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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 in normal B-lymphocytes derived from healthy donors. A) Isolated B-lymphocytes from normal donors were exposed to HDM2 SMIs for up to 48 h. Neither HDM2 SMI induced a significant decrease in cell viability compared to untreated cells (control). B) The inactive Nutlin-3 analog, Nutlin-3b, was ineffective in significantly decreasing cell viability in all 4 cell lines for up to 72 h. Columns and error bars represent Mean ± S.D. of three independent experiments.
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Figure 4: Effect of HDM2 inhibition in normal B-lymphocytes derived from healthy donors. A) Isolated B-lymphocytes from normal donors were exposed to HDM2 SMIs for up to 48 h. Neither HDM2 SMI induced a significant decrease in cell viability compared to untreated cells (control). B) The inactive Nutlin-3 analog, Nutlin-3b, was ineffective in significantly decreasing cell viability in all 4 cell lines for up to 72 h. Columns and error bars represent Mean ± S.D. of three independent experiments.

Mentions: Data shown in Figure 4A, indicate that neither HDM2 SMI significantly affected the viability of B-lymphocytes derived from normal donors exposed for up to 48 h. The inactive Nutlin-3 enantiomer, Nutlin-3b, did not show any significant reduction in the cell viability of cell lines, demonstrating the selectivity of each HDM2 SMI (Figure 4B).


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 in normal B-lymphocytes derived from healthy donors. A) Isolated B-lymphocytes from normal donors were exposed to HDM2 SMIs for up to 48 h. Neither HDM2 SMI induced a significant decrease in cell viability compared to untreated cells (control). B) The inactive Nutlin-3 analog, Nutlin-3b, was ineffective in significantly decreasing cell viability in all 4 cell lines for up to 72 h. 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 4: Effect of HDM2 inhibition in normal B-lymphocytes derived from healthy donors. A) Isolated B-lymphocytes from normal donors were exposed to HDM2 SMIs for up to 48 h. Neither HDM2 SMI induced a significant decrease in cell viability compared to untreated cells (control). B) The inactive Nutlin-3 analog, Nutlin-3b, was ineffective in significantly decreasing cell viability in all 4 cell lines for up to 72 h. Columns and error bars represent Mean ± S.D. of three independent experiments.
Mentions: Data shown in Figure 4A, indicate that neither HDM2 SMI significantly affected the viability of B-lymphocytes derived from normal donors exposed for up to 48 h. The inactive Nutlin-3 enantiomer, Nutlin-3b, did not show any significant reduction in the cell viability of cell lines, demonstrating the selectivity of each HDM2 SMI (Figure 4B).

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