Modeling Susceptibility to Chemotherapy-Induced Cardiotoxicity Using Human iPSCs

说明(申请人提供)：阿霉素是一种成熟和高效的化疗药物，通常用于治疗多种癌症，如淋巴瘤、白血病、卵巢癌、肺癌和乳腺癌，但其使用受到心脏毒性的限制。心脏毒性可从无症状的左心室射血分数降低到高度症状性心力衰竭(III至IV级)。约11%的患者发生阿霉素引起的急性心脏毒性(DIC)，高达36%的患者观察到可在治疗后长达10年的长期心脏毒性副作用。目前，我们无法预测哪些患者会出现心脏毒性。填补我们对DIC机制以及如何最好地预防DIC的知识空白的一个主要障碍是缺乏良好的人体模型，这是由于无法获得针对患者的特定人类心肌细胞样本，以及在体外分离和维护人类心肌细胞的困难。动物模型受到动物和人类心肌细胞之间显著功能差异的限制。这一障碍现在已经被人类诱导多能干细胞(IPSCs)的产生的最新进展所克服，在IPSCs中，患者的体细胞可以被重新编程为多能性并在体外无限期地保持。这些IPSC可以有效地分化为IPSC来源的心肌细胞(IPSC-CMS)并进行进一步的详细研究。在我们的初步研究中，我们开发并验证了一套用于评估人类IPSC-CMS中DIC的工具。我们已经证实，来源于DIC患者的IPSC-CMS在体外可以准确地概括易感表型。单核苷酸多态(SNP)研究发现了几个与DIC高度相关的SNP(P=10-9~10-5)。因此，在目标1中，我们将从100名接受阿霉素治疗的癌症患者中产生IPSC系，其中50人有心脏毒性，50人没有。在目标2中，我们将使用我们初步研究中建立的分析方法来评估DIC的易感性，并进行RNA-seq和eQTL定位以发现新的SNPs。在目标3中，我们将利用转录激活物样效应核酸酶(TALENS)将一个与DIC相关的SNP引入5个对照IPSC系中，并评估其对DIC易感性的影响。因此，R01提案的总体目标是使用患者特定的IPSC-CMS来帮助阐明DIC的分子机制。

DESCRIPTION (provided by applicant): Doxorubicin is a well-established and highly effective chemotherapy drug commonly used to treat multiple cancers such as lymphoma, leukemia, ovary, lung, and breast cancer, 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, which can manifest up to 10 years after treatment, are observed in up to 36% of patients. Currently, we cannot predict which patients will develop cardiotoxicity. A major hurdle in filling the significant gaps in our knowledge about the mechanisms of DIC and how best to prevent it is the lack of good human models, due to the inaccessibility of patient-specific human cardiomyocyte samples, and the difficulty in isolating and maintaining human cardiomyocytes in vitro. Animal models are limited by significant functional disparities between animal and human cardiomyocytes. This hurdle has now been overcome by the recent advances in the generation of human induced pluripotent stem cells (iPSCs), in which a patient's somatic cells can be reprogrammed to pluripotency and maintained indefinitely in vitro. These iPSCs can then be efficiently differentiated into iPSC-derived cardiomyocytes (iPSC-CMs) and further studied in detail. In our preliminary studies, we have developed and validated a set of tools for assessing DIC in human iPSC-CMs. We have established that iPSC-CMs, derived from patients who have developed DIC, accurately recapitulate the susceptibility phenotype in vitro. Single nucleotide polymorphism (SNP) studies have identified several SNPs that are predicted to be highly associated with DIC (P=10-9 to 10-5). Hence in Aim 1, we will generate iPSC lines from 100 cancer patients treated with doxorubicin, 50 of whom experienced cardiotoxicity and 50 did not. In Aim 2, we will use the assays established in our pilot studies to assess the susceptibility to DIC and perform RNA-seq and eQTL mapping to discover novel SNPs. In Aim 3, we will introduce a very well established DIC-related SNP into five control iPSC lines using transcription activator-like effector nucleases (TALENs) and assess the effect on DIC susceptibility. Hence the overall aim of this R01 proposal is to use patient-specific iPSC-CMs to help elucidate the molecular mechanisms of DIC.