title: CRISPR-Cas9-based target validation for p53-reactivating model compounds.
abstract: Inactivation of the p53 tumor suppressor by Mdm2 is one of the most frequent events in cancer, so compounds targeting the p53-Mdm2 interaction are promising for cancer therapy. Mechanisms conferring resistance to p53-reactivating compounds are largely unknown. Here we show using CRISPR-Cas9-based target validation in lung and colorectal cancer that the activity of nutlin, which blocks the p53-binding pocket of Mdm2, strictly depends on functional p53. In contrast, sensitivity to the drug RITA, which binds the Mdm2-interacting N terminus of p53, correlates with induction of DNA damage. Cells with primary or acquired RITA resistance display cross-resistance to DNA crosslinking compounds such as cisplatin and show increased DNA cross-link repair. Inhibition of FancD2 by RNA interference or pharmacological mTOR inhibitors restores RITA sensitivity. The therapeutic response to p53-reactivating compounds is therefore limited by compound-specific resistance mechanisms that can be resolved by CRISPR-Cas9-based target validation and should be considered when allocating patients to p53-reactivating treatments.
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.|Universities of Giessen and Marburg Lung Center (UGMLC), German Center for Lung Research (DZL), Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Wanzel
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.
authors.forename: Jonas B
authors.initials: JB
authors.lastname: Vischedyk
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.
authors.forename: Miriam P
authors.initials: MP
authors.lastname: Gittler
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.
authors.forename: Niklas
authors.initials: N
authors.lastname: Gremke
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.
authors.forename: Julia R
authors.initials: JR
authors.lastname: Seiz
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Hefter
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Noack
authors.affiliation: Universities of Giessen and Marburg Lung Center (UGMLC), German Center for Lung Research (DZL), Germany.|Max Planck Institute for Heart and Lung Research, Department of Lung Development and Remodeling, Bad Nauheim, Germany.|Department of Internal Medicine, Justus Liebig University, Giessen, Germany.
authors.forename: Rajkumar
authors.initials: R
authors.lastname: Savai
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Mernberger
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.
authors.forename: Joël P
authors.initials: JP
authors.lastname: Charles
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.
authors.forename: Jean
authors.initials: J
authors.lastname: Schneikert
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.
authors.forename: Anne Catherine
authors.initials: AC
authors.lastname: Bretz
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.|Genomics Core Facility, Philipps University, Marburg, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Nist
authors.affiliation: Institute of Molecular Oncology, Philipps University, Marburg, Germany.|Universities of Giessen and Marburg Lung Center (UGMLC), German Center for Lung Research (DZL), Germany.|Genomics Core Facility, Philipps University, Marburg, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Stiewe
mesh_terms: D064113:CRISPR-Cas Systems; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000235:genetics / Q000378:metabolism; D005663:Furans / Q000494:pharmacology*; D005786:Gene Expression Regulation; D016158:Genes, p53 / Q000502:physiology; D045325:HCT116 Cells / Q000187:drug effects; D006801:Humans; D058990:Molecular Targeted Therapy / Q000379:methods*; D009025:Morpholines / Q000494:pharmacology; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
chemical_list: D004268:DNA-Binding Proteins; C499905:FANCD2 protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D005663:Furans; D009025:Morpholines; C118989:NSC 652287; C485954:RAD18 protein, human; C546624:(5-(2,4-bis((3S)-3-methylmorpholin-4-yl)pyrido(2,3-d)pyrimidin-7-yl)-2-methoxyphenyl)methanol; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D002945:Cisplatin