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: Significant DEGs ranked by Wald stat were further analyzed with the GSEA Preranked tool in GSEA software (Broad Institute, version 4.3.2, build 13, RRID: SCR_003199; refs. , ) using the gene sets from the Molecular Signatures Database C2 (curated gene sets) collection (Broad Institute, version 2023.2, RRID: SCR_016863), supplemented with one gene set of the direct p53-targeted lncRNAs, which contains 86 lncRNAs that are upregulated by p53, each having a recurrent p53-binding site close to its transcription start site ( ).

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: 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: Significant DEGs ranked by Wald stat were further analyzed with the GSEA Preranked tool in GSEA software (Broad Institute, version 4.3.2, build 13, RRID: SCR_003199; refs. , ) using the gene sets from the Molecular Signatures Database C2 (curated gene sets) collection (Broad Institute, version 2023.2, RRID: SCR_016863), supplemented with one gene set of the direct p53-targeted lncRNAs, which contains 86 lncRNAs that are upregulated by p53, each having a recurrent p53-binding site close to its transcription start site ( ).

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

Journal: bioRxiv

Article Title: Complex I Drives Glutamine-Dependent TCA Cycle to Support Viability of MYC high Breast Cancer Cells

doi: 10.1101/2025.05.27.656356

Figure Lengend Snippet: A MYC protein expression across 15 TNBC cell lines determined using high content immunofluorescence imaging. Data: mean +/− SD. B Spearman correlation analysis for the correlation of MYC mRNA expression (Transcripts Per Kilobase Million/TMP values) and MYC protein expression in TNBC cells. C Hierarchical clustering of MYC activity gene sets. Left panel: Nine MYC target gene sets were derived from the MSigDB collections and an additional MYC-related gene set referred as GOGA_BC_SIGNATURE (GS-2) (from ) was included. Right panel: Hierarchical clustering of GSEA-based NES values for TNBC cell lines finds five similar gene sets (marked with red rectangle). MDA-MB-157 cell line was used as the reference cell line. D Spearman correlation between MYC V1V2 Normalized Enrichment Scores (NES) and Protein Expression Scores (PES) in TNBC cells. E Stratification of TNBC cells into MYC high and MYC low groups. TNBC cell lines were stratified into MYC high and MYC low groups according to the NES and FDR values from GSEA using MYC V1V2 gene set. The cut off for MYC high +++ group was arbitrarily set as NES>2, FDR<0.0001 and for MYC low 0 group NES<1.7, FDR>0.1. The NES and PES values correlated well except for HCC38, which was excluded from the analysis. F Differentially expressed Hallmark gene sets in MYC high (+++) versus MYC low (0) TNBC cell lines. G GSEA of mitochondrially localized MYC targets in MYC high (+++) versus MYC low (0) TNBC cell lines.

Article Snippet: MDA-MB-157 cells expressed low MYC protein and mRNA levels as well as the lowest score for MYC target signatures (Molecular Signature Database MSigDB; Broad Institute).

Techniques: Expressing, Immunofluorescence, Imaging, Activity Assay, Derivative Assay