Inhibition of DOT1L increases osteoclast fusion and resorption and aggravates bone loss

Osteoclasts are absorptive cells and play a critical role in homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role for epigenetic regulation of osteoclastogenesis. In this study, we investigated the role of DOT1L, which regulates gene expression epigenetically by histone H3K79 methylation during osteoclast formation. DOT1L and H3K79me2 levels were upregulated during osteoclast differentiation. Small molecule inhibitor- (EPZ5676 or EPZ004777) or short hairpin RNA-mediated reduction in DOT1L expression promoted osteoclast differentiation and resorption. DOT1L inhibition also increased osteoclast area and accelerated bone mass reduction in a mouse ovariectomy (OVX) model of osteoporosis. DOT1L inhibitors did not alter osteoblast differentiation in vitro and in vivo. Proteomics data, together with bioinformatics analysis, revealed that DOT1L inhibition altered reactive oxygen species (ROS) generation, autophagy activation, and cell fusion-related protein expression. ROS generation increased, and autophagy activation and cell migration ability enhancement were verified subsequently by flow cytometry and transwell migration assays. DOT1L inhibition increased NFATc1 nuclear translocation and NF-κB activation and strengthend osteoclast fusion and expression of resorption-related protein CD9, and MMP9 in osteoclasts derived from RAW264.7. Our findings support a new mechanism of DOT1L-mediated H3K79me2 epigenetic regulation of osteoclast differentiation, implicating DOT1L as a new therapeutic target for osteoclast dysregulation-induced disease.

破骨细胞（Osteoclasts）是一类骨吸收细胞，在稳态骨重塑与病理性骨吸收中发挥关键作用。越来越多的研究证据表明，破骨细胞生成的表观遗传调控具有重要作用。本研究探讨了DOT1L在破骨细胞形成过程中的作用——该因子通过组蛋白H3K79甲基化对基因表达进行表观遗传调控。破骨细胞分化过程中，DOT1L与H3K79me2的表达水平均会上调。使用小分子抑制剂（EPZ5676或EPZ004777）或短发夹RNA（short hairpin RNA, shRNA）介导DOT1L表达下调，可促进破骨细胞分化与骨吸收功能。在小鼠骨质疏松卵巢切除（ovariectomy, OVX）模型中，DOT1L抑制还可增大破骨细胞面积，加速骨量丢失。体外与体内实验均显示，DOT1L抑制剂不会改变成骨细胞的分化过程。蛋白质组学数据结合生物信息学分析显示，DOT1L抑制可调控活性氧（reactive oxygen species, ROS）生成、自噬激活以及细胞融合相关蛋白的表达。研究后续通过流式细胞术与Transwell迁移实验证实，ROS生成增多，同时自噬激活与细胞迁移能力增强。在RAW264.7细胞来源的破骨细胞中，DOT1L抑制可增强NFATc1的核转位与NF-κB激活，强化破骨细胞融合过程，并上调骨吸收相关蛋白CD9与基质金属蛋白酶9（MMP9）的表达。本研究结果揭示了DOT1L介导的H3K79me2表观遗传调控破骨细胞分化的全新机制，提示DOT1L可作为破骨细胞功能失调相关疾病的新型治疗靶点。