Search

// Francesca Vena 1, * , Ruochen Jia 1, * , Arman Esfandiari 1 , Juan J. Garcia-Gomez 1 , Manuel Rodriguez-Justo 2 , Jianguo Ma 3 , Sakeena Syed 3 , Lindsey Crowley 3 , Brian Elenbaas 3 , Samantha Goodstal 3 , John A. Hartley 1 and Daniel Hochhauser 1 1 Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, Paul O’Gorman Building, University College London, London WC1E 6DD, UK 2 Department of Research Pathology, UCL Cancer Institute, London EC1M6BQ, UK 3 EMD Serono Research and Development Institute, Billerica 01821, MA, USA * These authors have contributed equally to this work Correspondence to: Daniel Hochhauser, email: d.hochhauser@ucl.ac.uk Keywords: DNA damage; pancreatic cancer; ovarian cancer; targeted therapy; MEK inhibitors Received: January 08, 2018 Accepted: January 15, 2018 Published: January 22, 2018 ABSTRACT 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. In vivo 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.

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 p53WT (A375, WM35 and C8161) and three p53MUT (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 p53WT but not p53MUT 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.

Background The tumor suppressor p53 is a central hub in molecular signaling pathways that control the integrity of the human genome. The p53 protein functions as a transcription factor and increases the expression of many cellular genes which contribute to activation of cell cycle arrest, apoptosis and DNA repair. MDM2 is another important p53 target gene, and the MDM2 protein is capable of binding directly to p53 and directing it for degradation through the ubiquitin-dependent proteolytic pathway. Inhibition of MDM2 stabilizes p53 and MDM2 inhibitors are being explored clinically as therapies. Stabilization alone may not be enough to increase the activity of p53, and posttranslational modification of p53 by phosphorylation has been proposed to be an important contributory mechanism by which p53 becomes functionally active. Therefore maintaining the phosphorylated status of p53 in tumor cells may help to enhance its growth inhibitory and pro-apoptotic role. Wild type p53 – induced phosphatase (Wip1) is a serine – threonine phosphatase which dephosphorylates central players in the DNA damage response, including p53 and may be an additional target to enhance p53-dependent treatments. Therefore this work was focused on the effect of MDM2 (RG7388) and Wip1 (GSK2830371) inhibitors on MX-1 and MCF breast carcinoma cell lines. These two cell lines were recorded to have wild type TP53 status as well as high expression of Wip1. Trial design RG7388 and GSK2830371 were tested for growth inhibition on MX-1 and MCF-7 breast cancer cell lines using the sulforhodamine B (SRB) assay. The results were further confirmed and mechanism explored by western blotting using extracted protein from drug treated cell lines. Contradictory evidence regarding the TP53 mutation status of the MX-1 cell line was clarified by direct sequencing of MX-1 DNA. Results The MCF-7 cells responded to both RG7388 and GSK2830371 with GI50 value of 0.034 µM and 2.92 µM respectively. The MX-1 cells did not respond to either drug. The results of western blotting showed there was no expression of p53 in the MX-1 cell line. Failure to respond to RG7388and also no expression of p53 in western blotting made us suspicious about the TP53 status of the MX-1 cells. The direct sequencing results confirmed that there was a 5bp deletion in exon 4 of the TP53 gene of the MX-1 cells. The c.154_158delCAATG mutation creates a stop codon at the 54th aminoacid position and results in a truncated p53 protein (p.Gln52Valfs*3). Conclusion RG7388 and GSK2838371 showed cytotoxic effects on MCF-7 cells, whereas both RG7388 and GSK2838371 had no effect on the MX-1 cell line due to the truncated p53 and loss of p53 function. In conclusion, the potency of both drugs depends on the TP53 mutation status and they are likely to be mediated via p53-dependent growth inhibition and apoptosis. Further studies are needed to evaluate the combination effect of both drugs on TP53 wild type cell lines. Citation Format: Manoharan V, Lunec J, Esfandiari A, Mahdi A, Wu C-E, Zanjirband M, Karunanayake EH, Tennekoon KH, De Silva S. Cytotoxic potential of the RG7388 MDM2-p53 binding antagonist and the GSK2830371 WIP1 inhibitor on MX-1 and MCF-7 human breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-21.

MAPK and p14ARF⁻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 BRAFV600E and p53WT 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 BRAFV600E and p53WT 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.

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.

