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

Background: Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, therebycompromising p53 activity. Therefore, lymphoma is a suitable model for studying thetherapeutic value of disrupting the HDM2-p53 interaction by small-molecule inhibitors(SMIs). HDM2 have been developed and are under various stages of preclinical and clinicalinvestigation. Previously, we examined the anti-lymphoma activity of MI-319, the laboratorygrade 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 thepreclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines aswell as a cohort of patient-derived B-lymphcytes for its potential clinical use. Results: Preclinical assessment of MI-219 was evaluated by means of an in vitro and ex vivo approachand compared to Nutlin-3, the gold standard. Characterization of p53 activity and stabilitywere assessed by quantitative PCR, Western blot, and immunoprecipitation. Biologicaloutcome was measured using Trypan blue exclusion assay, Annexin V/PI, PARP andcaspase-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 ofapoptotic cell death. Using a cell free autoubiquitination assay, neither agent interfered withHDM2 E3 ligase function. MI-219 was more effective in upregulating wt-p53 stabilizationcompared to Nutlin-3. MI-219, but not Nutlin-3, enhanced the autoubiquitination anddegradation of HDM2. Conclusions: Our data reveals unexpected differences between MI-219 and the well-studied Nutlin-3 inlymphoma cell lines and patient samples. We suggest a novel mechanism for MI-219 thatalters the functional activity of HDM2 through enhanced autoubiquitination and degradation.Additionally, this mechanism appears to correspond to biological outcome. Our resultsprovide evidence that different classes of HDM2 SMIs elicit molecular events that extendbeyond HDM2-p53 dissociation which may be of biological and potentially therapeuticimportance.

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