Journal: Frontiers in Cell and Developmental Biology

Article Title: The regulation of pedicle initiation by androgens in sika deer (Cervus nippon)

doi: 10.3389/fcell.2025.1708732

Figure Lengend Snippet: Relative mRNA expression levels of CALR, SMC4, P53, MCM10 and SRCIN1, which were validated by Q-PCR analysis normalized to GAPDH. The data are expressed as mean ± SD. **: p < 0.01 (the androgen-activated AP vs. the androgen-unactivated AP, n = 3).

Article Snippet: Antibodies used in the Western blot analysis included: anti-GAPDH-loading control monoclonal antibody (bsm-33033M, Bioss, China), anti-SRCIN1 antibody (D153560, BBI life sciences, China), anti-Calreticulin polyclonal antibody (bs-5913R, Bioss, China), anti-SMC4 polyclonal antibody (bs-7728R, Bioss, China), anti-p53 protein (wt-p53) polyclonal antibody (bs-0033R, Bioss, China).

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: The regulation of pedicle initiation by androgens in sika deer (Cervus nippon)

doi: 10.3389/fcell.2025.1708732

Figure Lengend Snippet: Relative protein expression levels of CALR, p53, SMC4 and SRCIN1. (A) western blot verification of CALR, p53, SMC4 and SRCIN1 in the different AP tissues. (B) Relative expression levels of CALR, p53, SMC4 and SRCIN1 in the different AP tissues.The data are expressed as mean ± SD. **: p < 0.01 (the developed AP vs. the undeveloped AP, n = 3).

Article Snippet: Antibodies used in the Western blot analysis included: anti-GAPDH-loading control monoclonal antibody (bsm-33033M, Bioss, China), anti-SRCIN1 antibody (D153560, BBI life sciences, China), anti-Calreticulin polyclonal antibody (bs-5913R, Bioss, China), anti-SMC4 polyclonal antibody (bs-7728R, Bioss, China), anti-p53 protein (wt-p53) polyclonal antibody (bs-0033R, Bioss, China).

Techniques: Expressing, Western Blot

Journal: Communications Biology

Article Title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

doi: 10.1038/s42003-025-08413-0

Figure Lengend Snippet: a Differentially expressed TE subfamilies between GP5d and p53-KO GP5d cells. Scatter plot shows normalized RNA-seq read counts for TE subfamilies. Differentially expressed TE subfamilies are labeled by TE class. b Distinct TE subfamilies derepressed by CMEi in GP5d p53-KO cells. Expression changes for TE subfamilies (log2FC) were compared between different CME treatments in GP5d p53-KO cells. Significance symbols: **** indicates p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = non-significant |log2FC| < 1.5 or p > 0.05. c Loss of p53 is associated with stronger derepression of TEs by DNMTi-HDACi. Bar plots compare (i) the number of derepressed TE loci by DNMTi-HDACi in WT GP5d and two independent p53-KO clones and (ii) the number of derepressed TEs loci between LNCAP-1F5 p53-WT and p53-KO cells. d Rescue experiments showing that p53 reintroduction is associated with weaker derepression of TEs at the subfamily and locus levels by DNMTi-HDACi. Bar plot comparing the number of derepressed TE subfamilies and TE loci by DNMTi-HDACi in GP5d, GP5d p53-KO clones #1 and #2 and p53-transfected GP5d p53-KO clone #1. e Volcano plots showing the differentially expressed individual TE loci by DNMTi-HDACi in GP5d, OE19, and GP5d p53- KO cells. f Bar plot comparing p53 expression in GP5d and OE19 cells treated with DNMTi-HDACi vs. DMSO control (unpaired two-sided t-test). g Genome browser snapshot of the TP53 gene locus showing the ChIP-seq signals for H3K27ac and H3K4me3 and an RNA-seq signal track for both DMSO control and DNMTi-HDACi-treated GP5d cells. h Comparison of TE expression changes induced by DNMTi-HDACi treatment between TEs harboring p53REs with different strengths. Expressed TEs with p53REs were stratified into five grades from least to most likely p53REs with transactivation potential using p53retriever . Boxplots show the number of up- and downregulated TE loci upon DNMTi-HDACi for each grade, significantly differentially expressed loci are marked with red (Adjusted p < 0.05, |log2FC| > 1). Comparisons between grades were performed with one-sided Wilcoxon tests. Number of LINEs and LTRs loci for each grade are shown in Supplementary Data . Source data are provided as Supplementary Data .

