Translational Science

The MDM2/MDMX-p53 Antagonist PM2Radiosensitizes Wild-Type p53 TumorsDiana Spiegelberg1,2, Anja C. Mortensen1, Sara Lundsten1,Christopher J. Brown3, David P. Lane3,4, and Marika Nestor1

Abstract

Radiotherapy amplifies p53 expression in cancer cells withwild-type (wt) p53. Blocking the negative regulators MDM2and MDMX stabilizes p53 and may therefore potentiateradiotherapy outcomes. In this study, we investigate the effi-cacy of the novel anti-MDM2/X stapled peptide PM2 aloneand in combinationwith external gamma radiation in vitro andin vivo. PM2 therapy combined with radiotherapy elicitedsynergistic therapeutic effects compared with monotherapyin cells with wt p53 in both in vitro and in vivo assays, whereasthese effects did not manifest in p53 �/� cells. Biodistributionand autoradiography of 125I-PM2 revealed high and retaineduptake homogenously distributed throughout the tumor. Inmice carrying wt p53 tumors, PM2 combined with radiother-

apy significantly prolonged the median survival by 50%,whereas effects of PM2 therapy onmutant and p53 �/� tumorswere negligible. PM2-dependent stabilization of p53 wasconfirmed with ex vivo immunohistochemistry. These datademonstrate the potential of the stapled peptide PM2 as aradiotherapy potentiator in vivo and suggest that clinical appli-cation of PM2 with radiotherapy in wt p53 cancers mightimprove tumor control.

Significance: These findings contribute advances to cancerradiotherapy by using novel p53-reactivating stapled pep-tides as radiosensitizers in wild-type p53 cancers. Cancer Res;78(17); 5084–93. �2018 AACR.

IntroductionCurrently new drugs are developed at an extraordinary pace,

and as a result patients with cancer have far more treatmentoptions today than what existed even a decade ago. However,the ultimate goal of cancer research—"a cure for each cancerpatient"—remains out of reach. Today, we know that cancer isnot a single disease; instead, the term summarizes a class ofdifferent diseases where each patient-borne tumor has its veryown heterogeneous genetic setup (1). It has therefore becomeapparent that the existence of a single treatment that wouldbenefit all patients is very unlikely, barring the traditional radio-or chemotherapies targeting generic increase in cell growth. Amore realistic approach in cancer patient management is to usethe vast armamentarium of specific cancer drugs and therapies,and tailor the therapies to each individual patient based on theirindividual needs, that is, precision medicine (2). The precisetailoring of combining several, specific treatments can lead tosynergistic effects; by utilizing differentmechanisms of action that

harm the cancer cells at multiple sites. These combination treat-ments have the potential to improve the current clinical practice,by helping patients who do not respond to available therapies orsuffer intolerable side effects, as well as by decreasing the likeli-hood that resistant cells will develop (3). Furthermore, whencombining different treatment strategies, each treatment can beused at its optimal dose, thus reducing the risk of unacceptableadverse reactions as long as potential negative interactions such asincreased toxicity are accounted for.

One promising combination strategy is to combine radiother-apy with cancer drugs that exhibit radiosensitizing properties.Radiation, especially external beam radiotherapy with high-ener-gy photons (gamma and X-rays), is one of the most commontreatments for solid cancers; and hospitals in all parts of theworldpossess the necessary equipment. Radiotherapy is especially use-ful for eradicating cancer cells where surgery is difficult and aspost-surgical complementary treatment for removing remainingcells. However, radioresistant tumor cell sub-populations andtoxicity to normal tissues limit its efficacy. Overcoming thesenegatives would be clinically beneficial, for example, by findingdrugs that can radiosensitize tumor cells in a highly specificmanner (3).

Suitable candidates for radiosensitizing drugs are arguablythose that target radiation response mechanisms, such as stressresponse, cell/DNA repair, and apoptosis. One example is thetranscription factor p53, which is activated by cellular stress suchas exposure to ionizing radiation, hypoxia, and carcinogenicsubstances. In its wild-type (wt) state, p53 is an important tumorsuppressor and drives highly damaged or stressed cells into cell-cycle arrest or programmed cell death via several pathways (4).

In many tumors, neutralization and enhanced degradation ofwt p53 is a result of overexpression of E3 ubiquitin ligase (MDM)family members (5, 6). Induced p53 levels in the cell activatep53-dependent Mdm2 and MdmX gene transcription, leading to

1Department of Immunology, Genetics & Pathology, Uppsala University,Uppsala, Sweden. 2Department of Surgical Sciences, Uppsala University,Uppsala, Sweden. 3p53Lab, Agency for Science Technology and Research(A�STAR), Singapore, Singapore. 4Department of Microbiology, Tumor and CellBiology, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

D. Spiegelberg and A.C. Mortensen contributed equally to the article.

Corresponding Author: Marika Nestor, The Rudbeck Laboratory, UppsalaUniversity, 75185 Uppsala, Sweden. Phone: 46-70-234-1881; Fax: 46-18-471-3432; E-mail: Marika.Nestor@igp.uu.se

doi: 10.1158/0008-5472.CAN-18-0440

�2018 American Association for Cancer Research.

CancerResearch

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p53-MDM2/X complex formation and finally p53 degradation.This autoregulatory feedback loop controls p53 expression levelsin normal cells. In tumors however, the cell-cycle arrest andapoptotic responses are prevented, resulting in aggressive growth,increased radioresistance, chemotherapy resistance, and subse-quently a poor prognosis (7, 8). On the basis of our currentunderstanding, blocking the negative p53 regulator MDM2 byinhibiting the p53-MDM2 protein–protein interaction mayrestore and activate wt p53 signaling in tumors, thus re-enablingthe normal p53 mediated cell-cycle arrest and apoptosis.

