A Novel Combination Treatment Targeting L and MCL1 for ... · Small Molecule Therapeutics A Novel Combination Treatment Targeting BCL-XL and MCL1 for KRAS/BRAF-mutated and BCL2L1-ampliﬁed

Small Molecule Therapeutics

A Novel Combination Treatment TargetingBCL-XL and MCL1 for KRAS/BRAF-mutated andBCL2L1-amplified Colorectal CancersSung-Yup Cho1,2, Jee Yun Han2, Deukchae Na1,2,Wonyoung Kang2, Ahra Lee2,Jooyoung Kim2, Jieun Lee2, Seoyeon Min2, Jinjoo Kang2, Jeesoo Chae3,Jong-Il Kim3, Hansoo Park4,Won-Suk Lee5, and Charles Lee1,2,6

Abstract

Colorectal cancer is the third most commonly diagnosedcancer in the world, and exhibits heterogeneous characteristicsin terms of genomic alterations, expression signature, and drugresponsiveness. Although there have been considerable effortsto classify this disease based on high-throughput sequencingtechniques, targeted treatments for specific subgroups havebeen limited. KRAS and BRAF mutations are prevalent geneticalterations in colorectal cancers, and patients with mutations ineither of these genes have a worse prognosis and are resistant toanti-EGFR treatments. In this study, we have found that a

subgroup of colorectal cancers, defined by having either KRASor BRAF (KRAS/BRAF) mutations and BCL2L1 (encoding BCL-XL) amplification, can be effectively targeted by simultaneousinhibition of BCL-XL (with ABT-263) and MCL1 (with YM-155). This combination treatment of ABT-263 and YM-155 wasshown to have a synergistic effect in vitro as well as in in vivopatient-derived xenograft models. Our data suggest that com-bined inhibition of BCL-XL and MCL1 provides a promisingtreatment strategy for this genomically defined colorectal can-cer subgroup. Mol Cancer Ther; 16(10); 2178–90. �2017 AACR.

IntroductionColorectal cancer is the third most common cancer world-

wide and ranks as the fourth most prevalent cause of cancer-related deaths (1). Recent advances in high-throughputsequencing have enabled the molecular classification of colo-rectal cancers into hypermutated [microsatellite instability(MSI)/CpG island methylation phenotype (CIMP)-high] andnonhypermutated [microsatellite stable (MSS)/chromosomalinstable (CIN)] subgroups (2). In addition, gene expression

profiling studies have suggested further classification of thenonhypermutated group into epithelial proliferative and mes-enchymal/invasive stromal subgroups (3). Although these clas-sifications suggest potential avenues for targeted therapeutics,there has actually been limited success in targeted therapies forcolorectal cancers.

One subset of colorectal cancer cases for which targeted ther-apeutics will be particularly important are those patients withmutations in either the KRAS (30%–45% frequency) or BRAF(5%–15% frequency) genes. The prognosis for these patients issignificantly worse than patients without these mutations (4, 5).These cancers are resistant to currently employed anti-EGFRtreatments, such as panitumumab and cetuximab (6, 7), appar-ently due to activation of signaling pathways (includingMEK-ERKand PI3K pathways) that are important for cell survival and drugresistance (8, 9). Despite extensive attempts to regulate thesesignaling pathways, single drug treatments have shown minimaltherapeutic effect (10, 11). More recently, several combinationtreatment strategies have been pursued for colorectal cancerswith KRAS or BRAF (KRAS/BRAF) mutations. These combina-tion treatments are mainly based on MEK inhibitors (12, 13).Although some of these combination treatments are now underevaluation in clinical trials (13), it is still unclear which combina-tions will be clinically applicable. Furthermore, having alternatetreatment strategies for KRAS/BRAF–mutated colorectal cancerwill be important to mitigate colorectal cancer cases acquiringresistance to selected targeted therapies.

In this study,wehave found that asmuch as 21.7%of colorectalcancers can be categorized into a novel colorectal cancer subgroupdefined by KRAS/BRAF mutations and BCL2L1 (encoding BCL-XL) amplification. Concomitant treatment of a BCL-XL inhibitor,ABT-263, and an MCL1 inhibitor, YM-155, showed synergisticantitumor effects in both in vitro cell line models and in vivo

1Ewha Institute of Convergence Medicine, Ewha Womans University MokdongHospital, Seoul, Korea. 2Department of Life Science, Ewha Womans University,Seoul, Korea. 3Department of Biomedical Sciences, Seoul National UniversityCollege of Medicine, Seoul, Korea. 4Department of Biomedical Scienceand Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju,Korea. 5Department of Surgery, Gil Medical Center, Gachon University, Incheon,Korea. 6The Jackson Laboratory for Genomic Medicine, Farmington,Connecticut.

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

S.-Y. Cho and J.Y. Han contributed equally to this article.

Corresponding Authors: Charles Lee, The Jackson Laboratory for GenomicMedicine, 10 Discovery Drive, Farmington, CT 06032. Phone: 860-837-2474;Fax: 860-837-2398; E-mail: [email protected]; Hansoo Park, Department ofBiomedical Science and Engineering, Gwangju Institute of Science and Tech-nology (GIST), 123 Cheomdangwagi-ro, Buk-gu, Gwangju 61005, Korea. Phone:82-62-715-2114; E-mail: [email protected]; and Won-Suk Lee, Department ofSurgery, Gil Medical Center, Gachon University, 1198 Guwol-dong, Namdong-gu,Incheon 21565, Korea. Phone: 82-32-460-3216; Fax: 82-32-460-3009; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-16-0735

�2017 American Association for Cancer Research.

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patient-derived xenograft (PDX) models. These results propose anovel combination targeted treatment strategy for a subgroup ofrecalcitrant colorectal cancers.

Materials and MethodsCell culture

All of the human colorectal cancer cell lines, authenticatedusing DNA fingerprint analysis, were provided by the Korean CellLine Bank in 2015. Cells were passaged for fewer than 6 monthsafter resuscitation. Cellswere cultured inRPMI1640medium(LifeTechnologies) with 10% FBS (Life Technologies), penicillin (100U/mL; Life Technologies), and streptomycin (100 U/mL; LifeTechnologies). All cells were maintained in a humidified incu-bator with 5% CO2 at 37�C.

