The Novel SMAC Mimetic Birinapant Exhibits Potent Activity ... · the Smac tetrapeptide that binds...

Cancer Therapy: Preclinical

The Novel SMAC Mimetic Birinapant Exhibits Potent Activityagainst Human Melanoma Cells

Clemens Krepler1,2, Srinivas K. Chunduru7, Molly B. Halloran1,2, Xu He1,2, Min Xiao1,2, Adina Vultur1,2,JessieVillanueva2,3, YasuhiroMitsuuchi7, EricM.Neiman7,ChristopherBenetatos7,Katherine L.Nathanson4,6,Ravi K. Amaravadi5,6, Hubert Pehamberger8, Mark McKinlay7, and Meenhard Herlyn1,2

AbstractPurpose: Inhibitor of apoptosis proteins (IAP) promote cancer cell survival and confer resistance to

therapy.We report on the ability of secondmitochondria-derived activator of caspasesmimetic, birinapant,

which acts as antagonist to cIAP1 and cIAP2, to restore the sensitivity to apoptotic stimuli such as TNF-a in

melanomas.

Experimental Design: Seventeen melanoma cell lines, representing five major genetic subgroups of

cutaneous melanoma, were treated with birinapant as a single agent or in combination with TNF-a. Effectson cell viability, target inhibition, and initiation of apoptosis were assessed and findingswere validated in 2-

dimensional (2D), 3D spheroid, and in vivo xenograft models.

Results: When birinapant was combined with TNF-a, strong combination activity, that is, neither

compoundwas effective individually but the combinationwas highly effective, was observed in 12 of 18 cell

lines. This response was conserved in spheroid models, whereas in vivo birinapant inhibited tumor growth

without adding TNF-a in in vitro resistant cell lines. Birinapant combined with TNF-a inhibited the growth

of amelanoma cell line with acquired resistance to BRAF inhibition to the same extent as in the parental cell

line.

Conclusions: Birinapant in combination with TNF-a exhibits a strong antimelanoma effect in vitro.

Birinapant as a single agent shows in vivo antitumor activity, even if cells are resistant to single agent therapy

in vitro. Birinapant in combinationwith TNF-a is effective in amelanoma cell linewith acquired resistance to

BRAF inhibitors. Clin Cancer Res; 19(7); 1784–94. �2013 AACR.

IntroductionTreatment options for metastasized melanoma, a disease

with a low 5-year survival rate, have improved remarkablyin the last 2 years. After a decades-long period, in which thegold standard remained dacarbazine chemotherapy withmodest response rates (1), twonew therapies havebeenU.S.Food and Drug Administration-approved recently (2–4)and several are currently in late-stage clinical development(5). Among small-molecule inhibitors, the majority targets2 major pathways: the mitogen-activated protein kinase

(MAPK) and phosphatidylinositol 3-kinase (PI3K) path-ways. Therapy through modulating T-cell responses (4) isalso highly promising, but unfortunately for all new ther-apies, most patients eventually progress.

Antiapoptotic mechanisms are also frequently at work inmelanoma (6) and an attractive target for therapeutic inter-vention. For example, targeting the B-cell lymphoma 2 (Bcl-2) family of antiapoptotic proteins using Bcl-2 antisense (7)and Bcl-2 homology domain 3 (BH3) mimetics (8, 9) hasbeen promising preclinically, but will require additionalstudies to fully realize their potential in clinical trials.

One of the factors responsible for the difficulties inengaging the apoptotic cascade efficiently in melanoma isthe upregulation of conserved inhibitor of apoptosis pro-teins (IAP; refs. 10, 11). IAPs are a family of proteins definedby the presence of baculoviral IAP repeats. The bestdescribed members are XIAP, cIAP1, cIAP2, ML-IAP, andsurvivin. These proteins are associated with chemoresis-tance and poor outcome in many cancer types [reviewedin ref. (12)]. IAPs themselves are controlled by the secondmitochondria-derived activator of caspases (SMAC). SMACis released frommitochondria upononset of apoptosis (13)and binds directly to IAPs leading to their degradation (14,15). A number of IAP inhibitors, designed to function asSMACmimetics, have shown preclinical activity in a variety

Authors' Affiliations: 1TumorMicroenvironment andMetastasis Program,2Melanoma Research Center, and 3Molecular Oncogenesis Program, TheWistar Institute; 4Division of Translational Medicine and Human Genetics,5Department of Medicine, 6Abramson Cancer Center, Perelman School ofMedicine at the University of Pennsylvania, Philadelphia; 7TetraLogicPharmaceuticals, Malvern, Pennsylvania; and 8Department of Dermatolo-gy, Medical University Vienna, Vienna, Austria

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

Corresponding Author: Meenhard Herlyn, The Wistar Institute, 3601Spruce Street, Philadelphia, PA 19104. Phone: 215-898-3950; Fax: 215-898-0980; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-2518

