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Although DNA damage is intricately linked to metabolism, the metabolic alterations that occur in response to DNA damage are not well understood. We use a DNA repair–deficient model of ERCC1-XPF in Caenorhabditis elegans to gain insights on how genotoxic stress drives aging. Using multi-omic approach, we discover that nuclear DNA damage promotes mitochondrial β-oxidation and drives a global loss of fat depots. This metabolic shift to β-oxidation generates acetyl–coenzyme A to promote histone hyperacetylation and an associated change in expression of immune-effector and cytochrome genes. We identify the histone acetyltransferase MYS-1, as a critical regulator of this metabolic-epigenetic axis. We show that in response to DNA damage, polyunsaturated fatty acids, especially arachidonic acid (AA) and AA-related lipid mediators, are elevated and this is dependent on mys-1 . Together, these findings reveal that DNA damage alters the metabolic-epigenetic axis to drive an immune-like response that can promote age-associated decline.

In an aging population, intense interest has shifted toward prolonging health span. Mounting evidence suggests that cellular reactive species are propagators of cell damage, inflammation, and cellular senescence. Thus, such species have emerged as putative provocateurs and targets for senolysis, and a clearer understanding of their molecular origin and regulation is of paramount importance. In an inquiry into signaling triggered by aging and proxy instigator, hyperglycemia, we show that NADPH Oxidase (NOX) drives cell DNA damage and alters nuclear envelope integrity, inflammation, tissue dysfunction, and cellular senescence in mice and humans with similar causality. Most notably, selective NOX1 inhibition rescues age-impaired blood flow and angiogenesis, vasodilation, and the endothelial cell wound response. Indeed, NOX1i delivery in vivo completely reversed age-impaired hind-limb blood flow and angiogenesis while disrupting a NOX1-IL-6 senescence-associated secretory phenotype (SASP) proinflammatory signaling loop. Relevant to its comorbidity with age, clinical samples from diabetic versus nondiabetic subjects reveal as operant this NOX1-mediated vascular senescence and inflammation in humans. On a mechanistic level, our findings support a previously unidentified role for IL-6 in this feedforward inflammatory loop and peroxisome proliferator-activated receptor gamma (PPARγ) down-regulation as inversely modulating p65-mediated NOX1 transcription. Targeting this previously unidentified NOX1-SASP signaling axis in aging is predicted to be an effective strategy for mitigating senescence in the vasculature and other organ systems.

Nuclear DNA damage is intricately linked to cellular metabolism. However, the underlying mechanisms and full range of metabolic alterations that occur in response to persistent DNA damage are not well understood. Here, we use a DNA repair-deficient model of ERCC1-XPF in Caenorhabditis elegans (C. elegans), that accumulates physiologically relevant, endogenous DNA damage, to gain molecular insights on how persistent genotoxic stress drives biological aging. Using an integrated multi-omic approach, we discover that persistent genotoxic stress rewires lipid metabolism. In particular, nuclear DNA damage promotes mitochondrial β-oxidation and leads to a global loss of fat depots. This metabolic shift to β-oxidation generates acetyl-CoA and drives histone hyperacetylation. Concomitantly, we observe an associated change in gene expression of immune-effector and cytochrome (CYP) genes. We identify MYS-1, the ortholog of mammalian histone acetyltransferase TIP60, as a critical regulator of this metabolic-epigenetic axis. Moreover, we show that in response to persistent DNA damage, polyunsaturated fatty acids (PUFAs), especially arachidonic acid (AA) and AA-related lipid mediators are elevated. This elevation of PUFA species requires mys-1/Tip60. Together, these findings reveal that persistent nuclear DNA damage alters the metabolic-epigenetic axis to drive an immune-like response that can promote age-associated decline.

Although the link between DNA damage and aging is well accepted, the role of different DNA repair proteins on functional/physiological aging is not well-defined. Here, using Caenorhabditis elegans, we systematically examined the effect of three DNA repair genes involved in key genomic stability pathways. We assayed multiple health proxies including molecular, functional and resilience measures to define healthspan. Mutation in XPF-1/ERCC-1, a protein involved in nucleotide excision repair (NER), homologous recombination (HR) and interstrand crosslink (ICL) repair, showed the highest impairment of functional and stress resilience measures along with a shortened lifespan. brc-1 mutants, with a well-defined role in HR and ICL are short-lived and highly sensitive to acute stressors, specifically oxidative stress. In contrast, ICL mutants fcd-2 did not impact lifespan or most healthspan measures. Our efforts also uncover that DNA repair mutants show high sensitivity to oxidative stress with age, suggesting that this measure could act as a primary proxy for healthspan. Together, these data suggest that impairment of multiple DNA repair genes can drive functional/physiological aging. Further studies to examine specific DNA repair genes in a tissue specific manner will help dissect the importance and mechanistic role of these repair systems in biological aging.

