Combining ABCG2 Inhibitors with IMMU-132, an Anti Trop-2 … · an Anti–Trop-2 Antibody Conjugate...

Large Molecule Therapeutics

Combining ABCG2 Inhibitors with IMMU-132,an Anti–Trop-2 Antibody Conjugate of SN-38,Overcomes Resistance to SN-38 in Breastand Gastric CancersChien-Hsing Chang, Yang Wang, Maria Zalath, Donglin Liu, Thomas M. Cardillo,and David M. Goldenberg

Abstract

Sacituzumab govitecan (IMMU-132), an SN-38–conjugatedantibody–drug conjugate, is showing promising therapeuticresults in a phase I/II trial of patients with advanced Trop-2–expressing, metastatic, solid cancers. As members of the ATP-binding cassette (ABC) transporters confer chemotherapy resis-tance by active drug efflux, which is a frequent cause oftreatment failure, we explored the use of known inhibitors ofABC transporters for improving the therapeutic efficacy ofIMMU-132 by overcoming SN-38 resistance. Two humantumor cell lines made resistant to SN-38, MDA-MB-231-S120(human breast cancer) and NCI-N87-S120 (human gastriccancer), were established by continuous exposure of the paren-tal cells to stepwise increased concentrations of SN-38 andanalyzed by flow cytometry for functional activities of ABCG2and ABCB1, immunoblotting and qRT-PCR for the expression

of ABCG2 at both protein and mRNA levels, and MTS assaysfor the potency of SN-38 alone or in combination with amodulator of ABC transporters. MDA-MB-231-S120 andNCI-N87-S120 displayed reduced sensitivity to SN-38 in vitro,with IC50 values approximately 50-fold higher than parentalMDA-MB-231 and NCI-N87 cells. The increase in drug resis-tance of both S120 cell populations is associated with theexpression of functional ABCG2, but not ABCB1. Importantly,treatment of both S120 sublines with known ABCG2 inhibitors(fumitremorgin C, Ko143, and YHO-13351) restored toxicity ofSN-38, and the combination of YHO-13351 with IMMU-132increased the median survival of mice bearing NCI-N87-S120xenografts. These results provide a rationale for combinationtherapy of IMMU-132 and inhibitors of ABC transporters, suchas YHO-13351. Mol Cancer Ther; 15(8); 1910–9. �2016 AACR.

IntroductionMultidrug resistance (MDR) is a common cause of treatment

failure in cancer therapy (1–3). Thus, despite the notable successin treating diverse cancers, chemotherapeutics, including anti-body–drug conjugates (ADC), lose clinical activity over time, asexemplified by irinotecan (4), doxorubicin (5), paclitaxel (6),cisplatin (7), gemtuzumab ozogamicin (8), inotuzumab ozoga-micin (9), and others (1).

In general, the occurrence of drug resistance in cancer cellscan be intrinsic or acquired, with each type resulting from avariety of factors (10), such as decreased uptake of solubledrugs, activation of drug-detoxifying systems, modulation ormutation of drug targets, defective apoptosis pathways, andabove all, overexpression of one or more efflux pumps of theATP-binding cassette (ABC) superfamily (11). The human

genome comprises a total of 49 genes in the ABC superfamily(12), each assigned to one of seven subfamilies (A through G)based on the order and sequence homology of the transmem-brane (TM) domain and the nucleotide-binding folds (NBF).To date, ABCB1 (also known as MDR1 or P-gp), ABCC1 (alsoknown as MRP1), and ABCG2 (also known as BCRP, MXR, orABC-P) account for most studies on MDR (13–15).

Members of the ABC superfamily are transmembrane proteins,expressed either as a full- or half-transporter. A full-transportertypically contains two transmembrane (TM) domains and twonucleotide-binding folds (NBFs), with the TM domains partici-pating in substrate recognition and translocation across themem-brane, while the cytosolic NBFs provide the driving force fortransport via hydrolysis of the bound ATP. By contrast, a half-transporter has only one TMdomain andoneNBF, andmust formeither homodimers or heterodimers to be functional. A notableexample of a full-transporter is ABCB1, whose substrates includevinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes,irinotecan, and SN-38 (1). The five members of the ABCG sub-family are all half-transporters (12), of which ABCG2 hasbeen identified for its role in mediating cellular resistance toSN-38 (16, 17), as well as to tyrosine kinase inhibitors (18).

