nutlin-3a and Leukemia--Myeloid--Acute

Both the multi-kinase inhibitor sorafenib and the small molecule inhibitor of the MDM2/p53 interaction, nutlin-3, used alone, have shown promising anti-leukemic activity in acute myeloid leukemia cells. Thus, in this study we investigated the effect of the combination of sorafenib plus nutlin-3 in acute myeloid leukemia.. Primary acute myeloid leukemia blasts (n=13) and FLT3(wild-type)/p53(wild-type) (OCI-AML3), FLT3(mutated)/p53(wild-type) (MOLM), FLT3(mutated)/p53(mutated) (MV4-11), FLT3(wild-type)/p53(deleted) (HL60) or FLT3(wild-type)/p53(mutated) (NB4) acute myeloid cell lines were exposed to sorafenib, used alone or in association with nutlin-3 at a 1:1 ratio, in a range of clinically achievable concentrations (1-10 μM). Induction of apoptosis and autophagy was evaluated by transmission electron microscopy and by specific flow cytometry analyses. The levels of Mcl-1, p53 and Bak proteins were analyzed by western blotting. Knock-down of Bax and Bak gene expression was performed in transfection experiments with specific short interfering RNA.. The sorafenib+nutlin-3 drug combination exhibits synergistic cytotoxicity in primary acute myeloid leukemia blasts and in acute myeloid leukemia cell lines with maximal cytotoxicity in FLT3(mutated) MV4-11 and MOLM, followed by the FLT3(wild-type) OCI-AML3, HL60 and NB4 cell lines. The cytotoxic activity of sorafenib+nutlin-3 was characterized by an increase of both apoptosis and autophagy. Moreover, Bax and Bak showed prominent roles in mediating the decrease of cell viability in response to the drug combination in p53(wild-type) OCI-AML3 and p53(deleted) HL-60 cells, respectively, as demonstrated in transfection experiments performed with specific short interfering RNA.. Our data demonstrate that acute myeloid leukemia cells show a variable but overall good susceptibility to the innovative therapeutic combination of sorafenib+nutlin-3, which differentially involves the pro-apoptotic Bcl-2 family members Bax and Bak in p53(wild-type) and p53(deleted) cells.

Topics: Antineoplastic Agents; Drug Synergism; Female; fms-Like Tyrosine Kinase 3; HL-60 Cells; Humans; Imidazoles; Leukemia, Myeloid, Acute; Male; Niacinamide; Phenylurea Compounds; Piperazines; Sorafenib; Tumor Suppressor Protein p53

Pharmacological inhibition of MDM2/4, which activates the critical tumor suppressor p53, has been gaining increasing interest as a strategy for the treatment of acute myeloid leukemia (AML). While clinical trials of MDM2 inhibitors have shown promise, responses have been confined to largely molecularly undefined patients, indicating that new biomarkers and optimized treatment strategies are needed. We previously reported that the microRNA miR-10a is strongly overexpressed in some AML, and demonstrate here that it modulates several key members of the p53/Rb network, including p53 regulator MDM4, Rb regulator RB1CC1, p21 regulator TFAP2C, and p53 itself. The expression of both miR-10a and its downstream targets were strongly predictive of MDM2 inhibitor sensitivity in cell lines, primary AML specimens, and correlated to response in patients treated with both MDM2 inhibitors and cytarabine. Furthermore, miR-10a inhibition induced synergy between MDM2 inhibitor Nutlin-3a and cytarabine in both in vitro and in vivo AML models. Mechanistically this synergism primarily occurs via the p53-mediated activation of cytotoxic apoptosis at the expense of cytoprotective autophagy. Together these findings demonstrate that miR-10a may be useful as both a biomarker to identify patients most likely to respond to cytarabine+MDM2 inhibition and also a druggable target to increase their efficacy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Autophagy; Biomarkers, Tumor; Cell Line, Tumor; Cytarabine; Female; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mice; Mice, Inbred NOD; MicroRNAs; Piperazines; Proto-Oncogene Proteins c-mdm2

Early clinical trials using murine double minute 2 (MDM2) inhibitors demonstrated proof-of-concept of p53-induced apoptosis by MDM2 inhibition in cancer cells; however, not all wild-type

