Mdm2-p53 Signaling in Tissue Homeostasis and the DNA Damage Response: A Dissertation

The p53 transcription factor responds to various cellular stressors by regulating the expression of numerous target genes involved in cellular processes such as cell cycle arrest, apoptosis, and senescence. As these downstream pathways are harmful to the growth and development of normal cells when prolonged or deregulated, p53 activity needs to be under tight regulatory control. The Mdm2 oncoprotein is the chief negative regulator of p53, and many mouse models have demonstrated that absence of Mdm2 expression leads to constitutive p53 activation in a variety of cell types. While unregulated p53 can be deleterious to cells, functional p53 is essential for tumor suppression, as many human cancers harbor p53 mutations and p53 knockout mice rapidly develop spontaneous tumors. Therefore, the mechanisms that control p53 regulation by Mdm2 are critical to ensure p53 activity in the appropriate cellular context. Many genetically engineered mouse models have been created to analyze p53 and Mdm2 functions and these studies have yielded valuable insights into their physiological roles. This dissertation will describe the generation and characterization of novel mutant Mdm2 mouse models and their use to interrogate the roles of p53-Mdm2 signaling in tissue homeostasis and cell stress responses. Deletion of Mdm2 in epidermal progenitor cells of the skin and hair follicles resulted in progressive hair loss and decreased skin integrity, phenotypes that are characteristic of premature aging. Furthermore, p53 protein levels, p53 target gene expression, and cellular senescence were all upregulated in the skins of these mice, and epidermal stem cell numbers and function were diminished. These results indicate that Mdm2 is necessary to limit p53 activity in adult tissues to ensure normal stem cell function. Additional mouse models used to determine the role of Mdm2 phosphorylation will also be presented. DNA damage triggers an extensive cellular response, including activation of the ATM kinase. ATM activity is necessary for p53 protein stabilization and, therefore, p53 activation, but in vivo evidence suggests that phosphorylation of p53 itself had little effect on p53 stability. ATM was previously shown to phosphorylate MDM2 at serine residue 395 (394 in mice), and we generated knock-in mutant mouse models to study the role of this posttranslational modification in vivo. Absence of this phosphorylation site led to greatly diminished p53 stability and function in response to γ-irradiation and increased spontaneous tumorigenesis in mice. Conversely, a phosphomimic model demonstrated prolonged p53 activation in cells treated with γ-irradiation, which revealed that phosphorylation of this Mdm2 residue controls the duration of the DNA damage response. Therefore, these mouse models have uncovered new roles for the p53-Mdm2 regulatory axis in vivo and will be useful reagents in future studies of posttranslational modifications in oncogene and DNA damage-induced tumorigenesis

Understanding the Essex Junto: Fear, Dissent, and Propaganda in the Early Republic

Historians have never formed a consensus over the Essex Junto. In fact, though often associated with New England Federalists, propagandists evoked the Junto long after the Federalist Party’s demise in 1824. This article chronicles uses of the term Essex Junto and its significance as it evolved from the early republic through the 1840s

Human and mouse essentiality screens as a resource for disease gene discovery

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery. Discovery of causal variants for monogenic disorders has been facilitated by whole exome and genome sequencing, but does not provide a diagnosis for all patients. Here, the authors propose a Full Spectrum of Intolerance to Loss-of-Function (FUSIL) categorization that integrates gene essentiality information to aid disease gene discovery

Using Mouse Models to Explore MDM-p53 Signaling in Development, Cell Growth, and Tumorigenesis

The p53 transcription factor regulates the expression of numerous genes whose products affect cell proliferation, senescence, cellular metabolism, apoptosis, and DNA repair. These p53-mediated effects can inhibit the growth of stressed or mutated cells and suppress tumorigenesis in the organism. However, the various growth-inhibitory properties of p53 must be kept in check in nondamaged cells in order to facilitate proper embryogenesis or the homeostatic maintenance of adult tissues. This requisite inhibition of p53 is performed primarily by the MDM oncoproteins, Mdm2 and MdmX. These p53-binding proteins limit p53 activity both in normal cells and in stressed cells seeking to promote resolution of their p53-stress response. Many mouse models bearing genetic alterations in Mdm2 or MdmX have been generated to explore the function and regulation of MDM-p53 signaling in development, in tissue homeostasis, in aging, and in cancer. These models not only have demonstrated a critical need for Mdm2 and MdmX in normal cell growth and in development but more recently have identified the MDM-p53 signaling axis as a key regulator of the cellular response to a wide variety of genetic or metabolic stresses. In this review, we discuss what has been learned from various studies of these Mdm2 and MdmX mouse models and highlight a few of the many important remaining questions

ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice

DNA damage induced by ionizing radiation activates the ATM kinase, which subsequently stabilizes and activates the p53 tumor suppressor protein. Although phosphorylation of p53 by ATM was found previously to modulate p53 levels and transcriptional activities in vivo, it does not appear to be a major regulator of p53 stability. We have utilized mice bearing altered Mdm2 alleles to demonstrate that ATM phosphorylation of Mdm2 serine 394 is required for robust p53 stabilization and activation after DNA damage. In addition, we demonstrate that dephosphorylation of Mdm2 Ser394 regulates attenuation of the p53-mediated response to DNA damage. Therefore, the phosphorylation status of Mdm2 Ser394 governs p53 protein levels and functions in cells undergoing DNA damage