With the molecular mechanisms of intrinsic and acquired drugresistance in cancer increasingly being delineated, multipleapproaches to circumvent MDR have emerged. For example, thesensitivity of inotuzumab ozogamicin in ABCB1-expressing sub-lines of Daudi and Raji lymphomas could be restored effectively

Immunomedics, Inc. Morris Plains, New Jersey.

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

Dedicated to Ralph A. Reisfeld, PhD, on the celebration of his 90th birthday.

Corresponding Author: David M. Goldenberg, Immunomedics, Inc., 300 TheAmerican Road, Morris Plains, NJ 07950. Phone: 973-605-8200; Fax: 973-605-8282; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0219

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(8) August 20161910

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with PSC-833 (8), a second-generation modifier of ABCB1 (19).The use of a hydrophilic linker for conjugatingDM1 to antibodiesalso enabled such ADCs to evade ABCB1-mediated resistance(20), presumably due to the generation of a cytotoxic metabolitethat was better retained by the ABCB1-expressing cells. In addi-tion, targeting detoxifying enzymes, such as glutathione S-trans-ferase, with intracellularly activated prodrugs was found to bepromising (21). However, the strong rationale of using inhibitorsof ABC transporters to overcome MDR has met little success inclinical trials, which could be in part due to both imperfectinhibitors and inadequate study design, and is being addressedby developing newer agents with greater substrate specificity,higher potency, lower toxicity, and improved pharmacokineticproperties.

Sacituzumab govitecan, hereafter referred to as IMMU-132(Supplementary Fig. S1), is a Trop-2–targeting ADC of SN-38,the activemetabolite of irinotecan. IMMU-132 departs frommostADCs in its use of amoderately, not ultratoxic drug, its high drug-to-antibody ratio (DAR) without impairing target affinity andpharmacokinetics, and its selection of a pH-sensitive, cleavablelinker to confer cytotoxicity to both tumor and bystander cells(22–24). This novel ADC is currently in clinical trials for patientswith advanced triple-negative breast cancer (25), urothelial blad-der cancer (26), and other solid cancers. As these patients were allheavily pretreated with chemotherapy, the presence of acquiredresistancewith the expressionofMDRgenes is highly likely,whichmay affect the therapeutic outcome of IMMU-132. In this study,we explored the use of known inhibitors of ABC transporters forimproving the therapeutic efficacy of IMMU-132 by overcomingSN-38 resistance.

Materials and MethodsCell lines and cultures

Human cancer cell lines (MDA-MB-231, breast; NCI-N87,stomach; A549, lung; HCT15, colon) were purchased from theATCC with authentication by short tandem repeat profiling.Each cell line was maintained according to the recommenda-tions of ATCC and routinely tested for mycoplasma usingMycoAlert Mycoplasma Detection Kit (Lonza). The twoSN-38–resistant cell lines, MDA-MB-231-S120 and NCI-N87-S120, were established by continuous exposure of the parentalcells to stepwise increased concentrations of SN-38 from 6pmol/L to 120 nmol/L over a period of approximately 2 years.In addition, a revertant cell line (NCI-N87-S120-REV) wasobtained after culturing NCI-N87-S120 in SN-38–free mediumfor 4 months. The S120 clones were maintained in mediumcontaining 120 nmol/L of SN-38 but cultured in drug-freemedium for 7 to 14 days prior to any experiment. All cells weregrown at 37�C as monolayer cultures in a humidified atmo-sphere of 5% CO2.