Article Snippet: Next day cells were transfected with p53 expressing pIRES2-EGFP-p53 WT plasmid (addgene # 49242).

Techniques: RNA Sequencing, Labeling, Expressing, Clone Assay, Transfection, Control, ChIP-sequencing, Comparison

Journal: Communications Biology

Article Title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

doi: 10.1038/s42003-025-08413-0

Figure Lengend Snippet: a TF motif enrichment at TE sequences derepressed by DNMTi-HDACi in GP5d and OE19 cells. After performing motif enrichment analysis for individual motifs, similar motifs were combined into motif clusters from ref. . The representative TF clusters are labeled on the right. The minimum E-value found for an individual TF for each motif cluster was plotted in the final figure. b Boxplots comparing the expression of derepressed LTR12C in DMSO and DNMTi-HDACi GP5d, OE19 and GP5d-p53-KO cells (two-sided Wilcoxon paired test). The lower and upper hinges of the boxes represent the 25th to 75th percentiles, the midline is the median, and the whiskers extend from the hinges to the minimum and maximum values by 1.5 * interquartile range (IQR). c , Venn diagram showing the overlap between LTR12Cs derepressed by DNMTi-HDACi in GP5d and OE19 (shown in Fig. 4b). d Metaplots of ATAC-seq and CUT&TAG for H3K27me3, H3K4me1, RNAPII and Ser5p-RNAPII at derepressed LTR12Cs in DNMTi-HDACi OE19 cells (shown in Fig. 4b). Derepressed LTR12C elements show a poised chromatin state in OE19 cells, whereas LTR12C in GP5d cells were enriched with repressive H3K27me3 marks (see Supplementary Fig. ). e Heatmap showing the KAS-seq signal at derepressed LTR12C elements in DNMTi-HDACi OE19 cells (shown in Fig. 4b). f Volcano plot of nanopore sequencing data comparing CpG methylation levels at derepressed LTR12C elements (n = 499) in control and DNMTi-HDACi GP5d cells (shown in Fig. 4b). Significance was determined with a one-sided Fisher’s exact test. P -values were corrected with Benjamini-Hochberg method. g Heatmaps showing the ChIP-seq signals for H3K27ac and H3K4me3 in control and DNMTi-HDACi GP5d cells (shown in Fig. 4b). h Heatmaps showing the ChIP-seq signals for H3K27ac and H3K4me3 in control and DNMTi-HDACi OE19 cells (shown in Fig. 4b). i Derepressed LTR12C elements in DNMTi-HDACi GP5d and OE19 cells. Volcano plots show the changes in gene expression for derepressed LTR12C-associated genes for OE19 cells. Analysis of differentially expressed genes in OE19 cells with DNMTi-HDACi revealed significant upregulation of genes in the vicinity of derepressed LTR12C elements (±50 kb). In total, 102 out of 132 and 70 out of 89 differentially expressed genes were upregulated in GP5d and OE19 cells, respectively. j Genome browser snapshot of a derepressed LTR12C in control and DNMTi-HDACi GP5d cells. Panels show the ChIP-seq signals for H3K27ac, H3K4me3, and an RNA-seq signal track. k Genome browser snapshot of a derepressed LTR12C in control and DNMTi-HDACi OE19 cells. Each panel shows the ChIP-seq signals for H3K27ac, H3K4me3 and an RNA-seq signal track. Source data are provided as Supplementary Data .

Article Snippet: Next day cells were transfected with p53 expressing pIRES2-EGFP-p53 WT plasmid (addgene # 49242).

Techniques: Labeling, Expressing, Nanopore Sequencing, CpG Methylation Assay, Control, ChIP-sequencing, Gene Expression, RNA Sequencing

