Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Signature of chemokine ligands and (B) chemokine receptors in human ovarian cancer cell lines. After isolating total RNA from each cell line, PCR array was performed using a customized PCR array plate containing complementary sequences for human chemokine genes. Different colors indicate average cycle threshold with expression ranges from >35 to <25. (C) Protein expression of p53 and Mdm2 in ovarian cancer cell lines. Whole cell lysates were prepared and Western blot was carried out using antibodies specific to p53, Mdm2 and β-actin as loading control. Experiments were performed in duplicate and a representative result is shown. OV, OVCAR-3 cells; SK, SKOV-3 cells; A, A2780 cells; Ca, CaOV-3 cells; TOV, TOV-21G cells.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) TNF-induced chemokines in SKOV-3 cells. After isolating total RNA, PCR array was performed using a human chemokine PCR array plate. Dotted line indicates 2-fold increase; chemokines with a greater than 2-fold increase are recognized as TNF-induced chemokines. (B) Confirmation of p53 protein expression after transient transfection in SKOV-3 cells. After transfection of empty vector (EM) and p53 expression vector (p53), whole cell lysates were prepared and p53 expression was confirmed by Western blot. β-actin is used as a loading control. (C) Effect of p53 on TNF-induced chemokines. After overnight transfection of vectors, cells were treated with TNF (10 ng/ml) for 1 h and qRT-PCR was carried out using primers for CCL2, CXCL1, 2, 3 and 8. β-actin serves as normalization control. Different letters indicate significant differences (P≤0.05) within each chemokine group (ANOVA and Tukey's pairwise comparisons). Experiments were performed in triplicate and all data are shown as mean ± SE.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Nucleotide sequences of promoters for TNF-induced chemokines such as CCL20, CXCL1, 2, 3 and 8. These chemokine promoters contain one NF-κB site at the proximal region, except for CCL20, which has two NF-κB sites at the distal and proximal region. (B) Effect of p53 on NF-κB luciferase activity. After transfection of vectors or cotransfection with p65, cells were treated with TNF (10 ng/ml) for 4 h. (C) Effect of p53 on TNF-activated IκB. After transfection of empty vector or p53 in SKOV-3 (p53 null), OVCAR-3 (p53 mutant) and A2780 (p53 wild-type), cells were treated with TNF (10 ng/ml) for indicated times. β-actin serves as loading control. Experiments were performed in duplicate and a representative result is shown; numbers below are relative density values.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Mutagenesis, Control

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Accumulated effect of p53 on ubiquitylated proteins. After transient transfection of p53 in A2780, OVCAR-3 and SKOV-3 cells, whole cell lysates were prepared and Western blot was carried out using antibodies specific to ubiquitin, p21, IκB, p53 and β-actin (as loading control). Experiments were performed in duplicate and a representative result is shown. (B) Confirmation of p53 activity after transient transfection of p53. ELISA was performed in triplicate and data are shown as mean ± SE. Dark gray bars indicate significance (p<0.05, paired Student's t -test) within each cell line. (C) The effect of p53 on proteasome activity. Assays were performed in triplicate and data are shown as mean ± SE. Dark gray bars indicate significance (p<0.05, paired Student's t -test) within each cell line. (D) Effects of p53 on ubiquitination of IκB. Immunoprecipitated IκB was immunoblotted using ubiquitin antibody. Experiments were performed in duplicate and a representative result is shown.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Control, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: Effect of p53 on ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3) obtained from comparison between empty vector and p53 vector transfected ovarian cancer cells.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Ubiquitin Proteomics, Comparison, Plasmid Preparation, Transfection

