Search

Antibody-drug conjugates (ADCs) delivering pyrrolobenzodiazepine (PBD) DNA crosslinkers are currently being evaluated in clinical trials with encouraging results in Hodgkin and non-Hodgkin lymphomas. The first example of an ADC delivering a PBD DNA crosslinker (loncastuximab tesirine) has been recently approved by the U.S. Food and Drug Administration for the treatment of relapsed and refractory diffuse large B-cell lymphoma. There has also been considerable interest in mono-alkylating PBD analogues. We conducted a head-to-head comparison of a conventional PBD bis-imine and a novel PBD mono-imine. Key Mitsunobu chemistry allowed clean and convenient access to the mono-imine class. Extensive DNA binding studies revealed that the mono-imine mediated a type of DNA interaction that is described as "pseudo-crosslinking," as well as alkylation. The PBD mono-imine ADC demonstrated robust antitumor activity in mice bearing human tumor xenografts at doses threefold higher than those that were efficacious for the PBD bis-imine ADC. A single-dose toxicology study in rats demonstrated that the maximum tolerated dose of the PBD mono-alkylator ADC was approximately threefold higher than that of the ADC bearing a bis-imine payload, suggesting a comparable therapeutic index for this molecule. However, although both ADCs caused myelosuppression, renal toxicity was observed only for the bis-imine after repeated dosing, indicating possible differences in toxicological profiles that could influence tolerability and therapeutic index. These data show that mono-amine PBDs have physicochemical and pharmaco-toxicological properties distinct from their crosslinking analogues and support their potential utility as a novel class of ADC payload.

Supplementary figure S2: A) Western blot shows optimisation of anti-WIP1 antibody (F-10) using siRNA mediated knockdown in MCF-7 cells. Following 24 and 48 hours of siRNA mediated knockdown conditions, MCF-7 cells were treated with 10Gy IR, lysates were collected after 4 hours and analysed by western blotting. All the bands shown were knocked down in response to four different WIP1 targeting siRNA constructs (WIP1.1-4) compared to the universal control siRNA (Cont. siRNA). WIP1 knockdown also resulted in higher and persistent pp53Ser15 most notably in response to the WIP1.2 siRNA construct. WIP1.1 Sense: GUGCCAUAGUAAUCUGCAU; WIP1.2 Sense: GGUGUAGUCAUACCCUCAA; WIP1.3 Sense: GCCCUUCCUAUAAUAGUCA; WIP1.4 Sense: UUGGCCUUGUGCCUACUAA Universal control Sense: GCGCGCUUUGUAGGAUUCG. B) Nutlin-3 and RG7388 SRB growth inhibition curves in MCF-7 cells in the presence and absence of 2.5µM GSK2830371. C) On the left side two independent repeats of the western blot in Figure 2A showing degradation of WIP1 protein in MCF-7 cells over time following treatment with 2.5µM GSK2830371. Full-length WIP1 band optical density (OD) was normalised to actin OD over time.

Supplementary figure S4: A) Time-course of cell cycle distribution changes over 72 hours of treatment using FACS analysis. NGP and SJSA-1 cells were treated either with DMSO solvent control (Black), 2.5µM GSK2830371 (Blue), (GI50) Nutlin-3 (Red) or Nutlin-3 + GSK2830371 combination (Purple). HCT116+/+ cells were treated with 0.5 × Nutlin-3 GI50 in single and in combination treatment. B) Representative cell cycle distribution histograms for NGP and SJSA-1 cells indicating the increase in the proportion of Sub-G1events in response to 72 hours of treatment with GSK2830371 (WIP1i), Nutlin-3 and their combination.

