SMAC Mimetic Birinapant plus Radiation Eradicates Human Head … · Therapeutics, Targets, and Chemical Biology SMAC Mimetic Birinapant plus Radiation Eradicates Human Head and Neck

Therapeutics, Targets, and Chemical Biology

SMAC Mimetic Birinapant plus RadiationEradicates Human Head and Neck Cancers withGenomic Amplifications of Cell Death GenesFADD and BIRC2Danielle F. Eytan1,2,3, Grace E. Snow1,2, Sophie Carlson1, Adeeb Derakhshan1,2,3,Anthony Saleh1, Stephen Schiltz1, Hui Cheng1, Suresh Mohan1,2, Shaleeka Cornelius1,Jamie Coupar1, Anastasia L. Sowers4, Lidia Hernandez5, James B. Mitchell4,Christina M. Annunziata5, Zhong Chen1, and Carter Van Waes1

Abstract

Comparison of tumors fromTheCancerGenomeAtlas (TCGA)reveals that head and neck squamous cell carcinomas (HNSCC)harbor the most frequent genomic amplifications of Fas-associ-ated death domain (FADD), with or without Baculovirus inhib-itor of apoptosis repeat containingBIRC2 (cIAP1), affecting about30% of patients in association with worse prognosis. Here, weidentified HNSCC cell lines harboring FADD/BIRC2 amplifica-tions and overexpression by exome sequencing, RT-PCR, andWestern blotting. In vitro, FADD or BIRC2 siRNA knockdowninhibited HNSCC displaying amplification and increased expres-sion of these genes, supporting their functional importance inpromoting proliferation. Birinapant, a novel SMAC mimetic,sensitized multiple HNSCC lines to cell death by agonists TNFaor TRAIL and inhibited cIAP1>XIAP>IAP2. Combination ofbirinapant and TNFa induced sub-G0 DNA fragmentation in

sensitive lines and birinapant alone also induced significantG2–M cell-cycle arrest and cell death in UM-SCC-46 cells. Genetransfer and expression of FADD sensitized resistant UM-SCC-38cells lacking FADDamplification to birinapant and TNFa, support-ing a role for FADD in sensitization to IAP inhibitor and deathligands.HNSCCvaried inmechanismsof cell death, as indicatedbyreversal by inhibitors or protein markers of caspase-dependentapoptosis and/or RIPK1/MLKL-mediated necroptosis. In vivo, bir-inapant inhibited tumor growth and enhanced radiation-inducedTNFa, tumor responses, and host survival inUM-SCC-46 and -11Bxenograft models displaying amplification and overexpression ofFADDþ/� BIRC2. These findings suggest that combination ofSMAC mimetics such as birinapant plus radiation may be partic-ularly active in HNSCC, which harbor frequent FADD/BIRC2genomic alterations. Cancer Res; 76(18); 5442–54. �2016 AACR.

IntroductionHead and neck squamous cell carcinoma (HNSCC) is the sixth

most common cancer worldwide, with more than 600,000 newcases diagnosed each year and a 5-year survival rate of about 50%(1). The Cancer Genome Atlas (TCGA) analysis of 279 HNSCC

recently uncovered genomic alterations in cell death pathwaysaffecting approximately 40% of HNSCC (2–4). These includeabout 30% that harbor chromosome 11q13/22 amplificationsand overexpression of Fas-associated death domain (FADD), withor without Baculovirus inhibitor of apoptosis repeat containing(BIRC2/3) genes that encode cellular inhibitor of apoptosis pro-teins 1/2 (cIAP1/2). A mutually exclusive subtype (�10%) bearsmutations in caspase-8 (CASP8) together with oncogenes HRASorPIK3CA (2, 3). Significantly, amplifications of FADDwithout orwith BIRC2 are more often linked to the human papilloma virus–negative [HPV(�)] HNSCCs with worse prognosis (2).

Functionally, FADD, cIAP1/2, and CASP8 are critical compo-nents of the TNF receptor family signaling pathways that deter-mine cell death or survival (5–10). Binding of TNFa, TRAIL, orother death agonists to their receptors typically leads to cell deathvia FADD through the activation of caspase-8 and induction ofapoptosis, or alternatively, activation of RIP kinase 1 (RIPK1),MLKL, and induction of necroptosis (5–10). However, FADDoverexpression has also been implicated in cell growth in lungcancers and in primary tumors associated with lymph nodemetastases in HNSCC (11–13), suggesting that its function incancer is context-dependent. TNFa-FADD signaling and promo-tion of cell death via apoptosis or necrosis may be inhibited bycIAPs (14).

1Tumor Biology Section, Head and Neck Surgery Branch, NationalInstitute on Deafness and Other Communication Disorders, NIH,Bethesda, Maryland. 2NIH Medical Research Scholars Program/HHMI-NIH Scholars Research Program, Bethesda, Maryland. 3Cleve-land Clinic Lerner College of Medicine, Cleveland, Ohio. 4RadiationBiologyBranch,Center forCancerResearch,NationalCancer Institute,NIH, Bethesda, Maryland. 5Women's Malignancies Branch, Center forCancer Research, National Cancer Institute, NIH, Bethesda, Maryland.

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

D.F. Eytan, G.E. Snow, S. Carlson, A. Derakhshan, and A. Saleh contributedequally as first authors.

Corresponding Authors: Carter Van Waes, National Institute on Deafness andOther Communication Disorders, NIH, Building 10/Rm 7N240, 10 Center Dr.,Bethesda, MD 20891. Phone: 301-402-4216; Fax: 301-402-1140; E-mail:[email protected]; and Zhong Chen, [email protected]

doi: 10.1158/0008-5472.CAN-15-3317

�2016 American Association for Cancer Research.

