trastuzumab ft lapitinib

Preclinical Development

Dacomitinib (PF-00299804), an Irreversible Pan-HERInhibitor, Inhibits Proliferation of HER2-Amplified BreastCancer Cell Lines Resistant to Trastuzumab and Lapatinib

Ondrej Kalous1, Dylan Conklin1, Amrita J. Desai1, Neil A. O'Brien1, Charles Ginther1,Lee Anderson1, David J. Cohen1, Carolyn D. Britten1, Ian Taylor2, James G. Christensen2,Dennis J. Slamon1, and Richard S. Finn1

AbstractThehumanEGF (HER) family of receptors has beenpursued as therapeutic targets in breast cancer andother

malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a

significant number of patients donot respondordevelop resistance to these drugs.Herewe evaluate the in vitro

activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of

human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with

variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast

epithelial lines representing the knownmolecular subgroups of breast cancerwere treatedwith dacomitinib to

determine IC50 values.HER2-amplified lineswere farmore likely to respond to dacomitinib thannonamplified

lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated

with response.Dacomitinib reduced thephosphorylation ofHER2, EGFR,HER4,AKT, andERK in themajority

of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0–G1 arrest and an

induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and

acquired resistance to trastuzumab. Dacomitinibmaintained a high activity in lineswith acquired resistance to

lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative

effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers

resistant to trastuzumab and lapatinib. Mol Cancer Ther; 11(9); 1978–87. �2012 AACR.

IntroductionThe HER (ErbB) receptor family consists of 4 type I

receptor tyrosine kinases, which include the EGF recep-tor (EGFR, HER1, and ErbB-1), HER2 (HER2/neu,ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4), and theirassociated ligands, including EGF, neuregulins (NRG1-4), TGF-a, amphiregulin, betacellulin, heparin-bindingEGF-like growth factor and epiregulin. Ligand bindingto the extracellular domain leads to homodimerizationor heterodimerization of the receptors and autopho-sphorylation within the intracellular domain. Thisresults in the activation of signaling cascades involvedin mediating cell growth and differentiation. Unlike

other family members, HER2 has no known ligand and,under normal conditions, relies on forming heterodi-mers with other family members that have ligand inter-actions. In cancer, this tight regulation of HER familysignaling is disrupted and contributes to transformation(1).

HER2 amplification occurs in 20% to 25% of patientswith breast cancer. It is associated with a poor prognosisand is a validated target for therapy (2, 3). Trastuzumab(Herceptin; Genentech), a humanized monoclonal anti-body that binds to the extracellular domain of HER2,has been shown to improve survival in the metastaticand adjuvant settings (4–11), and lapatinib (Tykerb;GlaxoSmithKline), a small-molecule selective inhibitor ofthe HER2 and EGFR tyrosine kinases, is approved forHER2-positive advanced breast cancer that progressedafter trastuzumab-based therapy (12).

Pan-HER inhibitors were developed with the goal ofimproving therapeutic response and overcoming drugresistance that is seen with trastuzumab and lapatinib.Unlike lapatinib, which is a reversible inhibitor that com-petes with ATP at the binding sites (13), most pan-HERinhibitors bind to the kinases in a covalent and irreversibleform (14, 15). It is hypothesized that the prolonged sup-pression of multiple targets within the HER receptor

Authors' Affiliations: 1Department of Medicine, Division of Hematology/Oncology, Geffen School of Medicine at UCLA, Los Angeles; and 2PfizerGlobal Research and Development, Pfizer Inc., San Diego, California

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

CorrespondingAuthor:Richard S. Finn, Department ofMedicine, Divisionof Hematology/Oncology, Geffen School of Medicine at UCLA, 10833 LeConte Ave, 11-934 Factor Bldg, Los Angeles, CA 90095. Phone: 310-586-2091; Fax: 310-586-6830; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0730

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(9) September 20121978

on November 29, 2015. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 3, 2012; DOI: 10.1158/1535-7163.MCT-11-0730

http://mct.aacrjournals.org/

familymay overcome the problemswith adaptability andredundancy in signaling pathways.Dacomitinib (PF-00299804; chemical structure in Fig. 1)

is a second-generation irreversible pan-HER receptortyrosine kinase inhibitor (selectively inhibiting EGFR,HER2, and HER4) under clinical development (16, 17).Dacomitinib showedmarked activity in several xenograftmodels with variable levels of HER family receptors,including lung tumors resistant to EGFR small moleculetyrosine kinase inhibitors (16, 18). The primary aim of thisstudywas to describe the in vitro activity of dacomitinib ina panel of 47 human breast cancer and immortalizedbreast cell lines, to identify potential biomarkers ofresponse and/or resistance to guide clinical development.

