bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2020‒06‒21
fifty-five papers selected by
Kıvanç Görgülü
Technical University of Munich


  1. Cancer Cell. 2020 Jun 02. pii: S1535-6108(20)30260-9. [Epub ahead of print]
    Escobar-Hoyos LF, Penson A, Kannan R, Cho H, Pan CH, Singh RK, Apken LH, Hobbs GA, Luo R, Lecomte N, Babu S, Pan FC, Alonso-Curbelo D, Morris JP, Askan G, Grbovic-Huezo O, Ogrodowski P, Bermeo J, Saglimbeni J, Cruz CD, Ho YJ, Lawrence SA, Melchor JP, Goda GA, Bai K, Pastore A, Hogg SJ, Raghavan S, Bailey P, Chang DK, Biankin A, Shroyer KR, Wolpin BM, Aguirre AJ, Ventura A, Taylor B, Der CJ, Dominguez D, Kümmel D, Oeckinghaus A, Lowe SW, Bradley RK, Abdel-Wahab O, Leach SD.
      Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.
    Keywords:  GAP17; GTPase signaling; KRAS; RNA splicing; SF3B1; hnRNPK; oncogenes; p53; pancreatic cancer; splicing inhibitors
    DOI:  https://doi.org/10.1016/j.ccell.2020.05.010
  2. Cell Rep. 2020 Jun 16. pii: S2211-1247(20)30744-0. [Epub ahead of print]31(11): 107764
    Ozkan-Dagliyan I, Diehl JN, George SD, Schaefer A, Papke B, Klotz-Noack K, Waters AM, Goodwin CM, Gautam P, Pierobon M, Peng S, Gilbert TSK, Lin KH, Dagliyan O, Wennerberg K, Petricoin EF, Tran NL, Bhagwat SV, Tiu RV, Peng SB, Herring LE, Graves LM, Sers C, Wood KC, Cox AD, Der CJ.
      We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.
    Keywords:  ERK; FOSL1; KRAS; MYC; RAF; mesenchymal-to-epithelial transition; pancreatic cancer
    DOI:  https://doi.org/10.1016/j.celrep.2020.107764
  3. Cancer Res. 2020 Jun 17. pii: canres.3763.2019. [Epub ahead of print]
    Zhao Y, Wu Z, Chanal M, Guillaumond F, Goehrig D, Bachy S, Principe M, Ziverec A, Flaman JM, Collin G, Tomasini R, Pasternack A, Ritvos O, Vasseur S, Bernard D, Hennino A, Bertolino P.
      Pancreatic ductal adenocarcinoma (PDA) is a deadly and aggressive cancer. Understanding mechanisms that drive pre-neoplastic pancreatic lesions is necessary to improve early diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase (ALK4) have been demonstrated to favor PDA onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data mining and histological analyses performed on human and mouse tumor tissues reveal that Activin A is the major ALK4-ligand that drives PDA initiation. Activin A, which is absent in normal acinar-cells, was strongly induced during acinar-to-duct metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that was induced in precursor lesions. Blocking Activin A-signaling through the use of a soluble form of Activin receptor IIB (sActRIIB-Fc) and ALK4-knockout (KO) in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that Activin A acts as a protective senescence-associated secretory phenotype (SASP) factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-19-3763
  4. Nat Microbiol. 2020 Jun 15.
    Vallejo-Gracia A, Chen IP, Perrone R, Besnard E, Boehm D, Battivelli E, Tezil T, Krey K, Raymond KA, Hull PA, Walter M, Habrylo I, Cruz A, Deeks S, Pillai S, Verdin E, Ott M.
      Quiescence is a hallmark of CD4+ T cells latently infected with human immunodeficiency virus 1 (HIV-1). While reversing this quiescence is an effective approach to reactivate latent HIV from T cells in culture, it can cause deleterious cytokine dysregulation in patients. As a key regulator of T-cell quiescence, FOXO1 promotes latency and suppresses productive HIV infection. We report that, in resting T cells, FOXO1 inhibition impaired autophagy and induced endoplasmic reticulum (ER) stress, thereby activating two associated transcription factors: activating transcription factor 4 (ATF4) and nuclear factor of activated T cells (NFAT). Both factors associate with HIV chromatin and are necessary for HIV reactivation. Indeed, inhibition of protein kinase R-like ER kinase, an ER stress sensor that can mediate the induction of ATF4, and calcineurin, a calcium-dependent regulator of NFAT, synergistically suppressed HIV reactivation induced by FOXO1 inhibition. Thus, our studies uncover a link of FOXO1, ER stress and HIV infection that could be therapeutically exploited to selectively reverse T-cell quiescence and reduce the size of the latent viral reservoir.
    DOI:  https://doi.org/10.1038/s41564-020-0742-9
  5. MethodsX. 2020 ;7 100938
    Monteiro LB, Davanzo GG, de Aguiar CF, Moraes-Vieira PMM.
      The understanding of how different cell types adapt their metabolism in the face of challenges has been attracting the attention of researchers for many years. Recently, immunologists also started to focus on how the metabolism of immune cells can impact the way that immunity drives its responses. The presence of a pathogen or damage in a tissue changes severely the way that the immune cells need to respond. When activated, immune cells usually shift their metabolism from a high energy demanding status using mitochondria respiration to a glycolytic based rapid ATP production. The diminished amount of respiration leads to changes in the mitochondrial membrane potential and, consequently, generation of reactive oxygen species. Here, we show how flow cytometry can be used to track changes in mitochondrial mass, membrane potential and superoxide (ROS) production in live immune cells. ● This protocol suggests a quick way of evaluating mitochondrial fitness using flow cytometry. We propose using the probes MitoTraker Green and MitoTracker Red/ MitoSOX at the same time. This way, it is possible to evaluate different parameters of mitochondrial biology in living cells. ● Flow cytometry is a highly used tool by immunologists. With the advances of studies focusing on the metabolism of immune cells, a simplified application of flow cytometry for mitochondrial studies and screenings is a helpful clarifying method for immunology.
    Keywords:  Immunometabolism; Metabolic reprogramming; Mitochondrial function; Mitochondrial membrane potential
    DOI:  https://doi.org/10.1016/j.mex.2020.100938
  6. Dev Cell. 2020 Jun 02. pii: S1534-5807(20)30406-8. [Epub ahead of print]
    Jun H, Ma Y, Chen Y, Gong J, Liu S, Wang J, Knights AJ, Qiao X, Emont MP, Xu XZS, Kajimura S, Wu J.
      Maintaining energy homeostasis upon environmental challenges, such as cold or excess calorie intake, is essential to the fitness and survival of mammals. Drug discovery efforts targeting β-adrenergic signaling have not been fruitful after decades of intensive research. We recently identified a new beige fat regulatory pathway mediated via the nicotinic acetylcholine receptor subunit CHRNA2. Here, we generated fat-specific Chrna2 KO mice and observed thermogenic defects in cold and metabolic dysfunction upon dietary challenges caused by adipocyte-autonomous regulation in vivo. We found that CHRNA2 signaling is activated after acute high fat diet feeding and this effect is manifested through both UCP1- and creatine-mediated mechanisms. Furthermore, our data suggested that CHRNA2 signaling may activate glycolytic beige fat, a subpopulation of beige adipocytes mediated by GABPα emerging in the absence of β-adrenergic signaling. These findings reveal the biological significance of the CHRNA2 pathway in beige fat biogenesis and energy homeostasis.
    Keywords:  CHRNA2; beige fat; diet-induced thermogenesis; g-beige fat
    DOI:  https://doi.org/10.1016/j.devcel.2020.05.017
  7. Nature. 2020 Jun 17.
    Amor C, Feucht J, Leibold J, Ho YJ, Zhu C, Alonso-Curbelo D, Mansilla-Soto J, Boyer JA, Li X, Giavridis T, Kulick A, Houlihan S, Peerschke E, Friedman SL, Ponomarev V, Piersigilli A, Sadelain M, Lowe SW.
      Cellular senescence is characterized by stable cell-cycle arrest and a secretory program that modulates the tissue microenvironment1,2. Physiologically, senescence serves as a tumour-suppressive mechanism that prevents the expansion of premalignant cells3,4 and has a beneficial role in wound-healing responses5,6. Pathologically, the aberrant accumulation of senescent cells generates an inflammatory milieu that leads to chronic tissue damage and contributes to diseases such as liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis1,7. Accordingly, eliminating senescent cells from damaged tissues in mice ameliorates the symptoms of these pathologies and even promotes longevity1,2,8-10. Here we test the therapeutic concept that chimeric antigen receptor (CAR) T cells that target senescent cells can be effective senolytic agents. We identify the urokinase-type plasminogen activator receptor (uPAR)11 as a cell-surface protein that is broadly induced during senescence and show that uPAR-specific CAR T cells efficiently ablate senescent cells in vitro and in vivo. CAR T cells that target uPAR extend the survival of mice with lung adenocarcinoma that are treated with a senescence-inducing combination of drugs, and restore tissue homeostasis in mice in which liver fibrosis is induced chemically or by diet. These results establish the therapeutic potential of senolytic CAR T cells for senescence-associated diseases.