In the nematode C. elegans, insulin signaling regulates development and aging in response to the secretion of numerous insulin peptides. Here, we describe a novel, non-signaling isoform of the nematode insulin receptor (IR), DAF-2B, that modulates insulin signaling by sequestration of insulin peptides. DAF-2B arises via alternative splicing and retains the extracellular ligand binding domain but lacks the intracellular signaling domain. A daf-2b splicing reporter revealed active regulation of this transcript through development, particularly in the dauer larva, a diapause stage associated with longevity. CRISPR knock-in of mScarlet into the daf-2b genomic locus confirmed that DAF-2B is expressed in vivo and is likely secreted. Genetic studies indicate that DAF-2B influences dauer entry, dauer recovery and adult lifespan by altering insulin sensitivity according to the prevailing insulin milieu. Thus, in C. elegans alternative splicing at the daf-2 locus generates a truncated IR that fine-tunes insulin signaling in response to the environment.

NF-κB is a transcription factor activated in response to inflammatory, genotoxic and oxidative stress and important for driving senescence and aging. Ataxia-telangiectasia mutated (ATM) kinase, a core component of DNA damage response signaling, activates NF-κB in response to genotoxic and oxidative stress via post-translational modifications. Here we demonstrate that ATM is activated in senescent cells in culture and murine tissues from Ercc1-deficient mouse models of accelerated aging, as well as naturally aged mice. Genetic and pharmacologic inhibition of ATM reduced activation of NF-κB and markers of senescence and the senescence-associated secretory phenotype (SASP) in senescent Ercc1-/- MEFs. Ercc1-/Δ mice heterozygous for Atm have reduced NF-κB activity and cellular senescence, improved function of muscle-derived stem/progenetor cells (MDSPCs) and extended healthspan with reduced age-related pathology especially age-related bone and intervertebral disc pathologies. In addition, treatment of Ercc1-/∆ mice with the ATM inhibitor KU-55933 suppressed markers of senescence and SASP. Taken together, these results demonstrate that the ATM kinase is a major mediator of DNA damage-induced, NF-κB-mediated cellular senescence, stem cell dysfunction and aging and thus represents a therapeutic target to slow the progression of aging.

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1(−/−) mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl(4) and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1(−/−) cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1(−/−) cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats.

Nuclear factor κB (NF-κB) is a family of transcription factors important for regulating innate and adaptive immunity, cellular proliferation, apoptosis and senescence. The NF-κB family is comprised of five subunits, RelA/p65, RelB, C-Rel, p50 (p105/NF-κB1), and p52 (p100/NF-κB2). NF-κB activity goes up with age in multiple tissues. The two subunits RelA/p65 and p50 have been implicated in senescence and aging with genetic deletion of p65 and p50 reducing or increasing senescence respectively. Pharmacologic inhibition of NF-κB also extends health span and reduces senescence in mouse models of accelerated aging. In addition, NF-κB regulates expression of many of senescence associated secretory phenotype (SASP) factors released by certain types of senescent cells that drives loss of tissue homeostasis and secondary senescence. To measure NF-κB activity with aging in vivo, multiple methods can and need to be utilized including cellular localization of p65, EMSA analysis of NF-κB DNA binding, RNA in situ hybridization, and analysis of expression of NF-κB target genes. To colocalize NF-κB activation and senescence, p65 localization or transcriptional activity can be measured by immunostaining or RNA in situ hybridization for NF-κB regulated genes along with methods such as immunostaining for γH2AX or RNA in situ for senescence markers like p16INK4a and p21. These and related methods will be described in this chapter.