Mdm2-p53 signaling regulates epidermal stem cell senescence and premature aging phenotypes in mouse skin

The p53 transcription factor is activated by various types of cell stress or DNA damage and induces the expression of genes that control cell growth and inhibit tumor formation. Analysis of mice that express mutant forms of p53 suggest that inappropriate p53 activation can alter tissue homeostasis and life span, connecting p53 tumor suppressor functions with accelerated aging. However, other mouse models that display increased levels of wildtype p53 in various tissues fail to corroborate a link between p53 and aging phenotypes, possibly due to the retention of signaling pathways that negatively regulate p53 activity in these models. In this present study, we have generated mice lacking Mdm2 in the epidermis. Deletion of Mdm2, the chief negative regulator of p53, induced an aging phenotype in the skin of mice, including thinning of the epidermis, reduced wound healing, and a progressive loss of fur. These phenotypes arise due to an induction of p53-mediated senescence in epidermal stem cells and a gradual loss of epidermal stem cell function. These results reveal that activation of endogenous p53 by ablation of Mdm2 can induce accelerated aging phenotypes in mice

Mdm2 Phosphorylation Regulates Its Stability and Has Contrasting Effects on Oncogene and Radiation-Induced Tumorigenesis

ATM phosphorylation of Mdm2-S394 is required for robust p53 stabilization and activation in DNA-damaged cells. We have now utilized Mdm2S394A knockin mice to determine that phosphorylation of Mdm2-S394 regulates p53 activity and the DNA damage response in lymphatic tissues in vivo by modulating Mdm2 stability. Mdm2-S394 phosphorylation delays lymphomagenesis in Eμ-myc transgenic mice, and preventing Mdm2-S394 phosphorylation obviates the need for p53 mutation in Myc-driven tumorigenesis. However, irradiated Mdm2S394A mice also have increased hematopoietic stem and progenitor cell functions, and we observed decreased lymphomagenesis in sub-lethally irradiated Mdm2S394A mice. These findings document contrasting effects of ATM-Mdm2 signaling on p53 tumor suppression and reveal that destabilizing Mdm2 by promoting its phosphorylation by ATM would be effective in treating oncogene-induced malignancies, while inhibiting Mdm2-S394 phosphorylation during radiation exposure or chemotherapy would ameliorate bone marrow failure and prevent the development of secondary hematological malignancies

Phosphorylation of p53 serine 18 upregulates apoptosis to suppress Myc-induced tumorigenesis

ATM and p53 are critical regulators of the cellular DNA damage response and function as potent tumor suppressors. In cells undergoing ionizing radiation, ATM is activated by double-strand DNA breaks and phosphorylates the NH(2) terminus of p53 at serine residue 18. We have previously generated mice bearing an amino acid substitution at this position (p53S18A) and documented a role for p53 phosphorylation in DNA damage-induced apoptosis. In this present study, we have crossed E mu myc transgenic mice with our p53S18A mice to explore a role for ATM-p53 signaling in response to oncogene-induced tumorigenesis. Similar to DNA damage induced by ionizing radiation, expression of c-Myc in pre-B cells induces p53 serine 18 phosphorylation and Puma expression to promote apoptosis. E mu myc transgenic mice develop B-cell lymphoma more rapidly when heterozygous or homozygous for p53S18A alleles. However, E mu myc-induced tumorigenesis in p53S18A mice is slower than that observed in E mu myc mice deficient for either p53 or ATM, indicating that both p53-induced apoptosis and p53-induced growth arrest contribute to the suppression of B-cell lymphoma formation in E mu myc mice. These findings further reveal that oncogene expression and DNA damage activate the same ATM-p53 signaling cascade in vivo to regulate apoptosis and tumorigenesis

Aerobic glycolysis suppresses p53 activity to provide selective protection from apoptosis upon loss of growth signals or inhibition of BCR-Abl

Unlike the growth factor dependence of normal cells, cancer cells can maintain growth factor-independent glycolysis and survival through expression of oncogenic kinases, such as BCR-Abl. Although targeted kinase inhibition can promote cancer cell death, therapeutic resistance develops frequently, and further mechanistic understanding is needed. Cell metabolism may be central to this cell death pathway, as we have shown that growth factor deprivation leads to decreased glycolysis that promotes apoptosis via p53 activation and induction of the proapoptotic protein Puma. Here, we extend these findings to show that elevated glucose metabolism, characteristic of cancer cells, can suppress protein kinase Cdelta (PKCdelta)-dependent p53 activation to maintain cell survival after growth factor withdrawal. In contrast, DNA damage-induced p53 activation was PKCdelta independent and was not metabolically sensitive. Both stresses required p53 Ser(18) phosphorylation for maximal activity but led to unique patterns of p53 target gene expression, showing distinct activation and response pathways for p53 that were differentially regulated by metabolism. Consistent with oncogenic kinases acting to replace growth factors, treatment of BCR-Abl-expressing cells with the kinase inhibitor imatinib led to reduced metabolism and p53- and Puma-dependent cell death. Accordingly, maintenance of glucose uptake inhibited p53 activation and promoted imatinib resistance. Furthermore, inhibition of glycolysis enhanced imatinib sensitivity in BCR-Abl-expressing cells with wild-type p53 but had little effect on p53-null cells. These data show that distinct pathways regulate p53 after DNA damage and metabolic stress and that inhibiting glucose metabolism may enhance the efficacy of and overcome resistance to targeted molecular cancer therapies