    DOI:  https://doi.org/10.1038/s41586-020-2403-9
  8. Cancer Discov. 2020 Jun 16. pii: CD-19-1536. [Epub ahead of print]
    Han T, Goswami S, Hu Y, Tang F, Zafra MP, Murphy C, Cao Z, Poirier JT, Khurana E, Elemento O, Hechtman JF, Ganesh K, Yaeger R, Dow LE.
      The WNT pathway is a fundamental regulator of intestinal homeostasis and hyperactivation of WNT signaling is the major oncogenic driver in colorectal cancer (CRC). To date, there are no described mechanisms that bypass WNT dependence in intestinal tumors. Here, we show that while WNT suppression blocks tumor growth in most organoid and in vivo CRC models, the accumulation of CRC-associated genetic alterations enables drug resistance and WNT-independent growth. In intestinal epithelial cells harboring mutations in KRAS or BRAF, together with disruption of p53 and SMAD4, transient TGFB exposure drives YAP/TAZ-dependent transcriptional reprogramming and lineage reversion. Acquisition of embryonic intestinal identity is accompanied by a permanent loss of adult intestinal lineages, and long-term WNT-independent growth. This work identifies genetic and microenvironmental factors that drive WNT inhibitor resistance, defines a new mechanism for WNT-independent CRC growth and reveals how integration of associated genetic alterations and extracellular signals can overcome lineage-dependent oncogenic programs.
    DOI:  https://doi.org/10.1158/2159-8290.CD-19-1536
  9. Nat Struct Mol Biol. 2020 Jun 15.
    Wu T, Yoon H, Xiong Y, Dixon-Clarke SE, Nowak RP, Fischer ES.
      Controlled perturbation of protein activity is essential to study protein function in cells and living organisms. Small molecules that hijack the cellular protein ubiquitination machinery to selectively degrade proteins of interest, so-called degraders, have recently emerged as alternatives to selective chemical inhibitors, both as therapeutic modalities and as powerful research tools. These systems offer unprecedented temporal and spatial control over protein function. Here, we review recent developments in this field, with a particular focus on the use of degraders as research tools to interrogate complex biological problems.
    DOI:  https://doi.org/10.1038/s41594-020-0438-0
  10. Nat Cell Biol. 2020 Jun 15.
    Hsu JW, Bai M, Li K, Yang JS, Chu N, Cole PA, Eck MJ, Li J, Hsu VW.
      Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1-3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4-6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event.
    DOI:  https://doi.org/10.1038/s41556-020-0530-z
  11. Nat Commun. 2020 Jun 15. 11(1): 3018
    Perkail S, Andricovich J, Kai Y, Tzatsos A.
      Chronic pancreatitis represents a risk factor for the development of pancreatic cancer. We find that heterozygous loss of histone H2A lysine 119 deubiquitinase BAP1 (BRCA1 Associated Protein-1) associates with a history of chronic pancreatitis and occurs in 25% of pancreatic ductal adenocarcinomas and 40% of acinar cell carcinomas. Deletion or heterozygous loss of Bap1 in murine pancreata causes genomic instability, tissue damage, and pancreatitis with full penetrance. Concomitant expression of KrasG12D leads to predominantly intraductal papillary mucinous neoplasms and mucinous cystic neoplasms, while pancreatic intraepithelial neoplasias are rarely detected. These lesions progress to metastatic pancreatic cancer with high frequency. Lesions with histological features mimicking Acinar Cell Carcinomas are also observed in some tumors. Heterozygous mice also develop pancreatic cancer suggesting a haploinsufficient tumor suppressor role for BAP1. Mechanistically, BAP1 regulates genomic stability, in a catalytic independent manner, and its loss confers sensitivity to irradiation and platinum-based chemotherapy in pancreatic cancer.
    DOI:  https://doi.org/10.1038/s41467-020-16589-8
  12. Proc Natl Acad Sci U S A. 2020 Jun 15. pii: 202003346. [Epub ahead of print]
    Hoermayer L, Montesinos JC, Marhava P, Benková E, Yoshida S, Friml J.
      Wound healing in plant tissues, consisting of rigid cell wall-encapsulated cells, represents a considerable challenge and occurs through largely unknown mechanisms distinct from those in animals. Owing to their inability to migrate, plant cells rely on targeted cell division and expansion to regenerate wounds. Strict coordination of these wound-induced responses is essential to ensure efficient, spatially restricted wound healing. Single-cell tracking by live imaging allowed us to gain mechanistic insight into the wound perception and coordination of wound responses after laser-based wounding in Arabidopsis root. We revealed a crucial contribution of the collapse of damaged cells in wound perception and detected an auxin increase specific to cells immediately adjacent to the wound. This localized auxin increase balances wound-induced cell expansion and restorative division rates in a dose-dependent manner, leading to tumorous overproliferation when the canonical TIR1 auxin signaling is disrupted. Auxin and wound-induced turgor pressure changes together also spatially define the activation of key components of regeneration, such as the transcription regulator ERF115. Our observations suggest that the wound signaling involves the sensing of collapse of damaged cells and a local auxin signaling activation to coordinate the downstream transcriptional responses in the immediate wound vicinity.
    Keywords:  ERF115; auxin; cell division; cell expansion; regeneration
    DOI:  https://doi.org/10.1073/pnas.2003346117
  13. J Biol Chem. 2020 Jun 18. pii: jbc.RA120.012649. [Epub ahead of print]
    Shimizu T, Nakamura T, Inaba H, Iwasa H, Maruyama J, Arimoto-Matsuzaki K, Nakata T, Nishina H, Hata Y.
      The gene encoding the proto-oncogene GTPase RAS is frequently mutated in human cancers. Mutated RAS proteins trigger antiapoptotic and cell proliferative signals and lead to oncogenesis. However, RAS also induces apoptosis and senescence, which may contribute to the eradication of cells with RAS mutations. We previously reported that Ras association domain family member 6 (RASSF6) binds MDM2 proto-oncogene (MDM2) and stabilizes the tumor suppressor p53 and that the active form of KRAS promotes the interaction between RASSF6 and MDM2. We also reported that Unc-119 lipid-binding chaperone (UNC119A), a chaperone of myristoylated proteins, interacts with RASSF6 and regulates RASSF6-mediated apoptosis. In this study, using several human cancer cell lines, quantitative RT-PCR, RNAi-based gene silencing, and immunoprecipitation/-fluorescence and cell biology assays, we report that UNC119 interacts with the active form of KRAS and that the C-terminal modification of KRAS is required for this interaction. We also noted that the hydrophobic pocket of UNC119, which binds the myristoylated peptides, is not involved in the interaction. We observed that UNC119 promotes the binding of KRAS to RASSF6, enhances the interaction between RASSF6 and MDM2, and induces apoptosis. Conversely, UNC119 silencing promoted soft agar colony formation, migration, and invasiveness in KRAS-mutated cancer cells. We conclude that UNC119 promotes KRAS-mediated p53-dependent apoptosis via RASSF6 and may play a tumor-suppressive role in cells with KRAS mutations.
    Keywords:  GTPase; Human retina gene 4 (HRG4); KRAS; Ras association domain family member 6 (RASSF6); apoptosis; cancer; cell proliferation; cell signaling; p53; tumor suppressor gene
    DOI:  https://doi.org/10.1074/jbc.RA120.012649
  14. Aging Cell. 2020 Jun 14. e13172
    Zhao X, Li X, Shi X, Karpac J.
