Chemical Genomics Approach to Leukemia Differentiation

描述（由申请人提供）： 急性髓细胞白血病（AML）患者的长期生存率仍然很低，尽管在AML发病机制的理解进步。 然而，在急性早幼粒细胞白血病（APL）患者的化疗方案中加入分化治疗大大提高了他们的生存率。 实验证据表明，许多髓性白血病维持细胞成熟的分子机制。 不幸的是，很少有已知的分化的药理学触发因素，并且这些在其他骨髓恶性肿瘤的治疗中几乎没有显示出疗效。 我们假设存在替代的分化剂，它们可能既具有治疗潜力，又能深入了解分化的分子机制。 目前进行小分子文库筛选的方法是有限的。 为了鉴定髓系分化剂，我们设计了一种新的高通量筛选（HTS）方法。 在该方法中，基因表达模式作为分化表型的替代。 使用DNA微阵列确定区分原代人AML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。 这些特征被证实在造血分化的HL 60细胞系模型中是有区别的。 然后将它们简化为5基因多重RT-PCR测定。 PCR扩增产物的检测与一种新的方法，利用质谱。 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分化剂，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项增殖和分化试验进行评价，表达谱将用于表征其分子结果。 然后将在其他骨髓细胞系和原代患者细胞中测试它们的活性。 使用这些具有确认的分化能力的药物，我们将采用多方面的方法，通过DNA微阵列技术和机制-活性关系测试来探索髓系母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AML的潜在治疗药物。

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.