Several small molecules targeting the p53–MDM2 interaction(e.g., Nutlin-3, RITA, PRIMA-1, isoquinolin-1-one–based deriva-tives; PXN) have been developed in the recent years. One of themost intensively studied MDM2-antagonists is Nutlin-3,although several of the newer agents demonstrate a better oralbioavailability, lower toxicity and higher specific affinity to theN-terminal–binding site of MDM2 (9). All these small-moleculeMDM2 inhibitors have been shown to restore important tumor-suppressive functions such as cell-cycle arrest and apoptosis.However, overexpression of MDMX (also known as MDM4), astructural homologue of MDM2, can act as an MDM2 substitute,causing drug resistance (6, 10). MDMX has no ubiquitylationactivity, but it is able to inactivate p53 directly by binding to theN-terminus as well as indirectly by heterodimerization withMDM2assistingMDM2's ubiquitylating function (4). The limitedeffect of Nutlin-3 on MDMX-overexpressing cells among othershas prevented its translation to the clinic (11).

Consequently, molecules that selectively target MDM2 andMDMX are highly interesting. One example of a novel com-pound that targets both MDM2 and MDMX is the stapledpeptide PM2, which shows high affinity to both proteins anddisplays high in vivo stability resulting from a stapling process(12). Stapled peptides are emerging as a promising new classof compounds that selectively disrupt protein interactions.Stapled polypeptides are stabilized by introducing a chemicaltether to link amino acid side-chains together to create amacrocyclic compound. The resulting stapled peptide canexhibit radically changed properties from otherwise biological-ly inert peptides, such as increased cell penetration, proteaseresistance and target affinity (12).

The aim of this study was to evaluate the potential ofcombining the novel stapled peptide PM2 and ionizing radi-ation in vivo, using a panel of cancer cell lines and tumorxenografts of p53 wild-type, p53 knock-out (�/� HCT116) aswell as mutant p53 genotypes. PM2 therapy in combinationwith radiotherapy is a unique approach, and this is the firststudy to investigate the combination of the different treatmentsin an in vivo model.

Materials and MethodsCell lines

Cell lines used in the study were the human adenocarcinomacell lines HCT116 (wt p53), HCT116 (p53 �/�) obtained fromHorizon discovery, UK, the murine fibroblast cell line T22(wt p53), the pancreatic neuroendocrine cell line BON (mutantp53), provided by Prof. Townsend (The University of TexasMedical Branch, Texas University, Galveston, TX), and the humansquamous cell carcinoma cell lines UM-SCC-74A (wt p53) andUM-SCC-74B (wt p53), provided by Professor TE Carey (Univer-sity of Michigan). Authentication of the SCC cell lines was made

by DNA (STR) profiling. Cell cultures were routinely tested andfound to be negative for Mycoplasma. The cell lines in this studywere cultured for less than 6 months after delivery. Detailedinformation on cells and culture conditions is listed in theSupplementary Methods.

Drug and radiation treatmentPM2 (MW¼1462.75Da,Kd¼34.35�2.03nmol/L forMDM2

and45.73� 7.65 nmol/L forMDMX; ref. 13) and PM2-scrambledpeptides were synthesized and provided by the p53lab.Their respective sequences are Ac-TSFR8EYWALLS5-NH2 andTSLR8EYFALWS5-NH2. Stapled peptides were produced viaring closing metathesis (RCM) of the olefin bearing unnaturalamino acids R8 ((R)-2-(40octenyl) alanine) and S5 (and (S)-2-(40-pentenyl) alanine). Full details of the peptide synthesis meth-odology have been previously published (13). All peptides wereamidated at their C-terminus and acetylated at their N-terminus.Peptides were purified using HPLC to >95% purity and lyophi-lized. Ten mmol/L stock peptide solutions were prepared bydissolving the lyophilized powder in DMSO (13). IC50 valuesof PM2 have been measured at 0.94 � 0.04 mmol/L and 0.66 �0.06 mmol/L for MDM2 and MDMX, respectively (13). Radio-therapywas performed using a 137Cs g-ray irradiator at a dose-rateof 1 Gy/min (Best Theratronics Gammacell 40 Exactor). Fulldetails on PM2 drug administration and radiation treatments aregiven in the Supplementary Methods.

Western blot analysesWestern blottingwas performed to assess p53, p21, andMDM2

protein expression in HCT116, HCT116 p53 �/� and T22 cells asdescribed in the Supplementary Methods.

p53 activation assay and LDH release assayp53 transcriptional activation and LDH release profile by PM2

or scrambled peptide was assessed on T22 reporter cells asdescribed in the Supplementary Methods.

Cell viability assay (XTT), clonogenic survival, and solubilityassay

Cell viability and IC50 value was evaluated on HCT116 cellstreated with 0.1 to 40 mmol/L PM2 using the XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilidesalt) assay as described in the Supplementary Methods. Solu-bility of PM2 was assessed by using a standard additionapproach as described in the Supplementary Methods. Colonyforming ability (clonogenic survival) was tested as describedearlier (14) on cells being exposed to radiation doses of 0, 2, 4or 6 Gy and/or 20 mmol/L PM2. A more detailed descriptioncan be found in the Supplementary Methods.

Multicellular tumor spheroidsThree-dimensional multicellular tumor spheroids surrogates

non-vascularized micrometastases in vivo and is an especiallysuitable tool to investigate effects of radiation, because itharbors parameters that influence cellular responses to ionizingradiation such as hypoxia, local variations in cell signalingand cell proliferation as well as local pH variations and nutrientgradients (15). Consequently, in vitro therapy experiments wereperformed on 3D multicellular tumor spheroids, using wt p53,p53 �/�, and mutant p53 cancer cells as described in theSupplementary Methods.

PM2 Potentiates Radiotherapy in wt p53 Cancer

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Direct labeling of PM2 with the radionuclide 125IPM2 was radiolabeled with 125I using chloramine-T as

described in the Supplementary Methods. Labeling yield andstability in PBS and mouse serum was evaluated using instantthin layer chromatography as described in the SupplementaryMethods.

In vivo mouse xenograft modelsFemale nu/nu Balb/c mice (n ¼ 88) were housed under stan-

dard laboratory conditions and fed ad libitum. All experimentscomplied with Swedish law and were performed with permissionfrom the Uppsala Committee of Animal Research Ethics.

