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Cancer Biology and Translational Studies

Acquired Resistance to BET-PROTACs(Proteolysis-Targeting Chimeras) Caused byGenomic Alterations in Core Components of E3Ligase ComplexesLu Zhang1, Bridget Riley-Gillis2, Priyanka Vijay2, and Yu Shen1

Abstract

Proteolysis-targeting chimeras (PROTAC) are bifunction-al molecules that hijack endogenous E3 ubiquitin ligases toinduce ubiquitination and subsequent degradation of pro-tein of interest. Recently, it has been shown that PROTACswith robust in vitro and in vivo activities and, in some cases,drug-like pharmaceutical properties can be generated usingsmall-molecule ligands for the E3 ligases VHL and CRBN.These findings stoked tremendous enthusiasm on usingPROTACs for therapeutics development. Innate andacquired drug resistance often underlies therapeutic fail-ures, particularly for cancer therapy. With the PROTACtechnology progressing rapidly toward therapeutic applica-

tions, it would be important to understand whether andhow resistance to these novel agents may emerge. UsingBET-PROTACs as a model system, we demonstrate thatresistance to both VHL- and CRBN-based PROTACs canoccur in cancer cells following chronic treatment. However,unlike what was often observed for many targeted thera-peutics, resistance to BET-PROTACs did not result fromsecondary mutations that affect compound binding to thetarget. In contrast, acquired resistance to both VHL- andCRBN-based BET-PROTACs was primarily caused by geno-mic alterations that compromise core components of therelevant E3 ligase complexes.

IntroductionUbiquitination-mediated proteolysis is a centralmechanism of

protein homeostasis in cells. E3 ubiquitin ligases bind to definedsubstrates and mediate ubiquitination and degradation of theseproteins. Cullin-RING ubiquitin ligases (CRL) are the largestfamily of E3 ubiquitin ligases. CRLs are multicomponent com-plexes that minimally consist of a cullin, a RING finger protein,and a substrate recognition subunit (1). Proteolysis-targetingchimeras (PROTAC) are bifunctional molecules that hijackendogenous E3 ubiquitin ligase to cause ubiquitination andsubsequently degradation of proteins of interest (2). VHL andCereblon (CRBN) are the substrate recognition subunit of thecullin2 (CUL2)-containing VHL–CRL complex and the cullin4-containing CRBN–CRL complex, respectively (3, 4). Recently, ithas been shown that PROTACs with robust in vitro and in vivoactivities and, in some cases, drug-like pharmaceutical propertiescan be generated using small-molecule ligands for the E3 ligasesVHL andCRBN (5–9). PROTACs targeting the bromodomain andextraterminal domain proteins (BET-PROTAC) are the prototype

of these small-molecule PROTACs (5–7). BET-PROTACs triggerrapid and prolonged degradation of BET proteins with exception-al potencies, and exhibit robust antitumor activities in preclinicalmodels of leukemia, lymphoma, prostate cancer, and triple-negative breast cancers (5, 6, 10, 11).

Compared with traditional small-molecule inhibitors,PROTACs offer several advantages. For example, PROTACs canexert more rapid, potent, and durable inhibition of targets,abolish the scaffolding function of a protein, and turn a non-functional binder into functional degraders. In addition, thestringent conformational requirement and potential linker inter-action between target protein and E3 ligase within ternary com-plexmay allow PROTACs to offer an additional layer of selectivityover small-molecule inhibitors (12–15). These unique propertiesmake PROTACs a promising modality for the development ofnext-generation therapeutics. However, innate and acquired drugresistance is a common cause of therapeutic failure, particularlyfor cancer therapy. With the rapid advancement of the PROTACtechnology toward therapeutic applications, it would be impor-tant to understand whether and how drug resistance to thesenovel agents may emerge. In this study, using BET-PROTACs withboth VHL and CRBN ligands as a model system, we interrogatepotential mechanisms of acquired resistance to PROTACs incancer cell lines.

Materials and MethodsCells, compounds, and antibodies

SKM1,MV4:11, LNCaP, andOVCAR8 cell lines were purchasedfrom ATCC or DSMZ andmaintained by a Core Cell Line Facility.All cell lineswere tested forMycoplasmausingMycoAlertDetectionKit (Lonza) and authenticated using the Gene Print10 STR Kit

1Oncology Discovery, AbbVie Inc., North Chicago, Illinois. 2Genomic ResearchCenter, AbbVie Inc., North Chicago, Illinois.

