The p53 tumor suppressor retains its wild-type conformation and transcriptional activity in half of all human tumors, and its activation may offer a therapeutic benefit. However, p53 function could be compromised by defective signaling in the p53 pathway. Using a small-molecule MDM2 antagonist, nutlin-3, to probe downstream p53 signaling we find that the cell-cycle arrest function of the p53 pathway is preserved in multiple tumor-derived cell lines expressing wild-type p53, but many have a reduced ability to undergo p53-dependent apoptosis. Gene array analysis revealed attenuated expression of multiple apoptosis-related genes. Cancer cells with mdm2 gene amplification were most sensitive to nutlin-3 in vitro and in vivo, suggesting that MDM2 overexpression may be the only abnormality in the p53 pathway of these cells. Nutlin-3 also showed good efficacy against tumors with normal MDM2 expression, suggesting that many of the patients with wild-type p53 tumors may benefit from antagonists of the p53-MDM2 interaction.apoptosis ͉ inhibitor ͉ cell cycle ͉ tumor ͉ xenograft

CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G 2͞M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.apoptosis ͉ cancer therapy ͉ cell cycle ͉ cytokinesis ͉ replication origin T he cell cycle is a precisely controlled set of biochemical and morphological events driven by the sequential activation of cyclin-dependent kinases (CDKs) (1). Three CDKs and their activating cyclins (A, B, D, and E) play key roles in mammalian cell cycle regulation (2). It has been established that CDK4͞ cyclin D and CDK2͞cyclin E͞A promote the passage through G 1 and S phases, whereas CDK1͞cyclin B regulates the transition through late G 2 and mitosis (3). However, recent genetic and RNA interference studies in mammalian cells have revealed that CDK2 and CDK4 are not essential for cell cycle progression, thus leaving CDK1 as the only nonredundant cell cycle driver (4-6). Genetic studies in yeast and mammalian cells have established the critical role of CDK1 in mitosis, but the long list of its putative substrate proteins still is growing, and its precise functions in the context of the dividing cell are poorly understood (7,8). This deficiency is due, in part, to the lack of specific molecular tools for reversible modulation of CDK1 activity in vivo. Several potent small-molecule inhibitors have been reported, but their activity and cell cycle profiles are not consistent with specific CDK1 inhibition (9, 10). Here, we identify a selective and reversible inhibitor of the catalytic activity of human CDK1͞cyclin B1 and CDK1͞cyclin A complexes that allows synchronization of proliferating cells in the late G 2 phase and probing of CDK1 function in the cellular context. Results and DiscussionWe screened a diverse library of organic compounds for their ability to inhibit the catalytic activity of human CDK1͞cyclin B1. The hits then were tested for selectivity against CDK2͞cyclin E and CDK4͞cyclin D. A class of thiazolinone analogs emerged as a source of ATP-competitive CDK1 inhibitors that were then further optimized for potency, selectivity, and ability to modulate CDK1 in proliferating cells. One quinolinyl thiazolinone derivative, RO-3306, showed good potency, in vitro selectivity, and a cell cycle profile (G 2 ͞M arrest) consistent with CDK1 inhibition (Fig. 1A). RO-3306 inhibited CDK1͞cyclin B1 activity with K i of 35 nM, nearly 10-fold selectivity relative to CDK2͞ cyclin ...

The BRAF V600E mutation is common in several human cancers, especially melanoma. RG7204 (PLX4032) is a small-molecule inhibitor of BRAF V600E kinase activity that is in phase II and phase III clinical testing. Here, we report a preclinical characterization of the antitumor activity of RG7204 using established in vitro and in vivo models of malignant melanoma. RG7204 potently inhibited proliferation and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and ERK phosphorylation in a panel of tumor cell lines, including melanoma cell lines expressing BRAF V600E or other mutant BRAF proteins altered at codon 600. In contrast, RG7204 lacked activity in cell lines that express wild-type BRAF or non-V600 mutations. In several tumor xenograft models of BRAF V600E -expressing melanoma, we found that RG7204 treatment caused partial or complete tumor regressions and improved animal survival, in a dose-dependent manner. There was no toxicity observed in any dose group in any of the in vivo models tested. Our findings offer evidence of the potent antitumor activity of RG7204 against melanomas harboring the mutant BRAF V600E gene.

The p53 tumor suppressor is a key mediator of the cellular response to stress. Phosphorylation induced by multiple stress-activated kinases has been proposed to be essential for p53 stabilization, interaction with transcriptional co-activators, and activation of p53 target genes. However, genetic studies suggest that stress-activated phosphorylation may not be essential for p53 activation. We therefore investigated the role of p53 phosphorylation on six key serine residues (Ser 6 , Ser 15 , Ser 20 , Ser 37 , Ser 46 , and Ser 392 ) for p53 activation using nutlin-3, a recently developed small molecule MDM2 antagonist. We show here that nutlin does not induce the phosphorylation of p53. Comparison of the activity of unphosphorylated and phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide in HCT116 and RKO cells revealed no difference in their sequence-specific DNA binding and ability to transactivate p53 target genes and to induce p53-dependent apoptosis. We conclude that p53 phosphorylation on six major serine sites is not required for activation of p53 target genes or biological responses in vivo.

Recent studies have shown that activation of cell cycle checkpoints can protect normal proliferating cells from mitotic inhibitors by preventing their entry into mitosis. These studies have used genotoxic agents that act, at least in part, by activation of the p53 pathway. However, genotoxic drugs are known also to have p53-independent activities and could affect the sensitivity of tumor cells to antimitotic agents. Recently, we have developed the first potent and selective small-molecule inhibitors of the p53-MDM2 interaction, the nutlins, which activate the p53 pathway only in cells with wild-type but not mutant p53. Using these compounds, we show that p53 activation leads to G 1 and G 2 phase arrest and can protect cells from mitotic block and apoptosis caused by paclitaxel. Pretreatment of HCT116 and RKO colon cancer cells (wild-type p53) or primary human fibroblasts (1043SK) with nutlins for 24 hours followed by incubation with paclitaxel for additional 48 hours did not increase significantly their mitotic index and protected the cells from the cytotoxicity of paclitaxel. Cancer cells with mutant p53 (MDA-MB-435) responded to the same treatment with mitotic arrest and massive apoptosis. These results have two major implications for cancer therapy. First, p53-activating therapies may have antagonistic effect when combined with mitotic poisons. Second, pretreatment with MDM2 antagonists before chemotherapy of tumors with mutant p53 may offer a partial protection to proliferating normal tissues. (Cancer Res 2005; 65(5): 1918-24)