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(7) April 1, 20131784

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of cancer types (16–19). In contrast to XIAP, cIAP1 andcIAP2 can bind to caspases but lack binding domainscapable of inhibiting caspases (20). Previous studies haveshown that synthetic IAP antagonists are able to induceapoptosis in tumor cells primarily through inhibition ofcIAP1 and or cIAP2, which results in changes in TNFreceptor (TNFR) complex signaling. These effects weredependent on TNF-a signaling and caspase-8 activation(21). Moreover, SMAC mimetics can perturb NF-kB signal-ing downstream of TNFR (22).Birinapant is a novel dimeric SMACmimetic designed to

specifically target cIAP1 and cIAP2 for degradation, result-ing in a switch in TNFR signaling; activation of TNFR uponbinding of TNF-a in the presence of cIAP1 and cIAP2 leadsto NF-kB activation and increased cell proliferation. Incontrast, following cIAP1 and cIAP2 inhibition, TNF-asignaling leads to activation of caspase-8 and induction ofapoptosis (12).Immune cell infiltrates are often found in melanoma

lesions, leading to chronic inflammation and (among otherfactors) elevated levels of TNF-a (23, 24). TNF-a can thenbeused by melanoma cells to promote cell growth, invasion,and metastasis (25). Binding of TNF-a to its receptor leadsto the activation of TNFR complex-I, a membrane-localizedactivator of the canonical NF-kB pathway composed ofTNFRSF1A associated via death domain (TRADD), theubiquitin ligases TRAF2, TRAF5, cIAP1, and cIAP2, and theprotein kinase RIPK1 (26). This leads to increased cellproliferation via an ubiquitination cascade and down-stream activation of NF-kB. After SMAC mimetic-induceddegradation of cIAP1 and cIAP2, binding of TNF-a to itsreceptor leads to the formation of a death complex andactivation of caspases. This results in a reversal of the role ofTNF-a from promoting proliferation to inducing cell death(12).

In the present study, birinapant in combination withTNF-a led to a reduction of viability in the majority of17melanoma cell lines tested. This effect was dependent onTNF-a.

Our laboratory has previously published on melanomacell lines with acquired resistance to BRAF inhibitors (27).Notably, birinapant in combinationwith TNF-a elicited thesame antimelanoma effect in a parental andBRAF inhibitor-resistant cell line. This suggests a potential use of birinapantfor the treatment of patients with BRAF inhibitor-refractorymelanoma.

Materials and MethodsCell lines and reagents

Humanmelanoma cell lines were cultured in Dulbecco’smodified Eagle’s medium supplemented with 5% FBS andgrown at 37�C in 5% CO2. All cell lines were periodicallyauthenticated by DNA finger printing using Coriell’s micro-satellite kit and tested for mycoplasma by PCR.

Genomic DNA from samples were analyzed for muta-tions in BRAF, CDK4, CTNNB1, FBX04, KIT, MEK1, MEK2,MET, andNRAS by the nucleotide extension assay using theiPlex platform (Sequenom, Inc; ref. 28).

Birinapant was provided by TetraLogic. Birinapant is anovel bivalent, selective small-molecule peptidomimetic ofthe Smac tetrapeptide that binds with high affinity tomultiple members of the IAP family including XIAP andcIAP1. The dissociation constant (Kd) for XIAP and cIAP-1was determined to be 45 and <1 nmol/L, respectively (29).Birinapant was developed by TetraLogic Pharmaceuticalsand designed on the basis of the 4 N-terminal amino acidsof Smac/DIABLO (Ala- Val-Pro-Ile) and caspase-9 (Ala-Thr-Pro-Phe; ref. 30).

Human recombinant TNF-a was obtained from Invitro-gen. TNF-a monoclonal antibody (mAb) MAB610 wasobtained from R&D Systems.

cis-Diamineplatinum(II) dichloride (cisplatin) wasobtained from Sigma-Aldrich.

Immunoblot analysisFor immunoblot analyses, adherent cells were washed

with cold PBS containing 100 mmol/L Na3VO4, scraped,collected by centrifugation, and quick-frozen in dry icebefore lysis. Xenograft tumors were snap frozen in liquidnitrogen immediately after harvesting. Tumor chunks weregroundon liquidN2using aMM2mixermill (Retsch). Cellsand powder were lysed and equal amounts of protein (10–40 mg) was subjected to SDS-PAGE and proteins transferredonto PVDF membranes (Immobilon). Antibodies usedwere: cIAP1, AF8181; cIAP2, AF8171 (both R &D systems);PARP, 7150; GAPDH, 32233 (both Santa Cruz biotechnol-ogy); Caspase-8, 9746; NF-kBp65, 3031 (both Cell Signal-ing); XIAP, ADI-AAM-050 (ENZO); NFKB2 p100- p52,4882 (Cell Signaling); RIP1, 51-6559GR, RAC1, 610651(both BD Biosciences); ERK, sc-271270, pERK, sc 7976,(both Santa Cruz biotechnology). Membranes were probedwith primary antibodies overnight at 4�C, then furtherincubated with Alexa Fluor-labeled secondary antibodies

Translational RelevanceAlthough major advances have recently been made in

the treatment of malignant melanoma, this diseaseremains largely incurable once metastasized. The mainreason is the development of resistance, even to newtherapies recently introduced into the clinic or currentlyin late stage testing. In addition, the genetic heteroge-neity of melanoma often limits the use of effectivetargeted therapies to specific subsets of patients. Novelsmall-molecule inhibitors that could be applicable forall patients with melanoma, regardless of genetic sub-group,wouldbehighly beneficial to the current standardof care. We describe here a novel second mitochondria-derived activator of caspases mimetic small molecule,birinapant, which shows antitumor activity in majorsubgroups of cutaneous melanoma. Moreover, birina-pant could be useful in overcoming acquired resistanceto BRAF inhibitors, a patient population that is in urgentneed of new treatment strategies.

