Related Experiment Video
Updated: Mar 29, 2026
Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
CRISPR-Cas9-based target validation for p53-reactivating model compounds
Michael Wanzel1,2, Jonas B Vischedyk1, Miriam P Gittler1
1Institute of Molecular Oncology, Philipps University, Marburg, Germany.
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.
Insights
Compounds targeting the p53-Mdm2 interaction show promise in cancer therapy. However, resistance mechanisms vary by drug, impacting treatment effectiveness and patient selection for p53-reactivating therapies.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- Mdm2-mediated p53 inactivation is common in cancer, making p53-Mdm2 interaction inhibitors promising therapeutic agents.
- Understanding resistance mechanisms to p53-reactivating drugs is crucial for effective cancer treatment.
Purpose of the Study:
- To investigate compound-specific resistance mechanisms to p53-reactivating drugs.
- To validate drug targets using CRISPR-Cas9 technology.
- To determine patient allocation strategies for p53-reactivating therapies.
Main Methods:
- CRISPR-Cas9-based target validation in lung and colorectal cancer cell lines.
- Assessing cellular sensitivity to nutlin and RITA.
- Evaluating DNA damage induction and repair pathways.
- Utilizing RNA interference and pharmacological inhibitors (mTOR inhibitors) to target FancD2.
Main Results:
- Nutlin's efficacy depends on functional p53, while RITA sensitivity correlates with DNA damage induction.
- RITA-resistant cells exhibit cross-resistance to DNA crosslinking agents like cisplatin and enhanced DNA cross-link repair.
- Inhibition of FancD2 restores RITA sensitivity in resistant cells.
Conclusions:
- Resistance mechanisms to p53-reactivating compounds are drug-specific.
- CRISPR-Cas9 is valuable for target validation and understanding resistance.
- Patient selection for p53-reactivating therapies should consider these compound-specific resistance mechanisms.