Antibody–drug conjugates (ADC) delivering pyrrolobenzodiazepine (PBD) DNA cross-linkers are currently being evaluated in clinical trials, with encouraging results in Hodgkin and non–Hodgkin lymphomas. The first example of an ADC delivering a PBD DNA cross-linker (loncastuximab tesirine) has been recently approved by the FDA for the treatment of relapsed and refractory diffuse large B-cell lymphoma. There has also been considerable interest in mono-alkylating PBD analogs. We conducted a head-to-head comparison of a conventional PBD bis-imine and a novel PBD mono-imine. Key Mitsunobu chemistry allowed clean and convenient access to the mono-imine class. Extensive DNA-binding studies revealed that the mono-imine mediated a type of DNA interaction that is described as “pseudo cross-linking,” as well as alkylation. The PBD mono-imine ADC demonstrated robust antitumor activity in mice bearing human tumor xenografts at doses 3-fold higher than those that were efficacious for the PBD bis-imine ADC. A single-dose toxicology study in rats demonstrated that the MTD of the PBD mono-alkylator ADC was approximately 3-fold higher than that of the ADC bearing a bis-imine payload, suggesting a comparable therapeutic index for this molecule. However, although both ADCs caused myelosuppression, renal toxicity was observed only for the bis-imine, indicating possible differences in toxicologic profiles that could influence tolerability and therapeutic index. These data show that mono-amine PBDs have physicochemical and pharmacotoxicologic properties distinct from their cross-linking analogs and support their potential utility as a novel class of ADC payload.

Supplementary Table S1: A & B) List of genes with significantly increased mRNA levels in NGP cells 4 hours following treatment with 75nm RG7388 (GI50 in NGP cells) + 2.5µM GSK2830371. Genes were ordered based on p-value following Benjamini-Hotchberg correction for multiple testing. OR: Odds ratio.

Supplementary information to support article findings.

Reducing vertical motions of high-speed planing hulls in rough water is one of the most important factors that help a boat to become more operable, and will benefit the structure of the boat and the crew on board. In the recent decade, stepped planing hulls have been investigated with emphasis on their better performance in calm water than that of nonstepped planing hulls. However, there are still doubts about their performance in rough water. In this study, we investigate this problem by providing numerical simulations for motions of a double-stepped and a non-stepped planing hull in a vertical plane when they encounter head waves. The problem will be solved using the finite volume method and volume of fluid method. To this end, a numerical computational fluid dynamics code (STARCCM1) has been used. Accuracy of the numerical simulations is evaluated by comparing their outcome with available experimental data. The dynamic response of the investigated hulls has been numerically modeled for two different wave lengths, one of which is smaller than the boat length and the other which is larger than the boat length. Using the numerical simulations, heave and pitch motions as well as vertical acceleration are found. It has been found that at wave lengths larger than the boat length, heave amplitude decreases by 10–40%when two steps are added to the bottom of a vessel. It has also been observed that pitch of a planing hull is reduced by 18–32% in the presence of the two steps on its bottom. Finally, it has been observed that for wave lengths larger than the boat length, the maximum vertical acceleration decreases by a gravitational acceleration of about .2–.7.

Change in body shape characteristics is one of the ways to reduce the resistance and thereby increasing the speed of planing hulls. Creating the transverse steps is one of these variations. The main reason to use the steps in high-speed planing craft is that the wetted surface of the vessel is divided into small parts with higher width-length ratio in high velocities and in this situation, the generated lift force is more efficient. In this article, by performing a three-dimensional numerical solution, motion characteristics of a two-stepped planing hull with transverse steps in calm water have been examined. For this purpose, the vessel is free to trim and sinkage, and by using the morphing mesh approach, the numerical simulation continued until the equilibrium condition of the two-stepped planing hull is satisfied. Resistance, lift, trim angle, and wetted surface in various velocities have been computed and compared against existing experimental data. Analysis of considered two-stepped hull in calm water shows that the numerical solution for resistance, trim, and lift are relatively precise in comparison to model test data. Furthermore, various hull characteristics such as wetted length of keel, chine wetted length, spray angle and, ventilation length have been investigated. 1. Introduction In recent years, naval architectures have proposed significant modifications in planing hulls' design by creating steps, tunnel, and other geometric features. By considering these geometric variations, the hull wetted surface and, consequently, its resistance have been reduced and planing hulls velocity can be increased. In this regard, the step is one of the main hull's modifications which can lead to resistance reduction. Generally, lift of stepped planing hulls is distributed over the forward, middle, and stern plane of the hull (Fig. 1), and therefore, its ride quality will also be improved. Moreover, planing hulls with various step configurations have been used in high-speed craft since 1910, and various types of step with different shapes and arrangements have been used in planing hulls.