      The number, size, and composition of lipid droplets can be influenced by dietary changes that shift energy substrate availability. This diversification of lipid droplets can promote metabolic flexibility and shape cellular stress responses in unique tissues with distinctive metabolic roles. Using Drosophila, we uncovered a role for myocyte enhancer factor 2 (MEF2) in modulating diet-dependent lipid droplet diversification within adult striated muscle, impacting mortality rates. Muscle-specific attenuation of MEF2, whose chronic activation maintains glucose and mitochondrial homeostasis, leads to the accumulation of large, cholesterol ester-enriched intramuscular lipid droplets in response to high calorie, carbohydrate-sufficient diets. The diet-dependent accumulation of these lipid droplets also correlates with both enhanced stress protection in muscle and increases in organismal lifespan. Furthermore, MEF2 attenuation releases an antagonistic regulation of cell cycle gene expression programs, and up-regulation of Cyclin E is required for diet- and MEF2-dependent diversification of intramuscular lipid droplets. The integration of MEF2-regulated gene expression networks with dietary responses thus plays a critical role in shaping muscle metabolism and function, further influencing organismal lifespan. Together, these results highlight a potential protective role for intramuscular lipid droplets during dietary adaptation.
    Keywords:   Drosophila ; MEF2; carbohydrate; cyclin E; dietary adaptation; glucose; high calorie diet; lactate; lifespan; lipid droplet; lipid metabolism; muscle; survival
    DOI:  https://doi.org/10.1111/acel.13172
  15. Science. 2020 Jun 19. 368(6497): 1386-1392
    Klein IA, Boija A, Afeyan LK, Hawken SW, Fan M, Dall'Agnese A, Oksuz O, Henninger JE, Shrinivas K, Sabari BR, Sagi I, Clark VE, Platt JM, Kar M, McCall PM, Zamudio AV, Manteiga JC, Coffey EL, Li CH, Hannett NM, Guo YE, Decker TM, Lee TI, Zhang T, Weng JK, Taatjes DJ, Chakraborty A, Sharp PA, Chang YT, Hyman AA, Gray NS, Young RA.
      The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.
    DOI:  https://doi.org/10.1126/science.aaz4427
  16. Cell Metab. 2020 Jun 09. pii: S1550-4131(20)30303-X. [Epub ahead of print]
    Cheng X, Geng F, Pan M, Wu X, Zhong Y, Wang C, Tian Z, Cheng C, Zhang R, Puduvalli V, Horbinski C, Mo X, Han X, Chakravarti A, Guo D.
      Glioblastoma (GBM), a mostly lethal brain tumor, acquires large amounts of free fatty acids (FAs) to promote cell growth. But how the cancer avoids lipotoxicity is unknown. Here, we identify that GBM upregulates diacylglycerol-acyltransferase 1 (DGAT1) to store excess FAs into triglycerides and lipid droplets. Inhibiting DGAT1 disrupted lipid homeostasis and resulted in excessive FAs moving into mitochondria for oxidation, leading to the generation of high levels of reactive oxygen species (ROS), mitochondrial damage, cytochrome c release, and apoptosis. Adding N-acetyl-cysteine or inhibiting FA shuttling into mitochondria decreased ROS and cell death induced by DGAT1 inhibition. We show in xenograft models that targeting DGAT1 blocked lipid droplet formation, induced tumor cell apoptosis, and markedly suppressed GBM growth. Together, our study demonstrates that DGAT1 upregulation protects GBM from oxidative damage and maintains lipid homeostasis by facilitating storage of excess FAs. Targeting DGAT1 could be a promising therapeutic approach for GBM.
    Keywords:  DGAT1; ROS; acylcarnitine; fatty acids; glioblastoma; lipid droplets; lipotoxicity; mitochondria; oxidative stress; triglycerides
    DOI:  https://doi.org/10.1016/j.cmet.2020.06.002
  17. Cell. 2020 Jun 18. pii: S0092-8674(20)30686-3. [Epub ahead of print]
    Koundouros N, Karali E, Tripp A, Valle A, Inglese P, Perry NJS, Magee DJ, Anjomani Virmouni S, Elder GA, Tyson AL, Dória ML, van Weverwijk A, Soares RF, Isacke CM, Nicholson JK, Glen RC, Takats Z, Poulogiannis G.
      Oncogenic transformation is associated with profound changes in cellular metabolism, but whether tracking these can improve disease stratification or influence therapy decision-making is largely unknown. Using the iKnife to sample the aerosol of cauterized specimens, we demonstrate a new mode of real-time diagnosis, coupling metabolic phenotype to mutant PIK3CA genotype. Oncogenic PIK3CA results in an increase in arachidonic acid and a concomitant overproduction of eicosanoids, acting to promote cell proliferation beyond a cell-autonomous manner. Mechanistically, mutant PIK3CA drives a multimodal signaling network involving mTORC2-PKCζ-mediated activation of the calcium-dependent phospholipase A2 (cPLA2). Notably, inhibiting cPLA2 synergizes with fatty acid-free diet to restore immunogenicity and selectively reduce mutant PIK3CA-induced tumorigenicity. Besides highlighting the potential for metabolic phenotyping in stratified medicine, this study reveals an important role for activated PI3K signaling in regulating arachidonic acid metabolism, uncovering a targetable metabolic vulnerability that largely depends on dietary fat restriction.
    Keywords:  PIK3CA; PKCζ; arachidonic acid; cPLA2; cancer metabolism; diet; eicosanoids; fat restriction; iKnife; mTORC2
    DOI:  https://doi.org/10.1016/j.cell.2020.05.053
  18. J Cell Biol. 2020 Jul 06. pii: e201908087. [Epub ahead of print]219(7):
    Tang D, Sandoval W, Lam C, Haley B, Liu P, Xue D, Roy D, Patapoff T, Louie S, Snedecor B, Misaghi S.
      Accumulation of unfolded antibody chains in the ER triggers ER stress that may lead to reduced productivity in therapeutic antibody manufacturing processes. We identified UBR4 and UBR5 as ubiquitin E3 ligases involved in HC ER-associated degradation. Knockdown of UBR4 and UBR5 resulted in intracellular accumulation, enhanced secretion, and reduced ubiquitination of HC. In concert with these E3 ligases, PDIA3 was shown to cleave ubiquitinated HC molecules to accelerate HC dislocation. Interestingly, UBR5, and to a lesser degree UBR4, were down-regulated as cellular demand for antibody expression increased in CHO cells during the production phase, or in plasma B cells. Reducing UBR4/UBR5 expression before the production phase increased antibody productivity in CHO cells, possibly by redirecting antibody molecules from degradation to secretion. Altogether we have characterized a novel proteolysis/proteasome-dependent pathway involved in degradation of unfolded antibody HC. Proteins characterized in this pathway may be novel targets for CHO cell engineering.
    DOI:  https://doi.org/10.1083/jcb.201908087
  19. Cell Rep. 2020 Jun 16. pii: S2211-1247(20)30745-2. [Epub ahead of print]31(11): 107765
    Li X, Karras P, Torres R, Rambow F, van den Oord J, Marine JC, Kos L.
      Tumor cell plasticity, including transdifferentiation, is thought to be a key driver of therapy failure, tumor dormancy, and metastatic dissemination. Although melanoma cells have been shown to adopt various phenotypic features in vitro, direct in vivo evidence of metastatic cell plasticity remains sparse. Here, we combine lineage tracing in a spontaneous metastatic mouse model of melanoma, advanced imaging, and single-cell RNA sequencing approaches to search for pathophysiologically relevant melanoma cellular states. We identify melanoma cells in intravascular niches of various metastatic organs. These cells are quiescent, are negative for characteristic melanoma markers, and acquire endothelial cell features. We replicate the endothelial transdifferentiation (EndT) finding in another mouse model and provide evidence of EndT in BRAFV600E-metastatic biopsies from human lung, brain, and small intestine, thus highlighting the clinical relevance of these findings. The tumor-vasculature pattern described herein may contribute to melanoma dormancy within metastatic organs and represent a putative target for therapies.
    Keywords:  EndMT; dormancy; dormant niche; endothelial-mesenchymal transition; melanoma metastasis; melanoma mouse model; tumor vasculature; tumor-derived endothelial cells
    DOI:  https://doi.org/10.1016/j.celrep.2020.107765
  20. Cell Stress. 2020 May 11. 4(6): 114-146
    Grasso D, Zampieri LX, Capelôa T, Van de Velde JA, Sonveaux P.
      The rediscovery and reinterpretation of the Warburg effect in the year 2000 occulted for almost a decade the key functions exerted by mitochondria in cancer cells. Until recent times, the scientific community indeed focused on constitutive glycolysis as a hallmark of cancer cells, which it is not, largely ignoring the contribution of mitochondria to the malignancy of oxidative and glycolytic cancer cells, being Warburgian or merely adapted to hypoxia. In this review, we highlight that mitochondria are not only powerhouses in some cancer cells, but also dynamic regulators of life, death, proliferation, motion and stemness in other types of cancer cells. Similar to the cells that host them, mitochondria are capable to adapt to tumoral conditions, and probably to evolve to 'oncogenic mitochondria' capable of transferring malignant capacities to recipient cells. In the wider quest of metabolic modulators of cancer, treatments have already been identified targeting mitochondria in cancer cells, but the field is still in infancy.
