Articles de revues : « SAMDH1 »

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Ahn, Jinwoo, Caili Hao, Junpeng Yan, Maria DeLucia, Jennifer Mehrens, Chuanping Wang, Angela M. Gronenborn et Jacek Skowronski. « HIV/Simian Immunodeficiency Virus (SIV) Accessory Virulence Factor Vpx Loads the Host Cell Restriction Factor SAMHD1 onto the E3 Ubiquitin Ligase Complex CRL4DCAF1 ». Journal of Biological Chemistry 287, n^o 15 (23 février 2012) : 12550–58. http://dx.doi.org/10.1074/jbc.m112.340711.

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The sterile alpha motif and HD domain-containing protein-1 (SAMHD1) inhibits infection of myeloid cells by human and related primate immunodeficiency viruses (HIV and SIV). This potent inhibition is counteracted by the Vpx accessory virulence factor of HIV-2/SIVsm viruses, which targets SAMHD1 for proteasome-dependent degradation, by reprogramming cellular CRL4DCAF1 E3 ubiquitin ligase. However, the precise mechanism of Vpx-dependent recruitment of human SAMHD1 onto the ligase, and the molecular interfaces on the respective molecules have not been defined. Here, we show that human SAMHD1 is recruited to the CRL4DCAF1-Vpx E3 ubiquitin ligase complex by interacting with the DCAF1 substrate receptor subunit in a Vpx-dependent manner. No stable association is detectable with DCAF1 alone. The SAMHD1 determinant for the interaction is a short peptide located distal to the SAMHD1 catalytic domain and requires the presence of Vpx for stable engagement. This peptide is sufficient to confer Vpx-dependent recruitment to CRL4DCAF1 and ubiquitination when fused to heterologous proteins. The precise amino acid sequence of the peptide diverges among SAMHD1 proteins from different vertebrate species, explaining selective down-regulation of human SAMHD1 levels by Vpx. Critical amino acid residues of SAMHD1 and Vpx involved in the DCAF1-Vpx-SAMDH1 interaction were identified by mutagenesis. Our findings show that the N terminus of Vpx, bound to DCAF1, recruits SAMHD1 via its C terminus to CRL4, in a species-specific manner for proteasomal degradation.

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Martin-Gayo, Enrique, Taylor Hickman, Dina Pimenova, Florencia Pereyra, Eric Rosenberg, Mathias Lichterfeld et Xu Yu. « Cell-intrinsic HIV-1 immune responses in conventional dendritic cells from HIV-1 elite controllers (P6171) ». Journal of Immunology 190, n^o 1_Supplement (1 mai 2013) : 118.9. http://dx.doi.org/10.4049/jimmunol.190.supp.118.9.

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Abstract Introduction: Recent data suggest that in most HIV-1-infected individuals, cell-intrinsic immune responses of conventional dendritic cells (cDC) are blocked by the host proteins Samhd1 and Trex1. Elite controllers (EC) control HIV-1 replication in the absence of treatment, but immune defense mechanisms in these patients are not well understood. Here, we investigated cell-intrinsic immune responses to HIV-1 in these specific patients. Methods: PBMC from EC, untreated chronic progressors (CP), HAART-treated and HIV-1 negative subjects were ex vivo infected with HIV-1. Expression of viral replication products, type I interferons, Samhd1 and Trex1 were analyzed by qPCR. Results: cDC from HIV-1 negative persons were moderately susceptible to HIV-1, while cDC from EC and CP supported HIV-1 replication very weakly. However, in CP, HIV-1 replication was blocked at the level of early reverse transcription, likely as a result of high-level Samdh1 expression, while In EC, reverse transcription was unaltered, and restriction of viral replication mostly occurred at the level of integration. Functionally, these altered patterns of viral restriction in cDC from EC were associated with increased cellular activation, secretion of type I interferons and improved abilities to prime T cell responses. Conclusion: cDC from EC can mount cell-intrinsic immune responses against HIV-1, which may support the generation of highly effective HIV-1-specific T cell responses in these patients.

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Asadian, Peyman, et Dorothee Bienzle. « Interferon γ and α Have Differential Effects on SAMHD1, a Potent Antiviral Protein, in Feline Lymphocytes ». Viruses 11, n^o 10 (9 octobre 2019) : 921. http://dx.doi.org/10.3390/v11100921.

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Sterile alpha motif and histidine/aspartic domain-containing protein 1 (SAMHD1) is a protein with anti-viral, anti-neoplastic, and anti-inflammatory properties. By degrading cellular dNTPs to constituent deoxynucleoside and free triphosphate, SAMHD1 limits viral DNA synthesis and prevents replication of HIV-1 and some DNA viruses such as HBV, vaccinia, and HSV-1. Recent findings suggest SAMHD1 is broadly active against retroviruses in addition to HIV-1, such as HIV-2, FIV, BIV, and EIAV. Interferons are cytokines produced by lymphocytes and other cells that induce a wide array of antiviral proteins, including some with activity again lentiviruses. Here we evaluated the role of IFNs on SAMHD1 gene expression, transcription, and post-translational modification in a feline CD4+ T cell line (FeTJ) and in primary feline CD4+ T lymphocytes. SAMHD1 mRNA in FetJ cells increased in a dose-related manner in response to IFNγ treatment concurrent with increased nuclear localization and phosphorylation. IFNα treatment induced SAMHD1 mRNA but did not significantly alter SAMHD1 protein detection, phosphorylation, or nuclear translocation. In purified primary feline CD4+ lymphocytes, IL2 supplementation increased SAMHD1 expression, but the addition of IFNγ did not further alter SAMHD1 protein expression or nuclear localization. Thus, the effect of IFNγ on SAMHD1 expression is cell-type dependent, with increased translocation to the nucleus and phosphorylation in FeTJ but not primary CD4+ lymphocytes. These findings imply that while SAMH1 is inducible by IFNγ, overall activity is cell type and compartment specific, which is likely relevant to the establishment of lentiviral reservoirs in quiescent lymphocyte populations.

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Silberberg, Gilad, Bandana Vishwakarama, Brandon Walling, Chelsea Riveley, Alessandra Audia, Marianna Zipeto, Ido Sloma, Amy Wesa et Michael Ritchie. « Abstract 3907 : A pheno-multiomic integration analysis of primary samples of acute myeloid leukemia reveals biomarkers of cytarabine resistance ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 3907. http://dx.doi.org/10.1158/1538-7445.am2022-3907.

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Abstract The overall survival of patients diagnosed with Acute Myeloid Leukemia (AML) remains low. While initial responses to therapy are favorable, the duration of response is short and overcoming therapeutic resistance has proven difficult. A better understanding of the tumor cell biology and resistance mechanisms may shed light onto novel therapeutic targets that improve long-term outcome. In this study, we performed an exhaustive analysis to include deep tumor phenotyping, drug sensitivity profiling and comprehensive omic characterization. These datasets were included in integrative pharmaco-phenotypic-multiomic analyses to identify targets and biomarkers associated with cellular phenotype and drug response. Our results reveal that the major cellular discriminant within the cellular phenotype is CD34 expression, which associates with a high PDK-mediated metabolic profile and cytarabine sensitivity. Tumors exhibiting cytarabine resistance associate with a CD34-negative cellular phenotype and molecular characteristics such as MYC copy number gain, and increased expression of SAMDH1, FBP1 and TYMP proteins. Citation Format: Gilad Silberberg, Bandana Vishwakarama, Brandon Walling, Chelsea Riveley, Alessandra Audia, Marianna Zipeto, Ido Sloma, Amy Wesa, Michael Ritchie. A pheno-multiomic integration analysis of primary samples of acute myeloid leukemia reveals biomarkers of cytarabine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3907.

