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

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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.