Downregulation of HER3 by a Novel Antisense Oligonucleotide, EZN-3920… · 2013. 3. 28. · 2013...

Large Molecule Therapeutics

Downregulation of HER3 by a Novel AntisenseOligonucleotide, EZN-3920, Improves the Antitumor Activityof EGFR and HER2 Tyrosine Kinase Inhibitors in AnimalModels

Yaming Wu, Yixian Zhang, Maoliang Wang, Qi Li, Zhengxing Qu, Victoria Shi, Patricia Kraft, Steve Kim,Ying Gao, Jenny Pak, Stephen Youngster, Ivan D. Horak, and Lee M. Greenberger

AbstractAmong the four human EGF receptor (HER) family members (EGFR, HER2, HER3, HER4), HER3 is of

particular interest as it interacts with HER2 and EGFR via heterodimerization and is a key link to the

phosphoinositide 3-kinase (PI3K)/AKT signal transduction axis. Recent studies indicate that HER3 plays a

critical role in mediating resistance to agents that target EGFR or HER2. As HER3 lacks significant kinase

activity and cannot be inhibited by tyrosine kinase inhibitors, neutralizing antibodies and alternative inhibitors

of HER3 have been sought as cancer therapeutics. We describe here a locked nucleic acid (LNA)-based HER3

antisense oligonucleotide, EZN-3920, that specifically downmodulated the expression of HER3, which was

associated with growth inhibition. EZN-3920 effectively downmodulated HER3 expression, HER3-driven

PI3K/AKT signaling pathway, and growth in tumors derived from BT474M1 breast and HCC827 lung

carcinoma cell lines, which overexpress HER2 and EGFR, respectively. Furthermore, when EZN-3920 was

coadministered with gefitinib or lapatinib in xenograft tumor models, enhanced antitumor activity compared

with the effect of monotherapy was found. The effect was associated with a blockade of inducedHER3mRNA

expression caused by lapatinib or gefitinib treatment. Finally, EZN-3920 sustained its antiproliferative effect in

trastuzumab-resistant cells and three independently derived gefitinib-resistant cells. Our findings show that

downmodulation of HER3 by EZN-3920 leads to the suppression of tumor growth in vitro and in vivo,

suggesting that HER3 can be an effective target for the treatment of various cancers that have been activated

by HER3 alone or where HER3 activation is associated with EGFR or HER2 expression.Mol Cancer Ther; 12(4);

1–11. �2013 AACR.

IntroductionMembers of the human EGF receptor (HER) family of

receptor tyrosine kinases (RTK) designated as HER1(EGFR), HER2 (ErbB2 or p185HER2/neu), HER3, and HER4underlie the pathogenesis of many types of human cancer(1–4). Extracellular ligand binding initiates signaling bycausing specific homodimeric or heterodimeric receptorformation and activation of the cytoplasmic kinasedomain that phosphorylates tyrosines in the tail regionof each receptor. The phosphorylated HER membersbecome adocking station for various proteins and thereby

activate at least 3 major intracellular signaling cascades(RAS/ERK, AKT/PI3K/PTEN, and JAK/STAT), whichultimately regulate cell growth, differentiation, migra-tion, and survival.

HyperactivationofEGFRorHER2are alreadyknown tomediate tumor growth. Consistent with this, small mole-cules and antibodies that bind to ErbB1/EGFR (gefitinib,erlotinib, erbitux) and/or HER2 (e.g., trastuzumab, lapa-tinib) have already proven anti-cancer activity. However,in the past few years, new data have revealed that HER3plays a vital role in cancer growth and progression (5–8).HER3 is overexpressed in various cancers, includingbreast, colon, gastric, prostate, lung, hepatocellular, headand neck cancers, and melanoma, and is implicated intumorigenesis and prognosis (7–11). It is now clear thatheregulin binding to HER3 or HER3 dimerizationwith EGFR or HER2 leads to hyperphosphorylation ofcytoplasmic tail of HER3, which in turn results in AKTsignaling (12–14). Beyond this, HER3 can be hyperpho-sphorylated in patients with gefitinib-resistant cancer (4)as well as lapatinib- or trastuzumab-resistant tumor celllines via receptor tyrosine kinases (15, 16). The mRNA

Authors' Affiliations: Department of Pharmacology, Enzon Pharmaceu-ticals, Inc. Piscataway, New Jersey

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Yixian Zhang, Enzon Pharmaceuticals, Inc., 20Kingsbridge Road, Piscataway, NJ 08854. Phone: 732-980-4914; Fax:732-885-2950; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0838

�2013 American Association for Cancer Research.

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Published OnlineFirst February 8, 2013; DOI: 10.1158/1535-7163.MCT-12-0838

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encoding HER3 is also increased in response to treatmentof cellswithHER1 inhibitor (gefitinib), HER1/2 inhibitors(lapatinib), AKT inhibitors, phosphoinositide 3-kinase(PI3K) inhibitors, or anti-androgens (15–20).