Journal: Communications Biology

Article Title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

doi: 10.1038/s42003-025-08413-0

Figure Lengend Snippet: a Comparison of SETDB1 KO/inhibition-induced changes in the expression of TE subfamilies between six different cell lines. Differential expression analysis of RNA-seq data was performed with DESeq2. All TE subfamilies with absolute log2FC > 1.5 (treatment vs DMSO or A375 KO vs A375) and adjusted p < 0.05 in at least one CMEi treatment in one cell line were selected and their expression changes in log2FC was plotted. Rows and columns are clustered with hierarchical clustering. b Number of differentially expressed TE loci by SETDB1 KO/inhibition in A375, WT and p53-KO GP5d, OE19, and WT and p53-KO LNCaP-1F5 cells. c The proportion of TEs derepressed by SETDB1 KO/inhibition belonging to the major TE classes among the derepressed TEs by in A375, WT and p53-KO GP5d, OE19, and WT and p53-KO LNCaP-1F5 cells. d Number of Alu SINEs derepressed by SETDB1 KO/inhibition in A375, WT and p53-KO GP5d, OE19, and WT and p53-KO LNCaP-1F5 cells. Number above the bar represents the percentage of Alu SINEs in total derepressed TEs. e Motif enrichment analysis for TEs derepressed by SETDB1 KO/inhibition in A375, WT and p53-KO GP5d, OE19, and WT and p53-KO LNCaP-1F5 cells. The analysis was performed as described in Fig. . Source data are provided as Supplementary Data .

Article Snippet: Next day cells were transfected with p53 expressing pIRES2-EGFP-p53 WT plasmid (addgene # 49242).

Techniques: Comparison, Inhibition, Expressing, Quantitative Proteomics, RNA Sequencing

Journal: Communications Biology

Article Title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

doi: 10.1038/s42003-025-08413-0

Figure Lengend Snippet: a Boxplots showing a comparison of expression of transcriptionally active IR-Alu SINEs (total sum of RNA-seq reads for DMSO and DNMTi-HDACi ≥ 5, see “Methods” for details) in DMSO and DNMTi-HDACi GP5d, OE19, and GP5d p53-KO cells. Majority of IR-Alu SINEs are derepressed by DNMTi-HDACi (two-sided Wilcoxon paired test). The lower and upper hinges of the boxes represent the 25th to 75th percentiles, the midline is the median, and the whiskers extend from the hinges to the minimum and maximum values by 1.5 * IQR. b Heatmap showing the ChIP-seq signal for H3K27ac at IR-Alu SINEs in DMSO and DNMTi-HDACi GP5d and OE19 cells. c Comparison of normalized RNA-seq read counts for ADAR1 gene in DMSO and DNMTi-HDACi GP5d, OE19, and GP5d p53-KO cells. The graph shows mean ± SD values for three biological replicates (two-sided unpaired t-test). d Microscopy images for GP5d cells treated with DMSO or DNMTi-HDACi. DNA was stained with DAPI (blue), and dsRNA was stained using the J2 antibody (green). All scale bars are 20 μm. Cytoplasmic levels of dsRNA increased in DNMTi-HDACi treated GP5d cells as compared to the DMSO. e Microscopy images for dsRNA staining in OE19 cells treated with DMSO or DNMTi-HDACi, as shown in Fig. 6d. f Microscopy images for dsRNA staining in GP5d p53-KO cells treated with DMSO or DNMTi-HDACi, as shown in Fig. 6d. g Alu editing index (AEI) was calculated by using RNAeditingIndexer tool on RNA-seq data. Bar plots showing AEI for DMSO and DNMTi-HDACi GP5d, OE19, and GP5d p53-KO cells. The graph shows mean ± SD values for three biological replicates (two-sided unpaired t-test). h Bar plots comparing RIG1 gene expression in DMSO and DNMTi-HDACi GP5d, OE19, and GP5d p53-KO cells. The graph shows mean ± SD values for three biological replicates (two-sided unpaired t-test). i qRT-PCR data showing RIG-I mRNA expression in GP5d and OE19 cells treated with DMSO and DNMTi-HDACi (GAPDH normalized). The graph shows mean ± SD values for three biological replicates. Source data are provided as Supplementary Data .

Article Snippet: Next day cells were transfected with p53 expressing pIRES2-EGFP-p53 WT plasmid (addgene # 49242).

