Molecular Correlates of Methotrexate in Childhood ALL

描述（由申请人提供）：该项目是主要研究者，基础科学家和医生科学家之间长期合作的结果，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），抗代谢物，仍然是现代治疗方案的基石，为所有，并在关键的悬而未决的问题，有关这些药物的有效临床使用。虽然在治疗儿童ALL方面取得了重大进展，但这部分反映了近50%的患者亚组具有独特的良好预后。然而，其他患者的治疗成功却落后了。我们之前演示过：（a）人还原叶酸载体（hRFC），MTX和四氢叶酸的膜转运系统，在ALL淋巴母细胞中非常广泛（88倍）的表达，并且通常与MTX转运水平不成比例（B）在预后良好的ALL表型中升高的hRFC表达，包括超二倍体和t（12;21）;和（e）改变的hRFC mRNA剪接形式、hRFC编码序列中的基因序列变体和hRFC启动子中的高频序列缺失。启动研究以探索（d）亚甲基四氢叶酸还原酶（MTHFR）基因中的高频C677 T单核苷酸多态性和（e）多药耐药蛋白（MRP）家族对儿科ALL化疗反应和耐药性的贡献。在本申请中，我们建议使用原代ALL标本和移植到NOD-SCID小鼠的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）通过体外和体内测定，导致主要hRFC-B启动子在原代标本中广泛表达hRFC的分子机制;（iii）通过ALL和非ALL标本的高通量筛选和转染细胞中的功能分析，hRFC和MTHFR的基因和转录物序列变体作为MTX应答和抗性的决定因素的作用;和（iv）MRPs作为MTX和6-MP在ALL和转染细胞中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识别最有可能对抗代谢药物治疗有反应的患者和/或可能出现过度毒性的患者来促进儿童ALL的合理化疗。我们的研究还应该导致新的ALL疗法，并与抗叶酸药物治疗的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、心血管疾病和胎儿畸形的潜在贡献来看，我们的结果对人类健康和疾病应该具有深远的意义。

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.