SAMHD1 is a biomarker for cytarabine response and a therapeutic target in acute myeloid leukemia

Nature Medicine, Journal Year: 2016, Volume and Issue: 23(2), P. 250 - 255

Published: Dec. 19, 2016

Language: Английский

---

A G1‐like state allows HIV ‐1 to bypass SAMHD 1 restriction in macrophages

The EMBO Journal, Journal Year: 2017, Volume and Issue: 36(5), P. 604 - 616

Published: Jan. 25, 2017

Article25 January 2017Open Access Source DataTransparent process A G1-like state allows HIV-1 to bypass SAMHD1 restriction in macrophages Petra Mlcochova Division of Infection and Immunity, University College London, UK Search for more papers by this author Katherine Sutherland Sarah Watters Cosetta Bertoli MRC Laboratory Molecular Cell Biology, Rob AM de Bruin Jan Rehwinkel Medical Research Council Human Immunology Unit, Radcliffe Department Medicine, Weatherall Institute Oxford, Stuart J Neil Immunology, Inflammatory Disease, King's College, Gina M Lenzi Pediatrics, Center Drug Discovery, Emory School Atlanta, GA, USA Baek Kim Asim Khwaja Haematology, UCL, Matthew C Gage Christiana Georgiou Alexandra Chittka Simon Yona Mahdad Noursadeghi Greg Towers Ravindra K Gupta Corresponding Author [email protected] orcid.org/0000-0001-9751-1808 Africa Health Institute, KwaZulu Natal, South Information Mlcochova1, Sutherland1, Watters1, Bertoli2, Bruin2, Rehwinkel3, Neil4, Lenzi5, Kim5, Khwaja6, Gage7, Georgiou7, Chittka7, Yona7, Noursadeghi1, Towers1 *,1,8 1Division 2MRC 3Medical 4Division 5Department 6Research 7Division 8Africa *Corresponding author. Tel: +44 20 7679 2000; E-mail: The EMBO Journal (2017)36:604-616https://doi.org/10.15252/embj.201696025 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract An unresolved question is how achieves efficient replication terminally differentiated despite factor SAMHD1. We reveal inducible changes expression cell cycle-associated proteins MCM2 cyclins A, E, D1/D3 macrophages, without evidence DNA synthesis or mitosis. These are induced activation Raf/MEK/ERK kinase cascade, culminating upregulation CDK1 with subsequent T592 phosphorylation deactivation its antiviral activity. HIV infection limited these phase at single-cell level. Depletion decouples association between proteins, becoming highly susceptible HIV-1. observe both embryo-derived monocyte-derived tissue-resident frequencies approaching 20%, suggesting sustain vivo. Finally, we SAMHD1-dependent antiretroviral activity histone deacetylase inhibitors acting via p53 activation. data provide basis host-directed therapeutic approaches aimed limiting burden that may contribute curative interventions. Synopsis Here, show found G0- phases, latter permissive due canonical pathway which deactivates CDK1-mediated T592. Therefore, does not need encode antagonist. Macrophages transition classical G0 quiescent state, representing up 20% tissue macrophages. G0–G1 regulated pathway, associated increased protein, permissivity window infection. Histone possess Introduction SAMHD1, deoxynucleotide-triphosphate (dNTP) hydrolase, restricts reverse transcription (RT) through decreasing levels dNTPs (Goldstone et al, 2011; Lahouassa 2012; Schmidt 2015). position mediated cyclin-dependent kinases CDK1/2 (Cribier 2013; White 2013) actively dividing cells impairs dNTP hydrolase viral occur Arnold Some lentiviruses have evolved countermeasures against SAMHD1; example, HIV-2/SIVsm lineage encodes Vpx protein degrades otherwise SAMHD1-positive target (Kaushik 2009; Hrecka Laguette 2011). How pandemic strains achieve vivo Vpx-like has remained significant our understanding tropism pathogenesis (Watters 2013). dynamic non-dividing do culminate dependent on mitogen/growth factor-activated signalling sufficient deactivate potent Moreover, two distinct populations mice express profile, providing only an explanation ability high but also offering vital innate immune cells. Results Terminally stimulated enter observed culture human (MDM) foetal calf serum/FCS (stimulated cells), as opposed serum/HS (unstimulated led increase (Figs 1A–C EV1A–E). As expected, there was donor variation absolute (Fig EV1B). under stimulating conditions single-round VSV-G-pseudotyped virus 1A EV1A B) full-length infectious molecular clones 1B C, EV1C D), macrophage tropic viruses (BaL, YU-2), clinical isolates D) capsid mutations known alter interactions cyclophilin CPSF6 leading altered transcription, retargeted integration triggering sensing EV1E). enhancement post-entry step EV1F). effect FCS lost when charcoal-stripped used boiled serum/foetal serum mixture (1:1) used, existence heat stable stimulatory rather than inhibitory HS EV1A). Figure 1. Transitioning from MDM were cultured RPMI complemented MCSF 10% (unstim) (stim). infected expressing GFP, percentage quantified 48 h post-infection FACS (n = 3, mean ± s.e.m.; **P-value ≤ 0.01, unpaired t-test). BaL isolated 18 qPCR late RT products *P-value 0.05, Spreading MDM. Cells BaL, stained intracellular p24 FACS. Principal component analysis macrophage-associated transcripts compare relative clustering unstimulated range primary cells/cell lines. Diagram cellular functions genes (−log2(P-value) > 2) transcriptional compared Immunoblot expressed Star indicates non-specific band. This Western blot quantification Donor 1 Fig 2A. same blots 2A allow comparison different well