Experimental downmodulation of HER3 using geneticor pharmacologicmethods supports the notion thatHER3is an important driver in cancer growth. Consistent withthis, reducedHER3expressionby conditional knockout orLNA-based antisense molecule, designated as EZN-3920,in a polyoma-T model of breast cancer inhibits PI3Kactivation and tumor formation in this genetically engi-neered mouse model (21). In addition, siRNA and shorthairpin RNA (shRNA)-directed downmodulation ofHER3 resulted in tumor growth inhibition in lung ade-nocarcinoma (22) and decreased disease progression aswell as prolonged survival in xenograft mouse models ofovarian cancer (13) or of HER2-overexpressing breastcancer (5). Furthermore, a recent study showed the sen-sitivity of a subset of head and neck cancers to HER2kinase inhibitor is dependent on the co-expression ofHER3 and its ligand neuregulin-1 (14). In vitro, targetingHER3using siRNAs and antisense deoxyoligonucleotides(AS-ODNs) potently inhibits proliferation and promotesapoptosis in cells sensitive and insensitive to EGFR andHER2 inhibitors (5, 13, 17, 22, 23). Finally, a monoclonalantibody to HER3 inhibited tumor growth, especially incell lines that secrete heregulin, a ligand for HER3 (12).

In this study, we have further explored the use of theHER3 antisense oligonucleotide EZN-3920, which is anLNA-ASO. LNA-ASOs have shown very high bindingaffinity to mRNA, excellent potency for target mRNAdownmodulation, improved resistance to nuclease diges-tion, and excellent stability in plasma and tissues inpreclinical studies (24). Furthermore, LNA-ASOs simplyprepared in saline are highly effective in vitro and in vivo(25–29). It is shownhere that EZN-3920 suppressed tumorgrowth both in vitro and in vivo in tumors that co-expressHER1 or HER2 or develop resistance to HER1/2 inhibi-tors. The evidence suggests that the efficacy of EZN-3920alone or in combination with other anti-HER drugs suchas lapatinib, trastuzumab, or PI3K inhibitors may over-come emerging resistance to agents that target the HER/PI3K/AKT axis.

Materials and MethodsCell culture

Cell culture reagents were purchased from Invitrogenunless otherwise mentioned. All cell lines unless other-wise motioned were obtained from American Type Cul-tureCollection (ATCC) andweremaintained according toATCC’s recommendations in a medium supplementedwith 10% FBS at 37�C in a humidified atmosphere of 5%CO2 and 95% air. HCC827gr6 cancer cells were obtainedfrom Dr. Jeffrey Engelman’s laboratory (Department ofMedicine, Harvard Medical School, Charlestown, MA)grown in RPMI-1640 plus 10% FBS. Both PC9 and PC9GRcancer cells were obtained fromPasi A. J€anne’s laboratory(LoweCenter for ThoracicOncology, Dana-Farber Cancer

Institute, Boston, MA) and grown in RPMI-1640 contain-ing 10% FBS. BT474M1 breast cancer cells were obtainedfrom California Pacific Medical Center and maintainedin DMEM/F12 (1:1) containing 10% FBS. The 15PC3human prostate cancer cell line was provided by Dr. F.Bass (Neurozintuigen Laboratory, Academic MedicalCenter, Amsterdam, the Netherlands) and maintainedin Dulbeccos’ Modified Eagles’ Media (DMEM) plus10% FBS. Authentication of used cell lines was not doneby the authors.

Synthesis of oligonucleotideOligonucleotides were synthesized as described pre-

viously (26). EZN-3920 (50-TAGcctgtcacttCTC-30) is afully phosphorothioated oligonucleotide. Capital lettersdenote LNA monomers, and lowercase letters denoteDNA monomers. 50-Cy5.5–labeled EZN-3920 and ascrambled oligonucleotide, designated as EZN-3046 (50-CGCAgattagaaACCt-30) and abbreviated as EZN-SCR,also were made according to the method described pre-viously (29).

mRNA downmodulation in human xenograftsTwenty-four hours post-last dose, tumors were

harvested and cut into 3- to 5-mm3 pieces. Five to10 mg tumor sample was transferred into 1 mL lysis/binding solution in 1.5 mL Lysing Matrix D tubes andhomogenized in FastPrep (MP BIO). RNA purification,cDNA synthesis, and quantitative real-time PCR(qRT-PCR) were carried out as previously described(26). Targets and glyceraldehyde-3-phosphate dehydro-genase (GAPDH) mRNA were quantified using Taq-Man Assay Kits (Applied Biosystems) according to themanufacturer’s instructions. The following probeswere used for qRT-PCR: Hs00176538_m1 for HER3,4326317E for GAPDH, Hs00193306_m1 for EGFR1, andHs00170433_m1 for HER2.

Western blot analysisWestern blot analysis was conducted as previously

described (29). The antibodies used are as follows: HER3(sc-285), EGFR (sc-03), Akt (5G3, #2966), p-Akt (ser473,#4051), p-HER3(Tyr1289, #4791), p-p53 (Ser15, #9284), andp53 (1C12, #2524). All antibodies were derived from CellSignaling Technology, Inc.a-Tubulin (B-5-1-2, T5168) andanti-GAPDH-peroxidase (G9295) were purchased fromSigma. Anti-Rabbit IgG (H þ L) HRP conjugate (W4011)and anti-mouse IgG (Hþ L)HRP conjugate (W4021) werepurchased from Promega. GAPDH and a-tubulin servedas the loading controls.

Localization of Cy5.5-labeled EZN-3920 inBT474M1 tumors

Once the BT474M1 tumors reached an average vol-ume of 200 mm3, tumor-bearing mice were injected i.v.with 40 mg/kg unlabeled or Cy5.5-labeled EZN-3920 onday 0 (first day of dosing) and day 4. The tumors wereexcised 24 hours later and imaged whole by the

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Xenogen Lumina as described previously (29). Follow-ing imaging, tumors were cryopreserved with 4% para-formaldehyde, sectioned, and imaged with fluorescentmicroscopy.

Determination of the concentration of EZN-3920 intumorsAn LC/MS/MS system (29), 4000 QTrap mass spec-

trometer (AB Sciex) interfaced with an AcQuity UPLC(Waters), was used to determine EZN-3920 concentrationin tumor tissues. Briefly, analyte EZN-3920 and an EZN-4176 (internal standard) were eluted on Clarity 3u Oligo-RP column (50 � 2.00 mm2, Phenomenex) with a lineargradient of mobile phase A/B from 80/20 to 50/50 in 8minutes, and mobile phase A consisting of 0.4 mol/Lhexafluoro-2-propnol (HFIP) and 16.3 m mol/L triethylamine (TEA) in 5%methanol aqueous solution andmobilephase B consisting of 0.4 mol/L HFIP and 16.3 m mol/LTEA in 60% methanol. The flow rate was 0.15 mL/min.Column temperature was set at 65�C. The turbo IonSpraysource was operated with negative mode at 40�C. The iontransitions of m/z ¼ 651.5 (precursor ion) to m/z ¼ 95(product ion) for EZN-3920 and m/z ¼ 751.8 (precursorion) to m/z ¼ 95 (product ion) for EZN-3920 were mon-itored, and declustering potential and collision energywere optimized to DP ¼ �40 V and CE ¼ �100 V (EZN-3920), and DP ¼ �60 V and CE ¼ �100V (EZN-4176).ResultsSelection of EZN-3920 andmodels for in vivo studiesEZN-3920 was designed to bind with 100% comple-

mentary to HER3 mRNA and initiate RNase H–mediateddegradation of mRNA. An initial exploration of the activ-ity of EZN-3920 was one in an in vitro cell–based assaywhere EZN-3920 was added to cells in the presenceof transfection agent. It downmodulated HER3 mRNAin 15PC3 and Huh-7 cells with an IC50 of 0.24 and 0.36nmol/L, respectively (Supplementary Table S1). EZN-3920 also showed potent target and growth inhibition ina panel of cancer cell lines (Supplementary Table S2). In15PC3 prostate cancer cells, HER3 and p-AKT levels weredecreased (IC50 < 5 nmol/L; Supplementary Fig. S1A)and concomitantly caspase-3/7 activities increased (Sup-plementary Fig. S1B) in response to the treatment of EZN-3920. The data were consistent with the function of HER3in cancer cells. Recently, we also showed that EZN-3920was effective in downmodulating mRNA and HER3 pro-tein in the absence of lipofection (30). The effect wasspecific as EZN-3920 did not downmodulate EGFR orHER2 (see below). Beyond that, target inhibition intumors derived from 15PC3 cells after i.v. administrationofEZN-3920wasdetected (30). In addition, the compounddownmodulated HER3 and inhibited tumor growth in amouse model of mammary carcinoma driven by thepolyomavirus middle T oncogene (21). On the basis ofthis initial favorable activity, we decided to extend ourefficacy studies in additionalmodels inwhichHER3playsa key role in tumor development.

To find suitable models for in vivo studies, we screenedmultiple cell lines that may depend on HER3 for growth(4, 5, 13, 31–33) and tested the effect of EZN-3920 on thegrowth of these lines. As transfection is highly artificial asrecently reported (34) and does not represent the contextof our in vivo experiments in which no transfection sys-tems were used, the remainder of the in vitro studies wereconducted without lipofection. Table 1 shows that HER3was highly expressed in BT474M1, SKBR3, and HCC827when compared with OVCAR5 and A549. EZN-3920showed potent growth inhibition in BT474M1, SKBR3,and HCC827 cells (Table 1) but far less or no activity inOVCAR5 or A549 cells. This observation allowed us tofocus on the characterization of the effect of EZN-3920in BT474M1 and HCC827 models as, in our hands,SKBR3 is not tumorigenic in xenograft models (datanot shown), and the potency of EZN-3920 in OVCAR5and A549 was low. Beyond this, HER2 and EGFR aredrivers of cell growth in BT474M1 and HCC827, respec-tively, as the BT474M1 cell line overexpresses HER2 andHER3 knockdown resulted in tumor regression (5),whereas the HCC827 cell line overexpresses EGFR andis inhibited by gefitinib (35). Figure 1A shows that EZN-3920 effectively and specifically downmodulated HER3mRNA but not other family members, such as EGFRand HER2, in a dose-dependent manner in HCC827cells. The specificity was expected as EZN-3920 has 4and 3 base mismatches with the complementary regionof EGFR and HER2, respectively (SupplementaryTable S1) and such mismatches typically can result inan inability for downmodulating mRNA (26, 36). Thiseffect was not due to off-target effect of the oligonucle-otide backbone as the control oligonucleotide EZN-SCRfailed to downmodulate HER3 mRNA in 15PC3(Supplementary Fig. S2) or HCC827 cells (Fig. 1). InBT474M1 cells, EZN-3920 also showed specific down-modulation of the intended target HER3 but not EGFRnor HER2 (Fig. 1B). To make sure that mRNA down-modulation was associated with reduction of proteinexpression, we conducted Western blot analysis on the

Table 1. Relative HER3 expression andsensitivity to EZN-3920 of selected cell line

Tumortype Cell line

HER3expressiona IC50

b, mmol/L

Breast BT474M1 þþþ 1 � 0.1Breast SKBR3 þþþ 0.5 � 0.003NSCLC HCC827 þþ 0.5 � 0.04Ovarian OVCAR-5 þ 5 � 0.71NSCLC A549 þ/� >10aRelative HER3mRNA level measured by qRT-PCR:þ/�,Ct� 35; þ,30 < Ct < 35; þþ, 25 < Ct < 30; þþþ, Ct < 25.bCell growth (6 days) was measured by MTS assay asdescribed in Materials and Methods.

HER3 mRNA Antagonist

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lysates obtained from both cell lines after they weretreated with EZN-3920 or EZN-SCR. In both cell lines,treatment with EZN-3920 resulted in dose-dependentdownmodulation of HER3 protein and phospho (p)-HER3 levels (Fig. 1C). The reduced expression level ofHER3 was associated with deactivation of AKT path-way as p-AKT was also inhibited by EZN-3920 in adose-dependent manner. The effects were specific asAKT or tubulin levels were unchanged. The downmo-dulation of HER3 had no effect on HER2 or p-HER2,suggesting the effect of EZN-3920 was specific and theobserved decrease in HER3 protein level was sufficientto impact on the important downstream signaling suchas p-AKT.

Antitumor activity in xenograft modeIn vivo efficacy of EZN-3920 was first evaluated in the

HCC827 lung cancer model. EZN-3920 administered at30 and 40mg/kg given twice aweek for 4weeks inhibitedthe growth of HCC827 tumors by approximately 60% and80%, respectively, on day 28, whereas EZN-SCR did notinhibit tumor growth (Fig. 2A). To relate tumor growthinhibition to HER3 downmodulation, the effect of EZN-3920 on HER3 was tested in a separate short-term study.After 5 doses of EZN-3920 administered at 30 and40 mg/kg, 40% and 60% of the human HER3 mRNA intumorswas downmodulated, respectively, based on qRT-PCRanalysis (Fig. 2B). Furthermore,Westernblot analysisof each individual tumors revealed that HER3, p-HER3,

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Figure 1. Effect of EZN-3920 onHER3 mRNA and protein levels.A and B, specific downregulationof HER3 mRNA in HCC827 andBT474M1 cells. HCC827 cellswere treated with 0 to 3 mmol/Lof LNA for 3 days. HER3, EGFR,and HER2 mRNA levels weredetermined by RT-PCR usingTaqMan gene expression assays.Data aremean�SD; �,P < 0.05. C,EZN-3920 downmodulated HER3protein in HCC827 and BT474M1cells. HCC827 cells were treatedwith 0, 0.3, or 1 mmol/L EZN-3920for 2 days. Cellular protein levelswere determined by Western blotanalysis as described in Materialsand Methods.

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p-AKT, p-S6K, and cyclin D levels were significantlyinhibited (Fig. 2C). The Western blot analysis was furthersupport by immunohistochemical (IHC) data (Fig. 2D),which showed that the membrane staining of HER3 orp-HER3 was decreased in response to the treatment ofEZN-3920. Similar to what was observed in vitro, p-AKTlevel in both cytoplasm and nucleus of the tumors wasreduced (Fig. 2D). The inhibition of HER3 consequentlypromoted apoptosis (34) as revealed by terminal deoxy-nucleotidyl transferase–mediated dUTPnick end labeling(TUNEL) assay (Fig. 2E), confirming the importance ofHER3 in tumor growth and survival.The efficacy of EZN-3920 was explored in a second

xenograft model. In this case, tumors derived fromBT474M1breast cancer cellswereused asdownregulationof HER3 in this model has been shown to inhibit tumorgrowth (5). Similar to the HCC827 model, the growth of

BT474M1 was also significantly inhibited by EZN-3920 ina dose-dependent manner (Fig. 3A). The observed effectwas specific as EZN-SCR was not effective in inhibitingtumor growth. Furthermore, IHC analysis revealed thatthe growthwas associatedwith the inhibition of the target(Fig. 3B).

Accumulation of EZN-3920 and targetdownmodulation in tumors

To ascertain that the antitumor effect was associatedwith the presence of EZN-3920 in the tumors, we evalu-ated thepresence andduration ofEZN-3920 in establishedBT474M1 tumors using LC/MS/MS method (29). Figure4A shows that the concentration of EZN-3920 in thetumors reached 4 and 8 mmol/L 4 hours after a singledose of EZN-3920 and multiple doses of EZN-3920(q3dx3), respectively. The concentration of EZN-3920 in

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Figure 2. Specific antitumor effect of EZN-3920 in HCC827 lung cancer xenograft tumor model. A, EZN-3920 inhibited tumor growth in HCC827 lung cancerxenograft model. Once the tumors reached an average volume of 100 mm3, mice were administered 30 or 40 mg/kg EZN-3920 (q3dx9, i.v.) or thecontrol oligonucleotide EZN-SCR6 (30 mg/kg, q3d�9, i.v.; n ¼ 8 per group). �, P < 0.05 (except day 7) compared with either saline or EZN-SCR group.B, EZN-3920 downmodulated HER3 mRNA in HCC827 tumors. When tumors reached an average volume of 100 mm3, mice received treatment with30 or 40 mg/kg EZN-3920 (q3.5dx9, i.v; n ¼ 8 per group). �, P < 0.05 compared with saline group. Tumors were harvested 24 hours after the last dose. ThemRNA expression levels (% control � SEM) in tumors were determined by qRT-PCR. C, EZN-3920 downmodulated HER3 protein in HCC827 tumors.Half of the samples from C were analyzed by Western blot analysis for various proteins as indicated. D, EZN-3920 downmodulation of HER3, p-HER3,and p-AKT. Tumor-bearing mice were treated with the indicated amount of EZN-3920 (q3.5dx9). Twenty-four hours after the last dosing, samples wereharvested and subjected to IHC analysis. E, EZN-3920–induced apoptosis. Samples from D were analyzed by TUNEL assay.






the tumor remained unchanged during the 24-hour peri-od and decreased slightly after 48 hours. These concen-trations matched or exceed the level of EZN-3920 neededto inhibit cell growth in vitro (Fig. 1). We next asked if thecompoundwas, in fact, localized inside the cells. To locatethe compound inside the cells, we generated a Cy5.5-labeled EZN-3920 and determined that it was active asshown by HER3 mRNA downmodulation in the liver ofmice (Supplementary Fig. S3). Tumor-bearing mice werethen injectedwith 40mg/kg ofCy5.5-labeled EZN-3920 at0 and 4 hours.After 24 hours, animalswere sacrificed, andtumors were analyzed by fluorescence imaging.

As reported previously (29), a high-intensity signal inthe tumor was observed 24 hours posttreatment, indicat-ing localization of Cy5.5-EZN-3920 in tumors (Fig. 4B,bottom). To confirm the observation, we next carried outsectioning of the tumor andmicroscopically examined thetumors. Figure 4C shows that the compound, Cy5.5-EZN-3920 was present in tumor. Furthermore, the compoundwas primarily localized inside the cells as it was located

around the nuclei [detected by 40,6-diamidino-2-pheny-lindole DAPI staining], providing additional evidence tosupport previously shown target inhibition data.

EZN-3920 potentiates the effect of tyrosine kinaseinhibitor in tumor growth inhibition

Lapatinib, a small-molecule dual inhibitor of HER2 andEGFR tyrosine kinase activity, is currently used in thetreatment of HER2-positive breast cancer. Interestingly, itwas shown recently that inhibiting lapatinib causesincreases in HER3 mRNA as well as HER3 protein andp-HER3 levels through a negative feedback loop thatinvolves the PI3K/AKT pathway (15–20). This effect ulti-mately results in resistance to lapatinib. Consistent with

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Figure 3. Specific antitumor effect of EZN-3920 in BT474M1 breastcancer xenograft model. A, EZN-3920 inhibited tumor growth. Once thetumors reached an average volume of 100mm3, mice were administered30 or 40 mg/kg EZN-3920 (twice a week, q3.5dx9, i.v.) or the controloligonucleotide EZN-SCR (30 mg/kg, q3.5d�9, i.v.; n ¼ 8 per group).�, P < 0.05, all EZN-3920–treated groups versus either the saline orEZN-3046 group from day 14. B, EZN-3920 downmodulation of HER3and p-HER3. 24 hours after the last dose (40 mg/kg), samples wereharvested and subjected to IHC analysis.

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Figure 4. Presence of EZN-3920 in BT474M1 tumors. A, determination ofEZN-3920 in tumors by LC/MS/MS. After different time points after thelast dose, mice were sacrificed and tumors harvested. The concentrationdetermination of EZN-3920 in the tumors was measured. Data aremean � SEM (n ¼ 5). B and C, detection of Cy5.5-labeled EZN-3920 intumors. Tumor-bearing mice were given an i.v. injection on days 0 and 4.The tumors were excised 24 hours later and imaged whole by theXenogen Lumina (B) and then cryopreservedwith 4%paraformaldehyde,sectioned, and imaged with fluorescent microscopy (C). DAPI stainingwas also carried out to identify nuclei.

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this, a combination of lapatinib with a neutralizing HER3antibody AMG-888 showed better efficacy than eitheragent alone (15), and a combination of PI3K inhibitorBEZ235 with trastuzumab also resulted in a greater anti-tumor effect (16). Therefore,we reasoned that treatment ofcancer cells with lapatinib in the presence of EZN-3920should also yield improved efficacy over a single agent.We thus used BT474M1 as a model system. In BT474M1cells, lapatinib administered at a clinically achievablelevel (1 mmol/L; ref. 37) significantly induced HER3mRNA (Fig. 5A) and HER3 protein levels (Fig. 5B). How-ever, lapatinib-induced HER3 expression was effectivelyreduced by EZN-3920 in a dose-dependent manner(Fig. 5A and B), but HER3 mRNA expression was notreduced by the scrambled oligonucleotide control. Con-sequently, the combination of both agents exerted muchgreater growth inhibition than either agent alone (Fig.5C). The combinatorial effect appeared to be synergisticas a noneffective dose (5 mmol/L) of EZN-3920 with amoderately effective (40% growth inhibition with EZN-SCR) dose of lapatinib resulted in 60% growth inhibition(Fig. 5C). We next tested the effect of this combination ina tumor xenograft model. Tumor-bearing mice weretreated with lapatinib [100 mg/kg, intraperitoneally(i.p.)] in combination with a suboptimal dose of EZN-3920 (30 mg/kg). Lapatinib or EZN-3920 alone showedmoderate antitumor effect. However, when combined,the tumor growth was completely inhibited (Fig. 5D).Structure of gefitinib and lapatinib are shown in Fig. 5Gand H, respectively.To further support a combination approach, we con-

ducted a separate study using an EGFR small-moleculeinhibitor, gefitinib, which also induces HER3 (19). Simi-larly, while EZN-3920 or gefitinib exerted significantantitumor activities, the combination resulted in theregression of HCC827 tumor xenograft (Fig. 5E). Analysisof tumor samples (Supplementary Fig. S4) shows thatthere was a rapid increase in HER3 expression in tumorsafter mice was treated with gefitinib for only 12 hours.However, this induction was prevented by EZN-3920treatment (Supplementary Fig. S4). Moreover, as hetero-dimerization of HER2 with HER3 activates PI3K/AKTpathway, we next tested the combination of EZN-3920with a PI3KCA (PI3K constitutively active)-specific anti-sensemolecule EZN-4150 (30). The combination of these 2LNAs showedgreater antitumor activity than either agentadministered alone (Fig. 5F).

Antiproliferation activity of EZN-3920 in gefitinib-or trastuzumab-resistant cellsTo explore the potential use of EZN-3920 in drug-

resistant lung cancer, we next generated gefitinib-resis-tant HCC827 through chronic exposure of cells withincreasing amount of gefitinib over a 4-month period oftime. When the concentration reached 1 mmol/L, the cellswere cloned and tested for their responses to gefitinibtreatment. Figure 6A (top) shows that all clones werehighly resistant to the treatment of gefitinib, up to 10

mmol/L, whereas the IC50 for gefitinib in the parentalcells was approximately 10 nmol/L. The results wereconsistent with the published data (4). Interestingly, theclones showed enhanced sensitivity to EZN-3920 (Fig. 6A,bottom). The results are encouraging as it suggests thatEZN-3920 is capable of overcoming resistance induced bysmall-molecule inhibitors. To confirm the results, weobtained additional gefitinib-resistant cells and tested theeffect of EZN-3920 in these cells. Resistance to gefitinib inHCC827GR and PC9GR and were first validated (topof Fig. 6B and C). Interestingly, sensitivity to EZN-3920was retained in both HCC827GR and PC9GR (bottomof Fig. 6B and C). Beyond that, we also generated trastu-zumab-resistant BT474M1 (Fig. 6D, top) after chronicexposure of the cells to increasing concentration oftrastuzumab. Figure 6D (bottom) shows that the trastu-zumab clone was sensitive to the treatment of EZN-3920.Thus, all the resistant lines remained sensitive to EZN-3920 treatment, suggesting that HER3 plays a centric rolein the resistance of different modalities. This growthinhibition in the resistant cells was not simply associatedgeneral cytotoxicity but target inhibition based onmRNAassessment (Supplementary Fig. S5).

DiscussionUnlike most members of the HER family, HER3 has a

potent ability to activate downstream PI3K and Akt path-way once it dimerizes with HER2, mediate resistance toHER1 or HER2-targeted therapeutics, and play a criticalrole in cancer growth. Unlike HER1 or HER2, HER3 doesnot appear to have appreciable kinase activity (38–41), soATP-mimetic small-molecule inhibitorsmaynot be able toinhibit HER3-mediated tumor growth. On the other hand,inhibition of HER3 activity can also be achieved by anti-bodies that block ligand binding to HER3 (12). However,as the cytoplasmic tail of HER3 can be phosphorylatedand thereby hyperactivated by other growth factors (4),HER3 antibodies may not be effective in all patients, andresistance to these agents is highly likely, as the phos-phorylated tail of HER3 still engages the critical PI3Kpathway. Beyond this, isoforms of HER2 that are devoidof the extracellular domain and expressed in many breastcancers have been documented (42), and thus may pro-hibit the use of antibodies that target HER2 or HER2/3heterodimers. Therefore, an RNA antagonist to HER3offers a unique solution to controlling HER3-mediatedtumor growth. We show here that EZN-3920 is a specificinhibitor of HER3, as it downmodulated HER3 mRNA,protein, and consequently p-AKT levels (Figs. 1C and 2C).Furthermore, we showed that concomitant with HER3downmodulation, the basal level of p-AKT as well asneuregulin-1–induced p-AKT could be suppressed byEZN-3920 in FaDu head and neck cancer cells (Supple-mentary Fig. S6). Consistent with the inhibition of p-AKT,p-S6K was also inhibited in neuregulin-1–treated cells.More interestingly, we also noticed the reduction p-MAPK from cells treated with or without neregulin-1(Supplementary Fig. S6), suggesting that inhibition of






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Figure 5. EZN-3920 potentiated the effect of lapatinib, gefitinib, or PI3KCA inhibitor. A, EZN-3920 inhibited lapatinib-inducedHER3mRNA in BT474M1cancercells as determined by qRT-PCR. �, P < 0.05 compared with untreated group. B, EZN-3920 inhibited lapatinib-induced HER3 protein expression in BT474M1

Wu et al.





HER3 has the advantage to block both PI3K/AKT andERK pathways.Recently, it has been shown that the treatment of

lapatinib resulted in an increase in the p-HER3 level via

mRNA induction due to compensatory pathways (15–20).Consistentwith this,HER3mRNA is also inducedbyAKT(20) or PI3K inhibitor through derepression of FoxO3atranscription factor (15–16). This enhanced p-HER3 level

Figure 6. Gefitinib- or trastuzumab-resistant cells remained sensitive toEZN-3920. Cells were treated withdifferent concentrations of EZN-3920 or EZN-SCR controloligonucleotide for 6 days. Cellproliferation was determined by MTTassay. A, effect of EZN-3920 onHCC827 and gefitinib-resistantHCC827 cells. B, effect of EZN-3920on HCC827 and gefitinib-resistantHCC827GR cells. C, effect of EZN-3920 on PC9 and gefitinib-resistantPC9 cells. D, effect of EZN-3920 onBT474M1and trastuzumab-resistantBT474M1 cells. All data are mean �SD.

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cancer cells as determined by Western blot analysis. C, EZN-3920 potentiated the effect of lapatinib on the growth of BT474M1 cancer cells as determinedby MTT assay. Cells were treated with EZN-3920 for 24 hours followed with lapatinib for 48 hours.�, P < 0.05 compared with group of lapatinib plusEZN-SCR.D, combinationof EZN-3920with lapatinib inBT474M1breast cancer xenograftmodel.Micewere administered 30mg/kgEZN-3920 (every 3 andhalfdays, 9 doses, i.v.; n ¼ 8 per group), 100 mg/kg lapatinib (daily, 15 doses, orally), or both compounds. �, P < 0.05 compared with saline group; ��, P < 0.05compared with EZN-3920 group. E, combination with gefitinib in HCC827 NSCLC xenograft model. Mice were administered 30 mg/kg EZN-3920 (every 3and half days, 10 doses, i.v.;n¼ 8 per group), 15mg/kg gefitinib (daily, 10 doses, orally), or both compounds. �,P < 0.05 comparedwith saline group; ��,P < 0.05comparedwith the indicatedgefitinib group.F, combinationof EZN-3920withEZN-4150 inHCC827NSCLCxenograftmodel.Micewere administered30mg/kgEZN-3920 (every 3 and half days, 8 doses, i.v.; n ¼ 8 per group), 30 mg/kg EZN-4150 (every 3 and half days, 8 doses, i.v.), or both compounds. �, P < 0.05compared with saline group; ��, P < 0.05 compared with EZN-4150 or EZN-3920 group. G, structure of gefitinib. H, structure of lapatinib.






underscores the importance of HER3 in cell survival. Itis therefore logical to consider the combination of HER3antagonist such as EZN-3920 with lapatinib. Indeed,when we combine the 2, greater antitumor activity wasobserved than either agent alone. Our data are in agree-ment with the data published by Garrett and colleagues(15), which showed that a neutralizing HER3 antibodyAMG sensitized HER-positive BT474 breast cancercells to lapatinib both in vitro and in vivo. We observedthat the combination was not just unique to lapatinib.When we combined EZN-3920 with gefitinib or a spe-cific PI3KCA antisense molecule EZN-4150 in HCC827non–small cell lung cancer (NSCLC) xenograft model,greater efficacy was achieved than either agent alone. Itis worth noting that the benefit of combining agents totarget multiple members of the family was also recentlyshown by a two-in-one antibody against HER3 andEGFR (43). In our case, the improvement in efficacy isclearly an on-target effect as pretreatment of tumor-bearing mice with EZN-3920 suppressed a 3-fold induc-tion of HER3 mRNA by gefitinib (Supplementary Fig.S5). The combination of EZN-3920 with tyrosine kinaseinhibitors or inhibitors of the PI3K/AKT pathway maythus provide a novel and much more effective approachfor treating various cancers.

An inducible HER3 level plays an important role ingefitinib resistance (4, 17). Logically, inhibition of HER3should overcome the resistance. To test this hypothesis,the activity of EZN-3920 in gefitinib-resistant cells wasexamined in vitro. Gefitinib-resistant cells (HCC827R)were generated by selectingHCC827 lung carcinoma cellsfor resistance to the agent in vitro. HCC827R showedsignificant reduction in p-EGFR and EGFR levels (44),but unlike other HCC827 selected for resistance to gefiti-nib (4), this particular cell line does not amplify MET.EZN-3920 had better potency in HCC827R when com-pared with its parental cells, suggesting that HER3 stillplays a role in the survival of HCC827R.We subsequentlyshowed that cell lines with entirely different resistancemechanisms suchasPC9GR (T790Mmutation; ref. 45) andHCC827GR (MET amplification; ref. 4) were still sensitiveto EZN-3920 treatment. Furthermore, EZN-3920 showedequal if not more potent activity in a trastuzumab-resis-tant cell line, suggesting the ability of EZN-3920 to over-come resistance mediated through different mechanisms.

Collectively, these data suggest that downmodulationof HER3 by the antisense molecule EZN-3920 offers a

unique opportunity to confirmHER3-driven cancers. Thereported in vitro and in vivo activities are encouraging andsupportive of further development of this compound. Atthe moment, several anti-HER3 antibodies includingAMG-888 and MEHD7945A are being evaluated in clin-ical trials with such compounds with EZN-3920. It will beextremely important to develop a biomarker strategy toincrease success rates of clinical trials and maximizethe benefit to patients. Some preclinical data have beengenerated to suggest that the expression of ligand neur-egulin-1 is associated with the activation of HER3 inovarian (13) and head and neck cancer (14). Additionalpreclinical and clinical data aswell as data integrationwillhelp to provide a predictive biomarker for HER3-targetedtherapy.

Disclosure of Potential Conflicts of InterestL.M. Greenberger is employed (other than primary affiliation; e.g.,

consulting) in Bristol-Myers Squibb as Exec Director, Strategic Transac-tions and has ownership interest (including patents) in Enzon. Z. Qu isemployed (other than primary affiliation; e.g., consulting) in Enzon Phar-macueticals, Inc. as a scientist. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: Y. Wu, Y. Zhang, Z. Qu, V. Shi, I.D. Horak, L.M.GreenbergerDevelopment ofmethodology:Y.Wu,Y.Zhang,M.Wang, Z.Qu,V. Shi, S.Kim, Y. GaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Wu, Y. Zhang, M. Wang, Q. Li, Z. Qu, V. Shi,P. Kraft, S. Kim, J. PakAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): Y. Wu, Y. Zhang, M. Wang, Q. Li, Z. Qu, V.Shi, P. Kraft, S. Kim, J. Pak, L.M. GreenbergerWriting, review, and/or revision of themanuscript: Y.Wu, Y. Zhang, I.D.Horak, L.M. GreenbergerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Zhang, V. Shi, J. PakStudy supervision: Y. Wu, Y. Zhang, S. Youngster, I.D. Horak, L.M.Greenberger

AcknowledgmentsThe authors thank Hana Fainman for her critical reading and editing

of the manuscript.

Grant SupportThis work was supported by Enzon Pharmaceuticals.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 21, 2012; revised January 7, 2013; accepted January 28,2013; published OnlineFirst February 8, 2013.

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Published OnlineFirst February 8, 2013.Mol Cancer Ther Yaming Wu, Yixian Zhang, Maoliang Wang, et al. Modelsof EGFR and HER2 Tyrosine Kinase Inhibitors in AnimalOligonucleotide, EZN-3920, Improves the Antitumor Activity Downregulation of HER3 by a Novel Antisense

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Downregulation of HER3 by a Novel Antisense Oligonucleotide, EZN-3920… · 2013. 3. 28. · 2013...

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