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.

Although TP53 mutations are rare in B-cell chronic lymphocytic leukemia (CLL), Mdm2 overexpression has been reported as an alternative cause of p53 dysfunction. We investigated the potential therapeutic use of nongenotoxic p53 activation by a small-molecule antagonist of Mdm2, Nutlin-3a, in CLL. Nutlin-3a induced significant apoptosis in 30 (91%) of 33 samples from previously untreated patients with CLL; all resistant samples had TP53 mutations. Low levels of Atm (ataxia telangiectasia mutated) or high levels of Mdm2 (murine double minute 2) did not prevent Nutlin-3a from inducing apoptosis. Nutlin-3a used transcription-dependent and transcription-independent pathways to induce p53-mediated apoptosis. Predominant activation of the transcription-independent pathway induced more pronounced apoptosis than that of the transcription-dependent pathway, suggesting that activation of the transcription-independent pathway is sufficient to initiate p53-mediated apoptosis in CLL. Combination treatment of Nutlin-3a and fludarabine synergistically increased p53 levels, and induced conformational change of Bax and apoptosis in wild-type p53 cells but not in cells with mutant p53. The synergistic apoptotic effect was maintained in samples with low Atm that were fludarabine resistant. Results suggest that the nongenotoxic activation of p53 by targeting the Mdm2-p53 interaction provides a novel therapeutic strategy for CLL.

[1]  W. Deppert,et al.  Transcription-independent pro-apoptotic functions of p53. , 2005, Current opinion in cell biology.

[2]  M. Andreeff,et al.  Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

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

[4]  U. Moll,et al.  The MDM2-p53 interaction. , 2003, Molecular cancer research : MCR.

[5]  P. Feugier,et al.  Randomized comparison of fludarabine, CAP, and ChOP in 938 previously untreated stage B and C chronic lymphocytic leukemia patients. , 2001, Blood.

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

[7]  Yoichi Taya,et al.  ATM associates with and phosphorylates p53: mapping the region of interaction , 1999, Nature Genetics.

[8]  U. Moll,et al.  In Vivo Mitochondrial p53 Translocation Triggers a Rapid First Wave of Cell Death in Response to DNA Damage That Can Precede p53 Target Gene Activation , 2004, Molecular and Cellular Biology.

[9]  M. Dyer,et al.  p53 abnormalities in CLL are associated with excess of prolymphocytes and poor prognosis , 1997, British journal of haematology.

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

[11]  W. Plunkett,et al.  Fludarabine-mediated inhibition of nucleotide excision repair induces apoptosis in quiescent human lymphocytes. , 1996, Clinical cancer research : an official journal of the American Association for Cancer Research.

[12]  F. Mancini,et al.  p 53 Expression in B-Cell Chronic Lymphocytic Leukemia : A Marker of Disease Progression and Poor Prognosis , 1998 .

[13]  Martin Schuler,et al.  Direct Activation of Bax by p53 Mediates Mitochondrial Membrane Permeabilization and Apoptosis , 2004, Science.

[14]  M. Story,et al.  Induction of apoptotic cell death in chronic lymphocytic leukemia by 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosyl-2-fluoroadenine. , 1993, Blood.

[15]  Lisa L. Smith,et al.  Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions. , 2004, Blood.

[16]  H. Kantarjian,et al.  Deficiency of the ATM protein expression defines an aggressive subgroup of B-cell chronic lymphocytic leukemia. , 1998, Cancer research.

[17]  T. Kipps,et al.  Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation , 2005, Leukemia.

[18]  B. Cheson,et al.  Fludarabine compared with chlorambucil as primary therapy for chronic lymphocytic leukemia. , 2000, The New England journal of medicine.

[19]  A. Pettitt.  Mechanism of action of purine analogues in chronic lymphocytic leukaemia , 2003, British journal of haematology.

[20]  A. Pettitt,et al.  Purine analogues kill resting lymphocytes by p53‐dependent and ‐independent mechanisms , 1999, British journal of haematology.

[21]  T. Stankovic,et al.  Inactivation of ataxia telangiectasia mutated gene in B-cell chronic lymphocytic leukaemia , 1999, The Lancet.

[22]  A Benner,et al.  p53 gene deletion predicts for poor survival and non-response to therapy with purine analogs in chronic B-cell leukemias. , 1995, Blood.

