Levels of HdmX expression dictate the sensitivity of normal and transformed cells to Nutlin-3.

Hdm2 and HdmX coordinately regulate the stability and function of p53. Each is overexpressed in subsets of many different types of malignancy, and most of these subsets maintain wild-type p53. Nutlins, newly discovered small-molecule inhibitors of the Hdm2-p53 interaction, offer a novel strategy for therapy of tumors with wild-type p53. We now show that Nutlin-3 efficiently induces apoptosis and diminishes long-term survival of human fibroblasts transformed in vitro by Hdm2 but not HdmX. The resistance of cells overexpressing HdmX to Nutlin-3 is due to its inability to disrupt the p53-HdmX interaction, resulting in continued suppression of p53 activity. Although HdmX overexpression yielded cells resistant to Nutlin-3, ablation of HdmX expression by short hairpin RNA sensitized tumor cells to Nutlin-3-mediated cell death or arrest. Furthermore, deletion of the COOH-terminal RING finger domain of HdmX completely reversed the resistance to Nutlin-3, probably reflecting the requirement of the RING finger for interaction with Hdm2. Thus, the relative abundance of Hdm2 and HdmX and the specificity of Nutlin-3 for Hdm2 influence the sensitivity of cells to p53-dependent apoptosis or arrest in response to Nutlin-3. Our findings establish Hdm2 and HdmX as independent therapeutic targets with respect to reactivating wild-type p53 as a means for cancer therapy.

[1]  L. Vassilev,et al.  Nongenotoxic activation of the p53 pathway as a therapeutic strategy for multiple myeloma. , 2005, Blood.

[2]  Steffen Hauptmann,et al.  Significance of HDMX‐S (or MDM4) mRNA splice variant overexpression and HDMX gene amplification on primary soft tissue sarcoma prognosis , 2005, International journal of cancer.

[3]  Marina Konopleva,et al.  MDM2 antagonists induce p53-dependent apoptosis in AML: implications for leukemia therapy. , 2005, Blood.

[4]  A. Marchetti,et al.  Identification of an aberrantly spliced form of HDMX in human tumors: a new mechanism for HDM2 stabilization. , 2005, Cancer research.

[5]  T. Kuwana,et al.  PUMA Couples the Nuclear and Cytoplasmic Proapoptotic Function of p53 , 2005, Science.

[6]  H. Ovaa,et al.  Loss of HAUSP-mediated deubiquitination contributes to DNA damage-induced destabilization of Hdmx and Hdm2. , 2005, Molecular cell.

[7]  G. Stark,et al.  p130/p107/p105Rb-dependent transcriptional repression during DNA-damage-induced cell-cycle exit at G2 , 2005, Journal of Cell Science.

[8]  Petra de Graaf,et al.  Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Hui Wang,et al.  Novel antisense anti-MDM2 mixed-backbone oligonucleotides: proof of principle, in vitro and in vivo activities, and mechanisms. , 2005, Current cancer drug targets.

[10]  M. Protopopova,et al.  Small molecule RITA binds to p53, blocks p53–HDM-2 interaction and activates p53 function in tumors , 2004, Nature Medicine.

[11]  Zigang Dong,et al.  Post-translational modification of p53 in tumorigenesis , 2004, Nature Reviews Cancer.

[12]  Kristian Helin,et al.  Amplification of Mdmx (or Mdm4) Directly Contributes to Tumor Formation by Inhibiting p53 Tumor Suppressor Activity , 2004, Molecular and Cellular Biology.

[13]  Patrick Dumont,et al.  Mitochondrial p53 activates Bak and causes disruption of a Bak–Mcl1 complex , 2004, Nature Cell Biology.

[14]  R. Iggo,et al.  Regulation of p53 Stability and Function in HCT116 Colon Cancer Cells* , 2004, Journal of Biological Chemistry.

[15]  L. Vassilev,et al.  In Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2 , 2004, Science.

[16]  C. Cordon-Cardo,et al.  MDM2 and prognosis. , 2004, Molecular cancer research : MCR.

[17]  Guillermina Lozano,et al.  MDM2, an introduction. , 2003, Molecular cancer research : MCR.