Western blot analysisCell lysates were prepared using RIPA buffer (Thermo Scien-

tific) with protease inhibitor cocktail (Roche) and phosphataseinhibitor cocktail (Roche), incubated for 15 minutes in ice andcentrifuged at 16,800 � g for 10 minutes at 4�C. BCA method(Thermo Scientific) was used to determine the protein concen-tration. Proteins were resolved by SDS-PAGE and transferred topolyvinylidene difluoride membrane. After blocking using 5%skim milk, membranes were probed with anti-BCL-XL (Cell Sig-naling Technology), anti-MCL1 (SantaCruzBiotechnology), anti-cleaved caspase-3 (Cell Signaling Technology), anti-PARP (CellSignaling Technology), anti-survivin (Cell Signaling Technology),anti-caspase-8 (Cell Signaling Technology), anti-cleaved caspase-8 (Cell Signaling Technology), anti-caspase-9 (Cell SignalingTechnology), anti-BIM (Cell Signaling Technology), anti-AKT(Cell Signaling Technology), anti-phospho-AKT (Cell SignalingTechnology), anti-S6K (Cell Signaling Technology), anti-phos-pho-S6K (Cell Signaling Technology), anti-4EBP (Cell SignalingTechnology), anti-phospho-4EBP (Cell Signaling Technology),and anti-Actin (Sigma-Aldrich Corporation) antibody. The mem-branes were washed and incubated with horseradish peroxidase–conjugated secondary antibody, followed by enhanced chemilu-minescence development according to themanufacturer's instruc-tions (Pierce).

Droplet digital PCRThe extracted genomic DNA was restricted with EcoRI (New

England Biolabs) enzyme for 1 hour at 37�C. The PCR mixturewas assembled in 20-mL solution containing 1� droplet digitalPCR (ddPCR) supermix (Bio-Rad), 1� probe and primer premixfor determining BCL2L1 gene and internal control gene, RNase P(final concentration of 250 nmol/L for probe and 900 nmol/L foreach primer; Applied Biosystems), and 10 ng of the restrictedDNA. The reaction mixture and droplet generation oil (Bio-Rad)were loaded into the droplet generator (QX-200; Bio-Rad). Thedroplets were transferred to a 96-well PCR plate and PCR reactionwas performed as follows: enzyme activation for 10 minutes at95�C, 40 cycles of 94�C for 30 seconds, 60�C for 1 minute, and98�C for 10 minutes, followed by enzyme deactivation for 10minutes at 98�C and 4�C hold (performed with a ramp rate of2�C/sec in all steps). The PCR plate was placed in a droplet reader(Bio-Rad). After the reading, the copy number variation of targetgenes was analyzed by Quanta software (Bio-Rad) accompaniedby the droplet reader. The amplification threshold value was set at3.0 for tissues and cell lines.

Colorectal cancer patient sample collectionFrozen tissue and formalin-fixed paraffin-embedded (FFPE)

tissue samples of colorectal cancer were obtained from indivi-duals who underwent colorectal cancer surgery at Gachon Uni-versityGilMedical Center (Incheon, SouthKorea). TotalDNAwasextracted from frozen tissues using the QIAamp DNA Mini kit(Qiagen) for ddPCR, and FFPE tissue samples were used forhistologic analysis and IHC. All samples were obtained withinformed consent at the Gachon University Gil Medical Center,and the study was approved by the institutional review board inaccordance with the Declaration of Helsinki.

Histologic analysis and IHCAfter removal, tumor tissues were fixed in 10%neutral buffered

formalin and embedded in paraffin. Four-micrometer-thick con-secutive sections that covered the entire tumor were cut andmounted on silanized slides. Hematoxylin and eosin staining(H&E) was performed according to standard protocols. For IHC,slides were deparaffinized in xylene and rehydrated in a series ofgraded alcohols, and the antigen was retrieved in Antigenunmasking solutions (Vector Laboratories). Sections were thentreated with 3% of hydrogen peroxide to inhibit endogenousperoxidase. The sections were stained with primary antibodies[anti-BCL-XL (Cell Signaling Technology), anti-MCL1 (SantaCruzBiotechnology), anti-cleaved caspase-3 (Cell Signaling Technol-ogy), anti-Ki67 (Thermo Fisher Scientific)], followed by appro-priate secondary antibody, and then visualized by use of theappropriate substrate/chromogen (Diaminobenzidine, DAB)reagent according to the manufacturer's recommendations (Vec-tor Laboratories). TUNEL staining was performed using DeadEndColorimetric TUNEL System (Promega) according to the manu-facturer's instructions. Counterstaining was performed usingHematoxylin QS (Vector Laboratories). The intensities of stainwere assessed using "ImageJ" software (https://imagej.nih.gov/ij),which is a Java image processing and analysis program. Weutilized "ImageJ" software as previously reported by applyingsame threshold for each antibody staining and selecting "AnalyzeParticles" option (14).

Cell viability assay and estimation of combination indexFor the cell viability assays, about 5,000 cells in 96-well plates

were treated with indicated drugs for 72 hours and cell viabilitieswere estimated using the EzCytox WST assay kit according to themanufacturer's instructions (Daeil Lab). Cell viabilities wereestimated as relative values compared with untreated controls.Drug synergism was quantified by calculating combination index(CI) based on the multiple drug effect equation (15). The CI wasbased on themass action law in biophysics and biochemistry, andcalculated by the Chou–Talalay equation, which considers bothdrug potency and the shape of the dose–effect curve (15). The CIfor each concentration of drugs was calculated by CompuSynSoftware (ComboSyn Inc.) and a CI lower than 0.9 indicatessynergism.

Fluorocytometric analysisThe SW620 cells were seeded into 60-mm culture dishes (1 �

106 cells/well) and were treated with indicated drugs. The treatedcells were collected and washed in PBS. The apoptotic cellswere analyzed by FITC Annexin V Apoptosis Detection Kit (BDPharmingen) according to the manufacturer's protocol. The sus-pended cellswere treatedwith 10mLofAnnexinV-FITC and10mL

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of propidium iodide for 5–15 minutes in the dark. Stained cellswere analyzed with a FACScan flow cytometer (BectonDickinson).