Introduction MAPK and ARF-MDM2-p53 pathways are critical in cutaneous melanomas. This study examined the combination treatment of MEK inhibitor, trametinib, with MDM2 inhibitors, nutlin-3/RG7388/HDM201, in BRAFV600E mutant and p53 wild-type (p53WT) melanoma cell lines and explored the mechanistic basis of these responses. Material and methods Two BRAFV600E and p53WT melanoma cell lines, A375 and WM35, were treated with either trametinib or MDM2 inhibitors, or combinations. Results and discussions The combination treatment in both A375 and WM35 showed additive to synergistic effects by Chou-Talalay median effect analysis and combination index scores. The combination treatments induced higher levels of p53 target gene transcripts (CDKN1A(p21), MDM2, BAX, BBC3(PUMA), TP53I3(PIG-3)) and protein products (p21, MDM2), 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 mRNA and protein were down-regulated following pERK reduction by trametinib, but did not change after p53 activation by MDM2 inhibitors. Furthermore, suppression of DUSP6 by siRNA, or inhibition with the small molecule inhibitor, BCI, potentiated the effect of MDM2 inhibitors through increased ATM-dependent p53 phosphorylation, as demonstrated by complete reversal with the ATM inhibitor, KU55933. Conclusion Trametinib synergizes with MDM2 inhibitors through a DUSP6 mechanism in BRAFV600E and p53WT melanoma cells. DUSP6 regulation of p53 phosphorylation via ATM, could be a therapeutic target for combination with treatments involving activation of the ATM/p53 pathway.

Cutaneous melanoma is the most serious skin malignancy. The current study aimed to investigate the WIP1 inhibitor GSK2830371 and MDM2-p53 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. A panel of three p53WT (A375, WM35, C8161) and three p53MUT (WM164, WM35-R5R1, CHL-1) melanoma cell lines were used. GSK2830371 (≤10 μM) alone had no growth-inhibitory or cytotoxic effects on the cells, measured by sulforhodamine B (SRB) and clonogenic assays. In combination treatment GSK2830371 significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53WT but not p53MUT melanoma cells, indicating the potentiation worked in a p53-dependent manner (Table). Western blotting demonstrated GSK2830371 increased p53 stabilization through Ser15 phosphorylation and consequent Lys382 acetylation when it was combined with MDM2 inhibitors. These changes were ATM-mediated, shown by reversal with the ATM inhibitor (KU55933). Furthermore, GSK2830371 was demonstrated to slow down p53 degradation when de-novo protein synthesis was inhibited by cycloheximide. In qRT-PCR, nutlin-3 or RG7388 induced p53 transcriptional target genes (CDKN1A, MDM2, BAX, FAS, PUMA, TNFBSF10B, TP53INP1) and GSK2830371 enhanced the induction in p53WT but not p53MUT cells. In conclusion, 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 stabilization of p53 in cutaneous melanoma cells in a functional p53-dependent manner. Further studies in vivo are warranted to investigate the efficacy of this combination treatment. GI50 by sulforhodamine B (SRB) and LC50 by clonogenic assays in p53WT melanoma cellsCell linesGI50 (Mean + SEM)A375WM35C8161GSK2830371-+-+-+Nutlin-3 (µM)5.1 + 0.61.5 + 0.48.3 + 1.55.3 + 1.41.7 + 0.31.0 + 0.5p value0.0120.0100.049RG7388 (nM)228 + 3962 + 2377 + 58169 + 5746 + 015 + 5p value0.0250.0280.009HDM201 (nM)167 + 2541 + 9165 + 9877 + 5981 + 1149 + 10p value0.00460.1048< 0.001LC50 (Mean + SEM)A375WM35C8161GSK2830371-+-+-+Nutlin-3 (µM)2.0 + 0.90.8 + 0.50.5 + 0.10.4 + 0.10.9 + 0.20.4 + 0.1p value0.0400.07140.003RG7388 (nM)166 + 9554 + 37186 + 7868 + 2350 + 2221 + 10p value0.0060.0700.026HDM201 (nM)198 + 7015 + 411 + 38 + 3139 + 8519 + 5p value Citation Format: Chiao-En Wu, Arman Esfandiari, Yi-Hsuan Ho, Colin Shepherd, Ahmed Khairallah Mahdi, Erhan Aptullahoglu, John Wen-Cheng Chang, Penny Lovat, John Lunec. Inhibition of WIP1/PPM1D phosphatase by GSK2830371 potentiates the growth inhibitory and cytotoxic activity of MDM2 antagonists (nutlin-3, RG7388 and HDM201) in cutaneous melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2151. doi:10.1158/1538-7445.AM2017-2151