The SMAC Mimetic Birinapant Exhibits Potent Antimelanoma Activity

www.aacrjournals.org Clin Cancer Res; 19(7) April 1, 2013 1785




(IRDye 680LT goat-anti mouse, IRDye 800CW goat-antirabbit, donkey-anti mouse, donkey-anti rabbit, or donkey-anti goat IRDye 800CW or 680LT (LI-COR) for 1 hour andscanned using the Odyssey system (LI-COR).

In vitro drug sensitivity assaysFor monolayer cell culture assays, cells were allowed to

attach for 24 hours and subsequently incubated with bir-inapant and/or TNF-a for 24 or 72 hours. CellTiter 96AQueous Non-Radioactive Cell Proliferation Assay (MTS)assay (Promega) was conducted according to the manufac-turer’s description. For cell-cycle analysis, melanoma cellswere fixed in 70%ethyl alcohol and stainedwith propidiumiodide. Samples were subsequently analyzed with an EPICSXL (Beckman-Coulter) apparatus. Annexin V staining wasconducted with an Annexin V allophycocyanin conjugate(Invitrogen) according to the manufacturer’s description.Briefly, cells were treated with dimethyl sulfoxide control,birinapant, and/or TNF-a for 24 hours. Resuspended cellswere washed and incubated with the conjugate for 15minutes and Annexin-binding buffer was added. Sampleswere subsequently analyzed with an EPICS XL (Beckman-Coulter) apparatus.

451Lu and WM1366 melanoma cells were treated withbirinapant 1 mmol/L in combination with TNF-a 1 ng/mL.Cells were then incubated for 72 hours in the presence orabsence of Z-VAD-FMK (R & D systems) a pan-caspaseinhibitor. Proliferation was assessed using the MTS assay.

451Lu and WM1366 melanoma cells were treated withbirinapant 1 mmol/L in combination with TNF-a 1 ng/mL. Cells were then incubated for 72 hours in the pres-ence or absence of Necrostatin-1 (N9037, Sigma) a RIP1kinase inhibitor. Proliferation was assessed using the MTSassay.

RIPK1 gene expressionwas evaluated using commerciallyavailable gene expression assays from Life Technologies.Briefly, RNAwas isolated usingQiagen RNeasy kit and RNAwas quantified and normalized for addition to cDNA reac-tion. cDNA was created using cDNA kit from Life Technol-ogies (4368814) and real-time PCR was carried out forRIPK1 (assay ID Hs00169407_m1) and GUSB (assay IDHs99999908_m1) as a housekeeping gene. For analysis, therelative quantity of RIPK1 to GUSB was calculated.

For 3-dimensional (3D)melanoma spheroid assays, cellswere prepared as described previously (31, 32). Collagen-embedded spheroids were incubated with reagents for 72hours. Spheroids were imaged using a Leica TCS SP2 con-focalmicroscope after addition of a live/dead viability assaykit using the fluorescent dyes calcein AM and ethidiumhomodimer-1 (Invitrogen). To assess viability, spheroidswere grown in a 96-well format, and allowed to grow in thepresence of birinapant and/or TNF-a for 72 hours, thenAlamarBlue (Invitrogen)was added. Fluorescence wasmea-sured using an Envision Xcite plate reader (Perkin Elmer).All experiments were carried out in triplicate.

Tomeasure TNF-a levels in cell culture supernatants, cellswere grown for 3 days to 70% confluence. Supernatantswere collected and 200 mLwere tested in triplicate following

the manufacturer’s instructions using the BD OptEIAHuman TNF-a ELISA Kit II (BD).

Xenograft studiesAll animal experiments were carried out in accordance

with Wistar IACUC protocol 111954 in NUDE mice. Tenanimals each were inoculated s.c. with 1 � 106 451Lu or1205Lu human melanoma cells in a suspension of Matri-gel (BD Matrigel Basement Membrane Matrix, GrowthFactor Reduced)/complete media at a ratio of 1:1. Afterformation of palpable tumors, mice from both tumormodels were randomized into 2 groups. Both groups weretreated intraperitoneal 3 times/week with either vehiclecontrol or birinapant 30 mg/kg for 21 days. Birinapantwas dissolved in 12.5% Captisol (Ligand Pharmaceuti-cals) in distilled water. Tumor size was assessed twiceweekly by caliper measurement. Subsequently, satellitegroups of ten and fifteen mice were inoculated in thesame fashion with 451Lu and 1205Lu tumor cells, respec-tively. After tumors had reached a mean volume of 200mm3 animals were dosed with either birinapant or vehi-cle control as described above. After 48 hours and 2 doses,animals were sacrificed and tumors were harvested at 4time points after the last treatment. Tumor samples weresnap frozen in liquid nitrogen for subsequent proteinanalysis and preserved as formalin-fixed, paraffin embed-ded blocks for immunohistochemistry.

Paraffin-embedded 5 mmsections were stained with acti-vated caspase-3 mAb (5A1E, Cell Signaling). Briefly, afterdeparaffinization and endogen peroxidase blocking, anti-gen retrieval was conducted via heat mediation with citratebuffer. The antibody was diluted 1:100 in Superblock andincubated overnight at 4�C. After washing with Dulbecco’sPhosphate Buffered Saline slides were incubated for 30minutes in ABComplex/HRP conjugated Kit (DAKO). Fol-lowing a further 3 washing steps, the DAB-chromogensolution Kit (DAKO) was incubated on slides until visiblecolor reaction was observed. Slides were washed and coun-terstained in diluted (1:4 in aqua dest.) Harris’ hematoxlinsolution and mounted with Aquatex (Merck).