CancerResearch

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IAPs are degraded following binding by the second mito-chondria-derived activator of caspases (SMAC), an endogenousprotein released by mitochondria at the onset of apoptosis,(15). Targeting IAPs with small-molecule mimetics of SMACcan modulate cell death in cancer cells, giving rise to growinginterest in their potential as therapeutics (16). Birinapant is anovel bivalent peptidomimetic of SMAC, shown to preferen-tially target cIAP1, relative to cIAP2 and XIAP (17, 18). Birina-pant demonstrated antitumor activity in preclinical models ofhematologic cancers and solid tumors (19, 20). In early-phaseclinical trials, birinapant has demonstrated tolerability andsafety at active doses, with a prolonged plasma half-life of31 hours and tumor half-life of 52 hours (21). In a 5-armphase I/II dose escalation study, safety, tolerability, and phase IIdosing of birinapant in combination with different che-motherapies in patients with solid tumors were established(22). Interestingly, birinapant demonstrated prolonged pro-gression-free survival in previously relapsed or refractorypatients when combined with chemotherapies known to induceTNFa, such as irinotecan (21, 22). Notably, radiotherapy

induces TNFa and cytotoxicity (23, 24) and is a critical modal-ity in treatment of HNSCC.

Here we explore the potential and mechanisms by whichHNSCC harboring amplifications and overexpression of FADD,without or with BIRC2, may be sensitized to SMAC mimeticsalone or in combination with TNFa, TRAIL, or radiation.

Materials and MethodsTCGA data analysis

TCGA data were accessed through the cBioPortal for CancerGenomics (http://www.cbioportal.org/) and queried for FADD,BIRC2, BIRC3, andCASP8mutation and copy number alterationsfor all cancer studies (25, 26). Comparison for copy numbervariation between HPVþ and HPV� used one-sided Wilcoxonsigned rank test.

Cell lines and genomic alterationsThe 11 HPV�and 1 HPVþHNSCC cell lines were authenticated

by genotyping using 9 allelic markers (27) and obtained fromDr.

Figure 1.

Genetic alterations of cell deathpathway components in HNSCC andother cancer types from TCGA datasets.A, genetic alterations of FADD, BIRC2,BIRC3, and CASP8 for top 10 tumortypes. DNA amplification (red),mutation (green), and deletion (blue),or multiple defects (gray). B, OncoPrintshowing genetic and expressionalterations from 279 HNSCC samples.Vertical bars represent alterations forsingle specimens. Top bars, blue, HPV�;red, HPVþ. C, scatter plots, FADDamplification and overexpression ispredominantly associated with HPV�

tumors. P values are comparing HPVþ

and HPV� with one-sided Wilcoxonsigned rank test. D, Kaplan–Meiersurvival plots for FADD in 279 HNSCC inTCGA.

Birinapant and Radiation in HNSCC

www.aacrjournals.org Cancer Res; 76(18) September 15, 2016 5443




Figure 2.

Copy variation and expression of FADD, BIRC2/3, andCASP8 byHNSCC lines.A, chromosome 11q13/11q22 FADD/BIRC2 loci (red bars) copy variation in UM-SCC lines.Heatmaps show segmented copy number variations; red, gain; blue, loss; and white, no change. B, qRT-PCR data showing relative fold expression of FADD,BIRC2, BIRC3, and CASP8 mRNA in UM-SCC cell lines normalized to expression levels in primary human oral keratinocytes (HOK). UM-SCC-1 harboring a CASP8heterozygous Q107 nonsense mutant is indicated. �, P < 0.05; �� , P < 0.001 versus HOK, Student t test. C, Western blot of proteins from whole-cell lysates ofHOK and UM-SCC cell lines. Expressionwas quantified by densitometry comparison to HOK and b-tubulin as a loading control, except where �cIAP2 is undetectablein HOK.

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T.E. Carey at the University of Michigan (Ann Arbor, MI) in 2010.Stocks were preserved in �80�C, cultured as described (27), andused within 3 months.

DNA exome sequencing for copy number and mutationsgDNAwas obtainedwith aDNeasy Blood&Tissue kit (Qiagen)

from frozen cell pellets of UM-SCC lines. Whole-exome sequenc-ing with about 87� coverage was performed using SOLiD4platform (Applied Biosystems) and copy number alterations forFADD and BIRC2/3 genes were analyzed using CONTRA and IGAsoftware (28, 29). ACASP8mutationwas identified using ANNO-VAR (http://annovar.openbioinformatics.org/) and MutationAssessor (http://mutationassessor.org/r3/) using settings speci-fied (Supplementary Methods).

Western blotsUM-SCC cells were plated overnight before treatment with

birinapant � TNFa or TRAIL or 0.01% DMSO diluent control.At 12, 24, and 48 hours after treatment, whole-cell lysates werecollected, and electrophoresis and Western blotting were per-

formed, using antibodies, reagents, and conditions specified inSupplementary Methods.

Real-time quantitative PCRRNA isolation was performed using the RNeasy Mini Kit from

Qiagen and on snap-frozen tumor samples collected from miceafter 9 days of treatment using the mirVana miRNA Isolation KitfromAmbionbyLife Technologies (AM1561). TheHigh-CapacitycDNA Reverse Transcription Kit (Applied Biosciences) was usedper manufacturer's instructions. Thirty nanograms of cDNA wasamplified with primers and TaqMan Universal Master Mix in anABI Prism 7700 Sequence Detection System (Applied Biosys-tems). Relative gene expression was normalized to 18S control.Samples were assayed in triplicate and presented as the mean �SD. Significance was determined using the Student t test and P <0.05 was considered statistically significant.

Gene siRNA knockdown and overexpressionCells were transfected as described (30) with 50 nmol/L of

nontargeting control, BIRC2, BIRC3, and/or FADD siRNA, or

Figure 3.