// 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.

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 p53Ser15 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 p53Ser15, 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 p53Ser15 and its potential use as a biomarker for response to this combination regimen. Mol Cancer Ther; 15(3); 379–91. ©2016 AACR.

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.

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.

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

The mechanisms that determine p53 cell fate decision after its non-genotoxic activation by MDM2 inhibitors are poorly understood. Wild-type p53-inducible phosphatase-1 (WIP1/PPM1D) forms an autoregulatory loop with p53 and is involved in the homeostasis of the p53 stress response network. WIP1 is activated, gained/amplified and overexpressed in a range of p53 wild-type malignancies. We investigated cellular growth/proliferation of p53 wild-type (wt) and matched p53 mutant (mut)/null cell line pairs, differing in WIP1 genetic status, in response to Nutlin-3 + GSK2830371 (WIP1i)/WIP1 siRNA-mediated knockdown. We also assessed the effects of WIP1i or knockdown in combination with Nutlin-3 on p53 post-translational modifications, p53 downstream transcriptional targets and apoptosis. Phosphorylation of p53S15 (pp53S15) was used as a marker of WIP1 catalytic activity. WIP1-amplified MCF-7p53wt cells were very sensitive to the WIP1i, with a 50% growth inhibitory concentration (GI50) of 2.65μM + 0.54 (SEM). The three p53 wild-type and mutant/null cell line pairs showed relatively little sensitivity to single agent WIP1i (GI50>10μM). However the WIP1i, at a non-growth inhibitory dose (2.5μM) in vitro, potentiated their response to Nutlin-3 in a p53-dependent manner. HCT116p53+/+; WIP1L450fs*1/+ (L450fs*1 is a WIP1 activating mutation), NGPp53wt and SJSA-1p53wt cell lines showed a 2.4-fold (p = 0.007), 2.1-fold (p = 0.04) and 1.3-fold (p = 0.02) decrease in their Nutlin-3 GI50 values respectively in the presence of WIP1i at 2.5μM. In contrast Nutlin-3 GI50 did not change in HCT116p53-/−, N20R1p53Mut and SN40R2p53Mut, NGP and SJSA-1 Nutlin-3 resistant daughter cell lines respectively. WIP1 transient siRNA knockdown also resulted in potentiation of cells to Nutlin-3. In WIP1-non-amplified (WNA) cells treated with either Nutlin-3 at the GI50 dose or 2.5μM WIP1i, pp53S15 was not detected, however in combination, these drugs resulted in dramatic levels of pp53S15. Interestingly protein expression of p53 targets MDM2 and p21 were not affected in WNA cells by the increased p53S15 phosphorylation; whereas BAX expression was elevated. The Nutlin-3 + WIP1i/siRNA knockdown combination also resulted in a significant increase in cell death markers between 24-48Hrs post-treatment in a p53-dependent manner. Although the strongest WIP1i potentiation of Nutlin-3 was observed in HCT116p53+/+; WIP1L450fs*1/+ cells which possess an activating WIP1 mutation, the NGPp53wt and SJSA-1p53wt cell lines with wild-type WIP1 also showed potentiation. These findings highlight the mechanistic importance of the p53-WIP1 autoregulatory loop in cell fate determination after non-genotoxic p53 activation by Nutlin-3. Potent inhibitors of WIP1 may potentiate the response to other therapeutics aiming to activate p53 mediated tumour suppression. Citation Format: Arman Esfandiari, Nicola J. Curtin, John Lunec. Chemical inhibition or transient knockdown of wild-type p53 induced phosphatase 1 (WIP1/PPM1D) potentiates the response to MDM2 inhibitors in a p53-dependent manner. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1785. doi:10.1158/1538-7445.AM2015-1785