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

Nature Chemical Biology volume 12, pages 22–28 (2016)Cite this article

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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.

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Figure 1: p53-deficient cells are resistant to nutlin but not RITA.
Figure 2: RITA sensitivity correlates with induction of DNA damage.
Figure 3: RITA resistant cells are cross-resistant to cisplatin.
Figure 4: RITA resistance is mediated by FancD2.
Figure 5: RAD18 depletion sensitizes to RITA.
Figure 6: RITA resistance is overcome with mTOR inhibitors.

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Acknowledgements

We thank B. Vogelstein (John Hopkins University) for providing HCT116 p53−/− cells and A. Filmer for excellent technical assistance with next-generation sequencing. The authors are grateful to the members of the Stiewe laboratory for fruitful discussions and support of the project. This research was supported by Deutsche Forschungsgemeinschaft grants WA 2725/1-1 (M.W.), TRR81 (T.S.), STI 182/3-2 (T.S.) and STI 182/7-1 (T.S.); European Research Council grant P73CANCER 260431 (T.S.); Deutsche Krebshilfe grant 111250 (T.S.); Deutsche José Carreras Leukämie-Stiftung grant (T.S.), Von-Behring-Röntgen-Stiftung grant (T.S.), Rhön Klinikum AG grant (T.S.) and LOEWE Universities of Giessen and Marburg Lung Center grant (T.S.).

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Authors and Affiliations

  1. Institute of Molecular Oncology, Philipps University, Marburg, Germany

    Michael Wanzel, Jonas B Vischedyk, Miriam P Gittler, Niklas Gremke, Julia R Seiz, Mirjam Hefter, Magdalena Noack, Marco Mernberger, Joël P Charles, Jean Schneikert, Anne Catherine Bretz, Andrea Nist & Thorsten Stiewe

  2. Universities of Giessen and Marburg Lung Center (UGMLC), German Center for Lung Research (DZL), Germany

    Michael Wanzel, Rajkumar Savai & Thorsten Stiewe

  3. Department of Lung Development and Remodeling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

    Rajkumar Savai

  4. Department of Internal Medicine, Justus Liebig University, Giessen, Germany

    Rajkumar Savai

  5. Genomics Core Facility, Philipps University, Marburg, Germany

    Andrea Nist & Thorsten Stiewe

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  1. Michael Wanzel
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Contributions

M.W., J.B.V., M.P.G., N.G., M.H., M.N., J.P.C., J.R.S., J.S. and A.C.B. designed, performed and analyzed experiments. A.N. performed next-generation sequencing. M.M. performed bioinformatic data analysis. R.S. provided critical reagents. M.W. and T.S. wrote the manuscript with advice from all authors. M.W. and T.S. guided all aspects of this study.

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Correspondence to Thorsten Stiewe.

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Wanzel, M., Vischedyk, J., Gittler, M. et al. CRISPR-Cas9–based target validation for p53-reactivating model compounds. Nat Chem Biol 12, 22–28 (2016). https://doi.org/10.1038/nchembio.1965

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