Chromatin readers orchestrate doxorubicin-induced endothelial senescence and paracrine cardiotoxicity: towards epi-drugs in cardio-oncology
S A Mohammed, P Enz, N Atzemian, M V Mieghem, M Winkelbauer, Y Yundung, M Telesca, E Gorica, P Chansoria, A Mengozzi, O Dzemali, H Rodriguez, S Costantino, F Ruschitzka, F PaneniAbstract
Background
Anthracyclines such as doxorubicin (DOXO) are highly effective anticancer agents but induce cardiovascular toxicity. While DOXO-induced cardiomyocyte injury has been extensively studied, the contribution of non-cardiomyocytes populations, particularly endothelial cells (ECs), remains elusive1. Bromodomain and extra-terminal (BET) proteins, especially BRD4, act as chromatin readers translating histone acetylation into transcriptional activation, and are emerging a pivotal pleayers in cardiovascular disease.
Purpose
To investigatethe role BET epigenetic remodeling in DOXO-induced endothelial dysfunction.
Methods
Human aortic endothelial cells (ECs) were exposed to DOXO (50–500 nM) in the presence or in the absence of the selective BET inhibitor Apabetalone (RVX-208). Senescence markers and chromatin remodeling were assessed using RNA-seq, proteomics, ATAC-seq as well as BRD4 ChIP-seq[FP1] . Vascular function was evaluated ex vivo in mouse aortic rings and human internal mammary arteries. Paracrine effects of ECs were examined using endothelial-conditioned media applied to naïve ECs, cardiomyocytes, and iPSC-derived 3D cardiac spheroids.
Results
DOXO induced dose-dependent ECs senescence, shown by increased p53, p21, γH2AX and SA-β-gal positivity. RNA-seq revealed profound transcriptional reprogramming involving cell adhesion pathways, epigenetic regulators, and senescence associated secreted phenotype (SASP) factors. BET inhibition with RVX-208 suppressed p53/p21 induction, normalized SASP, restored endothelial morphology, and reduced SA-β-gal positivity (Fig. 1). Among BET proteins, BRD4 (but not BRD2/3) was the main driver of pro-senescent programs. ATAC- and ChIP-seq in DOXO-treated ECs demonstrated enhanced BRD4 occupancy at gene loci controlling senescence (Fig. 2), whereas RVX-208restored rejuvenation-associated genes (c-MYC, NANOG, KLF4). Conditioned media from DOXO-treated ECs induceda senescent phenotype in naive ECs and impaired migration and tube formation. Secretome proteomics revealed enrichment of SASP-associated mediators, indicating establishment of a pro-inflammatory paracrine milieu. This endothelial stress signal propagated beyond the vasculature, triggering stress responses in cardiomyocytes and destabilizing multicellular 3D cardiac spheroids. BET inhibition normalized maladaptive secretome changes and prevented ECs-driven cardiomyocyte dysfunction. Of note, ex vivo treatment with RVX-208 restored DOXO-impaired endothelial relaxation and sprouting in mouse arteries. Of clinical relevance, human internal mammary arteries exposed to DOXO exhibited BET upregulation and endothelial dysfunction, which was rescued by RVX-208.
Conclusions
BET-driven transcriptional programs are heavily implicated in DOXO-induced ECs damage. BET inhibitors may represent promising drugs to mitigate cardiovascular dysfunction in cardio-oncology patients.