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cyclohexanes; Female; Gene Expression Profiling; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mice; Mice, Nude; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Cancer cells show increased glycolysis and take advantage of this metabolic pathway to generate ATP. The TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits aerobic glycolysis and protects tumor cells from intracellular reactive oxygen species (ROS)-associated apoptosis. However, the function of TIGAR in glycolysis and survival of acute myeloid leukemia cells remains unclear.. We analyzed TIGAR expression in cytogenetically normal (CN-) AML patients and the correlations with clinical and biological parameters. In vivo and in vitro, we tested whether glycolysis may induce TIGAR expression and evaluated the combination effect of glycolysis inhibitor and TIGAR knockdown on human leukemia cell proliferation.. High TIGAR expression was an independent predictor of poor survival and high incidence of relapse in adult patients with CN-AML. TIGAR also showed high expression in multiple human leukemia cell lines and knockdown of TIGAR activated glycolysis through PFKFB3 upregulation in human leukemia cells. Knockdown of TIGAR inhibited the proliferation of human leukemia cells and sensitized leukemia cells to glycolysis inhibitor both in vitro and in vivo. Furthermore, TIGAR knockdown in combination with glycolysis inhibitor 2-DG led leukemia cells to apoptosis. In addition, the p53 activator Nutlin-3α showed a significant combinational effect with TIGAR knockdown in leukemia cells. However, TIGAR expression and its anti-apoptotic effects were uncoupled from overexpression of exogenous p53 in leukemia cells.. TIGAR might be a predictor of poor survival and high incidence of relapse in AML patients, and the combination of TIGAR inhibitors with anti-glycolytic agents may be novel therapies for the future clinical use in AML patients.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Case-Control Studies; Cell Line, Tumor; Cell Proliferation; Child; Deoxyglucose; Female; Gene Knockdown Techniques; Glycolysis; Heterografts; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Male; Mice; Middle Aged; Phosphofructokinase-2; Phosphoric Monoester Hydrolases; Piperazines; Prognosis; RNA, Small Interfering; Tumor Cells, Cultured; Young Adult

It has been shown that SOCS-1 plays an important role in the proper control of cytokine/growth factor responses and acts as a tumor suppressor in acute myeloid leukemias. Therefore, the objective of the present study was to evaluate the in vitro effect of treatment with Nutlin-3, a small molecule inhibitor of the MDM2/p53 interaction, on the expression of the suppressor of cytokine signaling 1 in primary acute myeloid leukemia cells and in myeloid cell lines with differential p53 status.. The expression of the suppressor of cytokine signaling 1 was quantitatively analyzed by real-time PCR in myeloid p53wild-type (OCI and MOLM) and p53null HL-60, leukemic cell lines, in patient-derived acute myeloid leukemia blasts, and in primary normal cell types, such as macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependence of the suppressor of cytokine signaling 1 upregulation that is induced by Nutlin-3 was analyzed in experiments performed using siRNA for p53, while the functional upregulation of the suppressor of cytokine signaling 1 was analyzed by assessing the levels of phosphorylated STAT-3.. Nutlin-3 significantly upregulated the transcription of the suppressor of cytokine signaling 1 in p53wild-type OCI and MOLM but not in p53deleted p53null HL60, myeloid leukemic cell lines, as well as in primary acute myeloid leukemia blasts. Conversely, and somewhat unexpectedly, Nutlin-3 did not modulate the suppressor of cytokine signaling 1 expression in primary normal macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependent upregulation of the suppressor of cytokine signaling 1 by Nutlin-3 was associated with the downregulation of phosphorylated STAT-3, a major molecular target of the suppressor of cytokine signaling 1.. Overall, our data suggest a potential role for the suppressor of cytokine signaling 1 as a therapeutic target of Nutlin-3 in p53 wild-type acute myeloid leukemias.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Cells, Cultured; Endothelial Cells; Humans; Imidazoles; Leukemia, Myeloid, Acute; Myeloid Cells; Piperazines; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Time Factors; Tumor Suppressor Protein p53; Up-Regulation