StatisticsIC50 values were calculated with GraphPad Prism 5

(GraphPad Software Inc.). Statistical significance was deter-mined using 2-sided Student t test. P < 0.05 was consideredto be significant.

ResultsThemajority of cell lines exhibited strong combinationactivity of birinapant and TNF-a

Reflecting the heterogeneity of melanoma, we used apanel of 17 human melanoma cell lines comprising majorgenetic subgroups of cutaneous melanoma, approximatelyreflecting the distribution seen in patients (ref. 33; Supple-mentary Table S1).

To test birinapant as a single agent and calculate the halfmaximal inhibitory concentration (IC50) values in this cellline panel, cells were treated with birinapant at 1, 10, 100,

Krepler et al.

Clin Cancer Res; 19(7) April 1, 2013 Clinical Cancer Research1786




and 1,000 nmol/L for 72 hours and cell viability wasassessed through the MTS assay. Only one cell line (WM9,BRAFV600E) out of 17 showed a response to birinapant (Fig.1, gray bars, TL32711). However, when birinapant wascombined with TNF-a (1 ng/mL), the majority of cell linesshowed birinapant IC50 values in the low nanomolar range(Fig. 1, black bars). This response was independent of thecell line genetic background. All cell lines were resistant toTNF-a alone (Supplementary Fig. S1).Three distinct response patterns following birinapant

addition were observed. For subsequent experiments, 4 celllineswere therefore selected on the basis of their response tobirinapant in combinationwith TNF-a: 1205Lu, resistant tothe combination therapy; WM9, sensitive to birinapant as asingle agent; and 2 cell lines sensitive to birinapant only incombination with TNF-a: 451Lu (BRAFV600E) andWM1366 (NRASQ61L; Fig. 2A).

cIAP1 and cIAP2 downregulation was observed in allresponse types, but apoptosis occurred only insensitive cell linesImmunoblot analysis showed inhibition of the birina-

pant target protein cIAP1 in all 4 cell lines. Although cIAP2wasupregulated in the 1205LuandWM1366 cell lines upontreatment with TNF-a, and was again degraded in thecombination with birinapant. No change in the levels ofXIAP could be observed (Fig. 2B). After treatment withbirinapant alone, apoptosis as assessed by cleavage of PARPcould be observed in the single-agent sensitive cell line only.Whenbirinapantwas combinedwith TNF-a, PARP cleavagewas now detectable in the combination-sensitive cell lines451Lu and WM1366 as well, corresponding to the growthinhibition observed in the MTS assay. No cleavage of PARPcould be observed in the resistant cell line 1205Lu, evenafter combination treatment. Notably, a decrease in NF-kBp65 protein levels could only be observed in the single-agent–sensitive cell line, WM9, after treatment with birina-pant alone or in combination with TNF-a. An increase inNF-kB2 p100 levels could be observed with TNF-a stimu-lation, and this could be brought back to baseline by the

combination of birinapant and TNF-a. The p52bandofNF-kB2 was not detectable. No change in the protein levels ofRIP1 kinase, which was found at similar levels in all 4 celllines, could be observed upon treatment with birinapant orTNF-a alone, but RIP1was depleted in 3 of 4 cell lines whenboth agents where combined (Fig. 2B).

We then confirmed the induction of apoptosis seen at theprotein level through cell-cycle analysis by propidiumiodide staining. A significant increase in sub-G1 fractions,indicative of apoptosis, was seen inWM9 cells when treatedwith birinapant alone; in 451Lu and WM1366 cells onlywhen birinapant was combined with TNF-a; but not in1205Lu cells, even after combination treatment (Fig. 2C).To confirm the indication of apoptosis seen in the cell-cycleanalysis, Annexin V staining was conducted. The relativeincrease in Annexin V–positive cells seen with birinapantalone only in WM9 and with birinapant in combinationwith TNF-a in WM9, 451Lu, and WM1366, but not in1205Lu cells, could confirm the pattern of apoptosis seenby cell-cycle analysis (Fig. 2D).

To investigate a possible role of MAPK and RAC1signaling in resistance to birinapant, phosphorylationstatus of ERK1/2 and protein levels of RAC1 upon treat-ment with birinapant at different time points in birina-pant-sensitive and -resistant cell lines were assessed. Wecould observe a marked decrease in phosphorylation ofERK1/2 only at 24 hours and this decrease was seen in abirinapant-sensitive as well as a birinapant-resistantcohort of cell lines. RAC1 protein was found at similarlevels in both sensitive and resistant cohorts and therewas no significant change upon birinapant treatment(Supplementary Fig. S2).

An immunoblot conducted in the 451Lu cell line showedthat birinapant could cause sustained cIAP1 protein degra-dation within one hour at a dose of 100 nmol/l, whereasXIAP levels were not affected (Fig. 3A).

To confirm that birinapant resistance was not caused byfailure of target degradation, we conducted immunoblotsfor cIAP1 and XIAP in a cohort of birinapant-TNF-acombination therapy–resistant cell lines and found cIAP1

Figure 1. Melanoma cell lines canbe sensitized to birinapant bycotreatment with TNF-a. Seventeenhuman melanoma cell linesrepresenting major mutationalsubgroups of cutaneous melanomawere treated with birinapant atconcentrations up to 1 mmol/L, alone(gray bars) or in combination withTNF-a 1 ng/mL (black bars). Valueson the y-axis represent IC50 values asassessed by the MTS proliferationassay at 72 hours. Data labelsindicate IC50 values frommean valueof biologic triplicates.






protein levels to be depressed to similar levels as seen inthe birinapant-sensitive cell line 451Lu (SupplementaryFig. S3).