Effects of BIRC2 and FADD siRNAknockdown or birinapant, with TNFa andTRAIL on UM-SCC cell lines. A, celldensity of UM-SCC-11A and UM-SCC-1measured by XTT at 3 daysposttreatment after control, BIRC2,BIRC3, and/or FADD siRNA knockdown,�20 ng/mL TNFa. Cell density, aspercent of control siRNA, no TNFa. Errorbars, SD of three replicates. Student ttest: � , P < 0.05; �� , P < 0.001 versuscontrol siRNA; ^, P < 0.05; ^^, P <0.001 versus control siRNA þ TNFa; þ,P < 0.05; þþ, P < 0.001 TNFa versus noTNFa. B, percent growth inhibition byXTT assays on day 3 posttreatment inUM-SCC cell panel treated with1 mmol/L birinapant, 20 ng/mL TNFa(top), 50 ng/mL TRAIL (bottom), or thecombinations. Nontreated andbirinapant controls for comparison withTNFa or TRAIL or their combinationswere performed in independentexperiments. Values normalized tonontreated cells for the sameexperiment, P < 0.05, � , compared withcontrol; bar, combination vs. birinapantalone, Student t test.






100 ng/well empty control pcDNA 3.1 or FADD vector plas-mids (gifts from Dr. Gabriel Nu~nez, University of Michigan;ref. 31), using 0.25 mL/well of Dharmafect Duo (GE Dharma-con) in 100 mL MEM media/well. After 24 hours, cells weretreated with birinapant (10 mmol/L) and/or TNFa (20 ng/mL),and viability was assessed via XTT assay 48 and 72 hoursfollowing treatment.

XTT and caspase assaysCells were plated in 96-well plates and treated with 0.01%

DMSO control or birinapant concentrations indicated � 20ng/mL TNFa (R&D systems). Cell density was measured byXTT Kits (Roche) and inhibitory concentration 50% (IC50) wasdetermined at 48 to 72 hours, as described (SupplementaryMethods). To differentiate between apoptotic and necroptoticcell death, cells were treated with birinapant � TNFa, TRAIL,ZVAD, ZIETD, or necrostatin (BD Pharmingen). Cells werecollected, lysed, and assayed for activity of caspases-3 and -8

using assay kits (Biovision) per manufacturer's protocol on amicroplate reader.

DNA cytofluometryCells were plated and treated over 24 to 72 hours with 0.01%

DMSO control, 20 ng/mL TNFa, or 1 mmol/L birinapant� TNFa,and prepared for flow cytometry using the Cycle TEST PLUS DNAReagent Kit (BDBiosciences), for analysis on the FACSCanto flowcytometer (BD Biosciences). Data from 10,000 cells per treatmentgroup and time point were analyzed using BD FACSDiva (BDBiosciences) software.

HNSCC xenograft studiesAll animal experiments were carried out under protocol 1322-

13 approved by the NIDCD Animal Care and Use Committee.Four- to 6-week-old female SCID/NCr-Balb/c mice and athymicnu/nu were obtained from Frederick Cancer Research and

Figure 4.

Effect of brinapant and TNFa on celldensity in FADD-deficient UM-SCC-38following overexpression of FADDvector, and on cell-cycle DNA in UM-SCC-46 and 1 with greater and weakerFADD expression. A, cellularproliferation in UM-SCC-38 followingtransfection with pcDNA 3.1 or FADDvector by XTT at 48 and 72 hoursposttreatment with 10 mmol/Lbirinapant and/or 20 ng/mL TNFa.Student t test, � , P < 0.05 for emptyvector versus FADD; þ, P < 0.05comparing FADD control versus FADDplus combination. B, UM-SCC-46 and -1cells were treated with 1 mmol/Lbirinapant with or without 20 ng/mLTNFa and harvested at 48 hoursposttreatment for DNA cell-cyclecytofluorometry. Histograms withpercentage of cells with sub-G0, G0–G1,S and G2–M DNA.

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Development Center (National Cancer Institute) and housedin a specific pathogen-free animal facility. Mice were injectedwith UM-SCC cells, randomized, and treated with birinapantand radiation as indicated and specified in SupplementaryMethods.

ImmunohistochemistryMice harboring UM-SCC-46 tumors were sacrificed after 11

days of control and birinapant treatment indicated. Tissue sam-ples were embedded in OCT, and frozen tissue sections wereprepared by Histoserv, Inc. Tissue sections were fixed using 4%

Figure 5.

Birinapant plus death ligands induced cell death in UM-SCC lines via caspase and/or necroptosis dependentmechanisms.A, cell lineswere treatedwith combinationsof 1 mmol/L birinapant, 20 ng/mL TNFa, 50 ng/mL TRAIL, 20 mmol/L ZVAD (pan-caspase inhibitor), 20 mmol/L ZIETD (caspase-8 inhibitor), or 20 mmol/Lnecrostatin-1 (RIPK1 inhibitor) and assessed for effect on cell density or caspase-3 and -8 activation. SE bars for three replicates, Student t test. � , P < 0.05.B, in independent experiments, UM-SCC-1, -46, and -11B cell lines were treated with 0.01% DMSO diluent control, or 1 mmol/L birinapant and 20 ng/mL TNFa (BþT),and whole-cell lysates procured 12, 24, and 48 hours after treatment were subjected to SDS-PAGE and Western blot analyses. Expression for each proteinwas quantified and normalized to both diluent control (¼1) and b-tubulin loading control for the respective lane for the blots shown for each cell line.






Figure 6.

Birinapant effects on tumor growth, survival, and molecular markers in human HNSCC xenograft models. A total of 5 � 106 UM-SCC-46 FADD or -11B FADDand BIRC2 amplified cells were injected subcutaneously into SCID/NCr-Balb mice. Mice were randomized (UM-SCC-46, n ¼ 15 mice/group; UM-SCC-11B, n ¼ 9mice/group) and treatment was initiated 15 and 35 days after tumor inoculation, respectively, when tumors reached 200 mm3. Control vehicle, 15 or 30 mg/kgbirinapant intraperitoneally was given every 3 days � 10 doses (double-ended arrow). (Continued on the following page.)

