p53 variants  (Addgene inc)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: In vitro CRISPR screening identified ULK1 as a combinatorial target with an MDM2 inhibitor. A MDM2 mRNA expression (log2(FPKM + 1)) among all paired samples from the TCGA grouped by cancer. Each point represents one sample. The P values are based on two-tailed Student’s t test. B Summary of the correlation between MDM2 expression and overall survival (OS) based on univariate Cox regression and Kaplan‒Meier analyses. Red indicates that MDM2 is a risk factor affecting the prognosis of cancer patients, and green represents protective factors. Only P values < 0.05 are shown. C Schematic diagram of the in vitro screening process used to identify novel drug combinations. D Dot plots showing gene-specific CRISPR viability scores (log fold change and RRA scores). The points ranked in the top ten are highlighted in blue. E Venn diagram showing the intersection of the top 15 genes ranked by the log fold change score and the top 15 genes ranked by the RRA score. F Heatmap of RNA-seq analysis of nine TP53 wild-type cancer cell lines. G Survival curves of APG-115 in nine TP53 wild-type cancer cell lines. H Correlation analysis between gene expression and the IC50 of APG-115. I Western blot showing ULK1 protein levels in A2780 cells and TOV21-G cells expressing sgRNAs targeting ULK1. J Cell viability was measured by an MTT assay. A2780 cells and TOV21-G cells expressing sgRNAs targeting ULK1 were treated with APG-115 for 72 h

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: In Vitro, CRISPR, Expressing, Two Tailed Test, RNA Sequencing Assay, Western Blot, MTT Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: P53 activation combined with ULK1 deficiency can initiate pyroptosis. A Evaluation of APG-115-induced pyroptosis in A2780 sgAAV1 and A2780 sgULK1 cells by phase contrast imaging. B Flow cytometric analysis of Annexin V-FITC and PI staining in A2780 ULK1 knockout cells following treatment with 10 µM APG-115 for 24 h. C LDH release was detected using an LDH Cytotoxicity Detection Kit (Beyotime) in A2780 ULK1 knockout cells following treatment with 10 µM APG-115 for 24 h. **** P < 0.0001. D, F Flow cytometric analysis of FITC staining, PI staining, and LDH release in A549 p53-overexpressing cells following treatment with TNFα + CHX for 24 h. **** P < 0.0001. E, G Flow cytometric analysis of FITC staining, PI staining, and LDH release in A2780 ULK1 knockout cells following treatment with TNFα + CHX for 24 h. H RNA was extracted from the indicated cells, and the expression of GSDMA-E was analyzed by qRT‒PCR. * P < 0.05, *** P < 0.001, **** P < 0.0001. I Western blot showing GSDME protein levels in indicated A2780 cells

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: Activation Assay, Imaging, Staining, Knock-Out, Expressing, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: P53 directly activates the transcription of GSDME. A Dot plot showing the differences in the mRNA expression of GSDME between patients with different TP53 mutation statuses. Each point represents one sample. B Bar plot showing the correlation between the mRNA expression of GSDME and that of TP53 in 1210 cell lines from the CCLE database grouped by organ system. C, D p53 affects the expression of GSDME. A2780 and A549 cells were transfected with the indicated plasmids, and the expression of GSDME was determined by immunoblotting. E, F RNA was extracted from the indicated cells, and the expression of GSDME was analyzed by qRT‒PCR. * P < 0.05, *** P < 0.001, **** P < 0.0001. G Illustration of the truncation fragments of the GSDME promoter. H, I Firefly luciferase activity was measured and normalized to that of Renilla luciferase as the internal control. ** P < 0.01, *** P < 0.001, **** P < 0.0001. J, K The published ChIP-seq dataset was reanalyzed via the UCSC Genome Browser. After p53 was activated with MDM2 inhibitors, a peak appeared in the GSDME promoter region. L The indicated HEK-293 T p53-overexpressing cells were subjected to a ChIP assay using an antibody against p53. Isotype-matched IgG was used as a negative control. ** P < 0.01. The data are representative of three independent experiments

