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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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Related in: MedlinePlus

Upregulation of p53 and p53-target proteins upon HDM2 inhibition. Cells were exposed to increasing concentrations of HDM2 SMIs for 24 h. Whole cell lysates were subjected to SDS-PAGE and probed for specific proteins. Western blots show changes in p53 and its target proteins after 24 h for the 4 cell lines studied. A) The two wt-p53 cell lines, WSU-FSCCL (A1) and KM-H2 (A2) are presented in the top panel. B) The two mt-p53 cell lines, WSU-DLCL2 (B1) and RL (B2) are presented in the lower panel. Representative blots are shown.
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Figure 5: Upregulation of p53 and p53-target proteins upon HDM2 inhibition. Cells were exposed to increasing concentrations of HDM2 SMIs for 24 h. Whole cell lysates were subjected to SDS-PAGE and probed for specific proteins. Western blots show changes in p53 and its target proteins after 24 h for the 4 cell lines studied. A) The two wt-p53 cell lines, WSU-FSCCL (A1) and KM-H2 (A2) are presented in the top panel. B) The two mt-p53 cell lines, WSU-DLCL2 (B1) and RL (B2) are presented in the lower panel. Representative blots are shown.

Mentions: HDM2 inhibitors upregulate expression of p53-dependent target proteins in wt-p53 cell lines after 24 h (Figure 5). Similar to that observed in patient’s samples, this time point best captured the differences between treatments and demonstrated the effects of MI-219 and Nutlin-3 in modifying the expression of p53, p21, cleaved PARP, cleaved Caspase-3 and Caspase-9 in wt-p53 cell lines. Once again, differences in response to these HDM2 SMIs were evident between the two wt-p53 cell lines (Figure 5A1 and A2). Of note, there was little change in p53 or p21 protein levels in mt-p53 cells (WSU-DLCL2 and RL) and did not show major evidence of PARP and Caspase-3 cleavage (Figure 5B1 and B2). These results show that HDM2 SMIs cannot effectively restore p53 activity to mt-p53 cell lines.


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 and p53-target proteins upon HDM2 inhibition. Cells were exposed to increasing concentrations of HDM2 SMIs for 24 h. Whole cell lysates were subjected to SDS-PAGE and probed for specific proteins. Western blots show changes in p53 and its target proteins after 24 h for the 4 cell lines studied. A) The two wt-p53 cell lines, WSU-FSCCL (A1) and KM-H2 (A2) are presented in the top panel. B) The two mt-p53 cell lines, WSU-DLCL2 (B1) and RL (B2) are presented in the lower panel. Representative blots are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Upregulation of p53 and p53-target proteins upon HDM2 inhibition. Cells were exposed to increasing concentrations of HDM2 SMIs for 24 h. Whole cell lysates were subjected to SDS-PAGE and probed for specific proteins. Western blots show changes in p53 and its target proteins after 24 h for the 4 cell lines studied. A) The two wt-p53 cell lines, WSU-FSCCL (A1) and KM-H2 (A2) are presented in the top panel. B) The two mt-p53 cell lines, WSU-DLCL2 (B1) and RL (B2) are presented in the lower panel. Representative blots are shown.
Mentions: HDM2 inhibitors upregulate expression of p53-dependent target proteins in wt-p53 cell lines after 24 h (Figure 5). Similar to that observed in patient’s samples, this time point best captured the differences between treatments and demonstrated the effects of MI-219 and Nutlin-3 in modifying the expression of p53, p21, cleaved PARP, cleaved Caspase-3 and Caspase-9 in wt-p53 cell lines. Once again, differences in response to these HDM2 SMIs were evident between the two wt-p53 cell lines (Figure 5A1 and A2). Of note, there was little change in p53 or p21 protein levels in mt-p53 cells (WSU-DLCL2 and RL) and did not show major evidence of PARP and Caspase-3 cleavage (Figure 5B1 and B2). These results show that HDM2 SMIs cannot effectively restore p53 activity to mt-p53 cell lines.

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