Abstract
Doxorubicin is a potent anticancer drug used to treat a variety of cancer types. However, its use is limited by doxorubicin-induced cardiotoxicity (DIC). A missense variant in the RARG gene (S427L; rs2229774) has been implicated in susceptibility to DIC in a genome wide association study. The goal of this study was to investigate the functional role of this RARG variant in DIC. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from patients treated with doxorubicin. iPSC-CMs from individuals who experienced DIC (cases) showed significantly greater sensitivity to doxorubicin compared to iPSC-CMs from doxorubicin-treated individuals who did not develop DIC (controls) in cell viability and optical mapping experiments. Using CRISPR/Cas9, we generated isogenic cell lines that differed only at the RARG locus. Genetic correction of RARG-S427L to wild type resulted in reduced doxorubicin-induced double stranded DNA breaks, reactive oxygen species production, and cell death. Conversely, introduction of RARG-S427L increased susceptibility to doxorubicin. Finally, genetic disruption of the RARG gene resulted in protection from cell death due to doxorubicin treatment. Our findings suggest that the presence of RARG-S427L increases sensitivity to DIC, establishing a direct, causal role for this variant in DIC.
Highlights
Doxorubicin is a potent anticancer drug used to treat a variety of cancer types
Karyotype analysis showed normal karyotypes in all cell lines with the exception of two passages of one cell line (DIC-023, Supplementary Fig. S2a,b),which showed a possible amplification of the minimal critical chromosomal region 12p, an abnormality often seen in iPSCs11,12
We further investigated the impact of RARG-S427L on doxorubicin-induced DNA double strand breaks (DSBs) and reactive oxygen species (ROS) production, both validated assays of doxorubicin-induced cardiotoxicity (DIC) in induced pluripotent stem cell (iPSC)-CMs7,8
Summary
Doxorubicin is a potent anticancer drug used to treat a variety of cancer types. its use is limited by doxorubicin-induced cardiotoxicity (DIC). A missense variant in the RARG gene (S427L; rs2229774) has been implicated in susceptibility to DIC in a genome wide association study. The goal of this study was to investigate the functional role of this RARG variant in DIC. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from patients treated with doxorubicin. Among the variants with the strongest genetic evidence is a non-synonymous coding variant (rs2229774, S427L) in the retinoic acid receptor gamma gene, RARG, which was identified in a genome-wide association study (GWAS) of DIC (odds ratio = 4.7, p = 5.9 10−8)[5]. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have emerged as a physiologically-relevant model system for studying DIC7,8, but to date the impact of specific genetic variants on DIC in this model has not been assessed. The goal of this study was to investigate the functional impact of RARG-S427L on DIC in isogenic, patient-derived iPSC-CMs
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