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: Expressing, Mutagenesis, Transfection, Western Blot, Luciferase, Activity Assay, Control, ChIP-sequencing, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: The synergistic induction of pyroptosis by p53 activation and ULK1 depletion depends on mitochondria quality control. A, B Immunoblot analysis of ULK1, TOMM20, HSPD1, TIM23 and GAPDH expression in indicated cells. The indicated A2780 cells were treated with10 µM APG-115 for 24 h. C Immunofluorescence images of A2780 cells with deletion of ULK1 and treatment with 10 µM APG-115 for 24 h. The cells were costained with MitoTracker Deep Red and DAPI. The white line represents 10 µm. D, E Flow cytometric analysis of FITC- and PI-stained control, siFUNDC1, and siPARK A2780 cells treated with 10 µM APG-115 for 24 h. F , G Survival fractions of control, siFUNDC1, and siPARK A2780 cells following treatment with 10 µM APG-115 for 24 h. H, I LDH release was detected in models of mitophagy deficiency or macroautophagy deficiency following treatment with 10 µM APG-115 for 24 h. ns: P > 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. J Schematic of mechanisms underlying the synergy between MDM2i and ULK1 deficiency in TP53 wild-type cells. See main text for a detailed description

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: Activation Assay, Control, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: Combined targeting of mitophagy and activation of p53 could be used to reverse platinum resistance. A GSVA scores of mitophagy-related genes in cisplatin-sensitive and cisplatin-resistant samples of four tumors from TCGA. The sensitivity of the tumor samples was calculated using the "pRRophetic" R package, and the optimal cutoff value of the ROC curve was selected as the cutoff point between sensitivity and resistance based on the Youden index. The P values were calculated using the Wilcoxon rank–sum test. B GSVA scores of pyroptosis-related genes in cisplatin-sensitive and cisplatin-resistant samples of four tumors from TCGA. C Illustration showing the process for generating cisplatin-resistant cell lines. D Survival fractions of the indicated cisplatin-resistant cells following treatment with cisplatin for 72 h. E Immunoblotting of ULK1, TOMM20, HSPD1, TIM23 and GAPDH in extracts of cisplatin-resistant cells. F, G GSDME expression in cisplatin-resistant cells. *** P < 0.001. H, I The indicated cisplatin-resistant cells were treated with TNFα + CHX for 24 h. ** P < 0.01, *** P < 0.001, **** P < 0.0001. J Survival fractions of the indicated cisplatin-resistant cells following treatment with cisplatin for 72 h

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: Activation Assay, Western Blot, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: Schematic of ULK1 deficiency and p53 activation promote pyroptosis by activating GSDME directly (By Figdraw)

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: Activation Assay

Journal: Cancer Research

Article Title: The Prolonged Half-Life of the p53 Missense Variant R248Q Promotes Accumulation and Heterotetramer Formation with Wild-Type p53 to Exert the Dominant-Negative Effect

