CRISPR-Cas9-based target validation for p53-reactivating model compounds

Nat Chem Biol. 2016 Jan;12(1):22-8. doi: 10.1038/nchembio.1965. Epub 2015 Nov 23.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Cisplatin / pharmacology
  • DNA Damage / drug effects
  • DNA Damage / genetics
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Drug Resistance, Neoplasm / drug effects*
  • Drug Resistance, Neoplasm / genetics
  • Fanconi Anemia Complementation Group D2 Protein / genetics
  • Fanconi Anemia Complementation Group D2 Protein / metabolism
  • Furans / pharmacology*
  • Gene Expression Regulation
  • Genes, p53* / physiology
  • HCT116 Cells / drug effects
  • Humans
  • Molecular Targeted Therapy / methods*
  • Morpholines / pharmacology
  • Proto-Oncogene Proteins c-mdm2 / metabolism
  • TOR Serine-Threonine Kinases / antagonists & inhibitors
  • TOR Serine-Threonine Kinases / metabolism
  • Ubiquitin-Protein Ligases


  • DNA-Binding Proteins
  • FANCD2 protein, human
  • Fanconi Anemia Complementation Group D2 Protein
  • Furans
  • Morpholines
  • NSC 652287
  • RAD18 protein, human
  • (5-(2,4-bis((3S)-3-methylmorpholin-4-yl)pyrido(2,3-d)pyrimidin-7-yl)-2-methoxyphenyl)methanol
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2
  • Ubiquitin-Protein Ligases
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • Cisplatin