Chemical Genomics Approach to Leukemia Differentiation

描述(申请人提供)：急性髓系白血病(AML)患者的长期存活率仍然很低，尽管对AML发病机制的了解取得了进展。然而，在急性早幼粒细胞白血病(APL)的化疗方案中加入分化治疗，大大提高了患者的存活率。实验证据表明，许多髓系白血病维持着细胞成熟的分子机制。不幸的是，很少有已知的分化的药理诱因，这些在治疗其他髓系恶性肿瘤方面显示出很少的疗效。我们假设存在替代的分化因子，它们可能既具有治疗潜力，又提供了对分化的分子机制的洞察。目前进行小分子文库筛选的方法是有限的。为了鉴定骨髓分化因子，我们设计了一种新的高通量筛选方法(HTS)。在这种方法中，基因表达模式作为分化表型的替代。利用DNA微阵列确定了原发人急性髓系白血病原始细胞与正常外周血中性粒细胞或单核细胞的基因表达特征。这些特征在HL60细胞系的造血分化模型中被证实是有区别的。然后将其简化为5基因多重RT-PCR方法。用一种新的质谱学方法检测了聚合酶链式反应扩增产物。在这个拟议的项目中，我们将利用这种筛选方法来确定新的候选AML分化因子，并优化剂量反应和动力学的暴露条件。这些化合物的生物活性将通过多种增殖和分化试验进行评估，并将使用表达谱来表征它们的分子后果。然后，它们的活性将在其他髓系细胞系和原发患者细胞中进行测试。利用这些已证实具有分化能力的药物，我们将采取多方面的方法，利用DNA微阵列技术和机制-活性关系测试来探索髓系原始细胞分化的潜在机制。这些候选化合物，以及对髓系白血病分化的更好理解，应该有助于指导我们寻找治疗AML的潜在药物。

DESCRIPTION (provided by applicant): Long-term survival for patients with acute myeloid leukemia (AML) remains poor despite advancement in the understanding of AML pathogenesis. However, the addition of differentiation therapy to chemotherapy regimens for patients with acute promyelocytic leukemia (APL) has greatly increased their survival. Experimental evidence suggests that many myeloid leukemias maintain the molecular machinery for cellular maturation. Unfortunately, there are few known pharmacological triggers of differentiation, and these have shown little efficacy in the treatment of other myeloid malignancies. We hypothesize that alternative differentiating agents exist that may have both therapeutic potential and provide insight into the molecular mechanisms of differentiation. Current methods of performing small molecule library screens are limited. To identify myeloid differentiating agents, we designed a new method of high throughput screening (HTS). In this method, a gene expression pattern served as a surrogate for the differentiated phenotype. A gene expression signature distinguishing primary human AML blasts from normal peripheral blood neutrophils or monocytes was determined using DNA microarrays. These signatures were confirmed to be discriminatory in an HL60 cell line model of hematopoietic differentiation. They were then simplified to a 5 gene multiplexed RT-PCR assay. PCR amplicon was detected with a novel method utilizing mass spectrometry. In this proposed project, we will utilize this screening method both to identify new candidate AML differentiating agents and to optimize exposure conditions for dose response and kinetics. The biological activity of these compounds will be evaluated with multiple assays for proliferation and differentiation, and expression profiling will be used to characterize their molecular consequences. Their activity will then be tested in other myeloid cell lines and in primary patient cells. Using those agents with a confirmed differentiation capacity, we will take a multifaceted approach to exploring potential mechanisms of myeloid blast differentiation with DNA microarray technology and mechanism-activity relationship testing. These candidate compounds, as well as an improved understanding of myeloid leukemia differentiation, should help direct us to potential therapeutic agents for AML.