Although p53 is found mutated in almost 50% of all cancers, p53 mutations in leukaemia are relatively rare. Acute myeloid leukaemia (AML) cells employ other strategies to inactivate their wild type p53 (WTp53), like the overexpression of the p53 negative regulators Mdm2 and Mdm4. As such, AMLs are excellent candidates for therapeutics involving the reactivation of their WTp53 to restrict and destroy cancer cells, and the Mdm2 antagonist nutlin-3 is one such promising agent. Using AML cell lines with WTp53, we identified stable and high levels of p53 in the OCI/AML-2 cell lines. We demonstrate that this nutlin-3 sensitive cell line overexpressed Mdm4 to sequester, stabilise and inhibit p53 in the cytoplasm. We also show that elevated Mdm4 competed with Mdm2-p53 interaction and therefore extended p53 half-life while preventing p53 transcriptional activity. Our results provide biochemical evidence on the dynamics of the p53-Mdm2-Mdm4 interactions in affecting p53 levels and activity, and unlike previously reported findings derived from genetically manipulated systems, AML cells with naturally high levels of Mdm4 remain sensitive to nutlin treatment.. Endogenously high levels of Mdm4 inhibit and sequester p53 in AML. High levels of Mdm4 do not block function of Mdm2 inhibitors in AML.

Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Humans; Imidazoles; Leukemia, Myeloid, Acute; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Tumor Suppressor Protein p53

The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy.. Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells.. Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML.

Topics: Acetylation; Antineoplastic Agents; Benzoquinones; Cell Line, Tumor; Drug Synergism; Gene Expression Regulation, Leukemic; Histones; HSP27 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Imidazoles; Lactams, Macrocyclic; Leukemia, Myeloid, Acute; Piperazines; Primary Cell Culture; Signal Transduction; Tumor Suppressor Protein p53

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 is a promising anti-cancer agent, which exhibits activity against a variety of cancers, including acute myeloid leukemia (AML). Previous studies have shown that Nutlin-3 variably induces apoptosis and cell cycle arrest in cancer cells while it shows low/absent cytotoxicity in normal cells. However, the reason for the selective pro-apoptotic activity in cancer cells with respect to normal counterparts is incompletely understood. In this study, we have compared the induction of several known target genes of p53 in two p53(wild-type) AML cell lines, OCI-AML3 and MOLM, in comparison with primary normal peripheral blood mononuclear cells (PBMC). Among several p53-target genes activated both in AML cell lines and normal PBMC (BBC3, BAX, MDM2, FAS, CDKN1A, GDF15, GADD45A, TNFRSF10B, TP53I3/PIG3), only TP53I3/PIG3 was selectively activated in MOLM and OCI-AML3, but not in PBMC. The important role of TP53I3/PIG3 in mediating the apoptotic activity of Nutlin-3 was underlined by knock-down experiments with siRNA specific for TP53I3/PIG3, which resulted in a significant decrease in the pro-apoptotic activity of Nutlin-3.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Leukemic; Gene Knockdown Techniques; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Leukocytes, Mononuclear; Piperazines; Proto-Oncogene Proteins; Tumor Suppressor Protein p53

Although TP53 mutations are rare in acute myeloid leukemia (AML), wild type p53 function is habitually annulled through overexpression of MDM2 or through various mechanisms including epigenetic silencing by histone deacetylases (HDACs). We hypothesized that co-inhibition of MDM2 and HDACs, with nutlin-3 and valproic acid (VPA) would additively inhibit growth in leukemic cells expressing wild type TP53 and induce p53-mediated apoptosis. In vitro studies with the combination demonstrated synergistic induction of apoptosis in AML cell lines and patient cells. Nutlin-3 and VPA co-treatment resulted in massive induction of p53, acetylated p53 and p53 target genes in comparison with either agent alone, followed by p53 dependent cell death with autophagic features. In primary AML cells, inhibition of proliferation by the combination therapy correlated with the CD34 expression level of AML blasts. To evaluate the combination in vivo, we developed an orthotopic, NOD/SCID IL2rγ(null) xenograft model of MOLM-13 (AML FAB M5a; wild type TP53) expressing firefly luciferase. Survival analysis and bioluminescent imaging demonstrated the superior in vivo efficacy of the dual inhibition of MDM2 and HDAC in comparison with controls. Our results suggest the concomitant targeting of MDM2-p53 and HDAC inhibition, may be an effective therapeutic strategy for the treatment of AML.

