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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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Reduction of cell survival in patient-derived lymphoma cells exposed to HDM2 SMIs. Box plots show percent survival compared to control for isolated primary lymphoma cells following exposure to increasing concentrations of MI-219 and Nutlin-3 at indicated time points. Survival is expressed as a percentage of live cells detected by Trypan blue exclusion relative to the total number of cells from each of the 11 patients ranging from 2 to 6 replicates per patient. The horizontal lines within the boxes represent the median while the upper and lower lines of the box endorse the 25th and 75th interquartile range (IQR). The upper- and lower-most lines extend to cover points within 1.5 times the IQR and circles outside of the lines indicate outliers. A mixed effects analysis of variance was used where the drug, concentration and time were defined as fixed effects; patient and replication were defined as random effects. Holm’s procedure was used to adjust for multiple comparisons. Significant overall differences between MI-219 and Nutlin-3 treatments were observed (p < 0.001).
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Figure 1: Reduction of cell survival in patient-derived lymphoma cells exposed to HDM2 SMIs. Box plots show percent survival compared to control for isolated primary lymphoma cells following exposure to increasing concentrations of MI-219 and Nutlin-3 at indicated time points. Survival is expressed as a percentage of live cells detected by Trypan blue exclusion relative to the total number of cells from each of the 11 patients ranging from 2 to 6 replicates per patient. The horizontal lines within the boxes represent the median while the upper and lower lines of the box endorse the 25th and 75th interquartile range (IQR). The upper- and lower-most lines extend to cover points within 1.5 times the IQR and circles outside of the lines indicate outliers. A mixed effects analysis of variance was used where the drug, concentration and time were defined as fixed effects; patient and replication were defined as random effects. Holm’s procedure was used to adjust for multiple comparisons. Significant overall differences between MI-219 and Nutlin-3 treatments were observed (p < 0.001).

Mentions: Enriched primary B-lymphocytes were analyzed for cell viability. Both MI-219 and Nutlin-3 induced concentration- and time-dependent decreases in cell viability in isolated and purified primary lymphoma cells. A comprehensive biostatistical analysis was performed on n = 11 lymphoma patient samples to determine whether there were significant differences between Nutlin-3 and MI-219 and the extent of their effects on cell viability. The mean cell survival for each combination of drug, concentration, and time is shown in Figure 1 along with standard errors. The results demonstrate that overall, MI-219 is significantly more effective (p < 0.001) at reducing the cell viability of primary lymphoma cells than Nutlin-3.


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)

Reduction of cell survival in patient-derived lymphoma cells exposed to HDM2 SMIs. Box plots show percent survival compared to control for isolated primary lymphoma cells following exposure to increasing concentrations of MI-219 and Nutlin-3 at indicated time points. Survival is expressed as a percentage of live cells detected by Trypan blue exclusion relative to the total number of cells from each of the 11 patients ranging from 2 to 6 replicates per patient. The horizontal lines within the boxes represent the median while the upper and lower lines of the box endorse the 25th and 75th interquartile range (IQR). The upper- and lower-most lines extend to cover points within 1.5 times the IQR and circles outside of the lines indicate outliers. A mixed effects analysis of variance was used where the drug, concentration and time were defined as fixed effects; patient and replication were defined as random effects. Holm’s procedure was used to adjust for multiple comparisons. Significant overall differences between MI-219 and Nutlin-3 treatments were observed (p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Reduction of cell survival in patient-derived lymphoma cells exposed to HDM2 SMIs. Box plots show percent survival compared to control for isolated primary lymphoma cells following exposure to increasing concentrations of MI-219 and Nutlin-3 at indicated time points. Survival is expressed as a percentage of live cells detected by Trypan blue exclusion relative to the total number of cells from each of the 11 patients ranging from 2 to 6 replicates per patient. The horizontal lines within the boxes represent the median while the upper and lower lines of the box endorse the 25th and 75th interquartile range (IQR). The upper- and lower-most lines extend to cover points within 1.5 times the IQR and circles outside of the lines indicate outliers. A mixed effects analysis of variance was used where the drug, concentration and time were defined as fixed effects; patient and replication were defined as random effects. Holm’s procedure was used to adjust for multiple comparisons. Significant overall differences between MI-219 and Nutlin-3 treatments were observed (p < 0.001).
Mentions: Enriched primary B-lymphocytes were analyzed for cell viability. Both MI-219 and Nutlin-3 induced concentration- and time-dependent decreases in cell viability in isolated and purified primary lymphoma cells. A comprehensive biostatistical analysis was performed on n = 11 lymphoma patient samples to determine whether there were significant differences between Nutlin-3 and MI-219 and the extent of their effects on cell viability. The mean cell survival for each combination of drug, concentration, and time is shown in Figure 1 along with standard errors. The results demonstrate that overall, MI-219 is significantly more effective (p < 0.001) at reducing the cell viability of primary lymphoma cells than Nutlin-3.

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