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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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Effect of HDM2 inhibition on p53-dependent gene expression. WSU-FSCCL cells were exposed to increasing concentration of Nutlin-3 and MI-219 for 12, 24 and 48 h. Baseline gene expression and after treatment were quantified by qRT-PCR relative to GAPDH using the ΔΔCt method and expressed as fold induction of gene expression relative to that in the untreated control. mRNA expression levels for p53 (A), HDM2 (B), p53AIP1 (C) and p21 (D). Coded colors represent different concentrations and are listed for each HDM2 SMI at the base of the page. Error bars plotted represent mean values ± SE performed in triplicate from two independently treated experiments.
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Figure 7: Effect of HDM2 inhibition on p53-dependent gene expression. WSU-FSCCL cells were exposed to increasing concentration of Nutlin-3 and MI-219 for 12, 24 and 48 h. Baseline gene expression and after treatment were quantified by qRT-PCR relative to GAPDH using the ΔΔCt method and expressed as fold induction of gene expression relative to that in the untreated control. mRNA expression levels for p53 (A), HDM2 (B), p53AIP1 (C) and p21 (D). Coded colors represent different concentrations and are listed for each HDM2 SMI at the base of the page. Error bars plotted represent mean values ± SE performed in triplicate from two independently treated experiments.

Mentions: To investigate the effects of HDM2 inhibition on p53 transcriptional regulation, we assessed the effect of SMI-mediated reactivity of p53 to enhance target gene expression levels using qRT-PCR. Additionally, we wanted to determine whether the increase in p53 was the result of newly transcribed p53 mRNA or the accumulation of p53 resulting from the HDM2-p53 disruption. Wt-p53 WSU-FSCCL cells exhibited increases in p53-target genes HDM2, p21, p53AIP1 upon HDM2 inhibition compared to control cells albeit with variable kinetics The results are presented in Figure 7. Of particular importance is that there was virtually no upregulation of p53 mRNA transcripts after treatment with HDM2 SMIs at 24 h, suggesting stabilized p53 protein is the result of HDM2 SMIs and not due to enhanced mRNA transcribed into protein. Interestingly, p53 transcripts mRNA increased 29-fold at 48 h in cells exposed to 10 μM MI-219 compared to a 2.5-fold increase in cells exposed to 10 μM Nutlin-3 for the same time period. Overall, MI-219 treatment demonstrated a surprisingly greater induction of p53-target genes compared to Nutlin-3. There was much higher and more sustained induction of HDM2 mRNA by MI-219 compared with Nutlin-3. Effect on HDM2 transcript peaked at 12 hours but was still remarkable at 24 hour in MI-219-treated cells. Both agents induced upregulation of p21 mRNA in WSU-FSCCL cells with overall higher induction by Nutlin-3 compared with MI-219. However, MI-219 effect was more evident at the earlier time points (12 and 24 hours) and at lower concentrations (2.5 and 5 μM) compared with Nutlin-3. The later induced a delayed induction (48 hours) of p21mRNA. p21 mRNA was increased up to 65-fold at 48 h in cells exposed to 10 μM MI-219 but was increased up to 252-fold at the same time period in cells exposed to 10 μM Nutlin-3. MI-219 was more effective than Nutlin-3 in inducing p53AIP1 mRNA, indicating greater induction of p53-dependent apoptosis genes.


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)

Effect of HDM2 inhibition on p53-dependent gene expression. WSU-FSCCL cells were exposed to increasing concentration of Nutlin-3 and MI-219 for 12, 24 and 48 h. Baseline gene expression and after treatment were quantified by qRT-PCR relative to GAPDH using the ΔΔCt method and expressed as fold induction of gene expression relative to that in the untreated control. mRNA expression levels for p53 (A), HDM2 (B), p53AIP1 (C) and p21 (D). Coded colors represent different concentrations and are listed for each HDM2 SMI at the base of the page. Error bars plotted represent mean values ± SE performed in triplicate from two independently treated experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Effect of HDM2 inhibition on p53-dependent gene expression. WSU-FSCCL cells were exposed to increasing concentration of Nutlin-3 and MI-219 for 12, 24 and 48 h. Baseline gene expression and after treatment were quantified by qRT-PCR relative to GAPDH using the ΔΔCt method and expressed as fold induction of gene expression relative to that in the untreated control. mRNA expression levels for p53 (A), HDM2 (B), p53AIP1 (C) and p21 (D). Coded colors represent different concentrations and are listed for each HDM2 SMI at the base of the page. Error bars plotted represent mean values ± SE performed in triplicate from two independently treated experiments.
Mentions: To investigate the effects of HDM2 inhibition on p53 transcriptional regulation, we assessed the effect of SMI-mediated reactivity of p53 to enhance target gene expression levels using qRT-PCR. Additionally, we wanted to determine whether the increase in p53 was the result of newly transcribed p53 mRNA or the accumulation of p53 resulting from the HDM2-p53 disruption. Wt-p53 WSU-FSCCL cells exhibited increases in p53-target genes HDM2, p21, p53AIP1 upon HDM2 inhibition compared to control cells albeit with variable kinetics The results are presented in Figure 7. Of particular importance is that there was virtually no upregulation of p53 mRNA transcripts after treatment with HDM2 SMIs at 24 h, suggesting stabilized p53 protein is the result of HDM2 SMIs and not due to enhanced mRNA transcribed into protein. Interestingly, p53 transcripts mRNA increased 29-fold at 48 h in cells exposed to 10 μM MI-219 compared to a 2.5-fold increase in cells exposed to 10 μM Nutlin-3 for the same time period. Overall, MI-219 treatment demonstrated a surprisingly greater induction of p53-target genes compared to Nutlin-3. There was much higher and more sustained induction of HDM2 mRNA by MI-219 compared with Nutlin-3. Effect on HDM2 transcript peaked at 12 hours but was still remarkable at 24 hour in MI-219-treated cells. Both agents induced upregulation of p21 mRNA in WSU-FSCCL cells with overall higher induction by Nutlin-3 compared with MI-219. However, MI-219 effect was more evident at the earlier time points (12 and 24 hours) and at lower concentrations (2.5 and 5 μM) compared with Nutlin-3. The later induced a delayed induction (48 hours) of p21mRNA. p21 mRNA was increased up to 65-fold at 48 h in cells exposed to 10 μM MI-219 but was increased up to 252-fold at the same time period in cells exposed to 10 μM Nutlin-3. MI-219 was more effective than Nutlin-3 in inducing p53AIP1 mRNA, indicating greater induction of p53-dependent apoptosis genes.

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