Journal: EMBO Molecular Medicine

Article Title: CDK6 protects epithelial ovarian cancer from platinum‐induced death via FOXO3 regulation

doi: 10.15252/emmm.201607012

Figure Lengend Snippet: Table reporting CDK6‐specific phosphorylation targets ordered by their PR score [adapted from Anders et al ]. The PR score represents the normalized phosphorylation levels of the indicated protein with respect to RB used as positive control (PR = 100). Experimental design of the loss‐of‐function screening performed on MDAH cells to evaluate the effect of silencing CDK6 phosphorylation targets. “ n ” indicates the number of independent experiments performed. Significance ( P ) was calculated by two‐sided, unpaired t ‐test. Graph reporting the results of the loss‐of‐function screening described in (B). Data represent the mean ± SD of three independent experiments each performed in triplicate and graphed as survival ratio between of CBDCA‐treated (140 μg/ml for 16 h) and untreated cells. Significance was calculated using two‐sided, unpaired t ‐test. Significant differences are evidenced by asterisks (** P < 0.01), and the exact P ‐values reported in Appendix Table S4 . Western blot analysis reporting the expression of FOXO3 in the indicated EOC cell lines. Actin was used as loading control. Co‐IP analysis of endogenous CDK4 with FOXO3 and cyclin D1 (upper left panel) in MDAH cells treated with CDDP 7.5 or 15 μg/ml for the indicated times. In the right panels, expression of the same proteins in the corresponding lysates (Input) is reported. In the lower left panels are reported the Co‐IP analyses of cyclin D1 and cyclin D3 with FOXO3 and CDK6 in cells treated for 3 or 6 h with 15 μg/ml of CDDP. In the right lower panels is reported the expression of the same proteins in the corresponding lysates (Input). Tubulin was used as loading control. Dose–response curve on MDAH cells transduced with control, cyclin D1, or cyclin D3 shRNAs and treated with increasing doses of CDDP for 16 h (three biological replicates). The resulted CDDP IC50 is reported in the table. Lower panels show the Western blot analyses of the expression of cyclins D1 and D3 in the transduced cells. Vinculin was used as loading control. Error bars represent SD. Source data are available online for this figure.

Article Snippet: In vitro kinase assays were performed by incubating cyclin D3‐CDK6 or cyclin D1‐CDK4 active kinase complexes (Signal Chem) with GST‐tagged FOXO3a full length (Abnova), GST‐FOXO3 mutants, or GST‐RB1 recombinant proteins, as substrates, for 30 min at 30°C in the presence of 1.5 μCi of γ 32 P‐ATP (PerkinElmer Life Sciences), as described (Berton et al , ; Fabris et al , ).

Techniques: Positive Control, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transduction

Journal: EMBO Molecular Medicine

Article Title: CDK6 protects epithelial ovarian cancer from platinum‐induced death via FOXO3 regulation

doi: 10.15252/emmm.201607012

Figure Lengend Snippet: In vitro phosphorylation assay performed using recombinant cyclin D1‐CDK4 complex and GST‐RB1 fragment or FOXO3 full length as substrates. The Coomassie staining of gel loaded with the same amount of recombinant proteins used in the kinase assay is shown. Arrows indicate the expected molecular weight of GST and FOXO3 recombinant proteins. In silico analysis performed using the GPS3.0 prediction software available online ( http://gps.biocuckoo.org/ ) of potentially phosphorylated sites on FOXO3 by CDK6/4. Pos. indicates the position of the phosphorylated serine. Peptide reports the sequence of the peptide containing the predicted phosphorylated serine (in red). The score indicates the probability of CDK6 or CDK4 to phosphorylate each indicated residue. The higher the score, the higher is the probability of phosphorylation. Schematic representation of FOXO3 predicted phosphorylation sites (with a score > 8 for CDK6 and/or CDK4) and FOXO3 deletion mutants used in this study (NT: N‐terminus, CT: C‐terminus). Sites predicted to be phosphorylated by CDK6, CDK4, or both CDKs with a score higher than 9 are reported in green, light blue, and orange, respectively. In vitro phosphorylation assays performed using recombinant cyclin D3‐CDK6 complex and the indicated FOXO3 deletion mutants or GST‐RB1 fragment as substrates. The Coomassie staining of companion gel is reported. Arrows indicate expected molecular weight of recombinant proteins. C2 = kinase reaction mix control. Amino acid sequence of FOXO3 region (residues 318–351) phosphorylated by CDK6 in vitro as experimentally determined using FOXO3 deletion mutants. Plots report the disorder and the surface exposure of the two peptides containing S325 (left) and S344 (right). Location of amino acids above the disordered (0.50 DC, dotted blue line) and surface exposure cutoff (0.25 EC, dotted orange line) indicates whether they fall in an intrinsically disordered (blue line) and surface‐exposed region (orange line). In the top part of the panel the highlighted residues S315 and S344 indicate the first and the last deleted amino acid in the D315‐344 deletion mutant. 325 highlighted the serine phosphorylated by CDK6. In vitro phosphorylation assays performed using recombinant cyclin D3‐CDK6 (upper panel) or cyclin D1‐CDK4 (lower panel) complexes and the indicated FOXO3 deletion mutants or GST‐RB1 fragment as substrates. C1 = kinase reaction mix plus recombinant cyclin D3‐CDK6; C2 = kinase reaction mix control. In vitro phosphorylation assays performed using recombinant cyclin D3‐CDK6 and the indicated FOXO3 deletion mutants fragment as substrates. The Coomassie staining of companion gel is reported. Arrows indicate expected molecular weight of recombinant proteins. C1 = kinase reaction mix plus recombinant cyclin D3‐CDK6. In vitro phosphorylation assays performed using recombinant cyclin D3‐CDK6 and, as substrates, the indicated FOXO3 deletion mutants with or without the substitution of serine 325 and/or 344 to alanine. The Coomassie staining of companion gel is reported. Arrows indicate expected molecular weight of recombinant proteins. C1 = kinase reaction mix plus recombinant cyclin D3‐CDK6. Source data are available online for this figure.