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Chen, Shuliang, Serena Bonifati, Zhihua Qin, Corine St. Gelais, Karthik M. Kodigepalli, Bradley S. Barrett, Sun Hee Kim et al. « SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-κB and interferon pathways ». Proceedings of the National Academy of Sciences 115, n^o 16 (2 avril 2018) : E3798—E3807. http://dx.doi.org/10.1073/pnas.1801213115.

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Sterile alpha motif and HD-domain–containing protein 1 (SAMHD1) blocks replication of retroviruses and certain DNA viruses by reducing the intracellular dNTP pool. SAMHD1 has been suggested to down-regulate IFN and inflammatory responses to viral infections, although the functions and mechanisms of SAMHD1 in modulating innate immunity remain unclear. Here, we show that SAMHD1 suppresses the innate immune responses to viral infections and inflammatory stimuli by inhibiting nuclear factor-κB (NF-κB) activation and type I interferon (IFN-I) induction. Compared with control cells, infection of SAMHD1-silenced human monocytic cells or primary macrophages with Sendai virus (SeV) or HIV-1, or treatment with inflammatory stimuli, induces significantly higher levels of NF-κB activation and IFN-I induction. Exogenous SAMHD1 expression in cells or SAMHD1 reconstitution in knockout cells suppresses NF-κB activation and IFN-I induction by SeV infection or inflammatory stimuli. Mechanistically, SAMHD1 inhibits NF-κB activation by interacting with NF-κB1/2 and reducing phosphorylation of the NF-κB inhibitory protein IκBα. SAMHD1 also interacts with the inhibitor-κB kinase ε (IKKε) and IFN regulatory factor 7 (IRF7), leading to the suppression of the IFN-I induction pathway by reducing IKKε-mediated IRF7 phosphorylation. Interactions of endogenous SAMHD1 with NF-κB and IFN-I pathway proteins were validated in human monocytic cells and primary macrophages. Comparing splenocytes from SAMHD1 knockout and heterozygous mice, we further confirmed SAMHD1-mediated suppression of NF-κB activation, suggesting an evolutionarily conserved property of SAMHD1. Our findings reveal functions of SAMHD1 in down-regulating innate immune responses to viral infections and inflammatory stimuli, highlighting the importance of SAMHD1 in modulating antiviral immunity.

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Kokaraki, Georgia, Ioanna Xagoraris, Pedro Farrajota Neves Da Silva, Lesley Ann Sutton, Raul Maia Falcão, Jorge Estefano Santana de Souza, Anders Österborg, Valtteri Wirta, Richard Rosenquist Brandell et Georgios Z. Rassidakis. « Mutations of the Novel Tumor Suppressor Gene SAMHD1 Are Frequent and Correlate with Decreased Protein Expression in Peripheral T-Cell Lymphomas (PTCL) ». Blood 138, Supplement 1 (5 novembre 2021) : 3515. http://dx.doi.org/10.1182/blood-2021-147428.

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Abstract Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which depletes the intracellular dNTP substrates and thus protects the host (human) cells from replication of viruses such as HIV. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS). In lymphoid malignancies, SAMHD1 gene mutations have been detected in a subset of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) resulting in decreased SAMHD1 mRNA levels and also mantle cell lymphoma (MCL) among B-cell neoplasms as well as in a subset (20%) of T-prolymphocytic leukemia (T-PLL). Therefore, SAMHD1 may play a role in oncogenesis as a tumor suppressor. In addition, SAMHD1 may confer resistance to cytarabine by hydrolysing their active triphosphate metabolites and its high protein levels correlate with poorer clinical outcome in acute myeloid leukemia. The mutation status of SAMHD1 gene and its expression patterns in peripheral T-cell lymphoma types is not known yet. The purpose of this study was to investigate SAMHD1 gene alterations using next generation sequencing and SAMHD1 protein expression in common types of PTCL. Methods: The study group included 81 adult patients with peripheral T-cell lymphomas (PTCL) including 26 patients with ALK+ anaplastic large cell lymphoma (ALCL), 20 ALK- ALCL, 13 angioimmunoblastic T-cell lymphomas (AILT) and 22 PTCL, not otherwise specified (NOS) with pre-treatment, formalin-fixed, paraffin-embedded (FFPE) tumor tissues available for immunohistochemical analysis. Double immunostaining (SAMHD1/CD68) was used to distinguish CD68+ histiocytes from the neoplastic T-cells. The Ventana autostainer and a previously validated monoclonal antibody for SAMHD1 (#A303-691A; Bethyl Laboratories, San Antonio, TX, USA) was utilized. The percentage of SAMHD1-positive cells was calculated by counting at least 500 tumor cells in each case. In a subset of 28 PTCLs, next generation sequencing (NGS) was performed using FFPE tissues and an enriched custom TruSight gene panel of 52 genes relevant to lymphoma biology. In addition, 3 control tissue samples were included in the analysis. The analysis pipeline was based on GATK best practices guidelines and all variants were annotated using Ensembl VEP v94.5. Freedom from progression (FFP) and overall survival (OS), were the clinical endpoints. Survival analyses were performed using the Kaplan-Meier method (log-rank test). Results: The expression level of SAMHD1 (percentage of positive neoplastic T-cells) varied significantly with AILT showing the highest level (median percentage 80%) as compared to ALK+ ALCL that showed the lowest level (median percentage 40%) of SAMHD1 expression (p=0.019, Kruskall-Wallis test). SAMHD1 mutations were detected for the first time in a subset of PTCL including 4/11 (36%) ALK+ ALCL, 1/5 (20%) ALK- ALCL, 3/6 (50%) AILT and 2/5 (40%) PTCL, NOS. The SAMHD1 gene alterations included missense mutations, nonsense (stopcodon) and splice region mutations. Importantly, reduced level (low percentage of positive tumor cells) of SAMHD1 protein expression was significantly associated with the presence of SAMHD1 mutations. More specifically, the median percentage of SAMHD1+ neoplastic T-cells was 80% in the PTCL group with wild-type SAMHD1 gene compared to 30% in the PTCL group with mutated SAMHD1 gene (p=0.01, Mann-Whitney U test), thus suggesting that alterations of SAMHD1 gene may represent a mechanism of SAMHD1 protein downregulation in a subset of PTCL. SAMHD1 expression or gene alterations did not correlate with FFP or OS in any PTCL histologic type, although the number of patients included in each group was not adequate to draw definite conclusions for prognostic significance. Conclusions: SAMHD1 gene mutations are frequently detected in a subset of PTCL and are associated with reduced expression of SAMHD1 protein. These findings reveal a novel mechanism (SAMHD1 mutations) of SAMHD1 downregulation in PTCL, and further support the tumor suppressor function of SAMHD1 gene in lymphomas. Disclosures Rosenquist Brandell: AbbVie: Honoraria; AstraZeneca: Honoraria; Illumina: Honoraria; Janssen: Honoraria; Roche: Honoraria.

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Xu, Bowen, Qianyi Sui, Han Hu, Xiangjia Hu, Xuchang Zhou, Cheng Qian et Nan Li. « SAMHD1 Attenuates Acute Inflammation by Maintaining Mitochondrial Function in Macrophages via Interaction with VDAC1 ». International Journal of Molecular Sciences 24, n^o 9 (26 avril 2023) : 7888. http://dx.doi.org/10.3390/ijms24097888.

