Common and rare variant associations with clonal haematopoiesis phenotypes.

Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1-5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications.

A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A.

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in aging and metabolism regulation. Targeted sequencing of SIRT6 locus in a population of 450 Ashkenazi Jewish (AJ) centenarians and 550 AJ individuals without a family history of exceptional longevity identified enrichment of a SIRT6 allele containing two linked substitutions (N308K/A313S) in centenarians compared with AJ control individuals. Characterization of this SIRT6 allele (centSIRT6) demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double-strand break repair, and more robustly kill cancer cells compared with wild-type SIRT6. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which was correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

The Emergency Medicine Group Standardized Letter of Evaluation as a Workplace-based Assessment: The Validity Is in the Detail.

INTRODUCTION: Interest is growing in specialty-specific assessments of student candidates based on clinical clerkship performance to assist in the selection process for postgraduate training. The most established and extensively used is the emergency medicine (EM) Standardized Letter of Evaluation (SLOE), serving as a substitute for the letter of recommendation. Typically developed by a program's leadership, the group SLOE strives to provide a unified institutional perspective on performance. The group SLOE lacks guidelines to direct its development raising questions regarding the assessments, processes, and standardization programs employ. This study surveys EM programs to gather validity evidence regarding the inputs and processes involved in developing group SLOEs. METHODS: A structured telephone interview was administered to assess the input data and processes employed by United States EM programs when generating group SLOEs. RESULTS: With 156/178 (87.6%) of Accreditation Council of Graduate Medical Education-approved programs responding, 146 (93.6%) reported developing group SLOEs. Issues identified in development include the following: (1) 84.9% (124/146) of programs limit the consensus process by not employing rigorous methodology; (2) several stakeholder groups (nurses, patients) do not participate in candidate assessment placing final decisions at risk for construct under-representation; and (3) clinical shift assessments don't reflect the task-specific expertise of each stakeholder group nor has the validity of each been assessed. CONCLUSION: Success of the group SLOE in its role as a summative workplace-based assessment is dependent upon valid input data and appropriate processes. This study of current program practices provides specific recommendations that would strengthen the validity arguments for the group SLOE.

Therapeutic targeting of Notch signaling and immune checkpoint blockade in a spontaneous, genetically heterogeneous mouse model of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer derived from the malignant transformation of T-cell progenitors. Outcomes remain poor for T-ALL patients who have either primary resistance to standard-of-care chemotherapy or disease relapse. Notably, there are currently no targeted therapies available in T-ALL. This lack of next-generation therapies highlights the need for relevant preclinical disease modeling to identify and validate new targets and treatment approaches. Here, we adapted a spontaneously arising, genetically heterogeneous, thymic transplantation-based murine model of T-ALL, recapitulating key histopathological and genetic features of the human disease, to the preclinical testing of targeted and immune-directed therapies. Genetic engineering of the murine Notch1 locus aligned the spectrum of Notch1 mutations in the mouse model to that of human T-ALL and confirmed aberrant, recombination-activating gene (RAG)-mediated 5' Notch1 recombination events as the preferred pathway in murine T-ALL development. Testing of Notch1-targeting therapeutic antibodies demonstrated T-ALL sensitivity to different classes of Notch1 blockers based on Notch1 mutational status. In contrast, genetic ablation of Notch3 did not impact T-ALL development. The T-ALL model was further applied to the testing of immunotherapeutic agents in fully immunocompetent, syngeneic mice. In line with recent clinical experience in T-cell malignancies, programmed cell death 1 (PD-1) blockade alone lacked anti-tumor activity against murine T-ALL tumors. Overall, the unique features of the spontaneous T-ALL model coupled with genetic manipulations and the application to therapeutic testing in immunocompetent backgrounds will be of great utility for the preclinical evaluation of novel therapies against T-ALL.

LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation.

Mice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS. Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies.

Relationship Between Institutional Standardized Letter of Evaluation Global Assessment Ranking Practices, Interviewing Practices, and Medical Student Outcomes.