Article Snippet: In vitro kinase assays were performed by incubating cyclin D3‐CDK6 or cyclin D1‐CDK4 active kinase complexes (Signal Chem) with GST‐tagged FOXO3a full length (Abnova), GST‐FOXO3 mutants, or GST‐RB1 recombinant proteins, as substrates, for 30 min at 30°C in the presence of 1.5 μCi of γ 32 P‐ATP (PerkinElmer Life Sciences), as described (Berton et al , ; Fabris et al , ).

Techniques: In Vitro, Phosphorylation Assay, Recombinant, Staining, Kinase Assay, Molecular Weight, In Silico, Software, Sequencing, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: CDK6 protects epithelial ovarian cancer from platinum‐induced death via FOXO3 regulation

doi: 10.15252/emmm.201607012

Figure Lengend Snippet: Percentage (left pie graph) and typical images of low (A) and high (B) CDK6 expression in a TMA containing 73 EOC samples. Co‐IP analysis of CDK6 with cyclin D1 and D3 in the indicated human EOC tumors. IgG = unrelated Ab. Lys = tumor cell lysates. Arrow points to the cyclin D3 band. CDDP IC50 of primary cultures derived from the indicated tumors, in the presence or not of PD (three biological replicates ± SD). In the lower panels, the Western blot analyses evaluating the expression of ATR, FOXO3, and CDK6 in the same primary cultures, treated as indicated, is shown. Kaplan–Meier survival curve evaluating the progression‐free survival (PFS) in the Tothill dataset, based on the expression of CDK6 (upper panel). The lower table summarizes the significance of CDK6 in predicting PFS in univariate and multivariate analysis (Cox regression) using all malignant cases in the dataset ( n = 218; events = 155; log‐rank test was used to calculate significance). HR = hazard ratio; CI = 95% confidence interval. Kaplan–Meier survival curve evaluating the significance of CDK6, CDK4, and the CDK6, FOXO3 and ATR combination, in predicting the PFS using the online tool KM Plotter ( n = 1,307; events = 484; log‐rank test). Source data are available online for this figure.

Article Snippet: In vitro kinase assays were performed by incubating cyclin D3‐CDK6 or cyclin D1‐CDK4 active kinase complexes (Signal Chem) with GST‐tagged FOXO3a full length (Abnova), GST‐FOXO3 mutants, or GST‐RB1 recombinant proteins, as substrates, for 30 min at 30°C in the presence of 1.5 μCi of γ 32 P‐ATP (PerkinElmer Life Sciences), as described (Berton et al , ; Fabris et al , ).

Techniques: Expressing, Co-Immunoprecipitation Assay, Derivative Assay, Western Blot

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: Discovery of MS28, the first cyclin D1 degrader. (A) Schematic representation of bridged PROTAC. The bridged PROTAC, which binds a bridge protein but not its binding partner (POI), recruits the protein complex into proximity with an E3 ligase, resulting in preferential polyubiquitination and subsequent degradation of the POI by the proteasome over the bridge protein. (B) Chemical structure of MS28. (C) Chemical structures of MS28N1 and MS28N2. (D) Western blot (WB) results of MS28, MS28N1, BSJ-03–204 (BSJ), MS140, and PB on degrading cyclin D1 and CDK4/6. Calu-1 cells were treated with the indicated compound at the indicated concentrations for 8 h. Results shown are representative of at least two independent experiments. (E) Binding affinities of MS28, MS28N1, MS28N2, and PB to CDK4 and CDK6 in biochemical assays. Results shown are the mean ± SD from duplicate experiments.

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: Binding Assay, Western Blot

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: MS28 preferentially degrades cyclin D1 over CDK4/6 in Calu-1 cells. (A) Left, MS28 degrades cyclin D1 first and CDK4/6 subsequently. Calu-1 cells were treated with DMSO or 3 μM of MS28 for the indicated time. Results shown are representative of three independent experiments. Right, quantification of relative cyclin D1, CDK4, and CDK6 abundance at each time point, following 3 μM MS28 treatment (four technical repeats from three biological repeats). P-values were calculated for each protein relative to its abundance at 0.5 h. ****P < 0.0001, ***P < 0.001, and **P < 0.01. The line above the bars indicates that all of the encompassed bars share the same p-value above the line. (B) MS28 concentration-dependently degrades cyclin D1. Calu-1 cells were treated with MS28 at the indicated concentration for 8 h. Results shown are representative of two independent experiments. (C) DC50 and Dmax values of MS28 in Calu-1 cells (calculated from the WB data in panel B and biological repeat). (D) MS28 did not change the mRNA levels of CCND1, CDK4, and CDK6 in RT-qPCR studies. Calu-1 cells were treated with DMSO, 3 μM MS28, or 3 μM PB for 4 h. mRNA levels are relative to DMSO control. Results are representative of two biological repeats.

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: Concentration Assay, Quantitative RT-PCR

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: MS28 effectively degrades cyclin D1 in a VHL-, CDK6-, and UPS-dependent manner. (A) Left, VHL KO rescues the MS28-induced degradation of cyclin D1 and CDK4/6. Calu-1 cells were transduced with lentivirus containing VHL-targeting sgRNA or an empty vector. Antibiotic-selected cells were checked for VHL expression (Top: WB results confirm CRISPR-mediated VHL KO in Calu-1 cells), followed by treatment with MS28 for 8 h. Right, quantification of cyclin D1 and CDK4/6 abundance in the indicated experimental groups. P-values for each protein were calculated between the mock and VHL KO groups (indicated by bar) within each concentration. (B) Left, CDK6 KD via siRNA rescues the MS28-induced degradation of cyclin D1. Calu-1 cells were transfected with CDK6-targeting siRNA for 2 days, followed by treatment with MS28 for 8 h. Right, quantification of cyclin D1 abundance in the indicated experimental groups. P-values were calculated between the control and CDK6 KD group within each MS28 concentration. (C) VHL coelutes with cyclin D1-CDK6 in the presence of MS28. His-tagged CDK6 was immobilized on cobalt agarose resin and incubated overnight along with cyclin D1. The VCB complex was added the next day with either DMSO or MS28. Pretreatment with MG132 (D) or MLN4924 (E) rescues the MS28-induced degradation of cyclin D1 and CDK4/6. Quantification of cyclin D1 and CDK4/6 abundance are listed aside. For each protein, p-values were calculated between groups indicated by the lines above the bars. Calu-1 cells were pretreated with MG132 or MLN4924 for 1 h, followed by treatment with MS28 for 8 h. WB results shown in panels A–E are representative of at least two independent experiments. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: Transduction, Plasmid Preparation, Expressing, CRISPR, Concentration Assay, Transfection, Incubation

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: MS28 is selective for cyclin D1/D3 and CDK4/6. (A) Left, MS28, but not MS28N1, degrades cyclin D1/3 and CDK4/6, and reduces the cyclin A2 level, but not cyclin D2/B1 and CDK2 in Calu-1 cells (4 h-treatment at 3 μM). WB data shown are representative of two biological repeats. Right, quantification of the relative abundance of cyclins and CDKs analyzed in the WB upon DMSO, MS28, or MS28N1 treatment. P-values were calculated relative to the DMSO control for each protein. ****P < 0.0001, ***P < 0.001, and **P < 0.01. (B) MS28 is selective for CDK6 over a panel of 57 other kinases at 1 μM concentration. Data are the means ± SD from duplicate experiments.