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Over-activation of Toll-like receptor 4 (TLR4) is the key mechanism in Gram-negative bacterial infection-induced sepsis. SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) inhibits multiple viruses, but whether it plays a role during bacterial invasion remains unelucidated. Monocyte-macrophage specific Samhd1 knockout (Samhd1−/−) mice and Samhd1−/− macrophage cell line RAW264.7 were constructed and used as research models to evaluate the role of SAMHD1 in TLR4-activated inflammation. In vivo, LPS-challenged Samhd1−/− mice showed higher serum inflammatory factors, accompanied with more severe inflammation infiltration and lower survival rate. In vitro, Samhd1−/− peritoneal macrophages had more activated TLR4 pathway upon LPS-stimulation, accompanied with mitochondrial depolarization and dysfunction and a higher tendency to be M1-polarized. These results could be rescued by overexpressing full-length wild-type SAMHD1 or its phospho-mimetic T634D mutant into Samhd1−/− RAW264.7 cells, whereas the mutants, dNTP hydrolase-function-deprived H238A and phospho-ablative T634A, did not exert the same effect. Lastly, co-IP and immunofluorescence assays confirmed that SAMHD1 interacted with an outer mitochondrial membrane-localized protein, voltage-dependent anion channel-1 (VDAC1). SAMHD1 inhibits TLR4-induced acute inflammation and M1 polarization of macrophages by interacting with VDAC1 and maintaining mitochondria function, which outlines a novel regulatory mechanism of TLR signaling upon LPS stimulation.

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Plitnik, Timothy, Mark E. Sharkey, Bijan Mahboubi, Baek Kim et Mario Stevenson. « Incomplete Suppression of HIV-1 by SAMHD1 Permits Efficient Macrophage Infection ». Pathogens and Immunity 3, n^o 2 (6 décembre 2018) : 197. http://dx.doi.org/10.20411/pai.v3i2.263.

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Background: Sterile alpha motif and histidine/aspartic acid domain-containing protein (SAMHD1) is a dNTP triphosphorylase that reduces cellular dNTP levels in non-dividing cells, such as macrophages. Since dNTPs are required for reverse transcription, HIV-2 and most SIVs encode a Vpx protein that promotes proteasomal degradation of SAMHD1. It is unclear how HIV-1, which does not appear to harbor a SAMHD1 escape mechanism, is able to infect macrophages in the face of SAMHD1 restriction.Methods: To assess whether HIV-1 had a mechanism to negate SAMHD1 activity, we compared SAMHD1 and dNTP levels in macrophages infected by HIV-1 and SIV. We examined whether macrophages infected by HIV-1 still harbored antiviral levels of SAMHD1 by assessing their susceptibility to superinfection by vpx-deleted SIV. Finally, to assess whether HIV-1 reverse transcriptase (RT) has adapted to a low dNTP environment, we evaluated SAMHD1 sensitivity of chimeric HIV-1 and SIV variants in which the RT regions were functionally exchanged.Results: Here, we demonstrate that HIV-1 efficiently infects macrophages without modulating SAMHD1 activity or cellular dNTP levels, and that macrophages permissive to HIV-1 infection remained refractory to superinfection by vpx-deleted SIV. Furthermore, through the use of chimeric HIV/SIV, we demonstrate that the differential sensitivity of HIV-1 and SIV to SAMHD1 restriction is not dictated by RT.Conclusions: Our study reveals fundamental differences between HIV-1 and SIV in the strategy used to evade restriction by SAMHD1 and suggests a degree of resistance of HIV-1 to the antiviral environment created by SAMHD1. Understanding how these cellular restrictions antagonize viral replication will be important for the design of novel antiviral strategies.Keywords: HIV-1/ macrophages/ SAMHD1

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Felip, Eudald, Lucía Gutiérrez-Chamorro, Maica Gómez, Edurne Garcia-Vidal, Margarita Romeo, Teresa Morán, Laura Layos et al. « Modulation of DNA Damage Response by SAM and HD Domain Containing Deoxynucleoside Triphosphate Triphosphohydrolase (SAMHD1) Determines Prognosis and Treatment Efficacy in Different Solid Tumor Types ». Cancers 14, n^o 3 (27 janvier 2022) : 641. http://dx.doi.org/10.3390/cancers14030641.

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SAMHD1 is a deoxynucleotide triphosphate (dNTP) triphosphohydrolase with important roles in the control of cell proliferation and apoptosis, either through the regulation of intracellular dNTPs levels or the modulation of the DNA damage response. However, SAMHD1′s role in cancer evolution is still unknown. We performed the first in-depth study of SAMHD1′s role in advanced solid tumors, by analyzing samples of 128 patients treated with chemotherapy agents based on platinum derivatives and/or antimetabolites, developing novel in vitro knock-out models to explore the mechanisms driving SAMHD1 function in cancer. Low (or no) expression of SAMHD1 was associated with a positive prognosis in breast, ovarian, and non-small cell lung cancer (NSCLC) cancer patients. A predictive value was associated with low-SAMHD1 expression in NSCLC and ovarian patients treated with antimetabolites in combination with platinum derivatives. In vitro, SAMHD1 knock-out cells showed increased γ-H2AX and apoptosis, suggesting that SAMHD1 depletion induces DNA damage leading to cell death. In vitro treatment with platinum-derived drugs significantly enhanced γ-H2AX and apoptotic markers expression in knock-out cells, indicating a synergic effect of SAMHD1 depletion and platinum-based treatment. SAMHD1 expression represents a new strong prognostic and predictive biomarker in solid tumors and, thus, modulation of the SAMHD1 function may constitute a promising target for the improvement of cancer therapy.

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Qin, Zhihua, Serena Bonifati, Corine St. Gelais, Tai-Wei Li, Sun-Hee Kim, Jenna M. Antonucci, Bijan Mahboubi et al. « The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells ». Journal of Biological Chemistry 295, n^o 6 (30 décembre 2019) : 1575–86. http://dx.doi.org/10.1074/jbc.ra119.010360.

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Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing WT SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-α, IFN-β, and TNF-α mRNA levels induced by Sendai virus infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in lipopolysaccharide-treated or Sendai virus–infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-β mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1's nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.

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An, Ni, Jianyuan Zhao et Shan Cen. « Interferon-Stimulated SAMHD1 Restricts Hepatitis C Virus Replication ». Proceedings 50, n^o 1 (13 juin 2020) : 42. http://dx.doi.org/10.3390/proceedings2020050042.

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Human SAMHD1 is an IFN-induced dNTP triphosphatase that is able to restrict HIV-1 replication, whereas its role in innate immunity against virus infection remains largely unexplored. In this work, we provided evidence that SAMHD1 functions as an anti-HCV host factor. We found that overexpression of SAMHD1 resulted in significant inhibition on the replication of HCV, but not other RNA viruses including influenza A virus and EV71. SAMHD1 knockdown partially relieved the inhibitory effect of IFN on HCV, suggesting its important role in the innate immune response against HCV. Mechanistic studies revealed that SAMHD1 targets viral RNA replication without impact on both protein translation and virus entry. Transcriptome analysis showed a broad inhibitory effect of SAMHD1 on host genes involved in cholesterol and fatty acid biosynthesis. In particular, SAMHD1 was shown to downregulate the mRNA abundance of SREBP1, a master transcriptional regulator of de novo lipid biosynthesis, impairing the formation of lipid droplets. Restoring intracellular lipid levels by either exogenous lipid addition or SREBP1 overexpression counteracted the restriction of HCV by SAMHD1, providing evidence that SAMHD1 inhibits the replication of HCV by suppressing host cholesterol and fatty acid biosynthesis. Together, these data unveil, for the first time, a novel antiviral mechanism of SAMHD1 and open new avenues for the development of novel anti-HCV therapeutics.

