Delayed-onset cardiotoxicity of anthracyclines is associated with dysregulation of FOXK1 targets

Background

Anthracyclines remain one of the most used chemotherapeutic agents in the treatment of various cancers. Despite the highly efficient anti-tumor activity, the use is limited by the associated cardiotoxicity. A subset of patients that receive anthracyclines present with dilatative cardiomyopathy several years after their initial exposition. The precise mechanisms underlying this delayed maladaptive response remains poorly understood.

Purpose

The purpose of this study was to gain a better understanding of the delayed maladaptive cardiac response to anthracyclines and thuswe hypothesized that an exposure to anthracyclines increases cardiac susceptible to future adverse stress response.

Methods

To test this, a ‘two-hit’ model using neonatal rat cardiomyocytes was developed. Cells treated with low doses of doxorubicin (Doxo) for 1 h followed by beta-adrenergic stimulation using Isoproterenol (Iso), five days later. A similar 'two-hit' model using a combination of Doxo+Iso was applied to a mouse model where C57B6 mice received doxorubicin and isoproterenol with a gap of two months.

Results

Neonatal rat cardiomyocytes treated with low doses of doxorubicin for 1 h followed by beta-adrenergic stimulation using Isoproterenol (Iso), five days later. Even after a five-day gap between the initial exposure to doxorubicin and isoproterenol, an increased expression of the pathological marker Nppb was observed, suggesting the presence of a memory of anthracycline exposure by the cardiomyocytes (Iso, fc=2.79; Doxo+Iso, fc=5.5 vs. control, p= 0.0241, <0.0001).

The in vivo study revealed that mice receiving a combination of Doxo+Iso with a gap of two months between the two treatments had significantly worse heart function compared to mice receiving either Doxo or Iso alone. RNA-seq led to the identification of FOXK1 as a potential transcriptional factor upstream of a maladaptive gene program regulated by Doxo+Iso compared to Doxo alone.

In addition, ATAC-seq of mitochondria isolated from hearts of mice unraveled a previously unrecognized regulatory region in the MT-RNR2 gene with reduced DNA accessibility upon doxorubicin exposure. An unbiased in vitro proteomic-interaction screen of this region again identified FOXK1 as an interacting protein, suggesting a role in direct regulation of mitochondrial function.

Conclusion

Ongoing studies employing a cardiomyocyte-specific, conditional FOXK1 knockout mouse models and targeted overexpression of FOXK1 in primary cells will aim to further elucidate the mechanistic role of FOXK1 in the long-term anthracycline toxicity.

Collectively, our findings identify FOXK1 as a novel mediator of long-term anthracycline-induced cardiac mitochondrial dysfunction, acting through both nuclear transcriptional control and direct regulation of mitochondrial gene accessibility.