Abstract PS13-22: Shedding light on the dark side of chemo: Post-GWAS functional studies of rs28714259 in anthracycline-induced cardiotoxicity

Anthracyclines are commonly used chemotherapies for breast cancer treatment, but can cause dose-related cardiotoxicities and lead to congestive heart failure (CHF) in 2% of patients. Several mechanisms of anthracycline’s cardiotoxic effect have been proposed, but the molecular pathogenesis is not fully understood. In addition, there are no clinically available biomarkers to predict cardiotoxicity. Previously we have identified and validated the association of a single nucleotide polymorphism (SNP), rs28714259 (G/A), with risk of anthracycline-induced CHF through genome-wide association study (GWAS) across three large phase III adjuvant breast cancer trials. rs28714259 (G/A) locates in a putative glucocorticoid receptor (GR) response element and the risk allele (A) is predicted to disrupt GR binding. The activation of GR signaling pathway by dexamethasone is known to protect cardiomyocytes from doxorubicin-induced apoptosis in rats. To investigate the role of rs28714259 in CHF post-GWAS, we set out to determine whether rs28714259 modulates GR signaling pathway through allele-specific GR enhancer activity. We cloned a 1kb DNA sequence on both sides of rs28714259, containing either the wildtype (G) or risk allele (A), into a luciferase reporter plasmid. Luciferase assay in iPSC-derived cardiomyocytes (iPSC-CMs) with GR activation by 100nM dexamethasone showed that cardiomyocytes transfected with wildtype construct had 60% increased activity compared to control vector with no enhancer. iPSC-CMs transfected with risk allele constructs did not show increased luciferase activity, suggesting that the A-allele disrupts GR-mediated transcriptional activation. Using electrophoretic mobility shift assay (EMSA) with nuclear extract from iPSC-CMs treated with dexamethasone, we observed a prominent band shift with either G- or A-allele probes. Furthermore, a supershift band was observed with GR antibody, confirming that GR indeed binds to the rs28714259 region. Notably, the band intensity of risk allele probes decreased by 50% compared to wildtype, suggesting weaker GR binding affinity to risk allele probes, consistent with reduced transcriptional activation. Finally, to identify genes differentially regulated by rs28714259 with anthracycline exposure, we performed RNA-Seq analysis on iPSC-CMs of each genotype. RNA-Seq data revealed that the top differentially regulated network was the death receptor pathway including: FADD, FAS, and Caspase-8; these effectors are known to induce apoptosis in response to doxorubicin. Moreover, 11 genes in the GR signaling pathway were also differentially regulated by rs28714259. Taken together, these findings suggest that the rs28714259 variant may possess allele-specific GR enhancer activity and differentially regulates genes involved in doxorubicin-induced apoptosis. Citation Format: Xi Wu, Gloria Xue, Fei Shen, Guanglong Jiang, Santosh Philips, Geneva Cunningham, Erica Cantor, Bryan P Schneider. Shedding light on the dark side of chemo: Post-GWAS functional studies of rs28714259 in anthracycline-induced cardiotoxicity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-22.