Cardioprotective SNPs in SLC28A3 and lncRNA SLC28A3-AS1 result in transcriptional changes and alternative splicing to reduce doxorubicin cytotoxicity

Cardiotoxicity is a therapeutic challenge for anthracycline-based treatments for solid tumors and leukemia. Genome-wide association studies have revealed that single nucleotide polymorphisms in the SLC28A3 locus (encoding Concentrative Nucleoside Transporter 3, CNT3) are significantly associated with reduced doxorubicin-induced cardiotoxicity. However, the mechanistic understanding of the functional effects of these genomic variants is lacking. We designed studies focused on clinically associated SNPs within SLC28A3 using minigenes, site-directed mutagenesis, splicing assays, modulation of SLC28A3 and its antisense long noncoding RNA (lncRNA, AS1), and doxorubicin transport and cytotoxicity measurements to gain more insight. We demonstrated that the cardioprotective synonymous SNP rs7853758 in the Ex14 coding region of SLC28A3 and the variant rs11140490 in Ex1 of its antisense lncRNA (SLC28A3-AS1) have functional consequences in regulating CNT3 transcript and protein expression using alterations in RNA levels and alternative splicing. Additionally, the deep intronic region of Int13, which harbors the SNP rs7030019, is critical for the splicing of CNT3 precursor mRNA at Ex13–14. Furthermore, we identified alternatively spliced variants of the AS1 lncRNA that differentially regulate CNT3 gene expression, doxorubicin transport, and cytotoxicity. Together, these findings suggest that antisense and splicing mechanisms may be exploited to modulate CNT3 function to reduce doxorubicin cytotoxicity, enabling the development of predictive biomarkers and chemotherapeutic management of anthracycline toxicities.