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

Corresponding Authors: Lu Zhang, AbbVie Inc., North Chicago, IL, 60064.Phone: 847-938-2212; Fax: 847-935-0014; E-mail: [email protected]; andYu Shen, [email protected]

Mol Cancer Ther 2019;18:1302–11

doi: 10.1158/1535-7163.MCT-18-1129

�2019 American Association for Cancer Research.

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(Promega) andmaintained in RPMI1640 mediumwith 10% FBS(Gibco). ABBV-075, ARV-771, and ARV-825 were synthesized atAbbVie by themethods according to (5, 6, 16). All antibodieswerepurchased from commercial sources as follows: antibodiesagainst BRD2/3/4 from Bethyl; antibodies against c-MYC, PARP,and VHL fromCell Signaling Technology; antibody against CRBNfrom Thermo Fisher Scientific; antibody against CUL2 fromAbcam; and antibody against b-actin from Sigma. O1R- andO3R-overexpressing cells were created by infection with pLOCor pLOC-CUL2 or pLOC-CRBN lentiviral particle (Dharmacon)in the presence of 10 mg/mL of polybrene. Cells were selected with10 mg/mL of blasticidin. Western blot analysis was performed toconfirm expression of these proteins in the cells.

Cell viability and caspase 3/7 activity assayCells were seeded in 96-well plates and incubated at 37�C in

an atmosphere of 5% CO2. Compounds were added at aseries of dilution after overnight incubation. After 3 daysincubation, Caspase-Glo3/7 luminescent and CellTiter-GloAssay (Promega) were performed according to the manufac-turer's instruction. Luminescence signal from each well wasmeasured using the EnSpire Luminometer (PerkinElmer), andthe data were analyzed using the GraphPad Prism Software(GraphPad Software Inc.).

qPCRTotal RNA was harvested using the RNeasy Plus Mini Kit

(Qiagen) and cDNA was made with the High-Capacity cDNAReverse Transcription Kit (Applied Biosystems). qPCR was per-formed with TaqMan Universal Master Mix and Probes (LifeTechnologies), and analyzed using the DDCt method. Data arepresented as mean � SD. The difference between two groups wasevaluated using the two-tailed Student t test. P values less than0.05 were considered statistically significant.

Western blot analysisCell lysates were prepared in Laemmli Buffer (Bio-Rad). Thirty

micrograms of total protein was resolved on a 7%–12%SDS-polyacrylamide gel and probed with corresponding primaryantibodies.

Whole-exome sequencingSamples were prepared in accordance with the manufacturer's

instructions for the KAPAHyper Prep and NimbleGen SeqCap EZMedExome kits. Briefly, 100 ng of DNA was sheared using aCovaris M220 sonicator (adaptive focused acoustics). DNA frag-ments were end-repaired, adenylated, ligated with KAPA dualindexing adapters, and amplified by 11 cycles of PCR with theKAPA hyper prep kit. Samples were pooled into a precapturelibrary and targeted capture was performed using the NimbleGenSeqCap EZMedExome capture probe set. Captured libraries werethen enriched by 14 cycles of PCR and final libraries wereevaluated using Qubit (Thermo Fisher Scientific) and AgilentTape Station and were sequenced on an Illumina NextSeqsequencer using 2 � 75 bp read length. The raw sequencing datais deposited at SRA (submission no. SUB5567117).

Exome sequencing reads were processed through the SentieonTNscope Pipeline (Sentieon Inc.) for alignment and somaticvariant calling (parental sample designated at "normal", O1Rand O3R designated as "tumor"). Quality of sequencing data wasassessed using Picard (http://broadinstitute.github.io/picard)

and MultiQC (17). SNV and indel variant calls were importedinto the VarSeq Software (Golden Helix) for filtering andannotation.

Somatic copy number calling was performed using VarScanv2.4.2 using tumor/normal mpileup from samtools v1.7 asinput (18). Parameters used were minimum segment size of100 bp, minimum coverage of 20 reads, minimum mappingquality of 20, and minimum base quality of 20. LOH in deletedregions were also visually confirmed by plotting minor allelefrequencies from germline variant calling with Sentieon jointHaplotypeCaller (Sentieon Inc.).