Antibodies, IMMU-132, and reagentsPolyclonal rabbit anti-ABCG2 antibody (#4477) was pur-

chased from Cell Signaling Technology and murine anti-gH2AX-AF488 (05-636-AF488) from EMD Millipore. Pheophor-bide A (PhA), fumitremorgin C (FTC), Ko143, YHO-13351,doxorubicin, paclitaxel, rhodamine 123, and verapamil wereobtained from Sigma. SN-38, purchased from Biddle SawyerPharma,was diluted to 1mmol/L inDMSOand stored in aliquotsat�20�C. Irinotecan-HCl injection was bought from Areva Phar-

maceuticals. The properties and preparation of IMMU-132 havebeen described previously (22–24).

Functional assays of ABC transportersFunctional assays of ABCG2, with PhA as the substrate and FTC

as the inhibitor, were performed essentially as described by Robeyand colleagues (27). Briefly, trypsinized cells were incubated with1 mmol/L PhA or with a combination of 1 mmol/L PhA and10 mmol/L FTC for 30 minutes at 37�C in 5% CO2 and washed.Subsequent incubations at 37�C for 1 hour were performed inPhA-free medium for cells treated only with PhA (to generate theefflux histogram) or in PhA-free medium containing 10 mmol/LFTC for cells treated with both PhA and FTC (to generate the FTC/efflux histogram). PhA fluorescence was measured on a FACS-Canto flow cytometer equipped with a 635-nm red diode laser.

Functional assays of ABCB1were performed as described abovefor ABCG2, with the substitution of rhodamine 123 for PhA andverapamil for FTC.

Western blottingCells were lysed in RIPA buffer (Cell Signaling Technology).

The protein concentration of the lysate was quantitated by thePierce BCA Protein Assay Kit (Thermo Fisher Scientific) using BSAas the standard. Equal amounts of lysate were loaded and sepa-rated by SDS-polyacrylamide gels (4%–20%) and transferredonto nitrocellulose membranes. The membranes were blockedwith 5% nonfat milk powder in TBS for 1 hour and probed withappropriate primary antibodies. After washing with TBS-T, themembrane was incubated with anti-rabbit IgG, HRP-linked anti-body (Cell Signaling Technology) and visualized using enhancedchemiluminescence.

In vitro cytotoxicitySensitivity to SN-38 or IMMU-132 was determined by an

MTS-based assay using CellTiter 96 AQueous One Solution(Promega). Briefly, cells were placed into wells of 96-well plates.Working solutions of IMMU-132 (at a concentration of 2,500nmol/L in SN-38 equivalents) and SN-38 at 2,500 nmol/L wereprepared from respective stock solutions in sterile media. Fromthese working solutions, serial 5-fold dilutions were made insterile media to yield final concentrations between 500 and0.0064 nmol/L in test wells. Plates were incubated at 37�C in ahumidified chamber with 5% CO2 for 96 hours, after which 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium was added and returned to theincubator until cells of the untreated control had an absorbancegreater than 1.0. Cytotoxicity was measured as a percent ofgrowth relative to untreated cells. Dose–response curves weregenerated from the mean of triplicate determinations, and IC50

values were calculated using GraphPad Prism Software V 6.05(Advanced Graphics Software).

The effect of ABCG2 inhibitors on the IC50 values of SN-38 forthe parental and resistant cell lines was determined from therespective dose–response curves obtained for SN-38 in the pres-ence of a nontoxic concentration of ABCG2 inhibitors.

Estimation of double-strand breaks with measurements ofgH2AX-stained cells

Cells (5 � 105 cells/mL per sample) were incubated with orwithout SN-38 (250nmol/L) at 37�C for the indicated times,fixed

Overcoming SN-38-ADC Drug Resistance

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in 4% formalin for 15minutes, thenwashed andpermeabilized in0.15% Triton X-100 in PBS for 15 minutes. After washing twicewith 1% BSA-PBS, the cells were incubated with murine anti-gH2AX-AF488 for 45 minutes at 4�C. The signal intensity ofgH2AX was measured by flow cytometry using a FACSCalibur(BD Biosciences).

