bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2021‒01‒24
77 papers selected by
Kıvanç Görgülü
Technical University of Munich

  1. J Biol Chem. 2020 Dec 18. pii: S0021-9258(17)50632-8. [Epub ahead of print]295(51): 17441-17459
      Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week-old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.
    Keywords:  ADMA; cachexia; cancer; l-NMMA; metabolomics; methylarginines; mitochondria; protein synthesis; protein turnover; skeletal muscle
  2. Nature. 2020 Dec 09.
      Compartmentalization of cellular material in droplet-like structures is a hallmark of liquid-liquid phase separation1,2, but the mechanisms of droplet removal are poorly understood. Evidence suggests that droplets can be degraded by autophagy3,4, a highly conserved degradation system in which membrane sheets bend to isolate portions of the cytoplasm within double-membrane autophagosomes5-7. Here we examine how autophagosomes sequester droplets that contain the protein p62 (also known as SQSTM1) in living cells, and demonstrate that double-membrane, autophagosome-like vesicles form at the surface of protein-free droplets in vitro through partial wetting. A minimal physical model shows that droplet surface tension supports the formation of membrane sheets. The model also predicts that bending sheets either divide droplets for piecemeal sequestration or sequester entire droplets. We find that autophagosomal sequestration is robust to variations in the droplet-sheet adhesion strength. However, the two sides of partially wetted sheets are exposed to different environments, which can determine the bending direction of autophagosomal sheets. Our discovery of this interplay between the material properties of droplets and membrane sheets enables us to elucidate the mechanisms that underpin droplet autophagy, or 'fluidophagy'. Furthermore, we uncover a switching mechanism that allows droplets to act as liquid assembly platforms for cytosol-degrading autophagosomes8 or as specific autophagy substrates9-11. We propose that droplet-mediated autophagy represents a previously undescribed class of processes that are driven by elastocapillarity, highlighting the importance of wetting in cytosolic organization.
  3. J Biol Chem. 2020 Dec 04. pii: S0021-9258(17)50489-5. [Epub ahead of print]295(49): 16743-16753
      Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.
    Keywords:  ERAD; ROS; SEL1L; calcium; cell death; cytochrome c; endoplasmic reticulum; endoplasmic reticulum stress (ER stress); endoplasmic reticulum-associated protein degradation (ERAD); hepatocyte death; liver; mitochondria; mitochondrial disease; mitochondrial permeability transition (MPT)
  4. J Biol Chem. 2020 Dec 11. pii: S0021-9258(17)50607-9. [Epub ahead of print]295(50): 17158-17168
      Cellular energy demands are met by uptake and metabolism of nutrients like glucose. The principal transcriptional regulator for adapting glycolytic flux and downstream pathways like de novo lipogenesis to glucose availability in many cell types is carbohydrate response element-binding protein (ChREBP). ChREBP is activated by glucose metabolites and post-translational modifications, inducing nuclear accumulation and regulation of target genes. Here we report that ChREBP is modified by proline hydroxylation at several residues. Proline hydroxylation targets both ectopically expressed ChREBP in cells and endogenous ChREBP in mouse liver. Functionally, we found that specific hydroxylated prolines were dispensable for protein stability but required for the adequate activation of ChREBP upon exposure to high glucose. Accordingly, ChREBP target gene expression was rescued by re-expressing WT but not ChREBP that lacks hydroxylated prolines in ChREBP-deleted hepatocytes. Thus, proline hydroxylation of ChREBP is a novel post-translational modification that may allow for therapeutic interference in metabolic diseases.
    Keywords:  ChREBP; carbohydrate function; glucose metabolism; glucose sensing; hepatocyte; hydroxyproline; post-translational modification (PTM); proline hydroxylation
  5. Aging Cell. 2021 Jan 22. e13309
      Aging-associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world's population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging-associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin-37 (IL-37) is a potent anti-inflammatory cytokine, and we present data demonstrating that IL-37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin-37 (IL-37) in aged mice reduces or prevents aging-associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL-37 expression decreases the surface expression of programmed cell death protein 1 (PD-1) and augments cytokine production from aged T-cells. Improved T-cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4+ T-cells and Lat in CD8+ T-cells when aged mice were treated with recombinant IL-37 (rIL-37) but not control immunoglobin (Control Ig). Importantly, IL-37-mediated rejuvenation of aged endogenous T-cells was also observed in aged chimeric antigen receptor (CAR) T-cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL-37 in boosting the function of aged T-cells and highlight its therapeutic potential to overcome aging-associated immunosenescence.
    Keywords:  CAR T-cells; PD-1; T-cells; aging; cytokines; inflammation; leukemia; signaling
  6. J Biol Chem. 2020 Dec 18. pii: S0021-9258(17)50663-8. [Epub ahead of print]295(51): 17877-17886
      The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.
    Keywords:  genetic disease; inherited; lipid; membrane; metabolism; phosphatidylcholine; phosphatidylethanolamine; phospholipid
  7. Trends Cancer. 2021 Jan 16. pii: S2405-8033(20)30338-1. [Epub ahead of print]
      The mechanisms governing the methylome profile of tumor suppressors and oncogenes have expanded with the discovery of oxidized states of 5-methylcytosine (5mC). Ten-eleven translocation (TET) enzymes are a family of dioxygenases that iteratively catalyze 5mC oxidation and promote cytosine demethylation, thereby creating a dynamic global and local methylation landscape. While the catalytic function of TET enzymes during stem cell differentiation and development have been well studied, less is known about the multifaceted roles of TET enzymes during carcinogenesis. This review outlines several tiers of TET regulation and overviews how TET deregulation promotes a cancer phenotype. Defining the tissue-specific and context-dependent roles of TET enzymes will deepen our understanding of the epigenetic perturbations that promote or inhibit carcinogenesis.
    Keywords:  5-hydroxymethylcytosine; carcinogenesis; epigenetics; ten-eleven translocation
  8. J Biol Chem. 2020 Dec 04. pii: S0021-9258(17)50485-8. [Epub ahead of print]295(49): 16691-16699
      Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin, and lipopolysaccharide. Manipulating the IκB kinase β activity coupled with in vivo and in vitro kinase assays demonstrated that IκB kinase β is a key serine/threonine kinase activated by autophagy stimuli and that it catalyzes phosphorylation of IGPR-1 at Ser220. The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase, which leads to phosphorylation of the major pro-autophagy proteins ULK1 and Beclin-1 (BECN1), increased LC3-II levels, and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer and suggest that IGPR-1 may serve as a promising therapeutic target.
    Keywords:  AMP-activated kinase (AMPK); IGPR-1; IKKβ; autophagy; cell adhesion molecule; cell surface receptor; cell–cell interaction; immunoglobulin-like domain; nutrient deprivation; post-translational modification (PTM); serine phosphorylation of IGPR-1; serine/threonine protein kinase
  9. J Biol Chem. 2020 Dec 04. pii: S0021-9258(17)50484-6. [Epub ahead of print]295(49): 16678-16690
      Large regions in tumor tissues, particularly pancreatic cancer, are hypoxic and nutrient-deprived because of unregulated cell growth and insufficient vascular supply. Certain cancer cells, such as those inside a tumor, can tolerate these severe conditions and survive for prolonged periods. We hypothesized that small molecular agents, which can preferentially reduce cancer cell survival under nutrient-deprived conditions, could function as anticancer drugs. In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as GSH and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing to apoptosis under nutrient-deprived conditions. Nutrient-deficient cancer cells are often deficient in GSH; thus, they are susceptible to redox system inhibitors. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment.
    Keywords:  auranofin; cancer therapy; chemical biology; drug discovery; drug screening; glutathione; metabolism; oxidation reduction (redox); oxidative stress; papyracillic acid; penicillic acid; redox regulation; thioredoxin
  10. J Biol Chem. 2020 Dec 25. pii: S0021-9258(17)50703-6. [Epub ahead of print]295(52): 18343-18354
      RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.
    Keywords:  ERK; GTPase Kras (KRAS); KRAS; PTPN2; cell proliferation; cell signaling; extracellular-signal-regulated kinase (ERK); plasma membrane; tyrosine phosphatase; tyrosine-protein phosphatase (tyrosine phosphatase)
  11. Nat Immunol. 2021 Jan 18.
      Metabolic programming controls immune cell lineages and functions, but little is known about γδ T cell metabolism. Here, we found that γδ T cell subsets making either interferon-γ (IFN-γ) or interleukin (IL)-17 have intrinsically distinct metabolic requirements. Whereas IFN-γ+ γδ T cells were almost exclusively dependent on glycolysis, IL-17+ γδ T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17+ γδ T cells selectively showed high lipid uptake and intracellular lipid storage and were expanded in obesity and in tumors of obese mice. Conversely, glucose supplementation enhanced the antitumor functions of IFN-γ+ γδ T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector γδ T cells and their manipulation in cancer immunotherapy.
  12. J Biol Chem. 2020 Dec 25. pii: S0021-9258(17)50699-7. [Epub ahead of print]295(52): 18284-18300
      Oxygen regulates hypoxia-inducible factor (HIF) transcription factors to control cell metabolism, erythrogenesis, and angiogenesis. Whereas much has been elucidated about how oxygen regulates HIF, whether lipids affect HIF activity is un-known. Here, using cultured cells and two animal models, we demonstrate that lipoprotein-derived fatty acids are an independent regulator of HIF. Decreasing extracellular lipid supply inhibited HIF prolyl hydroxylation, leading to accumulation of the HIFα subunit of these heterodimeric transcription factors comparable with hypoxia with activation of downstream target genes. The addition of fatty acids to culture medium suppressed this signal, which required an intact mitochondrial respiratory chain. Mechanistically, fatty acids and oxygen are distinct signals integrated to control HIF activity. Finally, we observed lipid signaling to HIF and changes in target gene expression in developing zebrafish and adult mice, and this pathway operates in cancer cells from a range of tissues. This study identifies fatty acids as a physiological modulator of HIF, defining a mechanism for lipoprotein regulation that functions in parallel to oxygen.
