Li Wei, Bi Chengfeng, Han Yating, Tian Tian, Wang Xianhuo, Bao Huijing, Xu Xiaoying, Zhang Xuhan, Liu Lu, Zhang Weiwei, Gao Hai, Wang Huaqing, Zhang Huilai, Meng Bin, Wang Xi, Fu Kai
Department of Lymphoma.
Department of Pathology, Sino-US Center for Lymphoma and Leukemia Research, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, Tianjin's Clinical Research Center for Cancer, Tianjin 300060, China.
Cancer Biol Med. 2019 Aug;16(3):530-541. doi: 10.20892/j.issn.2095-3941.2018.0380.
To explore the effect of dysregulation of epigenetic regulator EZH1 and EZH2 on the proliferation in MCL and the underlying mechanisms.
In this study, we elucidated the role of EZH1 and EZH2 overexpression by immunohistochemistry and correlated them to clinical outcome in 41 MCL patients. Quantitative real-time PCR and Western blot were applied to confirm the level of EZH1 and EZH2 in well-characterized MCL cell lines which were compared to those of naïve B cells. Then we manipulated the expression of EZH1 and EZH2 in MCL cells using CRISPR/Cas9 system to directly investigate their functional roles in MCL. We also evaluated the effect of two small molecule selective inhibitors, EPZ005687 and UNC1999, on MCL cell proliferation, cell cycle distribution and apoptosis . Finally, we performed RNA-sequencing (RNA-Seq) and Chromatin immunoprecipitation (ChIP) assay to further gain insight into the underlying molecular mechanisms.
We found that EZH2 protein is overexpressed in approximately half of this cohort of MCL cases. More importantly, the overexpression of EZH2 is associated with poor OS in the patients. Nevertheless, simple EZH2 depletion has little impact on the viability of MCL cells, predominantly because of the consequent up-regulation of EZH1. Consistently, UNC1999, a dual EZH1/2 inhibitor, unlike the EZH2 selective inhibitor EPZ005687, exerts a potent inhibitory effect on MCL cells. Furthermore, we discover and as the two important cell cycle regulators, the expression of which are repressed by EZH1/2 mediated epigenetic regulation and are restored by EZH1/2 dual inhibition.
Our study suggests that EZH2 participates in the pathogenesis of MCL which may serve as a potential biomarker for prognosis prediction. The dual inhibition of EZH1/2 is a promising therapeutic strategy for MCL.
探讨表观遗传调控因子EZH1和EZH2失调对套细胞淋巴瘤（MCL）增殖的影响及其潜在机制。
在本研究中，我们通过免疫组织化学阐明了EZH1和EZH2过表达的作用，并将其与41例MCL患者的临床结局相关联。应用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法（Western blot）来确认在特征明确的MCL细胞系中EZH1和EZH2的水平，并与原始B细胞进行比较。然后，我们使用CRISPR/Cas9系统操纵MCL细胞中EZH1和EZH2的表达，以直接研究它们在MCL中的功能作用。我们还评估了两种小分子选择性抑制剂EPZ005687和UNC1999对MCL细胞增殖、细胞周期分布和凋亡的影响。最后，我们进行了RNA测序（RNA-Seq）和染色质免疫沉淀（ChIP）分析，以进一步深入了解潜在的分子机制。
我们发现约一半的MCL病例中EZH2蛋白过表达。更重要的是，EZH2过表达与患者的总生存期（OS）较差相关。然而，单纯敲低EZH2对MCL细胞的活力影响不大，主要是因为随后EZH1上调。同样，与EZH2选择性抑制剂EPZ005687不同，双重EZH1/2抑制剂UNC1999对MCL细胞具有强大的抑制作用。此外，我们发现细胞周期蛋白依赖性激酶1（CDK1）和细胞周期蛋白B1（CCNB1）作为两个重要的细胞周期调节因子，其表达受到EZH1/2介导的表观遗传调控的抑制，并通过EZH1/2双重抑制得以恢复。
我们的研究表明，EZH2参与MCL的发病机制，可能作为预后预测的潜在生物标志物。EZH1/2双重抑制是一种有前景的MCL治疗策略。