Topics: Acetylation; Animals; Antigens, CD34; Apoptosis; Cell Line, Tumor; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, p53; Histone Deacetylase Inhibitors; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mice; Mice, Inbred NOD; Mice, SCID; Microscopy, Electron, Transmission; Piperazines; Proto-Oncogene Proteins c-mdm2; Valproic Acid

Topics: Antineoplastic Agents; Female; fms-Like Tyrosine Kinase 3; Humans; Imidazoles; Leukemia, Myeloid, Acute; Male; Niacinamide; Phenylurea Compounds; Piperazines; Sorafenib; Tumor Suppressor Protein p53

Fms-like tyrosine kinase-3 (FLT3) inhibitors have been used to overcome the dismal prognosis of acute myeloid leukemia (AML) with FLT3 mutations. Clinical results with FLT3 inhibitor monotherapy have shown that bone marrow responses are commonly less pronounced than peripheral blood responses. We investigated the role of p53 in bone marrow stromal cells in stromal cell-mediated resistance to FLT3 inhibition in FLT3 mutant AML. While the FLT3 inhibitor FI-700 induced apoptosis in FLT3 mutant AML cells, apoptosis induction was diminished under stromal coculture conditions. Protection appeared to be mediated, in part, by CXCL12 (SDF-1)/CXCR4 signaling. The protective effect of stromal cells was significantly reduced by pre-exposure to the HDM2 inhibitor Nutlin-3a. p53 activation by Nutlin-3a was not cytotoxic to stromal cells, but reduced CXCL12 mRNA levels and secretion of CXCL12 partially through p53-mediated HIF-1α down-regulation. Results show that p53 activation in stroma cells blunts stroma cell-mediated resistance to FLT3 inhibition, in part through down-regulation of CXCL12. This is the first report of Nutlin effect on the bone marrow environment. We suggest that combinations of HDM2 antagonists and FLT3 inhibitors may be effective in clinical trials targeting mutant FLT3 leukemias.

Topics: Adult; Apoptosis; Bone Marrow Cells; Chemokine CXCL12; Down-Regulation; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Leukemic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Leukemia, Myeloid, Acute; Mesenchymal Stem Cells; Mutation; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-mdm2; Pyridines; Pyrimidines; Tumor Cells, Cultured; Tumor Microenvironment; Tumor Suppressor Protein p53; Young Adult

Activation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a-induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Blast Crisis; Carrier Proteins; Caspase 6; Cell Line, Tumor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Leukemic; Gene Knockdown Techniques; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Mice; Mitochondrial Proteins; Piperazines; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Stromal Cells; Tumor Suppressor Protein p53; X-Linked Inhibitor of Apoptosis Protein

Treatment using Fms-like tyrosine kinase-3 (FLT3) inhibitors is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. We found that FI-700 immediately reduced antiapoptotic Mcl-1 levels and enhanced Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/internal tandem duplication cells through the Mcl-1/Noxa axis. FI-700 induced proteasome-mediated degradation of Mcl-1, resulting in the reduced ability of Mcl-1 to sequester proapoptotic Bim. Nutlin-3 induced Noxa, which displaced Bim from Mcl-1. The FI-700/Nutlin-3 combination profoundly activated Bax and induced apoptosis. Our findings suggest that FI-700 actively enhances p53 signaling toward mitochondrial apoptosis and that a combination strategy aimed at inhibiting FLT3 and activating p53 signaling could potentially be effective in AML.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Doxorubicin; fms-Like Tyrosine Kinase 3; G1 Phase; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Piperazines; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Pyridines; Pyrimidines; Tumor Suppressor Protein 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.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Calcitriol; Cell Differentiation; Cell Line, Tumor; Drug Evaluation, Preclinical; Drug Synergism; Gene Expression Regulation, Leukemic; Genes, p53; HL-60 Cells; Humans; Imidazoles; Leukemia, Myeloid, Acute; Monocytes; Piperazines; Proto-Oncogene Proteins c-mdm2; Up-Regulation

