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HDM2 antagonist MI-219 (spiro-oxindole), but not Nutlin-3 (cis-imidazoline), regulates p53 through enhanced HDM2 autoubiquitination and degradation in human malignant B-cell lymphomas.

Sosin AM, Burger AM, Siddiqi A, Abrams J, Mohammad RM, Al-Katib AM - J Hematol Oncol (2012)

Bottom Line: MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3.Additionally, this mechanism appears to correspond to biological outcome.Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Barbara Ann Karmanos Cancer Institute (KCI), Detroit, MI 48201, USA.

ABSTRACT

Background: Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, thereby compromising p53 activity. Therefore, lymphoma is a suitable model for studying the therapeutic value of disrupting the HDM2-p53 interaction by small-molecule inhibitors (SMIs). HDM2 have been developed and are under various stages of preclinical and clinical investigation. Previously, we examined the anti-lymphoma activity of MI-319, the laboratory grade of a new class of HDM2 SMI, the spiro-oxindole, in follicular lymphoma. Since then, MI-219, the clinical grade has become readily available. This study further examines the preclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines as well as a cohort of patient-derived B-lymphocytes for its potential clinical use.

Results: Preclinical assessment of MI-219 was evaluated by means of an in vitro and ex vivo approach and compared to Nutlin-3, the gold standard. Characterization of p53 activity and stability were assessed by quantitative PCR, Western blot, and immunoprecipitation. Biological outcome was measured using Trypan blue exclusion assay, Annexin V/PI, PARP and caspase-3 cleavage. Surprisingly, the overall biological effects of Nutlin-3 were more delayed (48 h) while MI-219 triggered an earlier response (12-24 h), predominantly in the form of apoptotic cell death. Using a cell free autoubiquitination assay, neither agent interfered with HDM2 E3 ligase function. MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3. MI-219, but not Nutlin-3, enhanced the autoubiquitination and degradation of HDM2.

Conclusions: Our data reveals unexpected differences between MI-219 and the well-studied Nutlin-3 in lymphoma cell lines and patient samples. We suggest a novel mechanism for MI-219 that alters the functional activity of HDM2 through enhanced autoubiquitination and degradation. Additionally, this mechanism appears to correspond to biological outcome. Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.

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Biological response of lymphoma cell lines to HDM2 SMIs. Nutlin-3 (Red bars) and MI-219 (Blue bars) represent cell viability for 4 cell lines exposed to increasing concentrations of HDM2 SMIs for up to 72 h. Comparison for overall differences across equivalent concentrations were significant between the two HDM2 SMIs (ANOVA; p = 0.001) for each time period (24 to 72 h) in both wt-p53 cell lines. Numerical values located under the graphs represent percentage of Annexin V positive cells for Nutlin-3 (red print) and MI-219 (blue print). MI-219- induced increases in Annexin V positive WSU-FSCCL cells at 24 and 48 h was significantly greater than that for Nutlin-3 (p = 0.0001) within the same cell line. For KM-H2 cells, the difference was significant only at 24 h (p = 0.026). Columns and error bars represent Mean ± S.D. of three independent experiments.
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Figure 3: Biological response of lymphoma cell lines to HDM2 SMIs. Nutlin-3 (Red bars) and MI-219 (Blue bars) represent cell viability for 4 cell lines exposed to increasing concentrations of HDM2 SMIs for up to 72 h. Comparison for overall differences across equivalent concentrations were significant between the two HDM2 SMIs (ANOVA; p = 0.001) for each time period (24 to 72 h) in both wt-p53 cell lines. Numerical values located under the graphs represent percentage of Annexin V positive cells for Nutlin-3 (red print) and MI-219 (blue print). MI-219- induced increases in Annexin V positive WSU-FSCCL cells at 24 and 48 h was significantly greater than that for Nutlin-3 (p = 0.0001) within the same cell line. For KM-H2 cells, the difference was significant only at 24 h (p = 0.026). Columns and error bars represent Mean ± S.D. of three independent experiments.

Mentions: A series of experiments were performed using both HDM2 SMIs in two wt-p53 and two mt-p53 lymphoma cell lines. As expected, cell death of mt-p53 cell lines RL and WSU-DLCL2 were not significantly affected by exposure to either HDM2 SMI at concentration up to 10 μM (Figure 3). In wt-p53 WSU-FSCCL and KM-H2 cells, the overall effect of MI-219-induced cell death was significantly greater than that of Nutlin-3. This was seen as early as 24 h following initiation of treatment (p = 0.0001). The apoptotic effect was more evident in the non-Hodgkin’s lymphoma WSU-FSCCL cell line than in the Hodgkin lymphoma KM-H2 cell line. MI-219 treatment in WSU-FSCCL cells led to complete elimination of cells at the end of 72 h at 5 μM and 10 μM concentrations unlike that seen with Nutlin-3 treatment at equivalent concentrations. An increase in the percentage of Annexin V positively stained cells over time reflected the decrease in viability for cell lines expressing wt-p53 (Figure 3; shown as Mean only because of space restriction). A summary of the Annexin V positive data for both wt-p53 cell lines is presented in Table 2.


