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.

Public Health Relevance

Doxorubicin is a highly effective chemotherapy drug. However, the use of doxorubicin is complicated by its well- established cardiotoxic side effects. Previously, we have shown that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) serve as an effective platform in modeling doxorubicin-induced cardiotoxicity (DIC) by recapitulating the clinical phenotype of DIC on a dish. In this proposal, we will identify the underlying mechanisms of DIC by employing a number of state-of-the-art approaches in genomics and epigenetics. The knowledge we collect here should be instrumental in developing novel patient-specific therapeutics to treat DIC.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL123968-06
Application #
9670811
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Luo, James
Project Start
2014-08-05
Project End
2022-03-31
Budget Start
2019-04-01
Budget End
2020-03-31
Support Year
6
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Stanford University
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Paik, David T; Tian, Lei; Lee, Jaecheol et al. (2018) Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells. Circ Res 123:443-450
Liu, Chun; Oikonomopoulos, Angelos; Sayed, Nazish et al. (2018) Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond. Development 145:
Churko, Jared M; Garg, Priyanka; Treutlein, Barbara et al. (2018) Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis. Nat Commun 9:4906
Chang, Alex C Y; Chang, Andrew C H; Kirillova, Anna et al. (2018) Telomere shortening is a hallmark of genetic cardiomyopathies. Proc Natl Acad Sci U S A 115:9276-9281
Zhang, Joe Z; Guo, Hongchao; Wu, Joseph C (2018) Applications of genetically engineered human pluripotent stem cell reporters in cardiac stem cell biology. Curr Opin Biotechnol 52:66-73
Ong, Sang-Ging; Lee, Won Hee; Zhou, Yang et al. (2018) Mining Exosomal MicroRNAs from Human-Induced Pluripotent Stem Cells-Derived Cardiomyocytes for Cardiac Regeneration. Methods Mol Biol 1733:127-136
Zhao, Ming-Tao; Shao, Ning-Yi; Hu, Shijun et al. (2017) Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells. Circ Res 121:1237-1250
Zhao, Ming-Tao; Chen, Haodong; Liu, Qing et al. (2017) Molecular and functional resemblance of differentiated cells derived from isogenic human iPSCs and SCNT-derived ESCs. Proc Natl Acad Sci U S A 114:E11111-E11120
Lee, Won Hee; Chen, Wen-Yi; Shao, Ning-Yi et al. (2017) Comparison of Non-Coding RNAs in Exosomes and Functional Efficacy of Human Embryonic Stem Cell- versus Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Stem Cells 35:2138-2149
Karakikes, Ioannis; Termglinchan, Vittavat; Cepeda, Diana A et al. (2017) A Comprehensive TALEN-Based Knockout Library for Generating Human-Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases. Circ Res 120:1561-1571

Showing the most recent 10 out of 36 publications