Nutlin-3 up-regulates the expression of Notch1 in both myeloid and lymphoid leukemic cells, as part of a negative feedback antiapoptotic mechanism.

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 significantly up-regulated the steady-state mRNA and protein levels of Notch1 in TP53(wild-type) (OCI, SKW6.4) but not in TP53(deleted) (HL-60) or TP53(mutated) (BJAB) leukemic cell lines. A direct demonstration that NOTCH1 was a transcriptional target of p53 in leukemic cells was obtained in experiments carried out with siRNA for p53. Moreover, inhibition of Notch1 expression using Notch1-specific siRNA significantly increased cytotoxicity in TP53(wild-type) leukemic cells. Of note, Nutlin-3 up-regulated Notch1 expression also in primary TP53(wild-type) B-chronic lymphocytic leukemia (B-CLL) cells and the combined use of Nutlin-3 plus pharmacological gamma-secretase inhibitors of the Notch signaling showed a synergistic cytotoxicity in both TP53(wild-type) leukemic cell lines and primary B-CLL cells. A potential drawback of gamma-secretase inhibitors was their ability to enhance osteoclastic maturation of normal circulating preosteoclasts induced by RANKL + M-CSF. Notwithstanding, Nutlin-3 completely suppressed osteoclastogenesis irrespective of the presence of gamma-secretase inhibitors. Taken together, these data indicate that the p53-dependent up-regulation of Notch1 in response to Nutlin-3 represents an antiapoptotic feedback mechanism able to restrain the potential therapeutic efficacy of Nutlin-3 in hematologic malignancies. Therefore, therapeutic combinations of Nutlin-3 + gamma-secretase inhibitors might potentiate the cytotoxicity of Nutlin-3 in p53(wild-type) leukemic cells.

[1]  P. Secchiero,et al.  MDM2 Antagonist Nutlin‐3 Suppresses the Proliferation and Differentiation of Human Pre‐Osteoclasts Through a p53‐Dependent Pathway , 2007, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[2]  M. Hilgarth,et al.  Induction of apoptosis by proteasome inhibitors in B-CLL cells is associated with downregulation of CD23 and inactivation of Notch2 , 2005, Leukemia.

[3]  T. Golde,et al.  TCR-Mediated Notch Signaling Regulates Proliferation and IFN-γ Production in Peripheral T Cells 1 , 2003, The Journal of Immunology.

[4]  I. Screpanti,et al.  Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells. , 2009, Blood.

[5]  P. Secchiero,et al.  Synergistic cytotoxic activity of recombinant TRAIL plus the non-genotoxic activator of the p53 pathway nutlin-3 in acute myeloid leukemia cells. , 2007, Current drug metabolism.

[6]  R. Fanin,et al.  Functional integrity of the p53-mediated apoptotic pathway induced by the nongenotoxic agent nutlin-3 in B-cell chronic lymphocytic leukemia (B-CLL) , 2006 .

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

[8]  L. Vassilev,et al.  MDM2 inhibitors for cancer therapy. , 2007, Trends in molecular medicine.

[9]  Xin Lu,et al.  Live or let die: the cell's response to p53 , 2002, Nature Reviews Cancer.

[10]  M. Dettke,et al.  Notch2 is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia. , 2002, Blood.

[11]  Hong Yang,et al.  Activation of p53 by MDM2 antagonists can protect proliferating cells from mitotic inhibitors. , 2005, Cancer research.

[12]  P. Secchiero,et al.  TRAIL inhibits osteoclastic differentiation by counteracting RANKL‐dependent p27Kip1 accumulation in pre‐osteoclast precursors , 2008, Journal of cellular physiology.

[13]  S. Salati,et al.  Signal control of hematopoietic stem cell fate: Wnt, Notch, and Hedgehog as the usual suspects , 2008, Current opinion in hematology.

[14]  G. Nilsson,et al.  Distinct and regulated expression of Notch receptors in hematopoietic lineages and during myeloid differentiation , 2001, European journal of immunology.

[15]  S. Hayashi,et al.  Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells. , 2003, Blood.

[16]  M. Nakao,et al.  Transcriptional Blockade Induces p53-dependent Apoptosis Associated with Translocation of p53 to Mitochondria* , 2005, Journal of Biological Chemistry.

[17]  P. Ojala,et al.  p53 Reactivation Kills KSHV Lymphomas Efficiently In Vitro and In Vivo: New Hope for Treating Aggressive Viral Lymphomas , 2007, Cell cycle.

[18]  M. Kaminski,et al.  Comprehensive biomarker and genomic analysis identifies p53 status as the major determinant of response to MDM2 inhibitors in chronic lymphocytic leukemia. , 2007, Blood.

[19]  M. Vitale,et al.  Thrombopoietin Enhances the αIIbβ3-Dependent Adhesion of Megakaryocytic Cells to Fibrinogen or Fibronectin Through PI 3 Kinase , 1997 .