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: Concentration Assay

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: MS28 effectively suppresses the downstream Rb-E2F pathway, proliferation, and tumorigenesis in cancer cells. (A) MS28 and PB significantly reduced mRNA levels of E2F target genes (CCNA2, CCNE1, CDC6, PCNA, and PLK1) in RT-qPCR studies. Calu-1 cells were treated with DMSO, 3 μM of MS28, or PB for 8 h. P-values were calculated in comparison to DMSO from two biological repeats. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05. (B) MS28 inhibits cell growth much more effectively than PB, BSJ, and MS28N1 in Calu-1 cells (treated with the indicated compound for 5 days). Data shown are the mean values ± SD from two biological repeats (each with three technical repeats). (C) MS28 suppresses clonogenicity of Calu-1 cells in a soft agar assay more effectively than PB, BSJ, and MS28N1. Images were taken at the end of the 20-day treatment. Each treatment group (at 0.3 μM) is representative of three independent experiments, each with at least five technical repeats. (D) MS28, not MS28N1, BSJ, or PB, effectively degrades cyclin D1 in NCI-H2110 cells (treated with the indicated compound at the indicated concentrations for 8 h). Results are representative of two biological repeats. (E) MS28, but not MS28N1, BSJ or PB, potently inhibits the growth of NCI-H2110 cells (5 days treatment, three biological repeats).

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: Quantitative RT-PCR, Soft Agar Assay

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: Human Primer Sequences Used in RT-qPCR Studies

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques:

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: sgRNA Sequences Used in CRISPR/Cas9-Mediated KO Studies

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: CRISPR, Sequencing

Journal: International Journal of Oncology

Article Title: Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer

doi: 10.3892/ijo.2017.4167

Figure Lengend Snippet: Effect of aspirin, salicylic acid, 2,3-DHBA, 2,6-DHBA and 2,4,6-THBA (at 0.5 mM) on CDK2, 4 and 6 enzyme activity. (A) Upper panel represents in vitro kinase assays showing the effect of aspirin, salicylic acid, 2,3-DHBA and 2,6-DHBA on CDK2 activity. Lower panel shows coomassie stained H1 histones. (B) Quantification of the blot in (A). (C) Upper panel represents in vitro kinase assays showing the dose-dependent effect of 2,4,6-THBA on CDK2 activity. Lower panel shows coomassie stained H1 histones. (D) Quantification of the blot in (C). (E) Upper panel represents the effect of aspirin, salicylic acid, 2,3-DHBA and 2,6-DHBA and 2,4,6-THBA on CDK4 activity. Lower panel shows coomassie stained retinoblastoma protein. (F) Quantification of the blot in (E). (G) Upper panel represents the effect of aspirin, salicylic acid, 2,3-DHBA and 2,6-DHBA (0.5 mM) and 2,4,6-THBA on CDK6 enzyme activity. Lower panel shows coomassie stained retinoblastoma protein. (H) Quantification of the blot in (G). * P<0.05, ** P<0.01, † P<0.001, ‡ P<0.001.

Article Snippet: CDK1/cyclin B1 active enzyme from New England Biolabs (NEB) (Ipswich, MA, USA), CDK1/cyclin B1, CDK2/cyclin A2, CDK4/cyclin D1, CDK6/cyclin D1, Retinoblastoma (C-term) and kinase buffer were purchased from SignalChem (Richmond, BC, Canada).

Techniques: Activity Assay, In Vitro, Staining

Journal: International Journal of Oncology

Article Title: Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer

doi: 10.3892/ijo.2017.4167

Figure Lengend Snippet: A model depicting how aspirin may preferentially act on colonic tissue to protect against CRC. We suggest that unabsorbed aspirin/salicylic acid in the stomach and upper intestine is passed on to the colon, taken up by the colonic epithelial cells, and metabolized by CYP450s to produce 2,3-DHBA and 2,5-DHBAs. These hydrophilic DHBAs may not easily cross the basolateral membrane of the epithelial cells and within these cells, they may accumulate to pharmacologically relevant concentrations, leading to the inhibition of CDK1 and CDK6, and exert anticancer effects. Colonic epithelial cells may also get exposed to salicylic acid metabolites generated by the gut microflora, and uptake of these HBAs by cells may also cause CDK inhibition. This additional layer of protection against tumor development may be unique to the colonic tissue allowing aspirin as a more effective drug against CRC. (?), Aspirin acetylates CDK1; however, how this modification affects its function is not clear at this stage.

Article Snippet: CDK1/cyclin B1 active enzyme from New England Biolabs (NEB) (Ipswich, MA, USA), CDK1/cyclin B1, CDK2/cyclin A2, CDK4/cyclin D1, CDK6/cyclin D1, Retinoblastoma (C-term) and kinase buffer were purchased from SignalChem (Richmond, BC, Canada).

Techniques: Inhibition, Generated, Modification