Original Article

Citation: Cell Death and Disease (2015) 6, e2035; doi:10.1038/cddis.2015.358
Published online 31 December 2015

Targeting RING domains of Mdm2–MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells
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W Wu1,4, C Xu1,4, X Ling1,4, C Fan1, B P Buckley2, M V Chernov2, L Ellis1, F Li1, I G Muñoz3 and X Wang1

  1. 1Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA
  2. 2Department of Stress Biology, Small Molecule Screening Core Facility, Roswell Park Cancer Institute, Buffalo, NY, USA
  3. 3Crystallography Unit, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid, Spain

Correspondence: X Wang, Department of Pharmacology and Therapeutics, Roswell Park Memorial, Elm & Carlton Streets, Buffalo, NY 14263, USA. Tel: +1 716 8457652; Fax: +1 716 8458857; E-mail: xinjiang.wang@roswellpark.org

4These authors contributed equally to this work.

Received 14 October 2015; Accepted 5 November 2015

Edited by G Raschellà

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Abstract

Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2–p53 interface or MdmX ((MDM4), mouse double minute 4)–p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2–MdmX really interesting new gene (RING)–RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2–MdmX RING–RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2–MdmX RING domain inhibitors)) that specifically inhibit Mdm2–MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2–MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2–MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.

Abbreviations:

DMSO, dimethyl sulfoxide; FCS, fetal calf serum; FRET, fluorescence resonance energy transfer; GST, glutathione S-transferase; HT, high-throughput; HTS, high-throughput screening; MDM2, mouse double minute 2; MDMX, (MDM4), mouse double minute 4; MMNi, MdmX and NEDD4-1 inhibitors; MMRi, Mdm2–MdmX RING domain inhibitors; NEDD4, neural precursor cell expressed developmentally downregulated protein 4; PARP, poly ADP ribose polymerase; PUMA, p53 upregulated modulator of apoptosis; RB, retinoblastoma protein; RING, really interesting new gene; RING–RING interaction, RING domain–RING domain interaction; WB, western blotting; WT, wild type