Transfection of small interfering RNAFor the transfection of small interfering RNA (siRNA) to down-

regulate target genes, specific siRNAs targeting for MCL1 andsurvivin were purchased from Genolution. Cells were transfectedusing Lipofectamine 2000 reagent (Invitrogen) according to themanufacturer's protocol. After 24 hours, cells were collected andplated in 96-well plates for drug test. The effects of siRNA knock-down were measured by Western blot analysis with the indicatedantibodies. The sequence of siRNA for MCL1 was 50- GUGC-CUUUGUGGCUAAACATT-30, for survivin was 50- AAGGCUGG-GAGCCAGAUGACGUU-30, and for GFP was 50- GACGUAA-ACGGCCACAAGUTT-30.

ImmunoprecipitationAfter treatment of indicated drugs, cell lysates were prepared

using immunoprecipitation (IP) buffer (Thermo Scientific) withprotease inhibitor cocktail (Roche) and phosphatase inhibitorcocktail (Roche), incubated for 15minutes on ice, and centrifugedat 16,800 � g for 10 minutes at 4�C. Protein G Dynabeads(Thermo Scientific) were incubated with anti-BIM for 2 hours at4�C with rotation. The antibody-bound magnetic beads wereincubated with the cell lysate for 2 hours at 4�C with rotation.The beadswerewashed for three timeswith IP buffer and eluted in4� SDS sample buffer (Bio-Rad) with boiling. Immunoprecipi-tated proteins were evaluated by Western blot analysis.

Real-time PCRTotal RNA from cells was extracted using RNeasy Plus Mini Kit

(Qiagen). Reverse transcription with 1 mg of total RNA wasperformed using Maxime RT PreMix (Intron Biotechnology).We performed real-time PCR using SYBR Green PCR MasterMix (Applied Biosystems) and checked MCL1, AKT, S6K, DR4,DR5, and TRAIL mRNA level normalized by GAPDH used asinternal control. The sequences of primers were as follows: forMCL1, 50-TAAGGACAAAACGGGACTGG-30 and 50- ACCAG-CTCCTACTCCAGCAA-30; for AKT, 50- TCTATGGCGCTGA-GATTGTG-30 and 50- CTTAATGTGCCCGTCCTTGT-30; for S6K,50- GTGGAGGAGAACTATTTATGCAGTTAGAAAGAG-30 and 50-TCCAGCGTCCCCAGGACCAGCTC-30; for DR4, 50-GGCTGAG-GACAATGCTCACA-30 and 50-TTGCTGCTCAGAGACGAAAGTG-30; for DR5, 50-GACTCTGAGACAGTGCTTCGATGA-30 and 50-CCATGAGGCCCAACTTCCT-30; for TRAIL, 50-GCTCTGGGC-CGCAAAAT-30 and 50-TGCAAGTTGCTCAGGAATGAA-30; and forGAPDH, 50- CGCTCTCTGCTCCTCCTGTT-30 and 50- CCATGG-TGTCTGAGCGATGT-30.

Downregulation of gene using CRISPR/Cas9 systemThe target sequence of DR5 with BsmI (New England Biolabs)

restriction site was cloned into the lentiCRISPR v2 plasmid(Addgene #52961). HEK293T were transfected using Lipofecta-mine 2000 reagent (Invitrogen) with lentiCRISPR v2 (DR5sgRNA), psPAX2, and VSV-G plasmids. After 48 hours, the super-natants were collected. SW620 cells were incubated with lentivi-rus-containing media for 48 hours and selected by puromycin (1mg/mL) for 72 hours. The effects of gene knockdown were mea-sured by Western blot analysis with the indicated antibodies.

RNA sequencing and gene ontology (GO) functionalenrichment analysis

Total RNA from cells after drug treatment was extracted usingRNeasy Plus Mini Kit (Qiagen). Whole-transcriptome expressionprofiles were generated by RNA sequencing using Mapsplice andRSEM with TCGA RNASeq v2 pipeline (https://wiki.nci.nih.gov/display/TCGA/RNASeqþVersionþ2; refs. 16, 17), and then calcu-lated reads counts for each gene were used for analyses of differ-entially expressed genes utilizing the DESeq2 packages (18).ClueGOplug-in (v2.2.5, http://www.ici.upmc.fr/cluego/) inCytos-cape software (v3.3.0, http://cytoscape.org/) was used to analyzeGO and functional groups in networks for upregulated genes incombination treatment. ClueGO is a tool combining GO terms tocreate functionally grouped annotations in a network (19). GOBiological Process database (http://www.geneontology.org/) wasused for functional enrichment analysis. Significantly enriched GOterms were calculated by a two-sided hypergeometric test with aBonferroni correction (P < 0.05) and the degree of connectivitybetween terms in the network is calculated using kappa statistics(kappascoreof0.4). TheRNAsequencingdatahavebeendepositedin the European Nucleotide Archive (accession no. PRJEB20153).

Animal experimentsMice were cared for according to institutional guidelines of the

Institutional Animal Care and Use Committee (IACUC) of theSeoul National University (No. 14-0016-C0A0). For patient-derived xenograft (PDX) models, the surgically resected tissueswereminced into pieces approximately 2mm in size and injectedinto the flanks of 4-week-old NOD/SCID/IL2g-receptor null(NSG) female mice. For cell line xenograft models, the flanks of4-week-oldNSG femalemicewere injected subcutaneouslywith 1� 106 cells in 100-mL PBS. Drug treatments began after tumorsreached approximately 200 mm3. Mice were randomly dividedinto four treatment groups consisting of 4–5 mice in each group:(i) vehicle only; (ii) ABT-263 only (Selleckchem, 100 mg/kg,daily), (iii) YM-155 only (Selleckchem, 5 mg/kg, daily), and (iv)ABT-737 plus YM-155. The vehicle for ABT-263 was 10% (v/v)ethanol (Merck), 30% (v/v) polyethylene glycol (Sigma), and60% (v/v) Phosal 50 PG (Lipoid), and the vehicle for YM-155was0.9% normal saline. ABT-263 was administered via oral injectionand YM-155 was subcutaneously administrated using micro-osmotic pump (Alzet model 1007D, Durect) for 21 days.

