Lan Xiaoying, Zhao Chong, Chen Xin, Zhang Peiquan, Zang Dan, Wu Jinjie, Chen Jinghong, Long Huidan, Yang Li, Huang Hongbiao, Carter Bing Z, Wang Xuejun, Shi Xianping, Liu Jinbao
Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
Department of Leukemia, Section of Molecular Hematology and Therapy, The University of Texas M.D. Anderson Cancer Center, Houston, TX, 77030, USA.
J Hematol Oncol. 2016 Nov 25;9(1):129. doi: 10.1186/s13045-016-0359-x.
Acquired imatinib (IM) resistance is frequently characterized by Bcr-Abl mutations that affect IM binding and kinase inhibition in patients with chronic myelogenous leukemia (CML). Bcr-Abl-T315I mutation is the predominant mechanism of the acquired resistance to IM. Therefore, it is urgent to search for additional approaches and targeting strategies to overcome IM resistance. We recently reported that nickel pyrithione (NiPT) potently inhibits the ubiquitin proteasome system via targeting the 19S proteasome-associated deubiquitinases (UCHL5 and USP14), without effecting on the 20S proteasome. In this present study, we investigated the effect of NiPT, a novel proteasomal deubiquitinase inhibitor, on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl.
Cell viability was examined by MTS assay and trypan blue exclusion staining assay in KBM5, KBM5R, K562, BaF3-p210-WT, BaF3-p210-T315I cells, and CML patients' bone marrow samples treated with NiPT. Cell apoptosis in CML cells was detected with Annexin V-FITC/PI and rhodamine-123 staining followed by fluorescence microscopy and flow cytometry and with western blot analyses for apoptosis-associated proteins. Expression levels of Bcr-Abl in CML cells were analyzed by using western blotting and real-time PCR. The 20S proteasome peptidase activity was measured using specific fluorogenic substrate. Active-site-directed labeling of proteasomal DUBs, as well as the phosphorylation of USP14 was used for evaluating the inhibition of the DUBs activity by NiPT. Mouse xenograft models of KBM5 and KBM5R cells were analyzed, and Bcr-Abl-related proteins and protein biomarkers related to proliferation, differentiation, and adhesion in tumor tissues were detected by western blots and/or immunohistological analyses.
NiPT induced apoptosis in CML cells and inhibited the growth of IM-resistant Bcr-Abl-T315I xenografts in nude mice. Mechanistically, NiPT induced decreases in Bcr-Abl proteins, which were associated with downregulation of Bcr-Abl transcription and with the cleavage of Bcr-Abl protein by activated caspases. NiPT-induced ubiquitin proteasome system inhibition induced caspase activation in both IM-resistant and IM-sensitive CML cells, and the caspase activation was required for NiPT-induced Bcr-Abl downregulation and apoptotic cell death.
These findings support that NiPT can overcome IM resistance through both Bcr-Abl-dependent and Bcr-Abl-independent mechanisms, providing potentially a new option for CML treatment.
在慢性粒细胞白血病（CML）患者中，获得性伊马替尼（IM）耐药常表现为Bcr-Abl突变，这些突变会影响IM的结合及激酶抑制作用。Bcr-Abl-T315I突变是获得性IM耐药的主要机制。因此，迫切需要寻找其他方法和靶向策略来克服IM耐药。我们最近报道，吡啶硫酮镍（NiPT）通过靶向19S蛋白酶体相关去泛素化酶（UCHL5和USP14）有效抑制泛素蛋白酶体系统，而不影响20S蛋白酶体。在本研究中，我们研究了新型蛋白酶体去泛素化酶抑制剂NiPT对携带Bcr-Abl-T315I或野生型Bcr-Abl的CML细胞的细胞存活或凋亡的影响。
通过MTS法和台盼蓝排斥染色法检测NiPT处理的KBM5、KBM5R、K562、BaF3-p210-WT、BaF3-p210-T315I细胞以及CML患者骨髓样本的细胞活力。用Annexin V-FITC/PI和罗丹明-123染色，随后通过荧光显微镜和流式细胞术检测CML细胞中的细胞凋亡，并通过蛋白质印迹分析凋亡相关蛋白。使用蛋白质印迹法和实时PCR分析CML细胞中Bcr-Abl的表达水平。使用特异性荧光底物测量20S蛋白酶体肽酶活性。蛋白酶体DUBs的活性位点定向标记以及USP14的磷酸化用于评估NiPT对DUBs活性的抑制作用。分析KBM5和KBM5R细胞的小鼠异种移植模型，并通过蛋白质印迹和/或免疫组织学分析检测肿瘤组织中与Bcr-Abl相关的蛋白以及与增殖、分化和黏附相关的蛋白质生物标志物。
NiPT诱导CML细胞凋亡，并抑制裸鼠中IM耐药的Bcr-Abl-T315I异种移植瘤的生长。机制上，NiPT诱导Bcr-Abl蛋白减少，这与Bcr-Abl转录下调以及活化的半胱天冬酶对Bcr-Abl蛋白的切割有关。NiPT诱导的泛素蛋白酶体系统抑制在IM耐药和IM敏感的CML细胞中均诱导半胱天冬酶活化，并且半胱天冬酶活化是NiPT诱导的Bcr-Abl下调和凋亡细胞死亡所必需的。
这些发现支持NiPT可通过依赖Bcr-Abl和不依赖Bcr-Abl的机制克服IM耐药，为CML治疗提供了潜在的新选择。