doi: 10.1158/0008-5472.CAN-24-1136

Figure Lengend Snippet: Characterization of the DNE exerted on WT p53 by the R248Q variant. A, Experimental schematic for the CRISPR/Ca9-mediated generation of human isogenic MOLM13- TP53 AML cell lines and their subsequent phenotypic and functional characterization. CRISPR-HDR, CRISPR-Cas9–mediated homology-directed repair; CRISPR-KO, CRISPR-Cas9–mediated gene knockout. B, Whole-cell lysates of isogenic MOLM13- TP53 AML cell lines of the indicated genotypes treated for 3 hours with either DMSO or 100 nmol/L Dauno were separated on a polyacrylamide gel and immunoblotted for p53, p21, and vinculin (representative blot of n = 3 independent experiments). C, Cell-cycle distribution of isogenic MOLM13- TP53 isogenic AML cell lines of the indicated genotypes treated for 6 hours with either DMSO or 100 nmol/L Dauno as determined by CytoPhase Violet staining ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SEM. **, P ≤ 0.01; ****, P ≤ 0.0001, one-way ANOVA, Tukey multiple comparison. D, Fraction of apoptotic MOLM13- TP53 isogenic AML cell lines as determined by Annexin V staining after treatment with either DMSO or 100 nmol/L Dauno for 72 hours ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SEM. *, P ≤ 0.05; ****, P ≤ 0.0001, two-way ANOVA, Tukey multiple comparison. E, Cell viability of MOLM13- TP53 isogenic AML cell lines was determined using the CellTiter-Glo reagent after being exposed to increasing concentrations of the indicated drugs for 72 hours ( n = 3 independent experiments). Symbols represent averages. Error bars, SD, Friedman test. F, Schematic depicting the experiment principle of the in vitro competition assay. Fluorescently labeled MOLM13- TP53 isogenic AML cell lines of various TP53 genotypes were cocultured at a 1:9 ratio in the presence of DMSO or 1 μmol/L nutlin-3a, and the relative fractions of each genotype were determined by flow cytometry every other day for 10 days. G, Heatmaps depict the results of the in vitro competition assays described in F . n = 3 independent experiments; color-coded averages are shown. d, day. H, Representative images of a colony-forming unit assay. MOLM13- TP53 isogenic AML cell lines of the indicated genotypes were cultured for 7 days in methylcellulose-based medium with DMSO or 2.5 μmol/L nutlin-3a. Images were taken using a Leica DMI6000B inverted microscope, and analysis was performed using the Colony Area ImageJ plugin, followed by manual counting of the colonies (representative images of n = 3 independent experiments). I, Counted colonies from colony-forming unit assay as described in H . n = 3 independent experiments. Bar graphs represent averages. Error bars, SEM. ****, P ≤ 0.0001, one-way ANOVA, Tukey multiple comparison. J, Quantification of the bioluminescent signal (i.e., total flux per second) for each group of mice 12 and 16 days after sublethal irradiation and subsequent injection with MOLM13- TP53 -GFP-luciferase isogenic AML cell lines (of the indicated genotypes). One week after injection, mice were treated with idasanutlin (Ida, 25 mg/kg of BW per day) or vehicle (Veh) for seven consecutive days. n = 7 animals per group. Symbols represent averages. Error bars, SD; ***, P ≤ 0.001, Mann–Whitney test. K, Survival analysis of NSG mice engrafted with MOLM13- TP53 -GFP-luciferase isogenic AML cell lines of the indicated genotypes and treated with idasanutlin (25 mg/kg of BW per day) or vehicle for seven consecutive days. n = 7 animals per group. ****, P ≤ 0.0001, Kaplan–Meier simple survival analysis. ns, nonsignificant.

Article Snippet: Afterward, mutagenized p53 variants were amplified and cloned into Cilantro 2 (a great gift from Benjamin Ebert, Addgene plasmid #74450, http://n2t.net/addgene:74450 , q RRID: Addgene_74450) while cutting out the included enhanced green fluorescent protein (EGFP) sequence.

Techniques: Variant Assay, CRISPR, Functional Assay, Gene Knockout, Staining, Comparison, In Vitro, Competitive Binding Assay, Labeling, Flow Cytometry, Colony-forming Unit Assay, Cell Culture, Inverted Microscopy, Irradiation, Injection, Luciferase, MANN-WHITNEY

Journal: Cancer Research

Article Title: The Prolonged Half-Life of the p53 Missense Variant R248Q Promotes Accumulation and Heterotetramer Formation with Wild-Type p53 to Exert the Dominant-Negative Effect