Uninfected exposed VSV-G GFP (HIV-1 exposed) MCM2, Geminin EdU incorporation (EdU added 50 prior analysis). On average, 104 each experiment recorded analysed using Hermes WiScan cell-imaging system ImageJ. cycle markers shown. quantitation content flow cytometry. Cycling THP-1 labelled propidium iodide (PI) CFSE loaded 4 days determine division/proliferation available online figure. Data [embj201696025-sup-0004-SDataFig1.pdf] Download figure PowerPoint Click here expand EV1. Enhanced susceptibility 3 additional (unstim), (stim), (CS), 5 min filtrated 0.45-μm filter (FCS boil), 1:1 mix (unstim:stim 1:1), (unstim:FCS boil 1:1) then HIV-1-expressing GFP. detected 12 donors (D1-D12) GFP; Single round panel (macrophage viruses: YU2; isolates: CH77, CH58, ZM247, WITO). numbers light microscopy. titres released determined indicator HeLa TZM-bl. Culture supernatants BaL-infected harvest day 2, 6 9 post-infection, filtered infect TZM-bl Luciferase measured 24 post-infection. (wt HIV-1; mutants P90A, N74D, G89V), All displayed similar (which normalised ˜100%). equal amounts (50 ng) BlaM-Vpr-containing h. CCF2/AM dye, fusion events cytometry BD LSR Fortessa gated 10,000 information: Graphs average n ≥ 0.05; 0.01; ***P-value 0.001, t-test. differential effects suggested approach uncovering mechanisms regulating transcriptomes cells, UNSTIM) STIM) aiming discover pathways higher permissivity. Comparison profiles predefined gene signature discriminates other types (Tomlinson 2012) clearly shows cluster together closely related myeloid 1D). they EV2). However, use ingenuity evaluate revealed enrichment number molecular/cellular regulation, growth proliferation, death survival 1E). top enriched encoding involved regulation 1F EV3A, Table EV1). observations validated level 1G). Stimulated showed D-type D1 D3, accumulate progress G1 (Baldin 1993; Sherr, 1993, 1996). Cyclin D2 below detectable E2F6 Geminin—all during entry (Coverley 2002; Fragkos Of note, CDK1—a key player progression—was upregulated along (minichromosome maintenance complex 1G H), origin licensing (but G0) 1996; Su O'Farrell, 1997; Tsuruga Musahl 1998; Stoeber 1998, 2001; Williams 1998). Importantly, p27 reduced following stimulation inhibitor decreases after re-entry (Sherr Roberts, 1999). EV2. Expression surface unaffected B. (CD68, CD14) M1 M2 (CD163, CD80, CD86 CD40) EV3. can be manipulated Ingenuity interaction nuclear demonstrates single dominant interacting proteins. Example acquisition system, automated microscopic platform. nuclei, active Click-iT® Alexa Fluor® 488 Imaging Kit. Scale bars: 10 μm. treated μM detect EdU-positive Quantification phases PI labelling. 7 changed into medium (10% FCS) days. CDK4/6 Palbociclib (1 μM) before virus; VLP-vpx time lysed immunoblotting. ImageJ s.e.m.). further absent G0/quiescent/terminally (i) all (MCM2) (Masai 2010), (ii) S G2/M (Geminin) (Fragkos 2015) (iii) specific therefore marker incorporation) 1H). platform EV3B) versus positive fivefold 10-fold S-G2-M Together low over 50-h period, suggest re-entered majority did S, 1H EV3C). confirmed divide staining 1I J, EV3D). able modulate measurable division synthesis. early consistent Transition regulate Given well-described Pauls 2014; Yan 2015), hypothesised might spontaneously To test this, examined knowing phosphorylates capacity restrict Welbourn raised pSAMHD1-T592 MDM, CDK2, CDK4 CDK6 2A). threefold fourfold EV3E), reported (Lahouassa 2012). Furthermore, exogenously degradation co-infection SIVmac virus-like particles bearing (VLP-vpx) 2B), depletion siRNA transfection 2C) infectivity specifically no change where phosphorylated thus already inactive 2. Bidirectional transitions shape SAMHD1-mediated A. three (D1, D2, D3) immunoblotting CDK order facilitate co-infected containing vpx (VLP-vpx). representative (ns) non-significant, C. transfected control pool siRNAs later D. Experimental model bidirectional G0–G1-like transitions. follows: (stim) described 4; [stim (day 3)] grown days; [unstim (3 days)] condition non-stimulating remaining E. BaL. CDKs, Graph example s.e.m. F. Proposed G, H. Unstimulated (G) (H) RAF (2 μM), B-RAF MEK1/2 (AS-703026, JAK 1–3 GSK3 PIM s.e.m., calculated triplicates, I. MEK/ERK (U0126, indicated Percentage non-significant; 2 [embj201696025-sup-0005-SDataFig2.pdf] probe reversibility unstimulating FCS, vice versa 2D E). Non-stimulating onwards 3), E] expressions (indicative returning state), decreased Conversely, EV3F) augmented MCM2/CDK1 re-entering state) phosphorylation. No CDK2 2E). reversible explore mapped responsible well-characterised 2F–I). identified involvement cascade 2F–I EV3G). B-Raf PLX4032 (active V600E mutant cancer cells) negative demonstrate specificity Raf inhibition 2G H). pharmacologically block putative signal activated MEK/ERK, U0126, reasoning would lead suppression manner 2F). Indeed, substantially inhibited loss correlated dephosphorylation, downregulation 2I). Critically, 2I) completely rescued U0126. inhibiting downstream illustrate, first time, regulates preferential targets next employed high-throughput co-localisation analysis, measure progression 3). (added infection) monitor 3A–E). non-stimulatory 3A–C G–J). illustrated expression, Stimulation simply MCM2-positive 3B, explains cultures 1A. even though EV4A), population synthesising minority (< 7%) statistic

Language: Английский

---

SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

Nature Medicine, Journal Year: 2016, Volume and Issue: 22(10), P. 1072 - 1074

Published: Oct. 1, 2016

Language: Английский

---

Transmission, Evolution, and Endogenization: Lessons Learned from Recent Retroviral Invasions

Microbiology and Molecular Biology Reviews, Journal Year: 2017, Volume and Issue: 82(1)

Published: Dec. 13, 2017

SUMMARY Viruses of the subfamily Orthoretrovirinae are defined by ability to reverse transcribe an RNA genome into DNA that integrates host cell during intracellular virus life cycle. Exogenous retroviruses (XRVs) horizontally transmitted between individuals, with disease outcome depending on interactions retrovirus and organism. When infect germ line cells host, they may become endogenous (ERVs), which permanent elements in subject vertical transmission. These ERVs sometimes remain infectious can themselves give rise XRVs. This review recent developments phylogenetic classification identification retroviral receptors elucidate origins evolution XRVs ERVs. We consider whether recurrently pressure shift receptor usage sidestep ERV interference. discuss how related undergo alternative fates different lineages after endogenization, koala (KoRV) receiving notable interest as a invader its line. KoRV is heritable but also infectious, provides insights early stages invasions well XRV generation from The relationship primate other placed context biogeography potential role bats rodents vectors for interspecies viral Combining studies extant “fossil” koalas Australasian species has broadened our understanding host-retrovirus interactions.