Techniques: Comparison, Expressing, RNA Sequencing, ChIP-sequencing, Microscopy, Staining, Gene Expression, Quantitative RT-PCR

Journal: Communications Biology

Article Title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

doi: 10.1038/s42003-025-08413-0

Figure Lengend Snippet: a SETDB1i/KO increases the expression of TE-chimeric transcripts. Boxplots show the expression of TE-chimeric transcripts in A375, GP5d, OE19, and GP5d p53-KO cells with and without SETDB1i/KO. Expression of TE-chimeric transcripts was analyzed with the TEprof2 pipeline . P -values were calculated with a two-sided Wilcoxon test (n = 53, 24, 144, and 141 differentially expressed TE-chimeric transcripts in A375, GP5d, GP5d p53-KO, and OE19, respectively). The lower and upper hinges of the boxes represent the 25th to 75th percentiles, the midline is the median, and the whiskers extend from the hinges to the minimum and maximum values by 1.5 * IQR. b Analysis of TE subfamilies from which the TE-chimeric transcripts are derived from upon SETDB1i/KO. The counts for TE-chimeric transcripts are log2-transformed. c DNMTi-HDACi increases the expression of TE-chimeric transcripts. Boxplots show the expression of TE-chimeric transcripts in DMSO and DNMTi-HDACi GP5d, GP5d p53-KO, and OE19 cells. P -values were calculated with a two-sided Wilcoxon test (n = 35, 175, and 193 for GP5d, OE19, and GP5d p53-KO cells, respectively). The boxplot features are as in Fig. 7a. d Cell type-specific expression of TE subfamilies forming TE-chimeric transcripts by DNMTi-HDACi in cancer cell lines. The counts for TE-chimeric transcripts are log2-transformed. e ISGs are upregulated by DNMTi-HDACi. ISG log2FCs are plotted for DNMTi-HDACi treated GP5d, OE19 and GP5d p53-KO cells. f SETDB1i and DNMTi-HDACi increased levels of Serine 477 phosphorylated IRF7. Western blot compares Ser477-phospho-IRF7 and total IRF7 protein levels in GP5d, OE19 and GP5d p53-KO cells treated with different CME inhibitors. g qRT-PCR data showing IFNα mRNA expression in GP5d and OE19 cells treated with DMSO, DNMTi-HDACi, and SETDB1i (GAPDH normalized). ISGs are upregulated by DNMTi-HDACi and SETDB1i in GP5d and OE19 cells. The graph shows mean ± SD values for three biological replicates. Source data are provided as Supplementary Data .

Article Snippet: Next day cells were transfected with p53 expressing pIRES2-EGFP-p53 WT plasmid (addgene # 49242).

Techniques: Expressing, Derivative Assay, Transformation Assay, Western Blot, Quantitative RT-PCR

Journal: Molecular cell

Article Title: A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

doi: 10.1016/j.molcel.2024.02.018

Figure Lengend Snippet: Figure 1. Class A JDPs prevent misfolding and aggregation of mutant p53 (A–C) Aggregation of R249S and R282W destabilized p53 mutants either alone (black) or upon addition of 2-fold molar excess of DNAJA1 (maroon), DNAJA2 (red), DNAJB1 (blue), DNAJB4 (purple), DNAJB2 (teal), DNAJB6 (green), DNAJC7 (yellow), or DNAJC8 (brown), monitored by light scattering. Data are means (n = 3). (D) Fluorescence anisotropy binding assays of Alexa Fluor 488-labeled DNAJA2, titrated with increasing concentrations of p53 WT (blue), R249S (pink), or R282W (orange), measured at 37C. R249S and R282W p53 mutants bind DNAJA2 with 4.4 ± 0.2 and 1.2 ± 0.1 mM affinity, while only a weak binding is detected for WT p53. Data are means ± SEM (n = 3). (E) Fluorescence anisotropy binding assays of Alexa Fluor 488-labeled DNAJA2, titrated with increasing concentrations of p53 WT at increasing temperatures (25C–44C; light to dark blue). DNAJA2 affinity for WT p53 increased with temperature, from 70 mM at 37C to 10.4 mM at 40C and 4.5 mM at 44C. Data are means ± SEM (n = 3). (F) Immunofluorescence staining of destabilized R249S (top) or R282W (bottom) p53 mutant overexpressed in the p53 null SaOS2 cell lines shows distinct accumulation of p53 in cytoplasmic foci, corresponding to aggregates.20 Co-expression of V5-DNAJA2, but not V5-DNAJB1, results in a significant increase in diffuse cytoplasmic staining of mutant p53. EV, empty vector. The crop images are the overlay of p53 signal with DAPI. Scale bars: 10 mm. (G) Quantification of cytoplasmic foci in cells overexpressing p53 WT, R249S, or R282W with and without DNAJA2 co-expression (representative images in Figures 1F and S1F). Data represent mean values ± SD (n = 3). ***p < 0.001 (Student’s t test). See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pET29b DNAJA2mono (1-353) F337D TEV cleavable His tag This paper N/A pcDNA5 DNAJA2 with V5 tag Hageman et al.82 Addgene 19519 pcDNA5 DNAJA2 DBH D169-182 with V5 tag This paper N/A pET29b DNAJA1 TEV cleavable His tag This paper N/A pET-sumo DNAJB1 His-SUMO tag Ulp1 cleavable Nillegoda et al.61 N/A pcDNA5 DNAJB1 with V5 tag Hageman et al.82 Addgene 19522 pET29b DNAJB2 His-SUMO tag Ulp1 cleavable This paper N/A pET29b DNAJB4 TEV cleavable His tag Faust et al.60 N/A pET-sumo DNAJB6 His-SUMO tag Ulp1 cleavable This paper N/A pET29b DNAJC7 TEV cleavable His tag Hou et al.83 N/A pET29b DNAJC8 His-SUMO tag Ulp1 cleavable This paper N/A pET-sumo Hsp70 His-SUMO tag Ulp1 cleavable Wentink et al.54 N/A pET29b Hsp110 TEV cleavable His tag Nillegoda et al.61 N/A pET-27b p53 DBD (94-293) Kitayner et al.84 N/A pcDNA3.1 p53 Loughery et al.85 Addgene 69003 pET-27b p53 R249S DBD (94-293) Suad et al.44 N/A pcDNA3.1 p53 R249S This paper N/A pET-27b p53 R282W DBD (94-293) Degtjarik et al.46 N/A pcDNA3.1 p53 R282W This paper N/A pET-Sumo tau C291S, C322S, L243C, T373C Irwin et al.50 N/A pET SUMO Ydj1 mono (111-351) F335D His-SUMO tag Ulp1 cleavable This paper N/A Ulp1 protease Produced in house N/A Tobacco Etch Virus Protease Produced in house N/A Software and algorithms Image Studio LI-COR https://www.licor.com/bio/image- studio/resources#is5-clx ImageJ Schneider et al.86 https://imagej.nih.gov/ij/ Huygens Professional Scientific Volume Imaging https://svi.nl/Huygens-Professional NMRFAM-SPARKY Goddard and Kneller87 https://nmrfam.wisc.edu/ nmrfam-sparky-distribution/ Topspin Bruker https://www.bruker.com/en/products- and-solutions/mr/nmr-software/topspin.html NMRPipe Delaglio et al.88 https://www.ibbr.umd.edu/nmrpipe/ CcpNmrAnalysis Skinner et al.89 https://ccpn.ac.uk/software/version-2/ Prism GraphPad https://www.graphpad.com/ scientific-software/prism/ Dynamics Wyatt Technology https://www.wyatt.com/products/ software/dynamics.html ASTRA Wyatt Technology https://www.wyatt.com/products/ software/astra.html UCSF ChimeraX Pettersen et al.90 https://www.cgl.ucsf.edu/ chimerax/download.html

Techniques: Mutagenesis, Fluorescence, Binding Assay, Labeling, Staining, Expressing, Plasmid Preparation