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Knecht, Kirsten M., Olga Buzovetsky, Constanze Schneider, Dominique Thomas, Vishok Srikanth, Lars Kaderali, Florentina Tofoleanu et al. « The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1 ». Proceedings of the National Academy of Sciences 115, n^o 43 (10 octobre 2018) : E10022—E10031. http://dx.doi.org/10.1073/pnas.1805593115.

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SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in noncycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analog drugs important for treating a variety of cancers, including acute myeloid leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We found that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogs tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regard to nucleotide analogs, which can be used to improve current cancer and antiviral therapies.

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Deutschmann, Janina, et Thomas Gramberg. « SAMHD1 … and Viral Ways around It ». Viruses 13, n^o 3 (2 mars 2021) : 395. http://dx.doi.org/10.3390/v13030395.

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The SAM and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase that plays a crucial role for a variety of different cellular functions. Besides balancing intracellular dNTP concentrations, facilitating DNA damage repair, and dampening excessive immune responses, SAMHD1 has been shown to act as a major restriction factor against various virus species. In addition to its well-described activity against retroviruses such as HIV-1, SAMHD1 has been identified to reduce the infectivity of different DNA viruses such as the herpesviruses CMV and EBV, the poxvirus VACV, or the hepadnavirus HBV. While some viruses are efficiently restricted by SAMHD1, others have developed evasion mechanisms that antagonize the antiviral activity of SAMHD1. Within this review, we summarize the different cellular functions of SAMHD1 and highlight the countermeasures viruses have evolved to neutralize the restriction factor SAMHD1.

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Ji, Xiaoyun, Ying Wu, Junpeng Yan, Jennifer Mehrens, Maria DeLucia, Caili Hao, Angela Gronenborn, Jacek Skowronski, Jinwoo Anh et Yong Xiong. « Kill HIV by Starvation–Mechanism of Allosteric Activation of SAMHD1 ». Acta Crystallographica Section A Foundations and Advances 70, a1 (5 août 2014) : C121. http://dx.doi.org/10.1107/s2053273314098787.

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SAMHD1, a deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase), prevents the infection of blood cells by retroviruses, including HIV, by depleting the cellular dNTP pool available for viral reverse transcription. SAMHD1 is a major regulator of cellular dNTP levels in mammalian cells. Mutations in SAMHD1 are associated with the autoimmune condition Aicardi Goutières Syndrome (AGS), whose clinical manifestations resemble congenital viral infection. The catalytic activity of SAMHD1 is regulated by allosteric binding of dGTP, which enables SAMHD1 monomers/dimers to assemble into the catalytically active tetrameric form. We have determined the crystal structure of the tetrameric human SAMHD1-dGTP complex. The structure reveals an elegant allosteric mechanism of activation via dGTP-induced assembly of the tetrameric complex from two inactive dimers. Intriguingly, GTP can also activate SAMHD1, and our data further show the binding promiscuity of other dNTPs at the allosteric site. These findings suggest a regulation system that may have a profound effect on the balancing of cellular dNTP pools. These results provide the basis for a mechanistic understanding of SAMHD1 function in HIV restriction, the pathogenesis of AGS, and regulation of cellular dNTP levels.

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Kim, Eui Tae, Tommy E. White, Alberto Brandariz-Núñez, Felipe Diaz-Griffero et Matthew D. Weitzman. « SAMHD1 Restricts Herpes Simplex Virus 1 in Macrophages by Limiting DNA Replication ». Journal of Virology 87, n^o 23 (25 septembre 2013) : 12949–56. http://dx.doi.org/10.1128/jvi.02291-13.

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Macrophages play important roles in host immune defense against virus infection. During infection by herpes simplex virus 1 (HSV-1), macrophages acquire enhanced antiviral potential. Restriction of HSV-1 replication and progeny production is important to prevent viral spread, but the cellular mechanisms that inhibit the DNA virus in macrophages are unknown. SAMHD1 was recently identified as a retrovirus restriction factor highly expressed in macrophages. The SAMHD1 protein is expressed in both undifferentiated monocytes and differentiated macrophages, but retroviral restriction is limited to differentiated cells by modulation of SAMHD1 phosphorylation. It is proposed to block reverse transcription of retroviral RNA into DNA by depleting cellular deoxynucleotide triphosphates (dNTPs). Viruses with DNA genomes do not employ reverse transcription during infection, but replication of their viral genomes is also dependent on intracellular dNTP concentrations. Here, we demonstrate that SAMHD1 restricts replication of the HSV-1 DNA genome in differentiated macrophage cell lines. Depleting SAMHD1 in THP-1 cells enhanced HSV-1 replication, while ectopic overexpression of SAMHD1 in U937 cells repressed HSV-1 replication. SAMHD1 did not impact viral gene expression from incoming HSV-1 viral genomes. HSV-1 restriction involved the dNTP triphosphohydrolase activity of SAMHD1 and was partially overcome by addition of exogenous deoxynucleosides. Unlike retroviruses, restriction of HSV-1 was not affected by SAMHD1 phosphorylation status. Our results suggest that SAMHD1 functions broadly to inhibit replication of DNA viruses in nondividing macrophages.

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Wu, Li. « Cellular and Biochemical Mechanisms of the Retroviral Restriction Factor SAMHD1 ». ISRN Biochemistry 2013 (17 juillet 2013) : 1–11. http://dx.doi.org/10.1155/2013/728392.

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Replication of HIV-1 and other retroviruses is dependent on numerous host proteins in the cells. Some of the host proteins, however, function as restriction factors to block retroviral infection of target cells. The host protein SAMHD1 has been identified as the first mammalian deoxynucleoside triphosphate triphosphohydrolase (dNTPase), which blocks the infection of HIV-1 and other retroviruses in non-cycling immune cells. SAMHD1 protein is highly expressed in human myeloid-lineage cells and CD4+ T-lymphocytes, but its retroviral restriction function is only observed in noncycling cells. Recent studies have revealed biochemical mechanisms of SAMHD1-mediated retroviral restriction. In this review, the latest progress on SAMHD1 research is summarized and the mechanisms by which SAMHD1 mediates retroviral restriction are analyzed. Although the physiological function of SAMHD1 is largely unknown, this review provides perspectives about the role of endogenous SAMHD1 protein in maintaining normal cellular function, such as nucleic acid metabolism and the proliferation of cells.

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Ezeonwumelu, Ifeanyi, Marc Castellví, Eudald Felip, Maria Pujantell, Edurne Garcia-Vidal, Eva Riveira-Muñoz, Roger Badia, Bonaventura Clotet, Mireia Margelí et Ester Ballana. « SAMHD1 Is a Modulator of Nucleos(t)ide Analogues’ Efficacy ». Proceedings 50, n^o 1 (15 juin 2020) : 58. http://dx.doi.org/10.3390/proceedings2020050058.