Statistical analysisStatistical calculations were performed using Prism5.0 (Graph-

Pad). Differences between multiple variables were assessed byone-way ANOVA with Tukey multiple comparison test. Thedifference of tumor growth rate betweendifferently treated groupswas analyzed by growth curve analysis, which is a multilevelregression technique designed for analysis of time course orlongitudinal data (20). Growth curve analysis was performedusing the R software package lme4. The difference was consideredsignificant if the P value was less than 0.05.

ResultsIncreased expression of BCL-XL protein in KRAS/BRAF-mutatedand BCL2L1-amplified colorectal cancers

Mutations in KRAS have been shown to increase protein expres-sion of BCL-XL (21, 22). Because KRAS and BRAF are in the samesignaling pathway (23), it is possible that BRAF mutations alsosimilarly increase protein expression of BCL-XL. Therefore, we

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investigated BCL-XL protein expression in colorectal cancer cell linesthat carry either KRAS or BRAF mutations versus having wild-typeKRAS/BRAF genes. As expected, the protein levels of BCL-XL wereseen to be elevated in most KRAS/BRAF-mutated colorectal cancercells (Fig. 1A and B; Supplementary Fig. S1A), which was confirmedusingKRAS/BRAF-mutated isogenic SW48cells (Supplementary Fig.S1B). We further determined the copy number of BCL2L1 in thecolorectal cancer cell lines examined using droplet digital PCR(ddPCR) and found five cell lines (5/13, 38.5%) that had increasedgene copy number of BCL2L1 (Fig. 1A). Among them, four cell lineswith bothKRAS/BRAFmutation and BCL2L1 amplification showedsignificantly increased BCL-XL expression levels byWestern blottingcompared with wild-type cells (Fig. 1A and B; Supplementary Fig.S1A).However, another antiapoptotic protein, BCL2,wasnotnearlyexpressed in these four cell lines (Supplementary Fig. S1C). More-over, KRAS/BRAF-mutated, BCL2L1-amplified colorectal cancer tis-sues from three unrelated patients also showed elevated BCL-XLexpression via immunostaining compared with colorectal cancertissues from patients with wild-type KRAS/BRAF (Fig. 1C). In ourcolorectal cancer cohort, we found simultaneousKRAS/BRAFmuta-tion and BCL2L1 amplification in 21.7% (5/23) of patients (Sup-plementary Fig. S2). Because BCL-XL proteins were highly expressedin colorectal cancers withKRAS/BRAFmutation and BCL2L1 ampli-fication, this subset of colorectal cancers could be considered forBCL-XL–targeted treatments (24).

A combination treatment targeting BCL-XL and MCL1 in KRAS/BRAF-mutated and BCL2L1-amplified colorectal cancer cells

Inhibition of BCL-XL alone does not appear to be effectivelycytotoxic as BCL-XL inhibitors lead to upregulation of MCL1,which in turn leads to cellular resistance to the original BCL-XL–targeted treatments (12, 25). Therefore, to effectively inhibit theactionof BCL-XL, itmay benecessary to inhibit bothBCL-XL aswellasMCL1.We identifiednine chemicals thatwere previously shownto downregulate MCL1 proteins (Supplementary Table S1) andcombined each of thesewith the BCL-XL–inhibiting drug, ABT-263(26), on a representativeKRAS/BRAF–mutated, BCL2L1-amplifiedcell line (SW620; Supplementary Fig. S3) aswell as a representativeKRAS/BRAF wild-type, BCL2L1-diploid cell line (SNU1684; Sup-plementary Fig. S4). The drug combination of ABT-263þ YM-155appeared to have a synergistic effect in the KRAS/BRAF–mutated,BCL2L1-amplified cell lineswith little cytotoxic or synergistic effectin theKRAS/BRAFwild-type and/or BCL2L1-diploid cell lines (Fig.2; Supplementary Fig. S5). However, KRAS/BRAF–mutated andBCL2L1-amplified SW620 cells treated with siRNA for BCL-XL didnot show the synergistic effect of ABT-263/YM-155 combination(Supplementary Fig. S6). In addition, concomitant treatment ofYM-155 abrogated the ABT-263–induced increase of MCL1 levelsonly in theKRAS/BRAF–mutated,BCL2L1-amplified cell lines (Fig.2; Supplementary Figs. S5, S7, and S8). The synergistic effect ofcombination treatment is partly attributed to the increase ofcellular apoptosis, which was validated by the observation ofincreased cleavage of PARP and caspase-3 (Fig. 2; SupplementaryFigs. S5, S7, and S8), and the increase of Annexin-V–positive andAnnexin-V/propidium iodide (PI) double-positive cells in FACSanalyses (Supplementary Fig. S9A–S9C).

Downregulation of MCL1 levels by combination treatment ofABT-263 and YM-155

YM-155 has two possible functions, to inhibit survivin and toinhibit MCL1 (27, 28). When combined with ABT-263, siRNA

experiments against MCL1 dramatically decreased the relative cellviability as compared with the siRNA control or siRNA to survivin(Fig. 3A; Supplementary Fig. S10A). This result suggests thattargeted cell death may have been occurred due to YM-1550saction against MCL1 rather than survivin.

When BCL-XL is inhibited, BIM (Bcl-2 interacting mediator ofcell death) is released, leading to mitochondrial-induced apo-ptosis in cells having increased BCL-XL expression (29, 30).However, BCL-XL inhibition also upregulates MCL1, which isthen capable of binding the free BIM molecules, preventingcellular apoptosis (12). To address the question of whetherinhibition of both BCL-XL and MCL1 leads to significant releaseof BIM molecules, we conducted immunoprecipitation experi-ments and found that the combination of YM-155 with ABT-263 led to the reduction of BCL-XL–BIM and MCL1–BIMcomplexes and increased amounts of free BIM within thetargeted cells (Fig. 3B; Supplementary Fig. S10B).