BACKGROUND: Emergency medicine (EM) program directors rely largely on the standardized letter of evaluation (SLOE) to help determine which applicants to interview in the face of an increasing number of applications. To further characterize the SLOE's role in the EM application process, particularly the global assessment (GA) ranking and its effect on interviewing practices and medical student outcomes, the leaders of EM programs were surveyed regarding their experiences in both generating and utilizing the SLOE. METHODS: Individuals on the Council of Emergency Medicine Residency Directors (CORD) and Clerkship Directors in Emergency Medicine (CDEM) Academy listservs were anonymously surveyed from March 21-30, 2015, with 18 questions in multiple-choice and fill-in-the-blank formats. RESULTS: There were 99 respondents. Only 39 respondents (39%) reported adhering strictly to SLOE guidelines by evenly placing their students into thirds (top, middle, lower) on the SLOE GA. Most respondents interviewed individuals ranked in the lower third. Programs adhering strictly to ranking guidelines were more likely to interview students in the lower third than those adhering loosely or not at all. There was no relationship between a program's self-reported adherence to the SLOE ranking guidelines and the number of unmatched students in EM during the 2014 and 2015 academic years. CONCLUSION: Many SLOE writers do not strictly adhere to CORD's SLOE writing guidelines when using the GA ranking, due to the fear of adversely impacting an applicant's ability to successfully match into EM. This calls into question the validity of the SLOE as it is currently used. However, this study suggests that adhering to recommended SLOE ranking guidelines is unlikely to substantially increase the risk that students will fail to match. If more evaluators were to adhere to the guidelines, the SLOE could become the valid evaluation instrument that graduate medical education has long been pursuing.

Effects of an unusual poison identify a lifespan role for Topoisomerase 2 in Saccharomyces cerevisiae.

A progressive loss of genome maintenance has been implicated as both a cause and consequence of aging. Here we present evidence supporting the hypothesis that an age-associated decay in genome maintenance promotes aging in Saccharomyces cerevisiae (yeast) due to an inability to sense or repair DNA damage by topoisomerase 2 (yTop2). We describe the characterization of LS1, identified in a high throughput screen for small molecules that shorten the replicative lifespan of yeast. LS1 accelerates aging without affecting proliferative growth or viability. Genetic and biochemical criteria reveal LS1 to be a weak Top2 poison. Top2 poisons induce the accumulation of covalent Top2-linked DNA double strand breaks that, if left unrepaired, lead to genome instability and death. LS1 is toxic to cells deficient in homologous recombination, suggesting that the damage it induces is normally mitigated by genome maintenance systems. The essential roles of yTop2 in proliferating cells may come with a fitness trade-off in older cells that are less able to sense or repair yTop2-mediated DNA damage. Consistent with this idea, cells live longer when yTop2 expression levels are reduced. These results identify intrinsic yTop2-mediated DNA damage as potentially manageable cause of aging.

Does the National Resident Match Program Rank List Predict Success in Emergency Medicine Residency Programs?

BACKGROUND: Emergency medicine (EM) residency programs use nonstandardized criteria to create applicant rank lists. One implicit assumption is that predictive associations exist between an applicant's rank and their future performance as a resident. To date, these associations have not been sufficiently demonstrated. OBJECTIVES: We hypothesized that a strong positive correlation exists between the National Resident Match Program (NRMP) match-list applicant rank, the United States Medical Licensing Examination (USMLE) Step 1 and In-Training Examination (ITE) scores, and the graduating resident rank. METHODS: A total of 286 residents from five EM programs over a 5-year period were studied. The applicant rank (AR) was derived from the applicant's relative rank list position on each programs' submitted NRMP rank list. The graduation rank (GR) was determined by a faculty consensus committee. GR was then correlated to AR using a Spearman's partial rank correlation. Additional correlations were sought with a ranking of the USMLE Step Score (UR) and the ITE Score (IR). RESULTS: Combining data for all five programs, weak positive correlations existed between GR and AR, UR, and IR. The majority of correlations ranged between. When comparing GR and AR, there was a weak correlation of 0.13 (p = 0.03). CONCLUSION: Our study found only weak correlations between GR and AR, UR, and IR, suggesting that those variables may not be strong predictors of resident performance. This has important implications for EM programs considering the resources devoted to applicant evaluation and ranking.

JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks.

The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6), promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB) repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

SIRT6 rescues the age related decline in base excision repair in a PARP1-dependent manner.

In principle, a decline in base excision repair (BER) efficiency with age should lead to genomic instability and ultimately contribute to the onset of the aging phenotype. Although multiple studies have indicated a negative link between aging and BER, the change of BER efficiency with age in humans has not been systematically analyzed. Here, with foreskin fibroblasts isolated from 19 donors between 20 and 64 y of age, we report a significant decline of BER efficiency with age using a newly developed GFP reactivation assay. We further observed a very strong negative correlation between age and the expression levels of SIRT6, a factor which is known to maintain genomic integrity by improving DNA double strand break (DSB) repair. Our mechanistic study suggests that, similar to the regulatory role that SIRT6 plays in DNA DSB repair, SIRT6 regulates BER in a PARP1-depdendent manner. Moreover, overexpression of SIRT6 rescues the decline of BER in aged fibroblasts. In summary, our results uncovered the regulatory mechanisms of BER by SIRT6, suggesting that SIRT6 reactivation in aging tissues may help delay the process of aging through improving BER.

SIRT6 represses LINE1 retrotransposons by ribosylating KAP1 but this repression fails with stress and age.

L1 retrotransposons are an abundant class of transposable elements that pose a threat to genome stability and may have a role in age-related pathologies such as cancer. Recent evidence indicates that L1s become more active in somatic tissues during the course of ageing; however the mechanisms underlying this phenomenon remain unknown. Here we report that the longevity regulating protein, SIRT6, is a powerful repressor of L1 activity. Specifically, SIRT6 binds to the 5'-UTR of L1 loci, where it mono-ADP ribosylates the nuclear corepressor protein, KAP1, and facilitates KAP1 interaction with the heterochromatin factor, HP1α, thereby contributing to the packaging of L1 elements into transcriptionally repressive heterochromatin. During the course of ageing, and also in response to DNA damage, however, we find that SIRT6 is depleted from L1 loci, allowing the activation of these previously silenced retroelements.

SIRT6: a promising target for cancer prevention and therapy.

Many of the pathologies associated with the aging process also contribute to tumor initiation, growth or metastasis. Insights from biogerontology may be instrumental for developing new therapies for cancer. This chapter highlights the rationale for combining biogerontology and cancer research to generate new strategies for cancer treatment. In particular, this chapter focuses on one gene, SIRT6, which has emerged as an important regulator of longevity in mammals and appears to have multiple biochemical functions, which antagonize tumor development and may be useful in cancer prevention and treatment.

IGF1R levels in the brain negatively correlate with longevity in 16 rodent species.

The insulin/insulin-like growth factor signaling (IIS) pathway is a major conserved regulator of aging. Nematode, fruit fly and mouse mutants with reduced IIS signaling exhibit extended lifespan. These mutants are often dwarfs leading to the idea that small body mass correlates with longevity within species. However, when different species are compared, larger animals are typically longer-lived. Hence, the role of IIS in the evolution of life history traits remains unresolved. Here we used comparative approach to test whether IGF1R signaling changes in response to selection on lifespan or body mass and whether specific tissues are involved. The IGF1R levels in the heart, lungs, kidneys, and brains of sixteen rodent species with highly diverse lifespans and body masses were measured via immunoblot after epitope conservation analysis. We report that IGF1R levels display strong negative correlation with maximum lifespan only in brain tissue and no significant correlations with body mass for any organ. The brain-IGF1R and lifespan correlation holds when phylogenetic non-independence of data-points is taken into account. These results suggest that modulation of IGF1R signaling in nervous tissue, but not in the peripheral tissues, is an important factor in the evolution of longevity in mammals.

