Epigenetic editing of chromatin readers rescues doxorubicin-induced endothelial senescence and vascular dysfunction

Abstract Background Cancer therapies (i.e. chemotherapy, CTX) have improved longevity and have led to a growing population of cancer survivors. Anthracyclines (i.e. Doxorubicin, DOXO) are first-line chemotherapy agents for several cancers which exert particularly toxic effects on the cardiovascular system leading to high cardiovascular morbidity. To date, no effective therapies are available to counteract CTX-induced cardiovascular toxicity. Epigenetic regulation of gene expression is emerging as a pivotal process underpinning cardiovascular disease. BET (bromodomain and extra-terminal) epigenetic reader proteins (namely BRD2 and BRD4) are pivotal fine tuners of gene expression by binding to specific acetylated lysine residues on histone tails. BET protein inhibitors (BETi), namely RVX-208, have shown to reset transcriptional programs thus rescuing disease phenotypes. Purpose To investigate whether BET proteins participate in DOXO-induced endothelial senescence and dysfunction. Methods Human aortic endothelial cells (HAECs) were exposed to different concentrations of DOXO (50nM-500nM) for 48 hours, in the presence of RVX-208 (5–20 µM) or vehicle (DMSO). RNA sequencing (RNAseq), proteomics, ATAC-seq, and ChIP-seq were performed to assess transcriptional and chromatin accessibility changes. To test the in vivo effects of BETi, C57BL/6J mice received a single intraperitoneal injection of DOXO (10 mg/kg) with or without pre-treatment with the selective BETi RVX-208 (150 mg/kg, oral gavage). The effects of RVX-208 on DOXO-related endothelial dysfunction were assessed ex-vivo in mouse aortic rings. Results DOXO-treated HAECs showed a dose-dependent increase in cell cycle inhibitors (p21 and p53) and DNA-damage response (gH2AX). Of interest, treatment with RVX-208 (15mM) prevented the upregulation of cell cycle inhibitors (p21 and p53) while rescuing alterations in cell morphology and reducing the number of SA-b gal positive cells. RNA-seq and proteomic analysis showed a marked deregulation of genes involved in inflammation, oxidative stress and mitochondrial dysfunction in DOXO-treated ECs, whereas RVX-208 prevented these transcriptional alterations. Aortas from DOXO-treated mice displayed an increase in the expression of cell cycle inhibitors which was mitigated by RVX-208. Of note, RVX-208 rescued DOXO-induced impairment of acetylcholine-dependent endothelial relaxation. In ECs, ATAC-seq and ChIP-seq showed that RVX-208 reduces the enrichment of BET proteins on the promoter of cell cycle inhibitors (P53 and P21), leading to their transcriptional repression. RVX-208-induced downregulation of p53 and p21 was associated with an increase in the expression of C-MYC, NANOG, and KLF4, genes involved in ECs rejuvenation. Conclusion Targeting BET proteins may prevent endothelial aging and vascular dysfunction in cancer patients undergoing cardiotoxic therapies.For image description, please refer to the figure legend and surrounding text. For image description, please refer to the figure legend and surrounding text.