Cancer Therapy: Clinical

Radiosensitization of Adenoid Cystic Carcinomawith MDM2 InhibitionPrashanth J. Prabakaran1, Amal M. Javaid1, Adam D. Swick1, Lauryn R.Werner1,Kwangok P. Nickel1, Emmanuel Sampene2,3, Rong Hu3,4, Irene M. Ong2,3,Justine Y. Bruce3,5, Gregory K. Hartig3,6, Aaron M.Wieland3,6, Jude Canon7,Paul M. Harari1,3, and Randall J. Kimple1,3

Abstract

Purpose: Adenoid cystic carcinoma (ACC) is a rare cancerarising from themajor or minor salivary gland tissues of the headand neck. There are currently no approved systemic agents orknown radiosensitizers for ACC. Unlike the more common headand neck squamous cell carcinomas that frequently harbor TP53mutations, ACCs contain TP53 mutations at a rate of <5%,rendering them an attractive target for MDM2 inhibition.

Experimental Design: We report the successful establishmentand detailed characterization of a TP53-WT ACC patient-derivedxenograft (PDX), which retained the histologic features of theoriginal patient tumor. We evaluated this model for response tothe MDM2 inhibitor AMG 232 as monotherapy and in combi-nation with radiotherapy.

Results: AMG 232 monotherapy induced modest tumorgrowth inhibition, and radiation monotherapy induced a tran-

sient tumor growth delay in a dose-dependent fashion. Strikingly,combination treatment of AMG232with radiotherapy (includinglow-dose radiotherapy of 2 Gy/fraction) induced dramatic tumorresponse and high local tumor control rates 3 months followingtreatment. Posttreatment analysis revealed that although bothAMG 232 and radiotherapy alone induced TP53 tumor-suppres-sive activities, combination therapy amplified this response withpotent induction of apoptosis after combination treatment.

Conclusions: These data identify that MDM2 inhibition canprovide potent radiosensitization in TP53-WTACC. In light of theabsence of effective systemic agents for ACC, the powerfulresponse profile observed here suggests that clinical trial evalu-ation of this drug/radiotherapy combinationmay bewarranted toimprove local control in this challenging malignancy. Clin CancerRes; 23(20); 6044–53. �2017 AACR.

IntroductionMalignancies of the salivary glands are a relatively rare and

diverse group of tumors, accounting for roughly 3% of all headand neck cancers (1). Adenoid cystic carcinoma (ACC) comprisesapproximately 10% of salivary gland tumors and is characterizedby a generally slow but unpredictable growth rate and a high rate

of eventual local and distant metastasis (1). These tumors arecategorized histologically as solid, tubular, or cribriform patterns,with the solid form typically demonstrating more aggressivedisease than the other types (2–6). Despite modern surgicaltreatments for patients with newly diagnosed ACC, both localand distant recurrences remain common, highlighting the needfor improved therapy options (7). Despite dozens of clinical trialsexamining drug therapies, there are no FDA-approved systemicagents for ACC, with conventional and molecular therapeuticseliciting crude response rates typically less than 10% to 20% (8–15). The most widely utilized regimen of cisplatin plus vinor-elbine has significant toxicities and a modestly higher responserate (16). The current standard of care remains surgical resectionand postoperative radiotherapy (1, 8), however, with a highpropensity for local and distant failure. The addition of cisplatinto adjuvant radiotherapy is currently being investigated in arandomized RTOG study enrolling multiple salivary gland his-tologies including ACC, but these results are not anticipated forseveral years.

The hallmark tumor suppressor protein p53 is a transcriptionfactor that regulates cell-cycle progression, senescence, DNArepair, and apoptosis to control tumor cell growth (17). TP53 ismutated in roughly 50%of all tumors (18, 19), but themajority ofACCs express wild-type (WT) p53. This is in stark contrast to headand neck squamous cell carcinoma in which TP53 mutations areseen in over 50% of cases. In ACC, TP53 mutations have beenidentified in approximately 5% of cases (20, 21). p53 proteinlevels are regulated by MDM2, an E3 ubiquitin ligase, which

1Department of Human Oncology, University of Wisconsin School of Medicineand Public Health Madison, Madison, Wisconsin. 2Department of Biostatistics,University ofWisconsin School of Medicine and Public Health Madison, Madison,Wisconsin. 3University of Wisconsin Carbone Cancer Center, University ofWisconsin School of Medicine and Public Health Madison, Madison, Wisconsin.4Department of Pathology, University of Wisconsin School of Medicine andPublic HealthMadison,Madison,Wisconsin. 5Department ofMedicine, UniversityofWisconsin School ofMedicine andPublic HealthMadison,Madison,Wisconsin.6Department of Surgery, University of Wisconsin School of Medicine and PublicHealth Madison, Madison, Wisconsin. 7Oncology Research, Amgen, Inc., Thou-sand Oaks, California.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

P.J. Prabakaran and A.M. Javaid contributed equally to this article.