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Nucleos(t)ide analogues are commonly used in the treatment of infectious disease and cancer. SAMHD1 is a deoxyribonucleotide (dNTP) triphosphohydrolase which is involved in the regulation of the intracellular dNTP pool, whose function has been linked to viral restriction, cancer development, and autoimmune disorders. Here, we evaluate SAMHD1 function on the antiviral and antiproliferative efficacy of a wide range of nucleos(t)ide analogues which are currently used to treat infections and cancer. The anti-HIV-1 and cytotoxic activity of compounds was assessed in primary and established cell lines in the presence or absence of SAMHD1. SAMHD1 effectively modified the anti-HIV-1 activity of all the nucleos(t)ide analogues tested, whereas sensitivity to a non-nucleoside inhibitor (nevirapine) or nucleoside phosphonates (cidofovir and tenofovir) was not affected. Interestingly, SAMHD1 could either enhance (gemcitabine, capecitabine, fluorouracil, and floxuridine) or inhibit (Ara-C, fludarabine, cladribine, clofarabine, and nelarabine) the antiviral potency of anticancer analogues, an effect that was not dependent on the specific nucleotide targeted. When cytotoxicity was evaluated, SAMHD1-dependent changes were less evident and were restricted to the increased efficacy of fluorouracil and floxuridine and reduced efficacy of nelarabine and ara-C in the presence of SAMHD1. In summary, our results demonstrate that SAMHD1 modifies the efficacy of a wide variety of nucleoside analogues which are used to treat infections, cancer, and other diseases. In addition, the anti-HIV activity of nucleos(t)ide analogues may represent a more sensitive measure of SAMHD1’s impact on drug efficacy. Thus, modulation of SAMHD1’s function may constitute a promising target for the improvement of multiple therapies.

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Castellví, Marc, Eudald Felip, Ifeanyi Ezeonwumelu, Roger Badia, Edurne Garcia-Vidal, Maria Pujantell, Lucía Gutiérrez-Chamorro et al. « Pharmacological Modulation of SAMHD1 Activity by CDK4/6 Inhibitors Improves Anticancer Therapy ». Cancers 12, n^o 3 (18 mars 2020) : 713. http://dx.doi.org/10.3390/cancers12030713.

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Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase involved in the regulation of the intracellular dNTP pool, linked to viral restriction, cancer development and autoimmune disorders. SAMHD1 function is regulated by phosphorylation through a mechanism controlled by cyclin-dependent kinases and tightly linked to cell cycle progression. Recently, SAMHD1 has been shown to decrease the efficacy of nucleotide analogs used as chemotherapeutic drugs. Here, we demonstrate that SAMHD1 can enhance or decrease the efficacy of various classes of anticancer drug, including nucleotide analogues, but also anti-folate drugs and CDK inhibitors. Importantly, we show that selective CDK4/6 inhibitors are pharmacological activators of SAMHD1 that act by inhibiting its inactivation by phosphorylation. Combinations of a CDK4/6 inhibitor with nucleoside or folate antimetabolites potently enhanced drug efficacy, resulting in highly synergic drug combinations (CI < 0.04). Mechanistic analyses reveal that cell cycle-controlled modulation of SAMHD1 function is the central process explaining changes in anticancer drug efficacy, therefore providing functional proof of the potential of CDK4/6 inhibitors as a new class of adjuvants to boost chemotherapeutic regimens. The evaluation of SAMHD1 expression in cancer tissues allowed for the identification of cancer types that would benefit from the pharmacological modulation of SAMHD1 function. In conclusion, these results indicate that the modulation of SAMHD1 function may represent a promising strategy for the improvement of current antimetabolite-based treatments.

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Merrien, Magali, Agata M. Wasik, Elin Ljung, Mohammad H. A. Morsy, Joana de Matos Rodrigues, Mattias Carlsten, Georgios Z. Rassidakis et al. « Clinical and biological impact of SAMHD1 expression in mantle cell lymphoma ». Virchows Archiv 480, n^o 3 (4 novembre 2021) : 655–66. http://dx.doi.org/10.1007/s00428-021-03228-w.

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AbstractSAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that restricts viral replication in infected cells and limits the sensitivity to cytarabine by hydrolysing its active metabolite, as recently shown in acute myeloid leukemia. Cytarabine is an essential component in the Nordic mantle cell lymphoma protocols (MCL2 and MCL3) for induction and high-dose chemotherapy treatment before autologous stem cell transplantation for younger patients with mantle cell lymphoma (MCL). We here investigated the expression of SAMHD1 in a population-based cohort of MCL (N = 150). SAMHD1 was highly variably expressed in MCL (range, 0.4% to 100% of positive tumor cells). Cases with blastoid/pleomorphic morphology had higher SAMHD1 expression (P = 0.028) and SAMHD1 was also correlated to tumor cell proliferation (P = 0.016). SAMHD1 expression showed moderate correlation to the expression of the transcriptional regulator SOX11 (P = 0.036) but genetic silencing of SOX11 and SAMHD1 by siRNA in MCL cell lines did not suggest mutual regulation. We hypothesized that expression of SAMHD1 could predict short time to progression in patients treated with Cytarabine as part of high-dose chemotherapy. Despite the correlation with known biological adverse prognostic factors, neither low or high SAMHD1 expression correlated to PFS or OS in patients treated according to the Nordic MCL2 or MCL3 protocols (N = 158).

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Daddacha, Waaqo, Dominique Monroe, Kristen Carver, Edidiong R. Usoro, Ahmet Alptekin, Hongyan Xu, Satoru Osuka, Ali S. Arbab et Daitoku Sakamuro. « Viral Particle-Mediated SAMHD1 Depletion Sensitizes Refractory Glioblastoma to DNA-Damaging Therapeutics by Impairing Homologous Recombination ». Cancers 14, n^o 18 (16 septembre 2022) : 4490. http://dx.doi.org/10.3390/cancers14184490.

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The current standard-of-care treatment for glioblastoma includes DNA damaging agents, γ-irradiation (IR) and temozolomide (TMZ). These treatments fail frequently and there is limited alternative strategy. Therefore, identifying a new therapeutic target is urgently needed to develop a strategy that improves the efficacy of the existing treatments. Here, we report that tumor samples from GBM patients express a high level of SAMHD1, emphasizing SAMHD1’s importance. The depletion of SAMHD1 using virus-like particles containing Vpx, VLP(+Vpx), sensitized two independent GBM cell lines (LN-229 and U-87) to veliparib, a well-established PARP inhibitor, and slowed cell growth in a dose-dependent manner. In the mouse GBM xenograft model, Vpx-mediated SAMHD1 depletion reduced tumor growth and SAMHD1 knockout (KO) improved survival. In combination with IR or TMZ, SAMHD1 KO and exposure to 50% growth inhibitory dose (gID50) of VLP(+Vpx) displayed a synergistic effect, resulting in impaired HR, and improved LN-229 cells’ sensitivity to TMZ and IR. In conclusion, our finding demonstrates that SAMHD1 promotes GBM resistance to treatment, and it is a plausible therapeutic target to improve the efficacy of TMZ and IR in GBM. Furthermore, we show that Vpx could be a potential therapeutic tool that can be utilized to deplete SAMHD1 in GBM.

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Zhu, Chun-Feng, Wei Wei, Xin Peng, Yu-Hui Dong, Yong Gong et Xiao-Fang Yu. « The mechanism of substrate-controlled allosteric regulation of SAMHD1 activated by GTP ». Acta Crystallographica Section D Biological Crystallography 71, n^o 3 (26 février 2015) : 516–24. http://dx.doi.org/10.1107/s1399004714027527.

