Partial resistance to HDAC inhibitors in FAPs of dystrophic muscles at late stages of disease is associated to epigenetic and transcriptional features of cellular senescence [ChIP-seq]

Pharmacological treatment of Duchenne Muscular Dystrophy (DMD) with histone deacetylase inhibitors (HDACi) is currently being tested in clinical trials. Pre-clinical studies performed in mdx mice - the mouse model of DMD - have shown that HDACi promote compensatory muscle regeneration, while inhibiting fibro-adipogenic degeneration, by targeting fibro-adipogenic progenitors (FAPs); however, these beneficial effects are restricted to early stages of disease progression. We show here that FAPs from late stage mdx mice exhibit epigenetic and transcriptional features of senescence that could not be fully reversed by HDACi. In particular, genome-wide increase in H3K9/14 acetylation at gene promoters of Senescence Associated Secretory Phenotype (SASP) genes was associated with their upregulation in late stage mdx FAPs. Treatment with the HDACi Trichostatin A (TSA) could inhibit SASP gene activation in FAPs, by decreasing H3K9/14 acetylation. Conversely, combinatorial decrease of H3K27 and/or H3K9/14 acetylation at promoters of genes required for cycle activation and progression was associated with their downregulation in FAPs from late stage mdx mice. However, these epigenetic and transcriptional alterations could not be reversed by TSA, due to a general resistance exhibited by FAPs from late stage mdx mice to HDACi-induced H3K9/14 hyperacetylation. Overall, this data reveal that disease-associated features of senescence develop in FAPs of DMD muscle through epigenetically distinct and pharmacologically dissociable events, and suggests that HDACi might at least retain anti- fibrotic and inflammatory activity at late stages of DMD, by repressing FAP-derived SASP. Integrative analysis of 2 different histone modifications (H3K9/14ac and H3K27ac ChIP-seq) and expression profiling (RNAseq) in FAPs derived from mdx mice at early and late stages of disease progression in response to TSA treatment. Comparative analysis of FAP transcriptome to other muscle-derived cells (MuSCs and Macrophages) to evaluate cell-specific features.

当前，针对杜氏肌营养不良症（Duchenne Muscular Dystrophy, DMD）采用组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitors, HDACi）开展的药物治疗，目前正处于临床试验阶段。此前在DMD的经典小鼠模型mdx小鼠中完成的临床前研究显示，HDACi可通过靶向脂肪成纤维祖细胞（fibro-adipogenic progenitors, FAPs），促进代偿性肌肉再生，同时抑制脂肪纤维化变性；但这类有益效应仅局限于疾病进展的早期阶段。本研究证实，晚期mdx小鼠的FAPs呈现出衰老相关的表观遗传与转录组特征，且此类特征无法通过HDACi完全逆转。具体而言，衰老相关分泌表型（Senescence Associated Secretory Phenotype, SASP）基因的启动子区域出现全基因组范围内的H3K9/14乙酰化水平升高，与晚期mdx小鼠FAPs中该类基因的上调表达密切相关。组蛋白去乙酰化酶抑制剂曲古抑菌素A（Trichostatin A, TSA）可通过降低H3K9/14乙酰化水平，抑制FAPs中的SASP基因激活。与之相反，细胞周期激活与进展所需基因的启动子区域出现H3K27和/或H3K9/14乙酰化的协同降低，与晚期mdx小鼠FAPs中此类基因的下调表达相关。但由于晚期mdx小鼠的FAPs普遍对HDACi诱导的H3K9/14高乙酰化产生抗性，TSA无法逆转此类表观遗传与转录组改变。综上，本研究数据表明，DMD肌肉组织中的FAPs会通过表观遗传层面的特异性差异与药理学层面的可解离事件，获得疾病相关的衰老特征；同时提示HDACi或可通过抑制FAPs分泌的SASP，在DMD疾病晚期仍保留抗纤维化与抗炎活性。本数据集整合分析了经TSA处理后，处于疾病早期与晚期阶段的mdx小鼠来源FAPs中的两种组蛋白修饰（H3K9/14ac与H3K27ac的染色质免疫共沉淀测序（ChIP-seq））数据，以及其表达谱（RNA测序（RNAseq））数据。此外，本数据集还对FAPs转录组与其他肌肉来源细胞（肌卫星细胞（Muscle Satellite Cells, MuSCs）与巨噬细胞（Macrophages））开展了比较分析，以评估其细胞特异性特征。