Molecular Glue Library Screening. The primary screen was performed in 384-well format using the Cell-Titer Glo (CTG) luminescent cell viability assay (Promega) as previously described.27 3,630 compounds from the St. Jude proprietary molecular glue library were screened against 9 human cancer cell lines (HD-MB03, MB004, MB002, MHH-CALL4, MOLM-13, TF-1, HEL, OCI-AML3, AML193). Each cell line was cultured in the complete medium recommended by the vendor and seeded in Corning 8804 BC white 384-well assay plates at densities of 1000, 1000, 1500, 7500, 1250, 156, 625, 1250, 1250 cells per well for HD-MB03, MB004, MB002, MHH-CALL4, MOLM-13, TF-1, HEL, OCI-AML, AML193, respectively. After overnight incubation at 37 °C in a humidified 5% CO2 incubator, cells were treated with compounds in dose-response format using a Pintool on a Biomek FXP Laboratory Automation Workstation (Beckman Coulter). After 72 h of incubation, cell proliferation was assessed using a Cell Titer-Glo (CTG) luminescent cell viability assay (Promega) according to the manufacturer’s instruction. Luminescence signal was measured using an EnVision plate reader (PerkinElmer).
Cell lines and cell culture. HD-MB03 cell line was obtained from Drs. Milde, Witt and Deubzer.51 HD-MB03 and MB004 cells were grown in neurobasal medium supplemented with glutamine, streptomycin, penicillin, B27, EGF, bFGF and heparin as previously described.52 MB002 and MB004 cells were obtained from Dr Yoon-Jae Cho.53 MB002 cells were cultured in 80% neurobasal medium supplemented with glutamine, streptomycin, penicillin, B27 and 20% conditioned media from previous passages; EGF, bFGF and heparin were also added to the medium. MHH-CALL4 cells were obtained from German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Germany) and were cultured in RPMI1640 medium supplemented with 10% FBS (Hyclone), Penicillin/Streptomycin (100 units/mL) and Glutamine (100 µM). Cell identity was confirmed by STR profiling using PowerPlex® Fusion System (Promega). MHH-CALL4, NALM-16, MHH-CALL-2 cells were obtained from German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Germany). PER-117 cells were obtained from Telethon Kids Institute (Perth, Australia). The cells were cultured in RPMI1640 medium supplemented with 10% FBS (Hyclone), Penicillin/Streptomycin (100 units/mL) and Glutamine (100 µM) except for MHH-CALL-2 cells where 20% FBS were used. MOLM-13 cells were obtained from DSMZ (ACC 554) and cultured in RPMI-1640 media (Gibco) supplemented with 20% FBS (R&D Systems) and 100 U/ml penicillin-streptomycin (Gibco). HNT-34 cells were purchased from DSMZ (ACC 600) and cultured in RPMI-1640 media supplemented with 20% FBS and 100 U/ml penicillin-streptomycin. Kasumi-3 cells were purchased from ATCC (CRL-2725) and cultured in RPMI-1640 media supplemented with 20% FBS and 100 U/ml penicillin-streptomycin. UCSD-AML1 cells were purchased from DSMZ (ACC 691) and cultured in RPMI-1640 media supplemented with 20% FBS, 100 U/ml penicillin-streptomycin, and 10 ng/ml human GM-CSF (PeproTech). TF-1 cells were purchased from DSMZ (ACC 334) and cultured in RPMI-1640 media supplemented with 20% FBS, 100 U/ml penicillin-streptomycin, and 5 ng/ml human GM-CSF. OCI-AML3 cells were purchased from DSMZ (ACC 690) and cultured in Alpha-MEM media (Invitrogen) supplemented with 20% FBS and 100 U/ml penicillin-streptomycin. HEL cells were obtained from Dr. Charles Mullighan and cultured in RPMI-1640 media supplemented with 10% FBS and 100 U/ml penicillin-streptomycin. AML193 cells were purchased from DSMZ (ACC 549) cultured in IMDM supplemented with 5% FBS, 100 U/ml penicillin-streptomycin, Insulin-Transferrin-Selenium (ITS-G, Gibco), and 5 ng/ml human GM-CSF. Cell identity was confirmed by STR profiling using PowerPlex® Fusion System (Promega).
Modeling SJ7095 in CK1α. Molecular modeling work was done using the Schrodinger Maestro molecular modeling package (Schrödinger Release 2019-4: LigPrep, Schrödinger, LLC, New York, NY, 2019). A lenalidomide bound complex structure was obtained from the PDB (PDB code: 5FQD).54 Only chains B (CRBN) and C (CK1α) were retained and the resulting structure was prepared using the Protein Preparation Wizard55 such that the missing loop Gln148-Glu153 in cereblon was reconstructed. The tool allowed hydrogen-bond optimization and restrained minimization of the complex (converge heavy atoms to RMSD 0.3 A). This structure was relaxed using a molecular dynamics simulation at 300K and 1.01325 bar pressure using a water-box. SPC waters and an orthorhombic boundary box with 15A buffer was chosen and the system was neutralized by adding Cl- ions. OPLS3e forcefield was used to simulate the system in GPU-accelerated Desmond (Schrödinger Release 2019-4: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2019. Maestro-Desmond Interoperability Tools, Schrödinger, New York, NY, 2019.). An NPT ensemble was used with timestep of 2 fs and Coulombic short range cutoff radius was set at 9Å. Upon completion of the simulation, the water molecules were removed and SJ7095 was docked in the place of the Lenalidomide molecule using Schrodinger Glide Extra Precision (XP) method56 and the docked complex was further simulated for 500 ns using the parameters defined earlier, to obtain the SJ7095-CRBN-CK1α complex.
Proteomics in MOLM-13. Each compound was tested in 3 wells of 2 million MOLM-13 cells each. Cells were treated in tissue culture-treated 6-well plates in 3 ml total media volume and incubated at 37°C for 4 hours. Cells were then collected and washed with DPBS. Washed cells were centrifuged at 400 RCF for 5 minutes at 4°C, supernatant was removed, and pellets were snap frozen in liquid nitrogen and stored at -80°C until sample submission.
Protein Digestion and Peptide Isobaric Labeling by TMT Reagents. The experiment was performed with a previously optimized protocol57 with slight modification. Cell pellets were lysed in lysis buffer (50 mM HEPES, pH 8.5, 8 M urea and 0.5% sodium deoxycholate). To profile whole proteome, the protein lysates (approximately 100 µg of protein per sample were proteolyzed with LysC (Wako) at an enzyme-to-substrate ratio of 1:100 (w/w) for 2 h at 21 oC. Following this the samples were diluted to a final 2 M Urea concentration, and further digested with trypsin (Promega) at an enzyme-to-substrate ratio of 1:50 (w/w) for at least 3 h. The peptides were reduced by adding 1 mM DTT for 30 min at 21oC followed by alkylation with 10 mM iodoacetamide for 30 min in the dark. The unreacted IAA was quenched with 30 mM DTT for 30 min. Finally, the digestion was stopped by adding trifluoroacetic acid (TFA) to 1%, desalted using C18 cartridges (Harvard Apparatus) and dried by speedvac. The purified peptides were resuspended in 50 mM HEPES (pH 8.5), labeled with TMT reagents (Thermo Scientific). The differentially labeled samples were pooled equally, desalted and dried for the subsequent peptide fractionation. Peptide Analysis by two-dimensional liquid chromatography-Tandem Mass Spectrometry (LC/LC-MS/MS) and MS Data analysis are described in the Supplementary Material.
Western blotting. Frozen pellets of ~1 million cells were lysed in 150 ul of SDS lysis buffer (60 mM Tris/HCl pH 7, 10% glycerol, 2% SDS, 5% beta mercaptoethanol, 0.02% bromophenol blue, 0.5% protease and phosphatase inhibitor cocktail (Sigma Aldrich)). When cells were resuspended, samples were heated for 3 minutes at 99°C then put on ice. Samples were then fragmented by sonication for 10 0.5-second pulses (550 Sonic Dismembrator, Fisher Scientific) and heated again at 99°C for 2 minutes. 16 μl of each sample was added to the wells of a 4-20% polyacrylamide gel (Bio-Rad). A mixture of 2 μl of Precision Plus Protein Unstained Standards and 2 μl of Precision Plus Protein All Blue Standards (Bio-Rad) was used for size marker. After electrophoresis, gels were transferred to nitrocellulose membrane using the Bio-Rad Trans-Blot Turbo system on the mixed molecular weight setting. Membranes were blocked for 1 hour in Li-Cor Intercept Blocking Buffer then probed overnight at 4°C with primary antibody in Li-Cor Intercept Antibody Diluent. The following morning, membranes were washed 3 times for 5 minutes with TBS + Tween-20, probed with fluorescent secondary antibodies (goat anti-rabbit IRDye 800CW and goat anti-mouse IRDye 680RD, Li-Cor) for 1 hour at room temperature in the dark, and washed again 3 times. Blots were imaged with the Li-Cor Odyssey CLx and analyzed using Image Studio Version 5.2.
Antibodies. Primary antibodies used:
rabbit anti-CK1α, ab206652, Abcam
rabbit anti-p21, 2947S, Cell Signaling Technology
mouse anti-beta actin, 3700S, Cell Signaling Technology
shRNA. MOLM-13 cells were transduced with shRNA lentiviral particles from non-targeting control (NTC) and 3 CK1ɑ shRNAs (CSNK1a1-a, CSNK1a1-b, CSNK1a1-c). Cells were then sorted after 3 days of culture for GFP+ cells. After sorting, 5.0E+03 cells were from each condition were harvested for immunoblot assay. An equal number of cells (5.0E+06) from each condition were then plated in triplicates. Cells were then grown for 13 days and change in GFP+ cells was assessed by flow cytometry at days 0, 3, 6, 10, and 13 pos-sort.
shRNA plasmid information
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Name:
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Company
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Catalog #
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Target Sequence
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NTC shRNA
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GeneCopoeia
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CSHCTR001-1-LVRU6GP
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GGTTGTGGACAACCATTTACT
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CK1ɑ-shRNA #1
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GeneCopoeia
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HSH059457-LVRU6GP-a
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GGTTGTGGACAACCATTTACT
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CK1ɑ-shRNA #2
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GeneCopoeia
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HSH059457-LVRU6GP-b
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GCAGAATTTGCGATGTACTTA
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CK1ɑ-shRNA #3
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GeneCopoeia
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HSH059457-LVRU6GP-c
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CCCTGAACCATCAATATGACT
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MOLM-13 CRBN Knock-out. CRBN-/- MOLM-13 cells were created using CRISPR-Cas9 technology. Briefly, 1 million MOLM-13 cells were transiently transfected with precomplexed ribonuclear proteins (RNPs) consisting of 400pmol of chemically modified sgRNA (Synthego), 135pmol of SpCas9 protein (St. Jude Protein Production Core), and 500ng of pMaxGFP (Lonza) via nucleofection (Lonza, 4D-Nucleofector™ X-unit) using solution SF and program EO100 in a 100ul cuvette according to the manufacturer’s recommended protocol. Transfected cells (GFP+) were single-cell sorted by flow cytometry (St. Jude Flow Cytometry and Cell Sorting Shared Resource) into 96-well tissue culture treated plates five days post nucleofection. Cells were clonally expanded and screened for the desired targeted modification (out-of-frame indels) via targeted deep sequencing using gene specific primers with partial Illumina adapter overhangs as previously described.58 Genotyping of clones was performed using CRIS.py.59 Knockout clones were identified as clones containing only out-of-frame indels. PowerPlex® Fusion System (Promega) was used to confirm final clone identification (Hartwell Center for Biotechnology at St. Jude). All clones tested negative for mycoplasma by the MycoAlertTMPlus Mycoplasma Detection Kit (Lonza). CRBN knockout confirmed by immunoblot (Supplementary Material).
Dependency assay. Dependency assay for CSNK1A1 was performed by nucleofecting precomplexed RNP (150pmol CSNK1A1 gRNA with 50pmol Cas9 protein) in 20ul P3 solution with program EO100. Two different gRNAs were tested separately (CAGE1215.CSNK1A1.g1 and CAGE1215.CSNK1A1.g7). Following nucleofection, cells were cultured at 37C, 5% CO2, and gDNA samples were taken at days 3, 7, 14, and 21 post transfection. Indel profiles were analyzed using CRIS.py. The sequences of the sgRNA spacers and associated genotyping primers are show in Table 1.
Table 1. The sequences of the CSNK1A1 sgRNA spacers and associated genotyping primers
Name
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Sequence (5’ to 3’)
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SM148.CRBN.g3 spacer
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UGUAUGUGAUGUCGGCAGAC
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CAGE1215.CSNK1A1.g1 spacer
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UUCUAGUCGCCGAGAUGACA
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CAGE1215.CSNK1A1.g7 spacer
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UUUACCUUUAGCCCUUGCCA
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Forward indexing primer (5'-3')
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AATGATACGGCGACCACCGAGATCTACAC(6bp index)ACACTCTTTCCCTACACGACGCTCTTC
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Reverse indexing primer (5'-3')
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CAAGCAGAAGACGGCATACGAGAT(10bp index)GTGACTGGAGTTCAGACGTGTGCTC
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SM148.hCRBN.DS.F2
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ctacacgacgctcttccgatctGCAGAGAGTGAGGAAGAAGATGA
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SM148.hCRBN.DS.R2
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cagacgtgtgctcttccgatctGCCCATGTCCTCATCCACAA
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CAGE1215.CSNK1A1.F
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ctacacgacgctcttccgatctCACCAAATAGTGTTCCCTCCTCA
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CAGE1215.CSNK1A1.R
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cagacgtgtgctcttccgatctTGGGTACCAGCTTACTGTCTCT
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CRBN Fluorescence Polarization Assay. In this competitive fluorescent polarization assay Cy5 conjugated lenalidomide analog (Cy5-O-Len)60 was used as the fluorescent probe. The assay cocktail was prepared by combining 6XHis-CRBN-DDB1 protein (200 nM) and Cy5-O-Len probe (30 nM) in 20 mM HEPES pH 7, 150 mM NaCl, 0.005% Tween-20 assay buffer. Compounds were transferred to a Corning 3821 black 384-well assay plate from a dose-response plate using a Labcyte Echo 650 Acoustic Liquid Handler (Beckman Coulter, USA). Then 20 mL of the assay cocktail was dispensed to wells of the assay plate with a Multidrop Combi Reagent Dispenser (Thermo Scientific, USA). The plates were incubated in the dark for 1 hour at room temperature and then read on an Envision plate reader (PerkinElmer, USA). IC50 values were determined using a proprietary software RISE (Robust Investigation of Screening Experiments), developed in house on the Pipeline Pilot platform (Biovia, v. 17.2.0). Data represent the mean of three independent determinations.
HiBiT-tagged Cell Culture. CSNK1A1-HiBiT HEK293 LgBiT stable CRISPR edited clonal cells (Promega, Cat. #CS3023104) and HiBiT-GSPT1 HEK293 LgBiT stable CRISPR edited clonal cells (Promega, Cat. #CS3023106) and HEK293 cells (ATCC, Cat. #CRL-1573) were maintained in DMEM (Gibco, Cat. #11995065) supplemented with 10% FBS (VWR, Cat.# 89510-194). IKZF1-HiBiT Jurkat LgBiT stable cells (Promega, Cat. #CS3023170) were maintained in RPMI-1640 (Gibco, Cat. #11875093) supplemented with 10% FBS.
Degradation and Cell Viability Assays. White 96-well assay plates (Corning, Cat. # 3017) were seeded with 30,000 cells/well in growth media for CSNK1A1-HiBiT HEK293 LgBiT stable and HiBiT-GSPT1 HEK293 LgBiT stable and incubated overnight at 37°C and 5% CO2. After the overnight incubation, the medium on the CSNK1A1-HiBiT HEK293 LgBiT stable and HiBiT-GSPT1 HEK293 LgBiT stable cells was aspirated off and replaced with CO2 independent medium (Gibco, Cat. #18045088) supplemented with 10% FBS and 1x Endurazine (Promega, Cat. #N2571) substrate. IKZF1-HiBiT Jurkat LgBiT stable cells were seeded at 60,000 cells/well in 50% growth media/50% CO2 independent medium supplemented with 10% FBS and 1x Endurazine substrate. Luminescence was allowed to equilibrate for all plates for 2.5 hours.
A dose response curve was generated by performing 3-fold dilutions of each compound including a DMSO only control in growth media. For live cell kinetic degradation assays, diluted compounds were added to CSNK1A1 and GSPT1 plates and placed in a GloMax Discover (Promega, Cat. #GM3000) preheated to 37°C or added to IKZF1 plates and placed in a CLARIOstar Plus (BMG LABTECH) preheated to 37°C. Luminescence was read every 5-15 minutes for 24 hours. At the end of all 24-hour cycles, CellTiter Glo 2.0 (Promega, Cat. #G9242) was added to the plates, incubated for at least 10 minutes and luminescence was read using the GloMax Discover.
Relative protein level was determined based on normalization to the DMSO control and reported at Fractional RLUs. Degradation rate, degradation maximums, and DC50 at 4 hours was calculated from Fractional RLUs as previously described39.
Live Cell Ternary Complex NanoBRET Assays. CSNK1A1-HiBiT HEK293 LgBiT stable and HiBiT-GSPT1 HEK293 LgBiT stable cells were transfected with a HaloTag-CRBN vector (Promega, Cat. #N269A) using Fugene HD (Promega, Cat. #E2311). HEK293 cells were transfected with a NanoLuc-IKZF1 and HaloTag-CRBN vector at a 1:100 ratio using Fugene HD and incubated overnight at 37°C with 5% CO2. Following overnight incubation, cells were replated in OptiMEM I Reduced Serum Medium, no phenol red (Gibco, Cat. #11058021) supplemented with 4% FBS, and HaloTag 618 ligand (Promega, Cat. #G9801). Plates were incubated overnight at 37°C with 5% CO2. On day 3, a dose response curve was generated by performing 3-fold dilutions of each compound including a DMSO only control in OptiMEM I Reduced Serum Medium, no phenol red supplemented with 4% FBS. Cells were treated with MG132 (Selleckchem, Cat. #S2619) for 30 minutes before being treated with the dilution series of compounds. Plates were incubated for 2 hours at 37°C with 5% CO2. After 2 hours, plates were read using NanoBRET Nano-Glo substate (Promega #N1573) on a GloMax Discover following manufacturer’s instructions. No ligand controls were subtracted from each sample and normalized to the no compound DMSO control to obtain Fractional BRET values. EC50 values were calculated for each compound curve.
Annexin-V staining for apoptosis. Wild type and CRBN-/- MOLM-13 cells were grown in 12-well plates (TPP) with 500,000 cells per well in 2 ml total media volume. Cells were treated with 1 μl/ml DMSO or 1 μM compound in triplicate, and incubated for 24 or 72 hours at 37°C. To prepare samples, this buffer was used: 10 mM HEPES, 0.14 M NaCl, 2.5 mM CaCl2 pH 7.4, 2% v/v FBS in UltraPure water (Invitrogen). After incubation, samples were collected in 5-ml tubes for flow cytometry (Falcon). Single-stain controls consisted of approximately 150 μl of multiple samples combined. Wells were washed with 1 ml of buffer that was added to the corresponding sample tube. Cells were centrifuged at 500 RCF for 5 minutes at 4°C to form a pellet. Supernatant was decanted and cells were washed again with 3 ml of buffer, then centrifuged. After decanting supernatant, 3 μl of APC-conjugated anti-Annexin-V antibody (BioLegend) was added to each tube in the residual buffer left behind. Samples were mixed by vortex and incubated at room temperature in the dark for 20 minutes. Samples were then washed and centrifuged a final time, decanting the supernatant and blotting the tubes to removed residual buffer. 50 μl of buffer with DAPI was added to all appropriate samples and mixed, then analyzed.
Oncolines® cancer cell lines. Cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) or German Collection of Microorganisms and Cell Cultures (DSMZ) (Braunschweig, Germany). All experiments were carried out within ten passages of the original vials. Authenticity of the cell lines was confirmed by short tandem repeat analysis at the respective provider.
Oncolines® SJ3149 cell viability assays. Cell viability assays were performed as previously described.61 In brief, cells were seeded in a 384-well plate at an optimized density. The starting cell number was determined after 24 hours incubation by adding ATPlite 1Step (PerkinElmer, Groningen, the Netherlands) to each well and recording luminescence on an Envision multimode reader (PerkinElmer, Waltham, MA). After addition of a duplicate 9-point dilution series of SJ3149, cells were incubated for another 72 hours, followed by cell counting using ATPlite 1Step. The dilution series ranged from 3.2 nM to 31,600 nM or was diluted 100-fold for the most sensitive cell lines to ideally capture the complete dose-response curve. Percentage viability was determined at each compound concentration by relating the observed signal to a vehicle-treated control, that is, medium containing 0.4% (v/v) DMSO. Compound IC50 values were calculated by fitting a 4-parameter logistic curve to the percentage viability values using IDBS XLfit5 (IDBS, Guildford, United Kingdom). The IC50 was limited to the maximum tested compound concentration when the IC50 exceeded the maximum tested concentration. All dose-response curves were visually inspected and submitted to an F-test, followed by invalidation of curves with F > 1.5. 10log(IC50[nmol/L]) values were used for bioinformatics analyses, unless otherwise indicated.
Oncolines® cell line genomics. Genomic status of 37 frequently altered cancer genes was retrieved from the COSMIC Cell Lines Project (v80) database62,63. Mutations, large deletions or amplifications, and gene translocations were considered relevant genomic alterations. Only non-synonymous alterations which directly altered the coding sequence were retained for further analysis. Furthermore, mutations were filtered for occurrence in primary patient samples. Mutation status of TP53 was retrieved from the DepMap database (version 22Q4). Considering the diverse spectrum of protein function-altering TP53 mutations, all non-synonymous mutations were retained for this gene (Extended Data Table).64 All 38 genes were altered in at least three cell lines. For each gene, the difference in average 10log IC50 between altered and wild-type cell lines was determined. Significance of the IC50 differences was determined by Type II analysis of variance (ANOVA). ANOVA p-values were subjected to Benjamini-Hochberg multiple testing correction, and associations with false discovery rate <20% were considered significant.
Cell line TP53 missense mutation status. TP53 missense mutations were retrieved from the DepMap database (version 22Q4). Only cell lines which had at least one missense mutation were selected for further analysis. Cell lines were grouped based on the specific TP53 residue position which was affected by the missense mutation. Missense mutation positions affected in at least three cell lines were selected for further analysis. Significance of IC50 difference was determined as described above.
Correlation analysis 120 reference anti-cancer agents. IC50 values for 120 anti-cancer reference agents profiled on 102 of the 115 cell lines were determined in a similar fashion as SJ3149 (Uitdehaag et al., 2016). Pearson correlations were calculated between SJ3149 10log IC50 values and those of the 120 reference agents, to determine the similarity of compound IC50 fingerprints.
Correlation analyses gene expression or gene dependency. Cell line RNA sequencing-based basal gene expression values (log2(TPM+1)) and CRISPR dependency scores were retrieved from the DepMap database (version 22Q2 and 22Q4, respectively). SJ3149 or nutlin-3a cell line drug response was related to gene expression or gene dependency by calculating Pearson correlations between the 10log IC50 values and either gene expression values or gene dependency scores in the same cell lines.
Protein Expression and Purification: Truncated human CRBN (StrepII-CRBN∆1-40) and human DDB1 (His6-DDB1∆BPB)65 were co-expressed in Trichoplusia ni High-Five insect cells using the BestBac 2.0 baculovirus expression system (Expression Systems). Human full-length CK1α (StrepII-CK1α) was synthesized and cloned into the pFastBac1 vector (Genscript) and expressed in High-Five insect cells using the Bac-to-Bac baculovirus expression system (Invitrogen). StrepII-CRBN∆1-40-His6-DDB1∆BPB cells were resuspended in 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM TCEP, 10% glycerol, 50 μM Ζinc Acetate, and SigmaFast protease cocktail inhibitor (Millipore Sigma). StrepII-CK1α cells were resuspended in 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.25 mM TCEP, 1 mM PMSF, 10% glycerol and SigmaFast protease cocktail inhibitor. Both StrepII-CRBN∆1-40-His6-DDB1∆BPB and StrepII-CK1α were lysed by sonication (2s ON, 2s OFF, 3 min) and clarified by centrifugation (136,000g) for 1.5 hours at 4°C and loaded onto StrepTactin Sepharose resin (IBA) equilibrated in 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 0.25 mM TCEP, and 10% glycerol. Resin was washed and proteins were eluted with equilibration buffer supplemented with 50 mM Biotin. The StrepII fusion tag of CK1α was cleaved using TEV protease (PPF). Both cleaved CK1α and StrepII-CRBN∆1-40-His6-DDB1∆BPB were buffer exchanged and purified by size exclusion chromatography (S200 10/300 Increase) in the presence of 50 mM HEPES pH 7.4, 200 mM NaCl, and 0.25 mM TCEP. Protein fractions were pooled and concentrated to 70 μM (StrepII-CRBN∆1-40-His6-DDB1∆BPB) and 80 μM (CK1α) for crystallization experiments.
Crystallization and Data Collection: For crystallization of the StrepII-CRBN∆1-40-His6-DDB1∆BPB-SJ3149-CK1α complex, 70 μM StrepII-CRBN∆1-40-His6-DDB1∆BPB was mixed and incubated with 35 μM SJ3149 before the addition of 80 μM CK1α. The mixture was incubated on ice for 1 h and subsequently centrifuged at 20,000g for 30 min at 4°C. 24-well sitting drop crystallization plates (Hampton) were set up by mixing protein 1:1 with reservoir solution containing 70 mM Tris pH 7.0, 140 mM MgCl2 and 10% (w/v) PEG8000 and plates were incubated at room temperature. Crystals appeared after 24 h and continued growing for 14 days via vapor diffusion. Crystals were cryo-protected in reservoir solution supplemented with 20% ethylene glycol and flash-cooled in liquid nitrogen. Diffraction data were collected at SER-CAT (beamline 22-ID) to a resolution of 3.45 Å.
Structure Determination and Model Building: Data collection and refinement statistics are provided in Table SB1. The P1 data was integrated and scaled in XDS, scaling two different datasets from the same crystal using XSCALE.66 The structure was solved by Molecular Replacement with 5FQD as the search model using Phaser.67 The asymmetric unit contained two ternary complexes, with ABC and DEF corresponding to DDB1, Cereblon and CK1alpha, respectively. SJ3149 is unambiguously modeled at the cereblon-CK1 alpha interface in the ABC quaternary complex. SJ3149 was not modeled in the DEF complex due to insufficient electron density. Model building and refinement were performed in COOT68 and Phenix69, respectively. The ABC complex electron density is of higher quality relative to DEF and is referred to throughout the manuscript.