Materials and MethodsCell lines, cell culture, and reagentsThe cell line panel included 44 breast cancer lines and 3

immortalized breast epithelial cell lines representing theknown molecular subgroups of breast cancer and hasbeendescribed in detail previously (19). The panel includ-ed MDA-MB-415, MDA-MB-134, HCC-1500, ZR-75-30,HCC-202, HCC-1419, HCC-38, HCC-70, HCC-1187,HCC-1806, HCC-1937, HCC-1954, MDA-MB-436, HCC-1569, Hs578t, HCC-1143, MDA-MB-175, BT-474, SK-BR-3,MDA-MB-361, UACC-893, UACC-812, UACC-732, T-47D, MDA-MB-453, MDA-MB-468, CAMA-1, MDA-MB-157, MCF-7, MDA-MB-435, ZR-75-1, BT-20, MDA-MB-231, BT-549, DU4475, HCC-1395, HCC-2218, 184A1,184B5, andMCF-10A thatwere purchased fromAmericanType Culture Collection. The cell lines EFM-192A, KPL-1,EFM-19, COLO-824, and CAL-51 were obtained from theGerman Tissue Repository DSMZ. Both cell line bankscarry out the cell lines authentication by short tandemrepeat analysis. The cell lines SUM-190 andSUM-225wereobtained from the University of Michigan (Ann Arbor,MI). Upon receipt, all cell lines were assessed for Myco-plasma contamination using amultiplex PCRmethod (20),and mitochondrial DNA from the cells was sequenced toconfirm their correct identity (21). Cell lines were thenexpanded and these procedures were repeated for all celllines before cryopreservation. All cell lineswere passagedfor less than 6 months before use in this study. Trastuzu-mab-resistant BT-474 (BT-474-TR) and SKBR3 (SK-BR-3-TR) cell lines (pools of resistant cells) were establishedafter serial passage in the continued presence of trastu-

zumab 105 mg/mL. Lapatinib-resistant BT-474 (BT-474-LR) and SK-BR-3 (SK-BR-3-LR) cell lines (pools of resis-tant cells) were established after serial passage in thepresence of gradually increasing concentrations of lapa-tinib (0.1–7 mmol/L). These lines were established in ourlaboratories, authenticated, and checked for Mycoplasmacontamination as described above.

HER2 amplification was defined as greater than 2HER2/neu FISH signals per chromosome 17 centromereFISH signal (22). A SpectrumOrange labeled HER2/neuprobe (ABBOTT Molecular) and SpectrumGreen labeledchromosome 17 alpha-satellite centromere probe wereused (ABBOTT Molecular).

Proliferation assaysCells were seeded in duplicate at 5� 103 to 5� 104 cells

per well in 24-well plates, and growth inhibition data wascalculated as described previously (19). Briefly, day afterplating, dacomitinib was added at 10 mmol/L and 2-folddilutions over 12 concentrations were carried out to gen-erate a dose–response curve. Control wells without thedrug were also seeded. The cells were counted on day 1when the drugwas added, aswell as after 6 dayswhen theexperiment ended. After the trypsinization cells wereplaced in an Isotone solution and immediately countedusing a Coulter Z1 particle counter (Beckman Coulter,Inc.). The suspension cultureswere counted using a Coul-ter Vi-Cell counter (Beckman Coulter, Inc.).

Microarray analysis of cell linesAgilent microarray analyses were developed for each

cell line as described previously (23, 24). These data areavailable with GEO accession number GSE18496.

Western blots and protein quantificationTodetermine the effect of dacomitinib on the expression

of analyzed proteins, cells in log-phase growth weretreated with 0.1 or 1 mmol/L dacomitinib and lysateswere taken as described (19). Details about antibodiesand immunoprecipitation techniques can be found inSupplementary Material.

Cell-cycle analysis and apoptosis studiesThe effects of dacomitinib on the cell cycle were

assessed using Nim-DAPI (40, 6-diamidino-2-phenylin-dole) staining (NPE Systems). The cells were plated

Figure 1. Chemical structures ofinvestigated molecules in this article. . H2O

Dacomitinib Lapatinib

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Dacomitinib (PF-00299804) and Breast Cancer

www.aacrjournals.org Mol Cancer Ther; 11(9) September 2012 1979




evenly in control and experimental wells, allowed togrow to log phase, and then treated with 100 nmol/L or1 mmol/Ldacomitinib for 48 hours or 5 days and analyzedas described previously (19).

For apoptosis, cells were plated evenly in control andexperimental wells, allowed to grow to log phase, andthen treatedwith 100 nmol/Lor 1mmol/Ldacomitinib for48 hours or 5 days and analyzed using Annexin-V–fluo-rescein isothiocyanate (FITC) as described (19).

Statistical analysisCochran–Mantel–Haenszel (CMH) c2 analysis was car-

ried out using the PROC FREQ function in SAS forWindows version 9.2 (SAS Institute, Inc.). Simple linearregression was also carried out in SAS using PROC REGfunction.

Pearson correlation coefficients and their correspond-ing P values were calculated using the PROC CORRfunction in SAS. Graphs were created in Microsoft Excel.

ResultsDacomitinib preferentially inhibits growth of HER-2–amplified breast cancer cell lines in vitro

Apanel of 44humanbreast cancer cell lines, representingluminal, nonluminal subtypes, and 3 immortalized breastepithelial lines (19), was used to test the antiproliferativeeffect of dacomitinib (PF-00299804). The calculated IC50

values for each cell line and its molecular classification, aswell as HER2 amplification status (by FISH) and estrogenreceptor (ER) status, were determined (Table 1 and Fig. 2).Sensitivitywasdefinedas IC50< 1mmol/L (the rationale forselecting this cut-off is discussed below).

As a group, the HER2-amplified cell lines were mostsensitive to growth inhibition by dacomitinib (IC50 < 1mmol/L in 14 of 16 lines; 87.5%) as compared with 5 of 28(17.9%) of HER2-nonamplified lines (excluding immor-talized lines).MDA-MB-453 andUACC-732were the onlyHER2-amplified lines resistant to the antiproliferativeeffect of the compound (IC50 > 1 mmol/L). The nonluminallines were the most resistant to this compound. c2 (CMH)analysis was carried out to compare the response classi-fication (response defined as IC50 < 1 mmol/L) in theHER2-amplified versus nonamplified groups of cell lines.HER2-amplified lines were far more likely to be classifiedas responders to dacomitinib [RR, 3.39; 95% confidenceinterval (CI), 1.82–6.33; P < 0.0001].

ER status correlated with response when treated as aseparate, independent variable (P ¼ 0.015). However, ERstatuswashighly cross-correlatedwithHER2 status.Aftercontrolling for HER2 status by stratified analysis, ERstatus was no longer a statistically significant predictorof response (P ¼ 0.17).

Quantitative HER2 expression associates withresponse to dacomitinib

We also aimed to explore the quantitative effect ofHER2 expression on response to dacomitinib, in compar-

ison with the qualitative amplified/nonamplified app-roach discussed above. Specifically, we wanted to deter-mine whether a higher relative expression of HER2mRNA is associated with a stronger response to dacomi-tinib, even within the HER2-amplified and nonamplifiedgroups. For this purpose, we ran a linear regressionanalysis using log(IC50) as the response variable andlog-ratio of expression by microarray (compared withmixed breast cancer RNA reference pool) as the predictorvariable. This analysis was carried out first on the entirepanel of lines, then separately on each group of HER2-amplified and nonamplified lines. The association wassignificant in all 3 analyses. (Full panel: b ¼ �1.07, SE ¼0.15, P < 0.0001, r¼�0.73; HER2 amplified: b¼�1.6, SE¼0.57, P ¼ 0.014, r ¼ �0.6; HER2-nonamplified: b ¼ �1.57,SE ¼ 0.6, P ¼ 0.014, r ¼ �0.44; Supplementary Fig. S1).However, the significance of this relationship in celllines with higher IC50 values (resistant) is unknown. Ina separate analysis for HER2-amplified lines, we foundthat the total HER2 protein levels (quantified by Westernblot) correlated with HER2 mRNA levels by microarray(r ¼ 0.67, P ¼ 0.004). Finally, we found an associationbetween the HER2 protein levels and response to daco-mitinib inHER2-amplified lines (b¼�1.99, SE¼ 0.55, P¼0.003, r ¼ �0.69; Supplementary Fig. S2).

All 4 HER family receptors were analyzed for theirrelationship between mRNA expression and IC50. Theassociation between HER2 expression and response todacomitinib was by far the strongest. In addition, therewas amarginal inverse correlation betweenHER3mRNAlevels and log(IC50) values (r ¼ �0.25, P ¼ 0.09). Nocorrelation was observed between the response to daco-mitinib and EGFRmRNA (r¼�0.07, P¼ 0.62) and HER4mRNA (r ¼ 0.05, P ¼ 0.75) levels, respectively (Supple-mentary Fig. S3).

Biochemical effects of dacomitinib on signaltransduction

The effects of dacomitinib onHER2, EGFR,HER4,AKT,and ERK activation were determined using Western blotanalysis of a subset of lines with variable levels of HERreceptors and sensitivities to dacomitinib.

After a 10-minute exposure to 100 nmol/L or 1 mmol/Lof dacomitinib,weobservedno effect on totalHER2,AKT,andERK inamajority of lines andaminordecrease in totalEGFR in several lines.Most of the selected lines expressedvery low baseline levels of total HER4 that mostly did notchange after the treatment with dacomitinib. Dacomitinibcaused an inhibition of HER2 phosphorylation in all lineswith detectable baseline phosphorylation (Fig. 3, densi-tometry in Supplementary Table S1). Dacomitinib causedan inhibition of EGFR phosphorylation in sensitive lines,but only 2 resistant lines (MDA-MB-453 and MDA-MB-231). Dacomitinib decreased the phosphorylation ofHER4 in sensitive lines with detectable baseline phos-phorylation. Dacomitinib inhibited phosphorylation ofAKT in all tested sensitive lines, but only 2 resistant lines(HCC-70 and MDA-MB-453). Similarly, dacomitinib

Kalous et al.

Mol Cancer Ther; 11(9) September 2012 Molecular Cancer Therapeutics1980




Table 1. The calculated IC50 for each cell line and its molecular classification

Cell line IC50 value (mmol/L) IC50 SE Breast cancer subtype HER2 status ER status

HCC-202 <0.005 n/a Luminal Amplified PositiveZR-75-30 <0.005 n/a Luminal Amplified PositiveMDA-MB-175 <0.005 n/a Luminal Normal PositiveHCC-2218 <0.005 n/a Luminal Amplified PositiveSUM-225 0.006 0.002 Luminal Amplified NegativeHCC-1419 0.006 0.002 Luminal Amplified Positive184A1 0.007 0.0003 Immortalized n/a NegativeMCF-10A 0.008 0.001 Immortalized n/a NegativeSK-BR-3 0.015 0.003 Luminal Amplified Negative

EFM-192A 0.016 0.002 Luminal Amplified PositiveUACC-893 0.017 0.006 Luminal Amplified PositiveBT-474 0.018 0.011 Luminal Amplified PositiveMDA-MB-361 0.03 0.01 Luminal Amplified PositiveUACC-812 0.04 0.00 Luminal Amplified PositiveHCC-1954 0.06 0.01 Basal Amplified Negative184B5 0.08 0.00 Immortalized n/a NegativeSUM-190 0.11 0.01 Luminal Amplified PositiveZR-75-1 0.24 0.12 Luminal Normal PositiveHCC-1500 0.53 0.30 Luminal Normal PositiveMDA-MB-415 0.67 0.07 Luminal Normal Positive

HCC-1143 0.73 0.05 Basal Normal NegativeHCC-1569 0.77 0.17 Post-EMT Amplified NegativeEFM-19 1.07 0.16 Luminal Normal PositiveCOLO-824 1.20 0.00 Basal Normal NegativeMDA-MB-157 1.36 0.08 Post-EMT Normal NegativeT-47D 1.41 0.69 Luminal Normal PositiveMDA-MB-468 1.42 0.03 Basal Normal NegativeHCC-70 1.44 0.06 Basal Normal NegativeHCC-1187 1.46 0.16 Basal Normal NegativeUACC-732 1.69 0.09 Luminal Amplified PositiveMDA-MB-134 1.74 0.17 Luminal Normal Positive

HCC-1395 1.77 0.64 Post-EMT Normal NegativeMDA-MB-453 2.00 0.11 Luminal Amplified NegativeHCC-1937 2.04 0.02 Post-EMT Normal NegativeHCC-1806 2.05 0.18 Basal Normal NegativeBT-549 2.22 0.03 Post-EMT Normal NegativeHCC-38 2.40 0.15 Basal Normal NegativeMDA-MB-436 2.56 0.00 Post-EMT Normal NegativeBT-20 2.58 0.29 Basal Normal NegativeCAMA-1 2.68 0.46 Luminal Normal PositiveCAL-51 2.76 0.46 Post-EMT Normal NegativeMDA-MB-435 2.92 0.00 Post-EMT Normal Negative

Hs578T 2.92 0.28 Post-EMT Normal NegativeDU-4475 3.55 0.99 Basal Normal NegativeMCF-7 3.67 0.35 Luminal Normal PositiveMDA-MB-231 4.23 0.11 Post-EMT Normal NegativeKPL-1 4.88 0.57 Luminal Normal Positive

NOTE: Included aremolecular subtype andHER2 and ER status. HER2-amplified cell lines weremost sensitive to the growth inhibitioneffectsof dacomitinib. Post-EMT, cell linesclassifiedas representingbreast cancers that hadundergonean epithelial-to-mesenchymaltransition.Abbreviation: n/a, not applicable.






caused a decrease in phosphorylation of ERK in all lines,except for resistant MDA-MB-231.

Effects of dacomitinib on cell cycle and apoptosisThe effects of dacomitinib on the cell cycle were

analyzed in a subset of both sensitive and resistant celllines. Cells were exposed to dacomitinib at 1 mmol/L for48 hours, and then flow cytometry was carried out usingNim-DAPI staining. We observed a significant G0–G1

cell-cycle arrest in the very sensitive HER2-amplifiedBT-474 and SK-BR-3 lines and less pronounced yetstatistically significant G0–G1 cell-cycle arrest in theresistant HER2-normal MCF-7 line. Dacomitinib causedno changes in cell cycle in the less sensitive ZR-75-1 andHCC-1143 lines and in the resistant MDA-MB-231 line(Fig. 4).

Similarly, the effect of dacomitinib on apoptosis wasdetermined in the same subset of lines. For this assay, cellswere exposed to 1 mmol/L of dacomitinib for 5 days andthen analyzed with a dual stain flow cytometry protocolusing Annexin-V–FITC and propidium iodide. A signif-icant increase in apoptosis was seen in the very sensitiveBT-474 and SK-BR-3 lines and in the less sensitive HCC-1143. The changes in ZR-75-1 (P ¼ 0.08) did not reach astatistical significance. No changes were observed inresistant lines (Fig. 5).

In a separate experiment, we analyzed the differ-ences in the cell cycle and apoptosis between the 48-hour and 5-day treatment with dacomitinib in thesensitive BT-474 and SK-BR-3 lines. There was nodifference in the cell cycle between these 2 time points.We observed no induction in apoptosis for SK-BR-3 at48 hours, which indicates that dacomitinib needsmore than 48 hours to induce apoptosis in this line. Inthe BT-474 line, we observed an induction of apoptosisat 48 hours, albeit at lesser degree than at 5 days(Supplementary Fig. S4).

Together, thesedata suggested that the antiproliferativeeffect of dacomitinib is mediated by both inhibition of thecell cycle and the induction of apoptosis.

Dacomitinib overcomes acquired resistance totrastuzumab and lapatinib in vitro

BT-474-trastuzumab- (BT-474-TR) and SK-BR-3-trastu-zumab–resistant (SK-BR-3-TR) cell lines were establishedin our laboratory after prolonged exposure of the parentalcell lines in medium with 105 mg/mL trastuzumab overseveral months (25). Despite resistance to trastuzumab,dacomitinib inhibited the proliferation of both these celllines with a similar potency as their parental cell lines(Table 2). Although the exact mechanism of trastuzumabresistance in these lines is unknown, they clearlymaintaindependence on HER signaling.

BT-474-lapatinib- (BT-474-LR) and SK-BR-3-lapatinib–resistant (SK-BR-3-LR) cell lines were developed in ourlaboratory after prolonged exposure of the parental celllines to progressively higher concentrations of lapatinibover several months. Unlike the trastuzumab-resistantcell lines, the sensitivity of these lines to dacomitinib wasless than their parental counterparts. For the BT-474-LR,there was a 1.7-fold increase in IC50, and for the SK-BR-3-LR, there was a 23-fold increase in IC50 (Table 2). How-ever, the decrease in sensitivity to dacomitinib in thelapatinib-resistant lines compared with their parentallines was much less pronounced than their decrease insensitivity to lapatinib. Specifically, there is an 89-fold and119-fold increase in lapatinib IC50 for BT-474-LR and SK-BR-3-LR comparedwith respective parental cell lines, andboth lapatinib-resistant cell lineswould still be consideredsensitive to dacomitinib with low IC50 values of 0.031 and0.339 mmol/L, respectively.

These data indicated that, in the HER2-amplified celllines, dacomitinib overcomes the acquired resistance to

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Figure 2. Inhibitory concentration and cell type. Bar graph of IC50 values (mmol/L) and cell type. Cell lines are color coded by subtype: dark blue bars (stripes),HER2 amplified; light blue, luminal; yellow, nonluminal/undergone an epithelial-to-mesenchymal transition; red, nonluminal; turquoise, immortalized. �, celllines with IC50 < 0.005 mmol/L.

Kalous et al.





trastuzumab and that it maintains a considerable anti-proliferative activity in the cell lines with acquired resis-tance to lapatinib.

Effects of dacomitinib on cell cycle, apoptosis, andsignal transduction in cell lines conditioned for theacquired resistance to trastuzumab and lapatinibWe analyzed the effect of dacomitinib on cell cycle and

apoptosis in the cell lines conditioned for acquired resis-tance to trastuzumab (BT-474-TR, SK-BR-3-TR) and lapa-tinib (BT-474-LR, SK-BR-3-LR). Cells were exposed to100 nmol/L or 1 mmol/L of dacomitinib for 48 hours (cellcycle) and 5 days (apoptosis).In the trastuzumab-resistant lines, dacomitinib causeda

G0–G1 arrest comparable with the parental lines. In lapa-tinib-resistant lines, the G0–G1 arrest caused by 1 mmol/Lof dacomitinib was similar to the parental lines. The effectwas less pronounced at 100 nmol/L of dacomitinib (Sup-plementary Fig. S5).

The effect of dacomitinib on apoptosis in trastuzumab-resistant lines was stronger than in the parental lines. Inlapatinib-resistant lines, dacomitinib at 100 nmol/L didnot induce apoptosis, and the effect at 1 mmol/L was lesspronounced than in the parental lines (SupplementaryFig. S6).

In addition, the effect of dacomitinib on the phosphor-ylation of HER2, EGFR, HER4, AKT, and ERK in theacquired resistant lines was analyzed by Western blots(Supplementary Fig. S7, densitometry in SupplementaryTable S2). Dacomitinib decreased the phosphorylation ofHER2, EGFR, AKT, and ERK in trastuzumab- and lapa-tinib-resistant lines. The expression of baseline total andphosphorylated HER4 was very low, making assessmentof dacomitinib effects difficult.

DiscussionUsing a broad panel of 44 human breast cancer cell lines

and 3 immortalized breast epithelial lines,we have shown

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Figure 3. The effects of dacomitinib on total and phosphorylated HER2, EGFR, HER4, AKT, and ERK. The effects of dacomitinib on total and phosphorylatedHER2, EGFR, HER4, AKT, and ERK were measured by Western blot as described in Materials and Methods. All cell lines were treated with 100 nmol/Lor 1 mmol/L dacomitinib for 10minutes. Cell lines are arranged frommost sensitive (low IC50; left) to least sensitive (high IC50; right). Dacomitinib had no effecton total HER2,AKT, andERK in amajority of lines andcaused aminor decrease in total EGFR in several lines.Most of the selected lines expressed low levels oftotal HER4, which remained mostly unchanged posttreatment. Dacomitinib inhibited HER2 phosphorylation in all lines with detectable baselinephosphorylation. Dacomitinib inhibited EGFR phosphorylation in sensitive lines, but only 2 resistant lines (MDA-MB-453 and MDA-MB-231). DacomitinibdecreasedHER4 phosphorylation in sensitive lineswith detectable baseline levels. Dacomitinib inhibited phosphorylation of AKT in all sensitive lines, but only2 resistant lines (HCC-70 and MDA-MB-453). Dacomitinib caused a decrease in phosphorylation of ERK in all lines, except for resistant MDA-MB-231.Densitometry data are available in Supplementary Table S1.






that HER2 amplification status was a strong predictor ofresponse to dacomitinib,with IC50 values below 1mmol/Lin the vast majority of HER2-amplified lines. MDA-MB-453 and UACC-732 were the only HER2-amplified linesthat were resistant to dacomitinib. Of note, these 2 lineshave some of the lowest levels of HER2 DNA copynumber by comparative genomic hybridization (data notshown) and HER2 mRNA among the HER2-amplifiedlines. In addition, these 2 lines express low baseline levelsof total and phosphorylated HER2 (25) and total andphosphorylated EGFR (data not shown). These 2 cell lineswere also previously shown to be resistant to trastuzumab(25) and lapatinib (25, 26). These findings may havepotential clinical implications for selecting the patientsfor clinical trials with dacomitinib.

Dacomitinib showedanantiproliferative activity super-ior to trastuzumab in vitro. The HER2-amplified linesSUM-225, HCC-1419, HCC-1954, UACC-893, and HCC-1569 were resistant to trastuzumab (25) but sensitive todacomitinib (IC50 < 1 mmol/L). These data suggest thatdacomitinib can potentially overcome de novo trastuzu-mab resistance. Lapatinib and dacomitinib generatedcomparable IC50 values in HER2-amplified lines; onlyHCC-1569 was resistant to lapatinib (IC50 > 1 mmol/L)but sensitive to dacomitinib (25). Similarly to trastuzumaband lapatinib, the nonluminal, HER2-nonamplified celllines were the most resistant to dacomitinib.

We have confirmed that dacomitinib acts by blockingthe phosphorylation of HER2, EGFR, and HER4 in breastcancer cell lines. The inhibition of HER2 phosphorylationwas dosedependent and specific toHER2-amplified lines.

The blockage of EGFR phosphorylation was more pro-nounced in HER2-amplified lines but was also present insome resistant lines. The inhibition of HER4 phosphory-lation was observed in several sensitive HER2-amplifiedlines. These effects lead to the inhibition of the PI3K/AKTsignaling pathway in sensitive lines and 2 resistant lines,as evidenced by a reduction of phosphorylated AKT. Wehave also observed a loss of phosphorylated ERK in mostlines, suggesting that dacomitinib also acts by inhibitingthe RAS/MAPKpathway. These effects of dacomitinib onHER receptors and downstream signaling pathways ulti-mately resulted in cell-cycle inhibition and apoptosis.

In this study, 1 mmol/L was chosen based on the dis-tribution of IC50 values in our dataset, which has biologicsignificance (i.e., there seemed to be a natural increase inthe IC50 distribution around the 1 mmol/L mark; cell linesbelow this cutoff point were mostly HER2 amplified;dacomitinib induced changes in cell cycle and apoptosisin cell lines with IC50 values below 1 mmol/L but notabove). In addition, phase I data was recently reported(17). Ingeneral, themaximumplasma levelsofdacomitinibin the phase I study were between 200 to 300 nmol/L andarewithin the range of the 1,000 nmol/L cut-off used here.

Trastuzumab and lapatinib have an important role inthe current clinical therapy of HER2-amplified breastcancer. Unfortunately, less than half of patients respondto monotherapy, and though the response rates increasewhen combined with chemotherapy, the responses areoften only temporary in a significant number of patients(4–11, 27–30). For these reasons, it is important to keepsearching for new therapies for this subset of patients.

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Figure 4. Effects of dacomitinibon cell cycle. A, sensitive celllines BT-474 and SK-BR-3(IC50 ¼ 0.02 mmol/L) show asignificant G0–G1 arrest and adecrease in the S-phase and G2

-phase fractions as compared withthe less sensitive ZR-75-1 andHCC-1143 (IC50 ¼ 0.24 and 0.73,respectively). B, resistant cell linesMCF-7 (the effect is lesspronounced yet statisticallysignificant) andMDA-MB-231 afterincubation with 1 mmol/Ldacomitinib for 2 days. Solid bars,control samples; striped bars,treated samples. Error barsrepresent SE for 2 separateexperiments. �, P < 0.05 comparedwith control.

Kalous et al.





Our findings suggest different, largely nonoverlappingmechanisms of resistance to dacomitinib, trastuzumab,and lapatinib. We found that 2 HER2-amplified cell linesthat we had conditioned for acquired resistance to tras-tuzumab (BT-474-TRandSK-BR-3-TR)werevery sensitiveto the antiproliferative effect of dacomitinib. This sensi-tivity to dacomitinib was comparable with the sensitivityobserved in their parental cell lines. To examine the effectof dacomitinib in the models of acquired resistance tolapatinib, we have generated 2 lines with acquired lapa-tinib resistance (BT-474-LR and SK-BR-3-LR). We foundthese 2 lines to be sensitive to dacomitinib, though theirsensitivity was less pronounced than in the parental lines.Several mechanisms of resistance to trastuzumab have

been proposed; however, their significance is not yet

clearly defined. Several studies have implicated anincreased activation of EGFR, HER3, and other receptortyrosine kinases (e.g., IGF-1R) and their ligands in resis-tance to trastuzumab (25, 31–35). Alterations in the PI3K/AKTpathway throughPIK3CAmutation (36, 37) and/or aloss of expression of the PTEN tumor suppressor (38, 39)have also been associated with resistance to trastuzumab.We have shown previously that, contrary to dacomitinib,the response to trastuzumab was not significantly asso-ciated with total HER2 protein levels (vs. amplification;ref. 25). A recent publication exploring mechanisms oftrastuzumab resistance (40) also supports the use of a pan-HER inhibitor in overcoming resistance to trastuzumab.

The mechanisms of lapatinib resistance are not wellunderstood either. Among the proposed mechanisms of

Figure 5. Effects of dacomitinib onapoptosis. A, sensitive cell lines BT-474 and SK-BR-3 show significantincrease in the percentage ofAnnexin-V–positive cells. Lesssensitive cell lines ZR-75-1 andHCC-1143 show less pronouncedincrease in Annexin-V–positive cells(statistically significant in HCC-1143). B, resistant cell lines MCF-7andMDA-MB-231shownochanges.Cells were incubated with 1 mmol/Ldacomitinib for 5 days. Solid bars,control samples; striped bars,treated samples. Error bars representSE for 2 separate experiments.�, P < 0.05 compared with control.

BT-474 (IC50 = 0.02 µmol/L) SK-BR-3 (IC 50= 0.02 µmol/L)

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lapatinib resistance areHER2mutations (41), activation ofprosurvival pathways through ER signaling (42), andoverexpression of membrane-bound receptor tyrosinekinase AXL (43). There are several possible explanationsfor the effect of dacomitinib in overcoming the acquiredresistance to lapatinib. Unlike lapatinib, dacomitinibbinds to the active site of kinases in a covalent andirreversible form, permanently blocking the kinase activ-ity. In addition to blocking HER2 and EGFR, dacomitinibalso inhibits HER4 kinase activity; however, the clinicalsignificance ofHER4 inhibition remains to be determined.We confirmed that dacomitinib blocks the phosphoryla-tion of HER4 in several sensitive lines. However, we didnot find a correlation between theHER4mRNA levels andthe response to dacomitinib.

In summary, this study shows that dacomitinib has astrong antiproliferative activity in HER2-amplified breastcancer cell lines and maintains this activity in HER2-amplified cell lines with de novo and acquired resistanceto trastuzumab and acquired resistance to lapatinib.Given the importance of finding new therapies fordrug-resistant breast cancer, these findings are a strongrationale for clinical development of this compound.

Disclosure of Potential Conflicts of InterestC.D. Britten received a commercial research grant from Pfizer Inc.

(major) and was paid for travel to a scientific meeting (minor, Pfizer). I.Taylor is an employee of Pfizer Inc. as Senior Director, has receivedcompensation (major) and has ownership interest in Pfizer Inc. (major).D.J. Slamon has received honoraria from Speakers Bureau of Genentech(minor), Sanofi-Aventis (minor), and GlaxoSmithKline (minor), has own-ership interest in Amgen (major), and is a consultant and an advisoryboardmember ofNovartis Pharmaceuticals (minor). Nopotential conflictsof interest were disclosed by the other authors.

AcknowledgmentsThe authors thank Veerauo Konkankit and Teodora Kolarova for their

excellent technical assistance and Dr. Habib Hamidi for reviewing themanuscript.

Grant SupportD.J. Slamon received Department of Defense Innovator Award

W81XWH-05-1-0395. The work is also funded by a gift to D.J. Slamon byThe Wittich Family Project for Emerging Therapies in Breast Cancer atUCLA’s Jonsson Comprehensive Cancer Center.

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

Received September 26, 2011; revised May 11, 2012; accepted June 1,2012; published OnlineFirst July 3, 2012.

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Table 2. Comparison of dacomitinib and lapatinib responses (IC50) in parental and trastuzumab- andlapatinib-resistant cell lines

Cell lineDacomitinibIC50 (mmol/L)

LapatinibIC50 (mmol/L)

DacomitinibIC50 fold changecompared withparental line

LapatinibIC50 fold changecompared withparental line

BT-474 0.018 � 0.011 0.016 � 0.011 – –

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SK-BR-3-TR 0.005a 0.039 � 0.010 0.33a 0.72SK-BR-3-LR 0.339 � 0.137 6.400 � 1.119 22.60 118.52

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Kalous et al.





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2012;11:1978-1987. Published OnlineFirst July 3, 2012.Mol Cancer Ther Ondrej Kalous, Dylan Conklin, Amrita J. Desai, et al. Resistant to Trastuzumab and LapatinibInhibits Proliferation of HER2-Amplified Breast Cancer Cell Lines Dacomitinib (PF-00299804), an Irreversible Pan-HER Inhibitor,

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