    Keywords:  apoptosis; mitochondrial biogenesis; mitophagy; oxidative phosphorylation (OXPHOS); reactive oxygen species (ROS); tricarboxylic acid (TCA) cycle; tumor metabolism
    DOI:  https://doi.org/10.15698/cst2020.06.221
  21. Cancer Res. 2020 Jun 19. pii: canres.3685.2019. [Epub ahead of print]
    Knochelmann HM, Dwyer CJ, Smith AS, Bowers JS, Wyatt MM, Nelson MH, Rangel Rivera GO, Horton JD, Krieg C, Armeson K, Lesinski GB, Rubinstein MP, Li Z, Paulos CM.
      The accessibility of adoptive T cell transfer therapies (ACT) is hindered by cost and time required for product development. Here we describe a streamlined ACT protocol using Th17 cells expanded only four days ex vivo. While shortening expansion compromised cell yield, this method licensed Th17 cells to eradicate large tumors to a greater extent than cells expanded longer term. Day 4 Th17 cells engrafted, induced release of multiple cytokines including IL-6, IL-17, MCP-1, and GM-CSF in the tumor-bearing host, and persisted as memory cells. IL-6 was a critical component for efficacy of these therapies via its promotion of long-term immunity and resistance to tumor relapse. Mechanistically, IL-6 diminished engraftment of FoxP3+ donor T cells, corresponding with robust tumor infiltration by donor effector over regulatory cells for the Day 4 Th17 cell product relative to cell products expanded longer durations ex vivo. Collectively, this work describes a method to rapidly generate therapeutic T cell products for ACT and implicates IL-6 in promoting durable immunity of Th17 cells against large, established solid tumors.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-19-3685
  22. Nat Commun. 2020 Jun 19. 11(1): 3148
    Son SM, Park SJ, Stamatakou E, Vicinanza M, Menzies FM, Rubinsztein DC.
      Macroautophagy ("autophagy") is the main lysosomal catabolic process that becomes activated under nutrient-depleted conditions, like amino acid (AA) starvation. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-conserved negative regulator of autophagy. While leucine (Leu) is a critical mTORC1 regulator under AA-starved conditions, how Leu regulates autophagy is poorly understood. Here, we describe that in most cell types, including neurons, Leu negatively regulates autophagosome biogenesis via its metabolite, acetyl-coenzyme A (AcCoA). AcCoA inhibits autophagy by enhancing EP300-dependent acetylation of the mTORC1 component raptor, with consequent activation of mTORC1. Interestingly, in Leu deprivation conditions, the dominant effects on autophagy are mediated by decreased raptor acetylation causing mTORC1 inhibition, rather than by altered acetylation of other autophagy regulators. Thus, in most cell types we examined, Leu regulates autophagy via the impact of its metabolite AcCoA on mTORC1, suggesting that AcCoA and EP300 play pivotal roles in cell anabolism and catabolism.
    DOI:  https://doi.org/10.1038/s41467-020-16886-2
  23. Mol Cell. 2020 Jun 18. pii: S1097-2765(20)30259-8. [Epub ahead of print]78(6): 1055-1069
    Marchi S, Giorgi C, Galluzzi L, Pinton P.
      Ca2+ ions are key second messengers in both excitable and non-excitable cells. Owing to the rather pleiotropic nature of Ca2+ transporters and other Ca2+-binding proteins, however, Ca2+ signaling has attracted limited attention as a potential target of anticancer therapy. Here, we discuss cancer-associated alterations of Ca2+ fluxes at specific organelles as we identify novel candidates for the development of drugs that selectively target Ca2+ signaling in malignant cells.
    Keywords:  IP(3)R; MCU complex; ORAI; TRP channels; mitochondrial permeability transition; store-operated calcium entry
    DOI:  https://doi.org/10.1016/j.molcel.2020.04.017
  24. Neuron. 2020 Jun 17. pii: S0896-6273(20)30400-1. [Epub ahead of print]106(6): 899-911
    Cho UH, Hetzer MW.
      In recent years, the nuclear pore complex (NPC) has emerged as a key player in genome regulation and cellular homeostasis. New discoveries have revealed that the NPC has multiple cellular functions besides mediating the molecular exchange between the nucleus and the cytoplasm. In this review, we discuss non-transport aspects of the NPC focusing on the NPC-genome interaction, the extreme longevity of the NPC proteins, and NPC dysfunction in age-related diseases. The examples summarized herein demonstrate that the NPC, which first evolved to enable the biochemical communication between the nucleus and the cytoplasm, now doubles as the gatekeeper of cellular identity and aging.
    DOI:  https://doi.org/10.1016/j.neuron.2020.05.031
  25. Cell Stress. 2020 May 14. 4(6): 147-150
    Chen Z, Berquez M, Luciani A.
      Dysregulation of the mitochondrial network in terminally differentiated cells contributes to a broad spectrum of disorders. Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of intermediary metabolism caused by the deficiency of methylmalonyl-CoA mutase (MMUT) - a mitochondrial enzyme that mediates the degradation of certain amino acids and lipids. The loss of MMUT activity triggers an accumulation of toxic endogenous metabolites causing severe organ dysfunctions and life-threatening complications. How MMUT deficiency instigates mitochondrial distress and tissue damage remains poorly understood. Using cell and animal-based models, we recently discovered that MMUT deficiency disables the PINK1-induced translocation of PRKN/Parkin to MMA-damaged mitochondria, impeding their delivery and subsequent dismantling by macroautophagy/autophagy-lysosome degradation systems (Luciani et al. Nat Commun. 11(1):970). This promotes an accumulation of damaged and/or dysfunctional mitochondria that spark epithelial distress and tissue damage. Using a systems biology approach based on drug-disease network perturbation modeling, we predicted targetable pathways, whose modulation repairs mitochondrial dysfunctions in patient-derived kidney cells and ameliorates disease-relevant phenotypes in mmut-deficient zebrafish. These results unveil a link between primary MMUT deficiency, defective mitophagy, and cell distress, offering promising therapeutic avenues for MMA and other mitochondria-related diseases.
    Keywords:  cell damage; inherited metabolic disorders; kidney tubule; metabolism; mitochondria; mitophagy; organelle quality control; oxidative stress
    DOI:  https://doi.org/10.15698/cst2020.06.222
  26. Proc Natl Acad Sci U S A. 2020 Jun 17. pii: 201916550. [Epub ahead of print]
    Jiang M, Chavarria TE, Yuan B, Lodish HF, Huang NJ.
      Phosphocholine phosphatase-1 (PHOSPHO1) is a phosphocholine phosphatase that catalyzes the hydrolysis of phosphocholine (PC) to choline. Here we demonstrate that the PHOSPHO1 transcript is highly enriched in mature brown adipose tissue (BAT) and is further induced by cold and isoproterenol treatments of BAT and primary brown adipocytes. In defining the functional relevance of PHOPSPHO1 in BAT thermogenesis and energy metabolism, we show that PHOSPHO1 knockout mice are cold-tolerant, with higher expression of thermogenic genes in BAT, and are protected from high-fat diet-induced obesity and development of insulin resistance. Treatment of mice with the PHOSPHO1 substrate phosphocholine is sufficient to induce cold tolerance, thermogenic gene expression, and allied metabolic benefits. Our results reveal a role of PHOSPHO1 as a negative regulator of BAT thermogenesis, and inhibition of PHOSPHO1 or enhancement of phosphocholine represent innovative approaches to manage the metabolic syndrome.
    Keywords:  PHOSPHO1; adipose; phosphocholine; thermogenesis
    DOI:  https://doi.org/10.1073/pnas.1916550117
  27. Mol Cell. 2020 Jun 18. pii: S1097-2765(20)30355-5. [Epub ahead of print]78(6): 1019-1033
    Bader JE, Voss K, Rathmell JC.
      The growing field of immune metabolism has revealed promising indications for metabolic targets to modulate anti-cancer immunity. Combination therapies involving metabolic inhibitors with immune checkpoint blockade (ICB), chemotherapy, radiation, and/or diet now offer new approaches for cancer therapy. However, it remains uncertain how to best utilize these strategies in the context of the complex tumor microenvironment (TME). Oncogene-driven changes in tumor cell metabolism can impact the TME to limit immune responses and present barriers to cancer therapy. These changes also reveal opportunities to reshape the TME by targeting metabolic pathways to favor immunity. Here we explore current strategies that shift immune cell metabolism to pro-inflammatory states in the TME and highlight a need to better replicate physiologic conditions to select targets, clarify mechanisms, and optimize metabolic inhibitors. Unifying our understanding of these pathways and interactions within the heterogenous TME will be instrumental to advance this promising field and enhance immunotherapy.