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formalin, permeabilized with methanol, and quenched with 3%H2O2. Samples were blocked using rabbit serum and stained withprimary and secondary antibodies specified in SupplementaryMethods using the VECTASTAIN Elite ABC Kit (Vector Laborato-ries). Slides were imaged using the Aperio ScanScope (LeicaBiosystems). Histoscores were calculated by ImageScope software(Leica Biosystems).

Statistical design for data analysisData fromXTT assays and colorimetric assays were presented as

mean � SD. For tumor growth analysis, significance was deter-mined using the Student t test and P < 0.05 was consideredstatistically significant. For survival analysis, the Gehan–Bre-slow–Wilcoxon test was used and significance set to 0.05 usingthe Bonferroni method.

ResultsFrequent genetic alterations of cell death signalingmolecules inHNSCC tumors

We surveyed the frequency of gene copy number, mutations,and expression alterations for FADD, BIRC2, BIRC3 on 11q13/22,and CASP8 in TCGA datasets, for 279 HNSCC tumor specimens,and other cancers (Fig. 1A). Compared with other cancer types,alterations of FADD are highest in HNSCC, with about 30%exhibiting amplifications. HNSCC is the second most commontumorwith alterations in theBIRC2/3 locus (8.6%). Alterations ofCASP8, predominantly mutations, are most frequent in HNSCC(11.2%). Including mRNA up- or downregulation, about 60% ofHPV�andHPVþHNSCC tumors harbor alterations for these genes(Fig. 1B). FADD amplification and increased RNA expression ispredominantly seen in HPV�tumors and is associated with earlyrecurrence and worse prognosis (Fig. 1C and D).

Genetic alterations of cell death signalingmolecules in HNSCCcell lines resemble HNSCC tumors

Because of the prevalence and association of FADD/BIRC2/3and CASP8 genomic alterations with HPV�HNSCC tumors, weused exome sequencing to examine the genomic alterations ofthese key cell death pathway components in a panel of 11 humanHPV�UM-SCC cell lines from patients with poor outcomes (Sup-plementary Table S1). Consistentwith TCGA, focal amplificationsof 11q13/22 loci for FADD and BIRC2/3were found in a subset ofUM-SCC lines (Fig. 2A). Real-time PCR confirmed an increase inFADD mRNA expression of more than 3-fold in UM-SCC-11A,-11B, -22B, and -46, as compared with primary human oralkeratinocytes (HOK; Fig. 2B). BIRC2 expression was increasedmore than 3-fold in UM-SCC-6, -11A, -11B. BIRC3 was moreweakly expressed despite coamplification. UM-SCC-1 wasfound to harbor a heterozygous CASP8 mutation (Q107 non-sense mutation, NS), confirmed recently (3). UM-SCC-11A/B,-22A/B, -38, and -74A/B harbor a common single-nucleotidevariant resulting in a K14R amino acid change in CASP8 ofunknown functional significance (data not shown). Correspond-ing FADD, cIAP1, cIAP2, andCASP8proteins analyzedbyWestern

blot in HOK and UM-SCC cell lines demonstrated differences inexpression largely consistent with the RT-PCR findings (Fig. 2C).Thus, differences in genomic alterations and expression of key celldeath pathway components in human tumor specimens in TCGAare represented among these cell lines.

Knockdown of FADD and BIRC2 inhibits growth and sensitizesHNSCC cells to TNFa

We examined the functional significance of FADD, BIRC2 and 3by siRNA knockdown alone or with TNFa in 2 cell lines differingin amplification and expression of these genes. In UM-SCC-11A,with relatively higher FADD and BIRC2 amplification and expres-sion, FADD and BIRC2 knockdown alone, and in combinationwith TNFa, decreased cell density as measured by XTT assay(Fig. 3A, left; P < 0.001). In UM-SCC-1 harboring lower ampli-fication and expression of FADD and BIRC2 with the heterozy-gous CASP8 Q107 nonsense mutation, weaker inhibition of celldensity was observed with knockdown in the absence or presenceof TNFa. (Fig. 3A, right). The combined effect of FADD andBIRC2knockdown resulted in similar reduction in cell density as knock-down with either individual gene (P < 0.001). BIRC3 knockdownalone had minimal effect, consistent with the low BIRC3 expres-sion. Knockdown of the targets bymore than 80%was confirmedin both lines (Supplementary Fig. S1). Together, these datasuggest that FADD and BIRC2 overexpression may contribute togrowth and/or TNFa resistance of HNSCC.

Effects of SMAC-mimetic birinapant, TNFa, or TRAIL on celldensity, cell cycle, or death in HNSCC cell lines

As antagonists of BIRCs/IAPs may inhibit cancer cells, wescreened the panel of UM-SCC cell lines for sensitivity to SMACmimetic and IAP inhibitor birinapant by XTT assay over a range ofconcentrations (1 nmol/L–10 mmol/L) used in previous studies(20). Figure 3B summarizes the inhibition of cell density of thepanel following treatment with birinapant or cell death agonistsTNFa or TRAIL alone and in combination, as assessed duringexponential growth on day 3, with the IC50 values provided inSupplementary Table S2. Multiple cell lines showed enhancedsensitivity to birinapant þ TNFa or TRAIL, including many thatshowed increased expression of FADD and/or other pathwaycomponents above. Birinapant alone was most active in UM-SCC-46, with increased expression of FADD, similar BIRC2, butlow CASP8 relative to normal HOK cells. An HPVþ cell line, UM-SCC-47, showed modest sensitivity to birinapant and deathligands. The least TNFa-sensitive cell lines included UM-SCC-9and 38, displaying weaker expression of FADD/BIRC2.

We established that gene transfer and overexpression of FADD,but not empty vector, enhanced sensitivity of the FADD-deficientline UM-SCC-38 to birinapant and TNFa, supporting a distinctfunction of FADD overexpression in sensitization to death ligandwhen combinedwith IAP inhibition (Fig. 4A). To further evaluatewhether decreased cell density observed was related to cell cycleand/or cytotoxic effects, DNA cytofluorometric analyses of sen-sitive UM-SCC-46 and less sensitive UM-SCC-1 cell lines were

(Continued.) A and B, UM-SCC46, birinapant treatment significantly inhibited tumor volume versus control vehicle (þ, P < 0.05; þþ, P < 0.01) and improvedmedian survival (58 vs. 39days).C andD,UM-SCC-11B, birinapant treatment significantly inhibited tumor volumeversus control vehicle (þ,P<0.05;þþ,P<0.01) andincreased median survival (Gehan–Breslow–Wilcoxon test and the Bonferroni method, P < 0.05). E, in vivo effects of birinapant treatment on cIAP1, cIAP2, XIAP,TUNEL, and Ki67 proliferation markers in UM-SCC-46 xenograft mouse model of HNSCC. Photomicrographs show immunostaining done on frozen sections oftumors harvested on treatment (day 25). Bar, 200 mm. Staining histoscores for untreated (black) and birinapant-treated mice (gray). Student t test: � , P < 0.01.






Figure 7.

Birinapant plus radiation induces tumor regression in xenografts and model of cell death pathways modulated by birinapant. A, a total of 2.5 � 106 UM-SCC-46cells were implanted into athymic nu/nu mice. Mice were randomized into four groups (vehicle control, 15 mg/kg birinapant, 10 � 2 Gy radiation, or combination,n ¼ 12 each) 7 days after tumor inoculation (day 7) when average tumor volume reached �200 mm3. Radiation beginning day 7: 2 Gy of radiation givenMonday to Friday for 2 weeks, to 20 Gy (red double-headed arrows). (Continued on the following page,)

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compared after treatment with birinapant plus TNFa for 48 hours(Fig. 4B; Supplementary Fig. S2A and S2B). UM-SCC-46 cellsoverexpressing FADD treatedwith birinapant alone, or birinapantand TNFa, exhibited strong increases in sub-G0DNA indicative ofcell death as well as increased G2–M accumulation. In contrast,treatment of UM-SCC-1 cells with weak FADD amplification/expression and heterozygous CASP8 mutation showed a weakerincrease in sub-G0 DNA and no accumulation in G2–M followingbirinapant or combination treatment. Birinapant plus TNFainduced increased sub-G0 and G2–M DNA in UM-SCC-46 cellsin independent experiments (Supplementary Fig. S2A and S2C).UM-SCC-11B expressing both increased FADD and BIRC2 dis-played increased sub-G0 DNA but no G2–M DNA accumulationwith the combination (Supplementary Fig. S2D).

Birinapant sensitizes to TNFa or TRAIL-induced cell deaththrough necroptosis and/or apoptosis

To further elucidate themechanism(s) leading to cell death, weanalyzed the effects of pan-caspase (ZVAD), caspase-8 (ZIETD),and RIPK1 (necrostatin) inhibitors on cell density in cell lineswith varying cell death pathway alterations (Fig. 5A). In UM-SCC-46, RIPK1 inhibition (necrostatin), but not caspase inhibition(ZVAD or ZIETD), reversed the effects of birinapant treatmentalone or with death agonists. These data support predominantlyRIPK1-mediated, caspase-independent, necroptotic cell death,consistent with the expression of high FADD, normal BIRC2, andlow CASP8 expression observed in UM-SCC-46. Conversely, inUM-SCC-1, the weaker inhibitory effect of birinapant plusdeath receptor agonists was blocked by both caspase inhibitorsZVAD or ZIETD, but not by necrostatin, supporting predom-inantly caspase-dependent activity. This cell line exhibitsweaker amplification and expression of FADD and reducedprotein expression of BIRC2 and heterozygous wt andmtCASP8. In UM-SCC-74B with normal FADD and BIRC2, butincreased expression of a CASP8 sequence variant, pan-caspaseinhibitor ZVAD, but not CASP8 inhibitor ZIETD or necrostatin,restored cell density in birinapant plus TNFa or TRAIL-treatedcells. UM-SCC-11B, which strongly expresses FADD, BIRC2,and CASP8, demonstrated effects by both caspase inhibitorsand necrostatin, supporting mixed mechanisms of cell death inthis cell line. In UM-SCC-11A, a cell line with high levels ofFADD and BIRC2 derived from a biopsy prior to chemotherapyin the same patient, we observed greater sensitivity with apattern of predominantly caspase-dependent apoptotic celldeath. To directly confirm the activation of caspases in UM-SCC-11A, caspase-3 and -8 enzyme assays were used to assessactivity with birinapant, TRAIL, or the combination. Birinapantplus TRAIL induced more than a 2-fold increase in bothcaspase-3 and caspase-8 activity in UM-SCC-11A, consistentwith the effects observed using the caspase inhibitors.

