Inhibition of the Akt/survivin pathway synergizes the antileukemia effect of nutlin-3 in acute lymphoblastic leukemia cells Ningxi Zhu, 1 Lubing Gu, 1 Fengzhi Li, 2 and Muxiang Zhou 1 1 Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia and 2 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York Abstract The phosphatidylinositol 3-kinase (PI3K)/Akt and p53 pathways play antiapoptotic and proapoptotic roles in cell death, respectively. Cancer cell growth and progression are associated with high levels of PI3K/Akt activation by loss of PTEN expression and the inactivation of p53 by MDM2 overexpression. We report that inhibition of PI3K/ Akt, either by the PI3K inhibitor Ly294002 or by expression of PTEN, synergized the ability of the MDM2 antagonist nutlin-3 to induce apoptosis in acute lympho- blasticleukemia(ALL).WeusedasetofALLcelllineswith wild-type p53 and MDM2 overexpression, but different status of PTEN expression/PI3K/Akt activation, to test the ability of nutlin-3 to induce p53 and apoptosis. Nutlin-3 activated p53 in all the ALL cell lines; however, induction of apoptosis was dependent on PTEN status. Nutlin-3 induced potent apoptosis in cells with PTEN expression but not in those without PTEN, suggesting that PTEN/ PI3K/Akt pathway may play a role in this process. Furthermore, nutlin-3 significantly down-regulated survi- vin expression in PTEN-positive cells but not in PTEN- negative cells. When these nutlin-3–resistant cells were either pretransfected with the PTEN gene or simultaneous- ly treated with the PI3K inhibitor Ly294002, survivin was down-regulated and sensitivity to nutlin-3 was increased. Furthermore, direct silencing of survivin by small interfer- ing RNA also increased the proapoptotic effect of nutlin-3 on the PTEN-negative, nutlin-3–resistant ALL cells. Our results suggest that Akt-mediated survivin up-regulation in PTEN-negative ALL cells may counteract the proapop- totic effect of nutlin-3, and indicate that a combination of MDM2 antagonist and PI3K/Akt inhibitor may be a promising approach for treating refractory ALL. [Mol Cancer Ther 2008;7(5):1101–9] Introduction It is well established that the inactivation of p53 plays a critical role in cell transformation and tumor cell growth (1). These p53-inactive cancers progress, developing resis- tance to apoptosis; therefore, cancer patients with inacti- vated p53 are usually resistant to the chemoradiation therapy that kills cancer cells through apoptotic pathways (2). Impairment of p53 function may be caused either by p53 gene mutations (3) or by changes in its primary negative regulator MDM2, which is amplified or overex- pressed in diverse human malignancies, including leuke- mia (4, 5). MDM2, a multifunctional oncoprotein, inactivates p53 through different mechanisms. First, MDM2 can bind the transcription domain of p53 and block its ability to activate gene transcription (6). Second, MDM2 functions as an E3 ligase, initiating the ubiquitination and degradation of p53 (7, 8). In addition, MDM2 can promote nuclear export of p53 and inhibit its acetylation (9). Given the importance of MDM2 in inactivating p53, disruption of the p53-MDM2 interaction seems to be an attractive approach in cancer therapy, at least in cancers having wild-type (wt) p53. Over the years, much effort has been made to disrupt interactions between MDM2 and p53 to facilitate wt p53 function. Strategies included the use of antibodies or inhibitory peptides directed at the MDM2 molecule, and either antisense oligonucleotides or small interfering RNA (siRNA) to inhibit MDM2 expression (10, 11). Recently, a potent and selective MDM2 antagonist called nutlin-3 was discovered (12). This nongenotoxic compound binds to the p53-binding pocket in the MDM2 protein, thus inhibiting the binding of p53 and activating the p53 pathway in cancer cells with wt p53. In fact, treatment with this small-molecule drug selectively induces cell cycle arrest, growth inhibition, and apoptosis in a variety of human cancers with wt p53, including prostate and lung carcinoma (13, 14), neuroblastoma (15), Hodgkin’s lymphoma (16), multiple myeloma (17), acute myelogenous leukemia (18), chronic lymphocytic leukemia (19, 20), and acute lymphoblastic leukemia (ALL; ref. 21). It has been shown that nutlin-3 is cytotoxic to cancer cells due to its ability to increase p53-mediated activity through both induction of proapoptotic factors and repression of antiapoptotic survivin (21). Survivin, a unique member of the inhibitor of apoptosis protein (IAP) family, plays an Received 2/25/08; accepted 3/26/08. Grant support: NIH grant R01 CA123490, Leukemia and Lymphoma Society grants 6249-05 and 6033-08, CURE Childhood Cancer, and Hope Street Kids. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Muxiang Zhou, Division of Hematology/Oncology, Department of Pediatrics, Emory University School of Medicine, 2015 Uppergate Drive, Atlanta, GA 30322. Phone: 404-727-1426; Fax: 404-727-4455. E-mail: mzhou@emory.edu Copyright C 2008 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-08-0179 1101 Mol Cancer Ther 2008;7(5). May 2008 on May 4, 2021. © 2008 American Association for Cancer Research. mct.aacrjournals.org Downloaded from