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SAMHD1 is the only known eukaryotic deoxynucleoside triphosphate triphosphohydrolase (dNTPase) and is a major regulator of intracellular dNTP pools. It has been reported to be a potent inhibitor of retroviruses such as HIV-1 and endogenous retrotransposons. Previous crystal structures have revealed that SAMHD1 is activated by dGTP-dependent tetramer formation. However, recent data have indicated that the primary activator of SAMHD1 is GTP, not dGTP. Therefore, how its dNTPase activity is regulated needs to be further clarified. Here, five crystal structures of the catalytic core of SAMHD1 in complex with different combinations of GTP and dNTPs are reported, including a GTP-bound dimer and four GTP/dNTP-bound tetramers. The data show that human SAMHD1 contains two unique activator-binding sites in the allosteric pocket. The primary activator GTP binds to one site and the substrate dNTP (dATP, dCTP, dUTP or dTTP) occupies the other. Consequently, both GTP and dNTP are required for tetramer activation of the enzyme. In the absence of substrate binding, SAMHD1 adopts an inactive dimer conformation even when complexed with GTP. Furthermore, SAMHD1 activation is regulated by the concentration of dNTP. Thus, the level of dNTP pools is elegantly regulated by the self-sensing ability of SAMHD1 through a novel activation mechanism.

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Farrajota Neves Da Silva, Pedro, Nikolaos Tsesmetzis, Ioanna Xagoraris, Agata Magdalena Wasik, Georgia Kokaraki, Evangelos Tzoras, Anders Österborg, Birgitta Sander, Nikolas Herold et George Rassidakis. « The Novel Tumor Suppressor SAMHD1 Is Differentially Expressed and Partly Regulated By MYC in Peripheral T-Cell Lymphomas (PTCL) ». Blood 132, Supplement 1 (29 novembre 2018) : 4130. http://dx.doi.org/10.1182/blood-2018-99-119229.

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Abstract Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which has been initially described to restrict human immunodeficiency virus type 1 (HIV-1) in certain cell types through depletion of intracellular dNTP substrates required for HIV-1 reverse transcription. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS) and have been identified as putative drivers of chronic lymphocytic leukemia resulting in decreased SAMHD1 mRNA and protein levels. More recently, SAMHD1 mutations have been reported in T-prolymphocytic leukemia (T-PLL). Based on these findings and the fact that SAMHD1 limits the dNTP pool in the cell, it may play a role in oncogenesis as a tumor suppressor. In addition, SAMHD1 may confer resistance to nucleoside-based chemotherapies by hydrolysing their active triphosphate metabolites, with cytarabine in acute myeloid leukemia being an example (Herold et al, Nat Med 2017; 23(2):256-263). The expression patterns and the potential role of SAMHD1 in the pathogenesis of peripheral T-cell lymphomas (PTCL) are not yet known. Methods: The patient cohort included 64 PTCLs of various histologic types which were diagnosed and treated at Karolinska University Hospital (Sweden). A control group of 4 reactive lymph nodes and 2 reactive tonsils was included in the study for comparison. All tissue samples were obtained prior to therapy. SAMHD1 expression was assessed by immunohistochemistry performed on a PTCL tissue microarray (TMA) with duplicate tumor cores from each case or full tissue sections using dual immunostaining (SAMHD1 / CD68) and a monoclonal antibody against SAMHD1 (Bethyl Laboratories, San Antonio, TX). At least 500 lymphoma cells were counted to calculate the percentage of SAMHD1-positive tumor cells. Overall survival (OS) was defined as time from diagnosis to death or last follow-up. Event-free survival (EFS) was defined as time from diagnosis to relapse, death, or last follow-up. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models. Two T-cell lymphomas cell lines (Mac1, Mac2A) were used as an in vitro system. As our preliminary findings from in silico analysis revealed potential binding sites for MYC on the SAMHD1 gene promoter, we hypothesized that MYC might regulate SAMHD1 expression. Therefore, the T-cell lymphoma cell lines were treated with the selective BET / MYC inhibitor JQ-1 or transiently transfected with a MYC-overexpressing plasmid or MYC gene-specific siRNA constructs, respectively. Western blot analysis was used to assess the protein levels. Results: SAMHD1 protein was expressed in reactive T-cells and histiocytes (CD68+) in all reactive lymphoid tissues (lymph nodes and tonsils) with strong staining intensity. SAMHD was differentially expressed among PTCL subtypes generally with weaker staining intensity as compared to normal T-cells and histiocytes, thus being positive in all (100%) angioimmunoblastic T-cell lymphomas (AILT), 67% PTCL-NOS, 45% ALK+ ALCL, 20% of ALK+ ALCL, and none (0%) of T-lymphoblastic lymphomas (p=0.0017, chi-square test). Among the SAMHD1- positive cases, the percentage of positive lymphoma cells ranged from 0 to 100% and its highest median was observed in AILT. SAMHD1 expression inversely correlated with CD30 expression (% CD30+ positive lymphoma cells) (p=0.0025, Mann-Whitney test). No significant associations between SAMHD1 levels and other clinicopathologic parameters or clinical outcome (EFS or OS) were found, however, the number of patients analyzed in each histologic subtype was limited. Inhibition of MYC activity by JQ-1 or MYC gene silencing with specific siRNA resulted in a substantial increase in the SAMHD1 protein level in T-cell lymphoma cell lines. Inversely, transient transfection of the cell lines with a MYC overexpressing plasmid resulted in decreased levels of SAMHD1. Taken together, the in vitro data suggest a possible MYC-associated regulation (repression) of SAMHD1 gene expression in T-cell lymphoma. Conclusions: SAMHD1 is shown for the first time to be differentially expressed among PTCL types and its regulation may involve MYC. Preliminary survival analysis shows no significant associations of SAMHD1 expression with EFS and OS in this cohort of PTCL, however, analysis of a larger PTCL study group is underway to draw definite conclusions. Disclosures Österborg: Gilead: Consultancy, Research Funding; Beigene: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Abbvie: Research Funding.

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Clifford, Ruth, Sam Ackroyd, Adam Burns, Adele Timbs, Jonathan Maelfait, Helene Dreau, Shirley Henderson et al. « SAMHD1, A Putative Tumour Suppressor, Is Recurrently Mutated in Chronic Lymphocytic Leukaemia, and Is Associated with Poor Risk Features ». Blood 120, n^o 21 (16 novembre 2012) : 713. http://dx.doi.org/10.1182/blood.v120.21.713.713.

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Abstract Abstract 713 Chronic lymphocytic leukaemia (CLL) has a highly variable clinical course. Recent whole genome sequencing (WGS) data reflects this heterogeneity revealing low level recurrent somatic mutations (Puente Nature 2011, Quesada Nat Gen 2011, Wang NEJM 2011, Schuh Blood 2012). Our WGS study of sequential samples from 3 patients revealed candidate founder mutations that were present in all cells at all time-points. We focus on one of the genes affected by founder mutations; SAMHD1. SAMHD1 has been identified as an anti-viral restriction factor that targets HIV-1 by blocking reverse transcription of viral RNA (Laguette Nature 2011, Hrecka Nature 2011). The recently elucidated triphosphohydrolase activity of SAMHD1 leads to depletion of deoxynucleotide triphosphates (dNTP) during reverse transcription, thus interrupting the viral replication cycle before integration into the genome (Goldstone Nature 2011). In constitutional disease, recessive mutations in SAMHD1 have been implicated in deregulation of the innate immune response and development of a congenital autoimmune encephalopathy, Aicardi–Gouti ères syndrome (AGS) (Crow Hum Mol Gen 2009). In our single institutional cohort of 100 1st line and relapsed CLL patients we identified 8 patients with acquired mutations in SAMHD1, of which 6 were chemorefractory (Table 1). This is much higher than the expected frequency of 25% chemorefractory patients in this cohort (Knight Leukemia 2012), implying a correlation between SAMHD1 mutations and poor outcome. In order to precisely establish the incidence of SAMHD1 mutations in patients requiring 1st line treatment, we sequenced 200 samples from patients recruited into first line UK clinical trials. We determined a mutation frequency of 3% (Table 1). Whole genome SNP arrays on our SAMHD1 mutated patients reveals monoalleleic deletions or copy neutral loss of heterozygosity at the SAMHD1 locus in 14 of 15 samples. TABLE 1: SAMHD1 mutated patients Sample Age IgHV mutation TP53 disruption Clinical Status Mutation 1 72 U Neg Pre Treatment c.-166G>T 2 72 M Neg Refractory M1K K523X 3 NK U Neg Refractory Y155C 4 65 U Neg Refractory R145X 5 NK NK NK Pre Treatment R145Q 6 63 U Neg Pre Treatment I136T 7 77 M Neg Refractory E355K 8 68 U Neg Refractory L431S 9 72 U Neg Refractory F545L 10 NK NK NK Pre Treatment W572X 11 69 NK NK Pre Treatment N/A 12 66 M Pos Refractory T365P 13 NK NK Neg Pre Treatment R371H 14 77 M Neg Refractory N/A 15 25 NK Neg AGS N/A U=Unmutated, M=Mutated. Next, we questioned whether patients with congenital SAMHD1 mutations are more susceptible to developing B-cell malignancies. We reviewed 20 patients with AGS and homozygous SAMHD1 mutations. Intriguingly, 2 of these patients have developed a B-cell malignancy. One of these patients presented at 1 month of age with features typical of AGS and has been subsequently diagnosed with CLL at the age of 25. Sequencing the SAMHD1 locus of both germline and CLL cells from this patient confirmed the homozygous 1609-1G4C mutation. We screened the patient's CLL cells for acquired mutations recently found to be recurrent in CLL. None of these genes were mutated suggesting the SAMHD1 germline mutation was sufficient to cause CLL. In addition, whole exome analysis is in progress for a more complete view of acquired mutations potentially contributing to CLL pathogenesis. To evaluate the interplay of recurrent somatic mutations in CLL in the context of our SAMHD1 mutations, we used a custom designed targeted sequencing panel (TruSeq Custom Amplicon, Illumina). SAMHD1 mutations were found exclusively in SF3B1 negative patients. Only one SAMHD1 patient had a TP53 mutation. To begin to functionally define the role of SAMHD1 mutations in CLL, we examined the impact of SAMHD1 mutations on SAMHD1 mRNA gene expression by quantitative PCR analysis of purified CLL cells and normal B cell controls. Expression in the mutated CLL cells was significantly lower compared to normal B cells. From this, we hypothesise that CLL cells with SAMHD1 mutations might show an increase in intracellular dNTP levels. We are currently evaluating the levels of dNTPs using a custom designed qPCR to measure dNTP incorporation onto template DNA. In conclusion, we provide the first evidence that the lentiviral restriction factor and dNTP triphosphohydrolase SAMHD1 acts as a tumour suppressor in human B cells. We propose that deregulation of the dNTP pool in B cells caused by mutations in SAMHD1 might contribute to lymphomagenesis. Disclosures: Ross: Illumina: Employment. Bentley:Illumina: Employment. Hillmen:Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Morrissey, Catherine, David Schwefel, Valerie Ennis-Adeniran, Ian A. Taylor, Yanick J. Crow et Michelle Webb. « The eukaryotic elongation factor eEF1A1 interacts with SAMHD1 ». Biochemical Journal 466, n^o 1 (6 février 2015) : 69–76. http://dx.doi.org/10.1042/bj20140203.

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We have demonstrated that SAMHD1 interacts with eukaryotic elongation factor 1A1 (eEF1A1). The interaction occurs in the cytoplasm and is increased in cells carrying mutant SAMHD1. Interactions with ubiquitination proteins support the possibility that eEF1A1 may target SAMHD1 to the proteasome.

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Zhang, Lei, Haojie Dong, Xin He, Amandeep Salhotra, Guido Marcucci, Yun Luo et Ling Li. « SAMHD1 Inhibitor Combined with Immune Checkpoint Inhibitor Elicits an Anti-AML Immunity ». Blood 142, Supplement 1 (28 novembre 2023) : 47. http://dx.doi.org/10.1182/blood-2023-182463.

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Despite advances in targeted therapy and use of molecular/cytogenetic risk stratification to guide treatment, clinical outcomes for patients with acute myeloid leukemia (AML) remain unfavorable. Harnessing the immune system with CAR-T or immune checkpoint inhibitors (ICIs) has been proven successful in other hematological malignancies and solid tumors. The use of these immunotherapies for the AML treatment remains challenging due to lack of selective target antigens or low expression of immune checkpoint proteins. Recently, one strategy proposed to transform AML from immune-cold to immune-hot is to trigger a type-I interferon (IFN-I) response via a dsDNA related cGAS/STING signaling or dsDNA related RIG-I/MDA5 signaling. We recently reported that the dNTP and Ara-CTP (active form of cytarabine [AraC]) hydrolase SAMHD1, whose expression levels are elevated in AML patients relative to normal donors, limits IFN-I and T cell responses in murine AML model ( Blood, 2022, 140 [Supplement 1]: 679-680). We also found that innate immune activation seen following SAMHD1 knockdown is associated with its catalytic activity inhibition in cancer cells, providing a rationale for the development of SAMHD1 inhibitor. To identify SAMHD1 inhibitor, we conducted a virtual screen on 8,000,000 compounds based on SAMHD1 crystal structure (PBD 6TXC). The top 500 hits were then assessed in SAMHD1-WT- or H233A (SAMHD1 loss-of-function mutant) -expressing Molm13 cells using a phenotypic screen for AraC sensitization. Specifically, a SAMHD1 inhibitor should sensitize SAMHD1-WT (S-WT) but not H233A (S-H233A) cells to AraC treatment. Among the top 20 hits from phenotypic screen, our HPLC-based cell-free and cell-based SAMHD1 enzymatic assay revealed Samin27 as the most potent hit in blocking SAMHD1 hydrolysis and promoting AraCTP accumulation. Therefore, we assessed the efficacy of Samin27 combined with AraC in vivo. Samin27 administration (25mg/kg i.p. qd, starting on day 8 for 7 days) enhanced AraC efficacy (50mg/kg i.p. qd, starting 8th day for 5 days) in NSG mice implanted with Molm13 cells, as evidenced by extended survival relative to AraC alone. Moreover, the physical interaction between Samin27 and SAMHD1 protein were confirmed in a Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance (NMR) titration assay. RNA-seq results revealed Samin27 ex-vivo treatment remarkably upregulated IFN-I response genes in Molm13 cells, consistent with transcriptome results of SAMHD1-knockdown (S-KD) Molm13 cells. SAMHD1 activity is known to restrict replication of LINE-1 (L1), which is the only autonomously active transposable elements (TE) in mammals; elevated retrotransposition induces DNA damage and promotes cytoplasmic DNA accumulation, resulting in an IFN-I response via cGAS signaling. Next, to explore the mechanism underlying SAMHD1-inhibition induced immunity, we asked if SAMHD1 loss promoted retrotransposition and upregulated TE transcripts in leukemia cells. Through re-analysis of RNA-seq data in Molm13 cells, we found SAMHD1 inhibition upregulated most L1 elements and LTR-containing endogenous retrovirus (ERV) subfamilies. To determine if Samin27 induced immunity via L1 upregulation, we employed 3TC treatment (3TC, the nucleoside reverse transcriptase inhibitor) to block L1 upregulation. 3TC treatment blocked Samin27-mediated IFN-I response genes upregulation. Given that IFN-I response can stimulate innate immune cells (macrophage and T cell) cross-priming, potentially rendering AML cells more susceptible to anti-CD47 monoclonal antibody (aCD47) treatment, we then assessed whether combining a SAMHD1 inhibitor with an aCD47 act synergistically. We first implanted murine MLL-AF9 (MA9) leukemia cells into WT B6 mice. Then we treated leukemic mice with isotype control (Ctrl), anti-CD47 (BE0283 [BioXCell], 10mg/kg, i.p. qd, 3 weeks), Samin27 (25mg/kg i.p. qd, 3 weeks) or combination. Notably, pharmacological inhibition of SAMHD1 remarkably inhibited leukemia progression, decreasing leukemia burden (Fig. A.B). In another cohort, combination treatment extended mouse survival (Fig. C). Collectively, our results may provide a rationale for combining Samin27 with aCD47 against AML population, where single anti-CD47 treatment shows little effects.

