Identification of Drug Transporter Genomic Variants and Inhibitors That Protect Against Doxorubicin-Induced Cardiotoxicity

Circulation. 2022 Jan 25;145(4):279-294. doi: 10.1161/CIRCULATIONAHA.121.055801. Epub 2021 Dec 7.

Abstract

Background: Multiple pharmacogenomic studies have identified the synonymous genomic variant rs7853758 (G > A, L461L) and the intronic variant rs885004 in SLC28A3 (solute carrier family 28 member 3) as statistically associated with a lower incidence of anthracycline-induced cardiotoxicity. However, the true causal variant(s), the cardioprotective mechanism of this locus, the role of SLC28A3 and other solute carrier (SLC) transporters in anthracycline-induced cardiotoxicity, and the suitability of SLC transporters as targets for cardioprotective drugs has not been investigated.

Methods: Six well-phenotyped, doxorubicin-treated pediatric patients from the original association study cohort were recruited again, and human induced pluripotent stem cell-derived cardiomyocytes were generated. Patient-specific doxorubicin-induced cardiotoxicity (DIC) was then characterized using assays of cell viability, activated caspase 3/7, and doxorubicin uptake. The role of SLC28A3 in DIC was then queried using overexpression and knockout of SLC28A3 in isogenic human-induced pluripotent stem cell-derived cardiomyocytes using a CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9). Fine-mapping of the SLC28A3 locus was then completed after SLC28A3 resequencing and an extended in silico haplotype and functional analysis. Genome editing of the potential causal variant was done using cytosine base editor. SLC28A3-AS1 overexpression was done using a lentiviral plasmid-based transduction and was validated using stranded RNA-sequencing after ribosomal RNA depletion. Drug screening was done using the Prestwick Chemical Library (n = 1200), followed by in vivo validation in mice. The effect of desipramine on doxorubicin cytotoxicity was also investigated in 8 cancer cell lines.

Results: Here, using the most commonly used anthracycline, doxorubicin, we demonstrate that patient-derived cardiomyocytes recapitulate the cardioprotective effect of the SLC28A3 locus and that SLC28A3 expression influences the severity of DIC. Using Nanopore-based fine-mapping and base editing, we identify a novel cardioprotective single nucleotide polymorphism, rs11140490, in the SLC28A3 locus; its effect is exerted via regulation of an antisense long noncoding RNA (SLC28A3-AS1) that overlaps with SLC28A3. Using high-throughput drug screening in patient-derived cardiomyocytes and whole organism validation in mice, we identify the SLC competitive inhibitor desipramine as protective against DIC.

Conclusions: This work demonstrates the power of the human induced pluripotent stem cell model to take a single nucleotide polymorphism from a statistical association through to drug discovery, providing human cell-tested data for clinical trials to attenuate DIC.

Keywords: CRISPR-Cas systems; cardiotoxicity; doxorubicin; human induced pluripotent stem cells; myocytes, cardiac.