Sirtuin 6 (SIRT6) rescues the decline of homologous recombination repair during replicative senescence.

Genomic instability is a hallmark of aging tissues. Genomic instability may arise from the inefficient or aberrant function of DNA double-stranded break (DSB) repair. DSBs are repaired by homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). HR is a precise pathway, whereas NHEJ frequently leads to deletions or insertions at the repair site. Here, we used normal human fibroblasts with a chromosomally integrated HR reporter cassette to examine the changes in HR efficiency as cells progress to replicative senescence. We show that HR declines sharply with increasing replicative age, with an up to 38-fold decrease in efficiency in presenescent cells relative to young cells. This decline is not explained by a reduction of the number of cells in S/G(2)/M stage as presenescent cells are actively dividing. Expression of proteins involved in HR such as Rad51, Rad51C, Rad52, NBS1, and Sirtuin 6 (SIRT6) diminished with cellular senescence. Supplementation of Rad51, Rad51C, Rad52, and NBS1 proteins, either individually or in combination, did not rescue the senescence-related decline of HR. However, overexpression of SIRT6 in "middle-aged" and presenescent cells strongly stimulated HR repair, and this effect was dependent on mono-ADP ribosylation activity of poly(ADP-ribose) polymerase (PARP1). These results suggest that in aging cells, the precise HR pathway becomes repressed giving way to a more error-prone NHEJ pathway. These changes in the processing of DSBs may contribute to age-related genomic instability and a higher incidence of cancer with age. SIRT6 activation provides a potential therapeutic strategy to prevent the decline in genome maintenance.

SIRT6 overexpression induces massive apoptosis in cancer cells but not in normal cells.

Emerging evidence suggests that Sirtuin 6 (SIRT6) functions as a longevity assurance gene by promoting genomic stability, regulating metabolic processes and attenuating inflammation. Here, we examine the effect of SIRT6 activation on cancer cells. We show that SIRT6 overexpression induces massive apoptosis in a variety of cancer cell lines but not in normal, non-transformed cells. This cell death requires the mono-ADP-ribosyltransferase but not the deacetylase activity of SIRT6 and is mediated by the activation of both the p53 and p73 apoptotic signaling cascades in cancer cells by SIRT6. These results suggest that SIRT6 is an attractive target for pharmacological activation in cancer treatment.

Repairing split ends: SIRT6, mono-ADP ribosylation and DNA repair.

The sirtuin gene family comprises an evolutionarily ancient set of NAD+ dependent protein deacetylase and mono-ADP ribosyltransferase enzymes. Found in all domains of life, sirtuins regulate a diverse array of biological processes, including DNA repair, gene silencing, apoptosis and metabolism. Studies in multiple model organisms have indicated that sirtuins may also function to extend lifespan and attenuate age-related pathologies. To date, most of these studies have focused on the deacetylase activity of sirtuins, and relatively little is known about the other biochemical activity of sirtuins, mono-ADP ribosylation. We recently reported that the mammalian sirtuin, SIRT6, mono-ADP ribosylates PARP1 to promote DNA repair in response to oxidative stress. In this research perspective we review the role of SIRT6 in DNA repair and discuss the emerging implications for sirtuin directed mono-ADP ribosylation in aging and age-related diseases.

SIRT6 promotes DNA repair under stress by activating PARP1.

Sirtuin 6 (SIRT6) is a mammalian homolog of the yeast Sir2 deacetylase. Mice deficient for SIRT6 exhibit genome instability. Here, we show that in mammalian cells subjected to oxidative stress SIRT6 is recruited to the sites of DNA double-strand breaks (DSBs) and stimulates DSB repair, through both nonhomologous end joining and homologous recombination. Our results indicate that SIRT6 physically associates with poly[adenosine diphosphate (ADP)-ribose] polymerase 1 (PARP1) and mono-ADP-ribosylates PARP1 on lysine residue 521, thereby stimulating PARP1 poly-ADP-ribosylase activity and enhancing DSB repair under oxidative stress.