Corresponding Author: Randall J. Kimple, Department of Human Oncology,University of Wisconsin School of Medicine, 3107 WIMR, 1111 Highland Avenue,Madison, WI 53705. Phone: 608-265-9156; Fax: 608-262-7224; E-mail:rkimple@humonc.wisc.edu

doi: 10.1158/1078-0432.CCR-17-0969

�2017 American Association for Cancer Research.

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targets p53 for proteasomal degradation (22). MDM2 inhibitorsare known to exhibit antitumor effects and are currently beingstudied in clinical trials (23–25). InACC, the promising efficacy ofan MDM2 inhibitor alone or in combination with cisplatin hasrecently been demonstrated in the treatment of ACC patient-derived xenografts (PDX; refs. 26, 27). In this study, we investi-gated the radiosensitizing effect of AMG 232, a picomolar affinitypiperidinone inhibitor of MDM2 that is currently in clinical trialsfor several tumor types. AMG 232 appears to synergize with p53-activating therapies, such as DNA-damaging agents, likely poten-tiating p53 signaling, leading to increased apoptosis anddecreased cell proliferation (28–30).

Among many antitumor effects, ionizing radiotherapy acti-vates p53 to induce its array of tumor-suppressive functions.AMG 232 and other MDM2 inhibitors have been shown toradiosensitize non–small cell lung cancer, breast cancer, coloncancer, melanoma, and prostate cancer cell lines that are p53WT, with more modest efficacy in models that harbor p53mutations (31, 32). Given the low rate of TP53 mutation inACC and promising preclinical data demonstrating a role forMDM2 inhibition in ACC, we hypothesized that the combi-nation of AMG 232 and radiotherapy would result inimproved tumor control than either treatment alone. Herein,we describe the characterization of an ACC PDX model, thepotent radiosensitizing effect of AMG 232 on this tumor, andanalysis investigating molecular mechanisms underlying thisradiosensitization.

Materials and MethodsMice

Six- to 8-week-old female NOD/SCID gamma (NSG, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice (The Jackson Laboratory) wereused for PDX establishment and tissue amplification. Six- to8-week-old female Hsd: athymic Nude-Foxn1nu(Harlan Labora-tories) were used for therapy studies. Mice were kept in theAssociation for Assessment and Accreditation of LaboratoryAnimal Care–approvedWisconsin Institute forMedical ResearchAnimal Care Facility, housed in specific pathogen-free roomsand had their clinical health evaluated at least twice weekly. Allstudies involving mice were carried out in accordance with aUniversity of Wisconsin Institutional Animal Care and UseCommittee–approved protocol.

PDX propagation and tumor harvestingThe UW-ACC-60 PDX was established and propagated as

described previously for head andneck squamous cell carcinomas(33, 34). TP53 sequencing was performed on total genomic DNAusing the Illumina TruSeq Cancer Amplicon panel and has beendeposited with the sequence read archive under BioProject ID:PRJNA381909.

Genetic testing of ACC PDX and patient donor tumorThe identity of the first and fifth passages of the ACC PDX was

confirmed to match its original patient donor tumor via shorttandem repeat (STR) testing. Total genomicDNAwas isolated fromflash-frozen tissue for all instances of the tumor using the QiagenDNeasy kit. An 18 loci STR assay (Promega PowerPlex 16 HSSystem #DC2100) was performed by the UW Translational Initia-tives in Pathology CORE facility. The sameDNA sampleswere usedto test forpresenceof theMYB-NFIBgene fusion frequentlydetectedin ACC via a PCR-based approach (35) using the following primersets: MYB-1910F 50-AGCTCCGTTTTAATGGC-30/NFIB-1096R 50-GGGTATAAATGCCTGCCGTT-30; MYB-1910F 50-AGCTCCGTTT-TAATGGC-30/ NFIB-1096R 50-GGGTATAAATGCCTGCCGTT-30,MYB- 1693F 50-GCAGGATGTGATCAAACAGG-30/ NFIB-1197R50-CCGGTAAGATGGGTGTCCTA-30. An approximately 750-bpDNA fragment containing all sequences involved in or adjacentto known fusion regions in MYB and NFIB was synthesized andserved as a positive control. Primers specific for b-actinwere used asa PCR reaction control.

Treatment and measurementWhen established tumors had reached sufficient size, tumor