doi: 10.1158/0008-5472.CAN-24-1136

Figure Lengend Snippet: R248Q impairs the ability of WT p53 to bind to promoter DNA and to transactivate expression of target genes. A, Immunofluorescence microscopy images showing p53 (magenta), DAPI (blue), and CellBrite Fix 640 membrane stain (green) in MOLM13- TP53 isogenic AML cell lines treated for 3 hours with 100 nM Dauno ( n = 3 independent experiments; one representative example is shown; confocal microscope Leica DMI6000B, 63× objective). Scale bar, 2.5 μm. B, ChIP followed by NGS in isogenic MOLM- TP53 AML cell lines of the indicated genotypes after treatment with DMSO or 100 nmol/L Dauno for 6 hours. Plots show genome-wide relative enrichment of p53 variants (WT or R248Q) over p53 null cells over TSS-proximal regions (−10 kb to first intron). C, Tornado plots of WT and R248Q ChIP-seq peaks over TSS-proximal regions (−10kb, first intron with a horizontal window of 1 kb around the peak center) in isogenic MOLM- TP53 AML cell lines of the indicated genotypes. Cells were treated as described in B . Plots show relative enrichment and mutual overlap of the peaks identified in each cell line. D, Representative Integrative Genome Viewer images depicting the ChIP-seq peaks for p53 over the TSS of CDKN1A or MDM2 in isogenic MOLM- TP53 AML cell lines of the indicated genotypes after treatment as described in B . E, Relative expression of CDKN1A and MDM2 transcripts normalized to ACTB in isogenic MOLM- TP53 AML cell lines of the indicated genotypes after treatment with DMSO or 100 nmol/L Dauno for 6 hours ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SD. ns, nonsignificant; ****, P ≤ 0.0001, one-way ANOVA, Tukey multiple comparison.

Article Snippet: Afterward, mutagenized p53 variants were amplified and cloned into Cilantro 2 (a great gift from Benjamin Ebert, Addgene plasmid #74450, http://n2t.net/addgene:74450 , q RRID: Addgene_74450) while cutting out the included enhanced green fluorescent protein (EGFP) sequence.

Techniques: Expressing, Immunofluorescence, Microscopy, Membrane, Staining, Genome Wide, ChIP-sequencing, Comparison

Journal: Cancer Research

Article Title: The Prolonged Half-Life of the p53 Missense Variant R248Q Promotes Accumulation and Heterotetramer Formation with Wild-Type p53 to Exert the Dominant-Negative Effect

doi: 10.1158/0008-5472.CAN-24-1136

Figure Lengend Snippet: Heterotetramers consisting of R248Q and WT p53 are transcriptionally inactive, but overexpression of WT p53 can overcome the DNE of R248Q. A, MOLM13- TP53 isogenic cell lines were treated with DMSO or 100 nmol/L Dauno for 3 hours, followed by collection of whole-cell protein lysates by using IGEPAL lysis buffer. Lysates were fixed with 0.03% glutaraldehyde and subsequently separated on a polyacrylamide gel under nonreducing conditions. The PVDF membrane was immunoblotted for p53 ( n = 3 independent experiments; one representative blot is shown). B, MOLM13- TP53 null or K562- TP53 null AML cell lines lentivirally transduced to stably express full-length R248Q, tetramerization-deficient R248Q (R248Q-L344A), or dimerization-deficient R248Q (R248Q-L344P or R248Q-OD lacking the OD of p53) were treated for 3 hours with either DMSO or 100 nmol/L Dauno. Glutaraldehyde-fixed whole-cell protein lysates were separated on a polyacrylamide gel and immunoblotted for p53 ( n = 3 independent experiments; representative blots are shown). C, Experimental schematic depicting the general principle of the flow cytometry–based FRET assay in K562- TP53 null AML cells. For experimental details, please refer to the Materials and Methods. D, Representative FACS plots showing results of the FRET assay in K562- TP53 null AML cells for the interaction between WT monomers (top), or WT and R248Q monomers (bottom). FRET donor and acceptor plasmids encoding p53 WT or R248Q variants either oligomerization-proficient (WT) or -deficient (WT-L344A, WT-L344P, or WT-OD) were co-electroporated into K562- TP53 null AML cells, followed by FRET measurement (FRET fluorescence measured using the 525/50-nm detector). E, Pooled results of the FRET assays in K562- TP53 null AML cells ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SD. ***, P ≤ 0.001; ****, P ≤ 0.0001, one-way ANOVA, Tukey multiple comparison. F, Experimental schematic and pooled results of the transcriptional reporter assay in K562- TP53 null -p21-GFP cells electroporated with plasmids encoding oligomerization-proficient or -deficient WT p53 variants ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SEM. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001, one-way ANOVA, Tukey multiple comparison. G, Experimental schematic and pooled results of the transcriptional reporter assay in K562- TP53 WT -p21-GFP cells electroporated with pRK5 plasmids encoding oligomerization-proficient or -deficient R248Q p53 variants ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SEM. **, P ≤ 0.01; ****, P ≤ 0.0001, one-way ANOVA, Tukey multiple comparison. H, Quantification of the bioluminescent signal (i.e., total flux per second) for each group of mice 12 and 16 days after sublethal irradiation and subsequent injection with MOLM13- TP53 +/− -GFP-luciferase AML cell lines overexpressing either R248Q (WT + R248Q) or oligomerization-deficient R248Q-L344P (WT + R248Q-L344P). Following, mice were treated with idasanutlin (Ida, 25 mg/kg of BW per day) or vehicle (Veh) for seven consecutive days ( n = 7 animals per group). Symbols represent averages. Error bars, SD. ***, P ≤ 0.001, Mann–Whitney test. I, Survival analysis of NSG mice engrafted with MOLM13- TP53 +/− -GFP-luciferase AML cell lines overexpressing either R248Q (WT + R248Q) or R248Q-L344P (WT + R248Q-L344P) and treated with idasanutlin (25 mg/kg of BW per day) or vehicle for seven consecutive days ( n = 7 animals per group). ****, P ≤ 0.0001, Kaplan–Meier simple survival analysis. J, Experimental schematic and pooled results of the transcriptional reporter assay in K562- TP53 R248Q -p21-GFP cells electroporated with the pRK5 plasmid encoding oligomerization-proficient WT p53 variant ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SEM. ****, P ≤ 0.0001, Welch t test. CNTR, control; FRET-Acc, FRET acceptor; FRET-Don, FRET donor. ns, nonsignificant.