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline. Keywords: Reactive oxygen species, Free radicals, Genotoxic stress, Oxidative lesions, Endogenous DNA damage, Cellular senescence, Aging

Aging is the primary risk factor for numerous chronic, debilitating diseases. These diseases impact quality of life of the elderly and consume a large portion of health care costs. The cost of age-related diseases will only increase as the world's population continues to live longer. Thus it would be advantageous to consider aging itself as a therapeutic target, potentially stemming multiple age-related diseases simultaneously. While logical, this is extremely challenging as the molecular mechanisms that drive aging are still unknown. Furthermore, clinical trials to treat aging are impractical. Even in preclinical models, testing interventions to extend healthspan in old age is lengthy and therefore costly. One approach to expedite aging studies is to take advantage of mouse strains that are engineered to age rapidly. These strains are genetically and phenotypically quite diverse. This review aims to offer a comparison of several of these strains to highlight their relative strengths and weaknesses as models of mammalian and more specifically human aging. Additionally, careful identification of commonalities amongst the strains may lead to the identification of fundamental pathways of aging.

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16Ink4a and p21Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1 −/∆ and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16Ink4a and p21Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1 −/∆ mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16Ink4a and p21Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1 −/Δ mice with a p16Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1 −/∆ and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster., Senescent cells contribute to aging and its associated morbidities. Senescent cells accumulate in vertebrates with aging. Here, we survey where (in what tissues) senescence occurs with aging in mice, by measuring p16Ink4a and p21Cip1 mRNA. A similar survey in Ercc1 −/Δ mice illustrates where (in what tissues) senescence occurs in vivo as a consequence of endogenous DNA damage.

Cellular senescence refers to the irreversible growth arrest of normally dividing cells in response to various types of stress. Cellular senescence is induced by telomere shortening due to repeated cell division, which causes a DNA damage response, as well as genotoxic, oxidative, and inflammatory stress. Strong mitogenic signaling, such as oncogene activation, also drives cells into a senescent state. Senescent cells express a specific subset of genes, termed the senescence-associated secretory phenotype (SASP), including pro-inflammatory factors, growth factors, and matrix metalloproteinases, which together promote non-cell autonomous, secondary senescence. Clearance of senescent cells that accumulate with age improves health span, implicating cellular senescence as a contributing factor to the aging process. Thus, there is a need for methods to identify and quantify cellular senescence, both in cultured cells and in vivo. Here, methods for the most well-characterized and widely used senescent assays are described, from cell morphology and senescence-associated β-galactosidase (SA-βgal) staining to nuclear biomarkers, SASP, and altered levels of tumor suppressors. © 2017 by John Wiley & Sons, Inc.

Ageing is defined as the progressive attrition of tissue/organ function resulting in an increased susceptibility to disease and death. The DNA mutation and damage theory of ageing posits that the accrual of genetic damage over time is the underlying cause of ageing. Evidence for this theory stems from the fact that numerous human progeroid syndromes are caused by inherited defects in genome maintenance mechanisms, linking excess genetic damage with accelerated ageing. These diseases have been modelled in mice and other organisms. However, the molecular mechanism by which genomic instability drives ageing is currently not known. The nematode, C. elegans, is a genetically tractable, well-studied model organism for investigating mechanisms of ageing and DNA repair pathways identified in mammalian systems are well conserved in the worm. Furthermore, proliferating and post-mitotic cells, which have distinct responses to genomic instability, are clearly delineated in the worm. Thus worms provide an opportunity to study the importance of genomic stability in each of these compartments in the context of a whole organism. Genomic instability can interfere with transcription, trigger apoptosis, attenuate proliferative capacity, and cause metabolic changes. Here, we first examine the DNA repair pathways that are conserved between worms and mammals, with an overview of the spatial and temporal activity of each of these repair pathways. This chapter then explores evidence from studies in the nematode that genomic instability, healthspan and organismal ageing are linked.

A chemical screen has identified a small molecule, piperlongumine (PL), as a compound that induces selective killing of cancer cells. Piperlongumine acts by increasing reactive oxygen species (ROS) levels in cancer cells. Although it is active against a number of tumour models in vivo irrespective of p53 status, it does not affect normal tissues, including rapidly proliferating non-tumour cells. This work suggests a novel strategy for eradicating cancer cells by targeting the ROS stress-response pathway, but further work will be needed to identify determinants of piperlongumine sensitivity in a wider range of cancers. Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage)1. Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells2. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress3,4,5.

Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress.

The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1(-/Δ) mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1(-/∆) mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.