Reverse transcription quantitative real-time PCRTotal RNA was extracted using RNeasy Mini Kit (Qiagen) and

reverse transcribed to complementary DNA using SuperScriptIV First-Strand Synthesis System (Life Technologies). qPCR wasperformed for each sample in triplicate on a Bio-Rad CFX96Real-Time System using the TaqMan Gene Expression Assay(with primer sets of Hs01053790_m1 for ABCG2 andHs99999905_m1 for GAPDH) and TaqMan Universal MasterMix II, all procured from Life Technologies. The mRNA levelof ABCG2 was normalized to that of GAPDH and expressed as2�DCt, where DCt ¼ [Ct (ABCG2) � Ct (GAPDH)], and Ct

(threshold cycle) is the number of PCR cycle correspondingto the intersection between an amplification curve and athreshold line determined for a target gene.

In vivo therapy studiesNCr female athymic nude (nu/nu) mice, 4 weeks old, were

purchased from Taconic Farms. NCI-N87 and NCI-N87-S120tumor xenografts were established by harvesting cells from tissueculture, making a 1:1 cell suspension in Matrigel (BD Bios-ciences), and injecting each mouse with a total of 1 � 107 cellssubcutaneously in the right flank. Tumor volume (TV) was deter-mined by measurements in two dimensions using calipers, withvolumes defined as: L�w2/2, where L is the longest dimension ofthe tumor and w the shortest. Mice were randomized into treat-ment groups of 9 to 10, and therapy begun when tumor volumeswere approximately 0.25 cm3. Mice bearing NCI-N87-S120tumors were treated with irinotecan (40 mg/kg i.v., every otherday for 5 times) or IMMU-132 (0.5 mg i.v. twice weekly for fourweeks). YHO-13351 (0.6 mg i.v.) was administered at the sametime the therapy started and again at 4 hours posttherapy. For theIMMU-132 þ YHO-13351 combination group, a third injectionof YHO-13351 was administered 24 hours post-IMMU-132administration. Control mice received each agent alone. ForYHO-13351 control mice, they were injected on the same sched-ule as when combined with irinotecan. Another group of micebearing parental NCI-N87 tumors served as a control for efficacyof IMMU-132 and irinotecan in tumors lacking the ABCG2pump.The lyophilized IMMU-132 was reconstituted and diluted insterile saline as required. Irinotecan-HCl injection was diluted insterile saline and the final dose based on bodyweight (40mg/kg).Mice were euthanized and deemed to have succumbed to diseaseonce tumors grew to >1.0 cm3 in size.

Statistical analysisStatistical analysis for the tumor growth datawas based onAUC

and survival time. Profiles of individual tumor growth wereobtained through linear curve modeling. An f test was employedto determine equality of variance between groups prior to statis-tical analysis of growth curves. A two-tailed t testwas used to assessstatistical significance between groups. As a consequence ofincompleteness of some of the growth curves due to deaths,statistical comparisons of AUC were only performed up to thetime at which the first animal within a group was sacrificed. Log-rank analysis to compare the Kaplan–Meier survival curves of twogroups was performed with GraphPad Prism V6.05. Significancewas set at P � 0.05.

ResultsEstablishment of SN-38–resistant cell lines

Table 1 summarizes the IC50 values of the parental cell lines(MDA-MB-231 and NCI-N87) and their SN-38–resistant coun-terparts (MDA-MB-231-S120 and NCI-N87-S120), as deter-mined for SN-38, doxorubicin and paclitaxel. Compared withthe parental cells, both S120 cells are about 50-fold moreresistant to SN-38 and relatively not cross-resistant to eitherdoxorubicin or paclitaxel. NCI-N87-S120 cells cultured for 3weeks or longer in SN-38–free medium gradually restoredtheir sensitivity to SN-38, resulting in a revertant cell line(NCI-N87-S120-REV) with reduced resistance to SN-38 (IC50 ¼50 nmol/L) over a period of 4 months.

Overexpression of functional ABCG2 in the S120 cell linesThe presence of ABCG2 in the S120 cells, but little, if any, in

the parental cells is shown by Western blot analysis (Fig. 1A)and corroborated by qRT-PCR, which indicate that virtually nomRNA transcripts of ABCG2 could be detected in MDA-MB-231and NCI-N87 cells (Supplementary Table S1). On the otherhand, the mRNA levels of ABCG2 relative to GAPDH inMDA-MB-231-S120 and NCI-N87-S120 are calculated to be27,408-fold and 167-fold higher than those in MDA-MB-231and NCI-N87, respectively (Fig. 1B). The expression of ABCG2was also confirmed in samples obtained from NCI-N87-S120,but not NCI-N87, xenografts (Fig. 1C).

That ABCG2 is functionally active in the two S120 cell popula-tions, but absent in the parental cells, is evident from the fourhistograms shown in Fig. 2 for PhA, which is a fluorescentsubstrate of ABCG2. In both parental cells, the intracellular levelsof PhA, as measured by the median fluorescence intensity (MFI),are relatively high and remainpractically the samewith orwithoutFTC, a potent mycotoxin (Supplementary Fig. S2) initially iden-tified for its effective reversal of resistance to mitoxantrone,doxorubicin, and topotecan in a multidrug-selected cell line. In

Table 1. Sensitivity of the parental and SN-38–resistant cell lines to selected drugs

IC50 (nmol/L)Drug NCI-N87 NCI-N87-S120 RFa MDA-MB-231 MDA-MB-231-S120 RFa

SN-38 4.3 � 0.7 211 � 39 49.1 4.8 � 1.7 248 � 79 51.7SN-38 þ YHO-13351b 3.9 � 0.5 6.4 � 1.6 1.6 2.4 16 � 11 6.7SN-38 þ Ko143b ND 1.3 � 0.4 ND ND ND NDDoxorubicin 32.2 � 7.5 74.6 � 3.3 2.3 10.9 � 0.6 43.1 � 4.6 4.0Paclitaxel 7.9 � 4.0 8.6 � 6.5 1.1 2.5 � 0.1 3.2 � 0.7 1.3

Abbreviation: ND, not determined.aRF is the resistant factor obtained as ½the IC50 mean of S120�

½the IC50 mean of parent�.bThe nontoxic concentrations used for YHO-13351 and Ko143 were 2 and 1 mmol/L, respectively.

Chang et al.

Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics1912




contrast, in either S120 cells, a high level of intracellular PhAsimilar to that in the parental cells could only be observedwith theaddition of FTC, whereas the omission of FTC resulted in a greaterthan 95% reduction of intracellular PhA. Similar results wereobtained in human lung cancer A549 cells, known to expressfunctional ABCG2 (28). In separate studies with rhodamine 123and verapamil as the substrate and the inhibitor for ABCB1,respectively, activity of ABCB1 was detected in human colorectalcancer HCT15 cells, which served as a positive control for expres-sing ABCB1 (29), but not in either NCI-N87-S120 or NCI-N87(Supplementary Table S2).

The activity of ABCG2 in the two S120–resistant cell lines alsowas demonstrated by comparing the levels of DNA double-strandbreaks (DSB) induced by SN-38with those in the parental cells, asmeasured by a flow cytometric assay for quantification of gH2AX,whose signal intensities directly correspond to the number ofDSBs formed (30). As shown in Fig. 3A, upon treatment with 250nmol/L of SN-38, the levels of gH2AX rose steadily in the parental,but not the resistant S120, cells, culminating, after 3 hours, in anincrease over the untreated controls that was about 2-fold forMDA-MB-231 and about 4-fold for NCI-N87 (Fig. 3B). ABCG2 isimplicated in preventing the increase of gH2AX in the S120 cellstreated with SN-38, as the addition of both FTC (10 mmol/L) andSN-38 (250 nmol/L) toMDA-MB-231-S120 could elevate gH2AXlevels with a comparable fold-increase with that of SN-38–treatedMDA-MB-231 (Fig. 3C). Similar results were obtained with NCI-N87-S120when treated with SN-38 or IMMU-132 in the presenceof FTC (Fig. 3D).

