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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.

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.

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