RNA sequencingEach cell line was sampled in triplicate and mRNA library

preparation from total RNA was conducted following the man-ufacturer's protocol for the Illumina TruSeq mRNA preparationkit. Briefly, 1 mg of total RNA was purified by using poly-T oligosattached to magnetic beads then fragmented by divalent cationsunder elevated temperature. The fragmented RNA underwent firststrand synthesis using reverse transcriptase and random primers.Second strand synthesis created the cDNA fragments using DNApolymerase I and RNaseH. The cDNA fragments then wentthrough end repair, adenylation of the 30 ends, and ligation ofadapters. The cDNA library was enriched using 15 cycles of PCRand purified. Final libraries were assessed using the AgilentBioanalyzer and Qubit (Thermo Fisher Scientific) assay methodsthen sequenced on an Illumina NextSeq sequencer using 2 � 75bp read length. The raw sequencing data is deposited at SRA(Submission no. SUB5567117).

RNA-sequencing reads were mapped to the human referencegenome (GRCh38) using STAR aligner and genes werequantified using featureCounts for all genes annotated inGencode v28 (19, 20). Quality of sequencing data was assessedusing Picard (http://broadinstitute.github.io/picard) andMultiQC (17). Genes with counts per million less than one intwo-thirds of samples or more were considered too lowlyexpressed and excluded. Differential gene expression (DGE) anal-ysis comparing parental versusO1R and parental versusO3R (n¼3 for each) was then performed using linear modeling in limmawith TMM normalization and voom transformation (21) DGEresults were plotted using GLIMMA (22). Sashimi plots wereprepared using integrated genome viewer (23).

TaqMan CNV confirmationConfirmation of exome copy number calls forCUL2 andCRBN

was performed using TaqMan copy number assays. Three prede-signed assays each forCUL2 andCRBN (Supplementary Table S1)were ordered alongwith the reference assays for TERT and RNasePgenes. Experiments were performed according to the manufac-turer's instructions using 20 ng DNA per reaction, 2� TaqMangenotyping master mix, and both a target (CUL2 or CRBN) andreference assay (TERT or RNaseP). Each assay combination wasrun in quadruplicate for parental, O1R, O3R, and a no templatecontrol. qPCR was run on the ABI 7500 and analyzed using theCopyCaller Software employing the DDCt method for normal-ization of copy calls relative to the reference assay and parentalsamples.

Sanger sequencingSequencing primers were designed using Primer3 software

(version 1.1.4, http://www.sourceforge.net) and were purchased

Defects in E3 Ligase Complex Cause Resistance to BET-PROTACs

www.aacrjournals.org Mol Cancer Ther; 18(7) July 2019 1303



http://broadinstitute.github.io/picard

http://broadinstitute.github.io/picard

http://www.sourceforge.net


from Integrated DNA Technologies (Supplementary Table S2).GenomicDNAwas amplified by singleplex PCR using the FailSafePCR System (Epicentre). Thermal cycling was performed with 40cycles [30 seconds at 98�C; 30 seconds at 62�C (�0.5�C eachcycle); 60 seconds at 72�C], followed by 25 cycles (30 seconds at

98�C; 30 seconds at 55�C; and 60 seconds at 72�C). Ampliconswere bidirectionally sequenced using Big Dye Terminator version1.1 technology on an ABI 3130xl System (Applied Biosystems).Sequence analysis was performed using the Sequencher softwareversion 5.4.6 (Gene Codes Corporation).

Figure 1.

Cancer cells develop drug resistance after chronic exposure to BET-PROTACs. A, Sensitivity to ARV-771 or ARV-825 of parental OVCAR8 (black) and resistantderivatives O1R (blue) and O3R (red) was assessed by CTG assays. Data represent mean� SD. N¼ 3. The table lists the IC50 value fold change of resistant cellsover parental cells for each compound. B, Cells were treated with indicated concentration of ARV-771 or ARV-825 for 48 hours and harvested for Westernblot analysis using cleaved PARP antibody. b-Actin was used as loading control. C, Caspase 3/7 assay was performed in parental and resistant cells treated witha series dilution of ARV-771 or ARV-825 for 24 hours. Data represent mean� SD. N¼ 3.

Zhang et al.

