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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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Upregulation of p53 protein predicts efficacy and biological response to MI-219 in primary lymphoma cells. A) Western blots of the upregulation of p53 and its target proteins upon HDM2 inhibition after 24 h in primary lymphoma cells isolated from four patients. B) Box plots show the cumulative biostatistical analyses of p53 target protein expression levels to estimate response predictors to HDM2 SMIs in primary B-lymphocytes isolated from lymphoma patients (n = 10). Western blots for each patient (n = 10) for each drug, dose, and time point for each protein detected. Quantification of Western blot bands (relative density) was calculated using ImageJ and normalized to internal control (GAPDH). Fold increase or decrease was calculated by standardizing each treatment as a ratio to the control. MI-219 is statistically more effective than Nutlin-3 (p = 0.001) overall regardless of p53 target protein or time point when main effects for drug, concentration, protein and time were fitted without interactions. Upregulation of p53 was statistically greater upon exposure to MI-219 than for Nutlin-3 at 24 h for equivalent concentration; 2.5 μM [*p = 0.05]; 5.0 μM [***p = 0.02] and 10 μM [**p = 0.03] shown in Figure 2-Bb).
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Figure 2: Upregulation of p53 protein predicts efficacy and biological response to MI-219 in primary lymphoma cells. A) Western blots of the upregulation of p53 and its target proteins upon HDM2 inhibition after 24 h in primary lymphoma cells isolated from four patients. B) Box plots show the cumulative biostatistical analyses of p53 target protein expression levels to estimate response predictors to HDM2 SMIs in primary B-lymphocytes isolated from lymphoma patients (n = 10). Western blots for each patient (n = 10) for each drug, dose, and time point for each protein detected. Quantification of Western blot bands (relative density) was calculated using ImageJ and normalized to internal control (GAPDH). Fold increase or decrease was calculated by standardizing each treatment as a ratio to the control. MI-219 is statistically more effective than Nutlin-3 (p = 0.001) overall regardless of p53 target protein or time point when main effects for drug, concentration, protein and time were fitted without interactions. Upregulation of p53 was statistically greater upon exposure to MI-219 than for Nutlin-3 at 24 h for equivalent concentration; 2.5 μM [*p = 0.05]; 5.0 μM [***p = 0.02] and 10 μM [**p = 0.03] shown in Figure 2-Bb).

Mentions: HDM2 inhibitors activate the p53 pathway in primary lymphoma cells. Both Nutlin-3 and MI-219 induced variable HDM2, p53 and p21 protein expression in purified patient-derived B-lymphocytes (Figure 2). Of special interest is the observation that MI-219, but not Nutlin-3, induced both higher and lower molecular weight species of HDM2. These molecular changes were best captured at 24 h and Western blots for 3 patients with SLL/CLL and 1 with MZL lymphoma are shown in Figure 2A. A statistical analysis summary for changes in the induction of p53-target proteins following exposure to HDM2 SMIs in patient samples is shown in Figure 2B. Cumulatively, MI-219 was more effective than Nutlin-3 (p = 0.001) in the upregulation of p53, p21, and HDM2 protein levels in primary B-lymphoma cells. At 24 h, expression of p53 protein was significantly induced with MI-219 compared to Nutlin-3 at all concentrations and was the largest contributor to the overall significant difference between the two treatments (Figure 2B).


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)

Upregulation of p53 protein predicts efficacy and biological response to MI-219 in primary lymphoma cells. A) Western blots of the upregulation of p53 and its target proteins upon HDM2 inhibition after 24 h in primary lymphoma cells isolated from four patients. B) Box plots show the cumulative biostatistical analyses of p53 target protein expression levels to estimate response predictors to HDM2 SMIs in primary B-lymphocytes isolated from lymphoma patients (n = 10). Western blots for each patient (n = 10) for each drug, dose, and time point for each protein detected. Quantification of Western blot bands (relative density) was calculated using ImageJ and normalized to internal control (GAPDH). Fold increase or decrease was calculated by standardizing each treatment as a ratio to the control. MI-219 is statistically more effective than Nutlin-3 (p = 0.001) overall regardless of p53 target protein or time point when main effects for drug, concentration, protein and time were fitted without interactions. Upregulation of p53 was statistically greater upon exposure to MI-219 than for Nutlin-3 at 24 h for equivalent concentration; 2.5 μM [*p = 0.05]; 5.0 μM [***p = 0.02] and 10 μM [**p = 0.03] shown in Figure 2-Bb).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Upregulation of p53 protein predicts efficacy and biological response to MI-219 in primary lymphoma cells. A) Western blots of the upregulation of p53 and its target proteins upon HDM2 inhibition after 24 h in primary lymphoma cells isolated from four patients. B) Box plots show the cumulative biostatistical analyses of p53 target protein expression levels to estimate response predictors to HDM2 SMIs in primary B-lymphocytes isolated from lymphoma patients (n = 10). Western blots for each patient (n = 10) for each drug, dose, and time point for each protein detected. Quantification of Western blot bands (relative density) was calculated using ImageJ and normalized to internal control (GAPDH). Fold increase or decrease was calculated by standardizing each treatment as a ratio to the control. MI-219 is statistically more effective than Nutlin-3 (p = 0.001) overall regardless of p53 target protein or time point when main effects for drug, concentration, protein and time were fitted without interactions. Upregulation of p53 was statistically greater upon exposure to MI-219 than for Nutlin-3 at 24 h for equivalent concentration; 2.5 μM [*p = 0.05]; 5.0 μM [***p = 0.02] and 10 μM [**p = 0.03] shown in Figure 2-Bb).
Mentions: HDM2 inhibitors activate the p53 pathway in primary lymphoma cells. Both Nutlin-3 and MI-219 induced variable HDM2, p53 and p21 protein expression in purified patient-derived B-lymphocytes (Figure 2). Of special interest is the observation that MI-219, but not Nutlin-3, induced both higher and lower molecular weight species of HDM2. These molecular changes were best captured at 24 h and Western blots for 3 patients with SLL/CLL and 1 with MZL lymphoma are shown in Figure 2A. A statistical analysis summary for changes in the induction of p53-target proteins following exposure to HDM2 SMIs in patient samples is shown in Figure 2B. Cumulatively, MI-219 was more effective than Nutlin-3 (p = 0.001) in the upregulation of p53, p21, and HDM2 protein levels in primary B-lymphoma cells. At 24 h, expression of p53 protein was significantly induced with MI-219 compared to Nutlin-3 at all concentrations and was the largest contributor to the overall significant difference between the two treatments (Figure 2B).

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