[20]  Chin-Tong Ong,et al.  NOTCH1 Regulates Osteoclastogenesis Directly in Osteoclast Precursors and Indirectly via Osteoblast Lineage Cells* , 2008, Journal of Biological Chemistry.

[21]  B. Dörken,et al.  Notch signaling in leukemias and lymphomas. , 2008, Current molecular medicine.

[22]  S. de Vos,et al.  Modeling Notch Signaling in Normal and Neoplastic Hematopoiesis: Global Gene Expression Profiling in Response to Activated Notch Expression , 2007, Stem cells.

[23]  M. Fujita,et al.  Regulation of Notch1 Gene Expression by p53 in Epithelial Cells , 2007, Molecular and Cellular Biology.

[24]  Taku Sato,et al.  Gamma-secretase inhibitors suppress the growth of leukemia and lymphoma cells. , 2007, Oncology reports.

[25]  T. Chou,et al.  Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. , 1984, Advances in enzyme regulation.

[26]  L. Vassilev,et al.  p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism. , 2008, Blood.

[27]  S. Pavey,et al.  Up-regulation of p21(WAF1/CIP1) by histone deacetylase inhibitors reduces their cytotoxicity. , 2001, Molecular pharmacology.

[28]  T. Park,et al.  Activated Notch1 interacts with p53 to inhibit its phosphorylation and transactivation , 2007, Cell Death and Differentiation.

[29]  P. Secchiero,et al.  TNF-related apoptosis-inducing ligand (TRAIL) blocks osteoclastic differentiation induced by RANKL plus M-CSF. , 2004, Blood.

[30]  Ying-hua Jin,et al.  Caspase 3-mediated Cleavage of p21 WAF1/CIP1 Associated with the Cyclin A-Cyclin-dependent Kinase 2 Complex Is a Prerequisite for Apoptosis in SK-HEP-1 Cells* , 2000, The Journal of Biological Chemistry.

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

[32]  Sathish Kumar Mungamuri,et al.  Survival signaling by Notch1: mammalian target of rapamycin (mTOR)-dependent inhibition of p53. , 2006, Cancer research.

[33]  T. Honjo,et al.  Conservation of the biochemical mechanisms of signal transduction among mammalian Notch family members , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Gabriel Pons,et al.  MDM2 antagonists activate p53 and synergize with genotoxic drugs in B-cell chronic lymphocytic leukemia cells. , 2006, Blood.

[35]  L. Jeffrey Medeiros,et al.  Inhibition of p53-Murine Double Minute 2 Interaction by Nutlin-3A Stabilizes p53 and Induces Cell Cycle Arrest and Apoptosis in Hodgkin Lymphoma , 2007, Clinical Cancer Research.

[36]  P. Secchiero,et al.  The MDM2 inhibitor Nutlins as an innovative therapeutic tool for the treatment of haematological malignancies. , 2008, Current pharmaceutical design.

[37]  P. Secchiero,et al.  Tumour necrosis factor‐related apoptosis‐inducing ligand sequentially activates pro‐survival and pro‐apoptotic pathways in SK‐N‐MC neuronal cells , 2003, Journal of neurochemistry.

[38]  A. Levine,et al.  The p53 pathway: positive and negative feedback loops , 2005, Oncogene.

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

[40]  F. Alimirah,et al.  Restoration of p53 expression in human cancer cell lines upregulates the expression of Notch1: implications for cancer cell fate determination after genotoxic stress. , 2007, Neoplasia.

[41]  L. Vassilev,et al.  Pharmacologic activation of p53-dependent and p53-independent apoptotic pathways in Hodgkin/Reed-Sternberg cells , 2007, Leukemia.

[42]  D. Dittmer,et al.  Functional p53 Signaling in Kaposi's Sarcoma-Associated Herpesvirus Lymphomas: Implications for Therapy , 2006, Journal of Virology.

[43]  L. Reiniger,et al.  Activity of the Notch‐signalling Pathway in Circulating Human Chronic Lymphocytic Leukaemia Cells , 2007, Scandinavian journal of immunology.

[44]  S. Leach,et al.  Notch in malignancy. , 2003, Cancer treatment and research.

[45]  T. Golde,et al.  TCR-mediated Notch signaling regulates proliferation and IFN-gamma production in peripheral T cells. , 2003, Journal of immunology.

[46]  M. Oren,et al.  The p53-Mdm2 module and the ubiquitin system. , 2003, Seminars in cancer biology.

[47]  L. Espinosa,et al.  The notch pathway positively regulates programmed cell death during erythroid differentiation , 2007, Leukemia.

[48]  Y. Okamoto,et al.  SurveyorTM nuclease-based detection of p53 gene mutations in haematological malignancy , 2007, Annals of clinical biochemistry.

[49]  Marina Konopleva,et al.  Mdm2 inhibitor Nutlin-3a induces p53-mediated apoptosis by transcription-dependent and transcription-independent mechanisms and may overcome Atm-mediated resistance to fludarabine in chronic lymphocytic leukemia. , 2006, Blood.