5-Azacytidine and MS-275 in Myeloid Malignancies

描述（申请人提供）：5-氮杂胞苷（5AC）是一种治疗骨髓增生异常综合征（MDS，缓解率50 - 60%）的高活性药物。5AC延缓MDS向急性髓性白血病（AML）的进展;然而，对5AC的反应的中位持续时间小于18个月。该药物的临床活性机制可能来自其对DNA甲基转移酶（DNMT）的强效抑制。细胞暴露于DNMT抑制剂（DNMTi）导致转录沉默基因的启动子区域中的CpG岛的甲基化逆转，并且可以导致这些基因的重新表达。甲基化基因的更稳健的再表达可以通过DNMTi与组蛋白脱乙酰酶抑制剂（HDACi）的组合来实现。MS-275，一种口服生物可利用的HDACi，诱导组蛋白超乙酰化，在给药后持续长达三周。这种新试剂提供了一个极好的机会来测试组合DNMTi和HDACi在骨髓恶性肿瘤中的概念。构成此快速试验应用基础的临床试验将确定皮下5AC和口服MS-275的最佳剂量和时间表。该申请要求提供资金进行实验室研究，旨在检验以下假设：5AC与MS-275的组合导致沉默基因的重新表达，并且这种重新表达与临床反应相关。患者将接受各种剂量和时间表的5AC和MS-275治疗（在每个治疗周期的第3天和第10天给药）。将对数据进行评价，以确定“临床最佳剂量”，该剂量可使组蛋白超乙酰化和基因再表达最大化，同时毒性最小。将使用定量rt-PCR监测AML和MDS中最常通过甲基化沉默的两种基因的表达：p15 INK 4 B和E钙粘蛋白（E-CAD）。将对分离的CD 34+细胞进行表达微阵列分析，以鉴定其表达在该药物组合反应中上调或下调的基因和分子通路，其可能与反应相关（无论是否甲基化）。将使用甲基化特异性PCR的实时PCR修饰和PCR产物的亚硫酸氢盐测序研究患者样本的启动子甲基化变化。染色质免疫沉淀试验将鉴定与p15和p21 WAF 1/CIP 1启动子序列相关的组蛋白乙酰化的变化。最后，Western分析将用于寻找磷酸化H2 AX（DNA损伤的标志物）的变化，以评估这些药物诱导DNA链断裂的程度。这项试验的初步数据很有希望，随后将直接进行5AC加MS-275与单独5AC相同方案的随机II期试验，以研究添加HDAC抑制剂的临床重要性。

DESCRIPTION (provided by applicant): 5-azacytidine (5AC) is a highly active agent for the treatment of myelodysplastic syndromes (MDS, response rate 50 - 60%). 5AC retards the progression of MDS to acute myeloid leukemia (AML); however, the median duration of response to 5AC is less than 18 months. The mechanism of clinical activity of this agent may derive from its potent inhibition of DNA methyltransferase (DNMT). Exposure of cells to DNMT inhibitors (DNMTi) leads to reversal of methylation of CpG islands in the promoter regions of transcriptionally silenced genes and can lead to re-expression of such genes. More robust re-expression of methylated genes may be accomplished by the combination of DNMTi with histone deacetylase inhibitors (HDACi). MS-275, an orally bioavailable HDACi, induces histone hyperacetylation sustained up to three weeks following drug administration. This new reagent provides an outstanding opportunity to test the concept of combined DNMTi and HDACi in myeloid malignancies. The clinical trial forming the basis of this Quick Trials application will identify an optimal doses and schedule of subcutaneous 5AC and oral MS-275. The application requests funds to perform laboratory studies aimed at testing the hypothesis that combinations of 5AC with MS-275 lead to re-expression of silenced genes, and that such re-expression is associated with clinical response. Patients will be treated with a variety of doses and schedules of 5AC and MS-275 (administered on days 3 and 10 of each treatment cycle). The data will be evaluated to identify a "clinically optimal dose" which maximizes histone hyperacetylation and gene re-expression with minimal toxicity. Quantitative rt-PCR will be used to monitor the expression of two genes which are most commonly silenced through methylation in AML and MDS: p15INK4B and Ecadherin (E-CAD). Expression microarrays will be performed on isolated CD34+ cells to identify genes and molecular pathways whose expression is up- or down-regulated in response to this combination of drugs which may be related to response (whether methylated or not). Patient samples will be studied for changes in promoter methylation using realtime PCR modifications of methylation-specific PCR, and bisulfite sequencing of PCR products. Chromatin immunoprecipitation assays will identify changes in acetylation of histones associated with promoter sequences in p15 and p21WAF1/CIP1. Finally, Western analysis will be used to seek changes in phosphorylated H2AX, a marker of DNA damage, to evaluate the extent to which these drugs induce DNA strand breaks. Promising preliminary data from this trial will be followed directly by a randomized Phase II trial of 5AC plus MS-275 versus the same schedule of 5AC alone to investigate the clinical importance of the addition of the HDAC inhibitor.