Inhibition of the proline metabolism rate-limiting enzyme P5CS allows proliferation of glutamine-restricted cancer cells.

Glutamine is a critical metabolite for rapidly proliferating cells as it is used for the synthesis of key metabolites necessary for cell growth and proliferation. Glutamine metabolism has been proposed as a therapeutic target in cancer and several chemical inhibitors are in development or in clinical trials. How cells subsist when glutamine is limiting is poorly understood. Here, using an unbiased screen, we identify ALDH18A1, which encodes P5CS, the rate-limiting enzyme in the proline biosynthetic pathway, as a gene that cells can downregulate in response to glutamine starvation. Notably, P5CS downregulation promotes de novo glutamine synthesis, highlighting a previously unrecognized metabolic plasticity of cancer cells. The glutamate conserved from reducing proline synthesis allows cells to produce the key metabolites necessary for cell survival and proliferation under glutamine-restricted conditions. Our findings reveal an adaptive pathway that cancer cells acquire under nutrient stress, identifying proline biosynthesis as a previously unrecognized major consumer of glutamate, a pathway that could be exploited for developing effective metabolism-driven anticancer therapies.

NRF2 activation induces NADH-reductive stress, providing a metabolic vulnerability in lung cancer.

Multiple cancers regulate oxidative stress by activating the transcription factor NRF2 through mutation of its negative regulator, KEAP1. NRF2 has been studied extensively in KEAP1-mutant cancers; however, the role of this pathway in cancers with wild-type KEAP1 remains poorly understood. To answer this question, we induced NRF2 via pharmacological inactivation of KEAP1 in a panel of 50+ non-small cell lung cancer cell lines. Unexpectedly, marked decreases in viability were observed in >13% of the cell lines-an effect that was rescued by NRF2 ablation. Genome-wide and targeted CRISPR screens revealed that NRF2 induces NADH-reductive stress, through the upregulation of the NAD+-consuming enzyme ALDH3A1. Leveraging these findings, we show that cells treated with KEAP1 inhibitors or those with endogenous KEAP1 mutations are selectively vulnerable to Complex I inhibition, which impairs NADH oxidation capacity and potentiates reductive stress. Thus, we identify reductive stress as a metabolic vulnerability in NRF2-activated lung cancers.

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.

SIRT6 is a key regulator of mitochondrial function in the brain.

The SIRT6 deacetylase has been implicated in DNA repair, telomere maintenance, glucose and lipid metabolism and, importantly, it has critical roles in the brain ranging from its development to neurodegeneration. Here, we combined transcriptomics and metabolomics approaches to characterize the functions of SIRT6 in mouse brains. Our analysis reveals that SIRT6 is a central regulator of mitochondrial activity in the brain. SIRT6 deficiency in the brain leads to mitochondrial deficiency with a global downregulation of mitochondria-related genes and pronounced changes in metabolite content. We suggest that SIRT6 affects mitochondrial functions through its interaction with the transcription factor YY1 that, together, regulate mitochondrial gene expression. Moreover, SIRT6 target genes include SIRT3 and SIRT4, which are significantly downregulated in SIRT6-deficient brains. Our results demonstrate that the lack of SIRT6 leads to decreased mitochondrial gene expression and metabolomic changes of TCA cycle byproducts, including increased ROS production, reduced mitochondrial number, and impaired membrane potential that can be partially rescued by restoring SIRT3 and SIRT4 levels. Importantly, the changes we observed in SIRT6-deficient brains are also occurring in aging human brains and particularly in patients with Alzheimer's, Parkinson's, Huntington's, and Amyotrophic lateral sclerosis disease. Overall, our results suggest that the reduced levels of SIRT6 in the aging brain and neurodegeneration initiate mitochondrial dysfunction by altering gene expression, ROS production, and mitochondrial decay.

Sirtuin 6 inhibition protects against glucocorticoid-induced skeletal muscle atrophy by regulating IGF/PI3K/AKT signaling.

Chronic activation of stress hormones such as glucocorticoids leads to skeletal muscle wasting in mammals. However, the molecular events that mediate glucocorticoid-induced muscle wasting are not well understood. Here, we show that SIRT6, a chromatin-associated deacetylase indirectly regulates glucocorticoid-induced muscle wasting by modulating IGF/PI3K/AKT signaling. Our results show that SIRT6 levels are increased during glucocorticoid-induced reduction of myotube size and during skeletal muscle atrophy in mice. Notably, overexpression of SIRT6 spontaneously decreases the size of primary myotubes in a cell-autonomous manner. On the other hand, SIRT6 depletion increases the diameter of myotubes and protects them against glucocorticoid-induced reduction in myotube size, which is associated with enhanced protein synthesis and repression of atrogenes. In line with this, we find that muscle-specific SIRT6 deficient mice are resistant to glucocorticoid-induced muscle wasting. Mechanistically, we find that SIRT6 deficiency hyperactivates IGF/PI3K/AKT signaling through c-Jun transcription factor-mediated increase in IGF2 expression. The increased activation, in turn, leads to nuclear exclusion and transcriptional repression of the FoxO transcription factor, a key activator of muscle atrophy. Further, we find that pharmacological inhibition of SIRT6 protects against glucocorticoid-induced muscle wasting in mice by regulating IGF/PI3K/AKT signaling implicating the role of SIRT6 in glucocorticoid-induced muscle atrophy.

DNA repair as a shared hallmark in cancer and ageing.

Increasing evidence demonstrates that DNA damage and genome instability play a crucial role in ageing. Mammalian cells have developed a wide range of complex and well-orchestrated DNA repair pathways to respond to and resolve many different types of DNA lesions that occur from exogenous and endogenous sources. Defects in these repair pathways lead to accelerated or premature ageing syndromes and increase the likelihood of cancer development. Understanding the fundamental mechanisms of DNA repair will help develop novel strategies to treat ageing-related diseases. Here, we revisit the processes involved in DNA damage repair and how these can contribute to diseases, including ageing and cancer. We also review recent mechanistic insights into DNA repair and discuss how these insights are being used to develop novel therapeutic strategies for treating human disease. We discuss the use of PARP inhibitors in the clinic for the treatment of breast and ovarian cancer and the challenges associated with acquired drug resistance. Finally, we discuss how DNA repair pathway-targeted therapeutics are moving beyond PARP inhibition in the search for ever more innovative and efficacious cancer therapies.

A non-dividing cell population with high pyruvate dehydrogenase kinase activity regulates metabolic heterogeneity and tumorigenesis in the intestine.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

Liquid biopsy reveals collateral tissue damage in cancer.

Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type-specific cfDNA methylation markers enabled the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.

Long-term analysis of antibodies elicited by SPUTNIK V: A prospective cohort study in Tucumán, Argentina.

BACKGROUND: Gam-COVID-Vac (SPUTNIK V) has been granted emergency use authorization in 70 nations and has been administered to millions worldwide. However, there are very few peer-reviewed studies describing its effects. Independent reports regarding safety and effectiveness could accelerate the final approval by the WHO. We aimed to study the long-term humoral immune response in naïve and previously infected volunteers who received SPUTNIK V. METHODS: Humoral immune responses, assayed by anti-SARS-CoV-2-spike-RBD IgG ELISA and neutralization assays, were measured in 602 healthcare workers at 0, 14, 28, 60 and 180 days after receiving SPUTNIK V between December 2020 and July 2021 in Tucumán, Argentina. FINDINGS: Seroconversion was detected in 97% of individuals after 28 days post-vaccination (dpv) (N = 405). Anti-RBD titers began to decrease after 60 dpv (N = 328), but remained detectable in 94% at 90 dpv (N = 224). At 180 dpv, anti-RDB titers persisted in 31% (N = 146). Previous infection triggered an increased immune response to the first dose and increased neutralization activity against variants of concern (VOC). Second doses in previously infected individuals further increased titers, even 90 dpv (N = 75). Basal antibody titers had more influence on post-vaccination anti-RBD responses than the time elapsed between diagnosis and vaccination (N = 274). INTERPRETATION: Data presented herein provides essential knowledge regarding the kinetics of antibodies induced by SPUTNIK V up to six months after immunization, and suggests that when considering one-dose vaccination policies for individuals with previous SARS-CoV-2 infection, serological studies to determine basal titers may be important, independent of when diagnosis occurred. FUNDING: Tucumán Public Health System (SIPROSA), Argentinean National Research Council (CONICET), National University of Tucumán (UNT).

Assessing kinetics and recruitment of DNA repair factors using high content screens.

Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library ("ChromORFeome" library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.

Elevated Humoral Immune Response to SARS-CoV-2 at High Altitudes Revealed by an Anti-RBD "In-House" ELISA.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a global pandemic with dramatic health and socioeconomic consequences. The Coronavirus Disease 2019 (COVID-19) challenges health systems to quickly respond by developing new diagnostic strategies that contribute to identify infected individuals, monitor infections, perform contact-tracing, and limit the spread of the virus. In this brief report, we developed a highly sensitive, specific, and precise "In-House" ELISA to correctly discriminate previously SARS-CoV-2-infected and non-infected individuals and study population seroprevalence. Among 758 individuals evaluated for anti-SARS-CoV-2 serology in the province of Tucumán, Argentina, we found a weak correlation between antibodies elicited against the RBD, the receptor-binding domain of the Spike protein, and the nucleocapsid (N) antigens of this virus. Additionally, we detected mild levels of anti-RBD IgG antibodies in 33.6% of individuals diagnosed with COVID-19, while only 19% showed sufficient antibody titers to be considered as plasma donors. No differences in IgG anti-RBD titers were found between women and men, neither in between different age groups ranging from 18 to 60. Surprisingly, individuals from a high altitude village displayed elevated and longer lasting anti-RBD titers compared to those from a lower altitude city. To our knowledge, this is the first report correlating altitude with increased humoral immune response against SARS-CoV-2 infection.

NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer.

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

SIRT6 transcriptionally regulates fatty acid transport by suppressing PPARγ.

Pathological lipid accumulation is often associated with enhanced uptake of free fatty acids via specific transporters in cardiomyocytes. Here, we identify SIRT6 as a critical transcriptional regulator of fatty acid transporters in cardiomyocytes. We find that SIRT6 deficiency enhances the expression of fatty acid transporters, leading to enhanced fatty acid uptake and lipid accumulation. Interestingly, the haploinsufficiency of SIRT6 is sufficient to induce the expression of fatty acid transporters and cause lipid accumulation in murine hearts. Mechanistically, SIRT6 depletion enhances the occupancy of the transcription factor PPARγ on the promoters of critical fatty acid transporters without modulating the acetylation of histone 3 at Lys 9 and Lys 56. Notably, the binding of SIRT6 to the DNA-binding domain of PPARγ is critical for regulating the expression of fatty acid transporters in cardiomyocytes. Our data suggest exploiting SIRT6 as a potential therapeutic target for protecting the heart from metabolic diseases.

A unique subset of glycolytic tumour-propagating cells drives squamous cell carcinoma.

Head and neck squamous cell carcinoma (SCC) remains among the most aggressive human cancers. Tumour progression and aggressiveness in SCC are largely driven by tumour-propagating cells (TPCs). Aerobic glycolysis, also known as the Warburg effect, is a characteristic of many cancers; however, whether this adaptation is functionally important in SCC, and at which stage, remains poorly understood. Here, we show that the NAD+-dependent histone deacetylase sirtuin 6 is a robust tumour suppressor in SCC, acting as a modulator of glycolysis in these tumours. Remarkably, rather than a late adaptation, we find enhanced glycolysis specifically in TPCs. More importantly, using single-cell RNA sequencing of TPCs, we identify a subset of TPCs with higher glycolysis and enhanced pentose phosphate pathway and glutathione metabolism, characteristics that are strongly associated with a better antioxidant response. Together, our studies uncover enhanced glycolysis as a main driver in SCC, and, more importantly, identify a subset of TPCs as the cell of origin for the Warburg effect, defining metabolism as a key feature of intra-tumour heterogeneity.

Nuclear metabolism and the regulation of the epigenome.

Cellular metabolism has emerged as a major biological node governing cellular behaviour. Metabolic pathways fuel cellular energy needs, providing basic chemical molecules to sustain cellular homeostasis, proliferation and function. Changes in nutrient consumption or availability therefore can result in complete reprogramming of cellular metabolism towards stabilizing core metabolite pools, such as ATP, S-adenosyl methionine, acetyl-CoA, NAD/NADP and α-ketoglutarate. Because these metabolites underlie a variety of essential metabolic reactions, metabolism has evolved to operate in separate subcellular compartments through diversification of metabolic enzyme complexes, oscillating metabolic activity and physical separation of metabolite pools. Given that these same core metabolites are also consumed by chromatin modifiers in the establishment of epigenetic signatures, metabolite consumption on and release from chromatin directly influence cellular metabolism and gene expression. In this Review, we highlight recent studies describing the mechanisms determining nuclear metabolism and governing the redistribution of metabolites between the nuclear and non-nuclear compartments.

Resveratrol: Friend or Foe?

In this issue of Molecular Cell, Benslimane et al. (2020) perform a CRISPR-Cas9 chemogenomic screen, identifying a network of DNA replication and genome integrity genes with the nutraceutical compound Resveratrol and its analog Pterostilbene, linking these compounds to the induction of DNA replication stress in mammalian cells.

Sirt6 deletion in bone marrow-derived cells increases atherosclerosis - Central role of macrophage scavenger receptor 1.

AIMS: Sirtuin 6 (Sirt6) is a NAD+-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrow-restricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model. METHODS AND RESULTS: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe-/- mice fed a high-cholesterol diet for 12 weeks (n = 12-14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels. CONCLUSIONS: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.