Published OnlineFirst May 7, 2019; DOI: 10.1158/1535-7163.MCT-18-1129 Cancer Biology and Translational Studies Molecular Cancer Therapeutics Acquired Resistance to BET-PROTACs (Proteolysis-Targeting Chimeras) Caused by Genomic Alterations in Core Components of E3 Ligase Complexes Lu Zhang1, Bridget Riley-Gillis2, Priyanka Vijay2, and Yu Shen1 Abstract Proteolysis-targeting chimeras (PROTAC) are bifunction- tions, it would be important to understand whether and al molecules that hijack endogenous E3 ubiquitin ligases to how resistance to these novel agents may emerge. Using induce ubiquitination and subsequent degradation of pro- BET-PROTACs as a model system, we demonstrate that tein of interest. Recently, it has been shown that PROTACs resistance to both VHL- and CRBN-based PROTACs can with robust in vitro and in vivo activities and, in some cases, occur in cancer cells following chronic treatment. However, drug-like pharmaceutical properties can be generated using unlike what was often observed for many targeted thera- small-molecule ligands for the E3 ligases VHL and CRBN. peutics, resistance to BET-PROTACs did not result from These findings stoked tremendous enthusiasm on using secondary mutations that affect compound binding to the PROTACs for therapeutics development. Innate and target. In contrast, acquired resistance to both VHL- and acquired drug resistance often underlies therapeutic fail- CRBN-based BET-PROTACs was primarily caused by geno- ures, particularly for cancer therapy. With the PROTAC mic alterations that compromise core components of the technology progressing rapidly toward therapeutic applica- relevant E3 ligase complexes. Introduction of these small-molecule PROTACs (5–7). BET-PROTACs trigger rapid and prolonged degradation of BET proteins with exception- Ubiquitination-mediated proteolysis is a central mechanism of al potencies, and exhibit robust antitumor activities in preclinical protein homeostasis in cells. E3 ubiquitin ligases bind to defined models of leukemia, lymphoma, prostate cancer, and triple- substrates and mediate ubiquitination and degradation of these negative breast cancers (5, 6, 10, 11). proteins. Cullin-RING ubiquitin ligases (CRL) are the largest Compared with traditional small-molecule inhibitors, family of E3 ubiquitin ligases. CRLs are multicomponent com- PROTACs offer several advantages. For example, PROTACs can plexes that minimally consist of a cullin, a RING finger protein, exert more rapid, potent, and durable inhibition of targets, and a substrate recognition subunit (1). Proteolysis-targeting abolish the scaffolding function of a protein, and turn a non- chimeras (PROTAC) are bifunctional molecules that hijack functional binder into functional degraders. In addition, the endogenous E3 ubiquitin ligase to cause ubiquitination and stringent conformational requirement and potential linker inter- subsequently degradation of proteins of interest (2). VHL and action between target protein and E3 ligase within ternary com- Cereblon (CRBN) are the substrate recognition subunit of the plex may allow PROTACs to offer an additional layer of selectivity cullin2 (CUL2)-containing VHL–CRL complex and the cullin4- over small-molecule inhibitors (12–15). These unique properties containing CRBN–CRL complex, respectively (3, 4). Recently, it make PROTACs a promising modality for the development of has been shown that PROTACs with robust in vitro and in vivo next-generation therapeutics. However, innate and acquired drug activities and, in some cases, drug-like pharmaceutical properties resistance is a common cause of therapeutic failure, particularly can be generated using small-molecule ligands for the E3 ligases for cancer therapy. With the rapid advancement of the PROTAC VHL and CRBN (5–9). PROTACs targeting the bromodomain and technology toward therapeutic applications, it would be impor- extraterminal domain proteins (BET-PROTAC) are the prototype tant to understand whether and how drug resistance to these novel agents may emerge. In this study, using BET-PROTACs with both VHL and CRBN ligands as a model system, we interrogate 1 2 Oncology Discovery, AbbVie Inc., North Chicago, Illinois. Genomic Research potential mechanisms of acquired resistance to PROTACs in Center, AbbVie Inc., North Chicago, Illinois. cancer cell lines. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Authors: Lu Zhang, AbbVie Inc., North Chicago, IL, 60064. Materials and Methods Phone: 847-938-2212; Fax: 847-935-0014; E-mail: [email protected]; and Yu Shen, [email protected] Cells, compounds, and antibodies SKM1, MV4:11, LNCaP, and OVCAR8 cell lines were purchased Mol Cancer Ther 2019;18:1302–11 from ATCC or DSMZ and maintained by a Core Cell Line Facility. doi: 10.1158/1535-7163.MCT-18-1129 All cell lines were tested for Mycoplasma using MycoAlert Detection Ó2019 American Association for Cancer Research. Kit (Lonza) and authenticated using the Gene Print10 STR Kit 1302 Mol Cancer Ther; 18(7) July 2019 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 7, 2019; DOI: 10.1158/1535-7163.MCT-18-1129 Defects in E3 Ligase Complex Cause Resistance to BET-PROTACs (Promega) and maintained in RPMI1640 medium with 10% FBS and MultiQC (17). SNV and indel variant calls were imported (Gibco). ABBV-075, ARV-771, and ARV-825 were synthesized at into the VarSeq Software (Golden Helix) for filtering and AbbVie by the methods according to (5, 6, 16). All antibodies were annotation. purchased from commercial sources as follows: antibodies Somatic copy number calling was performed using VarScan against BRD2/3/4 from Bethyl; antibodies against c-MYC, PARP, v2.4.2 using tumor/normal mpileup from samtools v1.7 as and VHL from Cell Signaling Technology; antibody against CRBN input (18). Parameters used were minimum segment size of from Thermo Fisher Scientific; antibody against CUL2 from 100 bp, minimum coverage of 20 reads, minimum mapping Abcam; and antibody against b-actin from Sigma. O1R- and quality of 20, and minimum base quality of 20. LOH in deleted O3R-overexpressing cells were created by infection with pLOC regions were also visually confirmed by plotting minor allele or pLOC-CUL2 or pLOC-CRBN lentiviral particle (Dharmacon) frequencies from germline variant calling with Sentieon joint in the presence of 10 mg/mL of polybrene. Cells were selected with HaplotypeCaller (Sentieon Inc.). 10 mg/mL of blasticidin. Western blot analysis was performed to confirm expression of these proteins in the cells. RNA sequencing Each cell line was sampled in triplicate and mRNA library Cell viability and caspase 3/7 activity assay preparation from total RNA was conducted following the man- Cells were seeded in 96-well plates and incubated at 37Cin ufacturer's protocol for the Illumina TruSeq mRNA preparation an atmosphere of 5% CO2. Compounds were added at a kit. Briefly, 1 mg of total RNA was purified by using poly-T oligos series of dilution after overnight incubation. After 3 days attached to magnetic beads then fragmented by divalent cations incubation, Caspase-Glo3/7 luminescent and CellTiter-Glo under elevated temperature. The fragmented RNA underwent first Assay (Promega) were performed according to the manufac- strand synthesis using reverse transcriptase and random primers. turer's instruction. Luminescence signal from each well was Second strand synthesis created the cDNA fragments using DNA measured using the EnSpire Luminometer (PerkinElmer), and polymerase I and RNaseH. The cDNA fragments then went the data were analyzed using the GraphPad Prism Software through end repair, adenylation of the 30 ends, and ligation of (GraphPad Software Inc.). adapters. The cDNA library was enriched using 15 cycles of PCR and purified. Final libraries were assessed using the Agilent qPCR Bioanalyzer and Qubit (Thermo Fisher Scientific) assay methods Total RNA was harvested using the RNeasy Plus Mini Kit then sequenced on an Illumina NextSeq sequencer using 2 Â 75 (Qiagen) and cDNA was made with the High-Capacity cDNA bp read length. The raw sequencing data is deposited at SRA Reverse Transcription Kit (Applied Biosystems). qPCR was per- (Submission no. SUB5567117). formed with TaqMan Universal Master Mix and Probes (Life RNA-sequencing reads were mapped to the human reference Technologies), and analyzed using the DDCt method. Data are genome (GRCh38) using STAR aligner and genes were presented as mean Æ SD. The difference between two groups was quantified using featureCounts for all genes annotated in evaluated using the two-tailed Student t test. P values less than Gencode v28 (19, 20). Quality of sequencing data was assessed 0.05 were considered statistically significant. using Picard (http://broadinstitute.github.io/picard) and MultiQC (17). Genes with counts per million less than one in Western blot analysis two-thirds of samples or more were considered too lowly Cell lysates were prepared in Laemmli Buffer (Bio-Rad). Thirty expressed and excluded. Differential gene expression (DGE) anal- micrograms of total protein was resolved on a 7%–12% ysis comparing parental versus O1R and parental versus O3R (n ¼ SDS-polyacrylamide gel and probed with corresponding primary 3 for each) was then performed using linear modeling in limma antibodies. with TMM normalization and voom transformation (21) DGE results were plotted using GLIMMA (22). Sashimi plots were Whole-exome sequencing prepared