Downregulation of MCL1 by YM-155 could be due to (i)decreased MCL1 mRNA expression, (ii) decreased MCL1 proteinsynthesis, or (iii) increased MCL1 protein degradation. We exam-ined MCL1 mRNA levels in two different KRAS/BRAF–mutated,BCL2L1-amplified cell lines (SW620 and HCT116) and found nosignificant decrease of MCL1 mRNA levels after treatment withYM-155 and after treatment with the combination of YM-155 andABT-263 (Fig. 3C). Next, we examined the rate of MCL1 proteindegradation in the SW620 cell line after treatmentwith YM-155 orcombination of YM-155 and ABT-263, and found no evidence foraccelerated MCL1 protein degradation in the presence of YM-155alone or combined with ABT-263 (Fig. 3D; Supplementary Fig.S10C). In addition, inhibition of proteosomal degradation didnot block the degradation of MCL1 by combination treatment ofABT-263 and YM-155 (Fig. 3E; Supplementary Fig. S10D).

Inhibition of mTOR pathway by combination treatment ofABT-263 and YM-155

To investigate whether downregulation of MCL1 is due todecreased MCL1 protein synthesis, we evaluated three proteins,S6K, eIF4E-binding protein 1 (4EBP1), and AKT, that are in themTOR pathway, which regulates protein translation of MCL1(31).We found that the combination treatment led to a deficiencyof phosphorylated and total S6K and AKT in KRAS/BRAF–mutat-ed, BCL2L1-amplified (SW620) cells (Fig. 4A; Supplementary Fig.S11A). In addition, the combination treatment led to the absenceof phosphorylated 4EBP1 in KRAS/BRAF–mutated, BCL2L1-amplified (SW620) cells (Fig. 4A; Supplementary Fig. S11A).However, these effects could not be observed in a KRAS/BRAFwild-type and BCL2L1-diploid cell line (SNU C1; Fig. 4A; Sup-plementary Fig. S11A). Taken together, this suggests that thedownregulation of MCL1 in these combination-targeted cells isin part due to the reduction of S6K, 4EBP1, and AKT, which mayinhibit the protein synthesis of MCL1.

Decreased levels of S6K protein does not appear to be due toinhibition of mRNA expression (Fig. 4B) or proteasomal degra-dation (Fig. 4C and D, Supplementary Fig. S11B and S11C), butrather via caspase-3–mediated protein degradation (Fig. 4E; Sup-plementary Fig. S11D). Downregulation of AKT was due to bothdecrease of mRNA expression (Fig. 4B) and decrease of proteinstability (Fig. 4C; Supplementary Fig. S11B) via caspase-3–medi-ated protein degradation (Fig. 4E; Supplementary Fig. S11D).Indeed, activation of caspase-3, which detected by cleavage ofPARP and caspase-3, preceded the degradation of AKT and S6K






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(Fig. 4F; Supplementary Fig. S11E). In addition, inhibition ofcaspase-3 restored MCL1 levels even in the presence of thecombination treatment of ABT-263 and YM-155 (Fig. 4E; Sup-plementary Fig. S11D).

Activation of death receptor pathway by combination treatmentof ABT-263 and YM-155

YM-155 was reported to activate death receptor pathway inpancreatic cancer cells (32), and we checked this possibility inour combination treatment for colorectal cancer cells. Cotreat-ment of YM-155 and ABT-263 leads to increased expressionof death receptor 4 (DR4)/DR5 and TRAIL (Fig. 5A and B), andactivated caspase-8 (Fig. 5C; Supplementary Fig. S12A) only inthe KRAS-mutated and BCL2L1-amplified cell line (SW620).In contrast, when we used CRISPR/Cas9 to specifically down-regulate DR5, sgRNA transfection deactivated caspase-3 andled to a subsequent increase of MCL1 protein levels in thepresence of the combination treatment of ABT-263 andYM-155 (Fig. 5D; Supplementary Fig. S12B). Taken together,cotreatment of ABT-263 and YM-155 appears to lead to

death receptor–mediated activation of caspase-3/8, inducingthe degradation of AKT and S6K, and resulting in translationalinhibition of MCL1. When both MCL1 and BCL-XL are effec-tively inhibited in KRAS/BRAF–mutated and BCL2L1-ampli-fied cells, free BIM molecules can trigger mitochondria-depen-dent cell apoptosis.

Transcriptome analysis of combination treatmentof ABT-263 and YM-155

To overview the global effect of each drug and combinationon KRAS/BRAF–mutated and BCL2L1-amplified cells, we inves-tigated the transcriptomic changes induced by drug treatmentusing RNA-seq. Total of 2,223 genes were significantly alteredby at least one treatment condition (P < 0.05; Fig. 6A; Supple-mentary Table S2). Compared with ABT-263 treatment, whichresulted in minimal gene expression changes (128 genes),YM-155 and combination treatment significantly alteredexpressions of 1,781 and 1,199 genes (P < 0.05), respectively,and exhibited a number of common altered genes (Fig. 6B).Gene Ontology (GO) functional enrichment analysis using

Figure 2.

Synergistic effect of ABT-263 and YM-155 only in colorectal cancer cells with both KRAS/BRAFmutation and BCL2L1 amplification. A–E, Combination cytotoxicityof ABT-263 and YM-155 in KRAS-mutated and BCL2L1-amplified cells (SW620; A), BRAF-mutated and BCL2L1-amplified cells (COLO205; B), KRAS/BRAF andBCL2L1 wild-type cells (SNU1684; C), KRAS-mutated and BCL2L1-diploid cells (LoVo; D), and KRAS/BRAF wild-type and BCL2L1-amplified cells (SNU C1; E).WST assays were used to examine the cell viability. Data are represented as mean values � SEM based on experiments that were performed in triplicate,and the regression lines were calculated by dose–response inhibition models in Prism 5.0. Combination index (CI) values were calculated at applied concentrations,and the pound signs represent synergistic effect of the two drugs (#, CI < 0.9; ##, CI < 0.6; ###, CI < 0.3). The Western blot images represent MCL1 levelsand the cleavage of caspase-3 (Casp3) and PARP after treatment of ABT-263 (1 mmol/L) and/or YM-155 (100 nmol/L) for 20 hours in each cell line. Triangles indicatecleaved fragments of PARP protein.

