DNA topoisomerase inhibition with the HIF inhibitor acriflavine promotes transcription of lncRNAs in endothelial cells

Sandra Seredinski; Frederike Boos; Stefan Günther; James A Oo; Timothy Warwick; Judit Izquierdo Ponce; Felix F Lillich; Ewgenij Proschak; Stefan Knapp; Ralf Gilsbach; Beatrice Pflüger-Müller; Ralf P Brandes; Matthias S Leisegang

doi:10.1016/j.omtn.2022.01.016

DNA topoisomerase inhibition with the HIF inhibitor acriflavine promotes transcription of lncRNAs in endothelial cells

Mol Ther Nucleic Acids. 2022 Jan 25:27:1023-1035. doi: 10.1016/j.omtn.2022.01.016. eCollection 2022 Mar 8.

Authors

Sandra Seredinski^{1

2}, Frederike Boos^{1

2}, Stefan Günther³, James A Oo^{1

2}, Timothy Warwick^{1

2}, Judit Izquierdo Ponce¹, Felix F Lillich⁴, Ewgenij Proschak⁴, Stefan Knapp⁴, Ralf Gilsbach^{1

2}, Beatrice Pflüger-Müller^{1

2}, Ralf P Brandes^{1

2}, Matthias S Leisegang^{1

2}

Affiliations

¹ Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
² German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany.
³ Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany.
⁴ Institute of Pharmaceutical Chemistry, Goethe University, 60438 Frankfurt, Germany.

Abstract

The transcription factor hypoxia-inducible factor 1 (HIF1) is an important driver of cancer and is therefore an attractive drug target. Acriflavine (ACF) has been suggested to inhibit HIF1, but its mechanism of action is unknown. Here we investigated the interaction of ACF with DNA and long non-coding RNAs (lncRNAs) and its function in human endothelial cells. ACF promoted apoptosis and reduced proliferation, network formation, and angiogenic capacity. It also induced changes in gene expression, as determined by RNA sequencing (RNA-seq), which could not be attributed to specific inhibition of HIF1. A similar response was observed in murine lung endothelial cells. Although ACF increased and decreased a similar number of protein-coding genes, lncRNAs were preferentially upregulated under normoxic and hypoxic conditions. An assay for transposase accessibility with subsequent DNA sequencing (ATAC-seq) demonstrated that ACF induced strong changes in chromatin accessibility at lncRNA promoters. Immunofluorescence showed displacement of DNA:RNA hybrids. Such effects might be due to ACF-mediated topoisomerase inhibition, which was indeed the case, as reflected by DNA unwinding assays. Comparison with other acridine derivatives and topoisomerase inhibitors suggested that the specific function of ACF is an effect of acridinium-class compounds. This study demonstrates that ACF inhibits topoisomerases rather than HIF specifically and that it elicits a unique expression response of lncRNAs.

Keywords: acriflavine; angiogenesis; chromatin; endothelial cells; hypoxia; long non-coding RNA; topoisomerase inhibitor.