Next, we investigated whether the observed cell deathwith birinapant at a dose of complete cIAP1 inhibition incombination with TNF-a was caspase dependent. Twocombination-sensitive cell lines (451LuandWM1366)weretreated with the combination of birinapant and TNF-awithorwithout additionofZ-VAD-FMK,apan-caspase inhibitor.Inbothcell lines, the inhibitionof caspases led toa completerestoration of proliferation (Fig. 3B). In a similar experi-ment, we then further explored the role of RIP1 kinase, an

essential part of the caspase-initiating complex activated byTNF-a. By treating the 2 cell lines as above, now in theabsence or presence of necrostatin-1, a RIP1 kinase inhib-itor, we could again reverse the effect of birinapant and TNF-a and restore cell proliferation (Fig. 3C).

As loss of RIP1 expression could therefore be a possiblemechanism of resistance to birinapant, we assessed RIP1expression in thewhole panel of cell lines. However, no lossor decrease in RIP1 expression in the 6 birinapant-resistantcell lines compared with the sensitive group could beobserved. This was also true after stimulation with TNF-a1 ng/mL (Fig. 3D).

Figure 2. Effect of birinapant aloneor in combination with TNF-a. Fourcell lines showing varyingresponses to birinapant as seen inthe whole panel of cell linesassessed: combination resistant(1205Lu), single-agent–sensitive(WM9), and combination-sensitive(451Lu and WM1366). A, dose–response curves after 72-hourincubation with birinapant(TL32711) at indicated doses aloneor in combination with TNF-a 1 ng/mL using the MTS assay. For thecell lines 451Lu and WM1366:P < 0.05 for doses �10 nmol/L.Data are represented as mean of 3or more experiments � SEM. B,immunoblots showing the samefour cell lines after 24 hoursincubation with birinapant1 mmol/L, TNF-a 1 ng/mL, or thecombination of both, comparedwith untreated controls. Levels oftarget IAP proteins, cleaved PARP,NF-kB p65, NF-kB2 p100- p52,and RIP1 were assessed. GAPDHwas included to ensure equalloading. C, relative number of cellsin the sub-G1 fraction as assessedby propidium iodide cell-cycleanalysis after 24-hour incubationwith birinapant 1 mmol/L, TNF-a1ng/Ml, or combination of both.Data are represented as mean �SEM (n ¼ 3 biologic replicates). D,relative number of Annexin V–positive cells as assessed by flowcytometry after 24-hour incubationwith birinapant 1 mmol/L, TNF-a 1ng/Ml, or combinationof both. Dataare represented as mean � SEM(n ¼ 3 biologic replicates).

Krepler et al.





Three-dimensional spheroid models confirmedresponses seen in adherent cell culturesCells grown in 3D spheroid cultures were previously

shown to have altered drug response profiles comparedwith adherent cell cultures (34), and are expected to moreaccurately predict in vivo efficacy due to similar architectureand microenvironmental signals (35, 36). The 4 previouslyselected cell lines were grown as 3D spheroids in a collagenmatrix and treatedwith birinapant alone or in combinationwith TNF-a. A live/dead fluorescent cell stain was used tovisually assess treatment effects using confocal microscopy(Fig. 4A): Spheroids of thebirinapant single-agent–sensitivecell line, WM9, did indeed show an extensive reduction inlive cells after addition of birinapant, but not after additionof TNF-a alone. The combination-sensitive cell lines, 451Luand WM1366, retained the same response patterns in 3Dcultures: both showed a marked decrease in live cells andincrease in dead cells only after treatment with birinapantin combination with TNF-a. In addition, the cell line that

was completely resistant to the combination treatment inadherent cell culture, 1205Lu, showed only slight growthretardation when grown as spheroids in the presence ofbirinapant in combination with TNF-a.

To objectively quantify viability in this model, weassessedmetabolic activity of spheroids after treatmentwithbirinapant in combination with TNF-a using Alamar Blue.The viability results mirrored the responses seen in the Live/Dead assay: a near total loss of viability in WM9, a dramaticdecrease in viability in the combination-sensitive cell lines(451Lu, WM1366), and only a slight reduction of viabilityin the 1205Lu cell line (Fig. 4B).

Birinapant inhibits tumor growth in melanomaxenotransplantation models as a single agent

To investigate whether birinapant could inhibit melano-ma tumor growth in an in vivo setting as a single agent, 2 celllines were selected for xenotransplantation experiments:both were in vitro birinapant single-agent–resistant, but

Figure 3. Effect of birinapant is dependent on kinases and RIP1. A, immunoblot showing the effect of birinapant on cIAP1 and XIAP protein levels at 3 differenttimepoints andconcentrations in themelanomacell line 451Lu.GAPDHwas included to ensure evenprotein loading.B, thecell lines451LuandWM1366weretreated with birinapant at a dose of 1 mmol/L in combination with TNF-a 1 ng/mL in the absence or presence of the pan-caspase inhibitor Z-VAD-FMK.Relative cell proliferation after 72 hours was assessed using the MTS assay. Data are represented as mean � SEM (n ¼ 3 biologic replicates),P <0.05. C, the cell lines 451Lu andWM1366were treatedwith birinapant at a dose of 1mmol/L in combinationwith TNF-a 1ng/mL in the absence or presenceof the RIP1 kinase inhibitor necrostatin-1 (Nec-1). Relative cell proliferation after 72 hours was assessed using the MTS assay. Data are represented asmean � SEM (n ¼ 3 biologic replicates), P < 0.05. D, RIPK1 RNA expression levels in the panel of cell lines shown in Supplementary Table S1.Cell lines are stratified into birinapant sensitive (with or without addition of TNF-a) and birinapant in combination with TNF-a resistant. The left panel showsuntreated cells and the right panel cells stimulated with TNF-a 1 ng/mL for 24 hours. Notice the difference in scale of the y-axes.






