Assessing the ability of hiCMs to recapitulate patient-specific doxorubicin-induced cardiotoxicity

项目总结 阿霉素是一种高效的化疗药物，通常用于约60%的儿童患者 有转移性实体瘤(肉瘤)、白血病和淋巴瘤。使用阿霉素的治疗方法有 并发其公认的心脏毒性副作用，影响大约16%的儿科 这可能会导致需要心脏移植的心力衰竭，并限制阿霉素的临床应用。尽管 在这一领域进行了50多年的研究，目前预测或预测的可能性仍然很小 预防心脏毒性。显然需要新的和创新的方法来克服这一点 障碍。候选基因关联研究和全基因组关联研究已经确定 多个单核苷酸多态(SNPs)与阿霉素诱导的统计相关性 心脏毒性(DIC)，然而这些SNPs的实验验证还不可行，因为 体外分离培养人心肌细胞。在我们最近的工作中，我们展示了特定于患者的 人诱导多能干细胞来源的心肌细胞(hiPSC-CM)是预测患者 DIC发生的可能性，首次证实DIC存在基因组基础。尽管GWAS 已经被证明是告知这些基因组碱基的一种强大的方法，它检测相关性而不是 因果关系和已识别的SNPs通常无法在后续研究中重复。在这里，我们假设 HiPSC-CMS可以在三种不同的模式下用于研究与DIC相关的遗传变异： 首先，发现新的预测性SNPs；其次，验证SNPs；以及第三，检测调制的SNPs 并确定心脏保护方法。在目标1中，我们将招募100名儿童癌症患者 接触阿霉素的人(50人有心脏毒性，50人没有)，评估 患者来源的HiPSC-CM对阿霉素的反应，以验证我们之前在大规模队列和 确认这是该领域的强大工具。在目标2中，我们将使用这100个特定于患者的线路来识别药物 反应差异表达数量性状基因座(DeQTL)，用基因组编辑验证这些变异， 并机械地检查导致DIC易感性的途径，集中在已知的 在心肌病、心肌保护和阿霉素代谢中的作用。在《目标3》中，我们将审问 40项现有DIC SNP研究的可重复性，使用CRISPR/Cas9敲除 对照等基因hPSC细胞系，评价hPSC-CM对阿霉素的反应。我们还将表演 基于慢病毒的组合排列全基因组CRISPR基因敲除筛选 它们调节了DIC的风险和对DIC的保护。最后，我们将利用上面的发现 发现/重新利用基因组信息的心脏保护药物来预防DIC。总而言之，这项工作将提供 美国为什么患者会经历DIC的遗传原理并提供了1，完全人类验证的SNP数据 临床应用；2、抗DIC的新型心脏保护药物。

PROJECT SUMMARY Doxorubicin is a highly effective chemotherapy drug commonly used in approximately 60% of pediatric patients with metastatic solid tumors (sarcomas), 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 cardioprotective methodologies. In Aim 1, we will recruit 100 pediatric cancer patients who were exposed to doxorubicin (50 who experienced cardiotoxicity and 50 who did not), assess the response of patient-derived hiPSC-CM to doxorubicin to validate our previous findings in a large cohort and confirm this as powerful tool in the field. In Aim 2, we will use these 100 patient-specific lines to identify drug response differential expression quantitative trait loci (deQTL), 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 knockout the gene of interest in a control isogenic hiPSC line then assess the response of hiPSC-CM to doxorubicin. We will also perform lentiviral-based pooled and arrayed whole genome CRISPR-mediated knockout screening to discover genes which modulate the risk of and protection from DIC. Finally, we will use the discoveries above to discover/repurpose genome-informed cardioprotective drugs to prevent DIC. 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.