Modeling Susceptibility to Chemotherapy-Induced Cardiotoxicity Using Human iPSCs

项目总结 阿霉素是一种久负盛名的高效化疗药物，常用于治疗多发性癌症 类型，但其使用受到心脏毒性的限制。心脏毒性可从左侧无症状的减少 心室射血分数到高度症状性心力衰竭(III至IV级)。急性阿霉素所致 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 病人的数量。然而，DIC的潜在机制在很大程度上仍然未知，阻碍了DIC的发展 治疗DIC的有效疗法。为此，在这项提案中，我们旨在使用最先进的方法来 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名接受阿霉素治疗的癌症患者的IPSC株，其中50人经历了心脏毒性，50人发生了 不。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。 导致DIC的多态(SNP)，相关SNP将引入或删除非DIC DIC或DIC患者的IPSC系，分别采用CRISPR基因编辑技术。我们将调查 功能和转录变化，并确定已识别的SNP是否导致疾病 表型。在目标2中，我们将在atac-seq、Chip-seq和ip-seq中执行表观遗传学技术的组合。 用质谱法鉴定转录调节因子拓扑异构酶II-β调控基因 已知可被阿霉素抑制。我们假设TOP2B控制着对心肌细胞至关重要的基因 收缩、新陈代谢和动态平衡，其表达在TOP2B被抑制时被破坏 阿霉素。这些实验的结果将揭示TOP2B调控的特定基因，这可能是 可作为DIC的潜在治疗靶点。在目标3中，我们将识别适合作为药物靶点的基因 使用CRISPR基因组筛选方法与DIC相关。这项新技术提供了一种独特的成本- 在IPSC中系统筛选阿霉素治疗失调的药物靶基因的有效机会- 不育系CMS。使用患者特定的IPSC-CMS，拟议的目标将使我们能够首次阐明基因 和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.