    Keywords:  fatty acid; hypoxia-inducible factor (HIF); lipoprotein; low-density lipoprotein; low-density lipoprotein (LDL); lysosomal acid lipase; mitochondria
  13. J Biol Chem. 2020 Dec 13. pii: S0021-9258(20)00163-5. [Epub ahead of print]296 100169
      The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and GSH metabolism. Previous reports proposed that mitochondrial reactive oxygen species production and disruption of the GSH pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now, it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2. Recently, we developed mitochondria-targeted reagents that can independently induce mitochondrial superoxide and hydrogen peroxide production mitoParaquat (MitoPQ) or selectively disrupt mitochondrial thiol homeostasis MitoChlorodinitrobenzoic acid (MitoCDNB). Using these reagents, here we have determined how enhanced generation of mitochondrial superoxide and hydrogen peroxide or disruption of mitochondrial thiol homeostasis affects activation of the Nrf2 system in cells, which was assessed by the Nrf2 protein level, nuclear translocation, and expression of its target genes. We found that selective disruption of the mitochondrial GSH pool and inhibition of its thioredoxin system by MitoCDNB led to Nrf2 activation, whereas using MitoPQ to enhance the production of mitochondrial superoxide and hydrogen peroxide alone did not. We further showed that Nrf2 activation by MitoCDNB requires cysteine sensors of Kelch-like ECH-associated protein 1 (Keap1). These findings provide important information on how disruption to mitochondrial redox homeostasis is sensed in the cytoplasm and signaled to the nucleus.
    Keywords:  MitoCDNB; MitoPQ; Nrf2; energy metabolism; reactive oxygen species (ROS); redox signaling; thiol oxidation
  14. Mol Cell. 2021 Jan 08. pii: S1097-2765(20)30951-5. [Epub ahead of print]
      Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.
    Keywords:  ALT; APB; BIR; BLM; PIAS4; PML; SUMO; phase separation; replication stress; telomere
  15. Nat Biotechnol. 2021 Jan 18.
      Single-cell transcriptomic analysis is widely used to study human tumors. However, it remains challenging to distinguish normal cell types in the tumor microenvironment from malignant cells and to resolve clonal substructure within the tumor. To address these challenges, we developed an integrative Bayesian segmentation approach called copy number karyotyping of aneuploid tumors (CopyKAT) to estimate genomic copy number profiles at an average genomic resolution of 5 Mb from read depth in high-throughput single-cell RNA sequencing (scRNA-seq) data. We applied CopyKAT to analyze 46,501 single cells from 21 tumors, including triple-negative breast cancer, pancreatic ductal adenocarcinoma, anaplastic thyroid cancer, invasive ductal carcinoma and glioblastoma, to accurately (98%) distinguish cancer cells from normal cell types. In three breast tumors, CopyKAT resolved clonal subpopulations that differed in the expression of cancer genes, such as KRAS, and signatures, including epithelial-to-mesenchymal transition, DNA repair, apoptosis and hypoxia. These data show that CopyKAT can aid in the analysis of scRNA-seq data in a variety of solid human tumors.
  16. Nat Cancer. 2020 Oct;1(10): 998-1009
      Metabolic reprogramming is a key hallmark of cancer, but less is known about metabolic plasticity of the same tumor at different sites. Here, we investigated the metabolic adaptation of leukemia in two different microenvironments, the bone marrow and the central nervous system (CNS). We identified a metabolic signature of fatty-acid synthesis in CNS leukemia, highlighting Stearoyl-CoA desaturase (SCD1) as a key player. In vivo SCD1 overexpression increases CNS disease, whilst genetic or pharmacological inhibition of SCD1 decreases CNS load. Overall, we demonstrated that leukemic cells dynamically rewire metabolic pathways to suit local conditions and that targeting these adaptations can be exploited therapeutically.
    Keywords:  SCD1; acute lymphoblastic leukemia; central nervous system; fatty acid synthesis; metabolic reprogramming
  17. J Biol Chem. 2020 Dec 18. pii: S0021-9258(17)50642-0. [Epub ahead of print]295(51): 17588-17601
      Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in aging. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount and sequence of mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify, and manipulate the properties of mtDNA within cells.
    Keywords:  aging; gene editing; microscopy; mitochondria; mitochondrial DNA (mtDNA); mitochondrial disease; mitophagy
  18. Cell Stem Cell. 2021 Jan 14. pii: S1934-5909(20)30601-9. [Epub ahead of print]
      In multiple myeloma, inflammatory and anti-viral pathways promote disease progression and cancer stem cell generation. Using diverse pre-clinical models, we investigated the role of interferon regulatory factor 4 (IRF4) in myeloma progenitor regeneration. In a patient-derived xenograft model that recapitulates IRF4 pathway activation in human myeloma, we test the effects of IRF4 antisense oligonucleotides (ASOs) and identify a lead agent for clinical development (ION251). IRF4 overexpression expands myeloma progenitors, while IRF4 ASOs impair myeloma cell survival and reduce IRF4 and c-MYC expression. IRF4 ASO monotherapy impedes tumor formation and myeloma dissemination in xenograft models, improving animal survival. Moreover, IRF4 ASOs eradicate myeloma progenitors and malignant plasma cells while sparing normal human hematopoietic stem cell development. Mechanistically, IRF4 inhibition disrupts cell cycle progression, downregulates stem cell and cell adhesion transcript expression, and promotes sensitivity to myeloma drugs. These findings will enable rapid clinical development of selective IRF4 inhibitors to prevent myeloma progenitor-driven relapse.
    Keywords:  CXCR4; IRF4; MYC; antisense oligonucleotide; bone marrow; cancer stem cells; cell cycle; interferon regulatory factor 4; multiple myeloma; translational research
  19. J Therm Biol. 2021 Jan;pii: S0306-4565(20)30561-1. [Epub ahead of print]95 102790
      With the increasing threat of climate change and the accompanying rise in the frequency and severity of extreme heat events, there are growing health concerns for heat-vulnerable elderly adults. Elderly adults are at increased risk of developing heat-related injuries, in part due to age-related declines in thermoregulatory and cellular function. Regarding the latter, the process of autophagy is activated as a cellular protective mechanism to counter heat-induced stress, but the extent that heat stress activates autophagy in elderly adults is not known. Further, the interplay between autophagy, the heat shock response (HSR), the acute inflammatory response, and apoptosis remains poorly understood in elderly adults. Therefore, the purpose of this study was to examine changes in autophagy, the HSR, inflammation, and apoptosis following increasing levels of ex vivo heat stress representative of physiologically relevant increases in body core temperatures (37-41 °C). Whole blood from 20 elderly adults (72 ± 4 years; 14 men, 6 women) was heated (via water immersion) to temperatures representative of normal resting conditions (normothermia; 37 °C), in addition to moderate and severe heat stress conditions (39, and 41 °C, respectively) for 90 min. Peripheral blood mononuclear cells (PBMC) were isolated and protein markers of autophagy, the HSR, acute inflammation, and apoptosis were examined. No significant increases in markers of autophagy or the HSR were observed following any temperature condition. However, an increase in acute inflammation was observed above baseline following moderate heat stress (39 °C), with further increases in inflammation and apoptosis observed during severe heat stress (41 °C). Our findings indicate that PBMCs from elderly adults do not exhibit increases in autophagy or the HSR following severe heat stress, potentially contributing to the elevated risk of cellular dysfunction seen in elderly adults during heat stress.
    Keywords:  Aging; Apoptosis; Autophagy; HSP70; HSP90; Heat shock response; Inflammation
  20. Nat Med. 2021 Jan 21.
      Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.
  21. J Biol Chem. 2021 Jan 19. pii: S0021-9258(21)00078-8. [Epub ahead of print] 100309
      Mitochondral DNA is located in organelle that house essential metablic reactions and contain high reactive oxygen species. Therefore, mitochondrial DNA suffers more oxidative damage than its nuclear counterpart. Formation of a repair enzyme complex is beneficial to DNA repair. Recent studies have shown that mitochondrial DNA polymerase (Pol γ) and poly(ADP-ribose) polymerase 1 (PARP1) were found in the same complex along with other mitochondrial DNA repair enzymes and mitochondrial PARP1 level is correlated with mtDNA integrity. However, the molecular basis for the functional connection between Pol γ and PARP1 has not yet been elucidated because cellular functions of PARP1 in DNA repair are intertwined with metabolism via NAD+ (nicotinamide adenosine dinucleotide), the substrate of PARP1 and a metabolic cofactor. To dissect the direct effect of PARP1 on mtDNA from the secondary perturbation of metabolism, we report here biochemical studies that recapitulated Pol γ PARylation observed in cells and showed that PARP1 regulates Pol γ activity during DNA repair in a metabolic cofactor NAD+ (nicotinamide adenosine dinucleotide)-dependent manner. In the absence of NAD+, PARP1 completely inhibits Pol γ, while increasing NAD+ levels to a physiological concentration that enables Pol γ to resume maximum repair activity. Because cellular NAD+ levels are linked to metabolism and to ATP production via oxidative phosphorylation, our results suggest that mtDNA damage repair is coupled to cellular metabolic state and the integrity of the respiratory chain.
    Keywords:  ADP-ribosylation; DNA polymerase; DNA repair; DNA synthesis; post-translational modification (PTM); protein-DNA interaction; protein-protein interaction; western blot
  22. J Cell Sci. 2021 Jan 22. pii: jcs247056. [Epub ahead of print]134(2):
      Autophagy is deregulated in many cancers and represents an attractive target for therapeutic intervention. However, the precise contributions of autophagy to metastatic progression, the principle cause of cancer-related mortality, is only now being uncovered. While autophagy promotes primary tumor growth, metabolic adaptation and resistance to therapy, recent studies have unexpectedly revealed that autophagy suppresses the proliferative outgrowth of disseminated tumor cells into overt and lethal macrometastases. These studies suggest autophagy plays unexpected and complex roles in the initiation and progression of metastases, which will undoubtedly impact therapeutic approaches for cancer treatment. Here, we discuss the intricacies of autophagy in metastatic progression, highlighting and integrating the pleiotropic roles of autophagy on diverse cell biological processes involved in metastasis.
    Keywords:  Autophagy; Cancer; Metastasis; Selective Autophagy
  23. Autophagy. 2021 Jan 17.
      Mitophagy formed the basis of the original description of autophagy by Christian de Duve when he demonstrated that GCG (glucagon) induced macroautophagic/autophagic turnover of mitochondria in the liver. However, the molecular basis of liver-specific activation of mitophagy by GCG, or its significance for metabolic stress responses in the liver is not understood. Here we show that BNIP3 is required for GCG-induced mitophagy in the liver through interaction with processed LC3B; an interaction that is also necessary to localize LC3B out of the nucleus to cytosolic mitophagosomes in response to nutrient deprivation. Loss of BNIP3-dependent mitophagy caused excess mitochondria to accumulate in the liver, disrupting metabolic zonation within the liver parenchyma, with expansion of zone 1 metabolism at the expense of zone 3 metabolism. These results identify BNIP3 as a regulator of metabolic homeostasis in the liver through its effect on mitophagy and mitochondrial mass distribution.
    Keywords:  BNIP3; LC3B; glucagon; hepatocyte; liver zonation; mitophagy; nutrient deprivation
  24. J Biol Chem. 2020 Dec 11. pii: S0021-9258(17)50599-2. [Epub ahead of print]295(50): 17060-17070
      CARM1 is a protein arginine methyltransferase (PRMT) that acts as a coactivator in a number of transcriptional programs. CARM1 orchestrates this coactivator activity in part by depositing the H3R17me2a histone mark in the vicinity of gene promoters that it regulates. However, the gross levels of H3R17me2a in CARM1 KO mice did not significantly decrease, indicating that other PRMT(s) may compensate for this loss. We thus performed a screen of type I PRMTs, which revealed that PRMT6 can also deposit the H3R17me2a mark in vitro. CARM1 knockout mice are perinatally lethal and display a reduced fetal size, whereas PRMT6 null mice are viable, which permits the generation of double knockouts. Embryos that are null for both CARM1 and PRMT6 are noticeably smaller than CARM1 null embryos, providing in vivo evidence of redundancy. Mouse embryonic fibroblasts (MEFs) from the double knockout embryos display an absence of the H3R17me2a mark during mitosis and increased signs of DNA damage. Moreover, using the combination of CARM1 and PRMT6 inhibitors suppresses the cell proliferation of WT MEFs, suggesting a synergistic effect between CARM1 and PRMT6 inhibitions. These studies provide direct evidence that PRMT6 also deposits the H3R17me2a mark and acts redundantly with CARM1.
    Keywords:  CARM1; PRMT6; arginine methylation; epigenetics; histone methylation; post-transcriptional regulation; post-translational modification (PTM); transcriptional coactivator
  25. JAMA Oncol. 2021 Jan 21.
      Importance: Clinical outcomes after curative treatment of resectable pancreatic ductal adenocarcinoma (PDA) remain suboptimal. To assess the potential of early control of systemic disease with multiagent perioperative chemotherapy, we conducted a prospective trial.Objective: To determine 2-year overall survival (OS) using perioperative chemotherapy for resectable PDA.
    Design, Setting, and Participants: This was a randomized phase 2 trial of perioperative chemotherapy with a pick-the-winner design. It was conducted across the National Clinical Trials Network, including academic and community centers all across the US. Eligibility required patients with Zubrod Performance Score of 0 or 1, confirmed tissue diagnosis of PDA, and resectable disease per Intergroup criteria.
    Interventions: Perioperative (12 weeks preoperative, 12 weeks postoperative) chemotherapy with either fluorouracil, irinotecan, and oxaliplatin (mFOLFIRINOX, arm 1) or gemcitabine/nab-paclitaxel (arm 2).
    Main Outcomes and Measures: The primary outcome was 2-year overall survival (OS), using a pick-the-winner design; for 100 eligible patients, accrual up to 150 patients was planned to account for cases deemed ineligible at central radiology review.
    Results: From 2015 to 2018, 147 patients were enrolled; 43 patients (29%) had ineligible disease, beyond resectability criteria, at central radiology review. There were 102 eligible and evaluable patients, 55 in arm 1 and 47 in arm 2, of whom the median (range) age was 66 (44-76) and 64 (46-76) years, respectively; 36 patients (65%) in arm 1 and 24 (51%) in arm 2 were men. In arm 1, 34 (62%) had Zubrod Performance Score of 0, while in arm 2, 31 (66%) did; and 44 (80%) in arm 1 and 39 (83%) in arm 2 had head tumors. Of 102 patients, 84% and 85% completed preoperative chemotherapy, 73% and 70% underwent resection, and 49% and 40% completed all treatment. Adverse events were expected hematologic toxic effects, fatigue, and gastrointestinal toxicities. Two-year OS was 47% (95% CI, 31%-61%) for arm 1 and 48% (95% CI, 31%-63%) for arm 2; median OS was 23.2 months (95% CI, 17.6-45.9 months) and 23.6 months (95% CI, 17.8-31.7 months). Neither arm's 2-year OS estimate was significantly higher than the a priori threshold of 40%. Median disease-free survival after resection was 10.9 months in arm 1 and 14.2 months in arm 2.
    Conclusions and Relevance: This phase 2 randomized clinical trial did not demonstrate an improved OS with perioperative chemotherapy, compared with historical data from adjuvant trials in resectable pancreatic cancer. Two-year OS was 47% with mFOLFIRINOX and 48% with gemcitabine/nab-paclitaxel for all eligible patients starting treatment for resectable PDA. The trial also demonstrated adequate safety and high resectability rates with perioperative chemotherapy, and challenges in quality control for resectability criteria.
    Trial Registration: Identifier: NCT02562716.
  26. Trends Biochem Sci. 2021 Jan 19. pii: S0968-0004(20)30323-6. [Epub ahead of print]
      Biomolecular condensates appear throughout the cell, serving many different biochemical functions. We argue that condensate functionality is optimized when the interactions driving condensation vary widely in affinity. Strong interactions provide structural specificity needed to encode functional properties but carry the risk of kinetic arrest, while weak interactions allow the system to remain dynamic but do not restrict the conformational ensemble enough to sustain specific functional features. To support our opinion, we describe illustrative examples of the interplay of strong and weak interactions that are found in the nucleolus, SPOP/DAXX condensates, polySUMO/polySIM condensates, chromatin, and stress granules. The common feature of these systems is a hierarchical assembly motif in which weak, transient interactions condense structurally defined functional units.
    Keywords:  biomolecular condensates; biophysics; chromatin; liquid-liquid phase separation; membraneless organelles; nucleolus; stress granules
  27. J Biol Chem. 2020 Dec 25. pii: S0021-9258(17)50694-8. [Epub ahead of print]295(52): 18226-18238
      Members of the B-cell lymphoma (BCL-2) protein family regulate mitochondrial outer membrane permeabilization (MOMP), a phenomenon in which mitochondria become porous and release death-propagating complexes during the early stages of apoptosis. Pro-apoptotic BCL-2 proteins oligomerize at the mitochondrial outer membrane during MOMP, inducing pore formation. Of current interest are endogenous factors that can inhibit pro-apoptotic BCL-2 mitochondrial outer membrane translocation and oligomerization. A mitochondrial-derived peptide, Humanin (HN), was reported being expressed from an alternate ORF in the mitochondrial genome and inhibiting apoptosis through interactions with the pro-apoptotic BCL-2 proteins. Specifically, it is known to complex with BAX and BID. We recently reported the fibrillation of HN and BAX into β-sheets. Here, we detail the fibrillation between HN and BID. These fibers were characterized using several spectroscopic techniques, protease fragmentation with mass analysis, and EM. Enhanced fibrillation rates were detected with rising temperatures or pH values and the presence of a detergent. BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. BID fibers display both types of secondary structure in the fiber, whereas BAX was converted entirely to β-sheets. The data show that two distinct segments of BID are incorporated into the fiber structure, whereas other portions of BID remain solvent-exposed and retain helical structure. Similar analyses show that anti-apoptotic BCL-xL does not form fibers with humanin. These results support a general mechanism of sequestration of pro-apoptotic BCL-2 proteins into fibers by HN to inhibit MOMP.
    Keywords:  B-cell lymphoma 2 (Bcl-2) family; BID; amyloid; apoptosis; conformational change; electron microscopy (EM); fibers; humanin; β-sheet
  28. Science. 2021 01 22. pii: eabc6663. [Epub ahead of print]371(6527):
      Dot1 (disruptor of telomeric silencing-1), the histone H3 lysine 79 (H3K79) methyltransferase, is conserved throughout evolution, and its deregulation is found in human leukemias. Here, we provide evidence that acetylation of histone H4 allosterically stimulates yeast Dot1 in a manner distinct from but coordinating with histone H2B ubiquitination (H2BUb). We further demonstrate that this stimulatory effect is specific to acetylation of lysine 16 (H4K16ac), a modification central to chromatin structure. We provide a mechanism of this histone cross-talk and show that H4K16ac and H2BUb play crucial roles in H3K79 di- and trimethylation in vitro and in vivo. These data reveal mechanisms that control H3K79 methylation and demonstrate how H4K16ac, H3K79me, and H2BUb function together to regulate gene transcription and gene silencing to ensure optimal maintenance and propagation of an epigenetic state.
  29. Elife. 2021 Jan 22. pii: e61170. [Epub ahead of print]10
      During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.