tissue was harvested fromNSGmice and passaged into nudemicevia subcutaneous implantation in bilateral rear flanks. Tumorvolume was assessed weekly with Vernier calipers and calculatedaccording to the equation V ¼ (p/6) � (large diameter) � (smalldiameter)2. These slow growing tumors required approximately 3months until average volume reached 250 mm3, at which timemice (n ¼ 6/group) were randomized into treatment groups:control (vehicle), AMG 232, radiotherapy, or combination ofradiotherapy þ AMG 232. Randomization resulted in a startingtumor size that was not statistically different across all groups atthe start of treatment. Radiotherapy at the specifieddose (2, 5, or 8Gy/fraction)was delivered twice per week for 4weeks for a total ofeight fractions. AMG 232was delivered every day concurrent withradiotherapy for 4 weeks by oral gavage at 50 mg/kg, a level thatfell within the range of doses used in prior studies in the samemouse strain (31). Additional earlier unpublished work hadhighlighted the clinical relevance of this dose showing that 50mg/kg produced drug exposure level in mice that is similar to theexposure level observed in human trials at well-tolerated doses.Radiotherapy was performed with a Precision Xray XRAD 320with 1Gy/minute delivered at 320KV/12.5mA at 50 cm SSDwitha beam hardening filter with HVL¼ 4mmCu. The delivered doserate was confirmed monthly by ionization chamber. Mice wereshielded with custom-built lead jigs to isolate exposure to the rearquarter of the body. Two hours following the initial treatment,additional tumor-bearing mice were sacrificed, and tumors wereharvested for biomarker analysis. Each tumor was divided in halffor formalin-fixed paraffin-embedded preservation and flashfreezing in liquid nitrogen. At the end of the study, 2 mice fromeither the vehicle or AMG 232 alone groups were retreated withvehicle or drug and 0, 2, or 8 Gy, and tumors were harvested 48

Translational Relevance

Adenoid cystic carcinoma is a rare form of salivary glandcancer in which localized tumor progression can significantlyharm quality of life. Adjuvant radiation is commonly used fornewly diagnosed disease, andmany patients eventually under-go reirradiation for palliation. Here, we demonstrate robustradiosensitization of ACC using an MDM2 inhibitor in apatient-derived xenograft model. The combination of MDM2inhibition and low-dose radiation causes tumor regression byreactivating p53, leading to growth arrest and apoptosis. Thesedata add to the growing literature, suggesting that MDM2inhibition is a promising approach in ACC, and suggest thatpatients with ACC could benefit from combination therapy ofAMG 232 and radiation.

AMG 232 þ Radiation in ACC

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hours posttreatment to investigate additional posttreatmenteffects (36).

Statistical analysis of PDX treatment studyA one-way ANOVA model was utilized, with the null hypoth-

esis that the groupmeans of control, AMG,AMGþ 2Gy, AMGþ 5Gy, and AMG þ 8 Gy are equal was tested globally. We investi-gated howwell themodel fit the data by reporting goodness-of-fitstatistics, including R-squared measures, which describe the per-centage of variation in the response explained by the model.Finally, we performed post hoc analysis to assess the effects ofpairwise comparisons between the groups (i.e., AMG vs. control,AMG vs. AMG þ 2 Gy, AMG vs. AMG þ 5 Gy, AMG vs. AMG þ 8Gy), using Tukey honestly significant difference post hoc test. Allstatistical analyses were performed using the two-sided alphasignificance level of 0.05, with STATA v14 software.

Thedoseof radiotherapy required to result in50% tumor control(TCD50)was calculatedatday125. Theproportionof injection siteswith no palpable tumor (tumor volume ¼ 0) was calculated. Theconfidence intervals of the proportion were used to set the range,the radiotherapy dose was converted to log10 format, and theresulting data points fit to a log(treatment) versus response curveusing a least squares fit. The log(TCD50) was compared by the extrasum-of-squares F test using GraphPad Prism v7.0a.

To assess antagonistic, additive, or synergistic effects, we usedthe fractional product method as described previously (37) oneweek after the end of treatment. Briefly, the observed fractionaltumor volume (FTV) is equal to the mean tumor volume of eachtreated group (AMG 232, radiation, or AMG 232 þ radiation)divided by the mean tumor volume of the control group. TheexpectedFTV from the combined treatment (FTVAMG 232þ radiation)is calculated by multiplying the observed FTVAMG 232 by theobserved FTVradiation. Dividing the expected FTVAMG 232 þ radiation

by the observed FTVAMG 232 þ radiation

yields a synergy assessment ratioin which a value >1 suggests that the combined treatments areeffectively synergistic, <1 antagonistic, and ¼1 additive.

Histology and IHCHematoxylin and eosin (H&E) staining was used for formal

pathologic analysis and to assess tumor quality. Additional slideswereprocessed by theUWTranslational Research inPathology labto assess for expression of p63, CD117, and CK5/6 by IHC.Staining for expression of p53, p-p53, and Ki-67 was performedby standard IHC techniques as described previously (12, 38). SeeSupplementary Table S1 for reagent details. A no primary anti-body control slide was stained with each condition to ensurespecificity of the reaction. Briefly, slides were baked in an 80

�C

oven for 20 minutes to melt paraffin and deparaffinized bysoaking in xylene three times for 5 minutes each. Slides werethen rehydrated in ethanol through soaking in decreasing con-centrations of ethanol (3� 100%, 2� 95%, 1� 80%, 1� 50%).Heat-induced epitope retrieval followed rehydration. Slides wereblocked for endogenous peroxidase activity and nonspecific anti-body interactions using 10%goat serum inPBS. Primary antibodyprepared in 1% goat serum was incubated overnight at 4�C(primary antibody dilutions per Supplementary Table S1). Thenext day, anti-rabbit or anti-mouse horseradish peroxidase–con-jugated secondary antibodies were applied to the slides for 30minutes followed by a 2-minute development with 3,30-diami-nobenzidine (DAB; #SK-4100; Vector Laboratories). Finally,slides were counterstained with hematoxylin, dipped in fresh

xylene and coverslipped with Cytoseal XYL (Thermo Fisher Sci-entific) and a number 1.5 coverslip. Where conducted, positivecell counts were performed using ImageJ with statistical analysisusing GraphPad Prism. Semiquantitative analysis of IHC stainingintensity was performed using ImageJ with the Colour Deconvo-lution plugin.

ImmunoblottingLysates were prepared from flash-frozen tumor by homogeni-

zationwith aDounce homogenizer inNP40 buffer supplementedwith protease/phosphatase inhibitor cocktail (Cell SignalingTechnologies #5872) followed by sonication. Equal amounts ofprotein were analyzed by SDS-PAGE (50 mg/well) and transferredto polyvinylidene difluoride membranes (Immobilon-FL). Pro-tein targets of interest were analyzed by specific primary anti-bodies, detected via incubationwith anti-mouse or anti-rabbit (asappropriate) near infrared–conjugated secondary antibodies(LiCOR), and imaged on a LiCOR Odyssey FC. Antibodies andsources are listed in Supplementary Table S1.

In situ hybridization by RNAscopeRNA levels were examined using the RNAscope 2.5 HD Detec-

tion Reagent – Brown (Advanced Cell Diagnostics) according tothemanufacturer's protocol. Slideswere baked at 60

�C for anhour

and then deparaffinized by incubating in xylene twice for 5minutes. Slides were then treated in 100% ethanol twice, for 1minute each, at room temperature, and then air dried. Heatedantigen retrieval was carried out using RNAscope 1� TargetRetrieval Reagents. RNAscope hydrogen peroxide was added tothe tissue sections, and the slides were incubated for 10 minutesand dried. Slides were then baked in the HybEZ Oven at 40

�C for

30 minutes. After baking, the Hs-CDKN1A hybridization probe(ACD 311401) was applied to the slides and incubated in theHybEZ oven for 2 hours at 40

�C. The probe was followed by the

application of Hybridization Amps 1–6. Slides were washedbetween the application of the probe and amps using 1� RNA-scope wash buffer. Signal detection was carried out using DAB;slides were counterstained using 50% hematoxylin, and washedwith 0.02% ammonia water. Slides were then dehydrated using70% ethanol, 95% ethanol (2�), incubated in xylene for 5minutes, and then coverslipped using Cytoseal XYL (ThermoFisher Scientific) and a number 1.5 coverslip.

Apoptotic body countThe xenograft specimens were fixed in 10% buffered formalin

andprocessed for routineparaffin sections. The5-mmsectionswerestainedbyhematoxylin andeosin. Thenumberof apoptotic bodiesperfieldwas countedwith a60�objective and10� eyepieceunderlight microscopy on hematoxylin and eosin–stained slides by ablinded pathologist (R. Hu). Areas adjacent to necrosis wereavoided. Apoptotic bodies were identified as shrunken cells withcompact, segregated and sharply delineated chromatin with adeeply eosinophilic cytoplasm. Five to 10 random fields wereexamined in each specimen depending on the size of tissue, andaverage was taken to represent the count for each condition.

ResultsEstablishment and characterization of ACC PDX

The PDX was established in NSG mice from a portion of asurgical specimen from a patient with a regional recurrence of

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ACC arising over 10 years after initial diagnosis. The PDXappeared approximately 3 months after implantation and wasable to be successfully passaged for additional analyses. Com-parison of H&E and IHC staining between the patient tumor andsubsequent passages was performed by a board-certified pathol-ogist specializing in head and neck cancer (R. Hu). Evaluation ofthe patient tumor (Fig. 1A) demonstrated an infiltrative tumorwith collagenous and fibrotic stroma that was infiltrated bylymphocytes and plasma cells. Blood vessels were present in thestroma and invaginated into the tumor lobules. The tumor had apredominantly cribriform growth pattern with smaller areas dis-playing a solid growth pattern. Intracystic spaces filled withbasophilic material were also identified. Comedonecrosis waspresent in less than 5% of the tumor mass. Evaluation of the firstand fifth passages of the PDX tumor (Fig. 1A) demonstrated asimilar growth pattern and morphology to the original tumorwith a predominantly cribriform pattern. The PDX tumors werewell circumscribed with scant collagenous stroma infiltrated by asmall number of macrophages and mast cells. Blood vessels wereobserved only in the stroma. TheNSGmice do not generate B andT cells, accounting for the lackof lymphocytes. ThePDX tissue alsodemonstrated greater comedonecrosis (25%–30%) relative to thepatient tissue. A biphasic appearance typical of ACCwas observedin both the patient primary and PDX tumors. The tumor wascomprised of a luminal epithelium forming scattered ductulesand abluminal (myoepithelial) cells, with abluminal cells form-ing the predominant cell type. This biphasic feature is highlightedby IHC studies of CD117, CK5/6, and p63: CD117 stains theluminal lining cells but not abluminal cells; p63 stains theabluminal cells only; CK5/6 stains abluminal cells weakly andstains luminal epithelium strongly. This luminal/abluminal pat-

