RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma
RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma
复制标题
DOI：
10.1073/pnas.1012351108
复制
发表时间：
2011-02-22
影响因子：
11.1
通讯作者：
Maris, John M.
中科院分区：
文献类型：
--
作者：
Cole, Kristina A.;Huggins, Jonathan;Maris, John M.
神经母细胞瘤是一种儿童癌症，尽管进行了密集的多模态治疗，但往往是致命的。为了确定这种疾病的治疗靶点，我们对蛋白激酶组进行了全面的功能丧失筛选。30种激酶在耗尽时显示出显著的细胞毒性，其中细胞周期检查点激酶1（CHK 1/CHEK 1）的丧失是最有效的。CHK 1 mRNA在MYC-神经母细胞瘤相关（MYCN）扩增（P < 0.0001）和高危（P = 0.03）肿瘤中表达更高。Western blotting结果显示，CHK 1在神经母细胞瘤细胞系中的共济失调毛细血管扩张反应激酶靶位点Ser 345和自磷酸化位点Ser 296处组成性磷酸化。这种模式也出现在8个高风险原发性肿瘤中的6个中，但在对照非神经母细胞瘤细胞系或8个低风险原发性肿瘤中的7个中没有。神经母细胞瘤细胞对两种CHK 1抑制剂SB 21807和TCS 2312敏感，中位IC 50值分别为564 nM和548 nM。相比之下，对照系具有高微摩尔IC 50值，表明CHK 1磷酸化与CHK 1抑制剂敏感性之间存在强相关性（P = 0.0004）。此外，细胞周期分析显示，CHK 1抑制神经母细胞瘤细胞在S期引起细胞凋亡，与其在复制叉进展中的作用一致。CHK 1抑制剂敏感性与总MYC（N）蛋白水平相关，在视网膜色素上皮细胞中诱导MYCN导致CHK 1磷酸化，当抑制时导致生长抑制。这些数据显示了功能性RNAi筛选鉴定神经母细胞瘤中易处理的治疗靶点并支持该疾病中CHK 1抑制策略的能力。
Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1mRNAexpression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC50 values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC50 values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.