Genomic Prediction of Doxorubicin-Induced Cardiotoxicity

项目摘要 蒽环类多柔比星用于约60%的转移性实体瘤儿童癌症患者 肿瘤（肉瘤）、母细胞瘤、白血病和淋巴瘤。使用阿霉素的治疗是复杂的， 公认的心脏毒性副作用，影响约16%的儿科患者，可导致心脏 需要心脏移植的衰竭，并限制了阿霉素的临床应用。尽管50多年来 尽管在这一领域的研究中，目前预测或预防心脏毒性的潜力仍然很小。那里 显然需要新颖和创新的方法来克服这一障碍。候选基因关联 研究和全基因组关联研究（GWAS）已经鉴定出许多单核苷酸多态性 （SNPs）与多柔比星诱导的心脏毒性（DIC）统计学相关，但实验验证 由于体外分离和培养人心肌细胞的困难，这些SNP的合成还不可行。 在我们最近的工作中，我们发现患者特异性的人诱导多能干细胞衍生的 心肌细胞（hiPSC-CM）是患者发生DIC可能性的有效预测因子，证实了 这是第一次发现弥散性血管内凝血的基因基础虽然GWAS已被证明是一种强大的方法， 告知这样的基因组碱基，它检测相关性而不是因果关系，并且识别的SNP通常不能 在后续研究中重复。在这里，我们假设hiPSC-CM可以用于三种不同的治疗。 研究DIC相关遗传变异的方法：首先，发现新的预测性SNP;其次， 验证SNP;第三，检查调节的途径并确定基因型特异性 心脏保护方法。在目标1中，我们将招募100名儿童癌症患者， 多柔比星，并评估体外患者来源的hiPSC-CM对多柔比星的反应，以验证我们先前的研究。 在一个具有不同生物学协变量的大型儿科队列中的发现，以验证该工具的功效。在目标2中， 将使用这100个患者特异性品系来鉴定药物反应差异表达数量性状基因座 （deQTL），评估生物学协变量，如剂量、年龄、性别、SF和癌症诊断， 加起来然后，我们将通过基因组编辑验证这些变体，并机械地检查途径 DIC易感性的原因集中在已知在心肌病，心脏保护， 和阿霉素代谢。在目标3中，我们将询问>40个现有DIC SNP的严谨性和再现性。 研究，使用CRISPR/Cas9编辑对照等基因hiPSC系中的感兴趣基因，然后评估响应 hiPSC-CM与阿霉素的比率。然后，我们将使用上述发现来发现/重新利用基因组信息 心脏保护药物以基因型特异性方式预防DIC。总之，这项工作将为我们提供 患者发生DIC的遗传学依据，并提供1个完全人类验证的SNP数据用于临床 应用，和2，新的心脏保护药物，以减轻DIC。

Project Summary The anthracycline doxorubicin used in approximately 60% of pediatric cancer patients with metastatic solid tumors (sarcomas), blastomas, leukemia, and lymphoma. Treatments using doxorubicin are complicated by its well-established cardiotoxic side effect, which affects approximately 16% of pediatric patients, can lead to heart failure requiring heart transplant, and limits doxorubicin’s clinical utilization. Despite more than 50 years of research in this field, there is still, at present, little potential for either predicting or preventing cardiotoxicity. There is an obvious need for novel and innovative approaches to overcome this hurdle. Candidate gene association studies and genome–wide association studies (GWAS) have identified many single nucleotide polymorphisms (SNPs) that are statistically correlated with doxorubicin–induced cardiotoxicity (DIC), yet experimental validation of these SNPs has not been feasible due to the difficulty in isolating and culturing human cardiomyocytes in vitro. In our recent work, we showed that patient–specific human induced pluripotent stem cell–derived cardiomyocytes (hiPSC–CM) are efficient predictors of a patient’s likelihood of developing DIC, confirming for the first time that there is a genomic basis to DIC. Although GWAS has proven to be a powerful methodology for informing such genomic bases, it detects correlation rather than causation, and identified SNPs commonly fail to be replicated in subsequent studies. Here, we hypothesize that hiPSC-CMs can be utilized in three different modalities to study genetic variants associated with DIC: firstly, to discover novel predictive SNPs; secondly, to validate SNPs; and thirdly, to examine the modulated pathways and determine genotype-specific cardioprotective methodologies. In Aim 1, we will recruit 100 pediatric cancer patients who were exposed to doxorubicin and assess the response of patient-derived hiPSC-CM to doxorubicin in vitro to validate our previous findings in a large pediatric cohort with diverse biological covariates to verify the power of this tool. In Aim 2, we will use these 100 patient-specific lines to identify drug response differential expression quantitative trait loci (deQTL), assessing biological covariates such as dose, age, sex, SF, and cancer diagnosis both individually and combined. We will then validate these variants with genome editing, and mechanistically examine pathways causative to DIC susceptibility concentrating on genes with known roles in cardiomyopathy, cardioprotection, and doxorubicin metabolism. In Aim 3, we will interrogate the rigor and reproducibility of >40 existing DIC SNP studies, using CRISPR/Cas9 to edit the gene of interest in control isogenic hiPSC lines then assess the response of hiPSC-CM to doxorubicin. We will then use the discoveries above to discover/repurpose genome-informed cardioprotective drugs to prevent DIC in a genotype-specific manner. In summary, this work will deliver us the genetic rationale for why patients experience DIC and provide 1, fully human validated SNP data for clinical application, and 2, novel cardioprotective drugs to attenuate DIC.