Pasetto Matteo, Antignani Antonella, Ormanoglu Pinar, Buehler Eugen, Guha Rajarshi, Pastan Ira, Martin Scott E, FitzGerald David J
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264; and.
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850.
Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):E1135-42. doi: 10.1073/pnas.1501958112. Epub 2015 Feb 23.
Immunotoxins (antibody-toxin fusion proteins) target surface antigens on cancer cells and kill these cells via toxin-mediated inhibition of protein synthesis. To identify genes controlling this process, an RNAi whole-genome screen (∼ 22,000 genes at three siRNAs per gene) was conducted via monitoring the cytotoxicity of the mesothelin-directed immunotoxin SS1P. SS1P, a Pseudomonas exotoxin-based immunotoxin, was chosen because it is now in clinical trials and has produced objective tumor regressions in patients. High and low concentrations of SS1P were chosen to allow for the identification of both mitigators and sensitizers. As expected, silencing known essential genes in the immunotoxin pathway, such as mesothelin, furin, KDEL receptor 2, or members of the diphthamide pathway, protected cells. Of greater interest was the observation that many RNAi targets increased immunotoxin sensitivity, indicating that these gene products normally contribute to inefficiencies in the killing pathway. Of the top sensitizers, many genes encode proteins that locate to either the endoplasmic reticulum (ER) or Golgi and are annotated as part of the secretory system. Genes related to the ER-associated degradation system were not among high-ranking mitigator or sensitizer candidates. However, the p97 inhibitor eeyarestatin 1 enhanced immunotoxin killing. Our results highlight potential targets for chemical intervention that could increase immunotoxin killing of cancer cells and enhance our understanding of toxin trafficking.
免疫毒素（抗体 - 毒素融合蛋白）靶向癌细胞表面抗原，并通过毒素介导的蛋白质合成抑制作用杀死这些细胞。为了鉴定控制这一过程的基因，通过监测间皮素导向的免疫毒素SS1P的细胞毒性，进行了一项RNAi全基因组筛选（每个基因三个小干扰RNA，共约22,000个基因）。SS1P是一种基于铜绿假单胞菌外毒素的免疫毒素，之所以被选用，是因为它目前正处于临床试验阶段，并且已在患者中产生了客观的肿瘤消退。选择高浓度和低浓度的SS1P，以便鉴定减轻剂和增敏剂。正如预期的那样，沉默免疫毒素途径中已知的必需基因，如间皮素、弗林蛋白酶、KDEL受体2或双氢酰胺途径的成员，可保护细胞。更令人感兴趣的是，观察到许多RNAi靶点增加了免疫毒素敏感性，这表明这些基因产物通常会导致杀伤途径效率低下。在顶级增敏剂中，许多基因编码定位于内质网（ER）或高尔基体的蛋白质，并被注释为分泌系统的一部分。与内质网相关降解系统相关的基因不在高级减轻剂或增敏剂候选之列。然而，p97抑制剂依维莫司汀1增强了免疫毒素的杀伤作用。我们的结果突出了化学干预的潜在靶点，这些靶点可以增加免疫毒素对癌细胞的杀伤作用，并增进我们对毒素运输的理解。