Sensitizing S120 cells to SN-38 with selected ABCG2 inhibitorsThe effect of two known ABCG2 inhibitors, Ko143 (31) and

YHO-13351 (32), on reversing the resistance of S120 cells toSN-38 was examined in vitro at a concentration not affecting thegrowth of either the parental or the S120 cells. As shown in Fig.4A–C of the representative dose–response curves, and in Table 1of the pertaining IC50 values, the addition of either ABCG2inhibitor, while conferring little impact on the sensitivity ofparental cells to SN-38, reduced the IC50 of SN-38 by more than90% in both resistant S120 cell lines. Limited studies also were

done for other ABCG2 inhibitors, such as FTC (33), cyclosporineA (34), and GF120918 (35), with results showing comparable orless potency (data not shown). Noting the reported instability ofKo143 in rat serum (36), YHO-13351was selected over Ko143 forin vivo evaluation.

Improved efficacy of IMMU-132 when combined withYHO-13351 in SN-38–resistant NCI-N87-S120 tumors

NCI-N87-S120 tumors grew slower in the mice than didparental NCI-N87 (Fig. 5A; P ¼ 0.005, AUC), with the mediansurvival for untreated animals more than 2-fold longer for themice bearingNCI-N87-S120 (P¼ 0.0006 vs. NCI-N87). Althoughtreatment with IMMU-132 or irinotecan provided no signi-ficant survival benefit to mice bearing NCI-N87-S120 tumors(Fig. 5B), both of these therapies resulted in a greater than 2-foldincrease in survival in mice bearing NCI-N87 (P < 0.0001;Fig. 5C). However, when IMMU-132 therapy was combined withYHO-13351 in mice bearing SN-38–resistant NCI-N87-S120,a significant 64% improvement in survival was achieved incomparison with untreated animals (P ¼ 0.0278). Althoughirinotecan plus YHO-13551 likewise improved the survivalof the mice, it did not reach significance (P ¼ 0.0852). As thein vitro assay showed there was no difference between parentalcell lines treated with SN-38 alone or in combination withYHO-13351, and the tumor xenograft samples of NCI-N87 wereabsent of ABCG2 by Western blot analysis (Fig. 1C), a combina-tion of IMMU-132 and YHO-13351 was not examined in micebearing NCI-N87.

DiscussionIMMU-132 is a first-in-class ADC made by conjugating the

moderately toxic drug, SN-38 (nanomolar potency), with apartially stable linker, site specifically and at a high DAR of 7.6,to a humanized antibody against Trop-2 expressed in manysolid cancers. Preclinical studies have demonstrated thatIMMU-132, in comparison with irinotecan, protects theIgG-bound SN-38 from glucuronidation, delivers much moreSN-38 (20- to 136-fold higher) to tumor xenografts, resulting in

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Overexpression of ABCG2 in the S120cells. A, Western blot analysis, showingthe expression of ABCG2 in the twoS120 cell lines, but little or none in theparental MDA-MB-231 and NCI-N87;b-actin served as the loading control.B, qRT-PCR results showing high levelsof ABCG2 mRNA (normalized toGAPDH mRNA) in the S120 cells. Thefold of increase was 27,408 for MDA-MB-231-S120 and 167 for NCI-N87-S120.C, expression of ABCG2 in NCI-N87-S120, but not NCI-N87, xenografts, asconfirmed by Western blot analysis.






improved pharmacokinetics and pharmacodynamics (37). Assuch, IMMU-132 provides a paradigm change that contraststhe prevailing approach of conjugating a low level of anultratoxic payload (picomolar potency) with a stable linkerto an antibody capable of internalization upon target engage-ment (38, 39). Importantly, the ongoing phase II studies withIMMU-132 as a single agent in patients with metastatic triple-negative breast cancer (mTNBC) who had received a median of5 (range ¼ 2 to 12) prior lines of therapy have shown aninterim objective response rate of 31% by RECIST 1.1 in 58evaluable patients (40), thus extending the results obtained inphase I trials, which indicated IMMU-132 had acceptabletoxicity and encouraging therapeutic activity in patients withdifficult-to-treat solid cancers (25). Promising initial resultshave also been reported for IMMU-132 administered topatients with platinum-resistant urothelial carcinoma (26).Whereas the phase II results observed in heavily pretreatedpatients with mTNBC have led the FDA to grant BreakthroughTherapy Designation to IMMU-132, those who showed earlyprogression of disease, thus failing IMMU-132, may reflectpossible drug resistance resulting from one or more preexisting

efflux pumps, as SN-38 is susceptible to multiple ABC trans-porters (1).

