Functional Validation of Doxorubicin-Induced Cardiotoxicity-Related Genes

Hananeh Fonoudi; Mariam Jouni; Romina B Cejas; Tarek Magdy; Malorie Blancard; Ning Ge; Disheet A Shah; Davi M Lyra-Leite; Achal Neupane; Mennat Gharib; Zhengxin Jiang; Yadav Sapkota; Paul W Burridge

doi:10.1016/j.jaccao.2023.11.008

Functional Validation of Doxorubicin-Induced Cardiotoxicity-Related Genes

JACC CardioOncol. 2024 Jan 23;6(1):38-50. doi: 10.1016/j.jaccao.2023.11.008. eCollection 2024 Feb.

Authors

Hananeh Fonoudi^{1

2}, Mariam Jouni^{1

2}, Romina B Cejas^{1

2}, Tarek Magdy^{1

2}, Malorie Blancard^{1

2}, Ning Ge^{1

2}, Disheet A Shah^{1

2}, Davi M Lyra-Leite^{1

2}, Achal Neupane³, Mennat Gharib^{1

2}, Zhengxin Jiang^{1

2}, Yadav Sapkota³, Paul W Burridge^{1

2}

Affiliations

¹ Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
² Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
³ Department of Epidemiology and Cancer Control, St. Jude Children's Hospital, Memphis, Tennessee, USA.

Abstract

Background: Genome-wide association studies and candidate gene association studies have identified more than 180 genetic variants statistically associated with anthracycline-induced cardiotoxicity (AIC). However, the lack of functional validation has hindered the clinical translation of these findings.

Objectives: The aim of this study was to functionally validate all genes associated with AIC using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Methods: Through a systemic literature search, 80 genes containing variants significantly associated with AIC were identified. Additionally, 3 more genes with potential roles in AIC (GSTM1, CBR1, and ERBB2) were included. Of these, 38 genes exhibited expression in human fetal heart, adult heart, and hiPSC-CMs. Using clustered regularly interspaced short palindromic repeats/Cas9-based genome editing, each of these 38 genes was systematically knocked out in control hiPSC-CMs, and the resulting doxorubicin-induced cardiotoxicity (DIC) phenotype was assessed using hiPSC-CMs. Subsequently, functional assays were conducted for each gene knockout on the basis of hypothesized mechanistic implications in DIC.

Results: Knockout of 26 genes increased the susceptibility of hiPSC-CMs to DIC. Notable genes included efflux transporters (ABCC10, ABCC2, ABCB4, ABCC5, and ABCC9), well-established DIC-associated genes (CBR1, CBR3, and RAC2), and genome-wide association study-discovered genes (RARG and CELF4). Conversely, knockout of ATP2B1, HNMT, POR, CYBA, WDR4, and COL1A2 had no significant effect on the in vitro DIC phenotype of hiPSC-CMs. Furthermore, knockout of the uptake transporters (SLC28A3, SLC22A17, and SLC28A1) demonstrated a protective effect against DIC.

Conclusions: The present findings establish a comprehensive platform for the functional validation of DIC-associated genes, providing insights for future studies in DIC variant associations and potential mechanistic targets for the development of cardioprotective drugs.

Keywords: GWAS; cardiomyocytes; doxorubicin; genomics; human induced pluripotent stem cells.

Abstract

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