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

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: The DNA damage response is important for CDT-dependent reduction in both cellular LC3-I and LC3-II levels HCT116 ( p53 +/+ ) cells ( A – E ) or HCT116 ( p53 −/− ) cells ( C ) were preincubated with KU-55933 (10 μM) ( A and B ), and then in the absence or presence of Cj -CDT (10 nM) ( A – C ), inactive CDT (containing Cj -CdtB H157G, 10 nM) ( A – C ), or Bleocin (10 μg/mL) ( D ). Alternatively, cell monolayers were incubated in the absence or presence of Nutlin-3 (20 μM) ( D and E ). After 24 h, monolayers were further incubated with CCCP (25 μM) ( A, C, and E ). Lysates were analyzed by immunoblot analysis for relative levels of LC3-I ( A, C, and E ), LC3-II ( A, C, and E ), β-actin ( A and C ), total p53 ( D ) p-p53 (S15) ( B – D ), and GAPDH ( B, D, and E ). The immunoblots are representative of those from 3 independent experiments ( n = 3). Densitometric analyses were combined from 3 independent experiments ( n = 3), and the relative levels of LC3-I, LC3-II, and p-p53 were calculated by dividing the intensity of immuno-specific bands corresponding to cellular LC3-I ( A, C, and E ), LC3-II ( A, C, and E ), and p-p53 ( B ) by the intensity of immuno-specific bands corresponding to controls β-actin ( A and C ) or GAPDH ( B and E ). The data represented by the black bars were each separately rendered relative to the data represented by the white bars, which were assigned an arbitrary value of 1.0. Error bars represent standard deviations. two-way ANOVA followed by Fisher’s LSD post hoc test ( A and C ), or two-tailed, unpaired Student’s t test ( B and E ) were used. Data are represented as mean ± SD. p < 0.05 indicates statistical significance (α = 0.05).

Article Snippet: For input analysis, a portion of the initial whole cell lysate (10%) was reserved, 6X Laemmli sample buffer, supplemented with β-mercaptoethanol, was added to generate a 1X concentration of Laemmli sample buffer in input lysates, and input lysates were fractionated by SDS-PAGE, using 12% polyacrylamide gels, at 110V for 90 min, and transferred to PVDF membranes at 100 V for 90 min. Membranes were blocked with 5% milk in TBS-T. Total cellular p53, LC3A/B, or β-actin levels were determined by immunoblot analysis using primary antibodies specific to p53 (#sc-126, Santa Cruz Biotechnology, USA), LC3A/B (#12741, Cell Signaling Technology, USA), and β-actin (#4970, Cell Signaling Technology, USA).

Techniques: Incubation, Western Blot, Two Tailed Test

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: DNA damage-dependent suppression of autophagy is linked to the ubiquitination and depletion of LC3 by a proteasome-dependent mechanism MEF Atg5 +/+ and MEF Atg5 −/− cells were incubated in the absence or presence of Cj -CDT (10 nM) ( A ). After 48 h, MEF cells were further incubated with rapamycin (20 μM) ( A ). After 3 h in the presence of rapamycin, cell lysates were examined for LC3-I, LC3-II, and β-actin ( A ). HCT116 cells ( B – E ), either transfected ( C ), or, not transfected ( B – E ) with the pCMV-Myc-LC3 overexpression plasmid, were incubated with Cj -CDT (10 nM) ( B – D ), Bleocin (10 μg/mL) ( B ), or Nutlin 3 (20 μM) ( E ), and, in the presence or absence of MG132 (380 nM) ( B – D ) or TAK-243 (100 nM) ( C ). After 24 h, HCT116 monolayers were further incubated with CCCP (25 μM) ( B ), while Nutlin-treated monolayers were further incubated with MG132 (380 nM) ( E ). After 3 h in the presence ( B ) or absence ( B to D ) of CCCP, or 4 h in the presence of MG132 ( E ), cell lysates were examined for LC3-I ( A – E ), LC3-II ( A – E ), Myc-LC3B ( C ), ubiquitin ( C ), total p53 ( E ), and β-actin ( B – E ). Alternatively, Myc-LC3B was immunoprecipitated with Myc-specific antibodies, and ubiquitinated Myc-LC3B was detected using Myc-specific and ubiquitin-specific antibodies ( C ). Intrinsic cellular LC3B was immunoprecipitated using LC3-specific antibodies,and ubiquitinated LC3B was detected using ubiquitin-specific antibodies ( D and E ). Immunoblots are representative of data collected from at least 3 biologically independent experiments ( n = 3). Densitometric analyses were combined from three independent experiments. The data shown by the black (filled) bars were each separately rendered relative to the data represented by the white (empty) bar, which was assigned an arbitrary value of 1.0. Error bars represent standard deviations. Statistical analyses of the data were conducted using two-tailed, unpaired Student’s t test ( A and E ) or two-way ANOVA, followed by Fisher’s LSD post hoc test ( B – D ). Data are represented as mean ± SD. p < 0.05 indicates statistical significance (α = 0.05).

Article Snippet: For input analysis, a portion of the initial whole cell lysate (10%) was reserved, 6X Laemmli sample buffer, supplemented with β-mercaptoethanol, was added to generate a 1X concentration of Laemmli sample buffer in input lysates, and input lysates were fractionated by SDS-PAGE, using 12% polyacrylamide gels, at 110V for 90 min, and transferred to PVDF membranes at 100 V for 90 min. Membranes were blocked with 5% milk in TBS-T. Total cellular p53, LC3A/B, or β-actin levels were determined by immunoblot analysis using primary antibodies specific to p53 (#sc-126, Santa Cruz Biotechnology, USA), LC3A/B (#12741, Cell Signaling Technology, USA), and β-actin (#4970, Cell Signaling Technology, USA).

Techniques: Ubiquitin Proteomics, Incubation, Transfection, Over Expression, Plasmid Preparation, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: DNA damage-dependent reduction in both cellular LC3-I and LC3-II levels is reversed by impairing the ATM-mediated stabilization of p53 HCT116 cells were incubated for 24 h with Cj -CDT (10 nM), or Bleocin (10 μg/mL). After 24 h, monolayers were further incubated in the absence or presence of the autophagy inducing compound CCCP (25 μM) ( B ), as well as the absence or presence of the ATM inhibitor, KU-55933 (10 μM). After an additional 3 h, cell lysates were examined, using immunoblot analyses, for total p53 ( A ), p-p53 (Ser15) ( A ), LC3-I ( B ), LC3-II ( B ), and β-actin ( A and B ). The immunoblots shown are representative from 3 biologically independent experiments ( n = 3). Densitometric analyses were combined from 3 immunoblots ( B ), and the relative levels of cellular LC3-I or LC3-II levels were calculated. The data shown by the black (filled) bars were each separately rendered relative to the data represented by the white (empty) bar, which was assigned an arbitrary value of 1.0. Error bars represent standard deviations of data combined from 3 biologically independent experiments ( n = 3). Statistical analyses of the data were conducted using two-way ANOVA, followed by Fisher’s LSD post hoc test. Data are represented as mean ± SD. p < 0.05 indicates statistical significance (α = 0.05).

Article Snippet: For input analysis, a portion of the initial whole cell lysate (10%) was reserved, 6X Laemmli sample buffer, supplemented with β-mercaptoethanol, was added to generate a 1X concentration of Laemmli sample buffer in input lysates, and input lysates were fractionated by SDS-PAGE, using 12% polyacrylamide gels, at 110V for 90 min, and transferred to PVDF membranes at 100 V for 90 min. Membranes were blocked with 5% milk in TBS-T. Total cellular p53, LC3A/B, or β-actin levels were determined by immunoblot analysis using primary antibodies specific to p53 (#sc-126, Santa Cruz Biotechnology, USA), LC3A/B (#12741, Cell Signaling Technology, USA), and β-actin (#4970, Cell Signaling Technology, USA).

Techniques: Incubation, Western Blot

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet: Model of the proposed p53-proteasome-LC3 axis for regulating the autophagic response in DNA damaged cells Autophagy suppression results from the proteasomal degradation of LC3 within DNA damaged cells as a mechanism for maintaining viability during the restoration of genome integrity ( A – D ). Upon DNA damage, DDR activation leads to the stabilization of p53, which is required for the ubiquitination of LC3 ( A ), thereby targeting LC3 for proteasome degradation ( B ). Available LC3 pools for autophagosome maturation are diminished leading to the production of fewer mature autophagosomes ( C ). Fewer mature autophagosomes results in reduced autophagic flux and preservation of pro-survival factors ( D ).

Article Snippet: For input analysis, a portion of the initial whole cell lysate (10%) was reserved, 6X Laemmli sample buffer, supplemented with β-mercaptoethanol, was added to generate a 1X concentration of Laemmli sample buffer in input lysates, and input lysates were fractionated by SDS-PAGE, using 12% polyacrylamide gels, at 110V for 90 min, and transferred to PVDF membranes at 100 V for 90 min. Membranes were blocked with 5% milk in TBS-T. Total cellular p53, LC3A/B, or β-actin levels were determined by immunoblot analysis using primary antibodies specific to p53 (#sc-126, Santa Cruz Biotechnology, USA), LC3A/B (#12741, Cell Signaling Technology, USA), and β-actin (#4970, Cell Signaling Technology, USA).

Techniques: Activation Assay, Ubiquitin Proteomics, Preserving

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet:

Article Snippet: For input analysis, a portion of the initial whole cell lysate (10%) was reserved, 6X Laemmli sample buffer, supplemented with β-mercaptoethanol, was added to generate a 1X concentration of Laemmli sample buffer in input lysates, and input lysates were fractionated by SDS-PAGE, using 12% polyacrylamide gels, at 110V for 90 min, and transferred to PVDF membranes at 100 V for 90 min. Membranes were blocked with 5% milk in TBS-T. Total cellular p53, LC3A/B, or β-actin levels were determined by immunoblot analysis using primary antibodies specific to p53 (#sc-126, Santa Cruz Biotechnology, USA), LC3A/B (#12741, Cell Signaling Technology, USA), and β-actin (#4970, Cell Signaling Technology, USA).

Techniques: Virus, Recombinant, Cell Recovery, Modification, Gentle, Flow Cytometry, Reverse Transcription, Bicinchoninic Acid Protein Assay, RNA Extraction, Western Blot, Cloning, Mutagenesis, Plasmid Preparation, Software, Cell Culture, Microscopy, Nucleic Acid Electrophoresis