In the current study, NCI-N87-S120 and MDA-MB-231-S120,the two sublines made resistant to SN-38, were shown to be 50-fold less responsive to SN-38 than their parental cells. Thesensitivity of NCI-N87-S120 to SN-38 could be restored to within5-fold of NCI-N87when propagated in vitrowithout SN-38 after aperiod of 3weeks or longer, suggesting a nongenetic origin of suchacquired resistance, which may or may not be clinically relevant.The presence of ABCG2 in the two S120 sublines, but not theirparents, was supported by several lines of evidence, including thedemonstration of active efflux of PhA; the detection of expressedprotein by Western blotting, which was corroborated with qRT-PCR ofmRNA; the increased accumulation of SN-38 by FTC usinggH2AX as a surrogate marker; and the reverted resistance byYHO-13351 or Ko143. Of note, both S120 sublines were foundnot to carry ABCB1 and neither phenotype was cross-resistant todoxorubicin or paclitaxel, similar to a previous observation (41)that the ABCG2-expressing, SN-38–resistant human colorectalHCT116-SN50 cancer subline showed no significant cross-resis-tance to doxorubicin (aswell as to 5-fluorouracil and oxaliplatin).

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Functional assay of ABCG2. Bothparental and S120 cells were incubatedwith 1 mmol/L PhA alone (blue) ortogether with 10 mmol/L FTC (orange)to generate the PhA efflux histogram.The MFI pertaining to each signal wasprovided in the histograms. In bothparental cells, which do not expressABCG2, the intracellular levels of PhAwere high and remained practically thesame with or without FTC. In the S120cells, the active efflux by ABCG2resulted in much lower levels ofPhA, which was restored with theaddition of FTC. MDA-MB-231(left upper panel), MDA-MB-231-S120(right upper panel), NCI-N87 (leftlower panel), NCI-N87-S120 (rightlower panel).

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gH2AX assay for DSB by flow cytometry. Cells were treated or not treated with SN-38 (250 nmol/L) for 3 hours, and the levels of gH2AX were monitoredhourly by flow cytometry and shown as MFI in a bar diagram (A) or as percentage of untreated (B). The effect of FTC (10 mmol/L) to increase the formationof DSB/gH2AX was shown for MDA-MB-231-S120 treated with SN-38 (C) and for NCI-N87-S120 treated for either SN-38 or IMMU-132 (D).






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Figure 4.

Dose–response curves of parental and S120 cells treated with different concentrations of SN-38 in the absence and presence of YHO-13351 or Ko143. Reversalof SN-38 resistance by YHO-13351 was shown for MDA-MB-231-S120 (A), NCI-N87-S120 (B), and by Ko143 for NCI-N87-S120 (C).


Chang et al.




1.00A

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Figure 5.

Efficacy of IMMU-132 in mice bearing SN-38–resistant NCI-N87-S120 gastric carcinoma xenograft. A, mean tumor growth curves for NCI-N87 and NCI-N87-S120xenografts. B, mice bearing NCI-N87-S120 SN-38–resistant human gastric tumors were treated with IMMU-132, irinotecan, YHO-13551, or combinations asindicated on the graph and described in Materials and Methods. C, mice bearing parental NCI-N87 tumors treated with IMMU-132 or irinotecan at the samedose and schedule as used in NCI-N87-S120 tumor-bearing animals. In the survival curves of B and C, the starting day of therapy (when tumor volumes reached�0.25 cm3) was marked as day 0. Mice were euthanized once tumors grew to >1.0 cm3 in size.