Tumor xenografts were formed by subcutaneous inoculation ofapproximately 1 � 106 wt p53 HCT116 (58 animals) or 1 � 106

�/�HCT116 (20 animals) cells suspended in 100 mL serum-freecell culture medium in the right posterior leg. One HCT116 wtp53mousewas excluded from the study due to lack of tumor take.In 10 mice, approximately 10 � 106 wt p53 UM-SCC-74B cellssuspended in 100 mL serum-free cell medium were injectedsubcutaneously into the left posterior leg and 5.5 � 106 mutantp53 BON cells suspended in 100 mL serum-free cell mediumwereinjected subcutaneously into the right posterior leg. After approx-imately 7 to 10 days tumors had established.

125I-PM2 biodistributionBiodistribution of 130 mmol/L 125I-PM2 in wt p53 HCT116

xenografts (n¼ 7)was studied ex vivo 6 (n¼ 3), 24 (n¼ 3), and 48hours (n ¼ 1) p.i. Animals were euthanized with a mixture ofketamine and xylazine followed by heart puncture. Blood wascollected, HCT116 tumors, thyroid (en bloc with larynx), heart,liver, kidneys, spleen, urinary bladder, colon, upper gastrointes-tinal tract, skin, bone and muscle were excised, weighed andmeasured in a gamma well-counter (1480 Wizard; Wallace Oy,Turku, Finland). Injection standards weremeasured for each timepoint. Radioactivity uptake in the organ was calculated as thepercentage of injected dose per gram of tissue (%ID/g). Thyroiduptake was calculated as the percentage of injected dose per organ(%ID/organ).

Digital autoradiographyDigital autoradiography on the wt p53 HCT116 xenografted

tumors was performed ex vivo following one injection of130 mmol/L 125I-PM2 as described in the SupplementaryMethods.

In vivo tumor growth and survivalApproximately 7 to 10 days after tumor inoculation, the

treatment was initiated according to the following schedule:wt p53 HCT116 control (n ¼ 11), 10� PM2 (n ¼ 6), 3 � 2 Gy(n ¼ 11), 10� PM2 and 3 � 2 Gy (n ¼ 7), 3� PM2 (n ¼ 7), 3�PM2 and 2 � 3 Gy (n ¼ 8), and �/� HCT116 control (n ¼ 5),10� PM2 (n ¼ 5), 3� 2 Gy (n¼ 5), 10� PM2 (n¼ 5) and 3� 2Gy group (n ¼ 5); see also descriptions in supplementary drugand radiation treatment section. All animals were euthanizedwith a mixture of ketamine and xylazine followed by heartpuncture when the tumor size reached a maximum of 1,000mm3. Tumor size (V ¼ 4pr1r2r3/3) and body weight wasmonitored in a blinded manner throughout the experiment.In an additional in vivo study, effects of PM2 treatment wasstudied in wt p53 UM-SCC-74B and mutant p53 BON xeno-grafts. PM2 treatment or mock treatment was performed for 10

consecutive days as described in supplementary drug andradiation treatment section (n ¼ 4 UM-SCC-74B control, n ¼5 all other groups). Tumor size and body weight was followedin a blinded manner during treatment, followed by euthaniza-tion at the end of treatment.

Ex vivo immunohistochemistryDrug and radiation treatment as described in the Supple-

mentary Data. Wt p53 HCT116 and �/� HCT116 tumors andliver were formalin fixated directly after dissection when tumorsize reached approximately 1,000 mm3 (n ¼ 18). Tumors werehematoxylin and eosin (H&E) stained and stained for p53expression, and livers were H&E stained as described in theSupplementary Methods. Wt p53 UM-SCC-74B tumors (n ¼ 6)and mutant p53 BON tumors (n ¼ 6) were formalin fixatedafter treatment and stained as described in the SupplementaryMethods.

Statistical analysisStatistical analyses were performed for analyses on IC50 value,

differences between treatment groups, Kaplan–Meier analysis,and combinative effects as described in the SupplementaryMethods.

ResultsPM2 characterizations—PM2 permeates the cell membrane,activates p53 transcription, and reduces cell viability in wt p53cells in a dose-dependent manner

PM2 was further characterized by assessing the LDH releaseprofile, p53 activation, p21 and MDM2 activity, solubility,IC50 value and dose–response (Supplementary Fig. S1A–S1C;Supplementary Fig. S2A–S2D). PM2 treatment in wt p53cells leads to p53 activation and induction of p21 andMDM2 activity (Supplementary Fig. S1A and S1C) but notin p53 �/�

–null cells (Supplementary Fig. S1B), nor with thecontrol PM2 scrambled peptide. Furthermore, the LDH releaseprofile confirmed intact cell membrane, demonstrating thatPM2 is permeating into the cell without disrupting the cellmembrane (Supplementary Fig. S2A). The solubility of PM2in PBS was determined to 54.02 mmol/L (Supplementary Fig.S2D). The IC50 value was determined to 12.26–19.35 mmol/L(95% CI) on HCT116 cells (Supplementary Fig. S2C), in linewith the p53 transcriptional activation assay (SupplementaryFig. S2B). Consequently, subsequent in vitro studies wereperformed at 20 mmol/L.

Western blotting—radiotherapy induces p53 expression, whichis stabilized by PM2 treatment

p53 protein expression was analyzed by Western blot analysis.Radiotherapy with 2 Gy induced p53 levels in wt p53 HCT116cells (Fig. 1A and B). The highest level was obtained 3 hours afterradiation exposure, 6.7 times higher than the control. After thistime point, p53 levels decreased. Therefore, PM2 therapy wasgiven 3 hours after radiotherapy. PM2monotherapywas also ableto induce p53 levels, where the highest p53 expressionwas seen atthe 6 hours time point.

PM2 therapy combined with radiotherapy resulted in the high-est p53 levels, reaching 8.8 times higher than controls. At 24 hoursafter radiotherapy, p53 levels of the single treatments haddropped significantly, whereas p53 levels of the combinationtreatment group remained stable.

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Clonogenic survival—low doses of PM2 radiosensitize wt p53cells in monolayer cell culture

Reponses to radiotherapy, selectivity of the inhibitor as well asefficacy of the combination treatments were first investigated in a2D colony-forming assay. The wt p53 HCT116 and�/�HCT116cells displayed similar responses to radiotherapy. The survivalfraction (SF) after treatment with a radiation dose of 4 Gywas SF4Gy ¼ 0.12 for wt p53 HCT116 and SF4Gy ¼ 0.08 for�/� HCT116 (Fig. 1C–E). PM2 treatment reduced the survivalby a factor of 4 at a radiation dose of 2 Gy and by a factor of 6 at4 and6Gy (Fig. 1CandE). Survival fractions of PM2and radiationtreated �/�HCT116 cells did not differ significantly at any of thetested concentrations (Fig. 1D and E).

Multicellular tumor spheroids—PM2 and radiotherapy reducetumor growth in wt p53 tumor-like cell culture

The efficacy of radiotherapy and PM2 treatment was investi-gated inmulticellular tumor spheroids, usingwt p53, p53�/�, andmutant p53 cancer cells.

In a first set of spheroid experiments, in vitro drug efficacy andpotential effects of DMSO levels were assessed. DMSO did notaffect cell viability at the levels used in the present study(Supplementary Fig. S3A). The in vitro drug efficacy assessmentdemonstrated that one weekly dose of 20 mmol/L PM2 resulted ina similar growth inhibitory effect as three repeated doses of20 mmol/L at 48 hours intervals (Supplementary Fig. S3B). Ther-apeutic effects of a single dose of PM2 with and without radio-therapy of 2 Gy were subsequently assessed in a panel of tumorspheroids (Supplementary Fig. S3C). Results demonstrated cleareffects of PM2 alone in all the wt p53 spheroids (HCT116,UM-SCC-74A, UM-SCC-74B), whereas no significant effect onspheroid growth in the mutant p53 spheroid model (BON) wasdetected. Furthermore, by combining PM2 treatment withradiotherapy, therapeutic effects were potentiated, significantlyreducing spheroid growth compared with either treatmentalone (Supplementary Fig. S3C).

Finally, the effects of fractionated PM2 and/or radiotherapyon wt 53 and �/� HCT116 tumor spheroids were assessed

Figure 1.

In vitro analysis of p53 expression and survival. A and B, p53 expression level analysis by Western blotting. (þ) PM2, treatment with 20 mmol/L PM2 3hours after radiotherapy; (�) PM2, treatment with dissolvent, DMSO; (þ) radiation, radiotherapy with 2 Gy; (�) radiation, pseudo-radiotherapy with 0 Gy.A, Representative membrane of wt p53 HCT116 cells treated with PM2 and radiotherapy. B, Quantitative analysis of Western blots. Radiotherapy inducesp53 expression, which is stabilized by PM2 treatment. n > 3,mean; error bars, SD.C–E,Clonogenic survival analysis of HCT116wt p53 (C) and�/�HCT116 (D) cells. n¼3; error bars, SD. HCT116wt p53 tumor cells responded to PM2 (20 mmol/L) therapy and radiotherapy (2, 4, 6 Gy) in a dose-dependentmanner. PM2 treatment did notaffect survival of �/� cells. E, Representative images of the cell colony formation after a radiation dose of 4 Gy and treatment with PM2.

PM2 Potentiates Radiotherapy in wt p53 Cancer

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Figure 2.

Multicellular tumor spheroid growth during fractionated PM2 therapy and/or radiotherapy. A–F, HCT116 wt p53 tumor spheroid analyses. PM2-treated tumorspheroids were treated with either 10� PM2 (A–C) or 3� PM2 (D and E) therapy and/or radiotherapy. B and E, Comparison of normalized spheroid size on day 10.Statistical significance, ���� ,P<0.0001; ��,P<0.001;mean, n¼4; error bars, 95% confidence interval.C,Representative pictures ofmulticellular HCT116wt p53 tumorspheroids. F, Synergistic effects displayed as combination index (CI) plot for combination of 3� PM2 or 10� PM2with radiotherapy for wt p53 spheroids.G–L,HCT116p53�/� tumor spheroid analyses. PM2-treated tumor spheroids were treated with either 10� PM2 (G–I) or 3� PM2 (J–K) therapy and/or radiotherapy.I, Representative pictures of multicellular HCT116 p53 �/� tumor spheroids. H and K, Comparison of normalized spheroid size on day 10. L, Additiveeffects displayed as combination index plot for combination of 3� PM2 or 10� PM2 with radiotherapy for �/� spheroids.

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(Fig. 2A–L). Characterizations of wt p53 HCT116 spheroids areshown in Fig. 2A–F. In wt p53 HCT116 spheroids, monother-apy with 3 � 2 Gy radiotherapy decreased the tumor growthsignificantly by about 50% of the control growth after 2 weeks.Furthermore, there was a significant difference between themonotherapies and combination treatments for both PM2treatment schedules, where the greatest effect and almostfull inhibition of cell growth was displayed in the combinationgroups (Fig. 2A–E). At day 10, monotherapy treatment with3� PM2 or 10� PM2 had decreased spheroid growth by 45%(P < 0.001) and 58% (P < 0.0001), respectively. Combinationtreatment with 3� PM2 and radiotherapy, as well as combi-nation treatment with 10� PM2 and radiotherapy, decreasedthe growth significantly by 70% (P < 0.0001) and 79 % (P <0.0001), respectively, compared with the controls (Fig. 2B andE). Chou-Talalays combination index (CI; ref. 16) indicatedsynergistic effects for both combination treatments (Fig. 2F; 3�PM2 and radiotherapy CIday 10 ¼ 0.68; 10� PM2 and radio-therapy CIday 10 ¼ 0.54). Characterizations of �/� HCT116spheroids are shown in Fig. 2G–L. Radiotherapy decreasedtumor growth in �/� HCT116 in a similar way as in wt p53cells (50%); however, PM2 treatment did not decrease cellproliferation (Fig. 2G–K). No synergistic effects were observedfor 3� PM2 and radiotherapy CI day 10 ¼ 0.88, or 10� PM2 andradiotherapy CIday 10 ¼ 0.94 in �/� spheroids as displayedin Fig. 2L.

In vivo biodistribution of 125I-PM2 –125I-PM2 is retained in

tumors for at least 48 hoursIn order of follow the distribution of PM2 within the body,

the stapled peptide was labeled with 125I and injected intomouse xenografts carrying wt p53 HCT116 tumors. 125I-PM2uptake was measured at 6, 24, and 48 hours after injection(Fig. 3A). The highest 125I-PM2 concentration of 23 %ID/g wasdetected in the tumor at 6 hours p.i. The 125I-PM2 tumor uptakeremained constant at approximately 20 %ID/g at 24 and48 hours p.i. 125I-PM2 uptake in liver and intestine peaked at6 hours with 7 %ID/g for both organs. At the later time points(24 and 48 hours), it had decreased to below 1 %ID/g. Traceruptake in the thyroid increased at 24 and 48 hours afterinjection, probably due to free iodine because the animals didnot receive any thyroid blocking agents. Serum stability evalua-tions of 125I-PM2 demonstrated that >95% of the label was stillattached to the peptide incubated in mouse serum at all timepoints assessed (up to 24 hours).

Autoradiography of wt p53 HCT116 tumors 24 hours afterinjection demonstrated a homogeneous distribution of the radi-olabeled peptide within the tumor (Fig. 3B).

Ex vivo immunohistochemistry—PM2 and radiotherapyincrease p53 levels in wt p53 tumors without inducing acutehepatotoxicity

Ex vivo immunohistochemistry analysis of wt p53 HCT116xenografted tumors revealed increased p53 expression levels in10� PM2 treated tumors (þþ) compared with control tumors(þ) for at least 48 hours after PM2 treatment, whereas �/�HCT116 xenografts did not show p53 expression (�) in any ofthe groups (Fig. 3C). However, the highest expression levels(þþþ) were present in combination treated wt p53 HCT116xenografts (Fig. 3C), demonstrating the high specificity of thetreatment.�/�HCT116 cells did not show p53 expression (�) in

anyof the groups (Fig. 3C).H&E stain ofmouse liver tissue treatedwith PM2 or radiotherapy did not show signs of acute toxicity: nonecrosis and inflammation was detected or the morphology waspreserved (Fig. 3D). Treatment of 3� PM2, 10� PM2 and/orradiotherapy did not affect animal weight. The increased p53expression after PM2 treatmentwas further validated in thewtp53UM-SCC-74B xenografts (Supplementary Fig. S4A), whereas themutant p53 BON xenografts demonstrated a strong staining thatwas not altered by PM2 treatment (Supplementary Fig. S4B).

In vivo mouse xenograft study—combination therapy of PM2and radiation reduces tumor growth in wt p53 tumors andprolongs survival

To investigate whether the radiosensitizing effects obtained inthe in vitro models would translate into an in vivo setting, wt p53HCT116 and�/�HCT116 tumors were investigated in a blindedin vivo mouse xenograft study. Mice carrying untreated wt p53HCT116 tumors demonstrated a median tumor size of 917 mm3

after a period of 10 days. Monotherapy with 10� PM2 or 3fractionated radiation doses of 2 Gy reduced the tumor growthsignificantly. At day 10 after treatment start, tumors that receivedmonotherapy with 10� PM2 or radiation reached half the size ofthe controls: 555mm3 and 526mm3, respectively (Fig. 4A and B).At the same time point, the combination treatment group thatreceived both 10� PM2 and radiation displayed the smallesttumors (368 mm3; Fig. 4A and B). The tumor sizes in thecombination group were significantly smaller than the tumorvolumes of the control and single treatment groups (P < 0.0001and P < 0.005). The same trend was observed for wt p53 tumorstreated with 3 doses of PM2; however, these effects were notstatistically significant (Fig. 4D and E). Combination index anal-ysis confirmed the in vitro results anddemonstrated synergy for thecombinations (Fig. 4F; 3� PM2 and radiotherapy CIday 10¼ 0.61;10� PM2 and radiotherapy CIday 10 ¼ 0.46).

Mice carrying �/� HCT116 tumors did not respond signif-icantly to PM2 treatment. Instead, the growth of PM2-treated�/� HCT116 tumors was almost identical to non-treatedcontrol animals. Radiation reduced tumor growth in a similarmanner as in the wt p53 animals (442 mm3 at day 10; Fig. 4Gand H). Furthermore, combination treatment did not benefitanimals with �/� tumors. The median tumor volume of PM2and radiation-treated tumors at day 10 (559 mm3) was notsignificantly different from the radiation monotherapy group(442 mm3; Fig. 4H). Radiotherapy as well as the 10 repeateddoses of PM2 were tolerated well by all mice without any signsof adverse effects. The wt p53-dependent effects of PM2 treat-ment were further confirmed in two additional xenograft mod-els, comparing tumor growth of 10� PM2-treated tumors andmock treated control tumors during treatment. In line with theHCT116 data, PM2 treatment reduced tumor growth in the wtp53 xenograft model UM-SCC-74B, but not in the mutated p53model BON (Supplementary Fig. S4C and S4D, respectively).

The survival proportions of this study revealed a significantadvantage and prolonged survival for mice in the combinationgroups carrying wt p53 HCT116 tumors (Fig. 4C and I; Table 1).The median survival (survival 50 %) for animals carrying wt p53tumors that did not receive treatment was 12 days. The mediansurvival for animals treated with 10� PM2 and radiotherapywas increased with 50% to 18 days. Furthermore, maximumsurvival of combination treated animals was almost twiceas long as control mice (control 14 days, combination 26

PM2 Potentiates Radiotherapy in wt p53 Cancer

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days; Fig. 4C; Table 1). Radiation treatment increased the mediansurvival in both wt HCT116 and �/� HCT116 tumors (16 days,15 days, respectively). Animals bearing �/� tumors did notbenefit from PM2 treatment, neither as monotherapy nor incombination with radiotherapy (Fig. 4I; Table 1).

DiscussionCombining cancer treatments that use different mechanisms of

action is a recognized concept to enhance treatment outcomes.Recent advances in the understanding of radiation effects and therole of the tumor-suppressor p53 allow for the design of morespecific combination treatments based on radiosensitization.

