5-Azacytidine and MS-275 in Myeloid Malignancies

5-氮杂胞苷和 MS-275 在骨髓恶性肿瘤中的应用

• 批准号: 6922848
• 负责人: STEVEN D GORE
• 金额: $ 25.86万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-15 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6922848
• 关键词: DNA methylation; acetylation; acute myelogenous leukemia; amidohydrolases; antineoplastics; azacitidine; chromatin immunoprecipitation; clinical research; clinical trial phase I; clinical trial phase II; combination chemotherapy; dosage; enzyme inhibitors; gene induction /repression; human subject; methyltransferase; microarray technology; myelogenous leukemia; neoplasm /cancer chemotherapy; neoplasm /cancer pharmacology; nucleic acid sequence; oral administration; patient oriented research; subcutaneous drug administration; western blottings

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.

描述（由申请人提供）：5-氮杂胞苷（5AC）是一种用于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 发展为急性髓系白血病 (AML)；然而，5AC 的中位反应持续时间少于 18 个月。该药物的临床活性机制可能源自其对 DNA 甲基转移酶 (DNMT) 的有效抑制。细胞暴露于 DNMT 抑制剂 (DNMTi) 会导致转录沉默基因启动子区域 CpG 岛的甲基化逆转，并可能导致此类基因的重新表达。通过 DNMTi 与组蛋白脱乙酰酶抑制剂 (HDACi) 的组合，可以实现甲基化基因更稳健的重新表达。 MS-275 是一种口服生物可利用的 HDACi，可诱导组蛋白过度乙酰化，并在给药后持续长达三周。这种新试剂为测试 DNMTi 和 HDACi 联合治疗骨髓恶性肿瘤的概念提供了绝佳的机会。构成此快速试验应用基础的临床试验将确定皮下注射 5AC 和口服 MS-275 的最佳剂量和时间表。该申请请求资金进行实验室研究，旨在测试以下假设：5AC 与 MS-275 的组合会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 天施用）。将评估数据以确定“临床最佳剂量”，以最小的毒性最大化组蛋白过度乙酰化和基因重新表达。定量 rt-PCR 将用于监测 AML 和 MDS 中最常通过甲基化沉默的两个基因的表达：p15INK4B 和 Ecadherin (E-CAD)。将在分离的 CD34+ 细胞上进行表达微阵列，以鉴定其表达上调或下调的基因和分子途径，以响应可能与反应（无论是否甲基化）相关的药物组合。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来研究患者样本的启动子甲基化变化。染色质免疫沉淀分析将鉴定与 p15 和 p21WAF1/CIP1 启动子序列相关的组蛋白乙酰化变化。最后，Western 分析将用于寻找磷酸化 H2AX（DNA 损伤标记）的变化，以评估这些药物诱导 DNA 链断裂的程度。在该试验获得有希望的初步数据后，将直接进行 5AC 加 MS-275 与单独 5AC 的相同方案的随机 II 期试验，以研究添加 HDAC 抑制剂的临床重要性。