451Lu did respond in vitro to the combination of birinapantwith TNF-a, whereas 1205Lu did not respond to the com-bination treatment in vitro. Both cell lines were inoculatedsubcutaneously in NUDE mice and allowed to form pal-pable tumors before being randomized into vehicle controland birinapant treatment groups.During 3weeks of dosing,birinapant showed an antitumor effect in both models,although the effect in the in vitro combination-sensitive celllinewasmore sustainedwith abrogation of tumor growth inthe birinapant-treated animals. In contrast, 1205Lu tumorsshowed a marked slowing of tumor growth, but not abro-gation of tumors (Fig. 5A).

In a subsequent in vivo experiment, we then went on toconfirm birinapant target inhibition in both models byimmunoblot of tumor lysates. Animals were again inocu-lated with both xenograft models and tumors allowed toform. Animals were then pretreated twice in an interval of48 hours and tumors were harvested 3, 6, 12, and 24 hoursafter the second dosing. Compared with vehicle control,cIAP1 protein was reduced to low levels 3 hours later, andthis effect was sustained for 24 hours in both models (Fig5B). Staining for activated caspase-3 in biopsies of the sametumors showed a modest increase in apoptotic cells in thebirinapant-treated animals compared with vehicle control,24 hours after treatment (Fig. 5C).

To further investigate the combination activity betweenbirinapant and TNF-a in vitro, the 4 cell lines previouslyselected on the basis of their response profiles were againused. All 4 cell lines had comparable levels of TNF-a incell culture supernatants (Supplementary Fig. S4) andadding high levels of TNF-a alone to all 4 cell lines hadno effect on cell proliferation (Supplementary Fig. S5).The one cell line sensitive to birinapant as a single agent,WM9, provided an opportunity to further investigate therole of TNF-a in this setting. We therefore added a TNF-a–blocking monoclonal antibody to WM9 cell culturesbefore treatment with birinapant. By gradually binding

endogenous TNF-a in the supernatant with increasingdoses of the antibody, it was possible to completelyabrogate the effect of birinapant in a dose-dependentmanner (Fig. 6A). This indicates that while no additionof exogenous TNF-awas necessary to observe a birinapanteffect in this cell line, the observed antitumor effect wasstill dependent on endogenous TNF-a.

Additional evidence on the causative role of TNF-a onthe effect of birinapant was provided by varying theschedule of combining birinapant with TNF-a: a combi-nation-sensitive cell line treated with birinapant for 36hours and subsequently incubated with TNF-a for 36hours, showed significant growth inhibition. Conversely,when treated first with TNF-a for 36 hours and then withbirinapant for 36 hours, cells were significantly less sen-sitive (Fig. 6B). Therefore, the combination activitybetween birinapant and TNF-a is schedule dependent.This corroborates the hypothesis that birinapant-mediat-ed IAP inhibition has to occur before the activation ofTNFR to induce an antitumor effect.

Previously, our group has generated human melanomacell lines with acquired resistance to BRAF inhibitors (27).The mechanism of resistance in these cell lines was RAFisoform switching and increased IGF-1R/PI3K signaling.Wecompared the cell lines 451Lu (BRAFV600E, BRAF inhibitorsensitive) and 451Lu-BR (BRAF inhibitor resistant) inregard to birinapant response. Cell viability was not affectedafter treatmentwith birinapant alone in neither the parentalnor the resistant cell line. When birinapant was combinedwith TNF-a, however, both cell lines showed a strongresponse that was identical between the sensitive and theresistant cell line (Fig. 6C).

Next, we investigated whether birinapant could sensitizemelanoma cells to chemotherapy. For this experiment, 2birinapant single-agent–resistant cell lineswere treatedwithcisplatin, a cytotoxic drug, with or without addition ofbirinapant. Cisplatin as a single agent reduced viability in

Figure 4. Effect of birinapant on melanoma cells grown as 3D spheroids. A, cell lines were grown as spheroids in a collagen mix for 72 hours with or withoutaddition of birinapant (bir) 1 mmol/L or TNF-a (TNF) 1 ng/mL or the combination of both. Spheroids were stained with a Live (green)/Dead (red) cell viability kitand imagedusing confocalmicroscopy. Experimentswere repeated3 times, each timeonespheroidper conditionwas imaged, representative imagesshown.B, spheroidsweregrown in 96-well plateswith one spheroid/well (n¼6 technical replicates). Spheroidswere then treated for 72 hourswith birinapant 1mmol/L,TNF-a 1 ng/mL, or the combination of both. Viability was assessed with the Alamar Blue assay. Data is shown relative to untreated control. Mean of biologictriplicates, error bars represent SEM.

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both melanoma cell lines (mean of both cell lines) atincreasing doses up to 20 mmol/L. Birinapant-mediatedcIAP1 degradation in combination with cisplatin signifi-cantly (P < 0.05) enhanced sensitivity to cisplatin in bothcell lines (Fig. 6D).

