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Novus Biologicals p53 mutant
P53 Mutant, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 25 article reviews

p53 mutant - by Bioz Stars, 2026-06

93/100 stars

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SIN1 controls AKT activity and TYMS levels following genotoxic stress. A Lysates from MDA-MB-231 cells stably expressing Sh control or small hairpin against SIN1, treated or not with 50 µM 5-FuDR or 100 µM 5-FU for 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B-D Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (5, 12) = 16.02, P < 0.001), p-P70SK (F(5,12) = 2.86, p = 0.06), P53 (F (5, 12) = 3.498, P < 0.05, P21 (F (5, 12) = 26.09, P < 0.0001), TYMS (F (5, 12) = 19.39, P < 0.001). E Bar graphs showing the percentage of Sh Ctrl and Sh SIN1 MDA-MB-231 cells at SubG1 after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 8) = 45.1, P < 0.0001). F Lysates of MCF7 cells stably expressing Sh control or small hairpin against SIN1 treated or not with 50 µM 5-FuDR for 24 h, then stained with the indicated antibodies. G-I Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in F. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 483.1, P < 0.0001), p-P70SK (F (3, 8) = 11.58, P < 0.01), P53 (F (3, 8) = 616.3, P < 0.0001), P21 (F (3, 8) = 251.3, P < 0.0001), TYMS (F (3, 8) = 97.82, P < 0.0001). J Bar graphs showing the percentage of MCF7 Sh Ctrl and Sh SIN1 cells at different phase of the cell cycle after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 18) = 11.89, ** P < 0.001, * P < 0.05)

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: SIN1 controls AKT activity and TYMS levels following genotoxic stress. A Lysates from MDA-MB-231 cells stably expressing Sh control or small hairpin against SIN1, treated or not with 50 µM 5-FuDR or 100 µM 5-FU for 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B-D Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (5, 12) = 16.02, P < 0.001), p-P70SK (F(5,12) = 2.86, p = 0.06), P53 (F (5, 12) = 3.498, P < 0.05, P21 (F (5, 12) = 26.09, P < 0.0001), TYMS (F (5, 12) = 19.39, P < 0.001). E Bar graphs showing the percentage of Sh Ctrl and Sh SIN1 MDA-MB-231 cells at SubG1 after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 8) = 45.1, P < 0.0001). F Lysates of MCF7 cells stably expressing Sh control or small hairpin against SIN1 treated or not with 50 µM 5-FuDR for 24 h, then stained with the indicated antibodies. G-I Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in F. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 483.1, P < 0.0001), p-P70SK (F (3, 8) = 11.58, P < 0.01), P53 (F (3, 8) = 616.3, P < 0.0001), P21 (F (3, 8) = 251.3, P < 0.0001), TYMS (F (3, 8) = 97.82, P < 0.0001). J Bar graphs showing the percentage of MCF7 Sh Ctrl and Sh SIN1 cells at different phase of the cell cycle after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 18) = 11.89, ** P < 0.001, * P < 0.05)

Article Snippet: Empty vector c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011; http://n2t.net/addgene:20011 ; RRID: Addgene_20011). pcDNA3 flag p53 was a gift from Thomas Roberts (Addgene plasmid # 10838; http://n2t.net/addgene:10838 ; RRID:Addgene 10838). p53 (dominant negative R175H mutant)-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64638; http://n2t.net/addgene:64638 ; RRID: Addgene_64638).

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Staining

P53 controls AKT phosphorylation and TYMS levels following genotoxic stress. A , C Lysates from MDA-MB-231 and MCF7 cells stably expressing Sh control or small hairpin against p53, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. B-D Bar graphs showing the results of densitometric analysis of AKT phosphorylation and TYMS protein levels under different conditions obtained in A and C. The data are presented as the means ± SEMs. B ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 5.51, P < 0.05), TYMS (F (3, 8) = 240.97, P < 0.0001). D ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 291.1, P < 0.001), TYMS (F (3, 8) = 25.51, P < 0.0001)

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: P53 controls AKT phosphorylation and TYMS levels following genotoxic stress. A , C Lysates from MDA-MB-231 and MCF7 cells stably expressing Sh control or small hairpin against p53, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. B-D Bar graphs showing the results of densitometric analysis of AKT phosphorylation and TYMS protein levels under different conditions obtained in A and C. The data are presented as the means ± SEMs. B ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 5.51, P < 0.05), TYMS (F (3, 8) = 240.97, P < 0.0001). D ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 291.1, P < 0.001), TYMS (F (3, 8) = 25.51, P < 0.0001)

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Control, Staining

SIN1 controls P53 transcriptional activity following genotoxic stress. A-D Bar graphs showing mRNA levels of TYMS and P53 target genes under the indicated conditions in MCF7 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. TYMS F (5, 12) = 17.18, P < 0.0001. CDKN1A F (5, 12) = 274.7, P < 0.0001. GAD45A F (5, 12) = 205.0, P < 0.0001. SESN2 F (5, 11) = 12.43, P = 0.0003. E–G Bar graphs showing mRNA levels of P53 target genes under the indicated conditions in MDA-MB-231 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. CDKN1A F(3,8) = 6.94, P = 0.012. GAD45A F (3, 8) = 22.4, P < 0.001. SESN2 F (3, 8) = 2.22, P = 0.16

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: SIN1 controls P53 transcriptional activity following genotoxic stress. A-D Bar graphs showing mRNA levels of TYMS and P53 target genes under the indicated conditions in MCF7 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. TYMS F (5, 12) = 17.18, P < 0.0001. CDKN1A F (5, 12) = 274.7, P < 0.0001. GAD45A F (5, 12) = 205.0, P < 0.0001. SESN2 F (5, 11) = 12.43, P = 0.0003. E–G Bar graphs showing mRNA levels of P53 target genes under the indicated conditions in MDA-MB-231 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. CDKN1A F(3,8) = 6.94, P = 0.012. GAD45A F (3, 8) = 22.4, P < 0.001. SESN2 F (3, 8) = 2.22, P = 0.16

Techniques: Activity Assay

Increase in TYMS levels alters P53 activity upon genotoxic stress. A Lysates from MCF7 cells stably expressing Sh control or small hairpin against SIN1 transiently transfected with TYMS expressing plasmid, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B Bar graphs showing the results of densitometric analysis of P53 protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. P53 F(7,17) = 44.62, P < 0.0001. C Bar graph displaying the proportion of cells at different stages in the cell cycle analyzed by flow cytometry. MCF7 sh control and sh SIN1 transfected as in A, then treated with or without 50 µM during 72 h. ANOVA, multiple comparisons: Dunnett test. G1 phase F (F (7, 24) = 25.65, P < 0.0001. See supplementary Table 2 for detailed statistics. D Lysates from MCF7 cells treated or not with FuDR for the indicated durations (hours). N = 3 biological replicates per condition. E Bar graph displaying the amount of SIN1 isoforms under the condition obtained in D. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. Long F(3,8) = 39.67, P < 0.001. Short Long F (3,8) = 26.06, P < 0.001

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: Increase in TYMS levels alters P53 activity upon genotoxic stress. A Lysates from MCF7 cells stably expressing Sh control or small hairpin against SIN1 transiently transfected with TYMS expressing plasmid, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B Bar graphs showing the results of densitometric analysis of P53 protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. P53 F(7,17) = 44.62, P < 0.0001. C Bar graph displaying the proportion of cells at different stages in the cell cycle analyzed by flow cytometry. MCF7 sh control and sh SIN1 transfected as in A, then treated with or without 50 µM during 72 h. ANOVA, multiple comparisons: Dunnett test. G1 phase F (F (7, 24) = 25.65, P < 0.0001. See supplementary Table 2 for detailed statistics. D Lysates from MCF7 cells treated or not with FuDR for the indicated durations (hours). N = 3 biological replicates per condition. E Bar graph displaying the amount of SIN1 isoforms under the condition obtained in D. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. Long F(3,8) = 39.67, P < 0.001. Short Long F (3,8) = 26.06, P < 0.001

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Transfection, Plasmid Preparation, Staining, Flow Cytometry

Figure 1. Immunohistochemistry Staining of p53 Mutant (High Power View, Magnification 400x). (A). Intense nuclear staining in immunopositive in ³10% of tumor cells was interpreted as positive (p53 mutant). (B). Weak nuclear staining was interpreted as negative as immunonegative (p53 wild-type)

Journal: Asian Pacific Journal of Cancer Prevention

Article Title: The Association between p53 Expression and Histopathology Grade of Astrocytoma

doi: 10.31557/apjcp.2025.26.7.2521

Figure Lengend Snippet: Figure 1. Immunohistochemistry Staining of p53 Mutant (High Power View, Magnification 400x). (A). Intense nuclear staining in immunopositive in ³10% of tumor cells was interpreted as positive (p53 mutant). (B). Weak nuclear staining was interpreted as negative as immunonegative (p53 wild-type)

Article Snippet: The research samples comprised Formalin-Fixed Paraffin-Embedded (FFPE) for Hematoxylin-Eosin (HE) and immunohistochemical staining with Anti-p53 mutant (M00001-3, Boster, Pleasanton, CA, USA).

Techniques: Immunohistochemistry, Staining, Mutagenesis

Functional characterization of p53-Y220C reactivator compounds and restoration of WT p53 function in cells. A, Structure of p53 reactivators and biochemical properties. The concentration of the test compound required to increase Y220C-DBD binding to the DNA from a consensus p53 response element by 1.5-fold (SC 150 ) was measured by the TR-FRET assay; the binding constant (Kd) was determined by surface plasmon resonance (SPR), and the structural stabilization shown by the Tm was determined through a thermal shift assay, as described in Methods. n.d., not determined. B, X-ray crystal structure of the Y220C−PC10709 complex, PDB code 9BR3 . The Y220C protein is shown as a gray cartoon representation with key residues highlighted as stick models. The hydrogen bonds between specific amino acids in the Y220C protein and PC10709 are indicated by dashed red lines. C, Induction of WT p53 antibody reactivity in cells. NUGC-3 cells were treated with PC14586 for 2 hours, followed by IP of p53 with either WT p53 (PAb1620) or mutant p53 (PAb240) cross-reacting antibodies. Changes in WT and mutant cross-reacting p53 levels were detected by Western blotting of the IP samples with the nonspecific 7F5 p53 antibody. D, Fold change in ELISA, normalized to the vehicle control of mutant (PAb240 antibody) and WT (PAb1620 antibody) p53 protein levels in NUGC-3 cells treated for 2 hours with increasing doses of the Y220C reactivator compounds. E, Biotinylated oligonucleotides corresponding to p53 response elements and a negative control (scramble) oligonucleotide were immobilized on an MSD plate and incubated with a cellular lysate from NUGC-3 cells treated with rezatapopt (PC14586; 2 hours). An increase in sequence-specific DNA binding of p53-Y220C was detected with a total p53 antibody. F, A 5-day MTT proliferation assay was performed as described in the Methods section using the four p53 reactivator compounds in NUGC-3 (p53-Y220C), T3M-4 (p53-Y220C), SJSA-1 (p53 WT), and NUGC-3 KO (p53 KO) cells. Proliferation rates and reactivator compound IC 50 s are presented for each cell line. G, The incorporation of EdU and IC 50 was measured following a 24-hour treatment with increasing doses of the reactivator compounds in the indicated cell lines. H, Quantitation of the expression of fluorescently tagged proteins at specific phases of the cell cycle in NUGC-3, T3M-4, and SJSA-1 cells stably expressing the Incucyte Cell Cycle Lentivirus after a 24-hour treatment with 5 µmol/L reactivator compounds and ( I ) increasing concentrations of rezatapopt (PC14586). J, PC14586 IC 50 values across various cell lines, including those harboring the TP53 Y220C mutation or other p53 hotspot mutations, p53 KO cell lines, and p53 WT cell lines, were obtained from a 5-day MTT assay. K, Western blot analysis of p21 and MDM2 protein expression in NUGC-3 and T3M-4 cells following 5-hour PC14586 treatment. Data in D–I were from three experiments, data in J were from two experiments, and graphs were plotted as mean ± SD.

Journal: Cancer Discovery

Article Title: Restoration of the Tumor Suppressor Function of Y220C-Mutant p53 by Rezatapopt, a Small-Molecule Reactivator

doi: 10.1158/2159-8290.CD-24-1421

Figure Lengend Snippet: Functional characterization of p53-Y220C reactivator compounds and restoration of WT p53 function in cells. A, Structure of p53 reactivators and biochemical properties. The concentration of the test compound required to increase Y220C-DBD binding to the DNA from a consensus p53 response element by 1.5-fold (SC 150 ) was measured by the TR-FRET assay; the binding constant (Kd) was determined by surface plasmon resonance (SPR), and the structural stabilization shown by the Tm was determined through a thermal shift assay, as described in Methods. n.d., not determined. B, X-ray crystal structure of the Y220C−PC10709 complex, PDB code 9BR3 . The Y220C protein is shown as a gray cartoon representation with key residues highlighted as stick models. The hydrogen bonds between specific amino acids in the Y220C protein and PC10709 are indicated by dashed red lines. C, Induction of WT p53 antibody reactivity in cells. NUGC-3 cells were treated with PC14586 for 2 hours, followed by IP of p53 with either WT p53 (PAb1620) or mutant p53 (PAb240) cross-reacting antibodies. Changes in WT and mutant cross-reacting p53 levels were detected by Western blotting of the IP samples with the nonspecific 7F5 p53 antibody. D, Fold change in ELISA, normalized to the vehicle control of mutant (PAb240 antibody) and WT (PAb1620 antibody) p53 protein levels in NUGC-3 cells treated for 2 hours with increasing doses of the Y220C reactivator compounds. E, Biotinylated oligonucleotides corresponding to p53 response elements and a negative control (scramble) oligonucleotide were immobilized on an MSD plate and incubated with a cellular lysate from NUGC-3 cells treated with rezatapopt (PC14586; 2 hours). An increase in sequence-specific DNA binding of p53-Y220C was detected with a total p53 antibody. F, A 5-day MTT proliferation assay was performed as described in the Methods section using the four p53 reactivator compounds in NUGC-3 (p53-Y220C), T3M-4 (p53-Y220C), SJSA-1 (p53 WT), and NUGC-3 KO (p53 KO) cells. Proliferation rates and reactivator compound IC 50 s are presented for each cell line. G, The incorporation of EdU and IC 50 was measured following a 24-hour treatment with increasing doses of the reactivator compounds in the indicated cell lines. H, Quantitation of the expression of fluorescently tagged proteins at specific phases of the cell cycle in NUGC-3, T3M-4, and SJSA-1 cells stably expressing the Incucyte Cell Cycle Lentivirus after a 24-hour treatment with 5 µmol/L reactivator compounds and ( I ) increasing concentrations of rezatapopt (PC14586). J, PC14586 IC 50 values across various cell lines, including those harboring the TP53 Y220C mutation or other p53 hotspot mutations, p53 KO cell lines, and p53 WT cell lines, were obtained from a 5-day MTT assay. K, Western blot analysis of p21 and MDM2 protein expression in NUGC-3 and T3M-4 cells following 5-hour PC14586 treatment. Data in D–I were from three experiments, data in J were from two experiments, and graphs were plotted as mean ± SD.

Article Snippet: The 96-well ELISA plates were coated with WT p53 (150 ng/well; PAb1620; Caprico Biotechnologies, Cat. # 102201, RRID: AB_3662135), mutant p53 (100 ng/well; PAb240; Novus Biologicals, Cat. # NB200-103, RRID: AB_10001083), or total p53 (3.13 ng/well; PAb1801; Novus Biologicals, Cat. # NB200-104, RRID: AB_10001307) antibodies and incubated overnight at 4°C.

Techniques: Functional Assay, Concentration Assay, Binding Assay, SPR Assay, Thermal Shift Assay, Mutagenesis, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Negative Control, Incubation, Sequencing, Proliferation Assay, Quantitation Assay, Expressing, Stable Transfection, MTT Assay

Selective restoration of WT p53 transcriptional responses by PC14586 in cells. A, Quantification of CDKN1A (p21) and MDM2 mRNA by qRT-PCR in p53-Y220C, other p53 hotspot mutants, p53 WT, and p53 KO cell lysates following 5 hours of treatment with rezatapopt (PC14586). Data for the top four cell lines were representative results of three biological repeats, data for other cell lines were from two biological repeats, and graphs were plotted as mean ± SD. A.U., arbitrary unit. B, Scatter plots showing expression levels of genes involved in the p53 signaling pathway in cells following treatment with rezatapopt (PC14586). RNA samples extracted from p53-Y220C–expressing cells (NUGC-3), p53 KO cells (NUGC-3 KO) treated with rezatapopt (PC14586; 5 µmol/L, 16 hours), and NUGC-3 KO_p53i cells induced with doxycycline (50 ng/mL, 12.5 hours) were profiled by the Qiagen RT 2 p53 pathway qRT-PCR panel. Scales are in log 10 (relative expression to housekeeping genes; n = 3). C, Scatter plots of log 2 (transcripts per million + 0.01) and volcano plots of −log 10 (FDR) versus log 2 (fold change) from RNA-seq analysis of expressed genes from p53-Y220C and p53 KO cell lines following DMSO and rezatapopt (PC14586; 5 µmol/L) treatment (16 hours). Genes enriched in gene sets of Fischer direct p53 targets (upregulated), Fischer DREAM Targets (downregulated), DEGs excluding the above-enriched genes (upregulated and downregulated), and non-DEGs were sequentially overlaid as indicated. D, Top enriched gene sets from GSEA of the Molecular Signatures Database C2 collection curated gene sets in the indicated RNA-seq data (NUGC-3 treated with 5 µmol/L PC14586 versus DMSO for 16 hours) from DEGs (Walden stat preranked). The number of overlapping genes from the upregulated DEGs enriched in both the Fischer direct p53 targets gene set, and each listed gene set was divided by the total number of genes in each listed gene set and then multiplied by 100 to obtain the overlap with Fischer_Direct_p53_Targets (%). Overlap with Fischer DREAM targets (%) was calculated in a similar manner, except using the downregulated DEGs. To calculate enrichment (%), the number of genes from the DEGs with the indicated direction of regulation enriched in each gene set was divided by the total number of genes in each listed gene set and multiplied by 100. Normalized enrichment scores (NES) and percentages (%) were plotted on the upper and lower x -axes. All gene sets shown had enrichment of FDR q value ( q ) <0.001. Note that the names of gene sets were shortened. See Supplementary File S11 for details. E, Examples of the GSEA enrichment plots from D . F, Schematic representation of p53 in the transcriptional repression of cell-cycle genes via retinoblastoma-E2F and DREAM complexes. CDK, cyclin-dependent kinase; RBL, retinoblastoma-like proteins p107 (RBL1) and p130 (RBL2).

Journal: Cancer Discovery

Article Title: Restoration of the Tumor Suppressor Function of Y220C-Mutant p53 by Rezatapopt, a Small-Molecule Reactivator

doi: 10.1158/2159-8290.CD-24-1421

Figure Lengend Snippet: Selective restoration of WT p53 transcriptional responses by PC14586 in cells. A, Quantification of CDKN1A (p21) and MDM2 mRNA by qRT-PCR in p53-Y220C, other p53 hotspot mutants, p53 WT, and p53 KO cell lysates following 5 hours of treatment with rezatapopt (PC14586). Data for the top four cell lines were representative results of three biological repeats, data for other cell lines were from two biological repeats, and graphs were plotted as mean ± SD. A.U., arbitrary unit. B, Scatter plots showing expression levels of genes involved in the p53 signaling pathway in cells following treatment with rezatapopt (PC14586). RNA samples extracted from p53-Y220C–expressing cells (NUGC-3), p53 KO cells (NUGC-3 KO) treated with rezatapopt (PC14586; 5 µmol/L, 16 hours), and NUGC-3 KO_p53i cells induced with doxycycline (50 ng/mL, 12.5 hours) were profiled by the Qiagen RT 2 p53 pathway qRT-PCR panel. Scales are in log 10 (relative expression to housekeeping genes; n = 3). C, Scatter plots of log 2 (transcripts per million + 0.01) and volcano plots of −log 10 (FDR) versus log 2 (fold change) from RNA-seq analysis of expressed genes from p53-Y220C and p53 KO cell lines following DMSO and rezatapopt (PC14586; 5 µmol/L) treatment (16 hours). Genes enriched in gene sets of Fischer direct p53 targets (upregulated), Fischer DREAM Targets (downregulated), DEGs excluding the above-enriched genes (upregulated and downregulated), and non-DEGs were sequentially overlaid as indicated. D, Top enriched gene sets from GSEA of the Molecular Signatures Database C2 collection curated gene sets in the indicated RNA-seq data (NUGC-3 treated with 5 µmol/L PC14586 versus DMSO for 16 hours) from DEGs (Walden stat preranked). The number of overlapping genes from the upregulated DEGs enriched in both the Fischer direct p53 targets gene set, and each listed gene set was divided by the total number of genes in each listed gene set and then multiplied by 100 to obtain the overlap with Fischer_Direct_p53_Targets (%). Overlap with Fischer DREAM targets (%) was calculated in a similar manner, except using the downregulated DEGs. To calculate enrichment (%), the number of genes from the DEGs with the indicated direction of regulation enriched in each gene set was divided by the total number of genes in each listed gene set and multiplied by 100. Normalized enrichment scores (NES) and percentages (%) were plotted on the upper and lower x -axes. All gene sets shown had enrichment of FDR q value ( q ) <0.001. Note that the names of gene sets were shortened. See Supplementary File S11 for details. E, Examples of the GSEA enrichment plots from D . F, Schematic representation of p53 in the transcriptional repression of cell-cycle genes via retinoblastoma-E2F and DREAM complexes. CDK, cyclin-dependent kinase; RBL, retinoblastoma-like proteins p107 (RBL1) and p130 (RBL2).

Techniques: Quantitative RT-PCR, Expressing, RNA Sequencing

Pharmacologic activation of WT p53 in p53-Y220C xenografts induced p53 signaling and inhibited tumor growth. A and B, PC14586 was administered orally at the indicated doses to p53-Y220C–expressing ( A ) NUGC-3 and ( B ) T3M-4 mouse xenografts. Analysis includes tumor volume (mm 3 ) measurements starting on day 1 of dosing (top left), AUC of tumor growth (top right), or percentage of body weight change (bottom). Each data point is the average tumor volume (left panel) or average percentage of body weight change (right). Percentage of TGI or regression is relative to starting volume. n = 10/group. Data shown are mean ± SEM. C, Fold change normalized to vehicle (V) control of mutant (PAb240 antibody), WT (PAb1620 antibody), and total (PAb1801 antibody) p53 protein levels using MSD in NUGC-3 xenograft tumors. Indicated time points are after the dose on day 4 of daily oral administration of vehicle at 25, 50, and 100 mg/kg PC14586. Consolidated vehicles from 7, 24, and 48 hours of 2QD × 1 treatments, n = 12; PC14586, n = 4/group. Data shown are mean ± SEM. Detectable plasma levels (μmol/L) are shown by the red symbol and designated on the right y- axis. D, Fold change in protein expression of p53 downstream targets using MSD (p21, MDM2) and normalized to vehicle control or ELISA (MIC-1) and normalized to vehicle control and tumor volume (mm 3 ) in tumor samples described in C . Data shown are mean ± SEM. E, Representative IHC images of Ki-67, p21, and MDM2 in the NUGC-3 xenograft model after 4 days of vehicle at 50 or 100 mg/kg PC14586 daily administration. Scale bars are 100 µm, 20× objective. Bar graphs represent the average percentage of positive stained cells on days 1 and 4 following the administration of 50 and 100 mg/kg PC14586. Symbols represent individual samples. Error bars show SEM. Statistical significance relative to vehicle was determined using one-way ANOVA with Dunnett’s multiple comparisons test (adjusted P value * < 0.01; *** 0.0001; **** <0.0001). QD, once daily.

Journal: Cancer Discovery

Article Title: Restoration of the Tumor Suppressor Function of Y220C-Mutant p53 by Rezatapopt, a Small-Molecule Reactivator

doi: 10.1158/2159-8290.CD-24-1421

Figure Lengend Snippet: Pharmacologic activation of WT p53 in p53-Y220C xenografts induced p53 signaling and inhibited tumor growth. A and B, PC14586 was administered orally at the indicated doses to p53-Y220C–expressing ( A ) NUGC-3 and ( B ) T3M-4 mouse xenografts. Analysis includes tumor volume (mm 3 ) measurements starting on day 1 of dosing (top left), AUC of tumor growth (top right), or percentage of body weight change (bottom). Each data point is the average tumor volume (left panel) or average percentage of body weight change (right). Percentage of TGI or regression is relative to starting volume. n = 10/group. Data shown are mean ± SEM. C, Fold change normalized to vehicle (V) control of mutant (PAb240 antibody), WT (PAb1620 antibody), and total (PAb1801 antibody) p53 protein levels using MSD in NUGC-3 xenograft tumors. Indicated time points are after the dose on day 4 of daily oral administration of vehicle at 25, 50, and 100 mg/kg PC14586. Consolidated vehicles from 7, 24, and 48 hours of 2QD × 1 treatments, n = 12; PC14586, n = 4/group. Data shown are mean ± SEM. Detectable plasma levels (μmol/L) are shown by the red symbol and designated on the right y- axis. D, Fold change in protein expression of p53 downstream targets using MSD (p21, MDM2) and normalized to vehicle control or ELISA (MIC-1) and normalized to vehicle control and tumor volume (mm 3 ) in tumor samples described in C . Data shown are mean ± SEM. E, Representative IHC images of Ki-67, p21, and MDM2 in the NUGC-3 xenograft model after 4 days of vehicle at 50 or 100 mg/kg PC14586 daily administration. Scale bars are 100 µm, 20× objective. Bar graphs represent the average percentage of positive stained cells on days 1 and 4 following the administration of 50 and 100 mg/kg PC14586. Symbols represent individual samples. Error bars show SEM. Statistical significance relative to vehicle was determined using one-way ANOVA with Dunnett’s multiple comparisons test (adjusted P value * < 0.01; *** 0.0001; **** <0.0001). QD, once daily.

Techniques: Activation Assay, Expressing, Control, Mutagenesis, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining

PC14586 restored multiple facets of dynamic WT p53 transcriptional responses in vivo . A, Time-dependent effects of PC14586 on gene expression of key p53 targets, CDKN1A , MDM2 , and BIRC5 , with repeat daily administration across 6 days in the NUGC-3 xenograft model. n = 4/group. Error bars show the SEM. The red symbols show plasma concentrations (µmol/L) and are designated on the right y -axis. B, Assessment of a panel of 84 p53 pathway genes measured by qRT-PCR. A representative number of upregulated and downregulated p53 target genes are shown across 6 days of 100 mg/kg PC14586 administration, with the mean value of log 2 (fold change) at each time point shown on the heatmap. See Supplementary File S12 for details. RNA samples extracted from tumor samples, as in A , were profiled by the Qiagen RT 2 p53 pathway qRT-PCR panel as described in the Methods section. Symbols represent the mean log 2 (fold change) for each. C, Volcano plots from RNA-seq analysis of expressed genes from NUGC-3 xenograft tumors following PC14586 (100 mg/kg) and vehicle treatment as indicated. Graphs were plotted as in , except that the p53-targeted lncRNA (upregulated) gene set was overlaid on top of the others. See Supplementary Fig. S5B for the corresponding scatter plots. In B and C, vehicle (consolidated), n = 12 and PC14586, n = 4 for each group, as in and and . D, Top enriched gene sets from GSEA of the Molecular Signatures Database C2 collection curated gene sets supplemented with Fischer p53-targeted lncRNA (86 genes; C2+) in the indicated RNA-seq data [rezatapopt (PC14586) for once daily (QD) ×6 at 24 hours] from DEGs. See Supplementary File S14 for details. Gene sets with a normalized enrichment score (NES) and an FDR q value ( q ) < 0.001 are highlighted in green, 0.001 to 0.05 are highlighted in orange, and 0.05 to 0.06 are highlighted in gray. Graphs were plotted as in . E, Updated schematic representation of p53 in regulating the cell cycle via retinoblastoma-E2F and DREAM complexes. Updated from , the lncRNA component was added to complement p21 in regulating the cell cycle. PINCR, p53-induced noncoding RNA.

Journal: Cancer Discovery

Article Title: Restoration of the Tumor Suppressor Function of Y220C-Mutant p53 by Rezatapopt, a Small-Molecule Reactivator

doi: 10.1158/2159-8290.CD-24-1421

Figure Lengend Snippet: PC14586 restored multiple facets of dynamic WT p53 transcriptional responses in vivo . A, Time-dependent effects of PC14586 on gene expression of key p53 targets, CDKN1A , MDM2 , and BIRC5 , with repeat daily administration across 6 days in the NUGC-3 xenograft model. n = 4/group. Error bars show the SEM. The red symbols show plasma concentrations (µmol/L) and are designated on the right y -axis. B, Assessment of a panel of 84 p53 pathway genes measured by qRT-PCR. A representative number of upregulated and downregulated p53 target genes are shown across 6 days of 100 mg/kg PC14586 administration, with the mean value of log 2 (fold change) at each time point shown on the heatmap. See Supplementary File S12 for details. RNA samples extracted from tumor samples, as in A , were profiled by the Qiagen RT 2 p53 pathway qRT-PCR panel as described in the Methods section. Symbols represent the mean log 2 (fold change) for each. C, Volcano plots from RNA-seq analysis of expressed genes from NUGC-3 xenograft tumors following PC14586 (100 mg/kg) and vehicle treatment as indicated. Graphs were plotted as in , except that the p53-targeted lncRNA (upregulated) gene set was overlaid on top of the others. See Supplementary Fig. S5B for the corresponding scatter plots. In B and C, vehicle (consolidated), n = 12 and PC14586, n = 4 for each group, as in and and . D, Top enriched gene sets from GSEA of the Molecular Signatures Database C2 collection curated gene sets supplemented with Fischer p53-targeted lncRNA (86 genes; C2+) in the indicated RNA-seq data [rezatapopt (PC14586) for once daily (QD) ×6 at 24 hours] from DEGs. See Supplementary File S14 for details. Gene sets with a normalized enrichment score (NES) and an FDR q value ( q ) < 0.001 are highlighted in green, 0.001 to 0.05 are highlighted in orange, and 0.05 to 0.06 are highlighted in gray. Graphs were plotted as in . E, Updated schematic representation of p53 in regulating the cell cycle via retinoblastoma-E2F and DREAM complexes. Updated from , the lncRNA component was added to complement p21 in regulating the cell cycle. PINCR, p53-induced noncoding RNA.

Techniques: In Vivo, Gene Expression, Clinical Proteomics, Quantitative RT-PCR, RNA Sequencing

Pharmacologic activation of WT p53 inhibited tumor growth and induced immunologic cell changes in tumors. A, Average tumor volume (mm 3 ; left), AUC analysis of tumor growth (middle), and percentage of body weight change (right) in C57Bl/6 mice bearing subcutaneous MT373 tumors treated with vehicle (V) or PC14374 at 150 or 300 mg/kg (2Q7D: two doses on day 1 only on a weekly dosing cycle). n =10/group. Data shown are mean ± SEM. Percentage of TGI (% TGI) or tumor regression is relative to tumor volume at study start. Statistical significance relative to the vehicle was determined using one-way ANOVA with Dunnett’s multiple comparisons tests (adjusted P value: **** <0.0001). B and C, Six days after inoculation with MT373 cells, mice were administered PC14374 orally at 150 or 300 mg/kg (2Q7D × 2 or 2Q7D × 3: two doses on day 1 only of a weekly dosing cycle for 2 or 3 weeks, respectively). B, Tumors were harvested 72 hours after last dose to analyze tumor-infiltrating lymphocytes by flow cytometry. Percentage of cell population change is normalized to CD45 + cells. Vehicle, n = 3/group; PC14374, n = 4/group. Error bars show SEM. Statistical significance relative to the vehicle was determined using one-way ANOVA with Dunnett’s multiple comparisons tests (adjusted P value: * ≤0.01; *** ≤0.001; **** ≤0.0001). C, Tumors harvested 72 hours after last dose were analyzed on the NanoString IO 360 panel for gene expression. Box plots represent fold change in the signature scores and the raw P value for the comparison between treatments for each cell signature (*, P ≤0.01; **, P ≤0.001). Vehicle control is treated as the baseline of the comparison. Vehicle, n = 3/group; PC14374, n = 4/group. Error bars show SEM. D, Mice bearing MT373 tumors were administered 75 mg/kg or 150 mg/kg PC14374 (2Q7D: two doses on day 1 only of a weekly dosing cycle), 200 µg anti–PD-1 (Q3D: dose every three days), or a combination of PC14374 and anti–PD-1 starting on day 6 after cell implantation. Tumor volume (mm 3 ) measurements started on day 1 of dosing. Kaplan–Meier curves are shown as percentage of survival with n = 10/group. The dashed vertical line represents dosing discontinuation at day 102. E, Mice bearing MT373 tumors were administered 150 mg/kg of PC14374 (2Q7D), 200 µg of anti–PD-1 (Bio X Cell Clone RMPI-14; Q3D), or 250 µg of anti-CD8 (Bio X Cell Clone 2.43) as single agents or in combination starting on day 6 after cell implantation. Groups dosed with anti-CD8 were pretreated 3 days prior to the study start. Tumor volume (mm 3 ) measurements started on day 1 of dosing. Kaplan–Meier curves are shown as percentage of survival with n = 10/group. The dashed vertical line represents dosing discontinuation on day 19. Statistical significance across all groups was determined using the log-rank (Mantel–Cox) test.

Journal: Cancer Discovery

Article Title: Restoration of the Tumor Suppressor Function of Y220C-Mutant p53 by Rezatapopt, a Small-Molecule Reactivator

doi: 10.1158/2159-8290.CD-24-1421

Figure Lengend Snippet: Pharmacologic activation of WT p53 inhibited tumor growth and induced immunologic cell changes in tumors. A, Average tumor volume (mm 3 ; left), AUC analysis of tumor growth (middle), and percentage of body weight change (right) in C57Bl/6 mice bearing subcutaneous MT373 tumors treated with vehicle (V) or PC14374 at 150 or 300 mg/kg (2Q7D: two doses on day 1 only on a weekly dosing cycle). n =10/group. Data shown are mean ± SEM. Percentage of TGI (% TGI) or tumor regression is relative to tumor volume at study start. Statistical significance relative to the vehicle was determined using one-way ANOVA with Dunnett’s multiple comparisons tests (adjusted P value: **** <0.0001). B and C, Six days after inoculation with MT373 cells, mice were administered PC14374 orally at 150 or 300 mg/kg (2Q7D × 2 or 2Q7D × 3: two doses on day 1 only of a weekly dosing cycle for 2 or 3 weeks, respectively). B, Tumors were harvested 72 hours after last dose to analyze tumor-infiltrating lymphocytes by flow cytometry. Percentage of cell population change is normalized to CD45 + cells. Vehicle, n = 3/group; PC14374, n = 4/group. Error bars show SEM. Statistical significance relative to the vehicle was determined using one-way ANOVA with Dunnett’s multiple comparisons tests (adjusted P value: * ≤0.01; *** ≤0.001; **** ≤0.0001). C, Tumors harvested 72 hours after last dose were analyzed on the NanoString IO 360 panel for gene expression. Box plots represent fold change in the signature scores and the raw P value for the comparison between treatments for each cell signature (*, P ≤0.01; **, P ≤0.001). Vehicle control is treated as the baseline of the comparison. Vehicle, n = 3/group; PC14374, n = 4/group. Error bars show SEM. D, Mice bearing MT373 tumors were administered 75 mg/kg or 150 mg/kg PC14374 (2Q7D: two doses on day 1 only of a weekly dosing cycle), 200 µg anti–PD-1 (Q3D: dose every three days), or a combination of PC14374 and anti–PD-1 starting on day 6 after cell implantation. Tumor volume (mm 3 ) measurements started on day 1 of dosing. Kaplan–Meier curves are shown as percentage of survival with n = 10/group. The dashed vertical line represents dosing discontinuation at day 102. E, Mice bearing MT373 tumors were administered 150 mg/kg of PC14374 (2Q7D), 200 µg of anti–PD-1 (Bio X Cell Clone RMPI-14; Q3D), or 250 µg of anti-CD8 (Bio X Cell Clone 2.43) as single agents or in combination starting on day 6 after cell implantation. Groups dosed with anti-CD8 were pretreated 3 days prior to the study start. Tumor volume (mm 3 ) measurements started on day 1 of dosing. Kaplan–Meier curves are shown as percentage of survival with n = 10/group. The dashed vertical line represents dosing discontinuation on day 19. Statistical significance across all groups was determined using the log-rank (Mantel–Cox) test.

Techniques: Activation Assay, Flow Cytometry, Gene Expression, Comparison, Control

Responses of two patients with solid tumors harboring TP53 Y220C mutations receiving rezatapopt. A–C Patient with advanced small cell lung carcinoma from the PYNNACLE phase I study. A, CT scans at baseline and 12 weeks showing a reduction in target lesions. B, TP53 Y220C VAF over time. C, Reduction in the sum diameter of target lesions over time (the dotted line represents the value that would constitute a PR; 54.6 mm). D–F Patient with advanced endometrial cancer from the PYNNACLE phase I study. D, CT scans at baseline and 11 weeks showing a reduction in target lesions. E, Cancer antigen 125 (CA-125) levels over time. F, Reduction in the sum diameter of target lesions over time (the dotted line represents the value that would constitute a PR; 39.9 mm). VAF, variant allele frequency; EOT, end of treatment.

Journal: Cancer Discovery

Article Title: Restoration of the Tumor Suppressor Function of Y220C-Mutant p53 by Rezatapopt, a Small-Molecule Reactivator

doi: 10.1158/2159-8290.CD-24-1421

Figure Lengend Snippet: Responses of two patients with solid tumors harboring TP53 Y220C mutations receiving rezatapopt. A–C Patient with advanced small cell lung carcinoma from the PYNNACLE phase I study. A, CT scans at baseline and 12 weeks showing a reduction in target lesions. B, TP53 Y220C VAF over time. C, Reduction in the sum diameter of target lesions over time (the dotted line represents the value that would constitute a PR; 54.6 mm). D–F Patient with advanced endometrial cancer from the PYNNACLE phase I study. D, CT scans at baseline and 11 weeks showing a reduction in target lesions. E, Cancer antigen 125 (CA-125) levels over time. F, Reduction in the sum diameter of target lesions over time (the dotted line represents the value that would constitute a PR; 39.9 mm). VAF, variant allele frequency; EOT, end of treatment.

Techniques: Variant Assay

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