An Organometallic Protein Kinase Inhibitor Pharmacologically Activates p53 and Induces Apoptosis in Human Melanoma Cells Keiran S.M. Smalley, 1 Rooha Contractor, 1 Nikolas K. Haass, 1 Angela N. Kulp, 1 G. Ekin Atilla-Gokcumen, 2 Douglas S. Williams, 2 Howard Bregman, 2 Keith T. Flaherty, 3 Maria S. Soengas, 4 Eric Meggers, 2 and Meenhard Herlyn 1 1 The Wistar Institute; 2 Department of Chemistry and 3 Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania; and 4 Department of Dermatology, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan Abstract Unlike other tumors, melanomas harbor wild-type (WT) p53 but exhibit impaired p53-dependent apoptosis. The mecha- nisms for the impaired p53 activation are poorly understood but may be linked to the high expression of the p53 sup- pressor Mdm2, which is found in >50% of melanoma lesions. Here, we describe an organometallic glycogen synthase kinase 3B (GSK3B) inhibitor (DW1/2) as a potent activator of p53 and inducer of cell death in otherwise highly chemoresistant melanoma cells. Using RNA interference and pharmacologic approaches, we show that p53 is required for the cytotoxic effects of this organometallic inhibitor. The DW1/2 compound was barely able to induce cell death in melanoma cells with p53 mutations, further confirming the requirement for p53- WT in the cytotoxic effects of the GSK3B inhibition. Mecha- nistic analysis of the p53-dependent cell death indicated an apoptotic mechanism involving depolarization of mitochon- drial membrane potential, caspase cleavage, and elevated NOXA expression. The effect of p53 was not simply due to passive up-regulation of protein expression as adenoviral- mediated overexpression of p53 was not able to induce cell death. Treatment of melanoma cells with DW1/2 was instead found to decrease levels of Mdm2 and Mdm4. The importance of Mdm2 down-regulation in DW1/2-induced apoptosis was confirmed by treating the p53-WT cells with the p53/Mdm2 antagonist Nutlin-3. Taken together, our data provide a new strategy for the pharmacologic activation of p53 in melanoma, which may be a viable approach for overcoming apoptotic resistance in melanoma and offer new hope for rational melanoma therapy. [Cancer Res 2007;67(1):209–17] Introduction New approaches to melanoma treatment are urgently needed (1) and the search is on to identify new targets for intervention. One target which has been little explored in melanoma therapy is the so-called ‘‘guardian of the genome,’’ the p53 tumor suppressor protein (2). Typically, p53 becomes activated in response to a range of stimuli, such as growth factor withdrawal, the acquisition of oncogenes, and DNA damage. Increased p53 activity is associated with cell cycle arrest, through increased expression of the cyclin- dependent kinase inhibitor p21 Waf-1 (3) and the induction of apoptosis, via the intrinsic mitochondrial pathway (4). Not surprisingly, the inactivation of the p53-dependent pathway is a key event in tumor initiation and progression. The loss of p53 activity is a major mechanism through which tumors become resistant to apoptosis, escape the control of their local microen- vironment, and become insensitive to many types of therapeutic intervention. The majority of cancers harbor point mutations in p53, most of which are in the central region of the protein responsible for DNA binding (5). Unlike other solid tumors, melanomas typically lack p53 mutations and retain expression of the wild-type (WT) protein, often at high levels (6–9). This is surprising, given the highly malignant nature of melanoma and its great resistance to therapeutic intervention. Evidence suggests that melanoma cells can tolerate high levels of transcriptionally active p53 (9) and have an abnormal p53 response following DNA damage (6). It is therefore likely that p53 is not a functional tumor suppressor in melanoma. One of the major mechanisms of p53 inactivation in melanoma is through the increased expression of Mdm2 (and its human homologue Hdm2), a nuclear protein that binds to, promotes the nuclear export of, and subsequently degrades p53 through its intrinsic E3 ubiquitin ligase activity (10). In addition to its role in proteasomal degradation, Mdm2 also inhibits the transcriptional activity of p53 activity through specific binding at key residues (11). More than 50% of primary invasive and metastatic melanoma samples overexpress Hdm2 at the protein level, and it is likely that this contributes to functional inactivation of p53 in melanoma (12). p53 function can also be negatively regulated by another protein, which is structurally related to Mdm2 called Mdm4 (and its human homologue HdmX; ref. 13). Although there is evidence that some melanoma cell lines overexpress Mdm4 (14), the exact relationship between p53 function and Mdm2/ Mdm4 expression remains to be unraveled (15). p53 can be pharmacologically activated in cancer cell lines through a variety of mechanisms, leading to the induction of apoptosis (16–19). Of these, two groups have identified glycogen synthase kinase (GSK) 3h as a major regulator of p53 localization and expression (20, 21). Although GSK3h has received little attention as a possible therapeutic target from the cancer research community, several very recent studies have suggested that inhibiting GSK3h may induce apoptosis in colorectal carcinoma cells through a p53-dependent pathway (18, 19). Classically, GSK3h was described as a key regulator of glycogen metabolism and is also known to regulate other processes, such as apoptosis in Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Keiran S.M. Smalley, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: 215-898-3951; E-mail: ksmalley@wistar.org. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1538 www.aacrjournals.org 209 Cancer Res 2007; 67: (1). January 1, 2007 Research Article