Journal: Molecular cell

Article Title: A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

doi: 10.1016/j.molcel.2024.02.018

Figure Lengend Snippet: Figure 2. Mapping the interaction of p53 R249S mutant with DNAJA2 chaperone (A) Overlay of two-dimensional 1H-15N HSQC spectra of 15N, 2H-labeled R249S DBD alone (black) and with 2-fold molar excess of deuterated (2H) DNAJA2 (red). (B) Cartoon representation of p53 R249S structure (PDB: 3D07), with residues showing significant binding to DNAJA2 (I/I0 < 0.4) colored red. (C) Microsecond exchange contributions (Rex,ms) to 15N transverse relaxation rates of DNAJA2-bound p53 obtained from measurement of residue-specific R2(2HxNz), R2(2HzNx), R2(2HxNx), and R1(2HzNz) relaxation rates (which should correlate with changes in chemical shifts between the bound and free p53 states25,26) plotted as a function of changes in chemical shifts between free p53 and unfolded, random coil27–29 values (D\random coil). p53 residues affected by DNAJA2 binding are colored red. Lack of correlation indicates that the DNAJA2-bound p53 is not found in an unfolded conformation. (D) Fluorescence anisotropy binding assays of Alexa Fluor 488-labeled DNAJA2, titrated with increasing concentrations of p53 R249S at increasing NaCl concentrations (50–300 mM; light to dark pink). DNAJA2 affinity for p53 R249S decreased with increase in ionic strength indicative of electrostatic interaction. Data are means ± SEM (n = 3). (E) Degree of solvent protection/accessibility, calculated based on H/D exchange experiments, for WT (left) and R249S (right) p53 DBD at 28C, colored on their corresponding crystal structures (WT, PDB: 2OCJ; R249S, PDB: 3D07). See also Figures S2 and S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pET29b DNAJA2mono (1-353) F337D TEV cleavable His tag This paper N/A pcDNA5 DNAJA2 with V5 tag Hageman et al.82 Addgene 19519 pcDNA5 DNAJA2 DBH D169-182 with V5 tag This paper N/A pET29b DNAJA1 TEV cleavable His tag This paper N/A pET-sumo DNAJB1 His-SUMO tag Ulp1 cleavable Nillegoda et al.61 N/A pcDNA5 DNAJB1 with V5 tag Hageman et al.82 Addgene 19522 pET29b DNAJB2 His-SUMO tag Ulp1 cleavable This paper N/A pET29b DNAJB4 TEV cleavable His tag Faust et al.60 N/A pET-sumo DNAJB6 His-SUMO tag Ulp1 cleavable This paper N/A pET29b DNAJC7 TEV cleavable His tag Hou et al.83 N/A pET29b DNAJC8 His-SUMO tag Ulp1 cleavable This paper N/A pET-sumo Hsp70 His-SUMO tag Ulp1 cleavable Wentink et al.54 N/A pET29b Hsp110 TEV cleavable His tag Nillegoda et al.61 N/A pET-27b p53 DBD (94-293) Kitayner et al.84 N/A pcDNA3.1 p53 Loughery et al.85 Addgene 69003 pET-27b p53 R249S DBD (94-293) Suad et al.44 N/A pcDNA3.1 p53 R249S This paper N/A pET-27b p53 R282W DBD (94-293) Degtjarik et al.46 N/A pcDNA3.1 p53 R282W This paper N/A pET-Sumo tau C291S, C322S, L243C, T373C Irwin et al.50 N/A pET SUMO Ydj1 mono (111-351) F335D His-SUMO tag Ulp1 cleavable This paper N/A Ulp1 protease Produced in house N/A Tobacco Etch Virus Protease Produced in house N/A Software and algorithms Image Studio LI-COR https://www.licor.com/bio/image- studio/resources#is5-clx ImageJ Schneider et al.86 https://imagej.nih.gov/ij/ Huygens Professional Scientific Volume Imaging https://svi.nl/Huygens-Professional NMRFAM-SPARKY Goddard and Kneller87 https://nmrfam.wisc.edu/ nmrfam-sparky-distribution/ Topspin Bruker https://www.bruker.com/en/products- and-solutions/mr/nmr-software/topspin.html NMRPipe Delaglio et al.88 https://www.ibbr.umd.edu/nmrpipe/ CcpNmrAnalysis Skinner et al.89 https://ccpn.ac.uk/software/version-2/ Prism GraphPad https://www.graphpad.com/ scientific-software/prism/ Dynamics Wyatt Technology https://www.wyatt.com/products/ software/dynamics.html ASTRA Wyatt Technology https://www.wyatt.com/products/ software/astra.html UCSF ChimeraX Pettersen et al.90 https://www.cgl.ucsf.edu/ chimerax/download.html

Techniques: Mutagenesis, Labeling, Binding Assay, Residue, Fluorescence, Solvent

Journal: Molecular cell

Article Title: A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

doi: 10.1016/j.molcel.2024.02.018

Figure Lengend Snippet: Figure 3. DNAJA2 recognizes misfolded p53 by the b-hairpin insertion in the ZFLR (A) Cartoon representation of DNAJA2 structural model, with the residues identified by NMR experiments to interact with p53 (Figures S3A and S3B) colored in violet. p53 binds to a b-hairpin insertion in the ZFLR. (B) Domain organization of wild-type DNAJA2 (top), DNAJA2 mutant lacking the b-hairpin (DNAJA2DBH; bottom), and a truncated monomeric DNAJA2 construct lacking the dimerization domain (DNAJA2mono; bottom). The ZFLR is colored yellow, the b-hairpin is colored violet, and the D169–182 deletion and its substitution with GGSGG sequence is represented as a gap in the ZFLR. (C) Light scattering measurements (optical density [OD] at 400 nm wavelength; OD 400 nm) following aggregation of R249S alone (black) or in the presence of WT DNAJA2 (red) or DNAJA2DBH mutant (violet). No aggregation-prevention activity was detected for the mutant DNAJA2 lacking the b-hairpin region. Data are means (n = 3). (D) Light scattering measurements (OD 400 nm) following aggregation of heat-denatured insulin alone (black) or in the presence of 3-fold molar excess of WT DNAJA2 (red) or DNAJA2DBH mutant (violet). Both DNAJA2 WT and DNAJA2DBH mutant fully suppress insulin aggregation over 5 h. Data are means (n = 3).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pET29b DNAJA2mono (1-353) F337D TEV cleavable His tag This paper N/A pcDNA5 DNAJA2 with V5 tag Hageman et al.82 Addgene 19519 pcDNA5 DNAJA2 DBH D169-182 with V5 tag This paper N/A pET29b DNAJA1 TEV cleavable His tag This paper N/A pET-sumo DNAJB1 His-SUMO tag Ulp1 cleavable Nillegoda et al.61 N/A pcDNA5 DNAJB1 with V5 tag Hageman et al.82 Addgene 19522 pET29b DNAJB2 His-SUMO tag Ulp1 cleavable This paper N/A pET29b DNAJB4 TEV cleavable His tag Faust et al.60 N/A pET-sumo DNAJB6 His-SUMO tag Ulp1 cleavable This paper N/A pET29b DNAJC7 TEV cleavable His tag Hou et al.83 N/A pET29b DNAJC8 His-SUMO tag Ulp1 cleavable This paper N/A pET-sumo Hsp70 His-SUMO tag Ulp1 cleavable Wentink et al.54 N/A pET29b Hsp110 TEV cleavable His tag Nillegoda et al.61 N/A pET-27b p53 DBD (94-293) Kitayner et al.84 N/A pcDNA3.1 p53 Loughery et al.85 Addgene 69003 pET-27b p53 R249S DBD (94-293) Suad et al.44 N/A pcDNA3.1 p53 R249S This paper N/A pET-27b p53 R282W DBD (94-293) Degtjarik et al.46 N/A pcDNA3.1 p53 R282W This paper N/A pET-Sumo tau C291S, C322S, L243C, T373C Irwin et al.50 N/A pET SUMO Ydj1 mono (111-351) F335D His-SUMO tag Ulp1 cleavable This paper N/A Ulp1 protease Produced in house N/A Tobacco Etch Virus Protease Produced in house N/A Software and algorithms Image Studio LI-COR https://www.licor.com/bio/image- studio/resources#is5-clx ImageJ Schneider et al.86 https://imagej.nih.gov/ij/ Huygens Professional Scientific Volume Imaging https://svi.nl/Huygens-Professional NMRFAM-SPARKY Goddard and Kneller87 https://nmrfam.wisc.edu/ nmrfam-sparky-distribution/ Topspin Bruker https://www.bruker.com/en/products- and-solutions/mr/nmr-software/topspin.html NMRPipe Delaglio et al.88 https://www.ibbr.umd.edu/nmrpipe/ CcpNmrAnalysis Skinner et al.89 https://ccpn.ac.uk/software/version-2/ Prism GraphPad https://www.graphpad.com/ scientific-software/prism/ Dynamics Wyatt Technology https://www.wyatt.com/products/ software/dynamics.html ASTRA Wyatt Technology https://www.wyatt.com/products/ software/astra.html UCSF ChimeraX Pettersen et al.90 https://www.cgl.ucsf.edu/ chimerax/download.html

