Introduction

TOP2 inhibitors (TOP2i) used in the treatment of breast cancers, including the anthracyclines (AC) Doxorubicin (DOX), Daunorubicin (DNR), Epirubicin (EPI), and the anthraquinone Mitoxantrone (MTX), can lead to cardiotoxicity in some women. It is unclear whether women would experience the same adverse effects from all drugs in this class. 

Methods

To gain insight into whether specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile, we studied the effects of treatment of DOX, DNR, EPI, MTX, and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from the same six females across a range of concentrations. We then selected a sub-lethal dose to elucidate primary transcriptional and cellular responses associated with treatment over 24 hours.

Results

While TRZ does not affect cell viability, all TOP2i induce cell death after 48 hours of treatment at concentrations observed in cancer patient serum. At 24 hours, all TOP2i affect calcium handling, a function critical for cardiomyocyte contraction. Global gene expression data show that drug treatment and exposure time account for most variation between samples, followed by the individual from which samples were derived. Thousands of genes respond after treatment with each of the TOP2i. We did not observe a gene expression response to TRZ. Response genes at 24 hours are associated with the p53 signaling pathway, DNA replication, and the cell cycle. Bayesian analysis across all drugs reveals three gene expression response signatures, which we denote as TOP2i early acute, early sustained, and late response genes. TOP2i early response genes are enriched in chromatin regulators, which have been implicated in mediating AC sensitivity across breast cancer patients. In line with variable responses to AC treatments in breast cancer patients, we observe an increase in transcriptional variability between individuals following 24 hours of AC treatment but not non-AC treatments.

To investigate potential genetic effects on drug response variability, we first identified a reported set of expression quantitative trait loci (eQTLs) that are uncovered specifically following DOX treatment in iPSC-CMs. DOX response eQTLs are enriched in genes that respond to all TOP2i compared to baseline eQTLs. Of the 142 DOX response QTLs that respond to DOX in our cells, 96% respond to at least one other TOP2i. Second, we identified eight genes in loci associated with AC toxicity by GWAS or TWAS. All of these genes, including RARG and SLC28A3, respond to at least two ACs. Consistent with the potential for these genes to be involved in cardiotoxicity, we find their expression to be correlated with the release of cardiotoxicity markers TNNI and LDH across individuals in response to AC treatment.

Conclusion

Our data thus demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes, including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity.