Destruction of DNA‐Binding Proteins by Programmable Oligonucleotide PROTAC (O'PROTAC): Effective Targeting of LEF1 and ERG

Abstract DNA‐binding proteins, including transcription factors (TFs), play essential roles in various cellular processes and pathogenesis of diseases, deeming to be potential therapeutic targets. However, these proteins are generally considered undruggable as they lack an enzymatic catalytic site or a ligand‐binding pocket. Proteolysis‐targeting chimera (PROTAC) technology has been developed by engineering a bifunctional molecule chimera to bring a protein of interest (POI) to the proximity of an E3 ubiquitin ligase, thus inducing the ubiquitination of POI and further degradation through the proteasome pathway. Here, the development of oligonucleotide‐based PROTAC (O'PROTACs), a class of noncanonical PROTACs in which a TF‐recognizing double‐stranded oligonucleotide is incorporated as a binding moiety of POI is reported. It is demonstrated that O'PROTACs of lymphoid enhancer‐binding factor 1 (LEF1) and ETS‐related gene (ERG), two highly cancer‐related transcription factors, successfully promote degradation of these proteins, impede their transcriptional activity, and inhibit cancer cell growth in vitro and in vivo. The programmable nature of O'PROTACs indicates that this approach is also applicable to destruct other TFs. O'PROTACs not only can serve as a research tool but also can be harnessed as a therapeutic arsenal to target DNA binding proteins for effective treatment of diseases such as cancer.

Scheme S2. Diagram for the synthesis of modified reverse strand
The reaction solution was diluted with DCM, washed with NaHCO 3 solution. The organic phase was

Synthesis of compound 3a-c:
Compound 2d-f (1.0 equiv) was dissolved in DCM and cooled to 0 o C, then TEA (1.5 equiv) and DMAP (0.01 equiv) was added. The mixture was stirred and Ac 2 O (1.5 equiv) was added slowly. The reaction was stirred at 0 o C for 1h. the reaction solution was washed with water, and the organic phase was dried with Na 2 SO 4 , filtered and concentrated. The residue was purified with flash chromatography (DCM:MeOH = 100:0 to 98:2), giving compound 3a-c.

Synthesis of compound 4a-c:
Compound 3a-c (1.0 equiv) was dissolved in THF and TBAF (1M in THF, 2.0 equiv) was added. The mixture was stirred at rt overnight. The solvent was removed and the residue was (1.0 equiv) was dissolved in anhydrous DCM, DIPEA (2.0 equiv) and Cl-POCEN i Pr 2 (1.5 equiv) was added.
The mixture was stirred at room temperature for 1 hour. Solvent was removed, and the residue was purified with flash chromatography (Hexane:Actone (5%TEA)=100:0 to 60:40), giving product as colorless oil.

Synthesis of compound mc4-5:
Compound 5a or 5b (1.0 equiv) and N-Hydroxysuccinimide (1.5 equiv) were mixed in DCM, cool to 0 o C, then EDCI (1.3 equiv) was added slowly. The mixture was stirred at RT overnight. The reaction was diluted with DCM and washed, with H 2 O and brine. The organic phase was dried with Na 2 SO 4 , filtered and concentrated, giving mc4-5 as yellow solid. Synthesis of compound mc6-7: Compound 5c or 2 was dissolved in DCM, TEA (2.0 equiv) and MsCl (1.2 equiv) were added, the mixture was stirred at RT for 2h. The reaction was added water, then extracted with DCM, the organic phase was dried and concentrated. The residue was dissolved in DCM MeOH/H 2 O and NaN 3 was added, then the mixture was heated to 70 o C overnight. Solvent was removed, to the residue was added water, then extracted with EA twice. The organic phase was concentrated and purified by flash chromatography (DCM: EA=100:0 to 85:15), giving compounds mc6-7.

Synthesis of oligonucleotides and annealing reaction
All oligonucleotides used in this study were synthesized by ExonanoRNA (Columbus, OH). For oligo annealing reaction, single-stranded forward and reverse oligonucleotides were mixed in an assembly buffer (10 mM Tris-HCl [pH7.5], 100 mM NaCl, 1 mM EDTA), and heated to 90 o C for 5 min, then slowly cooled down to 37 o C within 1 h. Double-stranded O'PROTACs were mixed well, aliquoted and stored at -20 o C for the future use.

Cell culture and transfection
RWPE-1, C4-2, LNCaP, 22Rv1, VCaP, PC-3 and DU145 prostate cancer cell line and 293T cell line were purchased from the American Type Culture Collection (ATCC). BPH1 cell line was kindly provided by Dr.
Simon Hayward at NorthShore University HealthSystem at Chigaco and LAPC4 cell line was kindly provided by Dr. Charles Sawyers at Memorial Sloan Kettering Cancer Center at New York. 293T cells were maintained in DMEM medium with 10% FBS. RWPE-1 cells were cultured in keratinocyte serum free medium supplemented with 0.05 mg/ml bovine pituitary extract, 5 ng/ml epidermal growth factor, and 100 U/ml penicillin -100 µg/ml streptomycin mixture. VCaP cells were cultured in RPMI medium with 15% FBS.

Western blot
Cell lysate was subjected to SDS-PAGE and proteins were transferred to nitrocellulose membranes (GE Healthcare Sciences). The membranes were blocked in Tris-buffered saline (TBS, pH 7.4) containing 5% non-fat milk and 0.1% Tween-20, washed twice in TBS containing 0.1% Tween-20, and incubated with primary antibody overnight at 4 °C, followed by secondary antibody for 1 h at room temperature. The proteins of interest were visualized using ECL chemiluminescence system (Thermo Fisher).
The cell lysate was incubated with Streptavidin Sepharose High Performance beads (GE Healthcare) overnight at 4 o C. The binding protein was eluted by elution buffer and subjected to western blot.

RNA extraction and RT-qPCR
RNA was extracted using TRIzol (Invitrogen) and reversely transcribed into cDNA with SuperScript III First-Strand Synthesis System (Promega). The quantitative PCR (qPCR) was performed in the iQ thermal cycler (Bio-Rad) using the iQ SYBR Green Supermix (Bio-Rad). Each sample was carried out in triplicate and three biological repeats were performed. The ΔCT was calculated by normalizing the threshold difference of a certain gene with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences are listed as following:

Immunofluorescent cytochemistry assay
PC-3 cells were seeded on the slides in 6-well plate overnight and reached to 60-70% of confluence and then transfected with LEF1 OP-V1 (0 nM or 100 nM). After 24 h, Cells were fixed by 4% paraformaldehyde and permeabilized with 0.05% Triton X-100. After 1h block at room temperature, cells were subjected to immunoblot with LEF1 antibody (#2230S, Cell Signaling Technology) at 4℃ overnight. After washing, cells were incubated with anti-rabbit Alexa Fluor® 594 (A-11012, Thermo Fishers) for 1 h at room temperature and mounted on the slides using the DAPI-containing counterstain solution (H-1200, Vector Laboratories) after washing. Images were taken by LSM 780 confocal microscope (Zeiss).

Cell growth assay
Cell viability was measured using the MTS assay according to the manufacture's instruction (Promega).
PC-3 and DU145 cells were transfected with LEF1 OP-V1 for 48 h and 1,000 cells were seeded in each well of 96-well plates with 100 μL of medium. After cells adhered to the plate, at indicated time points, cell culture medium was replaced with 1 × PBS and 10 μL of CellTiter 96R Aqueous One Solution Reagent (Promega) was added to each well. The plates were incubated for 2 h at 37 °C in a cell incubator. Microplate reader was used to measure absorbance of 490 nm in each well.

Nuclear extraction and electrophoretic mobility shift assay (EMSA)
Nuclear protein was extracted using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Cat# 78833, Thermo Fisher Scientific). EMSA was performed according to the manufacturer's instruction by using the biotin-labeled LEF1 or ERG OPROTAC as probes. For supershift assay, ERG or LEF1 antibodies were added into the cell nuclear extract mixed with the biotin-labelled OPROTAC probes and the mixture were incubated with for 1 h before loading into 6% of non-denatured polyacrylamide gel.

Three-dimensional (3D) culture
Twenty-thousands of VCaP cells were resuspended in 250 μl plain medium and seeded on the top of a thin layer of Matrigel Matrigel matrix (BD Bioscience) in a 24-well plate. After 30 min, when the cells were settled down, they were covered with a layer of 10% Matrigel diluted with DMEM/F12 medium. Cells were transfected with ERG OP-C-N1 (200 nM) and the medium was changed with fresh and warm DMEM/F12 plus 10% FBS medium every 2-3 days.

Mouse xenograft and drug treatment
The mouse experiments were approved by the Mayo Clinic Institutional Animal Care and Use Committee subcutaneously into the left flank of six-week-old SCID male mice. When the tumor volume reached approximately 75 mm 3 , mice were randomly divided into three groups for treatment with 1 × PBS, control OP or LEF1 OP-V1 (10 mg/kg in PEI solution) via tail vein injection every other day. The volume of xenografts and mouse body weight were measured every three days. After 18-day (for PC-3 tumors) or 21-day (for DU145 tumors) treatment, mice were euthanized and xenografts were harvested for the measurement of weight. One part of tissues was formalin fixed and paraffin-embedded (FFPE) for IHC analysis and the rest of the tissues was used for RNA and protein extraction for RT-qPCR and Western blot analysis, respectively.

Immunohistochemistry (IHC)
The FFPE xenograft tissues were cut consecutively at 4 micrometer for the IHC assay. The IHC staining was performed as previously reported [2] .

Statistical Analysis
Statistical analysis was performed with one-sided or two-sided paired Student's t-test for single comparison. P value < 0.05 is considered statistically significant. All values shown are expressed as means ± SD.