Activation of the p53 pathway by small-molecule-induced MDM2 and MDMX dimerization.

Activation of p53 tumor suppressor by antagonizing its negative regulator murine double minute (MDM)2 has been considered an attractive strategy for cancer therapy and several classes of p53-MDM2 binding inhibitors have been developed. However, these compounds do not inhibit the p53-MDMX interaction, and their effectiveness can be compromised in tumors overexpressing MDMX. Here, we identify small molecules that potently block p53 binding with both MDM2 and MDMX by inhibitor-driven homo- and/or heterodimerization of MDM2 and MDMX proteins. Structural studies revealed that the inhibitors bind into and occlude the p53 pockets of MDM2 and MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This mode of action effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. Dual MDM2/MDMX antagonists restored p53 apoptotic activity in the presence of high levels of MDMX and may offer a more effective therapeutic modality for MDMX-overexpressing cancers.

Tumor suppressor p53 status does not determine the differentiation-associated G1 cell cycle arrest induced in leukemia cells by 1,25-dihydroxyvitamin D3 and antioxidants.

Vitamin D derivatives can induce differentiation of human acute myeloid leukemia (AML) cells. Here, we investigated if the G1 cell cycle block associated with monocytic differentiation is modulated by the p53 status of the cells treated with 1,25D, alone or with plant antioxidants carnosic acid (C) or silibinin (S), and a p38 MAPK inhibitor SB202190 (SB), a combination (D-C/S-SB) previously shown to enhance differentiation of AML p53null cells. D-C/S-SB enhanced differentiation of OCI-AML3 (p53wt) and as expected HL60 (p53 null) cells, but not of MOLM-13 (p53wt) cells. Conversely, MOLM-13 (p53wt) cells treated with 1,25D and/or D-C/S-SB, resembled HL60 (p53 null) cells in rapid G1 block, while OCI-AML3 (p53wt) cells showed a delayed G1 block when treated in a similar way, indicating that there is no relationship between the p53 status and G1 block. Western blot analysis revealed that 1,25D and D-C/S-SB increased the inhibitory phosphorylation levels MEK-1 (P-Thr286), but decreased the levels of activated ERK1/2 (Thr202/Tyr204;Thr185/Tyr187), again without any apparent relationship to the p53 status. Interestingly, the increased levels of p21(Waf1/Cip1) were insufficient to promote a G1 block in this system, as only cell lines with increased levels of p27(Kip1) and p35Nck5a, an activator of Cdk5, showed a rapid G1 block. Overall, our data show that the p53-p21 axis is unlikely to have a role in differentiation-associated G1 block in AML cells with wt p53, and that this block is achieved by several, possibly co-operating but redundant pathways, that include inhibition of MEK-1 by p35Nck5a-activated Cdk5.

1,25-dihydroxyvitamin D3 enhances the apoptotic activity of MDM2 antagonist nutlin-3a in acute myeloid leukemia cells expressing wild-type p53.

The tumor suppressor p53 is often referred to as "the guardian of the genome" because of its central role in the cellular response to oncogenic stress and prevention of tumor development. Mutations of p53 in acute myeloid leukemia (AML) are rare but resistance to chemotherapy has been reported because of the deregulation of the p53 signaling and differentiation pathways. It is known that the interaction of the vitamin D metabolite 1,25-dihydroxyvitamin D(3) (1,25D) with its functional vitamin D receptor leads to differentiation, G(1) arrest, and increased cell survival in p53-null AML cells. However, there are no reports on the effect of 1,25D in leukemia cells expressing wild-type p53. Here, we examine vitamin D signaling in AML cells MOLM-13 and OCI-AML3 expressing wild-type p53 in the presence and absence of the MDM2 antagonist nutlin-3. We find that 1,25D alone induces monocytic differentiation in these cell lines similar to that seen in p53-null AML cells, suggesting that the presence of wild-type p53 is compatible with activation of vitamin D signaling. Combination of nutlin-3a with 1,25D accelerated programmed cell death, likely because of enhanced nutlin-induced upregulation of the proapoptotic PIG-6 protein and downregulation of antiapoptotic BCL-2, MDMX, human kinase suppressor of Ras 2, and phosphorylated extracellular signal-regulated kinase 2.

A new series of potent oxindole inhibitors of CDK2.

A novel series of oxindole-type inhibitors of CDK2 that have heteroatom substituted alkynyl moieties at their C-4 position is described. These novel 4-alkynyl-substituted inhibitors have superior potency relative to their parent compound in free enzyme and in cell based assays. The crystal structure of CDK2 in complex with one of these analogues was determined and gives insight to their increased potency. The biochemical evaluation of a representative derivative is also described.

Phosphorylation of p53 on key serines is dispensable for transcriptional activation and apoptosis.

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.