Mol Cancer Ther; 18(7) July 2019 Molecular Cancer Therapeutics1304




ResultsChronic exposure to BET-PROTACs leads to drug resistance incancer cell lines

Drug resistance often arises following chronic exposure tocancer therapeutic agents. To investigate whether PROTACs aresubject to similar drug resistance issues, we exposed two AML(SKM-1 andMV4:11) and two solid tumor (LNCaP andOVCAR8)cell lines to increasing concentrations of VHL- or CRBN-basedBET-PROTACs over a 4 month period. The establishment of theresistant cell lines is illustrated in Supplementary Fig. S1A.Although all the four cell lines underwent apoptosis after BET-PROTAC treatment, SKM1, MV4:11, and LNCaP cells were muchmore sensitive to these compounds compared with OVCAR8,likely due to the strong dependency on BET proteins in AML and

prostate cancer (refs. 6, 24, 25; Supplementary Fig. S2). While nostable resistant clones were obtained from SKM-1, MV4:11, orLNCaP cells incubated with either of the VHL- or CRBN-basedBET-PROTACs, several resistant clones emerged from theOVCAR8 cells. These resistant clones exhibited greater than40� IC50 increase over the parental cell line for the VHL-basedBET-PROTAC ARV-771 (6) or the CRBN-based BET-PROTACARV-825 (ref. 5; Fig. 1A).Withdrawing BET-PROTAC fromculturemedia for 2 months did not diminish resistance, indicating thatresistance to BET-PROTACs in these cells may result from stablegenetic changes (Supplementary Fig. S1B). In addition, the resis-tant clones maintained similar sensitivities as the parental celllines to the bromodomain inhibitor ABBV-075 (16), suggestingthat the BET bromodomains in these cells retain the ability of

Figure 2.

BET-PROTACs fail to induce BETprotein degradation in the resistantcells. A, Cells were treated withindicated concentration of ARV-771or ARV-825 for 16 hours andharvested forWestern blot analysisusing antibodies against BET familyproteins and c-Myc. b-Actin wasused as loading control. B, Cellswere treated with 0.05 mmol/L ofARV-771 or ARV-825 for indicatedtime and harvested forWesternblot analysis using antibodiesagainst BRD4. b-Actin was used asloading control.






binding BET inhibitors (Supplementary Fig. S1C). It is notewor-thy that the O1R cells, which are resistant to the VHL-basedBET-PROTAC ARV-771, remained sensitive to the CRBN-basedBET-PROTAC ARV-825. Conversely, the O3R cells, which areresistant to the CRBN-based BET-PROTAC ARV-825, remainedsensitive to the VHL-based BET-PROTAC ARV-771 (Fig. 1A).Consistent with what was observed in the proliferation assays,analysis of PARP cleavage and caspase activation further con-firmed that the O1R cells were resistant to ARV-771- but notARV-825–induced apoptosis, and the O3R cells were resistant toARV-825- but not ARV-771–induced apoptosis (Fig. 1B and C).Taken together, these results demonstrate that the O1R cells arespecifically resistant to the VHL-based BET-PROTAC (ARV-771),and the O3R cells are specifically resistant to the CRBN-basedBET-PROTAC (ARV-825).

BET-PROTACs fail to induce BET protein degradation in theresistant cells

It has been shown that BET-PROTACs inhibit cell growth andcause apoptosis by inducing degradation of BET family proteins.As expected, treatment of ARV-771 for 16 hours caused degrada-tion of BRD2, BRD3, and BRD4, and inhibited the well-established BET target gene Myc in the parental and the O3R cellsin a dose-dependentmanner (Fig. 2A). In contrast, ARV-771 failedto induce significant BETprotein degradation orMyc inhibition inthe O1R cells even at the very high concentration of 3 mmol/L.Similarly, treatment of ARV-825 for 16 hours caused BET proteindegradation andMyc inhibition in the parental andO1R cells at aslow as 30 nmol/L (Fig. 2A). However, ARV-825 did not degradeBRD2 or BRD4 protein efficiently at up to 3 mmol/L and onlypartially degraded BRD3 protein in O3R cells at higher

Figure 3.

Exome sequencing identifiedmutations and deletion in CUL2 gene. A, Amodel of CUL2 ligase. Adapted from Chen and colleagues (35). B, CUL2 somaticmutations identified through exome sequencing of parental and O1R samples. C, Sashimi plot of RNA-seq read coverage of CUL2 exons 11–14. Exon 12 skippingpresent in O1R replicates (red) but not in parental replicates (black). Solid peaks depict reads per kilobase per million; reads mappedwithin individual exons.Reads that bridge different exons are shown as arcs. Intron deletion and T435Hfs deletion indicated by blue and red arrows, respectively. D, CUL2 deletiondetected in O1R by copy number calling on exome-sequencing data (relative to parental). E, CUL2 deletion confirmation by TaqMan CNV assay.

