Senescence plays a key role in various physiological and pathological processes. We reported that injury-induced transient senescence correlates with heart regeneration, yet the multi-omics profile and molecular underpinnings of regenerative senescence remain obscure. Using proteomics and single-cell RNA sequencing, here we report the regenerative senescence multi-omic signature in the adult mouse heart and establish its role in neonatal heart regeneration and agrin-mediated cardiac repair in adult mice. We identified early growth response protein 1 (Egr1) as a regulator of regenerative senescence in both models. In the neonatal heart, Egr1 facilitates angiogenesis and cardiomyocyte proliferation. In adult hearts, agrin-induced senescence and repair require Egr1, activated by the integrin-FAK-ERK-Akt1 axis in cardiac fibroblasts. We also identified cathepsins as injury-induced senescence-associated secretory phenotype components that promote extracellular matrix degradation and potentially assist in reducing fibrosis. Altogether, we uncovered the molecular signature and functional benefits of regenerative senescence during heart regeneration, with Egr1 orchestrating the process. Zhang et al. show that Egr1 regulates transient senescence during neonatal heart regeneration and upon agrin-mediated cardiac repair in adult mice, acting downstream of the integrin-FAK-ERK-Akt1 axis in cardiac fibroblasts.

The p53 tumor suppressor, encoded by the TP53 gene, serves as a major barrier against malignant transformation. Patients with Li-Fraumeni syndrome (LFS) inherit a mutated TP53 allele from one parent and a wild-type TP53 allele from the other. Subsequently, the wild-type allele is lost and only the mutant TP53 allele remains. This process, which is termed loss of heterozygosity (LOH), results in only mutant p53 protein expression. We used primary dermal fibroblasts from LFS patients carrying the hotspot p53 gain-of-function pathogenic variant, R248Q to study the LOH process and characterize alterations in various pathways before and after LOH. We previously described the derivation of mutant p53 reactivating peptides, designated pCAPs (p53 Conformation Activating Peptides). In this study, we tested the effect of lead peptide pCAP-250 on LOH and on its associated cellular changes. We report that treatment of LFS fibroblasts with pCAP-250 prevents the accumulation of mutant p53 protein, inhibits LOH, and alleviates its cellular consequences. Furthermore, prolonged treatment with pCAP-250 significantly reduces DNA damage and restores long-term genomic stability. pCAPs may thus be contemplated as a potential preventive treatment to prevent or delay early onset cancer in carriers of mutant p53.

Tumor suppressor p53 (TP53) is frequently mutated in cancer, often resulting not only in loss of its tumor-suppressive function but also acquisition of dominant-negative and even oncogenic gain-of-function traits. While wild-type p53 levels are tightly regulated, mutants are typically stabilized in tumors, which is crucial for their oncogenic properties. Here, we systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. Most regulators of wild-type p53 also regulate p53 mutants, except for p53 R337H regulators, which are largely private to this mutant. Mechanistically, FBXO42 emerged as a positive regulator for a subset of p53 mutants, working with CCDC6 to control USP28-mediated mutant p53 stabilization. Additionally, C16orf72/HAPSTR1 negatively regulates both wild-type p53 and all tested mutants. C16orf72/HAPSTR1 is commonly amplified in breast cancer, and its overexpression reduces p53 levels in mouse mammary epithelium leading to accelerated breast cancer. This study offers a network perspective on p53 stability regulation, potentially guiding strategies to reinforce wild-type p53 or target mutant p53 in cancer.

Supplementary Figure Legend from hTERT-Immortalized Prostate Epithelial and Stromal-Derived Cells: an Authentic In vitro Model for Differentiation and Carcinogenesis

Supplementary Figure 2 from Transcriptional Programs following Genetic Alterations in p53, INK4A, and H-Ras Genes along Defined Stages of Malignant Transformation

Supplementary Figures 6-13 from p53 Regulates the Ras Circuit to Inhibit the Expression of a Cancer-Related Gene Signature by Various Molecular Pathways

Supplementary Figure S1 from hTERT-Immortalized Prostate Epithelial and Stromal-Derived Cells: an Authentic In vitro Model for Differentiation and Carcinogenesis

Supplementary Figure 4 from Transcriptional Programs following Genetic Alterations in <i>p53</i>, <i>INK4A</i>, and <i>H-Ras</i> Genes along Defined Stages of Malignant Transformation

Supplementary Materials & Methods from hTERT-Immortalized Prostate Epithelial and Stromal-Derived Cells: an Authentic In vitro Model for Differentiation and Carcinogenesis

Differential genes between pMSCs and their derived TLs (MSC TLs). The table contains genes that had at least 10 reads in the RNA sequencing analysis. CSC genes, ESC genes, shared target genes of Nanog, Sox2 and Oct4 or of Nanog, Sox2, Oct4, Dax1 and Nac1 and genes induced by the reprogramming process are indicated and can be filtered.