Article Snippet: Afterward, mutagenized p53 variants were amplified and cloned into Cilantro 2 (a great gift from Benjamin Ebert, Addgene plasmid #74450, http://n2t.net/addgene:74450 , q RRID: Addgene_74450) while cutting out the included enhanced green fluorescent protein (EGFP) sequence.

Techniques: Over Expression, Lysis, Membrane, Stable Transfection, Flow Cytometry, Fluorescence, Comparison, Reporter Assay, Irradiation, Injection, Luciferase, MANN-WHITNEY, Plasmid Preparation, Variant Assay, Control

Journal: Cancer Research

Article Title: The Prolonged Half-Life of the p53 Missense Variant R248Q Promotes Accumulation and Heterotetramer Formation with Wild-Type p53 to Exert the Dominant-Negative Effect

doi: 10.1158/0008-5472.CAN-24-1136

Figure Lengend Snippet: The longer protein half-life of R248Q leads to an increase of the R248Q:WT ratio. A, Bone marrow biopsies of patients with TP53 WT or TP53 R248Q -mutant AML, high-grade B-cell lymphoma (HGBCL), or early T precursor lymphoblastic leukemia (ETP-ALL) stained with hematoxylin and eosin (H&E) and p53 IHC ( n = 6 representative images; ×100 magnification) show strong nuclear p53 staining in neoplastic cells of TP53 R248Q mutated cases. B, Experimental schematic for the CRISPR/Ca9-mediated generation of human isogenic cancer cell lines with TP53 WT or R248Q alleles and their subsequent use for p53 quantification and half-life determination as shown in C and E . CRC, colorectal cancer; CRISPR-HDR, CRISPR-Cas9–mediated homology-directed repair; NSCLC, non–small cell lung cancer. C, Whole-cell lysates of isogenic MOLM13- TP53 , K562- TP5 3, A549- TP53 , and HCT116- TP53 isogenic cell lines with WT and R248Q alleles treated for 6 hours with either DMSO or 100 nM Dauno were separated on a polyacrylamide gel and immunoblotted for p53 and vinculin. P53 was quantified via ImageJ software using vinculin to normalize p53 levels ( n = 3–4 independent experiments; representative blots are shown). Bar graphs represent averages. Error bars, SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, two-way ANOVA, Tukey multiple comparison. D, NGS of cDNA from isogenic MOLM13- TP53 AML cell lines of the indicated genotypes. Number of NGS reads supporting the WT (gray) or R248Q (blue) allele are shown ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SD. Two-way ANOVA, Tukey multiple comparison. ns, nonsignificant; n.d., not detectable. E, Half-life determination of p53 WT and R248Q in untreated isogenic MOLM13- TP53 , K562- TP53 , A549- TP53 , and HCT116- TP53 isogenic cell lines. Whole-cell lysates of cycloheximide-treated cells were collected at the indicated time points and separated on a polyacrylamide gel and immunoblotted for p53 and vinculin. p53 was quantified using ImageJ software ( n = 3–4 independent experiments; representative blots are shown). Error bars, SEM. ****, P ≤ 0.0001, Mann–Whitney test.

