Activation of the p53 pathway by the MDM2 inhibitor nutlin-3a overcomes BCL2 overexpression in a preclinical model of diffuse large B-cell lymphoma associated with t(14;18)(q32;q21).
Cell cycle arrest was associated with upregulation of p21.Cell death was reduced when p53-dependent transactivation activity was inhibited by pifithrin-α (PFT-α), or PFT-μ inhibited direct p53 targeting of mitochondria.Nutlin-3a treatment in a xenograft animal lymphoma model inhibited growth of t(14;18)-positive DLBCL tumors, associated with increased apoptosis and decreased proliferation.
Affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
p53 is frequently wild type (wt) in diffuse large B-cell lymphoma (DLBCL) associated with t(14;18)(q32;q21) that overexpresses BCL2. Nutlin-3a is a small molecule that activates the p53 pathway by disrupting p53-MDM2 interaction. We show that nutlin-3a activates p53 in DLBCL cells associated with t(14;18)(q32;q21), BCL2 overexpression and wt p53, resulting in cell cycle arrest and apoptosis. Nutlin-3a treatment had similar effects on DLBCL cells of activated B-cell phenotype with wt p53. Cell cycle arrest was associated with upregulation of p21. Nutlin-3a-induced apoptosis was accompanied by BAX and PUMA upregulation, BCL-XL downregulation, serine-70 dephosphorylation of BCL2, direct binding of BCL2 by p53, caspase-9 upregulation and caspase-3 cleavage. Cell death was reduced when p53-dependent transactivation activity was inhibited by pifithrin-α (PFT-α), or PFT-μ inhibited direct p53 targeting of mitochondria. Nutlin-3a sensitized activation of the intrinsic apoptotic pathway by BCL2 inhibitors in t(14;18)-positive DLBCL cells with wt p53, and enhanced doxorubicin cytotoxicity against t(14;18)-positive DLBCL cells with wt or mutant p53, the latter in part via p73 upregulation. Nutlin-3a treatment in a xenograft animal lymphoma model inhibited growth of t(14;18)-positive DLBCL tumors, associated with increased apoptosis and decreased proliferation. These data suggest that disruption of the p53-MDM2 interaction by nutlin-3a offers a novel therapeutic approach for DLBCL associated with t(14;18)(q32;q21).
- Chromosomes, Human, Pair 14/genetics*
- Chromosomes, Human, Pair 18/genetics*
- Lymphoma, Large B-Cell, Diffuse/drug therapy/genetics/metabolism*
- Proto-Oncogene Proteins c-bcl-2/genetics/metabolism*
- Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors*/genetics/metabolism
- Translocation, Genetic/genetics*
- Tumor Suppressor Protein p53/genetics/metabolism*
- Antineoplastic Agents/pharmacology
- Blotting, Western
- Cell Cycle
- Cell Proliferation
- Disease Models, Animal
- Fluorescent Antibody Technique
- Mice, SCID
- RNA, Messenger/genetics
- Reverse Transcriptase Polymerase Chain Reaction
- Signal Transduction
- Tumor Cells, Cultured
- Xenograft Model Antitumor Assays
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fig3: Nutlin-3a induces apoptotic cell death of t(14;18)-positive diffuse large B-cell lymphoma (DLBCL) and ABC-type DLBCL cells through activation of the p53 pathway. (a) At 48 h after incubation with 10 μ nutlin-3a, a considerable increase in annexin V binding was observed in DLBCL cells with wild-type (wt) p53, indicating apoptotic cell death. No significant change was observed in Pfeiffer, BJAB and MS cells, which have mutant p53 (left panel). Annexin V binding corresponding to various concentrations of nutlin-3a treatment is depicted in the diagrams of the right panel (b). Microscopic examination of 4',6-diamidino-2-phenylindole-stained preparations of DoHH2 and MCA cell at 48 h after treatment with 5 μ nutlin-3a showed morphologic evidence of apoptosis, including nuclear condensation and fragmentation, whereas no such changes were observed in nutlin-3a-treated Pfeiffer and MS cells. (c) Western blot analysis of DLBCL cells with wt p53 after treatment with nutlin-3a showed increased levels of the proapoptotic proteins BAX and PUMA in all cell lines with wt p53, known transcriptional targets of p53. Also, substantially decreased levels of BCL-XL levels were observed after nutlin-3a treatment in DoHH2, OCI-LY10 and MCA cells, without much change in OCI-LY3 and EJ cells. Whereas the levels of total BCL2 remained constant, the p-Ser70BCL2 levels decreased dramatically after nutlin-3a treatment in all the cells with wt p53. In addition, western blot analysis demonstrated cleavage of caspase-3, accompanied by activation of caspase-9. By contrast, no significant changes in the levels of proapoptotic or antiapoptotic proteins, or in activation of caspase-3 or -9, were observed in nutlin-3a-treated Pfeiffer and MS cells. Lysates were prepared at 24 h following nutlin-3a treatment. (d) Coimmunoprecepitation of DoHH2 and MCA cell lysates treated with 10 μ of nutlin-3a or control (dimethyl sulfoxide) showed that nutlin-3a induced binding of BCL2 by p53 protein, suggesting that non-transcriptional mechanisms are also involved in nutlin-3a-induced cell death of t(14;18)-positive DLBCL cells. (e) Preincubation of DoHH2 and MCA cells with 25 μ pifithrin-α (PFT-α) or 4.8 μ PFT-μ (the maximum non-toxic dose) rescued a substantial number of nutlin-3a-treated cells from apoptotic cell death. PFT-μ, an agent that inhibits the interaction of p53 with antiapoptotic proteins without affecting p53 transactivation function, rescued a greater proportion of nutlin-3a-treated DoHH2 cells and a smaller proportion of MCA cells, whereas PFT-α rescued a larger proportion of nutlin-3a-treated-MCA cells and a smaller proportion of DoHH2 cells. (f) Treatment with a comparatively small dose of nutlin-3a (1 μ) enhanced dramatically the cytotoxicity of YC-137, a BH3 mimetic in DoHH2 and MCA cells, with no effect observed in Pfeiffer and MS cells. Combined treatment with nutlin-3a and YC-137 synergistically induced decreased viability of DoHH2 and MCA cells (average combination index (CI)=0.53 and CI=0.35, respectively).
To investigate the nature of nutlin-3a-induced suppression of cell viability, annexin V staining and flow cytometry were performed. As illustrated in Figure 3a, there was a concentration-dependent increase in annexin V binding in DoHH2, MCA, EJ, OCI-LY3 and OCI-LY10 cells, but not in Pfeiffer, MS and BJAB cells. At 24 h following treatment with 10 μ of nutlin-3a, annexin V binding was increased by ∼80, 74 and 58% in DoHH2, MCA and EJ cells, respectively. In OCI-LY3 and OCI-LY10 cells, annexin V binding was increased by 63 and 74%, respectively. No significant increase in annexin V binding was observed in nutlin-3a-treated Pfeiffer, BJAB and MS cells that harbor mt p53 (Figure 3a). Also, 4',6-diamidino-2-phenylindole staining and fluorescence microscopy demonstrated nuclear condensation and fragmentation, morphologic evidence of apoptotic cell death, in nutlin-3a-treated wt p53 cells (Figure 3b).