Language: Английский

---

Conserved Herpesvirus Protein Kinases Target SAMHD1 to Facilitate Virus Replication

Cell Reports, Journal Year: 2019, Volume and Issue: 28(2), P. 449 - 459.e5

Published: July 1, 2019

Highlights•SAMHD1 depletion facilitates EBV lytic replication•EBV protein kinase BGLF4 directly phosphorylates SAMHD1•BGLF4 phosphorylation of SAMHD1 inhibits its dCTPase and dTTPase activity•SAMHD1 is targeted by the conserved herpesvirus kinasesSummaryTo ensure a successful infection, herpesviruses have developed elegant strategies to counterbalance host anti-viral responses. Sterile alpha motif HD domain 1 (SAMHD1) was recently identified as an intrinsic restriction factor for variety viruses. Aside from HIV-2 related simian immunodeficiency virus (SIV) Vpx proteins, direct viral countermeasures against remain unknown. Using Epstein-Barr (EBV) primary model, we discover that SAMHD1-mediated antagonized BGLF4, member kinases encoded all herpesviruses. Mechanistically, find thereby deoxynucleotide triphosphate triphosphohydrolase (dNTPase) activity. We further demonstrate targeting common feature shared beta- gamma-herpesviruses. Together, our findings uncover immune evasion mechanism whereby exploit thwart defenses.Graphical abstract

Language: Английский

---

Dephosphorylation of the HIV-1 restriction factor SAMHD1 is mediated by PP2A-B55α holoenzymes during mitotic exit

Nature Communications, Journal Year: 2018, Volume and Issue: 9(1)

Published: June 4, 2018

Abstract SAMHD1 is a critical restriction factor for HIV-1 in non-cycling cells and its antiviral activity regulated by T592 phosphorylation. Here, we show that dephosphorylation at controlled during the cell cycle, occurring M/G 1 transition proliferating cells. Using several complementary proteomics biochemical approaches, identify phosphatase PP2A-B55α responsible rendering antivirally active. specifically targeted holoenzymes mitotic exit, line with observations key exit mammalian Strikingly, as HeLa or activated primary CD4 + T enter G phase, pronounced reduction of RT products observed upon infection dependent on presence dephosphorylated SAMHD1. Moreover, PP2A controls pT592 level monocyte-derived macrophages (MDMs). Thus, holoenzyme regulator to switch

Language: Английский

---

Selective inactivation of hypomethylating agents by SAMHD1 provides a rationale for therapeutic stratification in AML

Nature Communications, Journal Year: 2019, Volume and Issue: 10(1)

Published: Aug. 2, 2019

Abstract Hypomethylating agents decitabine and azacytidine are regarded as interchangeable in the treatment of acute myeloid leukemia (AML). However, their mechanisms action remain incompletely understood, predictive biomarkers for HMA efficacy lacking. Here, we show that bioactive metabolite triphosphate, but not functions activator substrate triphosphohydrolase SAMHD1 is subject to SAMHD1-mediated inactivation. Retrospective immunohistochemical analysis bone marrow specimens from AML patients at diagnosis revealed expression leukemic cells inversely correlates with clinical response decitabine, azacytidine. ablation increases antileukemic activity cell lines, primary blasts, xenograft models. acquire resistance partly by up-regulation. Together, our data suggest a biomarker stratified use hypomethylating potential target decitabine-resistant leukemia.

Language: Английский

---

SAMHD1 Suppression of Antiviral Immune Responses

Trends in Microbiology, Journal Year: 2018, Volume and Issue: 27(3), P. 254 - 267

Published: Oct. 15, 2018

Language: Английский

---

Human cytomegalovirus overcomes SAMHD1 restriction in macrophages via pUL97

Nature Microbiology, Journal Year: 2019, Volume and Issue: 4(12), P. 2260 - 2272

Published: Sept. 23, 2019

Language: Английский

---

Deficiency for SAMHD1 activates MDA5 in a cGAS/STING-dependent manner

The Journal of Experimental Medicine, Journal Year: 2022, Volume and Issue: 220(1)

Published: Nov. 8, 2022

Defects in nucleic acid metabolizing enzymes can lead to spontaneous but selective activation of either cGAS/STING or RIG-like receptor (RLR) signaling, causing type I interferon–driven inflammatory diseases. In these pathophysiological conditions, the DNA sensor cGAS and IFN production are linked damage. Physiological, tonic, signaling on other hand is essential functionally prime sensing pathways. Here, we show that low-level chronic damage mice lacking Aicardi-Goutières syndrome gene SAMHD1 reduced tumor-free survival when crossed a p53-deficient, not mismatch repair-deficient background. Increased did result higher levels interferon. Instead, found interferon response SAMHD1-deficient was driven by MDA5/MAVS pathway required functional priming through pathway. Our work positions upstream tonic Samhd1-deficient highlights an important role physiological innate immune priming.

Language: Английский

---