[23]  F. Kashanchi,et al.  Phosphorylation of p53 Serine 15 Increases Interaction with CBP* , 1998, The Journal of Biological Chemistry.

[24]  J. Byrd,et al.  Addition of rituximab to fludarabine may prolong progression-free survival and overall survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis of CALGB 9712 and CALGB 9011. , 2005, Blood.

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

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

[27]  A. López-Guillermo,et al.  Spontaneous and drug-induced apoptosis is mediated by conformational changes of Bax and Bak in B-cell chronic lymphocytic leukemia. , 2002, Blood.

[28]  John Calvin Reed,et al.  Drug-induced apoptosis in B-cell chronic lymphocytic leukemia: relationship between p53 gene mutation and bcl-2/bax proteins in drug resistance. , 1996, Oncogene.

[29]  T. Stankovic,et al.  Mutations in the ATM gene lead to impaired overall and treatment-free survival that is independent of IGVH mutation status in patients with B-CLL. , 2005, Blood.

[30]  T. Manshouri,et al.  Expression profile of MDM‐2 proteins in chronic lymphocytic leukemia and their clinical relevance , 1997, American journal of hematology.

[31]  D. Green,et al.  Transcription, apoptosis and p53: catch-22. , 2005, Trends in genetics : TIG.

[32]  Jean-Claude Martinou,et al.  Bid-induced Conformational Change of Bax Is Responsible for Mitochondrial Cytochrome c Release during Apoptosis , 1999, The Journal of cell biology.

[33]  S. Kato,et al.  Lack of correlation between p53-dependent transcriptional activity and the ability to induce apoptosis among 179 mutant p53s. , 2005, Cancer research.

[34]  D. Rose,et al.  Deoxyadenosine analogs induce programmed cell death in chronic lymphocytic leukemia cells by damaging the DNA and by directly affecting the mitochondria. , 2000, Blood.

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

[36]  K. Lee,et al.  P53, MDM-2, BAX and BCL-2 and drug resistance in chronic lymphocytic leukemia. , 1997, Leukemia & lymphoma.

[37]  Ash A. Alizadeh,et al.  Fludarabine treatment of patients with chronic lymphocytic leukemia induces a p53-dependent gene expression response. , 2004, Blood.

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

[39]  E. Koníková,et al.  Altered expression of p53 and MDM2 proteins in hematological malignancies. , 2003, Neoplasma.

[40]  A. Pettitt,et al.  p53 dysfunction in B-cell chronic lymphocytic leukemia: inactivation of ATM as an alternative to TP53 mutation. , 2001, Blood.

[41]  G. Packham,et al.  Chronic lymphocytic leukemia : revelations from the B-cell receptor , 2004 .

[42]  A. Gunasekera,et al.  Defining the p53 DNA‐binding domain/Bcl‐xL‐binding interface using NMR , 2004, FEBS letters.

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

[44]  F. Mancini,et al.  p53 expression in B-cell chronic lymphocytic leukemia: a marker of disease progression and poor prognosis. , 1998, Blood.

[45]  A. Pettitt,et al.  Relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation in chronic lymphocytic leukemia. , 2002, Blood.

[46]  M. Story,et al.  Induction of apoptotic cell death in chronic lymphocytic leukemia by 2-chloro-2′-deoxyadenosine and 9-β-D-arabinosyl-2-fluoroadenine , 1993 .

[47]  Hong Yang,et al.  Phosphorylation of p53 on Key Serines Is Dispensable for Transcriptional Activation and Apoptosis*♦ , 2004, Journal of Biological Chemistry.

[48]  W. Hiddemann,et al.  Multicentre prospective randomised trial of fludarabine versus cyclophosphamide, doxorubicin, and prednisone (CAP) for treatment of advanced-stage chronic lymphocytic leukaemia , 1996, The Lancet.

[49]  I. Pastan,et al.  Induction of caspase-dependent programmed cell death in B-cell chronic lymphocytic leukemia by anti-CD22 immunotoxins. , 2004, Blood.

[50]  E. Stavridi,et al.  ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins , 1998, Nature Genetics.

[51]  C. Pepper,et al.  Bcl-2 and bax expression and chlorambucil-induced apoptosis in the T-cells and leukaemic B-cells of untreated B-cell chronic lymphocytic leukaemia patients. , 2000, Leukemia research.

[52]  W. Deppert,et al.  Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress , 2006, Oncogene.