HDM2 antagonist Nutlin-3 disrupts p73-HDM2 binding and enhances p73 function

Abstract

Nutlin-3, a small molecule inhibitor, activates p53 by disrupting p53-HDM2 association. In this study, we found that Nutlin-3 suppressed cell growth and induced apoptosis in the absence of wild-type p53, suggesting a p53-independent mechanism for Nutlin-3-induced cell death. Like p53, its homolog p73 transactivates proapoptotic genes and induces cell death. Since HDM2, a key negative regulator of p53, also binds to and inhibits p73, we asked whether p73 could mediate Nutlin-3-induced apoptosis. We demonstrate that Nutlin-3 inhibits endogenous binding between the proapoptotic p73 isoform TAp73a and HDM2 in p53-null cells. Dissociation of p73 and HDM2 leads to increased p73 transcriptional activity with upregulation of p73 target genes noxa, puma and p21, as well as enhanced apoptosis. p73 knockdown by siRNA results in rescue of Nutlin-3-treated cells, indicating that Nutlin-3-induced apoptosis is, at least in part, p73 dependent. In addition, Nutlin-3 treatment increases TAp73a protein levels with prolongation of p73 half-life. These results provide the first evidence that Nutlin-3 disrupts endogenous p73-HDM2 interaction and enhances the stability and proapoptotic activities of p73 and thus, provides a rationale for the use of Nutlin-3 in the large number of human tumors in which p53 is inactivated.

E2F-1 transcriptional activity is a critical determinant of Mdm2 antagonist-induced apoptosis in human tumor cell lines

“…Indeed, we observed highly elevated levels of p73 in the highly sensitive cell lines with mutated Rb, whereas depletion of E2F-1 by siRNA transfection decreases the protein levels of p73 ( Figure 5B). Furthermore, as it has been shown that Nutlin-3 stabilizes p73 and induces p73-mediated apoptosis in p53À/À cells by disrupting p73-binding of Mdm2 (Lau et al, 2008), we also observed that Nutlin-3 inhibits the ability of Mdm2 to bind p73 and stabilizes p73 in wild-type p53 cells with mutated Rb (Supplementary Figure 4), resulting in a significant apoptotic response to Nutlin-3 ( Figure 3D). In contrast, depletion of p73 by siRNA transfection markedly suppresses Nutlin-3-induced apoptosis in these cell lines ( Figure 5C), suggesting that p73 is important for E2F-1-mediated apoptosis induced by Nutlin-3 in p53 wild-type cancer cells, particularly, with mutated Rb.…”

The therapeutic potential of p53 reactivation by nutlin-3a in ALK+ anaplastic large cell lymphoma with wild-type or mutated p53

“…47,48 Recent studies have shown that nutlin-3a, at very high concentrations, usually 420-30 mM, can inhibit the growth of cancer cells with deleted p53, or harbouring a nonfunctional, mt p53 gene, although the exact mechanism is incompletely understood. 35,36 It seems, depending on the in vitro system, that activation of other partners of Mdm2, including p73 or E2F1, may be involved in this phenomenon. 35,36 Accordingly, we showed here that nutlin-3a can enhance the cytotoxicity of doxorubicin chemotherapy against ALK þ ALCL cells harbouring nonfunctional mt p53, associated with increased p73 expression levels.…”

“…In addition, various studies have shown that very high doses of nutlin-3a, at the range of 20 mM to 430 mM, can induce cytotoxicity and inhibit the growth of cancer cells harbouring a nonfunctional mt p53 gene, through effects on partner proteins of Mdm2 other than p53, including the p53-related protein p73 and E2F1. [34][35][36] As these targets also can be affected by genotoxic chemotherapeutic agents, we investigated the potential effect of nutlin-3a on the anti-tumour activity of doxorubicin against ALK þ ALCL cells harbouring nonfunctional, mt p53.…”

Activation of p53 by MDM2 antagonists has differential apoptotic effects on Epstein–Barr virus (EBV)-positive and EBV-negative Burkitt's lymphoma cells

“…37 As p53 is rarely mutated in hematologic malignancies, many studies have been carried out on various types of tumor cell, including chronic lymphocytic leukemia B cells (B-CLL), 18,20 myeloma, 14 Kaposi sarcoma herpes virus (KHSV)-induced lymphoma 13,38 and Hodgkin's lymphoma. 19 In some cases, nutlin-3 is also able to induce apoptosis in p53-inactivated cells through a p73-dependent mechanism 39 and, as shown recently in a mantle cell lymphoma cell line carrying mutated p53, to synergize with drugs. 40 All these studies have suggested that nutlin-3 may indeed be considered, alone or in combination with various genotoxic drugs, as a novel therapeutic approach for patients.…”