Vorinostat Induces Apoptosis and Differentiation in Myeloid Malignancies: Genetic and Molecular Mechanisms

BackgroundAberrant epigenetic patterns are central in the pathogenesis of haematopoietic diseases such as myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Vorinostat is a HDACi which has produced responses in these disorders. The purpose of this study was to address the functional effects of vorinostat in leukemic cell lines and primary AML and MDS myeloid cells and to dissect the genetic and molecular mechanisms by which it exerts its action. Methodology/Principal FindingsFunctional assays showed vorinostat promoted cell cycle arrest, inhibited growth, and induced apoptosis and differentiation of K562, HL60 and THP-1 and of CD33+ cells from AML and MDS patients. To explore the genetic mechanism for these effects, we quantified gene expression modulation by vorinostat in these cells. Vorinostat increased expression of genes down-regulated in MDS and/or AML (cFOS, COX2, IER3, p15, RAI3) and suppressed expression of genes over-expressed in these malignancies (AXL, c-MYC, Cyclin D1) and modulated cell cycle and apoptosis genes in a manner which would favor cell cycle arrest, differentiation, and apoptosis of neoplastic cells, consistent with the functional assays. Reporter assays showed transcriptional effect of vorinostat on some of these genes was mediated by proximal promoter elements in GC-rich regions. Vorinostat-modulated expression of some genes was potentiated by mithramycin A, a compound that interferes with SP1 binding to GC-rich DNA sequences, and siRNA-mediated SP1 reduction. ChIP assays revealed vorinostat inhibited DNA binding of SP1 to the proximal promoter regions of these genes. These results suggest vorinostat transcriptional action in some genes is regulated by proximal promoter GC-rich DNA sequences and by SP1. ConclusionThis study sheds light on the effects of vorinostat in AML and MDS and supports the implementation of clinical trials to explore the use of vorinostat in the treatment of these diseases.

背景 表观遗传异常模式是骨髓增生异常综合征（myelodysplastic syndromes, MDS）、急性髓系白血病（acute myeloid leukaemia, AML）等造血系统疾病发病机制的核心环节。伏立诺他（vorinostat）是一种组蛋白去乙酰化酶抑制剂（HDACi），在这类疾病中已展现出治疗应答。本研究旨在探究伏立诺他对白血病细胞系、原代AML及MDS髓系细胞的功能效应，并解析其发挥作用的遗传与分子机制。
方法与主要结果 功能实验显示，伏立诺他可促进K562、HL60、THP-1细胞以及AML和MDS患者CD33+细胞的细胞周期阻滞，抑制细胞增殖，并诱导其凋亡与分化。为探究上述效应的遗传机制，我们对伏立诺他处理后这些细胞的基因表达调控情况进行了定量分析。伏立诺他可上调在MDS和/或AML中表达下调的基因（cFOS、COX2、IER3、p15、RAI3），并下调在这类恶性肿瘤中过表达的基因（AXL、c-MYC、Cyclin D1）；其对细胞周期与凋亡相关基因的调控模式可促进肿瘤细胞的周期阻滞、分化与凋亡，与功能实验结果一致。报告基因实验表明，伏立诺他对部分上述基因的转录调控作用由GC富集区域的近端启动子元件所介导。伏立诺他对部分基因的表达调控可被光神霉素A（mithramycin A）增强——该化合物可干扰SP1与GC富集DNA序列的结合，而通过小干扰RNA（siRNA）敲降SP1也可达到类似效果。染色质免疫沉淀（ChIP）实验显示，伏立诺他可抑制SP1与这些基因近端启动子区域的DNA结合。上述结果表明，伏立诺他对部分基因的转录调控作用受近端启动子GC富集DNA序列及SP1的调控。
结论 本研究阐明了伏立诺他在AML与MDS中的作用机制，支持开展临床试验以探索伏立诺他在这类疾病治疗中的应用。