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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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MI-219, but not Nutlin-3, enhances HDM2-mediated autoubiquitination and degradation. A) Immunoblot probed for ubiquitin represent autoubiquitination of recombinant His-HDM2 in the presence of Nutlin-3, MI-219 or MI-319 (laboratory grade MI-219) at IC50 or 50 μM for 1.5 h in a cell free autoubiquitination assay. Lane 1: no E2 enzyme; Lane 2: no ubiquitin; Lane 3: Control, no SMI. HDM2 SMIs were assessed at IC50 or 50 μM concentrations and were as follows; Lane 4–5, Nutlin-3; Lanes 6–7, MI-219; Lanes 8–9, MI-319; Lane 10: Disulfiram (10 μM). B) Western blot for HDM2 immunoprecipitated from WSU-FSCCL cells exposed to HDM2 SMIs for 12 h was then probed for with HDM2 to expose full length, ubiquitinated and degraded HDM2 species as described in Methods. C) Immunoprecipitated HDM2 from WSU-FSCCL cells exposed to HDM2 SMI for 16 h was immunoblotted with monoclonal anti-ubiquitin antibody to reveal ubiquitin associated-HDM2. Data are representative of 3 independent experiments.
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Figure 8: MI-219, but not Nutlin-3, enhances HDM2-mediated autoubiquitination and degradation. A) Immunoblot probed for ubiquitin represent autoubiquitination of recombinant His-HDM2 in the presence of Nutlin-3, MI-219 or MI-319 (laboratory grade MI-219) at IC50 or 50 μM for 1.5 h in a cell free autoubiquitination assay. Lane 1: no E2 enzyme; Lane 2: no ubiquitin; Lane 3: Control, no SMI. HDM2 SMIs were assessed at IC50 or 50 μM concentrations and were as follows; Lane 4–5, Nutlin-3; Lanes 6–7, MI-219; Lanes 8–9, MI-319; Lane 10: Disulfiram (10 μM). B) Western blot for HDM2 immunoprecipitated from WSU-FSCCL cells exposed to HDM2 SMIs for 12 h was then probed for with HDM2 to expose full length, ubiquitinated and degraded HDM2 species as described in Methods. C) Immunoprecipitated HDM2 from WSU-FSCCL cells exposed to HDM2 SMI for 16 h was immunoblotted with monoclonal anti-ubiquitin antibody to reveal ubiquitin associated-HDM2. Data are representative of 3 independent experiments.

Mentions: Neither class of agents inhibited the E3 ligase function of HDM2 in a cell-free ubiquitination assay (Figure 8A). Autoubiquitination of recombinant His-HDM2 was not inhibited by the addition of Nutlin-3, MI-219 or MI-319 (laboratory grade MI-219) at IC50 or even at much higher (50 μM) concentrations for 1.5 h. Disulfiram (10 μM) completely abrogated autoubiquitination and was included as a negative control.


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)

MI-219, but not Nutlin-3, enhances HDM2-mediated autoubiquitination and degradation. A) Immunoblot probed for ubiquitin represent autoubiquitination of recombinant His-HDM2 in the presence of Nutlin-3, MI-219 or MI-319 (laboratory grade MI-219) at IC50 or 50 μM for 1.5 h in a cell free autoubiquitination assay. Lane 1: no E2 enzyme; Lane 2: no ubiquitin; Lane 3: Control, no SMI. HDM2 SMIs were assessed at IC50 or 50 μM concentrations and were as follows; Lane 4–5, Nutlin-3; Lanes 6–7, MI-219; Lanes 8–9, MI-319; Lane 10: Disulfiram (10 μM). B) Western blot for HDM2 immunoprecipitated from WSU-FSCCL cells exposed to HDM2 SMIs for 12 h was then probed for with HDM2 to expose full length, ubiquitinated and degraded HDM2 species as described in Methods. C) Immunoprecipitated HDM2 from WSU-FSCCL cells exposed to HDM2 SMI for 16 h was immunoblotted with monoclonal anti-ubiquitin antibody to reveal ubiquitin associated-HDM2. Data are representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: MI-219, but not Nutlin-3, enhances HDM2-mediated autoubiquitination and degradation. A) Immunoblot probed for ubiquitin represent autoubiquitination of recombinant His-HDM2 in the presence of Nutlin-3, MI-219 or MI-319 (laboratory grade MI-219) at IC50 or 50 μM for 1.5 h in a cell free autoubiquitination assay. Lane 1: no E2 enzyme; Lane 2: no ubiquitin; Lane 3: Control, no SMI. HDM2 SMIs were assessed at IC50 or 50 μM concentrations and were as follows; Lane 4–5, Nutlin-3; Lanes 6–7, MI-219; Lanes 8–9, MI-319; Lane 10: Disulfiram (10 μM). B) Western blot for HDM2 immunoprecipitated from WSU-FSCCL cells exposed to HDM2 SMIs for 12 h was then probed for with HDM2 to expose full length, ubiquitinated and degraded HDM2 species as described in Methods. C) Immunoprecipitated HDM2 from WSU-FSCCL cells exposed to HDM2 SMI for 16 h was immunoblotted with monoclonal anti-ubiquitin antibody to reveal ubiquitin associated-HDM2. Data are representative of 3 independent experiments.
Mentions: Neither class of agents inhibited the E3 ligase function of HDM2 in a cell-free ubiquitination assay (Figure 8A). Autoubiquitination of recombinant His-HDM2 was not inhibited by the addition of Nutlin-3, MI-219 or MI-319 (laboratory grade MI-219) at IC50 or even at much higher (50 μM) concentrations for 1.5 h. Disulfiram (10 μM) completely abrogated autoubiquitination and was included as a negative control.

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