The expression of key cell death pathway proteins under con-ditions where treatment with birinapant plus TNFa induced celldeath were further examined at different time points in 3 of thelines displaying different patterns of cell death (Fig. 5B). Notably,expression of cIAP1, themost sensitive IAP targeted by birinapant,was similarly decreased in all 3 cell lines at 12 hours and remainedundetectable up to 48 hours after treatment with birinapant plusTNFa. In the 3 lines, both RIPK1 and RIPK3 showed decreasedexpression at later time points following drug treatment. UM-SCC-46 demonstrated a greater reduction in FADD and enhance-ment of necroptosis mediator MLKL expression from 12 to 48hours after treatment with birinapant (Fig. 5B), consistent withthe predominant rescue by necrostatin observed. Interestingly, all3 lines showed detectable cleavage of caspase-3, although bothUM-SCC-1 and -11B showed a relatively greater and sustainedincrease in CASP3 and CASP8 cleavage, consistent with predom-inant reversal by both pan- and caspase-8 inhibitors. Althoughbirinapant has been reported tohave a lower activity for cIAP2 andXIAP than cIAP1, we performed additional experiments to exam-ine the effects of birinapant on these IAPs, without andwith cIAP1siRNAknockdown inUM-SCC-46 in vitro. Birinapantwith controlsiRNA effectively inhibited cIAP1, partially inhibited XIAP, andweakly enhanced cIAP2 protein expression, whereas BIRC2 siRNAeffectively knocked down IAP1 but induced a compensatoryincrease in cIAP2 that was partially suppressed by combinationwith birinapant (Supplementary Fig. S3A). Consistent with thisevidence that birinapant also partially inhibits compensatoryinduction of IAP2 and XIAP, the drug had significantly greaterinhibitory effect than that observed with BIRC2/IAP1 knockdownalone, which was weakly attenuated by cIAP1 knockdown alone(Supplementary Fig. S3B). Together, these data provide evidencethat birinapant inhibition of IAPs anddeath agonists promote celldeath via apoptosis and/or necroptosis in HNSCC cell lines withvarying alterations affecting cell death components.

Birinapant inhibited tumor growth and prolonged survival inhuman HNSCC xenograft models with FADD without or withBIRC2 alterations

To evaluate the activity of birinapant in vivo, studies were doneusing UM-SCC-46 (increased FADD expression, normal BIRC2expression) andUM-SCC-11B (increasedFADDandBIRC2 expres-sion) cells inmurine xenograftmodels. SCIDmicewere implantedwith tumor cells, and treated with birinapant, 15 or 30 mg/kgintraperitoneally as reported (20), or control vehicle, when tumorsreached200mm3.Birinapant alone versus control had a significantinhibitory effect on tumor growth in both models (Fig. 6). For theUM-SCC-46 xenograftmodel, a significant differencewas observedin the mean tumor volumes of the control versus treated animalsthrough day 45 post-tumor cell injection (Fig. 6A). Improvementin median survival was observed, from 39 days for control to 58

(Continued.) Birinapant beginning day 8: 15 mg/kg birinapant intraperitoneally every 3 days for 10 doses (purple double-headed arrow). Statistical differenceversus control by Student t test: þ, P < 0.05. Error bars, SEM. B, survival analysis and significance determined by Gehan–Breslow–Wilcoxon test >100. Mediansurvival, 27 days (control), 42 days (birinapant, P ¼ 0.003), 80 days (radiation, P ¼ 0.0002), and combination group without deaths, to 100 days. C, photosof tumors of representativemice from each treatment group on day 21 (5 doses of birinapant and 20Gy radiation).D, relativemTNFamRNA expression in tumors onday 21 compared by qRT-PCR. Student t test: � , P < 0.05. Error bars, SEM. E, intrinsic cell death pathway, inducible by radiation DNA damage, and theextrinsic pathway, triggered by death receptor family ligands, can converge on caspase-3 to induce apoptosis. Death ligand binding via the extrinsic pathwayresults in a cytoplasmic complex that includes FADD, which may activate caspase-8/3, or RIP1/3/MLKL-mediated signaling and necroptosis. Cell death isblocked by IAPs. DNAdamage inducedmitochondrial release of SMACandSMACmimetic birinapant inhibits anddegrades IAPs (cIAP1, cIAP2, XIAP) to enhance TNF-mediated cell death.






days in the treated group (Fig. 6B). For UM-SCC-11B, prolongedtumor reduction was observed during and after treatment usingeither 30 or 15 mg/kg/d birinapant (Fig. 6C). Prolonged hostsurvival was observed in both treatment arms (Fig. 5D), with amedian of 82 days for control, 137 days for the 30mg/kg group (P¼ 0.08), and >180 days for the 15mg/kg group (P¼ 0.05). The 15and 30mg/kg doses of birinapant were well tolerated, and allmicemaintained normal body conditioning scores, and weight ontreatment (Supplementary Fig. S4A and S4B).

To examine the molecular and cellular effects associated withbirinapant treatment in vivo, the effects on the IAP cell deathpathway components demonstrated above were examined byimmunostaining of UM-SCC-46 tumor xenografts during treat-ment (day 25; Fig. 6E). In addition, markers of cell death (MLKL,TUNEL) and proliferation (Ki67) were also examined. Tumorimmunostaining was quantified by histoscores as described inMaterials and Methods. Birinapant treatment significantlyreduced cIAP1, cIAP2, and XIAP during sustained treatment,supporting contribution of inhibition of these IAPs to the activityin vivo. Necroptosis marker MLKL was significantly increased intumors during the treatment period. There were no significantdifferences in proliferation (Ki67) or apoptosis (TUNEL) markerstaining in tumor specimens at either time point, consistent withthe predominantly MLKL/necroptosis-dependent inhibitoryeffects observed in this model.

Combination of birinapant and radiation induced durabletumor regression and cure in the UM-SCC-46–derivedxenograft model