Zhang et al.





concentration (Fig. 2A). This is consistent with previous reports inother cancer types that BRD3 is more readily degraded by BET-PROTACs compared with BRD2 and BRD4 (6, 24, 26). Timecourse studies further established that extending BET-PROTACtreatment to 48 hours still could not trigger BRD4 degradation inthe resistant cells (Fig. 2B). These results collectively demonstratethat the O1R and O3R cells are resistant to BET protein degrada-tion inducedbyVHL-orCRBN-basedBET-PROTACs, respectively.The absence of cross resistance to both VHL- and CRBN-basedBET-PROTAC suggests that the proteasome degradation machin-ery downstream of protein ubiquitination remains functional inthese resistant cells.

Resistance to VHL-based BET-PROTAC is caused by CUL2 lossdue to multiple genomic alterations at the CUL2 locus

To unravel the mechanism of resistance to VHL-basedBET-PROTAC in the O1R cells, we examined the genomic andtranscriptional differences such as acquired mutations, geneexpression changes, and copy-number variation (CNV)between the parental and O1R cells using whole-exomesequencing and RNA-seq. No genomic or mRNA expressionalterations of BET family proteins or VHL were found in theO1R cells, and the protein level of VHL is comparable in theparental and the O1R cells (Supplementary Fig. S1D). Althoughthe protein levels of BRD4 and BRD3 are slightly decreased inO1R and O3R cells, there is still significant amount of these two

proteins, especially BRD4, detected (Supplementary Fig. S1D).Interestingly, the O1R cells were found to possess multiplegenomic alterations that impact the gene encoding CUL2, acritical component of the VHL–CRL complex (Fig. 3A). Thealterations include a frame-shift mutation predicted to causenonsense-mediated mRNA decay by introducing a prematurestop codon at position 442 in the cullin homology domain; anintronic mutation upstream of exon 12 with concomitantskipping of exon 12; and a large-scale deletion of 42 Mbencompassing the CUL2 gene (Fig. 3B–D). To investigatethe copy number of this region, we used SNP minor allelefrequencies across chromosome 10 in the parental, O1R, andO3R cells (Supplementary Fig. S3A). The pattern suggested thepresence of other chromosomal aberrations preexisting in theparental, making the estimation of exact copy number chal-lenging. Follow-up experiments confirmed the frame shift andthe intronic mutations by Sanger sequencing and the copynumber loss by TaqMan CNV analysis (Fig. 3E; SupplementaryFig. S3C and S3D). Consistent with the abnormalities at thegenomic level, RNA-seq revealed CUL2 to be one of the mostsignificantly downregulated genes in the O1R cells comparedwith parental cells (Supplementary Fig. S4A). qPCR andWestern blot analysis further established the significant reduc-tion of CUL2 mRNA and CUL2 protein in the O1R cellscompared with the parental cells (Fig. 4A and B). In contrast,the O3R cells (resistant to the CRBN-based BET-PROTAC) were

Figure 4.

Resistance toward VHL-based BETPROTAC ARV-771 derives fromdepletion of CUL2. A, qPCR analysisof CUL2mRNA level in parental,O1R, and O3R cells. Data representmean� SD. N¼ 3. B,Western blotanalysis of CUL2 protein level inparental, O1R, and O3R cells.b-Actin was used as loading control.Quantification of CUL2 protein levelis shown below. C,Western blotanalysis of CUL2 in O1R cellsexpressing empty vector or CUL2cDNA. b-Actin was used as loadingcontrol. D,Dose–response curvesfor ARV-771 in O1R cells expressingempty vector or CUL2 cDNA. Datarepresent mean� SD. N¼ 3. E,O1Rcells expressing empty vector orCUL2 cDNAwere treated withindicated concentration of ARV-771for 16 hours and harvested forWestern blot analysis. b-Actin wasused as loading control.






devoid of these genomic abnormalities in the CUL2 gene, andexpression of CUL2 was comparable with the parental cells(Fig. 4A and B).

The hypoxia inducible factors HIF-1A and HIF-2A are physio-logic substrates of the VHL–CRL complex. Under normoxic con-ditions, the VHL–CRL complex mediates HIF-1 ubiquitination

Figure 5.