In this study, we investigated the combination of ionizingradiotherapy with the novel drug candidate PM2 using bothin vitro and in vivo models, with the aim to radiosensitize wtp53 cancer cells by restoringwt p53 levels. This is thefirst time thatthe stapled peptide PM2 and radiotherapy has been investigatedtogether in an in vivo setting.

Previously, restoring wt p53 levels using anti-MDM2 therapyhas been tested in preclinical and clinical settings with modestsuccess rates (10). One possible reason is extensiveMDMX expres-sion, as well as alterations in the MDM2 protein. Nutlin-3 resis-tance, for example, has been discovered to depend on MDMXoverexpression, and mutations within the N-terminal domain ofMDM2aswell asmutations outside of the binding domain,whichcan allosterically modulate its binding properties (17). These

problems may be avoided using MDM2/X inhibitors like PM2.The recent review article by Tisato and colleagues (18) concludesthat the blocking of MDM2/X to activate the p53 pathway is anappealing and fruitful therapeutic strategy to treat cancer, partic-ularly for the management of hematological malignancies (e.g.,acute lymphoblastic and myelogenous leukemia, B-cell lympho-cytic leukemia etc.) that show low levels of TP53 mutations.

We hypothesize that activation and stabilization of the p53pathway byMDM2/X inhibition can also be a superior concept insolid diseases, when combined with second treatment strategieslike external beam radiotherapy. AsMDM2/X inhibition increasesp53 levels, and radiotherapy is specifically directed on the tumor,treatment efficacy could be enhanced and potential off-targeteffects minimized. An advantage of this specific combination isthat since wt p53 is still present in about 50%of solid cancer types(19), and modern hospitals are routinely equipped with radio-therapy facilities, this combination may yield a comparativelybroad target spectrum and general utility.

In the present study, in vitro characterizations of PM2 confirmedhigh selectivity of the stapled peptide to the wt p53 cell lines,demonstrating wt p53-dependent p53 activation, and inductionof p21 and MDM2 activity in wt p53 cells only (SupplementaryFig. S1). Colony formation assays verified the results and revealedsignificantly decreased cancer cell viability in the PM2 plus radio-therapy combination group compared with PM2 or radiationmonotherapy (Fig. 1). These results comply very well with thesubsequentmulticellular spheroid assay outcomes on cancer cells

Figure 3.

Ex vivo analysis. A, Biodistributionof 125I-PM2 as %ID/g, and thyroid as%ID/organ 6, 24, and 48 hoursafter injection. Error bars, SD. B,Representative autoradiographyimages of a wt p53 HCT116tumor 24 hours after 125I-PM2administration. PM2 displayed aneven distribution in the tumor. C, Exvivo immunohistochemistry imagesfrommice treated with either placebo,10� PM2 and/or 3 � 2 Gy of p53expression of wt p53 HCT116 tumorsand �/� HCT116 tumors andhematoxylin and eosin stain of mouseliver tissue (magnification, �40).

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with low (UM-SCC-74B; ref. 20) and moderate (HCT116) radi-ation resistance (21). The combination of low concentrations ofPM2 and radiation treatment demonstrated synergistic therapeu-tic effects and reduced tumor spheroid growth significantly (Fig. 2and Supplementary Fig. S3). Radiotherapy is known to elevatep53 levels, also evident from Western blot analysis and IHCanalysis (Figs. 1A and B and 3C), which in turn is stabilized byPM2 treatment. This codependence necessitates a treatment strat-egy with exact timing and a correct therapeutic window, as PM2should be preferably be present as MDM2 andMDMX expressionlevels peak.

To assess organ uptake and tumor distribution of PM2 in vivo,the stapled peptide was labeled with 125I, and was able todemonstrate a high and homogenous tumor uptake (Fig. 3A andB). Multiple studies have indicated that active transport has asignificant role in cellular uptake of stapled peptides, whereFAM labeled stapled peptides have been shown to be activelyendocytosed into cytoplasmic vesicles (22, 23). This is in linewithongoing work in the p53lab on PM2 and other stapled peptides,and with results reported by Yurlova and colleagues (24) dem-onstrating that inhibition of the MDM2:p53 interaction takesapproximately 30 to 60 minutes to occur, as opposed to

Figure 4.

In vivo mouse xenograft study. A–F, Mice carrying HCT116 wt p53 tumors treated with varying combinations of PM2 and radiation (n � 6 per group; error bars,95%CI).A–C, 10�PM2and/or3�2Gy radiation.DandE,3�PM2and/or3� 2Gy radiation.AandD,Tumor size (mm3)ofwtp53HCT116xenografts followedover time,until the first animal in each group was sacrificed. B and E, Tumor size (mm3) of wt p53 HCT116 xenografts at day 10 after treatment start. Statistical significance,� , P < 0.05. Growth of tumors treated with 10� PM2 and 3 � 2 Gy was significantly slower than the tumor growth in the single treatment groups. F, Synergisticeffects displayed as combination index (CI) plot of 3� PM2 and 10� PM2 and radiotherapy for wt p53 HCT116. G–I, Mice carrying �/� HCT116 tumors treated withcombinationsof PM2and radiation (n¼ 5 per group; error bars, 95%CI).G,Tumor size (mm3) of�/�HCT116 xenografts followedover time, until thefirst animal in eachgroup was sacrificed. H, Tumor size (mm3) of �/� HCT116 xenografts at day 10 after treatment start. No significant difference between the monotherapy andcombination treatment groups. C and I, Survival proportions of mice carrying HCT116 wt p53 (C) and�/� (I) tumors. Mice with wt p53 tumors treated with PM2 andradiation extended median survival with 50% from control mice and nearly doubled maximum survival. PM2 treatment had no survival advantage in �/� mice.

PM2 Potentiates Radiotherapy in wt p53 Cancer

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approximately 5 minutes for small-molecule antagonists of theinteraction. The LDH release profile in the present study furthersupported these conclusions, by verifying that PM2enters the cellswith no associated disruption of the lipid membrane bilayer(Supplementary Fig. S2). Furthermore, the 125I-PM2 biodistribu-tion data indicated that the iodinated peptide is retained andintact in the tumor cell for at least 48 hours p.i., as degradationwould result in free iodine, which is quickly released by the tumorcells by passive diffusion and excreted from the body. This wasfurther supported by the ex vivo IHC analyses, demonstratingincreased p53 staining in the PM2-treated wt p53 tumors com-pared with controls at least 48 hours after PM2 treatment(Fig. 3C), as well as the tumor spheroid drug activity assay(Supplementary Fig. S3), the Western blot analysis (Fig. 1) andserum stability assay, all suggesting that PM2 is intact and activefor at least 24 hours. These results were in line with previousstudies that have demonstrated that hydrocarbon stapling can bean efficient way to protect peptides from proteases and improvethe helical stability (23, 25), suggesting that PM2 remains activeand bound to MDM2 for at least 48 hours after treatment in thexenografted tumors, allowing for a therapeutic effect to occur.

The in vivo therapy studies validated the in vitro data in severalxenograft models, verifying a p53-dependent tumor response ofPM2 in vivo (Fig. 4; Table 1; Supplementary Fig. S4), and dem-onstrating a clear benefit of combining radiotherapy with PM2treatment on xenografted wt p53 HCT116 tumors (Fig. 4; Table1). In the mouse model with wt p53 tumors, PM2 therapy andradiation treatment significantly prolonged survival comparedwith the control and monotherapy groups. Mice undergoing thecombination treatment increasedmedian survival with 50% fromcontrol animals, and maximum survival was almost doubled.These encouraging results are likely underestimated because thechosen animal model does not account for immunogenic celldeath. The main mechanism of cell death and tumor reductionafter radiotherapy is generally due to DNA damage. Nevertheless,radiation exposure causes release of for example cytokines attract-ing immune cells and initiating a cascade of immunologicresponses that lead to inflammation and finally to immunogeniccell death (26). Furthermore, cell death caused by DNA damageleads to release of tumor-related antigens that can be recognizedby cells of the immune system, further amplifying the immunereactions (26). Therefore, the combination effect described heremight be even more pronounced in a clinical setting.

Moreover, byoptimizing theadministrationand formulationofthe different treatment regimes in patients, the efficacy of thecombination therapy can improve even further. Work is currentlybeing performed by our groups to enable optimal administrationroutes and formulation. A more soluble version of PM2 has beensuccessfully administered via intraperitoneal injections, wherestabilization of p53 in normal tissues and B16 melanoma allo-grafts 12 hours after injection was observed (ongoing study,personal communication). In addition, stapled peptides have also

been administered via intravenous injection using a lipid formu-lation in mice with p53 wt human tumors, resulting in dramaticreduction in tumor growth (ongoing study, personal communi-cation). Consequently, in the futurewepredict that these peptides,with appropriate formulation strategies, have the potential to besystemically delivered to patients, making their use in differentcancer therapiesmore widely applicable. Furthermore, the presentstudy highlights the importance of knowledge of the specifictumor phenotype in deciding treatment, as absence of p53 in�/�HCT116 cells completely negated any beneficial effects fromeither PM2 or combination treatment, as would be expected.

In the present study, repeated PM2 administration reduced wtp53 tumor growth significantly without any observed adverseeffects in the in vivo xenograft models (Fig. 3; SupplementaryFig. S4). This is important, because a serious problem discoveredin patients undergoing therapy with small-molecule MDM2antagonists was symptoms of gastrointestinal and hematologicaltoxicity (10). This can be potentially avoided by the use ofpeptides; however, in vivo half-life is usually a common problem.In PM2, this problem has been addressed by the use of stapling ofthe structure, which makes the peptide more resistant to prote-olysis (13). The use of peptides with drug-like properties has nowentered the clinics, even though for a long time the entire field ofdrug peptides was seen as largely academic. For example, inpatients with advanced neuroendocrine tumors where the use ofsurgery, external beam radiation and chemotherapy is limited,somatostatin analogs such as lanreotide and octreotide are rou-tinely given. By combining these peptides with radiotherapy, e.g.,by labeling these peptides with therapeutic radioactive nuclides,neuroendocrine tumors can be treated successfully (27). Theresults obtained with, for example, 177Lu-DOTATATE (Lutathera)are very encouraging in terms of tumor regression (28).

To conclude, the present in vivo study is the first of its kind,demonstrating prolonged survival of the PM2 treatment groups,and in particular in the combination group. PM2 therapy in itselfis promising, but it also has the potential to augment the activityof radiotherapy to a significant degree. The combination of PM2and radiotherapy could be a promising approach for improvingthe efficacy of radiotherapy in tumors with retained wt p53.Because PM2 is actively pursued as a new drug candidate, werecommend the inclusion of co-treatment regimes in furtherstudies performed in preclinical and clinical settings.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: D. Spiegelberg, A.C. Mortensen, D.P. Lane, M. NestorDevelopment of methodology: D. Spiegelberg, A.C. Mortensen, S. LundstenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Spiegelberg, A.C. Mortensen, S. Lundsten,C.J. Brown, M. Nestor

Table 1. Median and Maximum survival of wt p53 and �/� HCT116 mouse xenografts in days (d)

wt p53 Control 10� PM2 therapy 3� PM2 therapy Radiotherapy 10� PM2 þ radiotherapy 3� PM2 þ radiotherapy

Median survival (d) 12 12.5 11 16 18 16Maximum survival (d) 14 19 16 21 26 20

�/� p53 Control 10� PM2 therapy 3� PM2 therapy Radiotherapy 10� PM2 and radiotherapy 3� PM2 and radiotherapyMedian survival (d) 11 12 n/a 15 13 n/aMaximum survival (d) 13 13 n/a 18 14 n/a

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Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Spiegelberg, A.C. Mortensen, S. Lundsten,D.P. Lane, M. NestorWriting, review, and/or revision of the manuscript: D. Spiegelberg,A.C. Mortensen, S. Lundsten, C.J. Brown, D.P. Lane, M. NestorAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Lundsten, D.P. LaneStudy supervision: D.P. Lane, M. Nestor

AcknowledgmentsThe authors gratefully acknowledge support from the Swedish Research

Council (grant numbers 2013-30876-104113-30 and 2013-8807) and

The Swedish Cancer Society (grant numbers CAN 2015/1080 and CAN2015/385). The authors would also like to thank Nakul Ravi Raval forassistance with animal experiments.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 9, 2018; revised June 5, 2018; accepted July 9, 2018;published first July 19, 2018.

