Abstract 4086: RNAseq hiPSC-cardiomyocytes reveals altered expression of DNA damage and cell cycle genes in response to doxorubicin

Abstract

Abstract Introduction: Doxorubicin is a highly effective chemotherapeutic agent, but its use is limited due to cumulative and dose-dependent effects that can lead to irreversible heart damage. Despite efforts in understanding the mechanisms, the precise genes and pathways implicated in doxorubicin-induced cardiotoxicity still remain elusive. We hypothesized that by exploring time- and dose-dependent changes in gene expression in human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CM), we can identify genes and signaling pathways related to doxorubicin-induced cardiotoxicity. Methods: hiPSC-CM were treated with 0, 50, 150, or 450 nM of doxorubicin for 2, 7, or 12 days then subjected to RNA sequencing for assessment of gene expression. Sequenced reads were aligned to the human reference genome (GRCh37) with STAR, and a read count matrix was then generated with the R package GenomicAlignments. Global variation and differential gene expression analyses were performed using DESeq2 software (P<0.05). For time course analysis, log2 fold-changes in gene expression for pairwise comparisons between any two time points or doses were calculated using DESeq2 and a Time x Dosage interaction model. For Ingenuity Pathway Analysis of time course genes, input genes were selected based on a cutoff > 2.0 and <-2.0 average log2 fold-change in expression. Results: Hierarchical clustering of expression profiles of 40,861 genes produced by DESeq2 revealed 192 genes downregulated following doxorubicin exposure related to mitotic roles of polo-like kinase, cell cycle control chromosomal replication, and DNA damage checkpoint regulation pathways. This included a set of nine genes with the highest variation in expression, MCM5, PRC1, NUSAP1, CENPF, CCNB1, MELK, AURKB, RACGAP1, and TROAP, with functions in microtubule stabilization and cell cycle regulation. Following a 2-day exposure to doxorubicin, 242 genes, including the top nine genes, were differentially expressed between untreated and treated with enrichment in ribosomal processes, DNA repair and damage, and oxidation-reductase activity. Time course analysis revealed 289 genes between 0 nM and 150 nM on days 2, 7 and 12 with enrichment in fatty acid oxidation, MIF-mediated glucocorticoid regulation, and relaxin signaling pathways. Transcription regulator, SMARCA4, was identified as the only upstream regulator with elevated expression following a later recovery time (day 7 to day 12) compared to shorter or earlier time point comparisons at 150 nM dose. Conclusion: Overlapping genes and pathways identified in these analyses provide additional support for a role of reactive oxygen species and mitochondrial dysfunction linked to DNA damage in doxorubicin-induced cardiotoxicity. This study also warrants further investigation of SMARCA4 since it is a direct regulator of genes with overlapping functions described for the top nine genes. Citation Format: Monica E. Reyes, Jianzhong Ma, Megan L. Grove, Joann L. Ater, Alanna C. Morrison, Michelle A. Hildebrandt. RNAseq hiPSC-cardiomyocytes reveals altered expression of DNA damage and cell cycle genes in response to doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4086. doi:10.1158/1538-7445.AM2017-4086