Resistance to CRBN-based BET PROTAC is caused by the loss of Cereblon (CRBN) gene due to chromosomal deletion.A, CNV plot of chromosome 3 from O3Rcells. B, CRBN deletion confirmation by TaqMan CNV assay. C, qPCR analysis of CRBNmRNA level in parental, O1R, and O3R cells. Data represent mean� SD.N¼ 3. D,Western blot analysis of CRBN level in parental, O1R, and O3R cells. b-Actin was used as loading control. Quantification of CRBN protein level is shownbelow. E,Western blot analysis of CRBN in O3R cells expressing empty vector or CRBN cDNA. b-Actin was used as loading control. F, Dose–response curves forARV-825 in O3R cells expressing empty vector or CRBN cDNA. Data represent mean� SD. N¼ 3.G,O3R cells expressing empty vector or CRBN cDNAweretreated with indicated concentration of ARV-825 for 16 hours and harvested for Western blot analysis. b-Actin was used as loading control.

Zhang et al.





and degradation, consequently preventing HIF-1–dependenttranscription under normoxia (27). Gene set enrichment analysesrevealed significant upregulation and enrichment of the HIF1AandHIF2A pathways under normoxic conditions in the O1R cellscomparedwith the parental cells (Supplementary Fig. S4C). qPCRanalysis also demonstrated the upregulation of HIF-1 target genesSOD2, VEGF, and GLUT1 in the O1R cells compared with theparental cells or the O3R cells (Supplementary Fig. S4D). Theseresults collectively support that CUL2 loss in the O1R cellscompromises the function of the VHL–CRL complex.

To determine whether CUL2 loss directly contributes to theresistance to VHL-based BET-PROTAC, we created O1R-derivedcell lines that express exogenous CUL2 (Fig. 4C). As shownin Fig. 4D and E, overexpression of CUL2 in the O1R cellsresensitized the cells to ARV-771 in the cell proliferation assayand restored ARV-771–induced BET protein degradation,demonstrating that CUL2 loss is responsible for acquired resis-tance in O1R cells.

Resistance to CRBN-based BET PROTAC is caused by the loss ofCRBN gene due to chromosomal deletion

Whole-exome sequencing and RNA-seq were also carried outto determine the genomic and transcriptional differences suchas acquired mutations, gene expression changes, and CNVbetween the O3R cells and the parental or the O1R cells. Whileno genomic or expression alterations of BET family proteinswere found in the O3R cells, a 12 Mb deletion on chromosome3 that encompasses the CRBN gene was found in O3R but notin the O1R cells (Fig. 5A). On the basis of SNP minor allelefrequencies, copy number was estimated as three copies forchromosome 3p in the parental and the O1R cells and one copyin the region containing CRBN of the O3R cells (Supplemen-tary Fig. S3B). The estimated loss of two copies was consistentwith TaqMan CNV analysis, which demonstrated a greater than50% reduction in genomic DNA level relative to parental(Fig. 5B). Consistent with the CRBN gene deletion, RNA-seqdifferential expression analysis identified CRBN as one of themost significantly downregulated genes in the O3R cells com-pared with parental cells (Supplementary Fig. S4B). qPCR andWestern blot analysis confirmed significant downregulation ofthe CRBN mRNA and CRBN protein in the O3R cells (Fig. 5Cand D).

To determine whether the reduction of CRBN directly contri-butes to the resistance to CRBN-based BET-PROTAC in the O3Rcells, we created O3R-derived cell lines that express high levels ofCRBN (Fig. 5E). As shown in Fig. 5F and G, increasing CRBNexpression in the O3R cells resensitized the cells to ARV-825 inproliferation assays and restored ARV-825–induced BET proteindegradation.

DiscussionPROTACs have emerged as a promising novel modality for

the development of next-generation therapeutics. In this study,we reported that resistance to both VHL- and CRBN-basedBET-PROTACs can occur in cancer cells following chronictreatment. However, unlike what is often observed for othertargeted therapeutics, such as kinase inhibitors, cells that wereresistant to ARV-771 and ARV-825 did not contain secondarymutations that affect compound binding to the target.

