CBX3通过液-液相分离促进染色质修饰在多发性骨髓瘤耐药性产生中的机制研究

多发性骨髓瘤(MM)耐药性产生是临床治疗失败的主要原因，表观遗传学调控中的组蛋白修饰被认为是始动因素之一，但耐药性产生的组蛋白密码未知。我们利用SILAC标记的蛋白质组学分析硼替佐米(BTZ)耐药的MM细胞发现，一种参与染色质开放调控的CBX3蛋白增高且乙酰化水平降低；病人治疗前后标本中CBX3水平与临床预后具有密切相关性；在MM细胞中改变CBX3的表达导致其对BTZ的敏感性改变；CBX3含有蛋白质液相分离必需的内在无序结构域(IDR)，复合体解析发现其可招募参与DNA损伤修复的分子。我们提出CBX3是介导MM产生耐药性的重要表观遗传调控分子。本课题将从临床意义、细胞和动物实验探究CBX3蛋白液相分离和乙酰化修饰的机制、对染色质高级结构修饰和转录成瘾的调控机制，并设计筛选特异性抑制剂。本项目旨在探索MM耐药性产生最根本的转录调控机制，开发新分子靶点和特异性抑制剂，助力MM精准治疗。

The chemotherapy induced multidrug resistance is one of the most important reasons for the failure of Multiple Myeloma (MM) therapy in clinic, and chromatin modification mediated epigenetic regulation is believed to be one of the fundamental and initiative reason, however, the chromatin modification mechanisms, or the “chromatin codes” in myeloma drug resistance is still unknown. In our preliminary experiments, using the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) assay, we analyzed the changes in proteomics and acetylated proteins of two established bortezomib resistant MM cell lines (BR-MM), which were induced by increasing dosage of BTZ (starting at 0.5 nM and up to 5 nM during a 6 months period), and found that the CBX3, a nuclear-localized protein which regulates the switch of euchromosome and heterochromosome as well as the open access status of chromatin, was increased but the acetylation level was suppressed; clinically, CBX3 protein levels in tissue samples from the MM patients pre-treated and post-treated was negatively correlated with the efficacy of treatment response and bone disruptions; manipulation of CBX3 expression in MM cells changed the sensitivity to BTZ treatment, and the acetylation modification changed the CBX3 protein stability in BTZ-resistant MM cells; CBX3 complex in the BTZ-resistant cells was found to recruit partners that participate in DNA damage repair using co-immunoprecipitation assay, and this protein was also found to have the Intrinsically Disordered Regions (IDR) responsible for liquid-liquid phase separation. These results suggested that the CBX3 plays critical roles in the initiation of BTZ-induced drug resistance in MM cells, and changes in CBX3 level or protein modification will result in functional alternations in modification of the chromatin and transcriptome, which in turn regulates drug resistant genes as a consequence. In the current study, we will investigate the mechanism of CBX3 acetylation modification and the consequences on DNA damage repair as well as CBX3 liquid-liquid phase separation, chromosome conformation, open access status, and transcription addiction in the initiation of BTZ-induced drug resistance using in vitro and in vivo experiments, and try to design and screen a specific inhibitor of CBX3. The purpose of our study is to understand the fundamental and underlying mechanisms governing the initiation of BTZ-induced drug resistance in MM cells, and benefits the overcoming of refractory and relapse of patients with MM in clinic.