The BET protein inhibitor apabetalone (RVX-208) prevents doxorubicin-induced endothelial senescence

Abstract Background Accumulation of senescent endothelial cells (ECs) plays a pivotal role in cardiometabolic disorders and lifespan reduction(1). Yet, only a few senolytics are known to date, partly due to the poor grasp of the molecular mechanisms that control the senescence survival programme. Bromo-and extraterminal domain (BET) proteins, known epigenetic reader proteins, recognize acetylated histone tails and regulate gene transcription. RVX-208, an FDA-approved BET inhibitor, has shown to modulate transcriptional programs involved in inflammation, cancer, and renal disease(2). Yet, its potential role in EC senescence and cardio-oncology remains elusive. Purpose Our study aims to investigate whether pharmacological modulation of BET proteins by RVX-208 can mitigate doxorubicin-induced endothelial senescence. Methods Human aortic endothelial cells (HAECs) were exposed to different concentrations of Doxo (50nM-500nM) for 48 hours. Cell morphology changes, cell cycle arrest, senescence associated secreted phenotype (SASP) release, upregulation of SA-b gal activity, DNA damage response (gH2AX) and expression of p21, p16 and p53 were used as molecular markers of cellular senescence. Conditioned media was collected from Doxo-treated ECs and processed for molecular analyses or used for treatment of healthy, non-senescent ECs. To test whether RVX-208 exerts any senolytic or senomorphic effect, HAECs were treated with Doxo (100 nM) in presence or absence of RVX-208 (5mM-20mM) for 48 hrs. RNA-seq and proteomic profiling were performed in vehicle and Doxo-treated ECs. Results Doxo-treated ECs showed a dose-dependent increase in senescence markers (p16, p21 and p53) and DNA-damage response (gH2AX). FACS analysis revealed an accumulation of cells in G2/M phase in Doxo-treated ECs (100 nM). Of interest, treatment with RVX-208 (15mM) prevented the upregulation of senescent markers (p16, p21 and p53) while rescuing alterations in cell morphology and reducing the number of SA-b gal positive cells. RVX-208 prevented Doxo-induced upregulation of genes involved in inflammation, oxidative stress and mitochondrial dysfunction as shown by RNA-seq and proteomic analysis. Conditioned media from Doxo-treated ECs showed an enrichment of SASP-associated markers (IL6, CCL2, IL8, TIMP2 and PDGF-b) as shown by dot-blot arrays. Exposure of healthy ECs to conditioned media from senescent ECs recapitulated aging features. Notably, RVX-208 blunted the release of SASP markers (IL6, CCL2 and IL8) from Doxo-treated ECs. Mechanistically, we found enhanced BET protein occupancy (BRD4) with concomitant enrichment of activating chromatin marks (H3K27Ac and H3K4me) on the promoter of senescence genes COL1A2, CDKN1C and SESN3. Conclusion Targeting BET proteins with RVX-208 may offer a promising therapeutic strategy to prevent endothelial aging in cancer patients undergoing cardiotoxic therapies.