HDM2 antagonist MI-219 (spiro-oxindole), but not Nutlin-3 (cis-imidazoline), regulates p53 through enhanced HDM2 autoubiquitination and degradation in human malignant B-cell lymphomas.

Sosin AM, Burger AM, Siddiqi A, Abrams J, Mohammad RM, Al-Katib AM - J Hematol Oncol (2012)

Biological response of lymphoma cell lines to HDM2 SMIs. Nutlin-3 (Red bars) and MI-219 (Blue bars) represent cell viability for 4 cell lines exposed to increasing concentrations of HDM2 SMIs for up to 72 h. Comparison for overall differences across equivalent concentrations were significant between the two HDM2 SMIs (ANOVA; p = 0.001) for each time period (24 to 72 h) in both wt-p53 cell lines. Numerical values located under the graphs represent percentage of Annexin V positive cells for Nutlin-3 (red print) and MI-219 (blue print). MI-219- induced increases in Annexin V positive WSU-FSCCL cells at 24 and 48 h was significantly greater than that for Nutlin-3 (p = 0.0001) within the same cell line. For KM-H2 cells, the difference was significant only at 24 h (p = 0.026). Columns and error bars represent Mean ± S.D. of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473265&req=5

Figure 3: Biological response of lymphoma cell lines to HDM2 SMIs. Nutlin-3 (Red bars) and MI-219 (Blue bars) represent cell viability for 4 cell lines exposed to increasing concentrations of HDM2 SMIs for up to 72 h. Comparison for overall differences across equivalent concentrations were significant between the two HDM2 SMIs (ANOVA; p = 0.001) for each time period (24 to 72 h) in both wt-p53 cell lines. Numerical values located under the graphs represent percentage of Annexin V positive cells for Nutlin-3 (red print) and MI-219 (blue print). MI-219- induced increases in Annexin V positive WSU-FSCCL cells at 24 and 48 h was significantly greater than that for Nutlin-3 (p = 0.0001) within the same cell line. For KM-H2 cells, the difference was significant only at 24 h (p = 0.026). Columns and error bars represent Mean ± S.D. of three independent experiments.
Mentions: A series of experiments were performed using both HDM2 SMIs in two wt-p53 and two mt-p53 lymphoma cell lines. As expected, cell death of mt-p53 cell lines RL and WSU-DLCL2 were not significantly affected by exposure to either HDM2 SMI at concentration up to 10 μM (Figure 3). In wt-p53 WSU-FSCCL and KM-H2 cells, the overall effect of MI-219-induced cell death was significantly greater than that of Nutlin-3. This was seen as early as 24 h following initiation of treatment (p = 0.0001). The apoptotic effect was more evident in the non-Hodgkin’s lymphoma WSU-FSCCL cell line than in the Hodgkin lymphoma KM-H2 cell line. MI-219 treatment in WSU-FSCCL cells led to complete elimination of cells at the end of 72 h at 5 μM and 10 μM concentrations unlike that seen with Nutlin-3 treatment at equivalent concentrations. An increase in the percentage of Annexin V positively stained cells over time reflected the decrease in viability for cell lines expressing wt-p53 (Figure 3; shown as Mean only because of space restriction). A summary of the Annexin V positive data for both wt-p53 cell lines is presented in Table 2.

Bottom Line: MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3.Additionally, this mechanism appears to correspond to biological outcome.Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Barbara Ann Karmanos Cancer Institute (KCI), Detroit, MI 48201, USA.

ABSTRACT

Background: Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, thereby compromising p53 activity. Therefore, lymphoma is a suitable model for studying the therapeutic value of disrupting the HDM2-p53 interaction by small-molecule inhibitors (SMIs). HDM2 have been developed and are under various stages of preclinical and clinical investigation. Previously, we examined the anti-lymphoma activity of MI-319, the laboratory grade of a new class of HDM2 SMI, the spiro-oxindole, in follicular lymphoma. Since then, MI-219, the clinical grade has become readily available. This study further examines the preclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines as well as a cohort of patient-derived B-lymphocytes for its potential clinical use.

Results: Preclinical assessment of MI-219 was evaluated by means of an in vitro and ex vivo approach and compared to Nutlin-3, the gold standard. Characterization of p53 activity and stability were assessed by quantitative PCR, Western blot, and immunoprecipitation. Biological outcome was measured using Trypan blue exclusion assay, Annexin V/PI, PARP and caspase-3 cleavage. Surprisingly, the overall biological effects of Nutlin-3 were more delayed (48 h) while MI-219 triggered an earlier response (12-24 h), predominantly in the form of apoptotic cell death. Using a cell free autoubiquitination assay, neither agent interfered with HDM2 E3 ligase function. MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3. MI-219, but not Nutlin-3, enhanced the autoubiquitination and degradation of HDM2.

Conclusions: Our data reveals unexpected differences between MI-219 and the well-studied Nutlin-3 in lymphoma cell lines and patient samples. We suggest a novel mechanism for MI-219 that alters the functional activity of HDM2 through enhanced autoubiquitination and degradation. Additionally, this mechanism appears to correspond to biological outcome. Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.

Show MeSH
Related in: MedlinePlus