Radiation has previously been shown to induce TNFa (23).We examined the combinatorial effects of birinapant plusradiation in vivo using the UM-SCC-46 xenograft model, whichdisplayed sensitivity but were not cured by birinapant treat-ment alone. Tumor cells were injected subcutaneously into theupper leg of athymic nu/nu mice and treated with birinapant(15 mg/kg intraperitoneally) every 3 days, radiation 2 Gy dailyMonday to Friday for 2 weeks, or the combination when thetumors reached 200 mm3. Treatment with birinapant or radi-ation alone significantly delayed tumor growth (Fig. 7A) andimproved survival (Fig. 7B). Remarkably, birinapant combinedwith radiation cured the animals bearing UM-SCC-46–derivedxenografts, with no evidence of disease relapse to 130 days (Fig.7A and B). Representative differences in gross tumor volumeamong treatment arms were observed by day 21, without visualevidence of significant radiation dermatitis, contracture (Fig.7C) or weight loss (Supplementary Fig. S3C). As quantified byRT-PCR, TNFa expression in tumors was enhanced by thecombination of birinapant plus radiation versus either alone(Fig. 7D). To examine whether radiosensitization by birinapantobserved is due to intrinsic or TNFa-enhanced radiosensitivityof UM-SCC-46, cells were treated with 30 nmol/L birinapantimmediately after radiation, and replated after 24 hours forclonogenic assay. Interestingly, in vitro combination of birina-pant alone with radiation over a range of 0 to 9 Gy had nosignificant direct radiosensitizing effect over that due to radi-ation alone (Supplementary Fig. S5A), suggesting that theactivity observed in vivo is dependent on additional factor(s).As we hypothesized and observed that birinapant plus radia-tion potently induces endogenous TNFa in tumors in vivo (Fig.7D), we examined and confirmed that combination of TNFa (2ng/mL) with birinapant significantly enhances radiation sen-

sitivity at 3 Gy compared to the single agents (SupplementaryFig. S5B). Combination birinapant and radiation or radiationalone also significantly suppressed tumor growth in FADD- andBIRC2-overexpressing UM-SCC-11B xenografts, but not UM-SCC-1 xenografts displaying weaker FADD/BIRC2 copy gainand expression, and a heterozygous CASP8 mutation (Supple-mentary Fig. S6). Together, these findings support birinapantplus radiation as a therapeutic combination for HNSCC har-boring genomic amplification or increased expression of FADDwithout or with BIRC2.

DiscussionTCGA linked amplification of 11q13 containing FADD with

a major subset of HPV�HNSCC (2). 11q22 harboring BIRC2/3encoding IAP1/2 was often coamplified. CASP8 was mutated ina mutually exclusive subset. These molecules comprise corecomponents of the TNF receptor family signal pathways thatregulate the balance between cell death via apoptosis andnecroptosis or cell survival (Fig. 7E). SMAC mimetics promotedegradation of IAP proteins that inhibit the intrinsic andextrinsic cell death signaling pathways, potentially overcomingresistance to death ligands and radiation (16, 32). Here, wepresent evidence that HNSCC with genomic alterations inthese cell death pathway components are sensitive to combi-nations of a SMAC mimetic plus death ligands TNFa, TRAIL, orradiation.

Our TCGA analysis indicates that FADD amplification is morefrequent inHNSCC than other cancers and is associatedwith earlyrecurrence and worse prognosis. We observed increase in genecopy number and expression of FADD with or without BIRC2,whereas BIRC3 was minimally expressed, among a subset ofhuman HPV� HNSCC cell lines, consistent with HNSCC tumors(2). A heterozygous mutation of CASP8 was detected in only 1 of11 cell lines, approximating the frequency in tumors. Knockdownof FADD or BIRC2, but not BIRC3, inhibited and enhanced TNFasensitivity of UM-SCC-11A cells harboring amplification andincreased expression of FADD and BIRC2, which have beenimplicated in promoting cell proliferation, and resistance toTNFa-induced cell death (11–14).

Multiple HNSCC lines overexpressing one or more of these celldeath pathway components exhibited pharmacologic inhibitionby the SMAC mimetic birinapant in combination with TNFa orTRAIL. This inhibition accompanied a corresponding increase incell death, supportedby increased sub-G0DNA fragmentation, andreversibility by caspase and/or RIPK1/3 inhibitor(s). Birinapantplus TNFa promoted degradation of cIAP1, the product of BIRC2,and variably activated caspase and/or MLKL mediators of apopto-sis or necroptosis in different HNSCC. These findings implicateIAPs as criticalmediators of thewidespread resistanceofHNSCC toTNFa previously observed by our laboratory (33). Birinapantactivity was also associated with reproducible effects on G2–Mcell-cycle progression in UM-SCC-46 cells, similar to that reportedwith natural IAP inhibitor embelin in PC3 prostate cancer cells(32), and recently linked to the role of FADD in proliferation inlung carcinoma (34). In the latter study, the induction of mitosisand lung carcinogenesis depended upon activation of KRAS andCK1a phosphorylation of FADD during G2–M, where FADDinteracted with G2–M cell-cycle regulatory components PLK1,AURKA, and BUB1. Interestingly, UM-SCC-46 showed a greaterreduction in FADD by birinapant and TNFa during 48 hours


Eytan et al.




posttreatment when G2–M arrest was observed, compared withother lines lacking this effect. However, after sustained monother-apy in vivo, resumption of tumor growth was observed, and wedetected no difference in Ki67 staining. Whether there is sustainedG2–Mblock, or compensatorymechanism(s) of proliferationwithprolonged treatment in vivo, merits further investigation.Of poten-tially broader clinical relevance, birinapant combined with celldeath ligands was capable of inducing potent cytotoxicity inHNSCC lines that were derived from aggressive primary, metastat-ic, and recurrent tumors, that are oftenmutant or deficient for pro-death tumor suppressor TP53 (2).

