Hsp90 Inhibitor SNX-7081 Dysregulates Proteins Involved with DNA Repair and Replication and the Cell Cycle in Human Chronic Lymphocytic Leukemia (CLL) Cells

The proteomic effects of the Hsp90 inhibitor, SNX-7081, have been determined on the p53-mutated B-cell chronic lymphocytic leukemia (CLL) cell line, MEC1. Following SNX-7081 treatment (500 nM, 24 h), 51 proteins changed abundance by more than 2-fold (p < 0.05); 7 proteins increased while 44 proteins decreased. Proteins identified as differentially abundant by LC–MS/MS were validated by Western blotting (DDB1, PCNA, MCM2, Hsp90, Hsp70, GRP78, PDIA6, HLA-DR). RT-PCR showed that SNX-7081 unexpectedly modulates a number of these proteins in MEC1 cells at the mRNA level (PCNA, MCM2, Nup155, Hsp70, GRP78, PDIA6, and HLA-DR). Pathway analysis determined that 3 of the differentially abundant proteins (cyclin D1, c-Myc and pRb) were functionally related. p53 levels did not change upon SNX-7081 treatment of p53 wild-type Raji cells or p53-mutated MEC1 and U266 cells, indicating that SNX-7081 has a p53-independent mechanism. The decreases in DDB1, MCM2, c-Myc, and PCNA and increases of pRb and cyclin D1 were confirmed in MEC1, U266, Raji, and p53 null HL60 cells by Western blotting. These data suggest that SNX-7081 arrests the cell cycle and inhibits DNA replication and repair and provides evidence for the mechanism of the observed synergy between Hsp90 inhibitors and drugs that induce DNA strand breaks.本研究明确了热休克蛋白90（Hsp90）抑制剂SNX-7081对p53突变型B细胞慢性淋巴细胞白血病（CLL）细胞系MEC1的蛋白质组学影响。经500 nM SNX-7081处理24小时后，共有51种蛋白质的丰度发生2倍以上变化（p < 0.05），其中7种蛋白质丰度上调，44种下调。通过液相色谱-串联质谱（LC–MS/MS）鉴定得到的差异丰度蛋白质，经蛋白质印迹（Western blotting）验证，验证靶点包括DDB1、PCNA、MCM2、Hsp90、Hsp70、GRP78、PDIA6及HLA-DR。反转录聚合酶链反应（RT-PCR）结果显示，SNX-7081可在mRNA水平意外调控MEC1细胞中的多种上述蛋白质，涉及靶点包括PCNA、MCM2、Nup155、Hsp70、GRP78、PDIA6及HLA-DR。通路分析表明，3种差异丰度蛋白质（细胞周期蛋白D1（cyclin D1）、c-Myc及视网膜母细胞瘤蛋白（pRb））存在功能关联。在p53野生型Raji细胞，以及p53突变型MEC1与U266细胞中，经SNX-7081处理后p53水平未发生变化，表明SNX-7081的作用机制不依赖p53。通过蛋白质印迹验证，在MEC1、U266、Raji及p53缺失型HL60细胞中，均观察到DDB1、MCM2、c-Myc及PCNA丰度下调，pRb与细胞周期蛋白D1丰度上调。上述数据表明，SNX-7081可阻滞细胞周期并抑制DNA复制与修复，同时为Hsp90抑制剂与诱导DNA链断裂药物间的协同作用机制提供了实验依据。