should have far-reaching significance to human heath and disease.