[18]  S. Berberich,et al.  Overexpression of Mdm2 and MdmX fusion proteins alters p53 mediated transactivation, ubiquitination, and degradation. , 2003, Biochemistry.

[19]  Petr Pancoska,et al.  p53 has a direct apoptogenic role at the mitochondria. , 2003, Molecular cell.

[20]  M. Murphy,et al.  The codon 72 polymorphic variants of p53 have markedly different apoptotic potential , 2003, Nature Genetics.

[21]  P. Chène.  Inhibiting the p53–MDM2 interaction: an important target for cancer therapy , 2003, Nature Reviews Cancer.

[22]  A. Haas,et al.  MdmX Is a RING Finger Ubiquitin Ligase Capable of Synergistically Enhancing Mdm2 Ubiquitination* , 2002, The Journal of Biological Chemistry.

[23]  G. Hannon,et al.  Transformation of normal human cells in the absence of telomerase activation. , 2002, Cancer cell.

[24]  K. Helin,et al.  Mdm4 (Mdmx) Regulates p53-Induced Growth Arrest and Neuronal Cell Death during Early Embryonic Mouse Development , 2002, Molecular and Cellular Biology.

[25]  R. Ramirez-Solis,et al.  mdmx is a negative regulator of p53 activity in vivo. , 2002, Cancer research.

[26]  A. Jochemsen,et al.  Mutual Dependence of MDM2 and MDMX in Their Functional Inactivation of p53* , 2002, The Journal of Biological Chemistry.

[27]  Galina Selivanova,et al.  Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound , 2002, Nature Medicine.

[28]  N. Little,et al.  Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms , 2001, EMBO reports.

[29]  Valerie Reinke,et al.  Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53 , 2001, Nature Genetics.

[30]  Yolande F M Ramos,et al.  Aberrant expression of HDMX proteins in tumor cells correlates with wild-type p53. , 2001, Cancer research.

[31]  A. Levine,et al.  Surfing the p53 network , 2000, Nature.

[32]  N. Little,et al.  Hdmx stabilizes Mdm2 and p53. , 2000, The Journal of biological chemistry.

[33]  M. Jackson,et al.  MdmX Protects p53 from Mdm2-Mediated Degradation , 2000, Molecular and Cellular Biology.

[34]  D. George,et al.  Stabilization of the MDM2 Oncoprotein by Interaction with the Structurally Related MDMX Protein* , 1999, The Journal of Biological Chemistry.

[35]  M. Jackson,et al.  Constitutive mdmx expression during cell growth, differentiation, and DNA damage. , 1999, DNA and cell biology.

[36]  K. Shirouzu,et al.  MDM2 interacts with MDMX through their RING finger domains , 1999, FEBS letters.

[37]  W. Mercer,et al.  A Novel MDMX Transcript Expressed in a Variety of Transformed Cell Lines Encodes a Truncated Protein with Potent p53 Repressive Activity* , 1999, The Journal of Biological Chemistry.

[38]  Yolande F M Ramos,et al.  Comparative study of the p53-mdm2 and p53-MDMX interfaces , 1999, Oncogene.

[39]  J. Turchi,et al.  Mdm-2 phosphorylation by DNA-dependent protein kinase prevents interaction with p53. , 1997, Cancer research.

[40]  D. Lane,et al.  p53 protein stability in tumour cells is not determined by mutation but is dependent on Mdm2 binding , 1997, Oncogene.

[41]  A. Jochemsen,et al.  MDMX: a novel p53‐binding protein with some functional properties of MDM2. , 1996, The EMBO journal.

[42]  Guillermina Lozano,et al.  Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53 , 1995, Nature.

[43]  Lawrence A. Donehower,et al.  Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53 , 1995, Nature.

[44]  A. Levine,et al.  The mdm-2 gene is induced in response to UV light in a p53-dependent manner. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[45]  A. Levine,et al.  The p53-mdm-2 autoregulatory feedback loop. , 1993, Genes & development.

[46]  L. Donehower,et al.  Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours , 1992, Nature.

[47]  J. Levine,et al.  Surfing the p53 network , 2000, Nature.