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Effect of p53 on Mdm2 expression. After transient transfection of p53 in A2780, OVCAR-3 and SKOV-3 cells, whole cell lysates were prepared and Western blot was carried out using antibodies specific to Mdm2; and β-actin served as loading control. (B) Effects of p53 expression on p53 binding to p65 and IκB. After transient transfection of p53, immunoprecipitated (IP) p53 was immunoblotted (IB) using p65 or IκB antibody. (C) Effect of p53 on expression of various IKK isoforms. After transient transfection of p53, whole cell lysates were prepared and Western blot was carried out using antibodies specific to IKKα, IKKβ, IKKγ, IKKε; β-actin served as loading control. Experiments were performed in duplicate and a representative result is shown.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Western Blot, Control, Binding Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Effect of nutlin-3 on NF-κB luciferase activity. After transfection of vectors or cotransfection with p53, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for 4 h. Different letters indicate significant differences (P≤0.05) within each group (ANOVA and Tukey's pairwise comparisons). Experiments were performed in triplicate and all data are shown as mean ± SE. (B) Effect of nutlin-3 on TNF-activated IκB. After transfection of empty vector or p53 in SKOV-3 cells, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for indicated times. β-actin serves as loading control. Experiments were performed in duplicate and a representative result is shown. (C) Effect of nutlin-3 on TNF-induced chemokines. After overnight transfection of vectors, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for 1 h and qRT-PCR was carried out using primers for CCL2, CXCL1, 2, 3 and 8. β-actin serves as normalization control. Asterisk indicates significant differences (P≤0.05, paired Student's t -test) when compared to the presence of nutlin-3. Experiments were performed in triplicate and all data are shown as mean ± SE.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: Chronic inflammation promotes ovarian cancer progression via NF-κB signaling. Wild-type p53 reduces activity of the ubiquitin-proteasome system, resulting in low IκB degradation (blue line). This reduces NF-κB activity, inhibiting proinflammatory chemokine expression and attenuating the proinflammatory tumor microenvironment (blue arrow). On the other hand, p53 increases Mdm2 expression (dark arrow) in a feedback loop to compensate for the reduced activity of the ubiquitin-proteasome system. Loss of p53 observed frequently in advanced ovarian cancer triggers high proinflammatory chemokines by increasing NF-κB signaling which is composed of IκB and p65/p50 followed by a high IκB degradation (red arrow). Enhanced NF-κB activity results in potentiation of the proinflammatory tumor microenvironment for ovarian cancer progression such as peritoneal tumor dissemination and massive ascites. The imbalance between p53 and Mdm2 also contributes to increasing NF-κB signaling via the ubiquitin-proteasome system.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Ubiquitin Proteomics, Expressing

Journal: Biosensors

Article Title: Disposable Amperometric Immunosensor for the Determination of Human P53 Protein in Cell Lysates Using Magnetic Micro-Carriers

doi: 10.3390/bios6040056

Figure Lengend Snippet: Characteristics of electrochemical biosensors for the detection of p53 protein.

Article Snippet: A human total p53 DuoSet IC ELISA (from R and D Systems, Inc., Minnneapolis, MN, USA, Catalog Number DYC1043) was used to perform the ELISA assay.

Techniques: Transduction, Concentration Assay, Avidin-Biotin Assay

Journal: Biosensors

Article Title: Disposable Amperometric Immunosensor for the Determination of Human P53 Protein in Cell Lysates Using Magnetic Micro-Carriers

doi: 10.3390/bios6040056

Figure Lengend Snippet: Immunosensor selectivity towards p53 protein. Current values measured for 0.0 (dark blue bars) and 10.0 (light blue bars) ng·mL −1 human p53 standard in the absence (1) and in the presence of: 10.0 ng·mL −1 TNFα (2); 5.0 ng·mL −1 ErbB2 (3); 5.0 ng·mL −1 ERα (4); 5.0 ng·mL −1 PR (5); 5.0 mg·mL −1 BSA (6); 1.0 mg·mL −1 human IgG (7) and 0.1 mg·mL −1 human IgG (8). Supporting electrolyte, 0.05 M sodium phosphate solution, pH 6.0; E app = −0.20 V vs. the Ag pseudo-reference electrode. Other conditions are as described in (selected values column). S/B ratio (red •) are those obtained for each experimental point. Error bars estimated as triple that of the standard deviation ( n = 3).

Article Snippet: A human total p53 DuoSet IC ELISA (from R and D Systems, Inc., Minnneapolis, MN, USA, Catalog Number DYC1043) was used to perform the ELISA assay.

Techniques: Standard Deviation

Journal: International Journal of Preventive Medicine

Article Title: Combination of Salermide and Cholera Toxin B Induce Apoptosis in MCF-7 but Not in MRC-5 Cell Lines

doi:

Figure Lengend Snippet: The results of enzyme linked immunosorbent assay analysis based on the control index for acetylated and total P53 protein generated in Michigan Cancer Foundation (MCF-7) and MRC-5 cells were treated with and without drugs at different times. Cells were treated with combination of drugs for 0, 24, 48 and 72 h. Values are mean + standard error of triplicate experiments. (a) *** P < 0.001 versus all other groups in different time. ** P < 0.05 versus acetylated P53 in MCF-7 with combination of drugs treatment at 72 h. (b) * P < 0.001 versus all other groups in different time. ** P < 0.05 versus total P53 in MRC-5 without drugs treatment and with drugs treatment at time 48 and 72 h. No significant difference was shown in total and acetylated P53 content of other groups

Article Snippet: Acetylated and total P53 ELISA Kit was prepared by cell signaling technology and cell lyses were prepared at the first step.

Techniques: Enzyme-linked Immunosorbent Assay, Generated

Journal: Cell Discovery

Article Title: CD24–p53 axis suppresses diethylnitrosamine-induced hepatocellular carcinogenesis by sustaining intrahepatic macrophages

doi: 10.1038/s41421-017-0007-9

Figure Lengend Snippet: a Cell-based ELISA for p53 in macrophages. Macrophages were obtained as described in Figure . The p53 protein expression in macrophages was quantitated using a target specific primary antibody and HRP-conjugated secondary antibody detection agent, and the crystal violet provided whole cell staining that was used for cell number counts. The levels of p53 protein were normalized by total cell counts in each well with this formula of OD450/OD595. Each dot represents mean data from one mouse based on triplicate counts. Data from two independent experiments are presented. b Cd24 deficiency increased p21 mRNA expression after in vivo DEN (15 μg/g) treatment. Data from three independent experiments are pooled. c As in b , except that WT and Cd24 −/− macrophages were treated with 20 mM DEN in vitro. Data from two independent experiments are pooled. Statistical significance was determined by student’s t -test. d F4/80 staining for liver macrophages in DEN-treated mice. Cd24 +/+ Trp53 +/+ , Cd24 −/− Trp53 +/+ , Cd24 +/+ Trp53 −/− , and Cd24 −/− Trp53 −/− . Scale bar = 100 μm. e Integrated optical density of F4/80-positive cells quantified by Image-Pro Plus 6.0 software in a lower-power field of a microscopy. Twenty fields in each section were counted. Data from multiple experiments are presented. Statistical significances were analyzed by Student’s t -test

Article Snippet: The p53 protein expression in macrophages was quantitatively determined using Total p53 Cell-Based Colorimetric ELISA Kit (ImmunoWay, USA) according to the manufacturer’s instructions.

Techniques: In-Cell ELISA, Expressing, Staining, In Vivo, In Vitro, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2 *

doi: 10.1074/jbc.M114.626861

Figure Lengend Snippet: Crystal structure of SMYD2 in complex with LLY-507. A , SMYD2 is depicted as thin sticks in green , whereas the bound LLY-507 and S -adenosyl- l -homocysteine ( SAH ) are depicted as thick sticks in magenta and cyan , respectively. The F o − F c omit map electron density of LLY-507 in blue is contoured at 3σ. B , plot of SMYD2 interactions with LLY-507. C , structure of SMYD2 in complex with mono-methylated p53 peptide (Ref. ; Protein Data Bank code 3S7D ) with SMYD2 depicted as thin sticks in orange , and S -adenosyl- l -homocysteine and p53 peptide depicted as thick sticks in cyan and magenta , respectively. D , crystallographic and refinement statistics for the structure of SMYD2 in complex with LLY-507. R.m.s. , root mean square.

Article Snippet: SULFO-TAG TM total p53 detection antibody was added to the plate at a final concentration of 1.5 mg/ml in Antibody Dilution Buffer (Meso Scale Discovery) followed by shaking for 1 h at room temperature.

Techniques: Methylation

Journal: The Journal of Biological Chemistry

Article Title: LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2 *

doi: 10.1074/jbc.M114.626861

Figure Lengend Snippet: Chemical structure and biochemical profile of LLY-507. A , chemical structure of LLY-507. B , effect of LLY-507 on the methyltransferase activity of SMYD2 using p53(361–380) peptide as a substrate (IC 50 < 0.015 μ m ). Error bars represent S.D. C , thermostability melting curves of SMYD2 in the absence ( red ) or presence ( green ) of 100 μ m LLY-507. D , effect of LLY-507 at 1 ( light gray ), 10 ( dark gray ), and 50 μ m ( black ) against SMYD2 and 25 other protein or DNA methyltransferases.

Article Snippet: SULFO-TAG TM total p53 detection antibody was added to the plate at a final concentration of 1.5 mg/ml in Antibody Dilution Buffer (Meso Scale Discovery) followed by shaking for 1 h at room temperature.

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2 *

doi: 10.1074/jbc.M114.626861

Figure Lengend Snippet: LLY-507 inhibits SMYD2-mediated methylation of p53 Lys 370 in cells. A , Western blot showing concentration-dependent inhibition of p53 Lys 370 me1 following treatment with 0–2.5 μ m LLY-507 in HEK293 cells transiently transfected with FLAG-SMYD2 and FLAG-p53 (IC 50 < 1 μ m ). B , cell-based ELISA for p53 Lys 370 me1 of U2OS cells transfected with SMYD2 following treatment with LLY-507 for 15 h (IC 50 = 0.6 μ m ). C , concentration-dependent inhibition of p53 Lys 370 me1 by LLY-507 in KYSE-150 cells stably expressing SMYD2 as measured by a Meso Scale Discovery sandwich ELISA (IC 50 = 0.6 μ m ). D , Western blot showing a time-dependent decrease in p53 Lys 370 mono-methylation with 5 μ m LLY-507 treatment of KYSE-150 cells stably expressing FLAG-SMYD2. E , Western blot examining the protein levels of p53-Lys 370 me1 and p53 as well as α-tubulin, RNA polymerase ( pol ) II, and histone H2AX positive controls for cytoplasmic, soluble nuclear, and chromatin subcellular fractions, respectively, in KYSE-150 cells stably expressing FLAG-SMYD2. F , effect of LLY-507 on SMYD2 biochemical activity using histone H4(1–24) peptide as substrate (IC 50 = 31 n m ). G , relative intensities of methylated histone H3 or H4 peptides in SMYD2-overexpressing KYSE-150 cells treated with vehicle or 5 μ m LLY-507, identified and quantified using mass spectrometry. A full list of histone H3 and H4 peptides and their intensities is found in supplemental Table 5 . H , Western blot examining the protein levels of endogenous SMYD2 as well as α-tubulin, RNA polymerase ( Pol ) II, and histone H2AX positive controls for KYSE-150 cytoplasmic, soluble nuclear, and chromatin subcellular fractions, respectively. un , un-methylated; me1 , mono-methylated; me2 , di-methylated; me3 , tri-methylated; ac , acetylated.

Article Snippet: SULFO-TAG TM total p53 detection antibody was added to the plate at a final concentration of 1.5 mg/ml in Antibody Dilution Buffer (Meso Scale Discovery) followed by shaking for 1 h at room temperature.

Techniques: Methylation, Western Blot, Concentration Assay, Inhibition, Transfection, In-Cell ELISA, Stable Transfection, Expressing, Sandwich ELISA, Activity Assay, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2 *

doi: 10.1074/jbc.M114.626861

Figure Lengend Snippet: LLY-507 inhibits the proliferation of several ESCC, HCC, and breast cancer cell lines. A , effect of 3- or 7-day treatment with LLY-507 on the proliferation of ESCC, HCC, and breast cancer cell lines. Cells were treated for either 3–4 or 7 days with compound, and cell viability was then measured using CellTiter-Glo. B , concentration-response curves depicting effect of LLY-507 on 3- and 7-day growth of KYSE-150 cells. C , summary of the status of p53 or Rb in the ESCC, HCC, and breast cancer cell lines tested for the antiproliferative effects of LLY-507 derived from mining of the Cancer Cell Line Encyclopedia. D , mRNA expression analysis of hADR-α1A and SMYD2 in cell lines used in this study derived from mining of the Cancer Cell Line Encyclopedia. LOH , loss of heterozygosity.

Article Snippet: SULFO-TAG TM total p53 detection antibody was added to the plate at a final concentration of 1.5 mg/ml in Antibody Dilution Buffer (Meso Scale Discovery) followed by shaking for 1 h at room temperature.

Techniques: Concentration Assay, Derivative Assay, Expressing

Journal: Molecular Cancer Therapeutics

Article Title: KT-253, a Novel MDM2 Degrader and p53 Stabilizer, Has Superior Potency and Efficacy than MDM2 Small-Molecule Inhibitors

doi: 10.1158/1535-7163.MCT-24-0306

Figure Lengend Snippet: KT-253 is a potent and selective heterobifunctional MDM2 degrader with superior activity to MDM2/p53 small-molecule inhibitors. A, Chemical structure of KT-253. B, Volcano plots showing deep tandem mass tag proteomic analysis in RS4;11 ALL cells treated with KT-253 for indicated timepoints. No off-target protein degradation was observed. All significantly upregulated proteins are tumor suppressor protein p53 and its target genes as shown. A limma statistical package was used, and a weighted score was calculated that incorporates both fold change of replicate groups of KT-253-treated and DMSO-treated cells and significance for each protein. Proteins were deemed statistically significant on either tail when they fell outside of the cutoff function defined as described in the Methods section. C, HEK293-HiBiT cells treated for 4 hours at indicated concentrations of KT-253 or DS-3032. Nano-Glo HiBiT lytic detection assay was used to demonstrate picomolar MDM2 degradation potency with KT-253 relative to DMSO control. The half maximal degradation concentration (DC 50 ) was obtained by fitting the data with a four-parameter nonlinear-regression model in GraphPad Prism version 10.3.0 ( n = 3–4 biological replicates). D, CellTiter-Glo (CTG) assay was used to measure growth inhibition in RS4;11 cells treated with indicated concentrations of KT-253, DS-3032. KT-253 shows picomolar growth inhibition potencies compared with the five other SMIs (see Supplementary Table S3). The half maximal inhibitory concentration (IC 50 ) values were obtained by fitting the data using the four-parameter nonlinear-regression model in GraphPad Prism version 10.3.0 ( n = 3 biological replicates). E, CTG assay measuring growth inhibition of RS4;11 cells treated with KT-253, Compound 1 [a cereblon (CRBN)-dead KT-253 analog], or Compound 2 (KT-253 warhead; see Supplementary Fig. S1 for chemical structures). Right shift in potencies with CRBN-dead analog and KT-253 warhead indicates that growth inhibition potency by KT-253 is driven by the recruitment of CRBN and MDM2 ( n = 3 biological replicates). Dots indicate mean values, and error bars indicate standard deviation.

Article Snippet: p53 levels were quantified using total p53 assay kit [K150DBA-3, meso scale discovery] following the manufacturer’s protocol.

Techniques: Activity Assay, Detection Assay, Control, Concentration Assay, CTG Assay, Inhibition, Standard Deviation

Journal: Molecular Cancer Therapeutics

Article Title: KT-253, a Novel MDM2 Degrader and p53 Stabilizer, Has Superior Potency and Efficacy than MDM2 Small-Molecule Inhibitors

doi: 10.1158/1535-7163.MCT-24-0306

Figure Lengend Snippet: KT-253 overcomes p53/MDM2 feedback loop and shows differential pharmacology to MDM2 SMIs. The degrader advantage demonstrated using Western blot and targeted proteomic data. A, In RS4;11 cells, MDM2 levels are kept at undetectable levels with KT-253 treatment (IC 90 = 1.8 nmol/L) as indicated by Western blot analysis. However, when treated with DS-3032 SMI (IC 90 = 581.4 nmol/L), MDM2 levels are upregulated by activation of a positive feedback loop, potentially impairing p53 stabilization. β-Actin (ACTB) was used as a loading control. DMSO, dimethyl sulfoxide; IC 90 = 90% inhibition concentration. B, Targeted proteomic analysis of MDM2 levels in RS4;11 cells shows that KT-253 [150 nmol/L KT-253] can achieve greater than 90% degradation of MDM2 within 1 hour posttreatment, whereas MDM2 levels continued to increase 1 hour after treatment with DS-3032 (1 mmol/L DS-3032; N = 2 replicates per observation except where noted by § n = 1, n.d., not detected). C, Experimental design for washout experiments performed in RS4;11 cells. D, Caspase 3/7 activation relative to DMSO controls measured at indicated timepoints and concentrations post a washout at 4-hour treatment with KT-253 or DS-3032. Dots indicate mean values, and error bars indicate standard deviation ( n = 3 replicates per concentration; CTG, CellTiter-Glo; DMSO, dimethyl sulfoxide).

Article Snippet: p53 levels were quantified using total p53 assay kit [K150DBA-3, meso scale discovery] following the manufacturer’s protocol.

Techniques: Western Blot, Activation Assay, Control, Inhibition, Concentration Assay, Standard Deviation

Journal: Molecular Cancer Therapeutics

Article Title: KT-253, a Novel MDM2 Degrader and p53 Stabilizer, Has Superior Potency and Efficacy than MDM2 Small-Molecule Inhibitors

doi: 10.1158/1535-7163.MCT-24-0306

Figure Lengend Snippet: A single dose of KT-253 drives sustained tumor regression in ALL xenografts. A, Six cohorts ( n = 6) of size-matched RS4;11 xenograft model tumors (∼400 mm 3 at the start of treatment) were treated with KT-253 [administered intravenously (IV)] or DS-3032 [administered orally (PO)] or vehicle (administered intravenously). Tumor volumes were measured at indicated timepoints. Complete responses were observed with a single dose (SD) of KT-253 at 1 and 3 mg/kg. No complete responses were observed with clinically relevant dosing of DS-3032. Dots indicate mean values, and error bars indicate standard deviation. B, Kaplan–Meier survival analysis shows the median survival after an SD of KT-253 at 3 mg/kg was 50 versus 12 days for the clinically equivalent dosing regimen of DS-3032. QW × 3, once weekly for 3 weeks. C, Targeted proteomic analysis of tumors demonstrates robust degradation of MDM2 1 hour post dosing with KT-253. ACTB, β-Actin. This is associated with ( D ) activation of the p53 pathway as evidenced by a corresponding upregulation of proteomic biomarkers p53, p21, and PHLDA3. E, Immunohistochemical analysis of RS4;11 tumors demonstrates more robust activation of the p53 pathway and induction of cleaved caspase-3 (CC-3) following an SD of KT-253 (3 or 1 mg/kg) than following exposure-matched weekly dosing (h, hours; QD × 3, once daily for three consecutive days). Induction of CC-3 was not observed following treatment with the SMI DS-3032 ( n = 3 per group; scale bar, 100 μm). F, Plasma concentrations of KT-253 achieved with noted dosing regimens and the resulting tumor stasis or regression. Time over a certain threshold exposure of KT-253, shown with a dotted line, seems to be critical to achieving apoptotic cell fate commitment measured as accumulation of CC-3 in the xenografts (right). PK, pharmacokinetic. **, P < 0.01.

Article Snippet: p53 levels were quantified using total p53 assay kit [K150DBA-3, meso scale discovery] following the manufacturer’s protocol.

Techniques: Standard Deviation, Activation Assay, Immunohistochemical staining, Clinical Proteomics

Journal: Molecular Cancer Therapeutics

Article Title: KT-253, a Novel MDM2 Degrader and p53 Stabilizer, Has Superior Potency and Efficacy than MDM2 Small-Molecule Inhibitors