Article Snippet: Afterward, mutagenized p53 variants were amplified and cloned into Cilantro 2 (a great gift from Benjamin Ebert, Addgene plasmid #74450, http://n2t.net/addgene:74450 , q RRID: Addgene_74450) while cutting out the included enhanced green fluorescent protein (EGFP) sequence.

Techniques: Mutagenesis, Staining, CRISPR, Software, Comparison, MANN-WHITNEY

Journal: Cancer Research

Article Title: The Prolonged Half-Life of the p53 Missense Variant R248Q Promotes Accumulation and Heterotetramer Formation with Wild-Type p53 to Exert the Dominant-Negative Effect

doi: 10.1158/0008-5472.CAN-24-1136

Figure Lengend Snippet: A supraphysiologic protein abundance of R248Q is required for the DNE. A, Experimental schematic showing the principle of the p53 target degradation assay. B, Whole-cell lysates of iberdomide- and/or nutlin-3a–treated K562 cells with TP53 WT and degR248Q or K562 cells with TP53 WT and degGFP were separated on a polyacrylamide gel and immunoblotted for p53, p21, and vinculin ( n = 3 independent experiments; one representative blot is shown). C, Correlation between the WT p53 transcriptional activity as determined by the normalized p21/vinculin and the R248Q/WT ratio in K562 cells with TP53 WT and degR248Q. Cells were treated with 2.5 μmol/L nutlin-3a and increasing concentrations of iberdomide, whole-cell lysates were prepared, run on a polyacrylamide gel, immunoblotted for p53, p21, and vinculin, and bands were quantified using ImageJ software ( n = 3 independent experiments, pooled data, including averages and SEM are shown). D, Cell-cycle distribution of MOLM13 cells with WT TP53 and degR248Q or MOLM13 cells with WT TP53 and degGFP cells in the absence or presence of 1 μmol/L iberdomide and/or 2.5 μmol/L nutlin-3a for 24 hours as determined by CytoPhase Violet staining ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SEM. ****, P ≤ 0.0001, one-way ANOVA, Tukey multiple comparison. E, Fraction of apoptotic MOLM13 cells with WT TP53 and degR248Q or degGFP as determined by Annexin V staining in the absence or presence of 1 μmol/L iberdomide and/or 2.5 μmol/L nutlin-3a for 24 to 72 hours ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SEM. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001, two-way ANOVA, Tukey multiple comparison. F, Representative images of a colony-forming unit assay was performed in MOLM13 cells with WT TP53 and degR248Q or degGFP in methylcellulose-based medium containing DMSO, 5 μmol/L iberdomide, and/or 2.5 μmol/L nutlin-3a. After 7 days, images were taken using a Leica DMI6000 B inverted microscope, and analysis was performed using the Colony Area ImageJ plugin, followed by manual counting of the colonies (representative images of n = 3 independent experiments). G, Summary of counted colonies from the colony-forming unit assay as described in F ( n = 3 independent experiments). Bar graphs represent averages. Error bars, SEM. ****, P ≤ 0.0001, one-way ANOVA, Tukey multiple comparison. H, Results of in vitro competition assays of K562 cells with WT TP53 and degR248Q cocultured together with isogenic K562- TP53 WT cells (left) or K562- TP53 R248Q cells (right) in the absence or presence of 2.5 μmol/L nutlin-3a and/or 1 μmol/L iberdomide. The relative fractions of each genotype were determined by flow cytometry every other day for 8 days ( n = 3 independent experiments). Averages are shown. Error bars, SD. ****, P ≤ 0.0001, unpaired t test. I, The results of in vitro competition assays of MOLM13 cells with WT TP53 and degR248Q cocultured together with isogenic MOLM13 cells with WT TP53 (left) or MOLM13 cells with R248Q (right) in the absence or presence of 2.5 μmol/L nutlin-3a and/or 1 μmol/L iberdomide. The relative fractions of each genotype were determined by flow cytometry every other day for 8 days ( n = 3 independent experiments). Symbols indicate averages. Error bars, SD. ****, P ≤ 0.0001, unpaired t test; ns, nonsignificant.

