5-Azacytidine and MS-275 in Myeloid Malignancies

描述（由申请人提供）：5-氮杂胞苷（5AC）是一种治疗骨髓增生异常综合征（MDS，有效率50 - 60%）的高活性药物。5AC延缓MDS向急性髓系白血病（AML）发展；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于其对DNA甲基转移酶（DNMT）的有效抑制。细胞暴露于DNMT抑制剂（DNMTi）可导致转录沉默基因启动子区域CpG岛甲基化的逆转，并可导致这些基因的重新表达。通过将DNMTi与组蛋白去乙酰化酶抑制剂（HDACi）联合使用，可以实现更强的甲基化基因的再表达。MS-275是一种口服生物可利用的HDACi，在给药后可诱导组蛋白超乙酰化长达三周。这种新的试剂提供了一个很好的机会来测试联合DNMTi和HDACi在髓系恶性肿瘤中的概念。作为快速试验申请基础的临床试验将确定5AC皮下和MS-275口服的最佳剂量和时间表。该申请要求资金进行实验室研究，旨在测试5AC与MS-275联合导致沉默基因重新表达的假设，并且这种重新表达与临床反应相关。患者将接受5AC和MS-275的各种剂量和方案的治疗（在每个治疗周期的第3天和第10天给药）。将对数据进行评估，以确定“临床最佳剂量”，最大限度地提高组蛋白超乙酰化和基因再表达，同时将毒性降到最低。定量rt-PCR将用于监测两个基因的表达，这两个基因在AML和MDS中最常通过甲基化沉默：p15INK4B和Ecadherin （E-CAD）。表达微阵列将在分离的CD34+细胞上进行，以确定在这种药物组合下表达上调或下调的基因和分子途径，这些基因和分子途径可能与反应（无论甲基化与否）有关。将使用甲基化特异性PCR的实时PCR修饰和PCR产物的亚硫酸盐测序来研究患者样本启动子甲基化的变化。染色质免疫沉淀法将鉴定与p15和p21WAF1/CIP1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DNA损伤标记物磷酸化H2AX的变化，以评估这些药物诱导DNA链断裂的程度。从该试验中获得有希望的初步数据后，将直接进行5AC + MS-275与5AC单独治疗的随机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.