A mitochondria-regulated p53-CCF circuit integrates genome integrity with inflammation.

Genomic instability and inflammation are distinct hallmarks of aging, but the connection between them is poorly understood. Understanding their interrelationship will help unravel new mechanisms and therapeutic targets of aging and age-associated diseases. Here we report a novel mechanism directly linking genomic instability and inflammation in senescent cells, through a mitochondria-regulated molecular circuit that connects the p53 tumor suppressor and cytoplasmic chromatin fragments (CCF), a driver of inflammation through the cGAS-STING pathway. Activation or inactivation of p53 by genetic and pharmacologic approaches showed that p53 suppresses CCF accumulation and the downstream inflammatory senescence-associated secretory phenotype (SASP), independent of its effects on cell cycle arrest. p53 activation suppressed CCF formation by promoting DNA repair, reflected in maintenance of genomic integrity, particularly in subtelomeric regions, as shown by single cell genome resequencing. Activation of p53 by pharmacological inhibition of MDM2 in old mice decreased features of SASP in liver, indicating a senomorphic role in vivo . Remarkably, mitochondria in senescent cells suppressed p53 activity by promoting CCF formation and thereby restricting ATM-dependent nuclear DNA damage signaling. These data provide evidence for a mitochondria-regulated p53-CCF circuit in senescent cells that controls DNA repair, genome integrity and inflammatory SASP, and is a potential target for senomorphic healthy aging interventions.

Treatment of calcific arterial disease via enhancement of autophagy using GSK343.

Vascular calcification is a hallmark of atherosclerotic disease and serves as a strong predictor and risk factor for cardiovascular events. Growing evidence suggests that autophagy may play a protective role in early atherosclerosis. The precise effects of autophagy on VSMC-mediated calcification remain unknown. In this study, we utilized multi-omic profiling to investigate impaired autophagy at the transcriptional level as a key driver of VSMC calcification. Our findings revealed that impaired autophagy is an essential determinant of VSMC calcification. We observed that an osteogenic environment affects the open chromatin status of VSMCs, compromising the transcriptional activation of autophagy initiation genes. In vivo experiments involve pharmacological and genetic activation of autophagy using mouse models of spontaneous large (Mgp-/-) and small (Abcc6-/-) artery calcification. Taken together, these data advance our mechanistic understanding of vascular calcification and provide important insights for a broad range of cardiovascular diseases involving VSMC phenotype switch.

Apoptotic stress causes mtDNA release during senescence and drives the SASP.

Senescent cells drive age-related tissue dysfunction partially through the induction of a chronic senescence-associated secretory phenotype (SASP)1. Mitochondria are major regulators of the SASP; however, the underlying mechanisms have not been elucidated2. Mitochondria are often essential for apoptosis, a cell fate distinct from cellular senescence. During apoptosis, widespread mitochondrial outer membrane permeabilization (MOMP) commits a cell to die3. Here we find that MOMP occurring in a subset of mitochondria is a feature of cellular senescence. This process, called minority MOMP (miMOMP), requires BAX and BAK macropores enabling the release of mitochondrial DNA (mtDNA) into the cytosol. Cytosolic mtDNA in turn activates the cGAS-STING pathway, a major regulator of the SASP. We find that inhibition of MOMP in vivo decreases inflammatory markers and improves healthspan in aged mice. Our results reveal that apoptosis and senescence are regulated by similar mitochondria-dependent mechanisms and that sublethal mitochondrial apoptotic stress is a major driver of the SASP. We provide proof-of-concept that inhibition of miMOMP-induced inflammation may be a therapeutic route to improve healthspan.

Inhibition of PLK4 remodels histone methylation and activates the immune response via the cGAS-STING pathway in TP53-mutated AML.

