Modeling Susceptibility to Chemotherapy-Induced Cardiotoxicity Using Human iPSCs

项目摘要 多柔比星是一种公认的高效化疗药物，常用于治疗多种癌症 类型，但其使用受到心脏毒性的限制。肝毒性的范围可以从左心室无症状的减少 心室射血分数至高度症状性心力衰竭（III至IV级）。急性阿霉素诱导 约11%的患者发生心脏毒性（DIC），高达36%的患者观察到长期心脏毒性副作用 病人。然而，DIC的潜在机制在很大程度上仍不清楚，阻碍了DIC的发展。 DIC的有效治疗方法为此，在本提案中，我们的目标是使用最先进的方法， 基因组学和表观遗传学，以确定DIC的遗传和分子机制。在目标1中，我们将生成 来自100名接受多柔比星治疗的癌症患者的iPSC系，其中50名经历了心脏毒性，50名经历了心脏毒性。 没有从这些品系中，我们将进行RNA-seq和eQTL定位，以发现新的单核苷酸 基因多态性（SNP）负责DIC，相关的SNP将被引入或删除从非- DIC或DIC患者iPSC系分别用CRISPR基因编辑技术。我们将调查 功能和转录变化，并确定所鉴定的SNP是否是疾病的原因 表型在目标2中，我们将在ATAC-seq、ChIP-seq和IP-seq中进行表观遗传技术的组合。 质谱鉴定受拓扑异构酶II-β（TOP 2B）调控的基因， 已知被阿霉素抑制。我们假设TOP 2B控制心肌细胞关键基因， 收缩、代谢和稳态，其表达在TOP 2B抑制后被破坏， 阿霉素这些实验的结果将揭示由TOP 2B调控的特定基因，其可以是 作为DIC的潜在治疗靶点。在目标3中，我们将确定适合作为药物靶点的基因， 使用CRISPR基因组筛选方法，这项新技术提供了一个独特的和成本- 在iPSC中系统筛选多柔比星治疗失调的药物靶基因的有效机会- CM使用患者特异性iPSC-CM，所提出的目标将使我们能够首次阐明基因表达， 以及DIC的分子基础

PROJECT SUMMARY Doxorubicin is a well-established and highly effective chemotherapy drug commonly used to treat multiple cancer types, but its use is limited by cardiotoxicity. Cardiotoxicity can range from asymptomatic reduction in left ventricular ejection fraction to highly symptomatic heart failure (Class III to IV). Acute doxorubicin-induced cardiotoxicity (DIC) occurs in ~11% of patients and long-term cardiotoxic side effects are observed in up to 36% of patients. However, the underlying mechanisms of DIC remain largely unknown, hampering the development of effective therapeutics for DIC. To that end, in this proposal we aim to use state-of-the-art approaches in genomics and epigenetics to identify the genetic and molecular mechanisms of DIC. In Aim 1, we will generate iPSC lines from 100 cancer patients treated with doxorubicin, 50 of whom experienced cardiotoxicity and 50 did not. From these lines, we will perform RNA-seq and eQTL mapping to discover novel single nucleotide polymorphisms (SNPs) responsible for DIC, and the relevant SNPs will be introduced to or deleted from non- DIC or DIC patient iPSC lines, respectively, with the CRISPR gene editing technique. We will investigate the functional and transcriptional changes and determine whether the identified SNP is responsible for the disease phenotype. In Aim 2, we will perform a combination of epigenetic techniques in ATAC-seq, ChIP-seq, and IP- mass spectrometry to identify genes regulated by topoisomerase II-beta (TOP2B), a transcriptional regulator known to be inhibited by doxorubicin. We hypothesize that TOP2B controls genes critical for cardiomyocyte contraction, metabolism, and homeostasis, the expression of which is disrupted upon TOP2B inhibition by doxorubicin. Results from these experiments will reveal the specific genes regulated by TOP2B, which can be used as potential therapeutic targets of DIC. In Aim 3, we will identify genes suitable as drug targets that are related to DIC using the CRISPR genome screening approach. This novel technique offers a unique and cost- efficient opportunity to systemically screen for drug target genes dysregulated by doxorubicin treatment in iPSC- CMs. Using patient-specific iPSC-CMs, the proposed aims will allow us to elucidate for the first time the genetic and molecular basis for DIC.