DiscussionA delicate balance between inducers and inhibitors of

apoptosis exists in any given cell (37). In cancer cells,including melanoma, this equilibrium is often skewed infavor of survival, with IAPs being predominant (38, 39).Committing melanoma cells to apoptosis, which would bean ideal outcome for most therapies, therefore requiresadditional stimuli. Inhibition of IAPs for one can restorethe balance between survival and death signals, therebyfacilitating programmed cell death in melanoma.Chronic inflammation in the tumor microenvironment

ofmelanoma lesions often leads to elevated levels of TNF-a,at least in part, provided by the tumor-infiltrating immunecells such as macrophages (23–25). Levels of tumor-infil-tratingmacrophages have been shown to be associated withaggressive disease (40). All melanoma cell lines tested wereresistant to treatment with exogenous TNF-a as a single

agent. This was in line with previous clinical experiences,whereminimal antitumor effect and significant toxicity wasobserved (41, 42). Birinapant-mediated downregulation ofcIAP1 by itself did not induce any antitumor effect in vitro.As neither compound was effective alone, but both werehighly effective in combination, this satisfies the definitionof "coalism" according to the so-called Saariselk€a agreement(43, 44). This was confirmed on a subset of cell lines to beapoptosis-mediated and dependent on caspases and RIPK1activity.

Independent of mutational background (we assessedmelanoma cell lines from major genetic subgroups), 2patterns of responses emerged: cell lines that were eitherexquisitely sensitive or remarkably resistant to birinapant incombination with TNF-a at low or high doses of bothcompounds respectively. We confirmed downregulation ofcIAP1 target protein in resistant and sensitive cell lines byWestern blot analysis, but only the sensitive cell linesshowed PARP cleavage, indicative of apoptosis. This obser-vation could also be shown by increased sub-G1 cell frac-tions and increased Annexin V–positive cells, indicating anincrease in apoptotic cells, in the combination-sensitive celllines.

Figure 5. Effect of birinapant in vivo.A, NUDE mice bearing 451Lu (left) or1205Lu (right) xenografts weredosed 3 times per weekintraperitoneally with eitherbirinapant at 30 mg/kg or vehiclecontrol. Dosing was started afterformation of palpable tumors. Thegraphs show duration of treatment indays on the x-axes and fold changein tumor volume compared with firstday of dosing on the y-axes. In the451Lu xenografts, 2 animals of thebirinapant group had unmeasureabletumors at the end of study. Datarepresents mean � SEM (n ¼ 5/group), P < 0.05 at end of treatment.B, NUDE mice bearing 451Lu or1205Lu xenotransplants with ameantumor volume of approximately 200mm3 were dosed twice at a 48-hourinterval with either birinapant at 30mg/kg or vehicle control. At 4 timepoints (3, 6, 12, and 24 hours) afterlast dosing tumors were harvested.Immunoblots of tumor proteinlysates harvested at indicated timepoints are shown. Blots were probedfor levels of cIAP1, XIAP, and cIAP2(not measurable). GAPDH wasincluded to insure equal loading.Quantifications of cIAP1 bands areincluded as bar graphs. Datarepresents mean þ SEM. C, tumortissues of control and birinapant-treated animals were stained foractivated caspase-3.






As a subgroup of melanoma cell lines of all geneticbackgrounds tested was found to be resistant to birinapantin combination with TNF-a, we investigated whether levelsof phosphorylated ERK 1/2 or RAC1 were increased afterbirinapant treatment in these cell lines. These were recentlydescribed to play a role in resistance to SMACmimetics (45,46). Although we observed a decrease in ERK1/2 activationafter 24 hours of birinapant treatment in most cell linestested, we could not find any differential regulation of these2 proteins in the resistant cell lines as compared with thesensitive subset. Another possible mechanism of resistanceto SMAC mimetics is loss of RIP1 expression. We observedthe effect of birinapant to be dependent on RIP1 kinase inconcordance with previously published reports on com-pounds from the same class (47). As there was no differencein RIP1 expression between birinapant-resistant and -sen-sitive cell lines in the panel used in this study, RIP1 expres-sion is likely not a suitable biomarker to predict melanomaresponse to birinapant. Therefore, further investigation todefine a biomarker for this subgroup is warranted, as thiswould be highly useful in the clinical application ofbirinapant.

Ourobservations remained consistent across increasinglycomplex models from monolayer culture, to 3D matrixcultures, to humanmelanoma xenograft models. While the3D spheroid models closely mirrored the effects seen inadherent monolayer culture, the in vivo xenotransplanta-tion experiment results were reflecting the complexity of thein vivo setting. While in vitro 451Lu cells responded only tothe combination of birinapant and TNF-a, birinapant washighly active as a single agent in the in vivomodel abrogatingtumor growth. In addition, a cell line resistant to birinapantin vitro even in combinationwith TNF-a still showed slowertumor growth when treated with birinapant in vivo com-pared with vehicle-treated controls. This observation indi-cates the high complexity of melanoma growth in a tissuemicroenvironment providing a multitude of additionalstimuli. Together, these results indicate a potential effec-tiveness of birinapant as a single agent in patients withmelanoma.

