Differentially methylated gene regions (DMRs) induced by doxorubicin in heart: significance and clinical application

项目摘要 包括阿霉素在内的化疗用于治疗50%的儿科癌症患者， 阿霉素对肿瘤有效，11%的儿童患者接受阿霉素治疗后出现心脏功能障碍/衰竭 作为成年人迟发性阿霉素心脏毒性的机制尚不清楚， 目前FDA已批准用于儿童。我们独特的阿霉素心脏毒性大鼠模型结果 在7个月的潜伏期后，心脏功能不全，允许研究儿童心脏保护治疗 癌症（骨肉瘤）的免疫活性大鼠。在这个模型中，我们已经确定了一组差异 多柔比星治疗大鼠心脏中DNA甲基化区域（DMR），使用甲基-Seq靶富集 系统平台。我们假设阿霉素治疗改变了肿瘤细胞DNA甲基化的模式， 包括动物和人类患者的易感个体的心肌细胞。这种异常的DNA甲基化 可以作为阿霉素后损伤的DNA生物标志物。我们进一步假设DNA甲基化 通过有效的心脏保护剂治疗可以使关键基因调节区域的变化最小化， 减少心脏功能缺陷、心肌变性、间质纤维化和氧化应激DNA 损害在目标1中，我们将确定DNA中差异甲基化区域（DMR）的时间特征 阿霉素诱导的心脏和心脏保护剂如何调节DNA甲基化模式。使用 我们在初步研究中确定了一个由6个DMR组成的强大面板，我们将定义 阿霉素诱导的雄性和雌性大鼠DNA甲基化。我们还将测试心脏保护剂的效果 DNA甲基化，以验证DNA甲基化和心脏毒性之间的关系，并将确定 这些药物预防心脏病的潜在有效性，通过超声心动图监测，肌钙蛋白I 测量和组织学评分。我们还将确定海岸区域的DNA甲基化是否 代表可以将甲基化扩散到CpG岛并影响RNA表达的早期变化。提供 作为转化人类研究的桥梁，我们将研究阿霉素诱导的心脏DNA甲基化变化 免疫功能正常的大鼠的骨肉瘤。在目标2中，我们将确定人类心脏DNA是否来自 多柔比星治疗的患者与年龄匹配的对照患者相比具有差异甲基化的区域。 将来自多柔比星治疗的患者的库存冷冻心肌与年龄匹配的对照进行比较， Illumina Infinium人类甲基化450微珠芯片阵列以确定DMR模式。为了确定这些 最近发表的缺血性心脏病患者的DNA甲基化数据显示， 将与暴露于阿霉素的患者的DNA甲基化进行比较。我们的长期 目的（PA-19-111）是鉴定新的心脏特异性生物标志物，以帮助肿瘤学家和心脏病学家进行 心脏毒性的早期诊断方法，以便可以使用心脏保护策略。这是第一步 “概念验证”研究，以确定心脏组织中的DMR候选物，为未来的血清DMR研究做准备 在有记录的心脏阿霉素毒性的儿童或成人队列中，以建立诊断灵敏度。

Project Summary Chemotherapy that includes doxorubicin is used to treat 50% of all pediatric cancer patients, and while highly effective against tumors, 11% of patients treated as children with doxorubicin develop heart dysfunction/ failure as adults. Mechanisms of delayed doxorubicin cardiotoxicity are not clearly understood and no cardioprotective agent is currently FDA-approved for use in children. Our unique rat model of doxorubicin cardiotoxicity results in cardiac dysfunction after a seven-month latency, allowing the study of cardioprotective treatments in pediatric cancer (osteosarcoma) in immunocompetent rats. In this model, we have identified a set of differentially methylated regions of DNA (DMRs) in hearts of doxorubicin-treated rats, using a Methyl-Seq Target Enrichment System platform. We hypothesize that doxorubicin treatments alter patterns of DNA methylation in cardiomyocytes of susceptible individuals, including animals and human patients. This aberrant DNA methylation could serve as a DNA biomarker for post- doxorubicin damage. We further hypothesize that DNA methylation changes in critical gene-regulatory regions can be minimized by effective cardioprotective agent therapy, diminishing heart function deficits, myocardial degeneration, interstitial fibrosis and oxidative stress DNA damage. In Aim 1, we will determine the temporal features of differentially methylated regions (DMRs) in DNA induced by doxorubicin in heart and how cardioprotective agents modulate the DNA methylation pattern. Using a robust panel of 6 DMRs that we identified in preliminary studies, we will define the temporal development of doxorubicin-induced DNA methylation in male and female rats. We will also test effects of cardioprotective agents on DNA methylation, to verify relationships between DNA methylation and cardiotoxicity, and will determine potential effectiveness of these agents for preventing heart disease, monitored by echocardiography, troponin I measurements, and histology scores. We will also determine whether DNA methylation in shore regions represent early changes that can spread methylation into the CpG islands and affect RNA expression. To provide a bridge to translational human studies, we will examine doxorubicin-induced DNA methylation changes in hearts of immunocompetent rats bearing osteosarcoma. In Aim 2, we will determine whether human heart DNA from doxorubicin treated patients have differentially methylated regions compared to age matched control patients. Banked frozen myocardium from doxorubicin treated patients will be compared to age matched controls using the Illumina Infinium human methylation 450 beadchip array to determine DMR patterns. To determine if these patterns are doxorubicin specific, recently published DNA methylation data from ischemic heart disease patients (publically available) will be compared to DNA methylation from the doxorubicin-exposed patients. Our long-term goal (PA-19-111) is to identify novel heart specific biomarkers to assist oncologists and cardiologists with a method for early diagnosis of heart toxicity so that cardioprotection strategies can be used. This is the first step “proof of concept” study to identify DMRs candidates in heart tissues to prepare for future serum DMRs studies in pediatric or adult cohorts with documented heart doxorubicin toxicity to establish diagnostic sensitivity.