Figure 1.Upregulation of BCL-XL protein expressions in colorectal cancer with KRAS/BRAFmutation and BCL2L1 amplification. A, Protein expression in BCL-XL in colorectalcancer cell lines. Protein levels were estimated usingWestern blot analysis with actin as a loading control. The copy numbers of BCL2L1were determined by ddPCR,and the underlined numbers represent the BCL2L1-amplified samples (threshold cutoff of 3.0 copies). B, The quantified protein expression levels ofBCL-XL in colorectal cancer cell lines according to the status ofKRAS/BRAFmutation andBCL2L1 amplification. Thedensity of eachWestern blot band for BCL-XLwasmeasured by ImageJ software and normalized by actin expression levels. Asterisk indicates statistically significant differences (� , P < 0.05, one-way ANOVA,Tukey post hoc) compared with KRAS/BRAF wild-type and BCL2L1 dipolid cells. C, Protein expression of BCL-XL in colorectal cancer tissues. Protein levels wereevaluated by IHC using BCL-XL antibody (200�), and the relative staining intensities of BCL-XL expression were quantified by ImageJ software. Scale bar, 100 mm.Amp, amplification; MT, mutant; WT wild type.






Figure 3.

Molecular mechanisms of MCL1 downregulation by combination of ABT-263 and YM-155. A, The combined effect of MCL1 or survivin knockdown with ABT-263treatment in SW620 cells. The effect of the siRNA on protein expression wasmeasured usingWestern blot analysis (left), and cell viability was measured usingWSTassays (right). Data are represented as mean values � SEM based on experiments that were performed in triplicate, and the regression lines were calculated bydose–response inhibitionmodels in Prism 5.0. B, The interaction of BIMwith MCL1 and BCL-XL in SW620 cells treatedwith DMSO (control), ABT-263 (1 mmol/L), YM-155 (100 nmol/L), and combination of ABT-263 and YM-155. Cell lysates were immunoprecipitated by BIM (left) and MCL1 (middle) antibody, and BIM-boundMCL1 and BCL-XL were evaluated by Western blot analysis. Right, cell lysate input control. EL, extra-long; L, long; S, short. C, Relative transcript levels of MCL1 inSW620 and HCT116 cells treated with DMSO (control), ABT-263 (1 mmol/L), YM-155 (100 nmol/L), and combination of ABT-263 and YM-155. The mRNA levels werequantified by real-time PCR after 6 or 20 hours of treatment. Data are represented as mean values � SEM based on experiments that were performed in triplicate.D,Protein stability ofMCL1 under treatment of YM-155 (100nmol/L) and combination treatment ofABT-263 (1mmol/L) andYM-155 (100nmol/L). Theprotein levels ofMCL1 were evaluated after a 0- to 4-hour treatment with cycloheximide to inhibit protein translation (CHX; 100 mg/mL) in SW620. E, Effect of proteasomepathway inhibition onMCL1 protein levels under treatment of ABT-263 (1 mmol/L) and YM-155 (100 nmol/L). The protein levels of MCL1 were evaluated after 20 hoursof drug treatment with or without MG132 (10 mmol/L) in SW620, HCT116, and SNU C1 cells. Amp, amplification; MT, mutant.

Cho et al.





Figure 4.

Molecular mechanisms of inhibition of mTOR pathway by combination of ABT-263 and YM-155. A, The levels of proteins involved in mTOR pathway after drugtreatment. The levels of phosphorylated formand total proteinwere determined in SW620 (KRAS/BRAF-mutation andBCL2L1-amplification) and SNUC1 (wild-type)cells after 20 hours of DMSO (control), ABT-263 (1 mmol/L), YM-155 (100 nmol/L), and combination treatment. B, Relative transcript levels of AKT and S6K inSW620 and HCT116 cells treated with DMSO (control), ABT-263 (1 mmol/L), YM-155 (100 nmol/L), and combination of ABT-263 and YM-155. The mRNA levels werequantified by real-time PCR after 20 hours of treatment. Data are represented as mean values � SEM based on experiments that were performed intriplicate. C, Protein stability of AKT and S6K under treatment of ABT-263 (1 mmol/L) and YM-155 (100 nmol/L). The protein levels of AKT and S6K were evaluatedafter a 0- to 4-hour treatmentwith cycloheximide (CHX; 100 mg/mL) in SW620 cells.D, Effect of proteasome pathway inhibition onAKT and S6K protein levels undertreatment of ABT-263 (1 mmol/L) and YM-155 (100 nmol/L). The protein levels of AKT and S6K were evaluated after 20 hours of drug treatment with orwithout MG132 (10 mmol/L) in SW620 and HCT116 cells. E, Effect of pan-caspase inhibitor, Z-VAD-FMK treatment (left; 20 mmol/L) and caspase-3–specific inhibitor,Z-DEVD-FMK treatment (right; 20 mmol/L) on the protein levels of AKT, S6K, and MCL1 in SW620 cells. F, Time-course protein changes of PARP, cleaved caspase-3(Cleaved Casp3), AKT, S6K, and MCL1 after cotreatment with ABT-263 (1 mmol/L) and YM-155 (100 nmol/L).






Figure 5.

Molecular mechanisms of activation of death receptor pathway by combination of ABT-263 and YM-155. A, Relative transcript levels of DR4 and DR5 in colorectalcancer cells treated with DMSO (control), ABT-263 (1 mmol/L), YM-155 (100 nmol/L), and combination of ABT-263 and YM-155. The mRNA levels werequantified by real-time PCR after 20 hours of treatment. Data are represented as mean values� SEM based on experiments that were performed in triplicate. Amp,amplification; MT, mutant; WT, wild type. B, Relative transcript levels of TRAIL in colorectal cancer cells treated with DMSO (control), ABT-263 (1 mmol/L),YM-155 (100 nmol/L), and combination of ABT-263 and YM-155. The mRNA levels were quantified by real-time PCR after 20 hours of treatment. Data arerepresented as mean values � SEM based on experiments that were performed in triplicate. Amp, amplification; MT, mutant; WT, wild type. C, The cleavageof caspase-8 (Casp8) and caspase-9 (Casp9) in SW620 (KRAS/BRAF mutation and BCL2L1 amplification) and COLO320 (wild-type) cells treated withDMSO (control), ABT-263 (1 mmol/L), YM-155 (100 nmol/L), and combination of ABT-263 and YM-155. D, Effect of DR5 downregulation on ABT-263/YM-155-combination-induced activation of caspase-3 in SW620 cells. The cleavage of caspase-3 (Casp3) and PARP was evaluated using Western blotanalysis after 20-hour treatment of drugs.

Cho et al.





ClueGo (19) indicated that 737 genes, which were significantlyderegulated in combination treatment sample compared withother samples (Supplementary Table S3), are involved in sev-eral biological processes, including cell development, secretion,cell migration, regulation of signal transduction, cell surfacereceptor signaling pathway, and response to hypoxia (Fig. 6C;Supplementary Table S4). These results suggest that YM-155has more profound effects on gene expression compared withABT-263, and combination treatment of ABT-263 and YM-155produces stressful condition of cells by activating apoptosispathway and induced reactive hyperactivation of several cellu-lar processes especially in secretion, cell migration, and signal-ing transduction.

Efficacy of the ABT-263 and YM-155 combination treatment inin vivo patient-derived xenograft mice models

We established PDX mouse models from patient C033 (Fig.7A) andC178 (Supplementary Fig. S13A), known to be colorectalcancers with KRAS mutations (G13D for C033, G12V for C178)and BCL2L1 amplification (3 copies for C033, 4 copies for C178),for in vivo drug efficacy testing. These mice exhibited a synergistic

effect by the combined use of ABT-263 and YM-155 (Fig. 7A;Supplementary Fig. S13A). However, the synergistic effect of ABT-263 and YM-155 was not observed in PDX models from a KRASwild-type and BCL2L1-diploid patient (Supplementary Fig.S13B). We found similar synergistic effect by the combinationof ABT-263 and YM-155 in SW620 cell line xenograft models[KRAS-mutated (G12V) and BCL2L1-amplified (4 copies); Sup-plementary Fig. S14A], but not in Colo320HRS cell line xenograftmodels (KRAS wild-type and BCL2L1-diploid; SupplementaryFig. S14B). Histologic analyses showed increased expression ofMCL1 by the ABT-263 treatment, which was abrogated by con-comitant treatment with YM-155 (Fig. 7B). Furthermore, theconcentration of apoptotic cells was increased in tumor tissueafter the combination treatment based on TUNEL and cleavedcaspase-3 staining (Fig. 7B).However, therewas little difference incell proliferation in single-treated or combination treated cells, asexhibited by Ki67 immunostaining (Fig. 7B). These results areconsistent with the notion that the combination treatment ofABT-263 and YM-155 is effective in this colorectal cancer sub-group, genomically defined by KRAS/BRAF mutations andBCL2L1 amplification.

Figure 6.

Transcriptomic analysis of ABT-263 and YM-155 treatment. A, Gene expression profiling of KRAS/BRAF–mutated and BCL2L1-amplified cells (SW620)after 8 hours of treatment with DMSO (control), ABT-263 (1 mmol/L), YM-155 (100 nmol/L), and combination of ABT-263 and YM-155. Heatmap showshierarchical clustering of treated cells and 2,223 genes deregulated in response to treatment in at least one condition (P < 0.05). Each row representsa gene and each column a sample. Red and green indicate expression levels above and below the median of each gene expression across the samples,respectively. B, Venn diagram of genes differentially expressed after each treatment. Up, genes upregulated (P < 0.05); down: genes downregulated(P < 0.05). C, Network representation of enriched Gene Ontology (GO) biological processes (analyzed by ClueGO, P < 0.05), deregulated by combinationtreatment of ABT-263 and YM-155 compared with other three samples (control, ABT-263, and YM-155). The node size represents the term enrichmentsignificance, and the nodes are linked on the basis of their kappa score (�0.4), where the label of the terms with the most genes per group are shown.Functionally related groups partially overlap.






DiscussionAnti-EGFR agents, including cetuximab and panitumumab, are

effective for the treatment of some patients with colorectal cancer(33–35).However, these anti-EGFR treatments are less effective inpatients with KRAS or BRAF mutations (11, 36, 37). Therefore,there has been an urgent need to develop new therapeutic strat-egies for targeting KRAS/BRAF–mutated colorectal cancerpatients. In this study, we have identified a genomically definedsubgroup having KRAS/BRAF mutations and BCL2L1 amplifica-tion. This subgroup, found in as much as 21.7% of colorectalcancers examined, characteristically exhibits high expression ofBCL-XL. Here, we show that these colorectal cancers can beeffectively treated using a combination therapy targeting bothBCL-XL (with ABT-263) and MCL1 proteins (with YM-155). Wehave validated the efficacy of this cotreatment using in vitro celllines and in vivo PDX models.

To overcome drug resistance in colorectal cancer patients withKRAS/BRAF mutations, multiple combination treatmentapproaches have already been undertaken. Because KRAS/BRAFmutations result in the activation of MEK/MAPK pathway, MEK

inhibitor–based combination treatments have been most oftentested, with combination that include a PI3K inhibitor (38), anmTOR inhibitor (39), an AKT inhibitor (40), an IGF1R inhibitor(41), an STK33 inhibition (42), and a BCL-XL inhibitor (43). Inaddition, other combination strategies have been attempted,including a combination of BCL-XL and mTOR inhibitors (12),a combination of EGFR and PI3K inhibitors (44), and a combi-nation of proteasome and topoisomerase inhibitors (45). Amongthese combination approaches, no single effective therapy inclinic has been established. Furthermore, it is probable that evenKRAS/BRAF–mutated colorectal cancers exhibit heterogeneousresponses to specific combination treatments.

The novelty of our study is that the dual inhibition of BCL-XL

and MCL1 is consistently effective in colorectal cancer cells withboth KRAS/BRAF mutation and BCL2L1 amplification, not incolorectal cancer cells with KRAS/BRAF mutation and BCL2L1diploid. We showed that colorectal cancer cells with KRAS/BRAFmutation and BCL2L1 diploid exhibited diverse BCL-XL expres-sion levels (Fig. 1B), and combination of ABT-263 and YM-155was not highly effective in these cell lines (Fig. 2D; SupplementaryFig. S5D).On the contrary, colorectal cancer cells with bothKRAS/

Figure 7.

