The CML Epigenome - Implications for Therapy

由启动子甲基化和染色质修饰标记/介导的表观遗传学改变通过稳定地影响关键基因和途径的表达而促成肿瘤表型（疾病发病机制、进展、对治疗的抗性）。在CML中，初步数据表明，以启动子DNA甲基化为标志的表观遗传过程在疾病进展中以及对酪氨酸激酶抑制剂（TKI）的耐药性中起重要作用。表观遗传过程也可能影响对CML肿瘤细胞的免疫，例如通过掩蔽肿瘤抗原。基于此，我们提出以下假设：（一）表观遗传过程（DNA甲基化，组蛋白编码改变）有助于疾病进展（CP至AP和BP），以及对TKI的突变非依赖性耐药，通过稳定调节基因表达影响关键途径的功能，（ii）通过DNA甲基化抑制剂和组蛋白修饰药物靶向表观遗传沉默将补充TKI疗法， 晚期CML为了解决这些假设，我们提出了以下具体目标：（1）CML表观基因组的全球分析。使用甲基化CpG岛（MCA）和染色质免疫沉淀结合深度测序（在Solexa平台上），我们将确定CML细胞（与正常CD 34+细胞相比）在不同阶段（CP，AP，BP）和对TKI产生耐药性后的常见表观遗传异常。我们将使用标准技术和基因表达分析来验证选定的位点，并使用途径方法来研究CML中的功能性表观遗传异常。除了无监督的方法之外， 在PISK通路的鉴定中，我们还将具体检查PISK通路（与项目4的相互作用）和JAhC/STAT通路（与项目5的相互作用）以及潜在的免疫调节剂如肿瘤抗原（与项目2的相互作用）。(2)在前瞻性队列中选择表观遗传标记的预测和预后检验。在初步数据中，我们已经确定了CML进展的潜在DNA甲基化特征，以及TKI耐药。为此，我们将在参加TKI临床试验（与项目1的相互作用）以及干细胞移植治疗TKI耐药CML的所有阶段试验（与项目2的相互作用）的早期慢性期CML患者的前瞻性队列中测试该基因组的预测/预测价值。在随后的几年中，目标1中明确的基因/途径将在相同的队列中进行测试，以潜在地提高这些标志物的预测能力。(3)表观遗传作用药物治疗患者表观遗传参数的药效学分析。在这一特定目标中，我们将分析参加低甲基化药物地西他滨联合TKI达沙替尼临床试验（与项目1合作）和阿扎胞苷模拟抗CML免疫临床试验（项目2）的患者样本。我们将基线时的表观遗传模式与缓解可能性相关联，以及表观遗传调节（即药效学测量）与对药物/干细胞移植干预的缓解可能性相关联。该项目将提供关于CML表观遗传学改变、受影响的途径、预后意义以及表观遗传作用药物在该疾病治疗中的整合的确定性和全球性数据。

Epigenetic alterations marked/mediated by promoter methylation and chromatin modificafions contribute to neoplastic phenotypes (disease pathogenesis, progression, resistance to therapy) by stably affecting the expression of crifical genes and pathways. In CML, preliminary data suggest an important contribution of epigenetic processes marked by promoter DNA methylafion in the progression of disease, as well as resistance to tyrosine kinase inhibitors (TKIs). Epigenetic processes may also affect immunity against CML neoplastic cells, for example by masking tumor anfigens. Based on this, we propose the following hypotheses: (i) Epigenefic processes (DNA methylation, histone code alterations) contribute to disease progression (CP to AP and BP) in CML, as well as to mutation-independent resistance to TKIs, by affecting the function of crifical pathways through stable modulation of gene expression, (ii) Targefing epigenefic silencing by DNA methylafion inhibitors and histone modifying drugs will complement TKI therapy in advanced CML. To address these hypotheses, we propose the following specific aims: (1) Global analysis of the CML epigenome. Using Methylated CpG Island (MCA) and Chromatin Immunoprecipitation coupled with deep sequencing (on the Solexa platform), we will determine common epigenefic abnormalities in CML cells (compared to normal CD34+ cells) at various phases (CP, AP, BP) and after resistance to TKIs develop. We will validate selected loci using standard technology and gene expression analysis, and use a pathway approach to studying funcfional epigenetic anomalies in CML. In addifion to unsupervised approaches to pathway identificafion, we will also examine specifically the PISK pathway (an interaction with project 4) and the JAhC/STAT pathway (an interaction with project 5) and potential immunomodulators such as tumor anfigens (in an interaction with project 2). (2) Predicfive and prognostic tesfing of selected epigenefic marks in prospecfive cohorts. In preliminary data, we have Identified a potential DNA methylation signature of progression in CML, as well as TKI resistance. In this aim, we will test the prognosfic/predicfive value of this gene panel in prospecfive cohorts of patients with early chronic phase CML enrolled on clinical trials of TKI (an interaction with project 1), as well as trials of stem cell transplantation for TKI resistant CML at all phases (an interaction with project 2). In subsequent years, genes/pathways idenfified in aim 1 will be tested in the same cohorts to potenfially improve on the predicfive power of these markers. (3) Pharmacodynamic analysis of epigenefic parameters in patients treated with epigenetic acting drugs. In this specific aim, we will analyze samples from patients enrolled on a clinical trial ofthe hypomethylating agent decitabine in combinafion with the TKI dasatinib (collaborafion with project 1), and a clinical trial of azacitidine to sfimulate anfi-CML immunity (project 2). We will correlate epigenetic patterns at baseline with likelihood of response, as well as epigenetic modulation (i.e. pharmacodynamic measurement) with likelihood of response to the drug/stem cell transplant intervention. This project will provide definitive and global data on epigenetic alterations in CML, pathways affected, prognostic significance as well as integration of epigenetic acting drugs in the treatment of this disease.