Acute myeloid leukemia (AML) with TP53 mutation is one of the most lethal cancers and portends an extremely poor prognosis. Based on in silico analyses of druggable genes and differential gene expression in TP53-mutated AML, we identified pololike kinase 4 (PLK4) as a novel therapeutic target and examined its expression, regulation, pathogenetic mechanisms, and therapeutic potential in TP53-mutated AML. PLK4 expression was suppressed by activated p53 signaling in TP53 wild-type AML and was increased in TP53-mutated AML cell lines and primary samples. Short-term PLK4 inhibition induced DNA damage and apoptosis in TP53 wild-type AML. Prolonged PLK4 inhibition suppressed the growth of TP53-mutated AML and was associated with DNA damage, apoptosis, senescence, polyploidy, and defective cytokinesis. A hitherto undescribed PLK4/PRMT5/EZH2/H3K27me3 axis was demonstrated in both TP53 wild-type and mutated AML, resulting in histone modification through PLK4-induced PRMT5 phosphorylation. In TP53-mutated AML, combined effects of histone modification and polyploidy activated the cGAS-STING pathway, leading to secretion of cytokines and chemokines and activation of macrophages and T cells upon coculture with AML cells. In vivo, PLK4 inhibition also induced cytokine and chemokine expression in mouse recipients, and its combination with anti-CD47 antibody, which inhibited the "don't-eat-me" signal in macrophages, synergistically reduced leukemic burden and prolonged animal survival. The study shed important light on the pathogenetic role of PLK4 and might lead to novel therapeutic strategies in TP53-mutated AML.

Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Upregulation of PD-L1 in Senescence and Aging.

Cellular senescence is a stable form of cell cycle arrest associated with proinflammatory responses. Senescent cells can be cleared by the immune system as a part of normal tissue homeostasis. However, senescent cells can also accumulate in aged and diseased tissues, contributing to inflammation and disease progression. The mechanisms mediating the impaired immune-mediated clearance of senescent cells are poorly understood. Here, we report that senescent cells upregulate the immune checkpoint molecule PD-L1, the ligand for PD-1 on immune cells, which drives immune cell inactivation. The induction of PD-L1 in senescence is dependent on the proinflammatory program. Furthermore, the secreted factors released by senescent cells are sufficient to upregulate PD-L1 in nonsenescent control cells, mediated by the JAK-STAT pathway. In addition, we show that prolongevity intervention rapamycin downregulates PD-L1 in senescent cells. Last, we found that PD-L1 is upregulated in several tissues in naturally aged mice and in the lungs of idiopathic pulmonary fibrosis patients. Together, our results report that senescence and aging are associated with upregulation of a major immune checkpoint molecule, PD-L1. Targeting PD-L1 may offer new therapeutic opportunities in treating senescence and age-associated diseases.

Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection.

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS-mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells' self-nucleic acids can be employed as a "danger signal" to alarm the immune system.

SIRT1 - a new mammalian substrate of nuclear autophagy.

Macroautophagic/autophagic degradation of nuclear components (or nuclear autophagy) is a poorly understood area in autophagy research. We previously reported the nuclear lamina protein LMNB1 (lamin B1) as a nuclear autophagy substrate in primary human cells, stimulating the investigation of nuclear autophagy in the mammalian system. We recently reported the sirtuin protein SIRT1 as a new selective substrate of nuclear autophagy in senescence and aging. Upon senescence of primary human cells, SIRT1 degradation is mediated by a direct nuclear SIRT1-LC3 interaction, followed by nucleus-to-cytoplasm shuttling of SIRT1 and autophagosome-lysosome degradation. In vivo, SIRT1 is downregulated by lysosomes in hematopoietic and immune organs upon natural aging in mice and in aged human T cells. Our study identified another substrate of nuclear autophagy and suggests a new strategy to promote SIRT1-mediated health benefits by suppressing its autophagic degradation.Abbreviations: HSPC: hematopoietic stem and progenitor cells; NAD+: nicotinamide adenine dinucleotide; SASP: senescence-associated secretory phenotype.

SIRT1 is downregulated by autophagy in senescence and ageing.

SIRT1 (Sir2) is an NAD+-dependent deacetylase that plays critical roles in a broad range of biological events, including metabolism, the immune response and ageing1-5. Although there is strong interest in stimulating SIRT1 catalytic activity, the homeostasis of SIRT1 at the protein level is poorly understood. Here we report that macroautophagy (hereafter referred to as autophagy), a catabolic membrane trafficking pathway that degrades cellular components through autophagosomes and lysosomes, mediates the downregulation of mammalian SIRT1 protein during senescence and in vivo ageing. In senescence, nuclear SIRT1 is recognized as an autophagy substrate and is subjected to cytoplasmic autophagosome-lysosome degradation, via the autophagy protein LC3. Importantly, the autophagy-lysosome pathway contributes to the loss of SIRT1 during ageing of several tissues related to the immune and haematopoietic system in mice, including the spleen, thymus, and haematopoietic stem and progenitor cells, as well as in CD8+CD28- T cells from aged human donors. Our study reveals a mechanism in the regulation of the protein homeostasis of SIRT1 and suggests a potential strategy to stabilize SIRT1 to promote productive ageing.