IPA analysis of the differential genes between pMSCs and their derived TLs (MSC TLs). Biological function was determined for the genes that were downregulated in p53 Mut MSC TLs compared to their parental, p53 Mut pMSC. Biological function, Pathways, upstream regulators and networks were determined for the genes that were upregulated in p53 Mut MSC TLs compared to their parental. (The threshold of significant genes; padj<0.05, -2>FC>2, max reads>10)

Supplementary Figure 1. Mutant p53 enhanced the self-renewal capacity and oncogenicity of primary bone marrow MSCs. Supplementary Figure 2. Mutant p53 MSC derived tumor lines exhibited augmented tumorigenic capacity compared with their parental cells. Supplementary Figure 3. Transcriptional differences between pMSCs and their derived MSC TLs. Supplementary Figure 4. Knocking out mutant p53, by CRISPR gene editing system, in a MSC TL led to a reduction in tumor vascularization. Supplementary Figure 5. p53 Mut MSC TL sub-clones highly expressed ESC signature derived genes and the p53 Mut dependent genes in MSC TL sub-clones are enriched for chromatin transcriptional activation marks in ESCs. Supplementary Figure 6. The MSC-TLs ESC signature derived genes are associated with poor patient survival. Supplementary Figure 7. Six hotspot p53 mutations frequency in TCGA datasets. Supplementary Figure 8. ESC gene expression signature entailed in the MSC TLs is detected in human tumors harboring p53 missense mutations. Supplementary Table 1 List of QRT PCR primers. Supplementary Table 7 Description of TCGA samples.

Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome associated with germline TP53 pathogenic variants. Here, we perform whole-genome sequence (WGS) analysis of tumors from 22 patients with TP53 germline pathogenic variants. We observe somatic mutations affecting Wnt, PI3K/AKT signaling, epigenetic modifiers and homologous recombination genes as well as mutational signatures associated with prior chemotherapy. We identify near-ubiquitous early loss of heterozygosity of TP53 , with gain of the mutant allele. This occurs earlier in these tumors compared to tumors with somatic TP53 mutations, suggesting the timing of this mark may distinguish germline from somatic TP53 mutations. Phylogenetic trees of tumor evolution, reconstructed from bulk and multi-region WGS, reveal that LFS tumors exhibit comparatively limited heterogeneity. Overall, our study delineates early copy number gains of mutant TP53 as a characteristic mutational process in LFS tumorigenesis, likely arising years prior to tumor diagnosis.

Supplementary Figure 1 from Transcriptional Programs following Genetic Alterations in p53, INK4A, and H-Ras Genes along Defined Stages of Malignant Transformation

Supplementary Figure 1 from Mutant p53 Protects Cells from 12-O-Tetradecanoylphorbol-13-Acetate–Induced Death by Attenuating Activating Transcription Factor 3 Induction

Supplementary Tables 1-4 from Mutant p53 Protects Cells from 12-O-Tetradecanoylphorbol-13-Acetate–Induced Death by Attenuating Activating Transcription Factor 3 Induction

Supplementary Figure 3 from Transcriptional Programs following Genetic Alterations in <i>p53</i>, <i>INK4A</i>, and <i>H-Ras</i> Genes along Defined Stages of Malignant Transformation

Supplementary Figure Legends from Transcriptional Programs following Genetic Alterations in p53, INK4A, and H-Ras Genes along Defined Stages of Malignant Transformation

The MSC-TLs ESC signature derived genes are associated with poor patient survival. Kaplan-Meier analyses of the indicated data sets showing significant relationship between expression levels of genes, derived from the MSC-TLs ESC signature, and patient survival. Supplementary

Supplementary Figure 2 from Transcriptional Programs following Genetic Alterations in <i>p53</i>, <i>INK4A</i>, and <i>H-Ras</i> Genes along Defined Stages of Malignant Transformation