Publications by authors named "Arman Esfandiari"

6 Publications

  • Page 1 of 1

ATM Dependent DUSP6 Modulation of p53 Involved in Synergistic Targeting of MAPK and p53 Pathways with Trametinib and MDM2 Inhibitors in Cutaneous Melanoma.

Cancers (Basel) 2018 Dec 20;11(1). Epub 2018 Dec 20.

Northern Institute for Cancer Research, School of Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

MAPK and p14⁻MDM2⁻p53 pathways are critical in cutaneous melanomas. Here, synergistic combination of the MEK inhibitor, trametinib, with MDM2 inhibitors, nutlin-3/RG7388/HDM201, and the mechanistic basis of responses, for BRAF and p53 melanoma cells, are reported. The combination treatments induced higher levels of p53 target gene transcripts and protein products, resulting in increased cell cycle arrest and apoptosis compared with MDM2 inhibitors alone, suggesting trametinib synergized with MDM2 inhibitors via upregulation of p53-dependent pathways. In addition, DUSP6 phosphatase involvement was indicated by downregulation of its mRNA and protein following pERK reduction by trametinib. Furthermore, suppression of DUSP6 by siRNA, or inhibition with the small molecule inhibitor, BCI, at a dose without cytotoxicity, potentiated the effect of MDM2 inhibitors through increased ATM-dependent p53 phosphorylation, as demonstrated by complete reversal with the ATM inhibitor, KU55933. Trametinib synergizes with MDM2 inhibitors through a novel DUSP6 mechanism in BRAF and p53 melanoma cells, in which DUSP6 regulation of p53 phosphorylation is mediated by ATM. This provides a new therapeutic rationale for combination treatments involving activation of the ATM/p53 pathway and MAPK pathway inhibition.
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http://dx.doi.org/10.3390/cancers11010003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356368PMC
December 2018

MEK inhibition leads to BRCA2 downregulation and sensitization to DNA damaging agents in pancreas and ovarian cancer models.

Oncotarget 2018 Feb 22;9(14):11592-11603. Epub 2018 Jan 22.

Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, Paul O'Gorman Building, University College London, London WC1E 6DD, UK.

Targeting the DNA damage response (DDR) in tumors with defective DNA repair is a clinically successful strategy. The RAS/RAF/MEK/ERK signalling pathway is frequently deregulated in human cancers. In this study, we explored the effects of MEK inhibition on the homologous recombination pathway and explored the potential for combination therapy of MEK inhibitors with DDR inhibitors and a hypoxia-activated prodrug. We studied effects of combining pimasertib, a selective allosteric inhibitor of MEK1/2, with olaparib, a small molecule inhibitor of poly (adenosine diphosphate [ADP]-ribose) polymerases (PARP), and with the hypoxia-activated prodrug evofosfamide in ovarian and pancreatic cancer cell lines. Apoptosis was assessed by Caspase 3/7 assay and protein expression was detected by immunoblotting. DNA damage response was monitored with γH2AX and RAD51 immunofluorescence staining. antitumor activity of pimasertib with evofosfamide were assessed in pancreatic cancer xenografts. We found that BRCA2 protein expression was downregulated following pimasertib treatment under hypoxic conditions. This translated into reduced homologous recombination repair demonstrated by levels of RAD51 foci. MEK inhibition was sufficient to induce formation of γH2AX foci, suggesting that inhibition of this pathway would impair DNA repair. When combined with olaparib or evofosfamide, pimasertib treatment enhanced DNA damage and increased apoptosis. The combination of pimasertib with evofosfamide demonstrated increased anti-tumor activity in BRCA wild-type Mia-PaCa-2 xenograft model, but not in the BRCA mutated BxPC3 model. Our data suggest that targeted MEK inhibition leads to impaired homologous recombination DNA damage repair and increased PARP inhibition sensitivity in BRCA-2 proficient cancers.
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http://dx.doi.org/10.18632/oncotarget.24294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837749PMC
February 2018

Characterisation of the p53 pathway in cell lines established from TH-MYCN transgenic mouse tumours.