    DOI:  https://doi.org/10.1016/j.molcel.2020.05.034
  28. J Exp Med. 2020 Sep 07. pii: e20192080. [Epub ahead of print]217(9):
    Mahuron KM, Moreau JM, Glasgow JE, Boda DP, Pauli ML, Gouirand V, Panjabi L, Grewal R, Luber JM, Mathur AN, Feldman RM, Shifrut E, Mehta P, Lowe MM, Alvarado MD, Marson A, Singer M, Wells J, Jupp R, Daud AI, Rosenblum MD.
      Tumor-infiltrating CD8+ T cells mediate antitumor immune responses. However, the mechanisms by which T cells remain poised to kill cancer cells despite expressing high levels of inhibitory receptors are unknown. Here, we report that layilin, a C-type lectin domain-containing membrane glycoprotein, is selectively expressed on highly activated, clonally expanded, but phenotypically exhausted CD8+ T cells in human melanoma. Lineage-specific deletion of layilin on murine CD8+ T cells reduced their accumulation in tumors and increased tumor growth in vivo. Congruently, gene editing of LAYN in human CD8+ T cells reduced direct tumor cell killing ex vivo. On a molecular level, layilin colocalized with integrin αLβ2 (LFA-1) on T cells, and cross-linking layilin promoted the activated state of this integrin. Accordingly, LAYN deletion resulted in attenuated LFA-1-dependent cellular adhesion. Collectively, our results identify layilin as part of a molecular pathway in which exhausted or "dysfunctional" CD8+ T cells enhance cellular adhesiveness to maintain their cytotoxic potential.
    DOI:  https://doi.org/10.1084/jem.20192080
  29. Aging (Albany NY). 2020 Jun 13. 12
    Grosse L, Bulavin DV.
      Data obtained from genetically modified mouse models suggest a detrimental role for p16High senescent cells in physiological aging and age-related pathologies. Our recent analysis of aging mice revealed a continuous and noticeable accumulation of liver sinusoid endothelial cells (LSECs) expressing numerous senescence markers, including p16. At early stage, senescent LSECs show an enhanced ability to clear macromolecular waste and toxins including oxidized LDL (oxLDL). Later in life, however, the efficiency of this important detoxifying function rapidly declines potentially due to increased endothelial thickness and senescence-induced silencing of scavenger receptors and endocytosis genes. This inability to detoxify toxins and macromolecular waste, which can be further exacerbated by increased intestinal leakiness with age, might be an important contributing factor to animal death. Here, we propose how LSEC senescence could serve as an endogenous clock that ultimately controls longevity and outline some of the possible approaches to extend the lifespan.
    Keywords:  aging; lifespan; liver sinusoid endothelial cells; senescencee
    DOI:  https://doi.org/10.18632/aging.103492
  30. JCI Insight. 2020 Jun 18. pii: 133668. [Epub ahead of print]5(12):
    Schafer MJ, Zhang X, Kumar A, Atkinson EJ, Zhu Y, Jachim S, Mazula DL, Brown AK, Berning M, Aversa Z, Kotajarvi B, Bruce CJ, Greason KL, Suri RM, Tracy RP, Cummings SR, White TA, LeBrasseur NK.
      Produced by senescent cells, the senescence-associated secretory phenotype (SASP) is a potential driver of age-related dysfunction. We tested whether circulating concentrations of SASP proteins reflect age and medical risk in humans. We first screened senescent endothelial cells, fibroblasts, preadipocytes, epithelial cells, and myoblasts to identify candidates for human profiling. We then tested associations between circulating SASP proteins and clinical data from individuals throughout the life span and older adults undergoing surgery for prevalent but distinct age-related diseases. A community-based sample of people aged 20-90 years (retrospective cross-sectional) was studied to test associations between circulating SASP factors and chronological age. A subset of this cohort aged 60-90 years and separate cohorts of older adults undergoing surgery for severe aortic stenosis (prospective longitudinal) or ovarian cancer (prospective case-control) were studied to assess relationships between circulating concentrations of SASP proteins and biological age (determined by the accumulation of age-related health deficits) and/or postsurgical outcomes. We showed that SASP proteins were positively associated with age, frailty, and adverse postsurgery outcomes. A panel of 7 SASP factors composed of growth differentiation factor 15 (GDF15), TNF receptor superfamily member 6 (FAS), osteopontin (OPN), TNF receptor 1 (TNFR1), ACTIVIN A, chemokine (C-C motif) ligand 3 (CCL3), and IL-15 predicted adverse events markedly better than a single SASP protein or age. Our findings suggest that the circulating SASP may serve as a clinically useful candidate biomarker of age-related health and a powerful tool for interventional human studies.
    Keywords:  Aging; Cellular senescence
    DOI:  https://doi.org/10.1172/jci.insight.133668
  31. Mol Carcinog. 2020 Jun 20.
    Iwanaga R, Truong BT, Hsu JY, Lambert KA, Vyas R, Orlicky D, Shellman YG, Tan AC, Ceol C, Artinger KB.
      Melanoma is an aggressive, deadly skin cancer derived from melanocytes, a neural crest cell derivative. Melanoma cells mirror the developmental program of neural crest cells in that they exhibit the same gene expression patterns and utilize similar cellular mechanisms, including increased cell proliferation, epithelial-mesenchymal transition, and migration. Here we studied the role of neural crest regulator PRDM1 in melanoma onset and progression. In development, Prdm1a functions to promote neural crest progenitor fate, and in melanoma, we found that PRDM1 has reduced copy number and is recurrently deleted in both zebrafish and humans. When examining expression of neural crest and melanocyte development genes, we show that sox10 progenitor expression is high in prdm1a-/- mutants, while more differentiated melanocyte markers are reduced, suggesting that normally Prdm1a is required for differentiation. Data mining of human melanoma datasets indicates that high PRDM1 expression in human melanoma is correlated with better patient survival and decreased PRDM1 expression is common in metastatic tumors. When one copy of prdm1a is lost in the zebrafish melanoma model Tg(mitfa:BRAFV600E );p53-/- ;prdm1a+/- , melanoma onset occurs more quickly, and the tumors that form have a larger area with increased expression of sox10. These data demonstrate a novel role for PRDM1 as a tumor suppressor in melanoma.
    Keywords:  PRDM1; melanoma; neural crest cells; zebrafish
    DOI:  https://doi.org/10.1002/mc.23236
  32. Elife. 2020 Jun 16. pii: e56178. [Epub ahead of print]9
    Farnung L, Ochmann M, Cramer P.
      Chromatin remodelling plays important roles in gene regulation during development, differentiation and in disease. The chromatin remodelling enzyme CHD4 is a component of the NuRD and ChAHP complexes that are involved in gene repression. Here we report the cryo-electron microscopy (cryo-EM) structure of Homo sapiens CHD4 engaged with a nucleosome core particle in the presence of the non-hydrolysable ATP analogue AMP-PNP at an overall resolution of 3.1 Å. The ATPase motor of CHD4 binds and distorts nucleosomal DNA at superhelical location (SHL) +2, supporting the 'twist defect' model of chromatin remodelling. CHD4 does not induce unwrapping of terminal DNA, in contrast to its homologue Chd1, which functions in gene activation. Our structure also maps CHD4 mutations that are associated with human cancer or the intellectual disability disorder Sifrim-Hitz-Weiss syndrome.
    Keywords:  biochemistry; chemical biology; human; molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.56178
  33. Trends Cell Biol. 2020 Jun 16. pii: S0962-8924(20)30101-X. [Epub ahead of print]
    Bhattarai KR, Chaudhary M, Kim HR, Chae HJ.
      Recent work provides evidence for the new terminology, 'endoplasmic reticulum (ER) stress response or sensing failure', in relation to metabolic disease. We seek to identify and amass possible conditions of ER stress response failure in various metabolic and age-related pathogenesis, including obesity and diabetes.
    Keywords:  ER proteostasis; ER stress; ER stress response failure; aging; metabolic diseases; sXBP1
    DOI:  https://doi.org/10.1016/j.tcb.2020.05.004
  34. Oncogene. 2020 Jun 15.
    Wagner V, Gil J.