    Keywords:  cell biology; human
  30. Nat Metab. 2021 Jan;3(1): 33-42
      Mitochondrial diseases (MDs) are a heterogeneous group of disorders resulting from mutations in nuclear or mitochondrial DNA genes encoding mitochondrial proteins1,2. MDs cause pathologies with severe tissue damage and ultimately death3,4. There are no cures for MDs and current treatments are only palliative5-7. Here we show that tetracyclines improve fitness of cultured MD cells and ameliorate disease in a mouse model of Leigh syndrome. To identify small molecules that prevent cellular damage and death under nutrient stress conditions, we conduct a chemical high-throughput screen with cells carrying human MD mutations and discover a series of antibiotics that maintain survival of various MD cells. We subsequently show that a sub-library of tetracycline analogues, including doxycycline, rescues cell death and inflammatory signatures in mutant cells through partial and selective inhibition of mitochondrial translation, resulting in an ATF4-independent mitohormetic response. Doxycycline treatment strongly promotes fitness and survival of Ndufs4-/- mice, a preclinical Leigh syndrome mouse model8. A proteomic analysis of brain tissue reveals that doxycycline treatment largely prevents neuronal death and the accumulation of neuroimmune and inflammatory proteins in Ndufs4-/- mice, indicating a potential causal role for these proteins in the brain pathology. Our findings suggest that tetracyclines deserve further evaluation as potential drugs for the treatment of MDs.
  31. J Clin Invest. 2021 01 19. pii: 136055. [Epub ahead of print]131(2):
      Mitochondrial disorders represent a large collection of rare syndromes that are difficult to manage both because we do not fully understand biochemical pathogenesis and because we currently lack facile markers of severity. The m.3243A>G variant is the most common heteroplasmic mitochondrial DNA mutation and underlies a spectrum of diseases, notably mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). To identify robust circulating markers of m.3243A>G disease, we first performed discovery proteomics, targeted metabolomics, and untargeted metabolomics on plasma from a deeply phenotyped cohort (102 patients, 32 controls). In a validation phase, we measured concentrations of prioritized metabolites in an independent cohort using distinct methods. We validated 20 analytes (1 protein, 19 metabolites) that distinguish patients with MELAS from controls. The collection includes classic (lactate, alanine) and more recently identified (GDF-15, α-hydroxybutyrate) mitochondrial markers. By mining untargeted mass-spectra we uncovered 3 less well-studied metabolite families: N-lactoyl-amino acids, β-hydroxy acylcarnitines, and β-hydroxy fatty acids. Many of these 20 analytes correlate strongly with established measures of severity, including Karnofsky status, and mechanistically, nearly all markers are attributable to an elevated NADH/NAD+ ratio, or NADH-reductive stress. Our work defines a panel of organelle function tests related to NADH-reductive stress that should enable classification and monitoring of mitochondrial disease.
    Keywords:  Genetics; Intermediary metabolism; Metabolism; Mitochondria; Monogenic diseases; RET; HS6ST1; sE-selectin; integrated stress response; creatine; pyruvate; 2-hydroxybutyrate; alpha-hydroxybutyrate; lactoyl-amino acids; hydroxy-fatty acids; hydroxy-acylcarnitines
  32. Nat Rev Cancer. 2021 Jan 18.
      Metastasis formation is the major cause of death in most patients with cancer. Despite extensive research, targeting metastatic seeding and colonization is still an unresolved challenge. Only recently, attention has been drawn to the fact that metastasizing cancer cells selectively and dynamically adapt their metabolism at every step during the metastatic cascade. Moreover, many metastases display different metabolic traits compared with the tumours from which they originate, enabling survival and growth in the new environment. Consequently, the stage-dependent metabolic traits may provide therapeutic windows for preventing or reducing metastasis, and targeting the new metabolic traits arising in established metastases may allow their eradication.
  33. Cell Death Differ. 2021 Jan 18.
      In eukaryotic cells, macromolecular homeostasis requires selective degradation of damaged units by the ubiquitin-proteasome system (UPS) and autophagy. Thus, dysfunctional degradation systems contribute to multiple pathological processes. Ferroptosis is a type of iron-dependent oxidative cell death driven by lipid peroxidation. Various antioxidant systems, especially the system xc--glutathione-GPX4 axis, play a significant role in preventing lipid peroxidation-mediated ferroptosis. The endosomal sorting complex required for transport-III (ESCRT-III)-dependent membrane fission machinery counteracts ferroptosis by repairing membrane damage. Moreover, cellular degradation systems play a dual role in regulating the ferroptotic response, depending on the cargo they degrade. The key ferroptosis repressors, such as SLC7A11 and GPX4, are degraded by the UPS. In contrast, the overactivation of selective autophagy, including ferritinophagy, lipophagy, clockophagy and chaperone-mediated autophagy, promotes ferroptotic death by degrading ferritin, lipid droplets, circadian proteins, and GPX4, respectively. Autophagy modulators (e.g., BECN1, STING1/TMEM173, CTSB, HMGB1, PEBP1, MTOR, AMPK, and DUSP1) also determine the ferroptotic response in a context-dependent manner. In this review, we provide an updated overview of the signals and mechanisms of the degradation system regulating ferroptosis, opening new horizons for disease treatment strategies.
  34. Cancer Res. 2021 Jan 18. pii: canres.1760.2020. [Epub ahead of print]
      Ras proteins play a causal role in human cancer by activating multiple pathways that promote cancer growth and invasion. However, little is known about how Ras induces the first diagnostic features of invasion in solid tumors, including loss of epithelial integrity and breaching of the basement membrane. In this study, we found that oncogenic Ras strongly promotes the activation of hepsin, a member of the hepsin/TMPRSS type II transmembrane serine protease family. Mechanistically, the Ras-dependent hepsin activation was mediated via Raf-MEK-ERK signaling, which controlled hepsin protein stability through the heat shock transcription factor-1 stress pathway. In Ras-transformed three-dimensional mammary epithelial culture, ablation of hepsin restored desmosomal cell-cell junctions, hemidesmosomes, and basement membrane integrity and epithelial cohesion. In tumor xenografts harboring mutant KRas, silencing of hepsin increased local invasion concomitantly with accumulation of collagen IV. These findings suggest that hepsin is a critical protease for Ras-dependent tumorigenesis, executing cell-cell and cell-matrix pathologies important for early tumor dissemination.
  35. Elife. 2021 Jan 22. pii: e63645. [Epub ahead of print]10
      In budding yeast, the mitotic exit network (MEN), a GTPase signaling cascade, integrates spatial and temporal cues to promote exit from mitosis. This signal integration requires transmission of a signal generated on the cytoplasmic face of spindle pole bodies (SPBs; yeast equivalent of centrosomes) to the nucleolus, where the MEN effector protein Cdc14 resides. Here, we show that the MEN activating signal at SPBs is relayed to Cdc14 in the nucleolus through the dynamic localization of its terminal kinase complex Dbf2-Mob1. Cdc15, the protein kinase that activates Dbf2-Mob1 at SPBs, also regulates its nuclear access. Once in the nucleus, priming phosphorylation of Cfi1/Net1, the nucleolar anchor of Cdc14, by the Polo-like kinase Cdc5 targets Dbf2-Mob1 to the nucleolus. Nucleolar Dbf2-Mob1 then phosphorylates Cfi1/Net1 and Cdc14, activating Cdc14. The kinase-primed transmission of the MEN signal from the cytoplasm to the nucleolus exemplifies how signaling cascades can bridge distant inputs and responses.
    Keywords:  S. cerevisiae; cell biology; cell cycle; mitotic exit network; signal transduction
  36. Elife. 2021 Jan 19. pii: e59399. [Epub ahead of print]10
      It has been recognized for nearly a century that diet modulates aging. Despite early experiments suggesting that reduced caloric intake augmented lifespan, accumulating evidence indicates that other characteristics of the diet may be equally or more influential in modulating aging. We demonstrate that behavior, metabolism, and lifespan in Drosophila are affected by whether flies are provided a choice of different nutrients or a single, complete medium, largely independent of the amount of nutrients that are consumed. Meal choice elicits a rapid metabolic reprogramming that indicates a potentiation of TCA cycle and amino acid metabolism, which requires serotonin 2A receptor. Knockdown of glutamate dehydrogenase, a key TCA pathway component, abrogates the effect of dietary choice on lifespan. Our results reveal a mechanism of aging that applies in natural conditions, including our own, in which organisms continuously perceive and evaluate nutrient availability to promote fitness and well-being.
    Keywords:  D. melanogaster; genetics; genomics
  37. Science. 2021 Jan 21. pii: eabc1944. [Epub ahead of print]
      Detailed phylogenies of tumor populations can recount the history and chronology of critical events during cancer progression, such as metastatic dissemination. We applied a Cas9-based, single-cell lineage tracer to study the rates, routes, and drivers of metastasis in a lung cancer xenograft mouse model. We report deeply resolved phylogenies for tens of thousands of cancer cells traced over months of growth and dissemination. This revealed stark heterogeneity in metastatic capacity, arising from pre-existing and heritable differences in gene expression. We demonstrate that these identified genes can drive invasiveness, and uncovered an unanticipated suppressive role for KRT17 We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale.
  38. Science. 2021 01 22. 371(6527): 405-410
      Infection triggers expansion and effector differentiation of T cells specific for microbial antigens in association with metabolic reprograming. We found that the glycolytic enzyme lactate dehydrogenase A (LDHA) is induced in CD8+ T effector cells through phosphoinositide 3-kinase (PI3K) signaling. In turn, ablation of LDHA inhibits PI3K-dependent phosphorylation of Akt and its transcription factor target Foxo1, causing defective antimicrobial immunity. LDHA deficiency cripples cellular redox control and diminishes adenosine triphosphate (ATP) production in effector T cells, resulting in attenuated PI3K signaling. Thus, nutrient metabolism and growth factor signaling are highly integrated processes, with glycolytic ATP serving as a rheostat to gauge PI3K-Akt-Foxo1 signaling in the control of T cell immunity. Such a bioenergetic mechanism for the regulation of signaling may explain the Warburg effect.
  39. J Biol Chem. 2020 Nov 21. pii: S0021-9258(20)00076-9. [Epub ahead of print]296 100088
      Protein synthesis is an energetically costly cellular activity. It is therefore important that the process of mRNA translation remains in excellent synchrony with cellular metabolism and its energy reserves. Unregulated translation could lead to the production of incomplete, mistranslated, or misfolded proteins, squandering the energy needed for cellular sustenance and causing cytotoxicity. One-carbon metabolism (OCM), an integral part of cellular intermediary metabolism, produces a number of one-carbon unit intermediates (formyl, methylene, methenyl, methyl). These OCM intermediates are required for the production of amino acids such as methionine and other biomolecules such as purines, thymidylate, and redox regulators. In this review, we discuss how OCM impacts the translation apparatus (composed of ribosome, tRNA, mRNA, and translation factors) and regulates crucial steps in protein synthesis. More specifically, we address how the OCM metabolites regulate the fidelity and rate of translation initiation in bacteria and eukaryotic organelles such as mitochondria. Modulation of the fidelity of translation initiation by OCM opens new avenues to understand alternative translation mechanisms involved in stress tolerance and drug resistance.
    Keywords:  RNA modifications; folate metabolism; formylation; mitochondria; ribosome heterogeneity
  40. Nat Chem Biol. 2021 Jan 18.
      Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host's single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions.
  41. J Cell Sci. 2021 Jan 19. pii: jcs.244012. [Epub ahead of print]
      Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation and STIM1 (stromal interaction molecule-1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased Store Operated Calcium Entry (SOCE). In the pancreatic AR42J cells expressing the p.E152K variant, Ca2+-signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.
    Keywords:  Calcium signaling; Missense mutation; Modifier variant; Pancreatitis; Trypsin secretion
  42. Mol Cell. 2021 Jan 12. pii: S1097-2765(20)30958-8. [Epub ahead of print]
      Micronuclei are aberrant nuclear compartments that can form as a result of chromosome mis-segregation. Frequent loss of micronuclear envelope integrity exposes DNA to the cytoplasm, leading to chromosome fragmentation and immune activation. Here, we use micronuclei purification to show that the endoplasmic reticulum (ER)-associated nuclease TREX1 inhibits cGAS activation at micronuclei by degrading micronuclear DNA upon micronuclear envelope rupture. We demonstrate that the ER accesses ruptured micronuclei and plays a critical role in enabling TREX1 nucleolytic attack. TREX1 mutations, previously implicated in immune disease, untether TREX1 from the ER, disrupt TREX1 localization to micronuclei, diminish micronuclear DNA damage, and enhance cGAS activation. These results establish ER-directed resection of micronuclear DNA by TREX1 as a critical regulator of cytosolic DNA sensing in chromosomally unstable cells and provide a mechanistic basis for the importance of TREX1 ER tethering in preventing autoimmunity.
    Keywords:  APE1; BAF; STING; TREX1; cGAS; chromosome instability; chromothripsis; endoplasmic reticulum; micronuclei; nuclear envelope
  43. Cancer Metastasis Rev. 2021 Jan 20.
      Cancer is a pathology characterized by a loss or a perturbation of a number of typical features of normal cell behaviour. Indeed, the acquisition of an inappropriate migratory and invasive phenotype has been reported to be one of the hallmarks of cancer. The cytoskeleton is a complex dynamic network of highly ordered interlinking filaments playing a key role in the control of fundamental cellular processes, like cell shape maintenance, motility, division and intracellular transport. Moreover, deregulation of this complex machinery contributes to cancer progression and malignancy, enabling cells to acquire an invasive and metastatic phenotype. Metastasis accounts for 90% of death from patients affected by solid tumours, while an efficient prevention and suppression of metastatic disease still remains elusive. This results in the lack of effective therapeutic options currently available for patients with advanced disease. In this context, the cytoskeleton with its regulatory and structural proteins emerges as a novel and highly effective target to be exploited for a substantial therapeutic effort toward the development of specific anti-metastatic drugs. Here we provide an overview of the role of cytoskeleton components and interacting proteins in cancer metastasis with a special focus on small molecule compounds interfering with the actin cytoskeleton organization and function. The emerging involvement of microtubules and intermediate filaments in cancer metastasis is also reviewed.
    Keywords:  Anti-metastatic drugs; Cancer metastasis; Cytoskeleton; Invasion; Migration; Small molecule compounds
  44. Eur J Gastroenterol Hepatol. 2021 Jan 18.
      BACKGROUND: Early detection of pancreatic ductal adenocarcinoma (PDA) may improve survival. We previously developed a clinical prediction model among patients with new-onset diabetes to help identify PDAs 6 months prior to the clinical diagnosis of the cancer. We developed and internally validated a new model to predict PDA risk among those newly diagnosed with impaired fasting glucose (IFG).METHODS: We conducted a retrospective cohort study in The Health Improvement Network (THIN) (1995-2013) from the UK. Eligible study patients had newly diagnosed IFG during follow-up in THIN. The outcome was incident PDA diagnosed within 3 years of IFG diagnosis. Candidate predictors were factors associated with PDA, glucose metabolism or both.
    RESULTS: Among the 138 232 eligible patients with initial IFG diagnosis, 245 (0.2%) were diagnosed with PDA within 3 years. The median time from IFG diagnosis to clinical PDA diagnosis was 326 days (IQR 120-588). The final prediction model included age, BMI, proton pump inhibitor use, total cholesterol, low-density lipoprotein, alanine aminotransferase and alkaline phosphatase. The model achieved good discrimination [area under the curve 0.71 (95% CI, 0.67-0.75)] and calibration (Hosmer and Lemeshow goodness-of-fit test P > 0.05 in 17 of the 20 imputed data sets) with optimism of 0.0012662 (95% CI, -0.00932 to 0.0108771).
    CONCLUSIONS: We developed and internally validated a sequential PDA prediction model based on clinical information routinely available at the initial appearance of IFG. If externally validated, this model could significantly extend our ability to detect PDAs at an earlier stage.
  45. Proc Natl Acad Sci U S A. 2021 Jan 26. pii: e2022120118. [Epub ahead of print]118(4):
      In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.
    Keywords:  CRISPR-Cas9 screen; mTORC1; mitochondria
  46. Nat Rev Clin Oncol. 2021 Jan 20.
      Within the past decade, the field of immunotherapy has revolutionized the treatment of many cancers with the development and regulatory approval of various immune-checkpoint inhibitors and chimeric antigen receptor T cell therapies in diverse indications. Another promising approach to cancer immunotherapy involves the use of personalized vaccines designed to trigger de novo T cell responses against neoantigens, which are highly specific to tumours of individual patients, in order to amplify and broaden the endogenous repertoire of tumour-specific T cells. Results from initial clinical studies of personalized neoantigen-based vaccines, enabled by the availability of rapid and cost-effective sequencing and bioinformatics technologies, have demonstrated robust tumour-specific immunogenicity and preliminary evidence of antitumour activity in patients with melanoma and other cancers. Herein, we provide an overview of the complex process that is necessary to generate a personalized neoantigen vaccine, review the types of vaccine-induced T cells that are found within tumours and outline strategies to enhance the T cell responses. In addition, we discuss the current status of clinical studies testing personalized neoantigen vaccines in patients with cancer and considerations for future clinical investigation of this novel, individualized approach to immunotherapy.
  47. Nature. 2021 Jan 20.
      Ageing is characterized by the development of persistent pro-inflammatory responses that contribute to atherosclerosis, metabolic syndrome, cancer and frailty1-3. The ageing brain is also vulnerable to inflammation, as demonstrated by the high prevalence of age-associated cognitive decline and Alzheimer's disease4-6. Systemically, circulating pro-inflammatory factors can promote cognitive decline7,8, and in the brain, microglia lose the ability to clear misfolded proteins that are associated with neurodegeneration9,10. However, the underlying mechanisms that initiate and sustain maladaptive inflammation with ageing are not well defined. Here we show that in ageing mice myeloid cell bioenergetics are suppressed in response to increased signalling by the lipid messenger prostaglandin E2 (PGE2), a major modulator of inflammation11. In ageing macrophages and microglia, PGE2 signalling through its EP2 receptor promotes the sequestration of glucose into glycogen, reducing glucose flux and mitochondrial respiration. This energy-deficient state, which drives maladaptive pro-inflammatory responses, is further augmented by a dependence of aged myeloid cells on glucose as a principal fuel source. In aged mice, inhibition of myeloid EP2 signalling rejuvenates cellular bioenergetics, systemic and brain inflammatory states, hippocampal synaptic plasticity and spatial memory. Moreover, blockade of peripheral myeloid EP2 signalling is sufficient to restore cognition in aged mice. Our study suggests that cognitive ageing is not a static or irrevocable condition but can be reversed by reprogramming myeloid glucose metabolism to restore youthful immune functions.
  48. Cell Metab. 2021 Jan 18. pii: S1550-4131(20)30728-2. [Epub ahead of print]
      Glycolysis plays a central role in organismal metabolism, but its quantitative inputs across mammalian tissues remain unclear. Here we use 13C-tracing in mice to quantify glycolytic intermediate sources: circulating glucose, intra-tissue glycogen, and circulating gluconeogenic precursors. Circulating glucose is the main source of circulating lactate, the primary end product of tissue glycolysis. Yet circulating glucose highly labels glycolytic intermediates in only a few tissues: blood, spleen, diaphragm, and soleus muscle. Most glycolytic intermediates in the bulk of body tissue, including liver and quadriceps muscle, come instead from glycogen. Gluconeogenesis contributes less but also broadly to glycolytic intermediates, and its flux persists with physiologic feeding (but not hyperinsulinemic clamp). Instead of suppressing gluconeogenesis, feeding activates oxidation of circulating glucose and lactate to maintain glucose homeostasis. Thus, the bulk of the body slowly breaks down internally stored glycogen while select tissues rapidly catabolize circulating glucose to lactate for oxidation throughout the body.
    Keywords:  compartmentalized metabolism; glucose homeostasis; glycogen; glycolysis; glycolytic intermediates; glycolytic specialist; isotope tracing; metabolic heterogeneity; red muscle
  49. Nat Commun. 2021 01 20. 12(1): 470
      Healthy aging can be promoted by enhanced metabolic fitness and physical capacity. Mitochondria are chief metabolic organelles with strong implications in aging that also coordinate broad physiological functions, in part, using peptides that are encoded within their independent genome. However, mitochondrial-encoded factors that actively regulate aging are unknown. Here, we report that mitochondrial-encoded MOTS-c can significantly enhance physical performance in young (2 mo.), middle-age (12 mo.), and old (22 mo.) mice. MOTS-c can regulate (i) nuclear genes, including those related to metabolism and proteostasis, (ii) skeletal muscle metabolism, and (iii) myoblast adaptation to metabolic stress. We provide evidence that late-life (23.5 mo.) initiated intermittent MOTS-c treatment (3x/week) can increase physical capacity and healthspan in mice. In humans, exercise induces endogenous MOTS-c expression in skeletal muscle and in circulation. Our data indicate that aging is regulated by genes encoded in both of our co-evolved mitochondrial and nuclear genomes.
  50. J Cell Sci. 2021 Jan 19. pii: jcs.250670. [Epub ahead of print]
      Late endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Whereas these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the phosphatidylinositol 3-phosphate (PI3P) binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the lipid scramblase ATG9A, which drives expansion of nascent autophagosome membranes, from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. We propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.
    Keywords:  Autophagy; Endosome; Phosphoinositide; Recycling
  51. Nat Cell Biol. 2021 Jan 18.
      The response to poly(ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair and the abundance of lesions that trap PARP enzymes. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR-based screen, we identified the PAR-binding chromatin remodeller ALC1/CHD1L as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of breast cancer gene (BRCA)-mutant cells and enhanced sensitivity to PARPi by up to 250-fold, while overcoming several resistance mechanisms. ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of base damage repair factors. This resulted in an accumulation of replication-associated DNA damage, increased PARP trapping and a reliance on HR. These findings establish PAR-dependent chromatin remodelling as a mechanistically distinct aspect of PARPi responses and therapeutic target in HR-deficient cancers.
  52. Nat Rev Clin Oncol. 2021 Jan 19.
      The immune system has crucial roles in cancer development and treatment. Whereas adaptive immunity can prevent or constrain cancer through immunosurveillance, innate immunity and inflammation often promote tumorigenesis and malignant progression of nascent cancer. The past decade has witnessed the translation of knowledge derived from preclinical studies of antitumour immunity into clinically effective, approved immunotherapies for cancer. By contrast, the successful implementation of treatments that target cancer-associated inflammation is still awaited. Anti-inflammatory agents have the potential to not only prevent or delay cancer onset but also to improve the efficacy of conventional therapeutics and next-generation immunotherapies. Herein, we review the current clinical advances and experimental findings supporting the utility of an anti-inflammatory approach to the treatment of solid malignancies. Gaining a better mechanistic understanding of the mode of action of anti-inflammatory agents and designing more effective treatment combinations would advance the clinical application of this therapeutic approach.
  53. Elife. 2021 Jan 20. pii: e61980. [Epub ahead of print]10
      Little is known about the metabolic regulation of rare cell populations because most metabolites are hard to detect in small numbers of cells. We previously described a method for metabolomic profiling of flow cytometrically-isolated hematopoietic stem cells (HSCs) that detects 60 metabolites in 10,000 cells (Agathocleous et al., 2017). Here we describe a new method involving hydrophilic liquid interaction chromatography and high-sensitivity orbitrap mass spectrometry that detected 160 metabolites in 10,000 HSCs, including many more glycolytic and lipid intermediates. We improved chromatographic separation, increased mass resolution, minimized ion suppression, and eliminated sample drying. Most metabolite levels did not significantly change during cell isolation. Mouse HSCs exhibited increased glycerophospholipids relative to bone marrow cells and methotrexate treatment altered purine biosynthesis. Circulating human melanoma cells were depleted for purine intermediates relative to subcutaneous tumors, suggesting decreased purine synthesis during metastasis. These methods facilitate the routine metabolomic analysis of rare cells from tissues.
    Keywords:  mouse; regenerative medicine; stem cells
  54. Gut. 2021 Jan 19. pii: gutjnl-2020-323805. [Epub ahead of print]
    Keywords:  DNA microsatellite instability; immunotherapy; microsatellite instability; pancreas; pancreatic cancer
  55. EMBO Mol Med. 2021 Jan 18. e12836
      Lysosomal storage diseases are a group of metabolic disorders caused by deficiencies of several components of lysosomal function. Most commonly affected are lysosomal hydrolases, which are involved in the breakdown and recycling of a variety of complex molecules and cellular structures. The understanding of lysosomal biology has progressively improved over time. Lysosomes are no longer viewed as organelles exclusively involved in catabolic pathways, but rather as highly dynamic elements of the autophagic-lysosomal pathway, involved in multiple cellular functions, including signaling, and able to adapt to environmental stimuli. This refined vision of lysosomes has substantially impacted on our understanding of the pathophysiology of lysosomal disorders. It is now clear that substrate accumulation triggers complex pathogenetic cascades that are responsible for disease pathology, such as aberrant vesicle trafficking, impairment of autophagy, dysregulation of signaling pathways, abnormalities of calcium homeostasis, and mitochondrial dysfunction. Novel technologies, in most cases based on high-throughput approaches, have significantly contributed to the characterization of lysosomal biology or lysosomal dysfunction and have the potential to facilitate diagnostic processes, and to enable the identification of new therapeutic targets.
    Keywords:  autophagy; lysosomal biology; lysosomal storage diseases; lysosomes
  56. J Biol Chem. 2020 Dec 04. pii: S0021-9258(20)00123-4. [Epub ahead of print]296 100131
      TGR5, a G protein-coupled bile acid receptor, is expressed in various tissues and regulates several physiological processes. In the skeletal muscle, TGR5 activation is known to induce muscle hypertrophy; however, the effects on glucose and lipid metabolism are not well understood, despite the fact that the skeletal muscle plays a major role in energy metabolism. Here, we demonstrate that skeletal muscle-specific TGR5 transgenic (Tg) mice exhibit increased glucose utilization, without altering the expression of major genes related to glucose and lipid metabolism. Metabolite profiling analysis by capillary electrophoresis time-of-flight mass spectrometry showed that glycolytic flux was activated in the skeletal muscle of Tg mice, leading to an increase in glucose utilization. Upon long-term, high-fat diet challenge, blood glucose clearance was improved in Tg mice without an accompanying increase in insulin sensitivity in skeletal muscle and a reduction of body weight. Moreover, Tg mice showed improved age-associated glucose intolerance. These results strongly suggest that TGR5 ameliorated glucose metabolism disorder that is caused by diet-induced obesity and aging by enhancing the glucose metabolic capacity of the skeletal muscle. Our study demonstrates that TGR5 activation in the skeletal muscle is effective in improving glucose metabolism and may be beneficial in developing a novel strategy for the prevention or treatment of hyperglycemia.
    Keywords:  G protein–coupled receptor; aging; bile acid; diabetes; energy metabolism; muscle hypertrophy; obesity; skeletal muscle metabolism
  57. Trends Biochem Sci. 2021 Jan 15. pii: S0968-0004(20)30322-4. [Epub ahead of print]
      The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has been primarily characterized as an inflammatory mechanism in higher eukaryotes in response to cytosolic double-stranded DNA (dsDNA). Since its initial discovery, detailed mechanisms delineating the dynamic subcellular localization of its different components and downstream signaling have been uncovered, leading to attempts to harness its proinflammatory properties for therapeutic benefit in cancer. Emerging evidence, however, indicates that a crucial primordial function of STING is to promote autophagy, and that downstream interferon (IFN) signaling emerged recently in its evolutionary history. Furthermore, studies suggest that this pathway is a crucial regulator of cellular metabolism that potentially couples inflammation to nutrient availability. We focus on the evolutionarily conserved functions of STING, and we discuss how a broader understanding of this pathway can help us to better appreciate its potential role in cancer and harness it for therapeutic benefit.
    Keywords:  autophagy; inflammation; membrane trafficking; metabolism; metastasis; non-canonical NF-κB
  58. Cancer Res. 2021 Jan 21.
      Deregulation of the mRNA translational process has been observed during tumorigenesis. However, recent findings have shown that deregulation of translation also contributes specifically to cancer cell spread. During metastasis, cancer cells undergo changes in cellular state, permitting the acquisition of features necessary for cell survival, dissemination, and outgrowth. In addition, metastatic cells respond to external cues, allowing for their persistence under significant cellular and microenvironmental stresses. Recent work has revealed the importance of mRNA translation to these dynamic changes, including regulation of cell states through epithelial-to-mesenchymal transition and tumor dormancy and as a response to external stresses such as hypoxia and immune surveillance. In this review, we focus on examples of altered translation underlying these phenotypic changes and responses to external cues and explore how they contribute to metastatic progression. We also highlight the therapeutic opportunities presented by aberrant mRNA translation, suggesting novel ways to target metastatic tumor cells.
  59. Aging Cell. 2021 Jan 20. e13297
      The progressively increasing frailty, morbidity and mortality of aging organisms coincides with, and may be causally related to, their waning ability to adapt to environmental perturbations. Transcriptional responses to challenges, such as oxidative stress or pathogens, diminish with age. This effect is manifest in the declining function of the stress responsive transcription factor Nrf2. Protective gene expression programs that are controlled by the Drosophila Nrf2 homolog, CncC, support homeostasis and longevity. Age-associated chromatin changes make these genes inaccessible to CncC binding and render them inert to signal-dependent transcriptional activation in old animals. In a previous paper, we have reported that overexpression of the CncC dimerization partner Maf-S counteracts this degenerative effect and preserves organism fitness. Building on this work, we show here that Maf-S overexpression prevents loss of chromatin accessibility and maintains gene responsiveness. Moreover, the same outcome, along with an extension of lifespan, can be achieved by inducing CncC target gene expression pharmacologically throughout adult life. Thus, pharmacological or dietary interventions that can preserve stress responsive gene expression may be feasible anti-aging strategies.
    Keywords:  Nrf2; aging; chromatin; drosophila; oxidative stress; transcription
  60. Clin Cancer Res. 2021 Jan 21.
      PURPOSE: To investigate whether CEACAM7 represents a novel therapeutic target for treating pancreatic ductal adenocarcinoma (PDAC) and to generate CEACAM7-targeting CAR T cells to test this hypothesis.EXPERIMENTAL DESIGN: We identified CEACAM7 (CGM2), a member of the CEA family of proteins with expression restricted to the colon and pancreas, as a potential CAR T-cell target for PDAC. We probed a panel of PDAC tumor sections as well as patient-derived PDAC cell cultures for CEACAM7 expression. We generated CAR-targeting CEACAM7, and assessed antitumor efficacy of CEACAM7 CAR T cells using in vitro and in vivo models.
    RESULTS: We show here that CEACAM7 is expressed in a large subset of PDAC tumors, with low to undetectable expression in all normal tissues tested. CEACAM7 is also expressed in primary PDAC cultures isolated from patient-derived tumors, with high expression within the cancer stem cell-enriched subset. CAR T cells targeting CEACAM7 are capable of targeting antigen-expressing tumor cells, and mediate remission in patient-derived xenograft tumors.
    CONCLUSIONS: We identify CEACAM7 as a potential therapeutic target in PDAC and describe the development of CEACAM7-targeted CAR T cells with efficacy against PDAC.
  61. Sci Rep. 2021 Jan 18. 11(1): 1666
      Autophagy is a cellular degradation system contributing to homeostasis of tissue stem cells including haematopoietic stem cells (HSCs). It plays pleiotropic roles in HSC characteristics throughout life, but its stage-specific roles in HSC self-renewal are unclear. To investigate the effects of Atg5 deletion on stage-specific HSC functions, we compared the repopulating capacity of HSCs in Atg5f/f;Vavi-cre mice from postnatal day (P) 0-7 weeks of age. Interestingly, Atg5 deficiency led to no remarkable abnormality in the HSC self-renewal capacity at P0, but significant defects at P7, followed by severe defects. Induction of Atg5 deletion at P5 by tamoxifen administration to Atg5f/f;Rosa26-Cre-ERT2 mice resulted in normal haematopoiesis, including the HSC population, until around 1 year, suggesting that Atg5 in the early neonatal period was critical for haematopoiesis in adults. Mitochondrial oxidative stress was increased by Atg5 loss in neonatal HSC/progenitor cells. Although p62 had accumulated in immature bone marrow cells of Atg5f/f;Vavi-cre mice, p62 deletion did not restore defective HSC functions, indicating that Atg5-dependent haematopoietic regulation in the developmental period was independent of p62. This study proposes a critical role of autophagy in HSC protection against harsh environments in the early neonatal stage, which is essential for healthy long-term haematopoiesis.
  62. Mech Ageing Dev. 2021 Jan 14. pii: S0047-6374(21)00009-9. [Epub ahead of print] 111437
      Genetically modified mouse models of ageing are the living proof that lifespan and healthspan can be lengthened or shortened, and provide a powerful context in which to unravel the molecular mechanisms at work. In this study, we analysed and compared gene expression data from 10 long-lived and 8 short-lived mouse models of ageing. Transcriptome-wide correlation analysis revealed that mutations with equivalent effects on lifespan induce more similar transcriptomic changes, especially if they target the same pathway. Using functional enrichment analysis, we identified 58 gene sets with consistent changes in long- and short-lived mice, 55 of which were up-regulated in long-lived mice and down-regulated in short-lived mice. Half of these sets represented genes involved in energy and lipid metabolism, among which Ppargc1a, Mif, Aldh5a1 and Idh1 were frequently observed. Based on the gene sets with consistent changes, and also the whole transcriptome, the gene expression changes during normal ageing resembled the transcriptome of short-lived models, suggesting that accelerated ageing models reproduce partially the molecular changes of ageing. Finally, we identified new genetic interventions that may ameliorate ageing, by comparing the transcriptomes of 51 mouse mutants not previously associated with ageing to expression signatures of long- and short-lived mice and ageing-related changes.
    Keywords:  ageing; gene expression; lifespan; metabolism; mitochondria; mouse; progeria
  63. Matrix Biol. 2021 Jan 14. pii: S0945-053X(21)00004-4. [Epub ahead of print]
      Tissue repair and fibrosis, an abnormal form of repair, occur in most human organs in response to injury or inflammation. Fibroblasts play a major role in the normal repair process by differentiating into myofibroblasts that synthesize extracellular matrix (ECM) components and favor tissue remodeling to reestablish normal function and integrity. However, their persistent accumulation at the site of injury is a hallmark of fibrosis. Autophagy is a catabolic process that occurs in eukaryotic cells as a stress response to allow cell survival and maintenance of cellular homeostasis by degrading and recycling intracellular components. Recent advances identify autophagy as an important regulator of myofibroblast differentiation, tissue remodeling, and fibrogenesis. In this mini-review, we provide an overview of the interactions between autophagy, ECM, and fibrosis, and emphasize the molecular mechanisms involved in myofibroblast differentiation. We also describe the emerging concept of secretory autophagy as a new avenue for intercellular communication at the site of tissue injury and repair.
  64. Science. 2021 Jan 21. pii: eabe6959. [Epub ahead of print]
      Limited initial supply of SARS-CoV-2 vaccine raises the question of how to prioritize available doses. Here, we used a mathematical model to compare five age-stratified prioritization strategies. A highly effective transmission-blocking vaccine prioritized to adults ages 20-49 years minimized cumulative incidence, but mortality and years of life lost were minimized in most scenarios when the vaccine was prioritized to adults over 60 years old. Use of individual-level serological tests to redirect doses to seronegative individuals improved the marginal impact of each dose while potentially reducing existing inequities in COVID-19 impact. While maximum impact prioritization strategies were broadly consistent across countries, transmission rates, vaccination rollout speeds, and estimates of naturally acquired immunity, this framework can be used to compare impacts of prioritization strategies across contexts.
  65. J Biol Chem. 2020 Dec 25. pii: S0021-9258(17)50708-5. [Epub ahead of print]295(52): 18406-18425
      Mitochondria are specialized compartments that produce requisite ATP to fuel cellular functions and serve as centers of metabolite processing, cellular signaling, and apoptosis. To accomplish these roles, mitochondria rely on the genetic information in their small genome (mitochondrial DNA) and the nucleus. A growing appreciation for mitochondria's role in a myriad of human diseases, including inherited genetic disorders, degenerative diseases, inflammation, and cancer, has fueled the study of biochemical mechanisms that control mitochondrial function. The mitochondrial transcriptional machinery is different from nuclear machinery. The in vitro re-constituted transcriptional complexes of Saccharomyces cerevisiae (yeast) and humans, aided with high-resolution structures and biochemical characterizations, have provided a deeper understanding of the mechanism and regulation of mitochondrial DNA transcription. In this review, we will discuss recent advances in the structure and mechanism of mitochondrial transcription initiation. We will follow up with recent discoveries and formative findings regarding the regulatory events that control mitochondrial DNA transcription, focusing on those involved in cross-talk between the mitochondria and nucleus.
    Keywords:  DNA transcription; RNA polymerase; enzyme mechanism; enzyme structure; human mitochondrial RNA polymerase; mitochondria; mitochondrial DNA (mtDNA); mitochondrial DNA transcription; mitochondrial gene regulation; structure-function; transcription; transcription initiation factors; transcription regulation; yeast mitochondrial RNA polymerase
  66. Lancet Oncol. 2021 Jan 18. pii: S1470-2045(20)30693-8. [Epub ahead of print]
  67. Methods Mol Biol. 2021 ;2251 1-17
      Phosphoinositide (PPI) lipids are a crucial class of low-abundance signaling molecules that regulate many processes within cells. Methods that enable simultaneous detection of all PPI lipid species provide a wholistic snapshot of the PPI profile of cells, which is critical for probing PPI biology. Here we describe a method for the simultaneous measurement of cellular PPI levels by metabolically labeling yeast or mammalian cells with myo-3H-inositol, extracting radiolabeled glycerophosphoinositides, and separating lipid species on an anion exchange column via HPLC.
    Keywords:  PI3,4,5P3; PI3,4P2; PI3,5P2; PI3P; PI4,5P2; PI4P; PI5P; Phosphatidylinositol; Phosphoinositide lipids; PtdIns; Radioactive metabolic labeling
  68. EMBO J. 2021 Jan 18. e104296
      The IκB kinase (IKK)-NF-κB pathway is activated as part of the DNA damage response and controls both inflammation and resistance to apoptosis. How these distinct functions are achieved remained unknown. We demonstrate here that DNA double-strand breaks elicit two subsequent phases of NF-κB activation in vivo and in vitro, which are mechanistically and functionally distinct. RNA-sequencing reveals that the first-phase controls anti-apoptotic gene expression, while the second drives expression of senescence-associated secretory phenotype (SASP) genes. The rapidly activated first phase is driven by the ATM-PARP1-TRAF6-IKK cascade, which triggers proteasomal destruction of inhibitory IκBα, and is terminated through IκBα re-expression from the NFKBIA gene. The second phase, which is activated days later in senescent cells, is on the other hand independent of IKK and the proteasome. An altered phosphorylation status of NF-κB family member p65/RelA, in part mediated by GSK3β, results in transcriptional silencing of NFKBIA and IKK-independent, constitutive activation of NF-κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF-κB activation with important implications for genotoxic cancer treatment.
    Keywords:  DNA damage response; IκBα; NF-κB; SASP; senescence
  69. Biochem Biophys Res Commun. 2021 Jan 19. pii: S0006-291X(21)00058-9. [Epub ahead of print]542 17-23
      Nickel, the most frequent contact allergy cause, is widely used for various metallic materials and medical devices. Autophagy is an intracellular protein degradation system and contributes to metal recycling. However, it is unclear the functions of nickel in autophagy. We here demonstrated that NiCl2 induced microtubule-associated protein 1 light chain 3 (LC3)-II and LC3 puncta, markers of autophagosomes. Bafilomycin A1 (BafA1) treatment did not enhance LC3 puncta under NiCl2 stimulation, suggesting that NiCl2 did not induce autophagic flux. In addition, NiCl2 promotes the accumulation of SQSTM1/p62 and increased SQSTM1/p62 colocalization with lysosomal-associated membrane protein 1 (LAMP1). These data indicated that NiCl2 attenuates autophagic flux. Interestingly, NiCl2 induced the expression of the high-molecular-weight (MW) form of SQSTM1/p62. Inhibition of NiCl2-induced reactive oxygen species (ROS) reduced the high-MW SQSTM1/p62. We also showed that NiCl2-induced ROS activate transglutaminase (TG) activity. We found that transglutaminase 2 (TG2) inhibition reduced high-MW SQSTM1/p62 and SQSTM1/p62 puncta under NiCl2 stimulation, indicating that TG2 regulates SQSTM1/p62 protein homeostasis under NiCl2 stimulation. Our study demonstrated that nickel ion regulates autophagy flux and TG2 restricted nickel-dependent proteostasis.
    Keywords:  Autophagy; Lysosome; Nickel; Post-translational modification; SQSTM1/p62; Transglutaminase 2 (TG2)
  70. Endocrinology. 2021 Jan 18. pii: bqab012. [Epub ahead of print]
      Body energy homeostasis results from balancing energy intake and energy expenditure. Central nervous system administration of pituitary adenylate cyclase activating polypeptide (PACAP) dramatically alters metabolic function, but the physiologic mechanism of this neuropeptide remains poorly defined. PACAP is expressed in the mediobasal hypothalamus (MBH), a brain area essential for energy balance. Ventromedial hypothalamic nucleus (VMN) neurons contain, by far, the largest and most dense population of PACAP in the medial hypothalamus. This region is involved in coordinating the sympathetic nervous system in response to metabolic cues in order to re-establish energy homeostasis. Additionally, the metabolic cue of leptin signaling in the VMN regulates PACAP expression. We hypothesized that PACAP may play a role in the various effector systems of energy homeostasis, and tested its role by using VMN-directed, but MBH encompassing, AAV Cre injections to ablate Adcyap1 (gene coding for PACAP) in mice (Adcyap1MBHKO mice). Adcyap1  MBHKO mice rapidly gained body weight and adiposity, becoming hyperinsulinemic and hyperglycemic. Adcyap1  MBHKO mice exhibited decreased oxygen consumption (VO2), without changes in activity. These effects appear to be due at least in part to BAT dysfunction, and we show that PACAP-expressing cells in the MBH can stimulate BAT thermogenesis. While we observed disruption of glucose clearance during hyperinsulinemic/euglycemic clamp studies in obese Adcyap1  MBHKO mice, these parameters were normal prior to the onset of obesity. Thus, MBH PACAP plays important roles in the regulation of metabolic rate and energy balance through multiple effector systems on multiple time scales, which highlight the diverse set of fuctions for PACAP in overall energy homeostasis.
    Keywords:  Energy balance; Energy expenditure; glucose homeostasis; obesity; thermogenesis; ventromedial hypothalamus
  71. Aging Cell. 2021 Jan 20. e13296
      Cellular senescence is characterized by an irreversible cell cycle arrest and a pro-inflammatory senescence-associated secretory phenotype (SASP), which is a major contributor to aging and age-related diseases. Clearance of senescent cells has been shown to improve brain function in mouse models of neurodegenerative diseases. However, it is still unknown whether senescent cell clearance alleviates cognitive dysfunction during the aging process. To investigate this, we first conducted single-nuclei and single-cell RNA-seq in the hippocampus from young and aged mice. We observed an age-dependent increase in p16Ink4a senescent cells, which was more pronounced in microglia and oligodendrocyte progenitor cells and characterized by a SASP. We then aged INK-ATTAC mice, in which p16Ink4a -positive senescent cells can be genetically eliminated upon treatment with the drug AP20187 and treated them either with AP20187 or with the senolytic cocktail Dasatinib and Quercetin. We observed that both strategies resulted in a decrease in p16Ink4a exclusively in the microglial population, resulting in reduced microglial activation and reduced expression of SASP factors. Importantly, both approaches significantly improved cognitive function in aged mice. Our data provide proof-of-concept for senolytic interventions' being a potential therapeutic avenue for alleviating age-associated cognitive impairment.
    Keywords:  SASP; aging; brain; cognition; memory; neurodegeneration; senescence; senolytic; telomeres
  72. Nature. 2021 Jan;589(7842): 344-348
    Keywords:  Computer science; Mathematics and computing; Software; Technology
  73. Cell Mol Life Sci. 2021 Jan 19.
      Obesity has been linked to an increased risk of and a worse prognosis for several types of cancer. A number of interrelated mediators contribute to obesity's pro-tumor effects, including chronic adipose inflammation and other perturbations of immune cell development and function. Here, we review studies examining the impact of obesity-induced immune dysfunction on cancer risk and progression. While the role of adipose tissue inflammation in obesity-associated cancer risk has been well characterized, the effects of obesity on immune cell infiltration and activity within the tumor microenvironment are not well studied. In this review, we aim to highlight the impact of both adipose-mediated inflammatory signaling and intratumoral immunosuppressive signaling in obesity-induced cancer risk, progression, and metastasis.
    Keywords:  Chemoresistance; Immunotherapy; Leptin
  74. J Biol Chem. 2020 Nov 22. pii: S0021-9258(20)00050-2. [Epub ahead of print]296 100064
      Genetic mutations that affect telomerase function or telomere maintenance result in a variety of diseases collectively called telomeropathies. This wide spectrum of disorders, which include dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia, is characterized by severely short telomeres, often resulting in hematopoietic stem cell failure in the most severe cases. Recent work has focused on understanding the molecular basis of these diseases. Mutations in the catalytic TERT and TR subunits of telomerase compromise activity, while others, such as those found in the telomeric protein TPP1, reduce the recruitment of telomerase to the telomere. Mutant telomerase-associated proteins TCAB1 and dyskerin and the telomerase RNA maturation component poly(A)-specific ribonuclease affect the maturation and stability of telomerase. In contrast, disease-associated mutations in either CTC1 or RTEL1 are more broadly associated with telomere replication defects. Yet even with the recent surge in studies decoding the mechanisms underlying these diseases, a significant proportion of dyskeratosis congenita mutations remain uncharacterized or poorly understood. Here we review the current understanding of the molecular basis of telomeropathies and highlight experimental data that illustrate how genetic mutations drive telomere shortening and dysfunction in these patients. This review connects insights from both clinical and molecular studies to create a comprehensive view of the underlying mechanisms that drive these diseases. Through this, we emphasize recent advances in therapeutics and pinpoint disease-associated variants that remain poorly defined in their mechanism of action. Finally, we suggest future avenues of research that will deepen our understanding of telomere biology and telomere-related disease.
    Keywords:  dyskeratosis congenita; telomerase; telomerase assembly; telomerase recruitment; telomere; telomere shortening; telomeropathies
  75. Mol Biol Cell. 2021 Jan 21. mbcE20060390
      Acylglycerol Kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in AGK cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy and lactic acidosis. We mapped the proteomic changes in Sengers patient fibroblasts and AGKKO cell lines to understand the effects of AGK dysfunction on mitochondria. This uncovered downregulation of a number of proteins at the inner mitochondrial membrane, including many SLC25 carrier family proteins, which are predicted substrates of the complex. We also observed downregulation of SFXN proteins, which contain five transmembrane domains, and show that they represent a novel class of TIM22 complex substrate. Perturbed biogenesis of SFXN proteins in cells lacking AGK reduces the proliferative capabilities of these cells in the absence of exogenous serine, suggesting that dysregulation of one carbon metabolism is a molecular feature in the biology of Sengers syndrome.
  76. Mol Cancer Res. 2021 Jan 20. pii: molcanres.0926.2020. [Epub ahead of print]
      The β-linked N-acetyl-D-glucosamine (GlcNAc) is a post-translational modification of serine and threonine residues catalyzed by the enzyme O-GlcNAc transferase (OGT). Increased OGT expression is a feature of most human cancers and inhibition of OGT decreases cancer cell proliferation. Anti-proliferative effects are attributed to post-translational modifications of known regulators of cancer cell proliferation, such as MYC, FOXM1 and EZH2. In general, OGT amplifies cell-specific phenotype, for example, OGT overexpression enhances reprogramming efficiency of mouse embryonic fibroblasts into stem cells. Genome-wide screens suggest that certain cancers are particularly dependent on OGT, and understanding these addictions is important when considering OGT as a target for cancer therapy. The O-GlcNAc modification is involved in most cellular processes, which raises concerns of on-target undesirable effects of OGT targeting therapy. Yet, emerging evidence suggest that, much like proteasome inhibitors, specific compounds targeting OGT elicit selective anti-proliferative effects in cancer cells, and can prime malignant cells to other treatments. It is therefore essential to gain mechanistic insights on substrate specificity for OGT, develop reagents to more specifically enrich for O-GlcNAc modified proteins, identify O-GlcNAc 'readers' and develop OGT small molecule inhibitors. Here, we review the relevance of OGT in cancer progression and the potential targeting of this metabolic enzyme as a putative oncogene. Contrasting the functions of any candidate oncogene between normal and cancer cells is rarely done, but only by understanding the normal functions of a given factor, it is possible to understand these functions gone awry. Here we review oncogenic functions of OGT.