Techniques: Mutagenesis, Construct, Sequencing, Activity Assay

Journal: Molecular cell

Article Title: A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

doi: 10.1016/j.molcel.2024.02.018

Figure Lengend Snippet: Figure 4. Release of p53 monomers from the p53-DNAJA2 complex (A–C) Effect of various components of the Hsp70 chaperone system on the release of monomeric p53 mutants from preformed DNAJA2-p53 sequestration complexes. Misfolded p53 R249S (B) or R282W (C) mutants were incubated for 3 h at 37C alone (row 1) or in the presence of DNAJA2 WT (row 2) or DNAJA2 QPN (row 4). The formed DNAJA2-p53 complexes were then incubated with the indicated components of the Hsp70 system (rows 3–6), and the reactions were allowed to proceed at 25C. The final products of these reactions were separated into insoluble and soluble fractions, with the soluble fraction further separated by SEC. All of the fractions were blotted against p53. The elution volumes of molecular standards are indicated above, and the elution volume of untreated monomeric p53 is marked in red. Addition of Hsp70 refolding system to p53-DNAJA2 complexes is sufficient to efficiently release soluble p53 monomers. (D) (Top) 1H-15N HSQC NMR spectra of 15N-labeled p53 R249S DBD following sequestration by DNAJA2 and release by the Hsp70-system (fraction 19 in row 3 of B). (Bottom) Difference in chemical shifts between the untreated p53 R249S DBD and the DNAJA2/Hsp70/Hsp110-treated protein. No significant chemical shift perturbations are detected between the two samples, indicating that the chaperones do not affect the conformation of the released p53 mutant. (E) Interaction of untreated and chaperone-treated p53 WT, R249S, and R282W proteins with p21 promoter DNA as detected by EMSA. Both the WT-untreated p53 and the chaperone-treated protein bind to DNA, while the aggregated protein and p53 in complex with DNAJA2 do not. The position of the p53-p21 complex and unbound p21 DNA are indicated. See also Figures S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pET29b DNAJA2mono (1-353) F337D TEV cleavable His tag This paper N/A pcDNA5 DNAJA2 with V5 tag Hageman et al.82 Addgene 19519 pcDNA5 DNAJA2 DBH D169-182 with V5 tag This paper N/A pET29b DNAJA1 TEV cleavable His tag This paper N/A pET-sumo DNAJB1 His-SUMO tag Ulp1 cleavable Nillegoda et al.61 N/A pcDNA5 DNAJB1 with V5 tag Hageman et al.82 Addgene 19522 pET29b DNAJB2 His-SUMO tag Ulp1 cleavable This paper N/A pET29b DNAJB4 TEV cleavable His tag Faust et al.60 N/A pET-sumo DNAJB6 His-SUMO tag Ulp1 cleavable This paper N/A pET29b DNAJC7 TEV cleavable His tag Hou et al.83 N/A pET29b DNAJC8 His-SUMO tag Ulp1 cleavable This paper N/A pET-sumo Hsp70 His-SUMO tag Ulp1 cleavable Wentink et al.54 N/A pET29b Hsp110 TEV cleavable His tag Nillegoda et al.61 N/A pET-27b p53 DBD (94-293) Kitayner et al.84 N/A pcDNA3.1 p53 Loughery et al.85 Addgene 69003 pET-27b p53 R249S DBD (94-293) Suad et al.44 N/A pcDNA3.1 p53 R249S This paper N/A pET-27b p53 R282W DBD (94-293) Degtjarik et al.46 N/A pcDNA3.1 p53 R282W This paper N/A pET-Sumo tau C291S, C322S, L243C, T373C Irwin et al.50 N/A pET SUMO Ydj1 mono (111-351) F335D His-SUMO tag Ulp1 cleavable This paper N/A Ulp1 protease Produced in house N/A Tobacco Etch Virus Protease Produced in house N/A Software and algorithms Image Studio LI-COR https://www.licor.com/bio/image- studio/resources#is5-clx ImageJ Schneider et al.86 https://imagej.nih.gov/ij/ Huygens Professional Scientific Volume Imaging https://svi.nl/Huygens-Professional NMRFAM-SPARKY Goddard and Kneller87 https://nmrfam.wisc.edu/ nmrfam-sparky-distribution/ Topspin Bruker https://www.bruker.com/en/products- and-solutions/mr/nmr-software/topspin.html NMRPipe Delaglio et al.88 https://www.ibbr.umd.edu/nmrpipe/ CcpNmrAnalysis Skinner et al.89 https://ccpn.ac.uk/software/version-2/ Prism GraphPad https://www.graphpad.com/ scientific-software/prism/ Dynamics Wyatt Technology https://www.wyatt.com/products/ software/dynamics.html ASTRA Wyatt Technology https://www.wyatt.com/products/ software/astra.html UCSF ChimeraX Pettersen et al.90 https://www.cgl.ucsf.edu/ chimerax/download.html

Techniques: Incubation, Labeling, Mutagenesis

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

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).

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: Quantitative RT-PCR, Expressing, RNA Sequencing

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.

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

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.

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

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.

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

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.

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: Variant Assay