In other human cancer clones selected for resistance to SN-38 viacontinuous exposure of parental cell lines to the drug in culture,overexpression of ABCG2 has also been reported for the sublinesgenerated from MCF-7 (42, 43), MDA-MB-231 (43), the small-cell lung carcinoma PC-6 (17), the non–small cell lung adeno-carcinoma H23 (44), and the cervical carcinoma HeLa (45).Cross-resistance of these sublines to doxorubicin varied some-what, with the resistance ratio (IC50 in resistant subline divided byIC50 in the parental cell) being less than 1.3 for the sublines of PC-6 (17), 2.5 for the sublines of HeLa (45), and about 7.0 for thesubline of H23 (44). Whereas cross-resistance to doxorubicin wasnot determined for the SN-38–resistant sublines derived fromMCF-7 (42, 43) or MDA-MB-231 (43), these sublines remainedsensitive to vincristine (42), cisplatin (42, 43), anddocetaxel (43).

Althoughwe andothers have establishedABCG2as a key playerin reducing SN-38 sensitivity of various SN-38–resistant cancersublines, the potential involvement of DNA topoisomerase I(Top1), towhich SN-38 specifically binds and acts as an inhibitor,in the SN-38 resistance mechanism of such cancer cells, is lessdefined and remains a focus of continuous research, with thecurrent knowledge pointing to Top1 mutation (46, 47) anddegradation (48) as the two main roles of Top1 underlying themolecular mechanism of resistance to camptothecin in generaland SN-38 in particular.

When cultured in vitro, SN-38–resistant sublines of MCF-7 orMDA-MB-231 had longer doubling times than their parental cells(43). Thus, it is not surprising thatNCI-N87-S120 xenografts grewsignificantly slower in themice thanNCI-N87, which is consistentwith the notion that drug-resistant tumor sublines selected in vitrofrequently manifest less aggressive properties than their drug-sensitive parental cell lines (49). Nevertheless, in vivo studies showthat the NCI-N87-S120 xenograft retained ABCG2 expressionand was resistant to IMMU-132, yet its growth could be signi-ficantly subdued by IMMU-132 in combination with YHO-13351. A parallel study shows the parental xenograft was respon-sive to IMMU-132 or irinotecan, but with a shorter median

survival time. Together, these in vivo results suggest that suitableinhibitors that are tolerated well by the host animals can over-come ABC resistance and that the resistant tumor lines canbecome appreciably responsive to IMMU-132 and to a lesserextent to irinotecan. We are pursuing further work to address thefeasibility of preclinical testing for such drug resistance as apredictive bioassay (43, 44) to select patients who should receiveABC-blocking therapy with IMMU-132.Meanwhile, we are exam-ining the suitability of clinically tested tyrosine kinase inhibitors,some of which interfere with the functions of ABC transporters atnontoxic levels (50), to enhance the potency of IMMU-132 incancer cells that are intrinsically or made resistant to SN-38.

Disclosure of Potential Conflicts of InterestAll authors are current employees of Immunomedics, Inc., and have

stocks or stock options of Immunomedics, Inc. No other potential conflictsof interest were disclosed by the authors.

Authors' ContributionsConception and design: C.-H. Chang, T.M. Cardillo, D.M. GoldenbergDevelopment of methodology: C.-H. Chang, D. Liu, T.M. CardilloAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Wang, M. Zalath, D. Liu, T.M. CardilloAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, com-putational analysis): C.-H. Chang, Y. Wang, M. Zalath, D. Liu, T.M. CardilloWriting, review, and/or revision of the manuscript: C.-H. Chang, D. Liu,T.M. Cardillo, D.M. GoldenbergAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T.M. Cardillo, D.M. GoldenbergStudy supervision: C.-H. Chang, T.M. Cardillo, D.M. Goldenberg

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 April 8, 2016; revised May 11, 2016; accepted May 11, 2016;published OnlineFirst May 20, 2016.

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2016;15:1910-1919. Published OnlineFirst May 20, 2016.Mol Cancer Ther Chien-Hsing Chang, Yang Wang, Maria Zalath, et al. Breast and Gastric CancersAntibody Conjugate of SN-38, Overcomes Resistance to SN-38 in

Trop-2−Combining ABCG2 Inhibitors with IMMU-132, an Anti

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