      Cyclin-dependent kinases 4 and 6 (CDK4/6) phosphorylate and inhibit retinoblastoma (RB) family proteins. Hyperphosphorylated RB releases E2F transcription factors, activating a transcriptional program that initiates S phase. Due to the critical role that this pathway has in regulating cell cycle progression, inhibiting CDK4/6 is an attractive therapeutic strategy. Indeed, CDK4/6 inhibitors in combination with antiestrogens produce a significant benefit in patients with ER+/HER2- breast cancer. Clinical trials are currently investigating if the use of CDK4/6 inhibitors alone or in combination can be extended to other cancer types. Inhibition of CDK4/6 can result in different cell fates such as quiescence, senescence, or apoptosis. Senescence is a stress response that can be induced by stimuli that include oncogenic activation, chemotherapy, irradiation, and targeted therapies such as CDK4/6 inhibitors. Senescent cells undergo a stable cell cycle arrest and produce a bioactive secretome that remodels their microenvironment and engages the immune system. In this review, we analyze the therapeutic relevance of senescence induction by CDK4/6 inhibitors. We also discuss how different therapies, including checkpoint inhibitors and drugs targeting MEK or PI3K, can be used in combination with CDK4/6 inhibitors to reinforce or exploit senescence. Recently, a lot of effort has been put into identifying compounds that selectively kill senescent cells (termed senolytics). Thus, sequential treatment with senolytics might be an additional strategy to potentiate the antitumor effects of CDK4/6 inhibitors.
    DOI:  https://doi.org/10.1038/s41388-020-1354-9
  35. Elife. 2020 Jun 19. pii: e56189. [Epub ahead of print]9
    Shin S, Pang Y, Park J, Liu L, Lukas BE, Kim SH, Kim KW, Xu P, Berry DC, Jiang Y.
      Adipocytes arise from distinct progenitor populations during development and adult, but little is known about how developmental progenitors differ from adult progenitors. Here, we investigate the role of platelet-derived growth factor receptor alpha (PDGFRα) in the divergent regulation of the two different adipose progenitor cells (APCs). Using in vivo adipose lineage tracking and deletion mouse models, we found that developmental PDGFRα+ cells are adipogenic and differentiated into mature adipocytes, and the deletion of Pdgfra in developmental adipose lineage disrupted white adipose tissue (WAT) formation. Interestingly, adult PDGFRα+ cells do not significantly contribute to adult adipogenesis, and deleting Pdgfra in adult adipose lineage did not affect WAT homeostasis. Mechanistically, embryonic APCs require PDGFRα for fate maintenance, and without PDGFRα, they underwent fate change from adipogenic to fibrotic lineage. Collectively, our findings indicate that PDGFRα+ cells and Pdgfra gene itself are differentially required for WAT development and adult WAT homeostasis.
    Keywords:  cell biology; developmental biology; mouse
    DOI:  https://doi.org/10.7554/eLife.56189
  36. Nat Commun. 2020 Jun 19. 11(1): 3123
    Henrichs V, Grycova L, Barinka C, Nahacka Z, Neuzil J, Diez S, Rohlena J, Braun M, Lansky Z.
      Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.
    DOI:  https://doi.org/10.1038/s41467-020-16972-5
  37. Proc Natl Acad Sci U S A. 2020 Jun 15. pii: 201921554. [Epub ahead of print]
    Graves JD, Lee YJ, Liu K, Li G, Lin FT, Lin WC.
      Oxidative stress is a ubiquitous threat to all aerobic organisms and has been implicated in numerous pathological conditions such as cancer. Here we demonstrate a pivotal role for E2F1, a cell cycle regulatory transcription factor, in cell tolerance of oxidative stress. Cells lacking E2F1 are hypersensitive to oxidative stress due to the defects in cell cycle arrest. Oxidative stress inhibits E2F1 transcriptional activity, independent of changes in association with Rb and without decreasing its DNA-binding activity. Upon oxidative insult, SUMO2 is extensively conjugated to E2F1 mainly at lysine 266 residue, which specifically modulates E2F1 transcriptional activity to enhance cell cycle arrest for cell survival. We identify SENP3, a desumoylating enzyme, as an E2F1-interacting partner. Oxidative stress inhibits the interaction between E2F1 and SENP3, which leads to accumulation of sumoylated E2F1. SENP3-deficient cells exhibit hypersumoylation of E2F1 and are resistant to oxidative insult. High levels of SENP3 in breast cancer are associated with elevated levels of E2F targets, high tumor grade, and poor survival. Given the prevalence of elevated levels of SENP3 across numerous cancer types, the SENP3-E2F1 axis may serve as an avenue for therapeutic intervention in cancer.
    Keywords:  E2F1; SENP3; SUMO2; oxidative stress; sumoylation
    DOI:  https://doi.org/10.1073/pnas.1921554117
  38. Dev Cell. 2020 Jun 11. pii: S1534-5807(20)30413-5. [Epub ahead of print]
    Ding BS, Yang D, Swendeman SL, Christoffersen C, Nielsen LB, Friedman SL, Powell CA, Hla T, Cao Z.
      Here, we show that the liver-derived apolipoprotein M (ApoM) protects the lung and kidney from pro-fibrotic insults and that this circulating factor is attenuated in aged mice. Aged mouse hepatocytes exhibit transcriptional suppression of ApoM. This leads to reduced sphingosine-1-phosphate (S1P) signaling via the S1P receptor 1 (S1PR1) in the vascular endothelial cells of lung and kidney. Suboptimal S1PR1 angiocrine signaling causes reduced resistance to injury-induced vascular leak and leads to organ fibrosis. Plasma transfusion from Apom transgenic mice but not Apom knockout mice blocked fibrosis in the lung. Similarly, infusion of recombinant therapeutics, ApoM-Fc fusion protein enhanced kidney and lung regeneration and attenuated fibrosis in aged mouse after injury. Furthermore, we identified that aging alters Sirtuin-1-hepatic nuclear factor 4α circuit in hepatocytes to downregulate ApoM. These data reveal an integrative organ adaptation that involves circulating S1P chaperone ApoM+ high density lipoprotein (HDL), which signals via endothelial niche S1PR1 to spur regeneration over fibrosis.
    Keywords:  aging; endothelial cell; fibrosis; kidney repair; lipoprotein; lung regeneration; sphingosine-1-phosphate receptor; vascular barrier; vascular niche
    DOI:  https://doi.org/10.1016/j.devcel.2020.05.024
  39. EMBO Rep. 2020 Jun 15. e48779
    Li Y, Li M, Weigel B, Mall M, Werth VP, Liu ML.
      The nuclear lamina is essential for the structural integration of the nuclear envelope. Nuclear envelope rupture and chromatin externalization is a hallmark of the formation of neutrophil extracellular traps (NETs). NET release was described as a cellular lysis process; however, this notion has been questioned recently. Here, we report that during NET formation, nuclear lamin B is not fragmented by destructive proteolysis, but rather disassembled into intact full-length molecules. Furthermore, we demonstrate that nuclear translocation of PKCα, which serves as the kinase to induce lamin B phosphorylation and disassembly, results in nuclear envelope rupture. Decreasing lamin B phosphorylation by PKCα inhibition, genetic deletion, or by mutating the PKCα consensus sites on lamin B attenuates extracellular trap formation. In addition, strengthening the nuclear envelope by lamin B overexpression attenuates NET release in vivo and reduces levels of NET-associated inflammatory cytokines in UVB-irradiated skin of lamin B transgenic mice. Our findings advance the mechanistic understanding of NET formation by showing that PKCα-mediated lamin B phosphorylation drives nuclear envelope rupture for chromatin release in neutrophils.
    Keywords:  NET formation; PKCα; lamin B disassembly; nuclear envelope rupture
    DOI:  https://doi.org/10.15252/embr.201948779
  40. Immunol Rev. 2020 Jun 20.
    Anand PK.
      Inflammasomes are multi-protein complexes that regulate the cleavage of cysteine protease caspase-1, secretion of inflammatory cytokines, and induction of inflammatory cell death, pyroptosis. Several members of the nod-like receptor family assemble inflammasome in response to specific ligands. An exception to this is the NLRP3 inflammasome which is activated by structurally diverse entities. Recent studies have suggested that NLRP3 might be a sensor of cellular homeostasis, and any perturbation in distinct metabolic pathways results in the activation of this inflammasome. Lipid metabolism is exceedingly important in maintaining cellular homeostasis, and it is recognized that cells and tissues undergo extensive lipid remodeling during activation and disease. Some lipids are involved in instigating chronic inflammatory diseases, and new studies have highlighted critical upstream roles for lipids, particularly cholesterol, in regulating inflammasome activation implying key functions for inflammasomes in diseases with defective lipid metabolism. The focus of this review is to highlight how lipids regulate inflammasome activation and how this leads to the progression of inflammatory diseases. The key roles of cholesterol metabolism in the activation of inflammasomes have been comprehensively discussed. Besides, the roles of oxysterols, fatty acids, phospholipids, and lipid second messengers are also summarized in the context of inflammasomes. The overriding theme is that lipid metabolism has numerous but complex functions in inflammasome activation. A detailed understanding of this area will help us develop therapeutic interventions for diseases where dysregulated lipid metabolism is the underlying cause.
    Keywords:  NLRP3; cholesterol; homeostasis; inflammasome; lipids; metabolism
    DOI:  https://doi.org/10.1111/imr.12891
  41. Sci Transl Med. 2020 Jun 17. pii: eaav9760. [Epub ahead of print]12(548):
    De La Fuente A, Zilio S, Caroli J, Van Simaeys D, Mazza EMC, Ince TA, Bronte V, Bicciato S, Weed DT, Serafini P.
      Local delivery of anticancer agents has the potential to maximize treatment efficacy and minimize the acute and long-term systemic toxicities. Here, we used unsupervised systematic evolution of ligands by exponential enrichment to identify four RNA aptamers that specifically recognized mouse and human myeloid cells infiltrating tumors but not their peripheral or circulating counterparts in multiple mouse models and from patients with head and neck squamous cell carcinoma (HNSCC). The use of these aptamers conjugated to doxorubicin enhanced the accumulation and bystander release of the chemotherapeutic drug in both primary and metastatic tumor sites in breast and fibrosarcoma mouse models. In the 4T1 mammary carcinoma model, these doxorubicin-conjugated aptamers outperformed Doxil, the first clinically approved highly optimized nanoparticle for targeted chemotherapy, promoting tumor regression after just three administrations with no detected changes in weight loss or blood chemistry. These RNA aptamers recognized tumor infiltrating myeloid cells in a variety of mouse tumors in vivo and from human HNSCC ex vivo. This work suggests the use of RNA aptamers for the detection of myeloid-derived suppressor cells in humans and for a targeted delivery of chemotherapy to the tumor microenvironment in multiple malignancies.
    DOI:  https://doi.org/10.1126/scitranslmed.aav9760
  42. Sci Transl Med. 2020 Jun 17. pii: eabc4220. [Epub ahead of print]12(548):
    Goswami S, Chen Y, Anandhan S, Szabo PM, Basu S, Blando JM, Liu W, Zhang J, Natarajan SM, Xiong L, Guan B, Yadav SS, Saci A, Allison JP, Galsky MD, Sharma P.
      Immune checkpoint therapy (ICT) can produce durable antitumor responses in metastatic urothelial carcinoma (mUCC); however, the responses are not universal. Despite multiple approvals of ICT in mUCC, we lack predictive biomarkers to guide patient selection. The identification of biomarkers may require interrogation of both the tumor mutational status and the immune microenvironment. Through multi-platform immuno-genomic analyses of baseline tumor tissues, we identified the mutation of AT-rich interactive domain-containing protein 1A (ARID1A) in tumor cells and expression of immune cytokine CXCL13 in the baseline tumor tissues as two predictors of clinical responses in a discovery cohort (n = 31). Further, reverse translational studies revealed that CXCL13-/- tumor-bearing mice were resistant to ICT, whereas ARID1A knockdown enhanced sensitivity to ICT in a murine model of bladder cancer. Next, we tested the clinical relevance of ARID1A mutation and baseline CXCL13 expression in two independent confirmatory cohorts (CheckMate275 and IMvigor210). We found that ARID1A mutation and expression of CXCL13 in the baseline tumor tissues correlated with improved overall survival (OS) in both confirmatory cohorts (CheckMate275, CXCL13 data, n = 217; ARID1A data, n = 139, and IMvigor210, CXCL13 data, n = 348; ARID1A data, n = 275). We then interrogated CXCL13 expression plus ARID1A mutation as a combination biomarker in predicting response to ICT in CheckMate275 and IMvigor210. Combination of the two biomarkers in baseline tumor tissues suggested improved OS compared to either single biomarker. Cumulatively, this study revealed that the combination of CXCL13 plus ARID1A may improve prediction capability for patients receiving ICT.
    DOI:  https://doi.org/10.1126/scitranslmed.abc4220
  43. J Clin Invest. 2020 Jun 16. pii: 138392. [Epub ahead of print]
    Zhu X, Callahan MF, Gruber KA, Szumowski M, Marks DL.
      Cachexia, a devastating wasting syndrome characterized by severe weight loss with specific losses of muscle and adipose tissue, is driven by reduced food intake, increased energy expenditure, excess catabolism, and inflammation. Cachexia is associated with poor prognosis and high mortality, and frequently occurs in patients with cancer, chronic kidney disease, infection, and many other illnesses. There is no effective treatment for this condition. Hypothalamic melanocortins have a potent and long-lasting inhibitory effect on feeding and anabolism, and pathophysiological processes increase melanocortin signaling tone leading to anorexia, metabolic changes, and eventual cachexia. We utilized three rat models of anorexia and cachexia (LPS, methylcholanthrene sarcoma, and 5/6 subtotal nephrectomy) to evaluate efficacy of TCMCB07, a synthetic antagonist of the melanocortin-4 receptor. Our data show that peripheral treatment of TCMCB07 with intraperitoneal, subcutaneous, and oral administration increased food intake and body weight, and preserved fat mass and lean mass during cachexia and LPS-induced anorexia. Furthermore, administration of TCMCB07 diminished hypothalamic inflammatory gene expression in cancer cachexia. These results suggest that peripheral TCMCB07 treatment effectively inhibits central melanocortin signaling and therefore stimulates appetite and enhances anabolism, indicating that TCMCB07 is a promising drug candidate to treat cachexia.
    Keywords:  Cancer; Chronic kidney disease; Drug therapy; Metabolism; Therapeutics
    DOI:  https://doi.org/10.1172/JCI138392
  44. J Biol Chem. 2020 Jun 15. pii: jbc.RA120.012962. [Epub ahead of print]
    Abdisalaam S, Bhattacharya S, Mukherjee S, Sinha D, Srinivasan K, Zhu M, Akbay EA, Sadek HA, Shay JW, Aroumougame A.
      Defective DNA damage response (DDR) signaling is a common mechanism that initiates and maintains the cellular senescence phenotype. Dysfunctional telomeres activate DDR signaling, genomic instability, and cellular senescence, but the links among these events remains unclear. Here, using an array of biochemical and imaging techniques, including a highly regulatable CRISPR/Cas9 strategy to induce DNA double-strand breaks specifically in the telomeres, chromatin immunoprecipitation, telomere immunofluorescence, fluorescence in situ hybridization (FISH), micronuclei imaging, and the telomere shortest length assay (TeSLA), we show that chromosome mis-segregation due to imperfect DDR signaling in response to dysfunctional telomeres creates a preponderance of chromatin fragments in the cytosol, which leads to a premature senescence phenotype. We found that this phenomenon is caused not by telomere shortening, but by cyclic GMP-AMP synthase (cGAS) recognizing cytosolic chromatin fragments and then activating the stimulator of interferon genes (STING) cytosolic DNA-sensing pathway and downstream interferon signaling. Significantly, genetic and pharmacological manipulation of cGAS not only attenuated immune signaling, but also prevented premature cellular senescence in response to dysfunctional telomeres. The findings of our study uncover a cellular intrinsic mechanism involving the cGAS-mediated cytosolic self-DNA-sensing pathway that initiates premature senescence independently of telomere shortening.
    Keywords:  CRISPR/Cas; DNA damage; DNA damage response; chromatin; cyclic GMP-AMP synthase (cGAS); genome maintenance; micronuclei; senescence; telomere
    DOI:  https://doi.org/10.1074/jbc.RA120.012962
  45. Nat Chem Biol. 2020 Jun 15.
    Karim AS, Dudley QM, Juminaga A, Yuan Y, Crowe SA, Heggestad JT, Garg S, Abdalla T, Grubbe WS, Rasor BJ, Coar DN, Torculas M, Krein M, Liew FE, Quattlebaum A, Jensen RO, Stuart JA, Simpson SD, Köpke M, Jewett MC.
      The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.
    DOI:  https://doi.org/10.1038/s41589-020-0559-0
  46. Mitochondrion. 2020 Jun 13. pii: S1567-7249(20)30065-9. [Epub ahead of print]
    Castellani CA, Longchamps RJ, Sun J, Guallar E, Arking DE.
      Mitochondrial DNA copy number (mtDNA-CN) is a biomarker of mitochondrial function and levels of mtDNA-CN have been reproducibly associated with overall mortality and a number of age-related diseases, including cardiovascular disease, chronic kidney disease, and cancer. Recent advancements in techniques for estimating mtDNA-CN, in particular the use of DNA microarrays and next-generation sequencing data, have led to the comprehensive assessment of mtDNA-CN across these and other diseases and traits. The importance of mtDNA-CN measures to disease and these advancing technologies suggest the potential for mtDNA-CN to be a useful biomarker in the clinic. While the exact mechanism(s) underlying the association of mtDNA-CN with disease remain to be elucidated, we review the existing literature which supports roles for inflammatory dynamics, immune function and alterations to cell signaling as consequences of variation in mtDNA-CN. We propose that future studies should focus on characterizing longitudinal, cell-type and cross-tissue profiles of mtDNA-CN as well as improving methods for measuring mtDNA-CN which will expand the potential for its use as a clinical biomarker.
    Keywords:  clinical biomarker; complex disease; mitochondrial DNA; mtDNA
    DOI:  https://doi.org/10.1016/j.mito.2020.06.004
  47. Sci Immunol. 2020 Jun 19. pii: eabc4466. [Epub ahead of print]5(48):
    Bain CC, Gibson DA, Steers NJ, Boufea K, Louwe PA, Doherty C, González-Huici V, Gentek R, Magalhaes-Pinto M, Shaw T, Bajénoff M, Bénézech C, Walmsley SR, Dockrell DH, Saunders PTK, Batada NN, Jenkins SJ.
      Macrophages reside in the body cavities where they maintain serosal homeostasis and provide immune surveillance. Peritoneal macrophages are implicated in the etiology of pathologies including peritonitis, endometriosis, and metastatic cancer; thus, understanding the factors that govern their behavior is vital. Using a combination of fate mapping techniques, we have investigated the impact of sex and age on murine peritoneal macrophage differentiation, turnover, and function. We demonstrate that the sexually dimorphic replenishment of peritoneal macrophages from the bone marrow, which is high in males and very low in females, is driven by changes in the local microenvironment that arise upon sexual maturation. Population and single-cell RNA sequencing revealed marked dimorphisms in gene expression between male and female peritoneal macrophages that was, in part, explained by differences in composition of these populations. By estimating the time of residency of different subsets within the cavity and assessing development of dimorphisms with age and in monocytopenic Ccr2 -/- mice, we demonstrate that key sex-dependent features of peritoneal macrophages are a function of the differential rate of replenishment from the bone marrow, whereas others are reliant on local microenvironment signals. We demonstrate that the dimorphic turnover of peritoneal macrophages contributes to differences in the ability to protect against pneumococcal peritonitis between the sexes. These data highlight the importance of considering both sex and age in susceptibility to inflammatory and infectious diseases.
    DOI:  https://doi.org/10.1126/sciimmunol.abc4466
  48. Mol Cancer Ther. 2020 Jun 16. pii: molcanther.1161.2020. [Epub ahead of print]
    Roberts PJ, Kumarasamy V, Witkiewicz AK, Knudsen ES.
      Cyclin-dependent kinases 4 and 6 (CDK4/6) have emerged as important therapeutic targets. Pharmacological inhibitors of these kinases function to inhibit cell cycle progression and exert other important effects on the tumor and host environment. Due to their impact on the cell cycle, CDK4/6 inhibitors (CDK4/6i) have been hypothesized to antagonize the anti-tumor effects of cytotoxic chemotherapy in tumors that are CDK4/6 dependent. However, there are multiple preclinical studies that illustrate potent cooperation between CDK4/6i and chemotherapy. Furthermore, the combination of CDK4/6i and chemotherapy is being tested in clinical trials to both enhance anti-tumor efficacy and limit toxicity. Exploitation of the non-canonical effects of CDK4/6i could also provide an impetus for future studies in combination with chemotherapy. Thus, while seemingly mutually exclusive mechanisms are at play, the combination of CDK4/6 inhibition and chemotherapy could exemplify rational medicine.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-18-1161
  49. Immunity. 2020 Jun 16. pii: S1074-7613(20)30230-2. [Epub ahead of print]52(6): 957-970
    Blériot C, Chakarov S, Ginhoux F.
      Resident tissue macrophages (RTMs) have a broad spectrum of immune- and non-immune-related tissue-supporting activities. The roots of this heterogeneity and versatility are only beginning to be understood. Here, we propose a conceptual framework for considering the RTM heterogeneity that organizes the factors shaping RTM identity within four cardinal points: (1) ontogeny and the view that adult RTM populations comprise a defined mixture of cells that arise from either embryonic precursors or adult monocytes; (2) local factors unique to the niche of residence, evolving during development and aging; (3) inflammation status; and (4) the cumulative effect of time spent in a specific tissue that contributes to the resilient adaptation of macrophages to their dynamic environment. We review recent findings within this context and discuss the technological advances that are revolutionizing the study of macrophage biology.
    DOI:  https://doi.org/10.1016/j.immuni.2020.05.014
  50. Autophagy. 2020 Jun 16.
    Peña-Oyarzun D, Rodriguez-Peña M, Burgos-Bravo F, Vergara A, Kretschmar C, Sotomayor-Flores C, Ramirez-Sarmiento CA, de Smedt H, Reyes M, Perez W, Torres VA, Morselli E, Altamirano F, Wilson CAM, Hill JA, Lavandero S, Criollo A.
      Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a non-selective cation channel permeable to Ca2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2D509V, a Ca2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy.
    Keywords:  autophagy; beclin 1; calcium; polycystin-2; protein complex
    DOI:  https://doi.org/10.1080/15548627.2020.1782035
  51. Autophagy. 2020 Jun 19.
    Mesquita A, Glenn J, Jenny A.
      As one of the major, highly conserved catabolic pathways, autophagy delivers cytosolic components to lysosomes for degradation. It is essential for development, cellular homeostasis, and coping with stress. Reduced autophagy increases susceptibility to protein aggregation diseases and leads to phenotypes associated with aging. Of the three major forms of autophagy, macroautophagy (MA) can degrade organelles or aggregated proteins, and chaperone-mediated autophagy is specific for soluble proteins containing KFERQ-related targeting motifs. During endosomal microautophagy (eMI), cytoplasmic proteins are engulfed into late endosomes in an ESCRT machinery-dependent manner. eMI can be KFERQ-specific or occur in bulk and be induced by prolonged starvation. Its physiological regulation and function, however, are not understood. Here, we show that eMI in the Drosophila fat body, akin to the mammalian liver, is induced upon oxidative or genotoxic stress in an ESCRT and partially Hsc70-4-dependent manner. Interestingly, eMI activation is selective, as ER stress fails to elicit a response. Intriguingly, we find that reducing MA leads to a compensatory enhancement of eMI, suggesting a tight interplay between these degradative processes. Furthermore, we show that mutations in DNA damage response genes are sufficient to trigger eMI and that the response to oxidative stress is under the control of MAPK/JNK signaling. Our data suggest that, controlled by various signaling pathways, eMI allows an organ to react and adapt to specific types of stress and is thus likely critical to prevent disease.
    Keywords:  DNA damage; ER stress; ROS; autophagy; microautophagy; oxidative stress; proteostasis
    DOI:  https://doi.org/10.1080/15548627.2020.1783833
  52. ESMO Open. 2020 Jun;pii: e000683. [Epub ahead of print]4(Suppl 3):
    Tagliamento M, Bironzo P, Novello S.
      The immune surveillance system is complex and regulated by different actors. Programmed death protein-ligand 1 (PD-L1), the only approved biomarker in clinical practice, has proven to be imperfect in selecting patients to immune checkpoint inhibitors treatment. Therefore, new biomarkers, and new therapeutic targets, are needed to maximise the efficacy of immunotherapy. V-domain Ig Suppressor of T-cell Activation (VISTA) is a programmed death protein-1 (PD-1) homolog expressed on T cells and on antigen-presenting cells, which regulates processes of activation and repression of the immune system with not yet completely clarified mechanisms. Its blockage has demonstrated in vitro and in vivo antitumour activity. The clinical research of VISTA antagonists is ongoing. Particularly, CA-170, an orally delivered dual inhibitor of VISTA and PD-L1, has shown to have clinical efficacy in phase I and II clinical trials in different advanced solid tumour types. Further data are needed to define whether this drug class can become a new therapeutic option for patients with VISTA expressing cancers.
    Keywords:  VISTA; biomarkers; cancer; immune checkpoint; immunotherapy
    DOI:  https://doi.org/10.1136/esmoopen-2020-000683