In vivo efficacy ofABT-263 andYM-155treatment in patient-derivedxenograft (PDX) models with KRAS/BRAF mutation and BCL2L1amplification. A, The efficacy of ABT-263 and YM-155 in PDX models fromKRAS-mutated (G13D) and BCL2L1-amplified (3 copies) patient. The micewere treated with ABT-263 (100 mg/kg/day), YM-155 (5mg/kg/week), andcombination of ABT-263 and YM-155for 23 days (n ¼ 4). Average tumorsizes for each group are plotted (left),and representative tumors aftertreatment were shown (right). Thedifference of tumor growth ratebetween different drug-treatedgroups was analyzed by growth curveanalysis (asterisk indicates P¼0.026).Scale bar, 10mm.B, Immunohistologicanalysis of drug-treated residualtumors after treatment of ABT-263and YM-155 in PDX models. Therepresentative microscopic images ofH&E staining and immunohistologicanalysis are shown (200�). Therelative staining intensities of MCL1,TUNEL (terminal deoxynucleotidyltransferase dUTP nick end labeling),cleaved caspase-3, and Ki67 werequantified by ImageJ software. Scalebar, 100 mm.

Cho et al.





BRAF mutation and BCL2L1 amplification showed significantlyincreased BCL-XL expression levels (Fig. 1B), and combination ofABT-263 and YM-155 was consistently effective in these cell lines(Fig. 2A and B; Supplementary Fig. S5A and S5B). These resultssuggest that the colorectal cancer subtype with KRAS/BRAFmuta-tion can be divided into two subgroup according to BCL2L1 copynumber alteration (BCL2L1 diploid vs. BCL2L1-amplified), andcolorectal cancer cells with both KRAS/BRAF mutation andBCL2L1 amplification can be the target of our combinationtreatment.

Previous studies found that mutant KRAS increased the expres-sion levels of BCL-XL via ERK and STAT3-mediated pathways byboth transcriptional and posttranscriptional mechanisms (21,22). BCL-XL was also reported to be associated with the resistanceof MEK and PI3K inhibitors in KRAS-mutant cancers (43, 46).Therefore, overexpressed BCL-XL in colorectal cancer patientswithKRAS mutation is a feasible target of BCL-XL inhibitor–basedcombination treatment. In addition,MCL1 levelswere consideredto be a predictive marker for BCL-XL inhibitor, ABT-263 response(47). This study demonstrated that, when combined with ABT-263, the YM-155 activates a cell death receptor, leading to caspase-8 and caspase-3–mediated inhibition of the mTOR pathway bydegradation of AKT and S6K and subsequent downregulation ofMCL1 protein translation. Simultaneous inhibition of BCL-XL

and MCL1, using ABT-263 and YM-155, treatment showed cyto-toxic effects in glioblastoma and lung cancer cell lines (48).Recently, a specific MCL1 inhibitor, A-1210477, showed syner-gistic activity with ABT-263 in pancreas, bladder, lung cancer, andmyeloma cell lines (49), and this combination needs to beevaluated for colorectal cancers with KRAS/BRAF mutation andBCL2L1 amplification. Therefore, simultaneous targeting of BCL-XL andMCL1 is a judicious strategy for KRAS/BRAF–mutated andBCL2L1-amplified colorectal cancers.

Althoughwe suggested that ABT-263 and YM-155 inhibited theactivity of BCL-XL and the expression of MCL1, respectively, thesetwo drugs might have additional targets in inducing cell death.ABT-263 was known to inhibit other BCL2 family proteinsincluding BCL2. We found that colorectal cancer cell lines withKRAS/BRAF mutation and BCL2L1 amplification had lower pro-tein expression levels of BCL2 (Supplementary Fig. S1D), suggest-ing that the effect of BCL2 inhibition by ABT-263 was minimal incombination treatment of ABT-263 and YM-155. YM-155 wasoriginally developed as a survivin inhibitor, but we exhibited that

inhibition of survivin is dispensable for combination effect ofABT-263 and YM-155 via siRNA experiment (Fig. 3A). Therefore,the main targets of ABT-263 and YM-155 are BCL-XL and MCL1.However, these twodrugs canhave new targets that are not knownuntil now, and the effect on these new targets could improve thecombination effect of the drugs in the treatment of colorectalcancers.

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

Authors' ContributionsConception and design: S.-Y. Cho, J.Y. Han, D. Na, J.-I. Kim, H. Park,W.-S. Lee, C. LeeDevelopment of methodology: J.Y. Han, J. Lee, H. ParkAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.-Y. Cho, J.Y. Han, W. Kang, A. Lee, J. Kim, J. Lee,S. Min, J. KangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.-Y. Cho, J.Y. Han, J. Kim, J. ChaeWriting, review, and/or revision of the manuscript: S.-Y. Cho, J.Y. Han,H. Park, C. LeeAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.Y. Han, J. Kim, J. LeeStudy supervision: J.Y. Han, J.-I. Kim, H. Park, C. Lee

AcknowledgmentsThe authors would like to thank Drs. Asis Das and Dave Mellert for critical

reading of this manuscript.

Grant SupportThis work was supported by Gil Hospital Research Grant (GCU-2015-5092

and FRD20142802; to W.-S. Lee); the Korean Healthcare Technology R&Dproject through the Korean Health Industry Development Institute (KHIDI)funded by the Ministry of Health & Welfare, Republic of Korea (grant no.HI13C2148; to C. Lee); the International Research & Development Program ofthe National Research Foundation of Korea (NRF) funded by the Ministry ofScience, ICT & Future Planning (grant no. 2015K1A4A3047851; to C. Lee); andthe National Cancer Institute of the NIH (grant P30CA034196). C. Lee is adistinguished Ewha Womans University Professor supported in part by theEwha Womans University Research grant of 2016.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 2, 2016; revised March 27, 2017; accepted June 6, 2017;published OnlineFirst June 13, 2017.

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Cho et al.




2017;16:2178-2190. Published OnlineFirst June 13, 2017.Mol Cancer Ther Sung-Yup Cho, Jee Yun Han, Deukchae Na, et al.

-amplified Colorectal CancersBCL2L1-mutated and KRAS/BRAF and MCL1 for LA Novel Combination Treatment Targeting BCL-X

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