tern remained consistent acrossmultiple passages of the PDX. STRprofiling of the patient tumor and the same two passages of PDXconfirmed the genetic identity of the patient and xenograft tumors(Supplementary Table S2). A PCR-based approach was used totest for the MYB-NFIB fusion commonly observed in ACC: boththe original patient tumor and both PDX passages were found tobe negative for this genetic lesion (Supplementary Fig. S1).

As part of the standard characterization of our PDXs, tumortissue was isolated, gDNA was prepared, and Illumina HotSpotsequencing was performed as described previously (38) to deter-mine the TP53 status of this tumor. With over 50� coverage overthe most common sites of TP53 mutation, no oncogenic TP53SNPs or INDELs were observed in either the patient tumor or anysubsequent passage (Fig. 1B). A cancer risk–associated P72Ralteration was identified in greater than 95% of reads for thepatient and both PDX passages, consistent with a germline alter-ation (39). Given the fidelity of this PDXmodel to a typical TP53WT ACC tumor we hypothesized that it would be a good candi-date for MDM2 inhibition and radiation combination therapyand proceeded to test this treatment regimen.

Combination of AMG 232 with escalating radiotherapy doseWe investigated the ability of AMG 232 to radiosensitize ACC

in a pilot study using a single radiotherapy dose (5 Gy) deliveredtwice weekly over 4 weeks with concurrent daily AMG 232(Supplementary Fig. S2A). Tracking of mouse weights indicatedthat this treatment combinationwaswell tolerated by the animals(Supplementary Fig. S2B). The promising results of this study ledus to conduct a dose escalation experiment combining a singledose level of AMG 232 (50 mg/kg) with an escalating doseof radiotherapy of 2, 5, or 8 Gy/fraction for a total of 8 fractions

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Characterization of ACC PDX. A, H&E and IHC staining of the primary patient tumor, the first passage PDX, and the most recent (fifth passage) PDX. IHCdiagnostic markers for ACC included CD117, stains the luminal lining cells but not abluminal cells; p63 stains the abluminal cells only; CK5/6 stains abluminal cellsweakly and stains luminal epithelium strongly. B, Left, summary of nonsynonymous mutations identified by hotspot sequencing. Green, no oncogenicmutations identified; red, potentially oncogenic mutation identified. Right, coverage map of TP53 sequencing. Gray bars are histogram of counts for eachtarget amplicon. Orange bars are TP53 coding DNA sequence, with intervening noncoding regions indicated by dashed line. Blue boxes are targetamplicons included in the sequencing panel.

AMG 232 þ Radiation in ACC

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(Fig. 2A). AMG 232 alone hadminimal sustained effect on tumorgrowth, while the 3 radiotherapy alone groups slowed tumorgrowth in a dose-dependentmanner, but did not cause prolongedtumor regression. Strikingly, with as little as 2 Gy/fraction (16 Gytotal), the combination of AMG 232 and radiotherapy resulted inrapid and sustained tumor regression. To assess the extent oftumor control, mice were followed over 100 days after the end oftreatment, and still, no tumor regrowth was observed. Animalstreated with either 5 or 8 Gy/fraction demonstrated more rapidtumor shrinkage and also had no apparent tumor regrowth at theend of the study.

Statistical analysis was performed to determine whetherincreased radiotherapy dose produced an antitumor benefit.One-way ANOVA model was conducted to determine whetherthe mean volumes were different between five treatment groups:control, AMG 232, AMG 232þ 2 Gy, AMG 232þ 5 Gy, and AMG232þ 8Gy. Therewas a statistically significant difference betweengroups as determined by ANOVA (F(4,45) ¼ 14.47, P < 0.0001),with approximately 56% of the variance being explained by themodel. In addition, a Tukey post hoc test shows that mean scoreswere significantly lower in AMG þ 2 Gy group compared withAMG alone group [�0.657.54 � 160.118 (SD), P ¼ 0.002].Similarly, a statistically significant difference revealed a lowermean score for all the combination therapy groups comparedwith either the control or AMG 232 only groups. However, therewas no statistically significant difference between AMG þ 2 Gyversus AMGþ 5 Gy (P¼ 0.998) or AMGþ 5 Gy versus AMGþ 8Gy (P¼ 0.999). These data suggest that increasing doses from 2 to8 Gy within the combination arms were not statistically differentfrom each other. To further assess the ability of AMG 232 toimprove tumor control, a TCD50 calculation was performed (Fig.2B). Radiotherapy alone failed to control any tumors (TCD50 > 8Gy/fraction), while combination with AMG 232 significantly

decreased the TCD50 (<0.6 Gy/fraction, P < 0.0001) consistentwith radiosensitization. We used the fractional product methodsto calculate a synergy assessment ratio for AMG 232 and eachradiationdose. The ratio's calculated (2.1, 3.2, and3.3 for 2, 5, and8 Gy, respectively) are consistent with a finding of synergybetween radiation and AMG 232.

Radiotherapy and AMG 232 activate p53 and downstreamsignaling

We next investigated the molecular response to this treatmentregimen. The effect on p53 and p-p53 levels was examined byWestern blotting and IHC on xenograft tissues harvested 2 hoursposttreatment. Total p53 levels were fairly stable with someincrease at higher radiotherapy doses, while both AMG 232 orradiotherapy alone did activate p53 as shown by higher p-p53levels. The combination of radiotherapy and AMG 232 showedincreased p53 and p-p53 activation relative to radiotherapy orAMG 232 alone (Fig. 3A and B). Semiquantitative analysis of theIHC images confirmed increased p53 and p-p53 activation intreated tumors (Supplementary Fig. S3). DNA damage wasdetected by immunoblot for g-H2AX and KU80 2 hours post-radiotherapy; expression of g-H2AX was radiotherapy dosedependent, but the addition of AMG 232 had no effect. Littleeffect on KU80 was observed. Tumor samples harvested 48hours posttreatment revealed that AMG 232 treatment promot-ed elevated levels of MDM2 consistent with feedback that istypical after prolonged exposure to an inhibitor, with elevatedlevels of the P53 effector protein PUMA observed in all treatedtumors (Fig. 3A).

Downstream effects of p53 activation were evaluated byCDKN1A response (Fig. 3C). At 2 hours posttreatment, elevatedCDKN1A RNAwas identified by in situ hybridization (RNAscope)in both the AMG 232 and radiotherapy single modality

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Radiotherapy and AMG 232 combination therapy of ACC PDX. A, Growth curves representing tumor volume of the 8 treatment groups as a function of dayspostengraftment. Filled symbols represent mock/radiotherapy only groups; open symbols are AMG 232/radiotherapy combination groups. Radiotherapyfractions were delivered on days indicated by inverted triangles. AMG 232 or vehicle was delivered by oral gavage once per day over time period shown.B, TCD50 analysis of ACC PDX � AMG 232; data were fit to a log(treatment) versus response curve using a least squares fit. TCD50 with radiotherapy alone wasnot reached and can only be stated as >8 Gy. In combination with AMG 232, the TDC50 was calculated at 0.6 Gy with a 95% confidence intervals (CI) of 0.01to 1.5 Gy. Error bars, 95% CI.

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treatments with the combination presenting even greater expres-sion. This effect was confirmed at the protein level where thecombination produced elevated CDKN1A as seen by IHC. Semi-quantitative analysis of the IHC images showed adose-dependentelevation of CDKN1A with increasing radiation (SupplementaryFig. S3). Together, the results confirmed that the treatment reg-imen was inducing expected DNA damage and TP53-mediatedtumor-suppressive pathways.

Radiotherapy and AMG 232 cooperatively induceantiproliferative and proapoptotic responses

We next evaluated the growth and cell death impact of thesetreatments on the ACC PDX. Using tumor tissue harvested 48hours after a single dose of drug and/or radiotherapy, weimmunostained for the proliferation marker Ki-67 and countedpositive nuclei from multiple fields for each condition (Fig.4A). Relative to mock-treated animals, all treatments reducedcell proliferation, with a greater reduction and higher level of

significance for the high dose radiotherapy þ AMG 232 com-bination. We next immunostained for cleaved caspase-3, amarker of early stages of apoptosis, in tumors 48 hours afterre-treatment with a single dose of AMG 232 or radiotherapy(Fig. 4B). On the basis of counting of cleaved caspase-3–positive nuclei, neither AMG 232 nor 2 Gy radiotherapy aloneincreased apoptosis in these tissues. Although the 8 Gy fractiondid have higher counts, it did not reach statistical significance.Consistent with the rapid shrinkage of the tumors in the AMG232 þ radiotherapy arms, both of these treatments presentedsignificantly elevated caspase staining in a dose-dependentmanner. We analyzed later stages of apoptosis by countingapoptotic bodies in multiple high-power fields for the samesamples. In this analysis, only the AMG 232 þ 8 Gy treatmentproduced a statically significant increase in apoptosis, with theAMG 232 þ 2 Gy counts slightly elevated after this singlefraction (Fig. 4C). Overall, these results are consistent with anantiproliferative, growth-delay impact of the AMG 232 and

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Figure 3.

TP53 pathway activation in response to radiotherapy and MDM2 inhibition. A, Left, immunoblot for p53, p-p53, g-H2AX, and Ku80 of ACC PDX tissue 2 hoursfollowing AMG 232, radiotherapy, or combination treatment; right, immunoblot of MDM2 and PUMA 48 hours following AMG 232, radiotherapy, orcombination treatment. B, Representative fields of IHC staining for p53 and p-p53 of ACC tumor samples 2 hours post-radiotherapy and AMG 232 treatment. Scalebars, 20 mm. C, Representative fields of in situ RNA staining for CDKN1A (top) and CDKN1A protein (a.k.a. p21) IHC (bottom) of ACC tumor samples 2 hours post-radiotherapy and AMG 232 treatment. Scale bars, 20 mm.

AMG 232 þ Radiation in ACC

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Figure 4.

Proliferation and apoptosis markers following radiotherapy and AMG 232 treatment. A, Ki-67 immunostaining of PDX samples harvested 48 hoursposttreatment. Images are representative of 20� fields; scale bars, 10 mm. Treatment was scored by counting Ki-67–positive nuclei from three 20� fields percondition. Bar plots, mean countswith SD; statistical analysis by one-wayANOVAwith Tukeymultiple comparisons.B,Cleaved caspase-3 staining and quantificationof 48-hour posttreatment tumor samples. Images are representative of 40� fields; scale bars, 20 mm. Cleaved caspase-3–positive nuclei within the tumorwere counted for three 40� fields for duplicate tumor samples; stromal regions were ignored. Cells presenting cytoplasmic or background staining were notincluded in counts. Bars are mean of all fields scored for a given condition with SD; statistical analysis by one-way ANOVA with Tukey multiple comparisons.C, Quantification of apoptotic bodies in 48-hour posttreatment tumor samples in H&E-stained slides. Five to ten 60� fields were counted for duplicatetumor samples. Representative images are shown for mock and AMG 232 þ 8 Gy treatments (scale bars, 20 mm) with inset images with arrows highlightingapoptotic bodies (scale bars, 5 mm). Bars are mean of all fields scored for a given condition with SD; statistical analysis by one-way ANOVA with Tukeymultiple comparisons. ���� , P < 0.0001; �� , P < 0.01; � , P < 0.05.

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radiotherapy single-modality treatments, but a cell death–inducing, tumor-shrinking effect of the combination.

DiscussionACC patients have limited treatment options beyond surgery

and radiotherapy, and long-termprognosis remains poor. There isa need for novel therapeutic options to improve care, particularlyfor patients with metastatic disease. The rarity of the disease, thelack of robust preclinical models, and the absence of effectivesystemic therapies to date remains a major challenge for ACCpatients. We developed and characterized a PDX model (UW-ACC-60) representing one of only a handful available for sus-tained preclinical investigation of ACC (27, 40). This modelrepresents the most prevalent type of ACC seen in patients, thecribriform subtype and maintains its histologic features overmultiple passages.

Targeted sequencing demonstrated that this model, like themajority of ACCs, does not contain a TP53 function alteringmutation, making it amenable for treatment approaches that relyon functional p53. The low rate of TP53 mutations seen in ACChas led other groups to investigate the efficacy of MDM2 inhibi-tors as potential systemic therapies given alone, or in combinationwith cisplatin. Nor and colleagues used PDXs established at theirinstitution to demonstrate that an MDM2 inhibitor, MI-773,slowed growth of ACC both alone and when combined withcisplatin (26, 27). They showed that MDM2 inhibition led to p53activation, induction of apoptosis, tumor growth delay, and couldprevent the recurrence of surgically resected xenografted tumors.

On the basis of the critical need for innovative approaches toimprove local control for patients both newly diagnosed withACC and dealing with local–regional recurrences, we began thisproject to investigate potential radiosensitizers of ACC. Buildingon thework byNor and colleagues (26, 27), and our success usingAMG 232 to radiosensitize lung squamous cell carcinoma inaddition to cell lines derived from colon, breast, sarcoma, andmelanoma (31), we combined AMG 232 with radiotherapy inACC.We hypothesized that radiation-induced p53 activity wouldbe enhanced by inhibiting MDM2, a negative regulator of p53.This combination would thus take advantage of the low rate ofTP53 mutation seen in ACC. The combination of radiotherapyand AMG 232 resulted not only in growth delay typically seen inxenograft studies, but also complete regression of the tumors thatpersisted for months after the conclusion of treatment. Althoughboth MDM2 inhibition or radiotherapy alone were sufficient toactivate p53 signaling, as single modalities, they had modesteffects on tumor growth. Combination treatment decreased Ki-67 staining and induced apoptosis consistent with not onlygrowth delay but also the observed tumor regression. AMG232 þ radiotherapy resulted in greater p53 pathway inductionthan either treatment alone as demonstrated by upregulation ofp53 target genes and increased protein expression. Given the keyrole of p53 as a tumor suppressor, these findings are not unex-pected and in fact complement what both we, and Nor andcolleagues, have previously demonstrated regarding the mecha-nism of MDM2 inhibition as an anticancer therapy.