We observed differences in sensitivity to death ligands andpredominant cell death pathways and mechanisms upon com-parison of HNSCC harboring different alterations in FADD,BIRC2, and CASP8. Evidence for caspase-dependent apoptosis,RIPK/MLKL-dependent necroptosis, or mixedmechanisms of celldeath were observed by comparing the effects of reversible inhi-bitors and establishedmarkers of caspase and/or RIPK-dependentpathways. Birinapant and TNFa-mediated cytotoxicity of UM-SCC-11B with FADD/BIRC2 amplification and normal CASP8expressionwas reversed by pan-caspase andCASP8 inhibitors andshowed increased caspase-3 or -8 cleavage, supporting predom-inantly caspase-dependent mechanisms of apoptosis. In contrast,inhibition of UM-SCC-46 was predominantly reversed by theRIPK1 inhibitor necrostatin and associated with induction ofMLKL in vitro and in vivo, supporting necroptosome-dependentinhibition in thisHNSCCoverexpressing FADDbut low caspase-8protein. Since caspase-8 has been shown to inhibit necrosis whilepromoting apoptosis (35), low CASP8 expression could explainthe predominantly necroptotic cell death seen in UM-SCC-46.Interestingly, UM-SCC-74B, expressing a common K14R aminoacid variant of CASP8, and lower FADDandBIRC2, demonstratedsensitivity to birinapant and death ligands reversible by pan-caspase but not caspase-8 inhibitors, consistent with caspase-8–independent cell death mechanisms. Further experiments pro-vided evidence that birinapant can also partially inhibit XIAPexpression, which could potentially enhance other caspase-dependent mechanisms. Compared with these lines, UM-SCC-1 with lower amplification and expression of FADD/BIRC2 and aheterozygous CASP8mutation was relatively less sensitive in vitroand in vivo. Inhibition ofUM-SCC-1 and 11Bwas reversed by bothcaspase and necroptosis inhibitors and demonstrated cleavedcaspase and basal MLKL expression, supporting mixed mechan-isms. Raulf and colleagues found evidence for mixed apoptoticand necroptotic cell death in response to a SMAC mimetic inHNSCC cell line HSC3, and not others, but the genomic altera-tions were undefined (36). Together, these observations suggestthat the status of FADD, BIRC2, CASP8 and other components ofthe cell deathmachinerymay affect sensitivity to these agents andmerit further investigation as potential predictive biomarkers forresponse in future clinical studies of SMAC mimetics in HNSCC.

Our in vivo preclinical studies demonstrate that two indepen-dentmodels with genomic alterations in FADD�/þBIRC2 ampli-fication exhibited significant sensitivity and prolonged survivalfollowing birinapant treatment alone, despite differences inintrinsic sensitivity in vitro. Birinapant alone induced endogenousTNFa mRNA in vivo, but the incomplete suppression of tumorgrowth with birinapant monotherapy suggested that this levelalone may be insufficient to potentiate tumor regression. Birina-pant plus radiation led to more durable responses in UM-SCC-46and 11B xenograft models. Furthermore, the combination of

birinapant plus radiationmore potently enhanced the expressionof endogenousmurine TNFa comparedwith either therapy alone.While birinapant alone did not significantly enhance the effects ofradiation in vitro, addition of TNFa enhanced radiosensitizationin clonogenic assay. Because TNFa is induced in tumors followingradiation treatment (23), studying the addition of birinapant plusradiation holds considerable clinical relevance to this standardtreatment for HNSCC.

Recently, combinatorial activity of different SMAC mimeticswith radiation and induction of TNFa has been demonstrated inlung cancer and HNSCC cell lines (37, 38). Partial response inUM-SCC-1 xenografts in the latter study was observed, similar toour results. The relative contribution of weaker amplification ofFADD/BIRC2 and/or heterozygous mutation of CASP8 to theresistance to birinapant plus TNFa or radiation in thismodelmaymerit future investigation. Together, these observations under-score the importance of assessing genomic alterations, expression,and the molecular and antitumor effects of targeting cell deathpathways with SMAC mimetics in preclinical studies and futureclinical trials in HNSCC and other cancers. FADD/BIRC2 altera-tions may represent an Achilles' heel for this subset of HNSCCand for clinical investigation with SMAC mimetics.

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

Authors' ContributionsConception and design: D.F. Eytan, G.E. Snow, A. Saleh, J.B. Mitchell, C.M.Annunziata, Z. Chen, C. Van WaesDevelopment of methodology: D.F. Eytan, S. Carlson, A. Saleh, L. Hernandez,J.B. MitchellAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D.F. Eytan, G.E. Snow, S. Carlson, A. Derakhshan,A. Saleh, S. Schiltz, S. Mohan, S. Cornelius, A.L. Sowers, Z. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.F. Eytan, G.E. Snow, S. Carlson, A. Derakhshan,A. Saleh, S. Schiltz, H. Cheng, Z. Chen, C. Van WaesWriting, review, and/or revision of the manuscript: D.F. Eytan, G.E. Snow,S. Carlson, A. Saleh, S. Mohan, J.B. Mitchell, C.M. Annunziata, Z. Chen, C. VanWaesAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.F. Eytan, S. Carlson, J.F. Coupar, Z. Chen,C. Van WaesStudy supervision: A. Saleh, Z. Chen, C. Van Waes

AcknowledgmentsBirinapant was provided to NIDCD for research under a Materials Transfer

Agreement with the National Cancer Institute and TetraLogic Pharmaceuticals.This study utilized the high-performance computational capabilities of theBiowulf Linux cluster at the NIH, Bethesda, MD (http://biowulf.nih.gov).Thanks toBarbaraConley and James F. Battey for reading andhelpful commentson this article.

Grant SupportThe studywas supported byNIDCD intramural projects ZIA-DC-000016, 73,

74 (C. VanWaes, Z. Chen, S. Carlson, A. Saleh, S. Schiltz, H. Cheng, S. Cornelius,J.F. Coupar) and the NIH-Medical Research Scholars Program (D.F. Eytan, G.E.Snow, S. Mohan, A. Derakhshan).

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 December 10, 2015; revised June 7, 2016; accepted June 26, 2016;published OnlineFirst July 28, 2016.






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2016;76:5442-5454. Published OnlineFirst July 28, 2016.Cancer Res Danielle F. Eytan, Grace E. Snow, Sophie Carlson, et al.

BIRC2 and FADDGenes and Neck Cancers with Genomic Amplifications of Cell Death SMAC Mimetic Birinapant plus Radiation Eradicates Human Head

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SMAC Mimetic Birinapant plus Radiation Eradicates Human Head … · Therapeutics, Targets, and Chemical Biology SMAC Mimetic Birinapant plus Radiation Eradicates Human Head and Neck