Int J Oncol 2018 Mar 31;52(3):967-977. Epub 2018 Jan 31.

Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne NE1 7RU, UK.

Cell lines established from the TH-MYCN transgenic murine model of neuroblastoma are a valuable preclinical, immunocompetent, syngeneic model of neuroblastoma, for which knowledge of their p53 pathway status is important. In this study, the Trp53 status and functional response to Nutlin-3 and ionising radiation (IR) were determined in 6 adherent TH-MYCN transgenic cell lines using Sanger sequencing, western blot analysis and flow cytometry. Sensitivity to structurally diverse MDM2 inhibitors (Nutlin-3, MI-63, RG7388 and NDD0005) was determined using XTT proliferation assays. In total, 2/6 cell lines were Trp53 homozygous mutant (NHO2A and 844MYCN+/+) and 1/6 (282MYCN+/-) was Trp53 heterozygous mutant. For 1/6 cell lines (NHO2A), DNA from the corresponding primary tumour was found to be Trp53 wt. In all cases, the presence of a mutation was consistent with aberrant p53 signalling in response to Nutlin-3 and IR. In comparison to TP53 wt human neuroblastoma cells, Trp53 wt murine control and TH-MYCN cell lines were significantly less sensitive to growth inhibition mediated by MI-63 and RG7388. These murine Trp53 wt and mutant TH-MYCN cell lines are useful syngeneic, immunocompetent neuroblastoma models, the former to test p53-dependent therapies in combination with immunotherapies, such as anti-GD2, and the latter as models of chemoresistant relapsed neuroblastoma when aberrations in the p53 pathway are more common. The spontaneous development of Trp53 mutations in 3 cell lines from TH-MYCN mice may have arisen from MYCN oncogenic driven and/or ex vivo selection. The identified species-dependent selectivity of MI-63 and RG7388 should be considered when interpreting in vivo toxicity studies of MDM2 inhibitors.
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http://dx.doi.org/10.3892/ijo.2018.4261DOI Listing
March 2018

Targeting negative regulation of p53 by MDM2 and WIP1 as a therapeutic strategy in cutaneous melanoma.

Br J Cancer 2018 02 12;118(4):495-508. Epub 2017 Dec 12.

Northern Institute for Cancer Research, School of Medicine, Newcastle University, Paul O'Gorman Building, Framlington Place, Newcastle-upon-Tyne NE2 4HH, UK.

Background: Cutaneous melanoma is the most serious skin malignancy and new therapeutic strategies are needed for advanced melanoma. TP53 mutations are rare in cutaneous melanoma and hence activation of wild-type p53 is a potential therapeutic strategy in cutaneous melanoma. Here, we investigated the WIP1 inhibitor, GSK2830371, and MDM2-p53 binding antagonists (nutlin-3, RG7388 and HDM201) alone and in combination treatment in cutaneous melanoma cell lines and explored the mechanistic basis of these responses in relation to the genotype and induced gene expression profile of the cells.

Methods: A panel of three p53 (A375, WM35 and C8161) and three p53 (WM164, WM35-R and CHL-1) melanoma cell lines were used. The effects of MDM2 and WIP1 inhibition were evaluated by growth inhibition and clonogenic assays, immunoblotting, qRT-PCR gene expression profiling and flow cytometry.

Results: GSK2830371, at doses (⩽10 μM) that alone had no growth-inhibitory or cytotoxic effects on the cells, nevertheless significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53 but not p53 melanoma cells, indicating the potentiation worked in a p53-dependent manner. The siRNA-mediated knockdown of p53 provided further evidence to support the p53 dependence. GSK2830371 increased p53 stabilisation through Ser15 phosphorylation and consequent Lys382 acetylation, and decreased ubiquitination and proteasome-dependent degradation when it was combined with MDM2 inhibitors. These changes were at least partly ATM mediated, shown by reversal with the ATM inhibitor (KU55933). GSK2830371 enhanced the induction of p53 transcriptional target genes, cell cycle arrest and apoptosis.