The association of Rad18 with Polη is crucial for efficient translesion synthesis and DNA damage tolerance. Rad18–Polη interactions and UV tolerance depend on JNK-dependent Rad18 phosphorylation. These results provide a new mechanism by which SAPK signaling promotes genome maintenance., The E3 ubiquitin ligase Rad18 chaperones DNA polymerase η (Polη) to sites of UV-induced DNA damage and monoubiquitinates proliferating cell nuclear antigen (PCNA), facilitating engagement of Polη with stalled replication forks and promoting translesion synthesis (TLS). It is unclear how Rad18 activities are coordinated with other elements of the DNA damage response. We show here that Ser-409 residing in the Polη-binding motif of Rad18 is phosphorylated in a checkpoint kinase 1–dependent manner in genotoxin-treated cells. Recombinant Rad18 was phosphorylated specifically at S409 by c-Jun N-terminal kinase (JNK) in vitro. In UV-treated cells, Rad18 S409 phosphorylation was inhibited by a pharmacological JNK inhibitor. Conversely, ectopic expression of JNK and its upstream kinase mitogen-activated protein kinase kinase 4 led to DNA damage–independent Rad18 S409 phosphorylation. These results identify Rad18 as a novel JNK substrate. A Rad18 mutant harboring a Ser → Ala substitution at S409 was compromised for Polη association and did not redistribute Polη to nuclear foci or promote Polη−PCNA interaction efficiently relative to wild-type Rad18. Rad18 S409A also failed to fully complement the UV sensitivity of Rad18-depleted cells. Taken together, these results show that Rad18 phosphorylation by JNK represents a novel mechanism for promoting TLS and DNA damage tolerance.

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

The Ser/Thr Rho kinase 1 (ROCK1) is known to have major roles in a wide range of cellular activities, including those involved in tumour metastasis and apoptosis. Here we identify an indispensable function of ROCK1 in metabolic stress-induced autophagy. Applying a proteomics approach, we characterize Beclin1, a proximal component of the phosphoinositide 3-kinase class III lipid-kinase complex that induces autophagy, as an interacting partner of ROCK1. Upon nutrient deprivation, activated ROCK1 promotes autophagy by binding and phosphorylating Beclin1 at Thr119. This results in the specific dissociation of the Beclin1-Bcl-2 complex without affecting the Beclin1-UVRAG interaction. Conversely, inhibition of ROCK1 activity increases Beclin1-Bcl-2 association, thus reducing nutritional stress-mediated autophagy. Genetic knockout of ROCK1 function in mice also leads to impaired autophagy as evidenced by reduced autophagosome formation. These results show that ROCK1 acts as a prominent upstream regulator of Beclin1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy.

The nuclear genome decays as organisms age. Numerous studies demonstrate that the burden of several classes of DNA lesions is greater in older mammals than in young mammals. More challenging is proving this is a cause rather than a consequence of aging. The DNA damage theory of aging, which argues that genomic instability plays a causal role in aging, has recently gained momentum. Support for this theory stems partly from progeroid syndromes in which inherited defects in DNA repair increase the burden of DNA damage leading to accelerated aging of one or more organs. Additionally, growing evidence shows that DNA damage accrual triggers cellular senescence and metabolic changes that promote a decline in tissue function and increased susceptibility to age-related diseases. Here, we examine multiple lines of evidence correlating nuclear DNA damage with aging. We then consider how, mechanistically, nuclear genotoxic stress could promote aging. We conclude that the evidence, in toto, supports a role for DNA damage as a nidus of aging.

TP53 is the most frequently mutated gene in human cancer, and small-molecule reactivation of mutant p53 function represents an important anticancer strategy. A cell-based, high-throughput small-molecule screen identified chetomin (CTM) as a mutant p53 R175H reactivator. CTM enabled p53 to transactivate target genes, restored MDM2 negative regulation, and selectively inhibited the growth of cancer cells harboring mutant p53 R175H in vitro and in vivo. We found that CTM binds to Hsp40 and increases the binding capacity of Hsp40 to the p53 R175H mutant protein, causing a potential conformational change to a wild-type-like p53. Thus, CTM acts as a specific reactivator of the p53 R175H mutant form through Hsp40. These results provide new insights into the mechanism of reactivation of this specific p53 mutant., United States. National Institutes of Health (GM108415), United States. National Institutes of Health (CA142805), United States. National Institutes of Health (CA149477), United States. National Institutes of Health (CA80058), United States. National Institutes of Health (GM086258)