hiPSC-Cardiomyocytes to Screen Variants Predictive of Doxorubicin Cardiotoxicity

hiPSC-心肌细胞筛选预测阿霉素心脏毒性的变异体

• 批准号: 8909180
• 负责人: Daniel Bernstein
• 金额: $ 23.21万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-08 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8909180
• 关键词: Adult; Adverse effects; Affect; Anthracyclines; Antineoplastic Agents; Antineoplastic Protocols; Biological Assay; Biological Models; Calcium; Candidate Disease Gene; Cardiac; Cardiac Myocytes; Cardiotonic Agents; Cardiotoxicity; Cardiovascular Diseases; Cell Line; Cell Survival; Cell physiology; Cells; Chemotherapy-Oncologic Procedure; Child; Childhood; Childhood Cancer Treatment; Clinical; Data; Development; Disease; Dose; Doxorubicin; Drug toxicity; Echocardiography; Exposure to; Gene Mutation; Genes; Genetic; Genetic Polymorphism; Genomic approach; Genomics; Glucuronosyltransferase; Health; Heart; Heart failure; Human; In Vitro; Incidence; Lead; Life; Linkage Disequilibrium; Long-Term Survivors; Malignant Childhood Neoplasm; Malignant Neoplasms; Methods; Mitochondria; Modeling; Molecular; Nuclear Receptors; Nucleosides; Odds Ratio; Oncologist; Patients; Pharmaceutical Preparations; Pharmacogenomics; Phase; Physicians; Population; Predisposition; Probability; Production; Protocols documentation; RARG gene; Report (document); Reporting; Retinoic Acid Receptor; Risk; Survival Rate; Testing; Toxic effect; Transcription Coactivator; Translations; Validation; Variant; abstracting; analog; base; cell injury; childhood cancer survivor; clinical care; clinical practice; cohort; drug efficacy; follow-up; genetic variant; genome wide association study; high risk; improved; induced pluripotent stem cell; loss of function; malignant breast neoplasm; meetings; mortality; new technology; nuclease; oncology; pharmacogenetic testing; predictive modeling; risk variant; screening; tool; transcription factor

DESCRIPTION (provided by applicant): Doxorubicin, although one of the oldest anti-cancer agents, is highly effective in treating a wide range of cancers and is still utilized in 70% of all childhood cancer treatments. However, its utility is limited by its cardiac toxicity, occurring in p to 65% of long-term survivors of childhood cancer. Children are more susceptible to this life-threatening side effect than adults. We have found two genetic variants associated with dramatically altered risk of doxorubicin cardiotoxicity. One protective variant is in the gene SLC28A3, an anti-cancer drug transporter and one risk variant is in RARG, a nuclear receptor and transcription factor that alters expression of other genes. Although these studies represent an advance in using a patient's genetics to guide doxorubicin usage (pharmacogenomics), the true effect of these gene variants is far from proven. Additional criteria that must be met include (1) Confirmation in other patient cohorts; (2) Validation, using a model system, that the gene variant alters cardiotoxicity; (3) Validation of a mechanism for its effects (e.g. does a loss-of-function change in a drug transporter lead to decreased intracellular drug levels and decreased toxicity); and (4) Demonstration that reversion of the variant to the normal (wild-type) gene rescues the altered toxicity effect. Patient-derived hiPSC-CMs (human induced pluripotent stem cell-derived cardiomyocytes) represent a novel technology which has been applied to understanding disease mechanisms and to screening drugs for toxicity. Although hiPSC-CMs do not replicate all aspects of mature cardiomyocytes, we show that hiPSC-CMs from patients who have had doxorubicin cardiotoxicity show increased doxorubicin damage compared to cells from patients without cardiotoxicity. We hypothesize that hiPSC-CMs represent a model platform for studying the validity and mechanisms of gene variants in regulating doxorubicin cardiotoxicity. Aim 1: To develop hiPSC lines with the gene variant in SLC28A3 and examine for decreased susceptibility to doxorubicin cardiotoxicity. Cells will be derived (a) directly from patients with the gene variant; and (b) by genetically inducing the same gene alterations in a control hiPSC line. Doxorubicin toxicity will be quantified by assays of cell function and viabilit. Aim 2: To develop hiPSC lines with the candidate gene variant in RARG and examine for increased susceptibility to in vitro doxorubicin cardiotoxicity. Aim 3: To explore the mechanism(s) by which each variant alters doxorubicin cardiotoxicity. (a) Expression of each candidate gene will be increased or decreased in a control hiPSC-CM line; (b) The variant will be reverted to normal (wild-type) in hiPSC-CMs from patients with each variant (c) We will then explore the specific mechanisms by which each variant affects doxorubicin cardiotoxicity. Aim 4: To utilize our platform to validate additional high risk hits. We will duplicate the above studies or other variants, chosen by meeting a strict definition of high probability and replicability.

描述（由申请人提供）：多柔比星，虽然是最古老的抗癌剂之一，但在治疗各种癌症方面非常有效，并且仍然在70%的癌症中使用。 儿童癌症治疗然而，它的效用受到其心脏毒性的限制，发生在65%的儿童癌症长期幸存者中。儿童比成人更容易受到这种危及生命的副作用的影响。我们已经发现了两种与阿霉素心脏毒性风险显著改变相关的遗传变异。一种保护性变体是在基因SLC 28 A3中，一种抗癌药物转运蛋白，一种风险变体是在RARG中，RARG是一种核受体和转录因子，可以改变其他基因的表达。尽管这些研究代表了利用患者遗传学来指导阿霉素使用（药物基因组学）的进步，但这些基因变异的真正作用远未得到证实。必须满足的其他标准包括：（1）在其他患者队列中得到证实;（2）使用模型系统验证基因变异改变心脏毒性;（3）验证其作用机制（例如，药物转运蛋白的功能丧失变化是否会导致细胞内药物水平降低和毒性降低）;和（4）证明变体回复到正常（野生型）基因挽救了改变的毒性效应。 患者来源的hiPSC-CM（人诱导多能干细胞来源的心肌细胞）代表了一种新技术，其已被应用于理解疾病机制和筛选药物的毒性。虽然hiPSC-CM不能复制成熟心肌细胞的所有方面，但我们发现，与没有心脏毒性的患者的细胞相比，患有阿霉素心脏毒性的患者的hiPSC-CM显示出增加的阿霉素损伤。我们假设hiPSC-CM代表了研究基因变体在调节阿霉素心脏毒性中的有效性和机制的模型平台。目的1：开发具有SLC 28 A3中的基因变体的hiPSC系，并检查对阿霉素心脏毒性的降低的易感性。细胞将（a）直接来源于具有基因变体的患者;和（B）通过在对照hiPSC系中遗传诱导相同的基因改变。将通过细胞功能和活力测定来定量阿霉素毒性。目的2：开发具有RARG中的候选基因变体的hiPSC系，并检查对体外多柔比星心脏毒性的敏感性增加。目的3：探索每种变体改变阿霉素心脏毒性的机制。(a)在对照hiPSC-CM系中，每个候选基因的表达将增加或减少;（B）在来自具有每个变体的患者的hiPSC-CM中，变体将恢复正常（野生型）。（c）然后，我们将探索每个变体影响多柔比星心脏毒性的具体机制。目标4：利用我们的平台验证额外的高风险命中。我们将重复上述研究或其他变体，通过满足高概率和可重复性的严格定义来选择。

项目成果

Mitochondrial remodeling: Rearranging, recycling, and reprogramming.

• DOI: 10.1016/j.ceca.2016.04.006
• 发表时间: 2016-08
• 期刊: Cell calcium
• 影响因子: 4
• 作者: Gottlieb RA;Bernstein D
• 通讯作者: Bernstein D

Thalidomide treatment prevents chronic graft rejection after aortic transplantation in rats - an experimental study.

• DOI: 10.1111/tri.13004
• 发表时间: 2017-11
• 期刊: Transplant international : official journal of the European Society for Organ Transplantation
• 作者: Miller KK;Wang D;Hu X;Hua X;Deuse T;Neofytou E;Renne T;Velden J;Reichenspurner H;Schrepfer S;Bernstein D
• 通讯作者: Bernstein D

A coding variant in RARG confers susceptibility to anthracycline-induced cardiotoxicity in childhood cancer.

• DOI: 10.1038/ng.3374
• 发表时间: 2015-09
• 期刊: Nature genetics
• 影响因子: 30.8
• 作者: Aminkeng F;Bhavsar AP;Visscher H;Rassekh SR;Li Y;Lee JW;Brunham LR;Caron HN;van Dalen EC;Kremer LC;van der Pal HJ;Amstutz U;Rieder MJ;Bernstein D;Carleton BC;Hayden MR;Ross CJ;Canadian Pharmacogenomics Network for Drug Safety Consortium
• 通讯作者: Canadian Pharmacogenomics Network for Drug Safety Consortium