Radiosensitization and xenograft cures were observed withtotal radiotherapy doses as low as 16 Gy delivered over 4 weeks.Although additional studies confirming the efficacy of AMG 232in combination with low-dose radiotherapy would be needed,these results are encouraging. Many ACC patients live years after

completion of adjuvant radiotherapy and often are faced with thedifficult decision to undergo repeated operations and/or reirra-diation due to local and/or regional tumor progression. This workraises the possibility that a radiosensitizer could be combinedwith focal radiation to improve local tumor control. Using mod-ern techniques, highly conformal radiation can be easily deliveredto sites of measureable disease while limiting the volume ofnormal tissues receiving appreciable radiotherapy dose. Althoughthis combination would need to be carefully studied to ensurereasonable normal tissue toxicity, we did not identify any signif-icant toxicity in the mice treated with combination therapy.

We acknowledge several limitations of this study. As withmostcurrent PDX studies, we completed these experiments in immu-nocompromised animals, thus limiting our ability to identifyimmunologic mechanisms, and focused the study on intrinsicradiosensitivity. It is possible that the addition of immune-medi-ated cell death driven by radiotherapy and AMG 232 wouldrequire even lower doses to achieve similar results. Althoughthere are several other described ACC PDX models (27, 28), thestudies in this article clearly utilize only a single in vivo modeldeveloped from a single patient. The fact that the histology of thismodel (cribriform subtype) is themost common type of ACC andthat the mutational profile is consistent with recently publishedsequencing of ACC patients suggests that these results may havemore broad implications, but additional work is needed toconfirm these findings. Finally, because no ACC-immortalizedcell lines are publicly available (41), and we have thus far beenunable to establish a reliable in vitromodel, our experiments werelimited to in vivo studies. We have an active tissue donationprotocol ongoing and are seeking to generate additional PDXsand derive cell lines or 3D culture models of ACC to further theseand future investigations.

In conclusion, we have demonstrated robust radiosensitizationof an ACCPDX by combining theMDM2 inhibitor AMG232withrelatively low doses of radiotherapy. Physicians who care for ACCpatients knowall toowell thedesperateneed forbothnewsystemicagents as well as improved local control for these patients. Giventhe low frequency of TP53 mutation in ACC, tumor characteriza-tion or patient selection criteria to identify patients with wild-typeTP53 may be unnecessary for a clinical trial. This work adds to thegrowing evidence that targeting MDM2, alone or in combinationwith another anticancer therapy, in patients with ACC may be asuccessful treatment strategy. Through continuedwork,wemayyetbe able to improvequality of life, andpotentially improve survival,in patients living with ACC.

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

Authors' ContributionsConception and design: P.J. Prabakaran, A.M. Javaid, A.D. Swick, L.R. Werner,E. Sampene, J. Canon, R.J. KimpleDevelopment of methodology: P.J. Prabakaran, A.M. Javaid, A.D. Swick,L.R. Werner, R.J. KimpleAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P.J. Prabakaran, A.M. Javaid, A.D. Swick, L.R. Werner,R. Hu, G.K. Hartig, P.M. Harari, R.J. KimpleAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.J. Prabakaran, A.M. Javaid, A.D. Swick, E. Sampene,I.M. Ong, P.M. HarariWriting, review, and/or revision of the manuscript: A.M. Javaid, A.D. Swick,L.R.Werner, E. Sampene, I.M. Ong, J.Y. Bruce, G.K. Hartig, A.Wieland, J. Canon,P.M. Harari, R.J. Kimple

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Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P.J. Prabakaran, A.M. Javaid, K.P. Nickel,R.J. KimpleStudy supervision: A.D. Swick, R.J. Kimple

AcknowledgmentsWe would like to thank Ella Ward and University of Wisconsin Research

Pathology facility for tissue processing and histology services. We also wouldlike to acknowledge the University of Wisconsin Translational Research Initia-tives in Pathology laboratory, in part supported by the UW Department ofPathology and Laboratory Medicine and UWCCC grant P30 CA014520, for useof its facilities and services including STR analysis. The author(s) thank theUniversity of Wisconsin Biotechnology Center DNA Sequencing Facility forproviding the hotspot sequencing facilities and services.

Grant SupportThis work was supported in part by a Department of HumanOncology Seed

Grant and the Karl Harter Scholarship Fund (to R.J. Kimple), CA160639 (toR.J. Kimple), a PhRMA Foundation Postdoctoral Fellowship in TranslationalMedicine and Therapeutics (to A.D. Swick), University of Wisconsin CarboneCancer Center Support Grant (P30 CA014520), and Wisconsin Head and NeckSPORE Grant (NIHP50 DE026787).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 6, 2017; revised May 24, 2017; accepted June 23, 2017;published OnlineFirst June 28, 2017.

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2017;23:6044-6053. Published OnlineFirst June 28, 2017.Clin Cancer Res   Prashanth J. Prabakaran, Amal M. Javaid, Adam D. Swick, et al.   InhibitionRadiosensitization of Adenoid Cystic Carcinoma with MDM2

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