Conclusions: GSK2830371, a WIP1 inhibitor, at doses with no growth-inhibitory activity alone, potentiated the growth-inhibitory and cytotoxic activity of MDM2 inhibitors by increasing phosphorylation, acetylation and stabilisation of p53 in cutaneous melanoma cells in a functional p53-dependent manner.
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http://dx.doi.org/10.1038/bjc.2017.433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830592PMC
February 2018

TP53 mutant MDM2-amplified cell lines selected for resistance to MDM2-p53 binding antagonists retain sensitivity to ionizing radiation.

Oncotarget 2016 Jul;7(29):46203-46218

Newcastle Cancer Centre, Northern Institute for Cancer Research, Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom.

Non-genotoxic reactivation of the p53 pathway by MDM2-p53 binding antagonists is an attractive treatment strategy for wild-type TP53 cancers. To determine how resistance to MDM2/p53 binding antagonists might develop, SJSA-1 and NGP cells were exposed to growth inhibitory concentrations of chemically distinct MDM2 inhibitors, Nutlin-3 and MI-63, and clonal resistant cell lines generated. The p53 mediated responses of parental and resistant cell lines were compared. In contrast to the parental cell lines, p53 activation by Nutlin-3, MI-63 or ionizing radiation was not observed in either the SJSA-1 or the NGP derived cell lines. An identical TP53 mutation was subsequently identified in both of the SJSA-1 resistant lines, whilst one out of three identified mutations was common to both NGP derived lines. Mutation specific PCR revealed these mutations were present in parental SJSA-1 and NGP cell populations at a low frequency. Despite cross-resistance to a broad panel of MDM2/p53 binding antagonists, these MDM2-amplified and TP53 mutant cell lines remained sensitive to ionizing radiation (IR). These results indicate that MDM2/p53 binding antagonists will select for p53 mutations present in tumours at a low frequency at diagnosis, leading to resistance, but such tumours may nevertheless remain responsive to alternative therapies, including IR.
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http://dx.doi.org/10.18632/oncotarget.10073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216791PMC
July 2016

Chemical Inhibition of Wild-Type p53-Induced Phosphatase 1 (WIP1/PPM1D) by GSK2830371 Potentiates the Sensitivity to MDM2 Inhibitors in a p53-Dependent Manner.

Mol Cancer Ther 2016 Mar 1;15(3):379-91. Epub 2016 Feb 1.

Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom.

Sensitivity to MDM2 inhibitors is widely different among responsive TP53 wild-type cell lines and tumors. Understanding the determinants of MDM2 inhibitor sensitivity is pertinent for their optimal clinical application. Wild-type p53-inducible phosphatase-1 (WIP1) encoded by PPM1D, is activated, gained/amplified in a range of TP53 wild-type malignancies, and is involved in p53 stress response homeostasis. We investigated cellular growth/proliferation of TP53 wild-type and matched mutant/null cell line pairs, differing in PPM1D genetic status, in response to Nutlin-3/RG7388 ± a highly selective WIP1 inhibitor, GSK2830371. We also assessed the effects of GSK2830371 on MDM2 inhibitor-induced p53(Ser15) phosphorylation, p53-mediated global transcriptional activity, and apoptosis. The investigated cell line pairs were relatively insensitive to single-agent GSK2830371. However, a non-growth-inhibitory dose of GSK2830371 markedly potentiated the response to MDM2 inhibitors in TP53 wild-type cell lines, most notably in those harboring PPM1D-activating mutations or copy number gain (up to 5.8-fold decrease in GI50). Potentiation also correlated with significant increase in MDM2 inhibitor-induced cell death endpoints that were preceded by a marked increase in a WIP1 negatively regulated substrate, phosphorylated p53(Ser15), known to increase p53 transcriptional activity. Microarray-based gene expression analysis showed that the combination treatment increases the subset of early RG7388-induced p53 transcriptional target genes. These findings demonstrate that potent and selective WIP1 inhibition potentiates the response to MDM2 inhibitors in TP53 wild-type cells, particularly those with PPM1D activation or gain, while highlighting the mechanistic importance of p53(Ser15) and its potential use as a biomarker for response to this combination regimen.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785723PMC
March 2016