Mitochondria-to-nucleus retrograde signaling drives formation of cytoplasmic chromatin and inflammation in senescence.

Cellular senescence is a potent tumor suppressor mechanism but also contributes to aging and aging-related diseases. Senescence is characterized by a stable cell cycle arrest and a complex proinflammatory secretome, termed the senescence-associated secretory phenotype (SASP). We recently discovered that cytoplasmic chromatin fragments (CCFs), extruded from the nucleus of senescent cells, trigger the SASP through activation of the innate immunity cytosolic DNA sensing cGAS-STING pathway. However, the upstream signaling events that instigate CCF formation remain unknown. Here, we show that dysfunctional mitochondria, linked to down-regulation of nuclear-encoded mitochondrial oxidative phosphorylation genes, trigger a ROS-JNK retrograde signaling pathway that drives CCF formation and hence the SASP. JNK links to 53BP1, a nuclear protein that negatively regulates DNA double-strand break (DSB) end resection and CCF formation. Importantly, we show that low-dose HDAC inhibitors restore expression of most nuclear-encoded mitochondrial oxidative phosphorylation genes, improve mitochondrial function, and suppress CCFs and the SASP in senescent cells. In mouse models, HDAC inhibitors also suppress oxidative stress, CCF, inflammation, and tissue damage caused by senescence-inducing irradiation and/or acetaminophen-induced mitochondria dysfunction. Overall, our findings outline an extended mitochondria-to-nucleus retrograde signaling pathway that initiates formation of CCF during senescence and is a potential target for drug-based interventions to inhibit the proaging SASP.

Histone Acetyltransferase p300 Induces De Novo Super-Enhancers to Drive Cellular Senescence.

Accumulation of senescent cells during aging contributes to chronic inflammation and age-related diseases. While senescence is associated with profound alterations of the epigenome, a systematic view of epigenetic factors in regulating senescence is lacking. Here, we curated a library of short hairpin RNAs for targeted silencing of all known epigenetic proteins and performed a high-throughput screen to identify key candidates whose downregulation can delay replicative senescence of primary human cells. This screen identified multiple new players including the histone acetyltransferase p300 that was found to be a primary driver of the senescent phenotype. p300, but not the paralogous CBP, induces a dynamic hyper-acetylated chromatin state and promotes the formation of active enhancer elements in the non-coding genome, leading to a senescence-specific gene expression program. Our work illustrates a causal role of histone acetyltransferases and acetylation in senescence and suggests p300 as a potential therapeutic target for senescence and age-related diseases.

Senescence Elicits Stemness: A Surprising Mechanism for Cancer Relapse.

Cellular senescence is traditionally viewed as a permanent form of cell cycle arrest that restrains tumorigenesis. In a recent study in Nature, however, Milanovic et al. (2018) challenge this conventional view, showing that senescence can counterintuitively promote cancer stemness and tumor aggressiveness. This finding suggests that attacking senescence can be exploited in cancer therapy.

Combinatorial genetics in liver repopulation and carcinogenesis with a in vivo CRISPR activation platform.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR-associated 9 (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high-throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type-specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto-oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene-inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors. CONCLUSION: The in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).

Cytoplasmic chromatin triggers inflammation in senescence and cancer.

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

Vps34 regulates Rab7 and late endocytic trafficking through recruitment of the GTPase-activating protein Armus.

The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34-/- mouse embryonic fibroblasts (MEFs), transferrin recycling and EEA1 membrane localization were unaffected despite elevated Rab5-GTP levels. Strikingly, a large increase in Rab7-GTP levels, an accumulation of enlarged late endosomes, and decreased EGFR degradation were observed in Vps34-deficient cells. The hyperactivation of Rab7 in Vps34-deficient cells stemmed from the failure to recruit the Rab7 GTPase-activating protein (GAP) Armus (also known as TBC1D2), which binds to PI(3)P, to late endosomes. Protein-lipid overlay and liposome-binding assays reveal that the putative pleckstrin homology (PH) domain in Armus can directly bind to PI(3)P. Elevated Rab7-GTP led to the failure of intraluminal vesicle (ILV) formation and lysosomal maturation. Rab7 silencing and Armus overexpression alleviated the vacuolization seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking.

Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53's transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma.