Functional Validation of Doxorubicin-Induced Cardiotoxicity-Related Genes.

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.