References1. Marusyk A, Polyak K. Tumor heterogeneity: causes and consequences.

Biochim Biophys Acta 2010;1805:105.2. Garraway LA,Verweij J, BallmanKV. Precisiononcology: anoverview. JClin

Oncol 2013;31:1803–5.3. Al-Lazikani B, Banerji U, Workman P. Combinatorial drug therapy for

cancer in the post-genomic era. Nat Biotech 2012;30:679–92.4. HoeKK,VermaCS, LaneDP.Drugging the p53pathway: understanding the

route to clinical efficacy. Nat Rev Drug Discov 2014;13:217–36.5. SchillingD,D€uwelM,MollsM,Multhoff G. Radiosensitization ofwildtype

p53 cancer cells by theMDM2-inhibitor PXN727 is associated with alteredheat shock protein 70 (Hsp70) levels. Cell Stress Chaperones 2013;18:183–91.

6. Bernal F, Wade M, Godes M, Davis TN, Whitehead DG, Kung AL, et al.A Stapled p53 Helix overcomes HDMX-mediated suppression of p53.Cancer Cell 2010;18:411–22.

7. Gudkov AV, Komarova EA. The role of p53 in determining sensitivity toradiotherapy. Nat Rev Cancer 2003;3:117–29.

8. Ventura A, Kirsch DG, McLaughlin ME, Tuveson DA, Grimm J, Lintault L,et al. Restoration of p53 function leads to tumour regression in vivo. Nature2007;445:661.

9. Rothweiler U, Czarna A, Krajewski M, Ciombor J, Kalinski C, Khazak V,et al. Isoquinolin-1-one Inhibitors of the MDM2–p53 Interaction. Chem-MedChem 2008;3:1118–28.

10. Burgess A, Chia KM,Haupt S, ThomasD,Haupt Y, LimE. Clinical overviewof MDM2/X-targeted therapies. Front Oncol 2016;6:7.

11. Parrales A, Iwakuma T. Targeting oncogenicmutant p53 for cancer therapy.Front Oncol 2015;5:288.

12. Wei SJ, Chee S, Yurlova L, Lane D, Verma C, Brown C, et al. Avoiding drugresistance through extended drug target interfaces: a case for stapledpeptides. Oncotarget 2016;7:32232–46.

13. Brown CJ, Quah ST, Jong J, Goh AM, Chiam PC, Khoo KH, et al. Stapledpeptides with improved potency and specificity that activate p53.ACS Chem Biol 2013;8:506–12.

14. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenicassay of cells in vitro. Nat Protoc 2006;1:2315–9.

15. Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA. Spheroid-based drugscreen: considerations and practical approach. Nat Protoc 2009;4:309–24.

16. Chou T-C. Drug combination studies and their synergy quantificationusing the Chou-Talalay Method. Cancer Res 2010;70:440–6.

17. Wei SJ, Joseph T, Chee S, Li L, Yurlova L, Zolghadr K, et al. Inhibition ofNutlin-resistant HDM2 mutants by stapled peptides. PLoS ONE 2013;8:e81068.

18. Tisato V, Voltan R, Gonelli A, Secchiero P, Zauli G. MDM2/X inhibitorsunder clinical evaluation: perspectives for the management of hematolog-ical malignancies and pediatric cancer. J Hematol Oncol 2017;10:133.

19. Duffy MJ, Synnott NC, McGowan PM, Crown J, O'Connor D, GallagherWM. p53 as a target for the treatment of cancer. Cancer Treat Rev2014;40:1153–60.

20. Spiegelberg D, Mortensen AC, Selvaraju RK, Eriksson O, Stenerl€ow B,Nestor M. Molecular imaging of EGFR and CD44v6 for prediction andresponse monitoring of HSP90 inhibition in an in vivo squamous cellcarcinoma model. Eur J Nucl Med Mol Imaging 2016;43:974–82.

21. Spiegelberg D, Dascalu A, Mortensen AC, Abramenkovs A, Kuku G, NestorM, et al. The novel HSP90 inhibitor AT13387 potentiates radiation effectsin squamous cell carcinoma and adenocarcinoma cells. Oncotarget2015;6:35652–66.

22. Thean D, Ebo JS, Luxton T, Lee XEC, Yuen TY, Ferrer FJ, et al. Enhancingspecific disruption of intracellular protein complexes by hydrocarbonstapled peptides using lipid based delivery. Sci Rep 2017;7:1763.

23. Bernal F, Tyler AF, Korsmeyer SJ, Walensky LD, Verdine GL. Reactivation ofthe p53 tumor suppressor pathway by a stapled p53 peptide [J. Am. Chem.Soc. 2007, 129, 2456�2457]. J Am Chem Soc 2007;129:2456–7.

24. Yurlova L, Derks M, Buchfellner A, Hickson I, Janssen M,Morrison D, et al.The fluorescent two-hybrid assay to screen for protein–protein interactioninhibitors in live cells: targeting the interaction of p53 with Mdm2 andMdm4. J Biomol Screen 2014;19:516–25.

25. Chang YS, Graves B, Guerlavais V, Tovar C, Packman K, To K-H, et al.Stapled a�helical peptide drug development: a potent dual inhibitor ofMDM2 and MDMX for p53-dependent cancer therapy. Proc Natl Acad SciU S A 2013;110:E3445–E54.

26. Park B, Yee C, Lee K-M. The effect of radiation on the immune response tocancers. Int J Mol Sci 2014;15:927–43.

27. Kwekkeboom DJ, Krenning EP. Peptide receptor radionuclide therapy inthe treatment of neuroendocrine tumors. Hematol Oncol Clin North Am2016;30:179–91.

28. KamBLR, Teunissen JJM, Krenning EP, deHerderWW, Khan S, van Vliet EI,et al. Lutetium-labelled peptides for therapy of neuroendocrine tumours.Eur J Nucl Med Mol Imaging 2012;39:103–12.

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