Birinapant was effective as a single agent in vitro onlyin one of the 17 cell lines tested. We therefore investi-gated the role of TNF-a signaling on the birinapant effectin this cell line. Blocking endogenous TNF-a in the

Figure 6. The role of TNF-a in the effectiveness of birinapant in vitro. A, TNF-a is required for in vitro growth inhibition: Themelanoma cell lineWM9was treatedwith birinapant at doses indicated on the bottom x-axis for 72 hours. The same cell line was treated with birinapant at a fixed dose of 1 mmol/L in combinationwith a monoclonal blocking antibody binding to TNF-a (TNF-a mAb) at increasing doses as indicated on the top x-axis. Viability was assessed after72 hours via the MTS assay; absolute values are shown on the y-axis. Data represent mean of 3 experiments � SEM. B, combination activity betweenbirinapant and TNF-a is schedule dependent: 451Lu cells were incubated either with birinapant at indicated doses for 36 hours and subsequently TNF-a1 ng/mL for 36 hours (birinapant � TNF-a), with TNF-a 1 ng/mL for 36 hours and subsequently birinapant at indicated doses (TNF-a � birinapant),or the combination of both for full 72 hours. Cell viability was assessed relative to untreated controls. Data representsmean of 3 experiments�SEM. C, effectof the combination therapy on a BRAF inhibitor–resistant cell line. A cell line with acquired resistance to BRAF inhibition (451Lu-BR) shows theidentical response to birinapant as its parental cell line (451Lu). Cells were treated with birinapant at indicated doses either alone (451Lu, 451Lu-BR) or incombination with TNF-a 1 ng/mL (451Lu þ TNF-a, 451Lu-BR þ TNF-a) for 72 hours. Cell viability is shown relative to untreated controls. Data representsmean of 3 experiments �SEM. D, effect of cisplatin on melanoma viability in the absence or presence of birinapant. The birinapant-resistant cell lines 451LuandWM1366were treated with cisplatin in increasing doses up to 20 mmol/L as a single agent or in combination with birinapant at a fixed dose of 100 nmol/L.Cell viability is shown relative to untreated controls. Data represents mean of both cell lines � SEM.

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supernatant completely abrogated the effect of birina-pant in a dose-dependent fashion, thereby showing thedependency of birinapant on concurrent TNF-a stimu-lation in vitro.This dependency was furthermore schedule-specific, as

we could show in a cell line sensitive to birinapant only incombination with TNF-a. The effect of the combinationtherapy was preserved when sequentially added in thefollowing order: first birinapant, with downregulation ofcIAP1 and cIAP2, and subsequent TNF-a, with activation ofthe TNFR complex 2 and the apoptotic cascade. Addingthese 2 compounds in the reverse order diminished theireffectiveness significantly.With the prospect of increased numbers of patients with

melanoma acquiring resistance to BRAF inhibitors, thequestion whether birinapant might be a feasible second-line therapy for such cases was explored. Indeed, a cell linewith acquired resistance to BRAF inhibition did not alter itssensitivity profile to birinapant when compared with theparental cell line. This suggests birinapant as a possiblesecond-line therapy in patients with acquired resistance toBRAF inhibitors.Although targeted therapy with small molecules is show-

ing impressive results in melanoma therapy, cytotoxicagents are still playing a major role in this disease whenpatients are either not eligible for kinase inhibitors andimmunotherapy, or have relapsed on these drugs. We havetherefore investigated the role of birinapant in sensitizingmelanoma to cisplatin, a DNA-damaging agent. Althoughthe results seen in this study are encouraging, more combi-nations and schedules will have to be explored as thesestudies are currently ongoing.

Disclosure of Potential Conflicts of InterestE.M.Neiman is employed (other thanprimary affiliation; e.g., consulting)

in Tetralogic Pharmaceuticals as a Research Scientist. M. McKinlay hasownership interest (including patents) in TetraLogic Common Stock. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: C. Krepler, S.K. Chunduru, R. Amaravadi, M.McKinlay, M. HerlynDevelopment of methodology: C. Krepler, S.K. Chunduru, M. McKinlay,M. HerlynAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): C. Krepler, M.B. Halloran, X. He, M. Xiao, J.Villanueva, E.M. Neiman, K.L. NathansonAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): C. Krepler, X. He, M. Xiao, Y. Mitsuuchi,C. Benetatos, K.L. Nathanson, R. Amaravadi, M. McKinlayWriting, review, and/or revision of the manuscript: C. Krepler, S.K.Chunduru, A.M. Vultur, Y. Mitsuuchi, C. Benetatos, K.L. Nathanson, R.Amaravadi, H. Pehamberger, M. HerlynAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A.M. Vultur, K.L. NathansonStudy supervision: M. Herlyn

AcknowledgmentsThe authors thank the Microscopy Facility managed by James E. Hayden,

the Flow Cytometry Facility managed by Jeffrey S. Faust, the Animal Facilitymanaged by Denise DiFrancesco, and Protein Expression and MolecularScreening Facility managed by David C. Schultz, all at the Wistar Institute.

Grant SupportC. Krepler is a MAX KADE foundation postdoctoral research exchange

grant recipient. The studieswere fundedbyNIHgrantsCA25874,CA047159,CA 114046, and CA10815, and the Dr. Miriam and Sheldon G. AdelsonMedical Research Foundation.

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked advertise-ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 6, 2012; revised January 23, 2013; accepted January 24,2013; published OnlineFirst February 12, 2013.

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2013;19:1784-1794. Published OnlineFirst February 12, 2013.Clin Cancer Res Clemens Krepler, Srinivas K. Chunduru, Molly B. Halloran, et al. against Human Melanoma CellsThe Novel SMAC Mimetic Birinapant Exhibits Potent Activity

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