Although the protein levels of BRD3 and BRD4 are slightlydecreased in O1R and O3R cells, there is still significant amountof these two proteins, especially BRD4, detected. Consideringthat BRD4 is the major player in c-Myc regulation among thethree BRD proteins, this subtle change in BRD4 protein level isnot likely to contribute to the acquired drug resistance (28).Rather, resistance to both classes of BET-PROTACs was primar-ily attributed to genomic alterations that impact core compo-nents of the corresponding E3 ligase complexes. In cells thatwere resistant to ARV-771 or ARV-825, the proteasome degra-dation machinery also remained intact. The preference oftargeting the E3 ligases components over the proteasomemachinery for resistance development is intriguing. We suspectthat the particular vulnerability of E3 ligase components forresistance development may relate to the redundancy and/ornonessentiality of these components for cell survival, whilecompromising proteasome function in cells could be lethal orsignificantly compromise cell fitness. The multiple genomicalterations at the CUL2 locus suggest the existence of strongselection pressure for the cells to abolish CUL2 function in theVHL–CRL complex for resistance development. Interestingly, incells that are resistant to the CRBN-based BET-PROTACARV-825, resistance was primarily attributed to CRBN deletionand no genomic/transcription alterations were observed forCUL4, the CUL2 equivalent of the CRBN–CRL complex. CRBNappears to be a common point of resistance development forCRBN-targeting agents. It has been shown that deletion ofCRBN was the primary cause of resistance to iMiDs in myelomacells (29). The expression level of CRBN has also been iden-tified as a prognostic or predictive biomarker for patients withmultiple myeloma receiving iMiDs (30–32). In addition tomultiple myeloma, both genetic alterations and differentialexpression of CUL2 and CRBN have been observed in manyother cancer types based on The Cancer Genome Atlas dataset(refs. 33, 34; Supplementary Fig. S5). Interestingly, cell lineswith lower CUL2 (Toledo and U266B1) or CRBN (TOV112Dand HCT116) expression are less sensitive to ARV-771 andARV-825, respectively (Supplementary Fig. S6A and S6B).Importantly, siRNA-mediated knockdown of CUL2 or CRBNconferred resistance to ARV-771 and ARV-825 in LNCaP cells,suggesting that there is an association between the expressionlevel of CUL2 or CRBN and sensitivity to VHL- or CRBN-basedPROTACs (Supplementary Fig. S6C and S6D). However, theexpression level of these two proteins may not be the onlydetermining factor for PROTAC sensitivity. The fact thatonly the OVCAR8 cell line readily developed resistance to thesecompounds may be attributed to the genetic background ofthe parental cells. Consistently, OVCAR8 cells harbor an ATMmutation (p.V613L) and exhibit a mutational signature asso-ciated with double-stranded break repair defects with elevatednumbers of larger indels (>3 bp; Supplementary Fig. S7). Theseunderlying genetic attributes may play a role in the differentialability of the OVCAR8 cells to gain resistance to theBET-PROTACs compared with other cell lines tested in thisstudy.

In summary, we report here, for the first time, the mechanismsof acquired resistance to PROTACs in cancer cell lines. Theseresults highlight the critical involvement of E3 ligase complexes inresistance development and lay the foundation for futureinvestigation.






Disclosure of Potential Conflicts of InterestY. Shen has ownership interest (including stock, patents, etc.) in AbbVie. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: L. Zhang, B. Riley-Gillis, Y. ShenDevelopment of methodology: L. Zhang, B. Riley-GillisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Zhang, B. Riley-GillisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Zhang, B. Riley-Gillis, P. VijayWriting, review, and/or revision of the manuscript: L. Zhang, B. Riley-Gillis,P. Vijay, Y. ShenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. ZhangStudy supervision: L. Zhang, Y. Shen

AcknowledgmentsWewould like to thank Erin Murphy, Areej Ammar, Elina Dilmukhametova,

and Ken Idler from the AbbVie Genomic Technologies team for help inpreparing the samples for sequencing analysis. Thank you to Arne Grundstadfrom the AbbVie Computational Genomics team for help in processing theexome sequencing data. A special thank you to Relja Popovic from the AbbViePharmacogenomics team for key discussions guiding the initiation and designof the project.

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 October 2, 2018; revised January 28, 2019; accepted May 2, 2019;published first May 7, 2019.

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2019;18:1302-1311. Published OnlineFirst May 7, 2019.Mol Cancer Ther Lu Zhang, Bridget Riley-Gillis, Priyanka Vijay, et al. E3 Ligase ComplexesChimeras) Caused by Genomic Alterations in Core Components of Acquired Resistance to BET-PROTACs (Proteolysis-Targeting

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