title: Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity.
abstract: 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.
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264; and.
authors.forename: Matteo
authors.initials: M
authors.lastname: Pasetto
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264; and.
authors.forename: Antonella
authors.initials: A
authors.lastname: Antignani
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850.
authors.forename: Pinar
authors.initials: P
authors.lastname: Ormanoglu
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850.
authors.forename: Eugen
authors.initials: E
authors.lastname: Buehler
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850.
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Guha
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264; and pastani@mail.nih.gov fitzgerd@helix.nih.gov.
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850.
authors.forename: Scott E
authors.initials: SE
authors.lastname: Martin
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264; and pastani@mail.nih.gov fitzgerd@helix.nih.gov.
authors.forename: David J
authors.initials: DJ
authors.lastname: FitzGerald
mesh_terms: D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D034622:RNA Interference
chemical_list: D000922:Immunotoxins
keywords: RNAi; genome; immunotoxin; screen; toxin