doi: 10.1158/1535-7163.MCT-24-0306

Figure Lengend Snippet: KT-253 potently stabilizes p53, leading to induction of apoptosis in vitro in hematologic and solid tumor lines. A, Mesoscale discovery platform analysis of p53 levels in RS4;11 ALL cells 2 hours posttreatment with KT-253 or DS-3032 at indicated concentrations, relative to DMSO-treated cells, shows rapid and potent stabilization of p53 protein levels with KT-253. Dose–response data were fitted using a four-parameter nonlinear-regression model in GraphPad Prism version 10.3.0. Dots indicate mean values, and error bars indicate standard deviation ( n = 2 biological replicates). B, RS4;11 cells treated for 8 hours with indicated compounds and indicated concentrations, followed by qRT-PCR analysis, show dose-dependent, potent, and acute induction of p53 target gene ( MDM2 , GDF15 , CDKN1A , GADD45A , TNFRSF10B , FAS , and BBC3 ). Fold change in mRNA levels for each target gene was calculated relative to housekeeping gene IPO8 using the ΔΔCt method . Dose–response data were fitted using a four-parameter nonlinear-regression model in GraphPad Prism version 10.3.0. Dots indicate mean values, and error bars indicate standard deviation ( n = 3 replicates). C, CellTiter-Glo (CTG) assay shows potent growth inhibition across a panel of p53 WT hematologic cell lines treated with increasing concentrations of KT-253. The half maximal inhibitory concentration (IC 50 ) values, shown in Supplementary Table S4, were calculated by fitting the data using a four-parameter nonlinear-regression model in GraphPad Prism version 10.3.0 ( n = 2 biological replicates). Caspase activation was measured using Caspase-Glo 3/7 assay. D, Cells were treated for 4 hours followed by two washouts using PBS and allowed to grow in complete media for additional 20 hours. Quantification of flow cytometry data on cell cycle distribution and apoptosis in RS4;11 and MV4;11 cells show depletion of S-phase cells and potent induction of apoptosis with KT-253 treatment versus DS-3032. Scatter plots indicate that a large subset of p53 WT ( E ) hematologic and ( F ) solid tumor cell lines show potent growth inhibition (96-hour CTG assay) and induction of apoptosis (48-hour caspase 3/7 activity assay). Cell panels were treated with KT-253 across a concentration range of 1 to 10,000 nmol/L. Induction of apoptosis in a given cell line is shown as a green circle at its corresponding IC 50 value. Lack of caspase activation is shown in red.

Article Snippet: p53 levels were quantified using total p53 assay kit [K150DBA-3, meso scale discovery] following the manufacturer’s protocol.

Techniques: In Vitro, Standard Deviation, Quantitative RT-PCR, CTG Assay, Inhibition, Concentration Assay, Activation Assay, Caspase-Glo Assay, Flow Cytometry, Activity Assay

Journal: Molecular Cancer Therapeutics

Article Title: KT-253, a Novel MDM2 Degrader and p53 Stabilizer, Has Superior Potency and Efficacy than MDM2 Small-Molecule Inhibitors

doi: 10.1158/1535-7163.MCT-24-0306

Figure Lengend Snippet: A single dose of KT-253 drives sustained tumor regression in MV4;11 AML xenografts and demonstrates strong antileukemic activity in patient-derived xenograft models of systemic AML. A, Five cohorts ( n = 6) of size-matched MV4;11 AML xenograft model tumors (∼300 mm 3 at the start of treatment) were treated with KT-253 (administered intravenously) or DS-3032 (administered orally) or vehicle (administered intravenously). Tumor volumes were measured at indicated timepoints. A single 3 mg/kg dose of KT-253 led to complete responses in five out of six animals. No complete responses were observed with clinically relevant dosing of DS-3032. B, Kaplan–Meier survival analysis shows that the median survival after a single dose (SD) of KT-253 at 3 mg/kg was >180 versus 16 days for the clinically equivalent dosing regimen of DS-3032 (QW × 3, once weekly for 3 weeks). C, Immunohistochemical analysis of MV4;11 tumors demonstrates robust activation of the p53 pathway and induction of cleaved caspase-3 (CC-3) following an SD of KT-253 but not following exposure-matched weekly dosing. Induction of CC-3 following treatment with the SMI DS-3032 was modest (QD × 3, once daily for three consecutive days, scale bar = 100 μm). D, AML patient-derived xenograft model CTG-2227 showing percent hCD45 + cells in peripheral blood (PB) and bone marrow (BM), and hCD34 + leukemic stem blasts in peripheral blood 42 days posttreatment initiation indicate robust reduction in tumor burden. Animals were dosed on days 0, 21, and 41, and samples were collected 24 hours post the last dose (*, P < 0.05; **, P < 0.01).

Article Snippet: p53 levels were quantified using total p53 assay kit [K150DBA-3, meso scale discovery] following the manufacturer’s protocol.

Techniques: Activity Assay, Derivative Assay, Immunohistochemical staining, Activation Assay