Abstract 4516: A new class of MDM2 inhibitors cause growth inhibition and stabilize wt p53 in lymphoma cells but do not interfere with MDM2 E3 ligase activity

Abstract

Abstract The p53 tumor suppressor protein function is impaired in over 50% of all cancers via inactivating mutations. However, the frequency of p53 mutations is much lower in hematological malignancies. Most lymphomas have wild-type (wt) p53, mutant (mt) p53 is found in less than 30% of all cases. The ubiquitin E3 ligase MDM2 plays a central role in cancer development and progression and is typically overexpressed in lymphomas. MDM2 expression destabilizes functional wt p53 by causing its polyubiquitination and subsequent degradation in the proteasome. Small-molecule inhibitors (SMI) that can block the MDM2-p53 protein-protein interaction and restore p53 function are a promising therapeutic strategy for the treatment of cancers retaining wt p53 that is degraded by overexpression of MDM2. By utilizing two wt p53 and two mt p53 lymphoma cell lines whose p53 status was confirmed by DNA sequencing, we studied the growth inhibitory potential of two different classes of MDM2 SMIs: Nutlin-3, a cis-imidazoline, and MI-219, a new spiro-oxindole MDM2 SMI. Both trypan blue exclusion and MTT assays demonstrated IC50s between 2 and 4 µM in wt p53 cells in vitro. Mutant p53 cells displayed IC50s greater than 10 µM. Growth inhibition was time- and dose-dependent. Additional flow cytometric analyses empolying Annexin V and propidium iodide staining illustrated increasing apoptosis with cells decreasing in G2/M and S phase that also showed time- and dose-dependency. The MDM2 SMI MI-219 showed a much more prominent cell cycle arrest compared to Nutlin-3. Western blotting demonstrated a stabilization of wt p53 and proapoptotic proteins such as p21, Bcl-2, and Bax. In order to determine whether MDM2 SMIs affect the E3 ligase RING-finger domain of MDM2, in vitro autoubiquitination assays were performed using recombinant MDM2 with various concentration of drug. MDM2 autoubiquitination was not affected, even at a concentration as high as 50 µM. These results show that MDM2 autoubiquitination and thus, its degradation remains unaffected in the presence of MDM2 inhibitors that interfere with the p53-MDM2 binding site. This feature of MDM2 SMI is attractive since inhibition of MDM2 autoubiquitination would lead to its stabilization and could negate effects on p53. Our data suggest that MI-219 would be a useful treatment for lymphomas expressing wt p53 and that this should be explored in further preclinical and future clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4516.