Activation of the phosphatidylinositol-3 kinase/Akt/mammalian target of the rapamycin (PI3K/Akt/mTOR) pathway and inactivation of wild-type p53 by murine double minute 2 homologue (Mdm2) overexpression are frequent molecular events in acute myeloid leukemia (AML). We investigated the interaction of PI3K/Akt/mTOR and p53 pathways after their simultaneous blockade using the dual PI3K/mTOR inhibitor PI-103 and the Mdm2 inhibitor Nutlin-3. We found that PI-103, which itself has modest apoptogenic activity, acts synergistically with Nutlin-3 to induce apoptosis in a wild-type p53-dependent fashion. PI-103 synergized with Nutlin-3 to induce Bax conformational change and caspase-3 activation, despite its inhibitory effect on p53 induction. The PI-103/Nutlin-3 combination caused profound dephosphorylation of 4E-BP1 and decreased expression of many proteins including Mdm2, p21, Noxa, Bcl-2 and survivin, which can affect mitochondrial stability. We suggest that PI-103 actively enhances downstream p53 signaling and that a combination strategy aimed at inhibiting PI3K/Akt/mTOR signaling and activating p53 signaling is potentially effective in AML, where TP53 mutations are rare and downstream p53 signaling is intact.

Topics: Apoptosis; Drug Synergism; Furans; Gene Expression Regulation; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mitochondrial Proteins; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-mdm2; Pyridines; Pyrimidines; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Aberrant expression of Aurora kinases and inactivation of wild-type p53 by Mdm2 overexpression are frequent molecular events in acute myelogenous leukemia (AML), and preclinical data for inhibition of Aurora kinases or Mdm2 are promising. However, it remains largely unknown whether the viability of cells exposed to Aurora kinase inhibitors depends on the p53 status. We investigated the interaction of Aurora kinases and p53 pathways after their simultaneous blockades using a small-molecule pan-Aurora kinase inhibitor, MK-0457, and a selective small-molecule antagonist of Mdm2, Nutlin-3. We found that MK-0457, which itself activates p53 signaling, acts synergistically with Nutlin-3 to induce apoptosis in wild-type p53 AML cell lines OCI-AML-3 and MOLM-13 but not in p53-null HL-60 cells. MK-0457 and Nutlin-3 showed synergism in inducing p53, conformational change of Bax and Deltapsi(m) loss, suggesting an involvement of p53-mediated mitochondrial apoptosis. Nutlin-3 constrained endoreduplication after Aurora inhibition via activation of a p53-dependent postmitotic checkpoint and p21 induction in pseudo-G1 cells. Our findings provide the molecular rationale for concomitant targeting of Aurora kinases and Mdm2 in AML where TP53 mutations are rare and downstream p53 signaling is mostly intact.

Topics: Apoptosis; Aurora Kinases; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; DNA, Neoplasm; Drug Resistance, Neoplasm; Drug Synergism; G1 Phase; Gene Duplication; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mitochondria; Mitosis; Piperazines; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Acute myeloid leukemia (AML) cells are relatively resistant to tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL). We previously reported that triptolide, a potent anticancer agent from a Chinese herb, decreases XIAP in leukemic cells. We evaluated the combination of triptolide and TRAIL and found synergistic promotion of apoptosis in AML cells. XIAP-overexpressing U937 cells (U937XIAP) were more resistant to TRAIL than U937neo cells, and inhibition of XIAP with the small-molecule inhibitor 1396-11 enhanced TRAIL-induced apoptosis, implying XIAP as a resistance factor in AML. Furthermore, triptolide increased DR5 levels in OCI-AML3, while the DR5 increase was blunted in p53-knockdown OCI-AML3 and p53-mutated U937 cells, confirming a role for p53 in the regulation of DR5. In support of this finding, disruption of MDM2-p53 binding with subsequent increase in p53 levels by nutlin3a increased DR5 levels and sensitized OCI-AML3 cells to TRAIL. The combination of 1396-11 plus nutlin3a plus TRAIL was more effective than either the 1396-11 and TRAIL or nutlin3a and TRAIL combinations in OCI-AML3 cells, further supporting the role of triptolide as a sensitizer to TRAIL-induced apoptosis in part by independent modulation of XIAP expression and p53 signaling. Thus, the combination of triptolide and TRAIL may provide a novel strategy for treating AML by overcoming critical mechanisms of apoptosis resistance.

Topics: Aniline Compounds; Antineoplastic Agents, Alkylating; Apoptosis; Diterpenes; Drug Synergism; Epoxy Compounds; Female; Humans; Imidazoles; Leukemia, Myeloid, Acute; Male; Phenanthrenes; Piperazines; Proto-Oncogene Proteins c-mdm2; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Suppressor Protein p53; U937 Cells; X-Linked Inhibitor of Apoptosis Protein

Activation of the Raf/MEK/ERK pathway and inactivation of wild-type p53 by Mdm2 overexpression are frequent molecular events in acute myelogenous leukemia (AML). We investigated the interaction of Raf/MEK/ERK and p53 pathways after their simultaneous blockades using a selective small-molecule antagonist of Mdm2, Nutlin-3a, and a pharmacologic MEK-specific inhibitor, PD98059. We found that PD98059, which itself has minimal apoptogenic activity, acts synergistically with Nutlin-3a to induce apoptosis in wild-type p53 AML cell lines OCI-AML-3 and MOLM-13. Interestingly, PD98059 enhanced nuclear proapototic function of p53 in these cells. In accordance with the activation of transcription-dependent apoptosis, PD98059 treatment promoted the translocation of p53 from the cytoplasm to the nucleus in OCI-AML-3 cells, in which p53 primarily initiates transcription-independent apoptosis when cells are treated with Nutlin-3a alone. The critical role of p53 localization in cells with increased p53 levels was supported by enhanced apoptosis induction in cells cotreated with Nutlin-3a and the nuclear export inhibitor leptomycin B. PD98059 prevented p53-mediated induction of p21 at the transcriptional level. The repressed expression of antiapototic p21 also seemed to contribute to synergism between PD98059 and Nutlin-3a because (a) the synergistic apoptogenic effect was preserved in G(1) cells, (b) p53-mediated induction of p21 was preferentially seen in G(1) cells, (c) PD98059 strongly antagonized p21 induction by Nutlin-3a, and (d) cells with high p21 levels were resistant to apoptosis. This is the first report showing that the Raf/MEK/ERK pathway regulates the subcellular localization of p53 and the relative contribution of transcription-dependent and transcription-independent pathways in p53-mediated apoptosis.

Topics: Apoptosis; Cell Line, Tumor; Cell Nucleus; Drug Synergism; Fatty Acids, Unsaturated; Flavonoids; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinase Kinases; Piperazines; Proto-Oncogene Proteins c-mdm2; raf Kinases; Transcription, Genetic; Tumor Suppressor Protein p53

To potentiate the response of acute myeloid leukemia (AML) to TRAIL cytotoxicity, we have adopted a strategy of combining nutlin-3, a potent non-genotoxic activator of the p53 pathway, with recombinant TRAIL. The rationale for using such a combination was that deletions and/or mutations of the p53 gene occur in only 5-10% of AML and that TRAIL and nutlin-3 activate the extrinsic and intrinsic pathways of apoptosis, respectively. TRAIL induced a rapid increase of apoptosis when added to OCI M4-type and MOLM M5-type AML cells, carrying a wild-type p53, as well as to NB4 M3-type AML, carrying a mutated p53. On the other hand, the small molecule activator of the p53 pathway nutlin-3 induced p53 accumulation, cell cycle arrest and a slow progressive increase of apoptosis in OCI and MOLM but not in NB4. Of note, nutlin-3 up-regulated the surface expression of TRAIL-R2 and synergized with TRAIL in inducing apoptosis in OCI and MOLM as well as in primary M4-type and M5-type AML blasts, but not in NB4 cells. Moreover, while nutlin-3 up-regulated the expression of cyclin dependent kinase inhibitor p21, a p53-target gene mediating cell cycle block and showing anti-apoptotic activity, the simultaneous addition of TRAIL plus nutlin-3 induced the caspase-dependent cleavage of p21. The relevance of p21 down-regulation for sensitizing AML cells to apoptosis was underscored in knocking-down experiments with small interfering RNAs. Our data suggest that the combined treatment of nutlin-3 plus TRAIL might offer a novel therapeutic strategy for AML.

Topics: Antineoplastic Agents; Apoptosis; Caspases; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mutation; Piperazines; Receptors, TNF-Related Apoptosis-Inducing Ligand; Recombinant Proteins; RNA Interference; RNA, Small Interfering; Time Factors; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Up-Regulation

Disruption of Mdm2-p53 interaction activates p53 signaling, disrupts the balance of antiapoptotic and proapoptotic Bcl-2 family proteins and induces apoptosis in acute myeloid leukemia (AML). Overexpression of Bcl-2 may inhibit this effect. Thus, functional inactivation of antiapoptotic Bcl-2 proteins may enhance apoptogenic effects of Mdm2 inhibition. We here investigate the potential therapeutic utility of combined targeting of Mdm2 by Nutlin-3a and Bcl-2 by ABT-737, recently developed inhibitors of protein-protein interactions. Nutlin-3a and ABT-737 induced Bax conformational change and mitochondrial apoptosis in AML cells in a strikingly synergistic fashion. Nutlin-3a induced p53-mediated apoptosis predominantly in S and G2/M cells, while cells in G1 were protected through induction of p21. In contrast, ABT-737 induced apoptosis predominantly in G1, the cell cycle phase with the lowest Bcl-2 protein levels and Bcl-2/Bax ratios. In addition, Bcl-2 phosphorylation on Ser70 was absent in G1 but detectable in G2/M, thus lower Bcl-2 levels and absence of Bcl-2 phosphorylation appeared to facilitate ABT-737-induced apoptosis of G1 cells. The complementary effects of Nutlin-3a and ABT-737 in different cell cycle phases could, in part, account for their synergistic activity. Our data suggest that combined targeting of Mdm2 and Bcl-2 proteins could offer considerable therapeutic promise in AML.

Topics: Animals; Annexin A5; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; G1 Phase; G2 Phase; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mice; Mitochondria; Mitosis; Mutant Proteins; Nitrophenols; Phosphorylation; Piperazines; Protein Conformation; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; S Phase; Sulfonamides; Tumor Suppressor Protein p53

Although TP53 mutations are rare in acute myeloid leukemia (AML), inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2 (murine double minute 2). Recently, small-molecule antagonists of MDM2, Nutlins, have been developed that inhibit the p53-MDM2 interaction and activate p53 signaling. Here, we study the effects of p53 activation by Nutlin-3 in AML cells. Treatment with MDM2 inhibitor triggered several molecular events consistent with induction of apoptosis: loss of mitochondrial membrane potential, caspase activation, phosphatidylserine externalization, and DNA fragmentation. There was a positive correlation in primary AML samples with wild-type p53 between baseline MDM2 protein levels and apoptosis induced by MDM2 inhibition. No induction of apoptosis was observed in AML samples harboring mutant p53. Colony formation of AML progenitors was inhibited in a dose-dependent fashion, whereas normal CD34+ progenitor cells were less affected. Mechanistic studies suggested that Nutlin-induced apoptosis was mediated by both transcriptional activation of proapoptotic Bcl-2 family proteins, and transcription-independent mitochondrial permeabilization resulting from mitochondrial p53 translocation. MDM2 inhibition synergistically enhanced cytotoxicity of cytosine arabinoside and doxorubicin in AML blasts but not in normal hematopoietic progenitor cells. p53 activation by targeting the p53-MDM2 interaction might offer a novel therapeutic strategy for AML that retain wild-type p53.

Topics: Adult; Aged; Apoptosis; Caspases; Cell Nucleus; Cytoplasm; Doxorubicin; Enzyme Activation; Female; Humans; Imidazoles; Leukemia, Myeloid, Acute; Male; Middle Aged; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Tumor Cells, Cultured; Tumor Suppressor Protein p53