Article Snippet: Afterward, mutagenized p53 variants were amplified and cloned into Cilantro 2 (a great gift from Benjamin Ebert, Addgene plasmid #74450, http://n2t.net/addgene:74450 , q RRID: Addgene_74450) while cutting out the included enhanced green fluorescent protein (EGFP) sequence.

Techniques: Quantitative Proteomics, Degradation Assay, Activity Assay, Software, Staining, Comparison, Colony-forming Unit Assay, Inverted Microscopy, In Vitro, Flow Cytometry

Journal: Cancer Research

Article Title: The Prolonged Half-Life of the p53 Missense Variant R248Q Promotes Accumulation and Heterotetramer Formation with Wild-Type p53 to Exert the Dominant-Negative Effect

doi: 10.1158/0008-5472.CAN-24-1136

Figure Lengend Snippet: Therapeutic efficacy of lowering the increased R248Q:WT protein ratio in vivo . A, Experimental workflow for in vivo p53-targeted R248Q degradation xenograft assay. MOLM13 cells with WT TP53 with degradable BFP (degBFP) or R248Q (degR248Q) as well as GFP-luciferase were transplanted into sublethally irradiated NSG mice. After 5 to 7 days, treatment with vehicle (Veh), iberdomide (Iber), and/or idasanutlin (Ida) at the indicated concentrations was initiated and continued for 7 to 10 days, respectively. Leukemic burden was assessed by bioluminescent imaging 12 and 16 days after injection, and survival was monitored. B, Representative bioluminescent images of mice treated according to the experimental workflow depicted in A . C, Quantification of the bioluminescent signal (i.e., total flux per second) for each group of mice 12 and 16 days after sublethal irradiation and injection of MOLM13 cells with WT TP53 and degR248Q or degBFP and GFP-luciferase. Mice were treated as described in A ( n = 7 animals per group). Symbols represent averages. Error bars, SD. ***, P ≤ 0.001, Mann–Whitney test. D, Survival analysis of NSG mice engrafted with MOLM13 cells with WT TP53 and degR248Q or degBFP and GFP-luciferase and treated as described in A ( n = 7 animals per group). ****, P ≤ 0.0001, Kaplan–Meier simple survival analysis.

Article Snippet: Afterward, mutagenized p53 variants were amplified and cloned into Cilantro 2 (a great gift from Benjamin Ebert, Addgene plasmid #74450, http://n2t.net/addgene:74450 , q RRID: Addgene_74450) while cutting out the included enhanced green fluorescent protein (EGFP) sequence.

Techniques: Drug discovery, In Vivo, Xenograft Assay, Luciferase, Irradiation, Imaging, Injection, MANN-WHITNEY

Journal: Cancer Research

Article Title: The Prolonged Half-Life of the p53 Missense Variant R248Q Promotes Accumulation and Heterotetramer Formation with Wild-Type p53 to Exert the Dominant-Negative Effect

doi: 10.1158/0008-5472.CAN-24-1136

Figure Lengend Snippet: Proposed models for DNE exerted by R248Q on WT p53. A, Visual summary of the findings of the present study demonstrating that the increased R248Q:WT ratio is a critical determinant of the DNE of R248Q and a putative therapeutic target. B, Hypothetical model (model A, top) and proposed model (model B, bottom) for the oligomerization of R248Q and WT p53 monomers in cells with monoallelic TP53 R248Q missense mutations based on the findings of the present study and refs. and .

Article Snippet: Afterward, mutagenized p53 variants were amplified and cloned into Cilantro 2 (a great gift from Benjamin Ebert, Addgene plasmid #74450, http://n2t.net/addgene:74450 , q RRID: Addgene_74450) while cutting out the included enhanced green fluorescent protein (EGFP) sequence.

Techniques: