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MDM2 antagonist nutlin-3a sensitizes tumors to V-ATPase inhibition.

ABSTRACT: Treating cancer is one of the big challenges of this century and it has become evident that single chemotherapeutic treatment is rarely effective. As tumors often carry multiple mutations using combination therapy which addresses different targets seems therefore more beneficial. One of the most frequently mutated genes in tumors is the tumor suppressor p53. Significant work has been put in the development of p53 activators, which are now in clinical studies against diverse cancers. Recently, we could show that inhibition of V-ATPase, a multisubunit proton pump, by archazolid induces p53 protein levels in cancer cells. In this study, we provide evidence that the combination of archazolid with the p53 activator nutlin-3a is synergistically inducing cell death in different p53 wild type tumor cell lines. Mechanistically, this effect could presumably be attributed to reduction of glycolysis as TIGAR mRNA levels were increased and glucose uptake and Glut1 protein levels were reduced. In addition, combination treatment highly activated pro-apoptotic pathways including IGFBP3 and Bax inducing caspase-9 and PARP cleavage. Remarkably, combination of archazolid and nutlin-3a was more efficient in reducing tumor growth compared to single dose treatment in a U87MG mouse model in vivo. Hence, our findings suggest the combination of archazolid and nutlin-3a as a highly promising strategy for the treatment of p53 wild type tumors.

SUBMITTER: Schneider LS

PROVIDER: S-EPMC5423188 | biostudies-other | 2016 Aug

REPOSITORIES: biostudies-other

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Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a.

Project description:In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in combinatorial therapies. WIP1, encoded from PPM1D, is a negative regulator of p53. We evaluated PPM1D expression and explored the therapeutic efficacy of WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in AML cell lines and primary samples. PPM1D transcript levels were higher in young patients compared with older ones and in core-binding-factor AML compared with other cytogenetic subgroups. In contrast, its expression was reduced in NPM1-mutated (mut, irrespective of FLT3-ITD status) or TP53-mut cases compared with wild-type (wt) ones. Either Nut-3a, and moderately WIP1i, as single agent decreased cell viability of TP53-wt cells (MV-4-11, MOLM-13, OCI-AML3) in a time/dosage-dependent manner, but not of TP53-mut cells (HEL, KASUMI-1, NOMO-1). The drug combination synergistically reduced viability and induced apoptosis in TP53-wt AML cell line and primary cells, but not in TP53-mut cells. Gene expression and immunoblotting analyses showed increased p53, MDM2 and p21 levels in treated TP53-wt cells and highlighted the enrichment of MYC, PI3K-AKT-mTOR and inflammation-related signatures upon WIP1i, Nut-3a and their combination, respectively, in the MV-4-11 TP53-wt model. This study demonstrated that WIP1 is a promising therapeutic target to enhance Nut-3a efficacy in TP53-wt AML.

| S-EPMC8067413 | biostudies-literature

Pharmacological inhibition of WIP1 sensitizes acute myeloid leukemia cells to MDM2 inhibitor Nutlin-3a

Project description:The wild-type p53-inducible protein phosphatase WIP1, encoded by the PPM1D gene, is a negative regulator of p53 and is involved in cell cycle control and DNA damage stress-response. In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in a number of combination strategies. We investigated the therapeutic potential of its inhibition through the selective WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in a panel of AML cell lines and performed gene expression analysis of single agent and combined treatments. A p53-related signature was significantly upregulated in the TP53-wildtype MV-4-11 AML cell line. Moreover, drug-specific transcriptional changes likely contribute to the synergistic combination effect.

2021-05-06 | GSE156182 | GEO

Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3.

Project description:The MDM2-p53 pathway has a prominent oncogenic function in the pathogenesis of various cancers. Nutlin-3, a small-molecule antagonist of MDM2-p53 interaction, inhibits proliferation in cancer cells with wild-type p53. Herein, we evaluate the expression of MDM2, both the full length and a splicing variant MDM2-A, and the sensitivity of Nutlin-3 in different cancer cell lines. Included are seven cell lines with wild-type p53 (four mesothelioma, one breast cancer, one chondrosarcoma, and one leiomyosarcoma), two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one mesothelioma cell line harboring a p53 point mutation. Nutlin-3 treatment increased expression of cyclin D1, MDM2, and p53 in cell lines with wild-type p53. Additive effects were observed in cells containing wild-type p53 through coordinated attack on MDM2-p53 binding and cyclin D1 by lentivirual shRNA knockdown or small molecule inhibition, as demonstrated by immunoblots and cell viability analyses. Further results demonstrate that MDM2 binds to cyclin D1, and that an increase in cyclin D1 expression after Nutlin-3 treatment is correlated with expression and ubiquitin E3-ligase activity of MDM2. MDM2 and p53 knockdown experiments demonstrated inhibition of cyclin D1 by MDM2 but not p53. These results indicate that combination inhibition of cyclin D1 and MDM2-p53 binding warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53.

| S-EPMC5078041 | biostudies-literature

MDM2 antagonist nutlin-3a reverses mitoxantrone resistance by inhibiting breast cancer resistance protein mediated drug transport.

Project description:Breast cancer resistance protein (BCRP; ABCG2), a clinical marker for identifying the side population (SP) cancer stem cell subgroup, affects intestinal absorption, brain penetration, hepatobiliary excretion, and multidrug resistance of many anti-cancer drugs. Nutlin-3a is currently under pre-clinical investigation in a variety of solid tumor and leukemia models as a p53 reactivation agent, and has been recently demonstrated to also have p53 independent actions in cancer cells. In the present study, we first report that nutlin-3a can inhibit the efflux function of BCRP. We observed that although the nutlin-3a IC(50) did not differ between BCRP over-expressing and vector control cells, nutlin-3a treatment significantly potentiated the cells to treatment with the BCRP substrate mitoxantrone. Combination index calculations suggested synergism between nutlin-3a and mitoxantrone in cell lines over-expressing BCRP. Upon further investigation, it was confirmed that nutlin-3a increased the intracellular accumulation of BCRP substrates such as mitoxantrone and Hoechst 33342 in cells expressing functional BCRP without altering the expression level or localization of BCRP. Interestingly, nutlin-3b, considered virtually "inactive" in disrupting the MDM2/p53 interaction, reversed Hoechst 33342 efflux with the same potency as nutlin-3a. Intracellular accumulation and bi-directional transport studies using MDCKII cells suggested that nutlin-3a is not a substrate of BCRP. Additionally, an ATPase assay using Sf9 insect cell membranes over-expressing wild-type BCRP indicated that nutlin-3a inhibits BCRP ATPase activity in a dose-dependent fashion. In conclusion, our studies demonstrate that nutlin-3a inhibits BCRP efflux function, which consequently reverses BCRP-related drug resistance.

| S-EPMC3108438 | biostudies-literature

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

Project description: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.

| S-EPMC2868102 | biostudies-literature

Structure of a stapled peptide antagonist bound to nutlin-resistant Mdm2.

Project description:As key negative regulator of the p53 tumour suppressor, Mdm2 is an attractive therapeutic target. Small molecules such as Nutlin have been developed to antagonise Mdm2, resulting in p53-dependent death of tumour cells. We have recently described a mutation in Mdm2 (M62A), which precludes binding of Nutlin, but not p53. This Nutlin-resistant variant is not, however, refractory to binding and inhibition by stapled peptide antagonists targeting the same region of Mdm2. A detailed understanding of how stapled peptides are recalcitrant to Mdm2 mutations conferring Nutlin-resistance will aid in the further development of potent Mdm2 antagonists. Here, we report the 2.00 Å crystal structure of a stapled peptide antagonist bound to Nutlin resistant Mdm2. The stapled peptide relies on an extended network of interactions along the hydrophobic binding cleft of Mdm2 for high affinity binding. Additionally, as seen in other stapled peptide structures, the hydrocarbon staple itself contributes to binding through favourable interactions with Mdm2. The structure highlights the intrinsic plasticity present in both Mdm2 and the hydrocarbon staple moiety, and can be used to guide future iterations of both small molecules and stapled peptides for improved antagonists of Mdm2.

| S-EPMC4130638 | biostudies-literature

MYCN sensitizes neuroblastoma to the MDM2-p53 antagonists Nutlin-3 and MI-63.

Project description:MYCN amplification is a major biomarker of poor prognosis, occurring in 25-30% of neuroblastomas. MYCN has contradictory roles in promoting cell growth and sensitizing cells to apoptosis. We have recently shown that p53 is a direct transcriptional target of MYCN in neuroblastoma and that p53-mediated apoptosis may be an important mechanism of MYCN-induced apoptosis. Although p53 mutations are rare in neuroblastoma at diagnosis, the p53/MDM2/p14(ARF) pathway is often inactivated through MDM2 amplification or p14(ARF) inactivation. We hypothesized that reactivation of p53 by inhibition of its negative regulator MDM2, using the MDM2-p53 antagonists Nutlin-3 and MI-63, will result in p53-mediated growth arrest and apoptosis especially in MYCN-amplified cells. Using the SHEP Tet21N MYCN-regulatable system, MYCN(-) cells were more resistant to both Nutlin-3 and MI-63 mediated growth inhibition and apoptosis compared with MYCN(+) cells and siRNA-mediated knockdown of MYCN in four MYCN-amplified cell lines resulted in decreased p53 expression and activation, as well as decreased levels of apoptosis following treatment with MDM2-p53 antagonists. In a panel of 18 neuroblastoma cell lines treated with Nutlin-3 and MI-63, the subset amplified for MYCN had a significantly lower mean GI(50) value (50% growth inhibition) and increased caspase 3/7 activity compared with the non-MYCN-amplified group of cell lines, but p53 mutant cell lines were resistant to the antagonists regardless of MYCN status. We conclude that amplification or overexpression of MYCN sensitizes neuroblastoma cell lines with wild-type p53 to MDM2-p53 antagonists and that these compounds may therefore be particularly effective in treating high-risk MYCN-amplified disease.

| S-EPMC3191119 | biostudies-literature

Nutlin-3a, an MDM2 antagonist and p53 activator, helps to preserve the replicative potential of cancer cells treated with a genotoxic dose of resveratrol.

Project description:Resveratrol is a natural compound that has been intensely studied due to its role in cancer prevention and potential as an anti-cancer therapy. Its effects include induction of apoptosis and senescence-like growth inhibition. Here, we report that two cancer cell lines (U-2 OS and A549) differ significantly in their molecular responses to resveratrol. Specifically, in U-2 OS cells, the activation of the p53 pathway is attenuated when compared to the activation in A549 cells. This attenuation is accompanied by a point mutation (458: CGA→TGA) in the PPM1D gene and overexpression of the encoded protein, which is a negative regulator of p53. Experimentally induced knockdown of PPM1D in U-2 OS cells resulted in slightly increased activation of the p53 pathway, most clearly visible as stronger phosphorylation of p53 Ser37. When treated with nutlin-3a, a non-genotoxic activator of p53, U-2 OS and A549 cells both responded with substantial activation of the p53 pathway. Nutlin-3a improved the clonogenic survival of both cell lines treated with resveratrol. This improvement was associated with lower activation of DNA-damage signaling (phosphorylation of ATM, CHK2, and histone H2AX) and higher accumulation of cells in the G1 phase of the cell cycle. Thus, the hyperactivation of p53 by nutlin-3a helps to preserve the replicative potential of cells exposed to resveratrol.

| S-EPMC3723979 | biostudies-literature

MDM2 Inhibitor, Nutlin 3a, Induces p53 Dependent Autophagy in Acute Leukemia by AMP Kinase Activation.

Project description:MDM2 (mouse double minute 2) inhibitors that activate p53 and induce apoptosis in a non-genotoxic manner are in clinical development for treatment of leukemias. P53 can modulate other programmed cell death pathways including autophagy both transcriptionally and non-transcriptionally. We investigated autophagy induction in acute leukemia by Nutlin 3a, a first-in-class MDM2 inhibitor. Nutlin 3a induced autophagy in a p53 dependent manner and transcriptional activation of AMP kinase (AMPK) is critical, as this effect is abrogated in AMPK -/- mouse embryonic fibroblasts. Nutlin 3a induced autophagy appears to be pro-apoptotic as pharmacological (bafilomycin) or genetic inhibition (BECLIN1 knockdown) of autophagy impairs apoptosis induced by Nutlin 3a.

| S-EPMC4595506 | biostudies-literature

MDM2 antagonist nutlin-3 displays antiproliferative and proapoptotic activity in mantle cell lymphoma.

Project description:PurposeMantle cell lymphoma (MCL) has one of the poorest prognoses of the non-Hodgkin's lymphomas, and novel therapeutic approaches are needed. We wished to determine whether Nutlin-3, a novel small-molecule murine double minute 2 (MDM2) antagonist that efficiently activates TP53, might be effective in inducing cell death in MCL.Experimental designMCL cell lines with known TP53 status were treated with Nutlin-3, and biological and biochemical consequences were studied. Synergies with the prototypic genotoxic agent doxorubicin and the novel proteasome inhibitor bortezomib were assessed.ResultsNutlin-3 resulted in a reduction in cell proliferation/viability (IC50 < 10 micromol/L), an increase in the apoptotic fraction, and cell cycle arrest in wild-type (wt) TP53 Z-138 and Granta 519 cells. These effects were accompanied by TP53 accumulation and induction of TP53-dependent proteins p21, MDM2, Puma, and Noxa. Cell cycle arrest was characterized by suppression of S phase and an increase in the G0-G1 and G2-M fractions and accompanied by suppression of total and phosphorylated retinoblastoma protein and a decrease in G2-M-associated proteins cyclin B and CDC2. The combination of Nutlin-3 with doxorubicin or bortezomib was synergistic in wt-TP53 MCL cells. Nutlin-3 also induced cell cycle arrest and reduced cell viability in the mutant TP53 MINO cells but at a significantly higher IC50 (22.5 micromol/L). These effects were associated with induction of the TP53 homologue p73, slight increases in p21 and Noxa, and caspase activation. Nutlin-3 and bortezomib synergistically inhibited cell growth of MINO.ConclusionThese findings suggest that the MDM2 antagonist Nutlin-3 may be an effective agent in the treatment of MCL with or without wt-TP53.

| S-EPMC7322626 | biostudies-literature

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