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Coggins, Si’Ana A., Bijan Mahboubi, Raymond F. Schinazi et Baek Kim. « SAMHD1 Functions and Human Diseases ». Viruses 12, n^o 4 (31 mars 2020) : 382. http://dx.doi.org/10.3390/v12040382.

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Deoxynucleoside triphosphate (dNTP) molecules are essential for the replication and maintenance of genomic information in both cells and a variety of viral pathogens. While the process of dNTP biosynthesis by cellular enzymes, such as ribonucleotide reductase (RNR) and thymidine kinase (TK), has been extensively investigated, a negative regulatory mechanism of dNTP pools was recently found to involve sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1, SAMHD1. When active, dNTP triphosphohydrolase activity of SAMHD1 degrades dNTPs into their 2′-deoxynucleoside (dN) and triphosphate subparts, steadily depleting intercellular dNTP pools. The differential expression levels and activation states of SAMHD1 in various cell types contributes to unique dNTP pools that either aid (i.e., dividing T cells) or restrict (i.e., nondividing macrophages) viral replication that consumes cellular dNTPs. Genetic mutations in SAMHD1 induce a rare inflammatory encephalopathy called Aicardi–Goutières syndrome (AGS), which phenotypically resembles viral infection. Recent publications have identified diverse roles for SAMHD1 in double-stranded break repair, genome stability, and the replication stress response through interferon signaling. Finally, a series of SAMHD1 mutations were also reported in various cancer cell types while why SAMHD1 is mutated in these cancer cells remains to investigated. Here, we reviewed a series of studies that have begun illuminating the highly diverse roles of SAMHD1 in virology, immunology, and cancer biology.

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Monit, Christopher, Elizabeth R. Morris, Christopher Ruis, Bart Szafran, Grant Thiltgen, Ming-Han Chloe Tsai, N. Avrion Mitchison et al. « Positive selection in dNTPase SAMHD1 throughout mammalian evolution ». Proceedings of the National Academy of Sciences 116, n^o 37 (26 août 2019) : 18647–54. http://dx.doi.org/10.1073/pnas.1908755116.

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The vertebrate protein SAMHD1 is highly unusual in having roles in cellular metabolic regulation, antiviral restriction, and regulation of innate immunity. Its deoxynucleoside triphosphohydrolase activity regulates cellular dNTP concentration, reducing levels below those required by lentiviruses and other viruses to replicate. To counter this threat, some primate lentiviruses encode accessory proteins that bind SAMHD1 and induce its degradation; in turn, positive diversifying selection has been observed in regions bound by these lentiviral proteins, suggesting that primate SAMHD1 has coevolved to evade these countermeasures. Moreover, deleterious polymorphisms in humanSAMHD1are associated with autoimmune disease linked to uncontrolled DNA synthesis of endogenous retroelements. Little is known about how evolutionary pressures affect these different SAMHD1 functions. Here, we examine the deeper history of these interactions by testing whether evolutionary signatures in SAMHD1 extend to other mammalian groups and exploring the molecular basis of this coevolution. Using codon-based likelihood models, we find positive selection in SAMHD1 within each mammal lineage for which sequence data are available. We observe positive selection at sites clustered around T592, a residue that is phosphorylated to regulate SAMHD1 activity. We verify experimentally that mutations within this cluster affect catalytic rate and lentiviral restriction, suggesting that virus–host coevolution has required adaptations of enzymatic function. Thus, persistent positive selection may have involved the adaptation of SAMHD1 regulation to balance antiviral, metabolic, and innate immunity functions.

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Jiang, Huinan, Chuan Li, Zhuogang Liu et Shengjing Hospital. « Expression and Relationship of SAMHD1 with Other Apoptotic and Autophagic Genes in Acute Myeloid Leukemia Patients ». Acta Haematologica 143, n^o 1 (21 août 2019) : 51–59. http://dx.doi.org/10.1159/000500822.

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Background: SAM domain- and HD domain-containing protein 1 (SAMHD1) is a cellular enzyme which is responsible for blocking replication in viruses and participates in the progression of many cancers. Objective: The aim of this study was to correlate the expression level of SAMHD1 with other apoptotic and autophagic genes in acute myeloid leukemia (AML) patients. Methods: In the present study, mRNA levels of SAMHD1 with other apoptotic and autophagic-related genes were evaluated in patients who were newly diagnosed with AML. Results: SAMHD1, Bcl-xl, Bax, Bak, XIAP, and cIAP1 were downregulated in the AML group compared to the non-AML group (p < 0.05). SAMHD1 expression did not correlate with the other genes, while most apoptotic genes were positively correlated with each other. SAMHD1 expression was not associated with the blood routine or blast percentage of the AML patients, while Bax, Bak, cIAP2, and LC3 were significantly correlated with white blood cells. No statistically significant differences were found between the studied genes and prognosis stratifications, but Bcl-xl, Bak, cIAP1, and Mcl-1, LC3 were expressed at lower levels in the unfavorable AML group compared to the controls. Conclusion: SAMHD1 and Bcl-xl, Bax, Bak, XIAP, and cIAP1 were downregulated in AML patients, while there were no significant differences in the clinical characteristics and prognosis with reference to SAMHD1 expression.

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Ahn, Jinwoo. « Functional organization of human SAMHD1 and mechanisms of HIV-1 restriction ». Biological Chemistry 397, n^o 4 (1 avril 2016) : 373–79. http://dx.doi.org/10.1515/hsz-2015-0260.

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Abstract Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) is a triphosphohydrolase that catalyzes the conversion of deoxyribonucleoside triphosphate to deoxyribonucleoside and triphosphate. SAMHD1 has been a recent focus of study since it was identified as a potent human immunodeficiency virus-1 (HIV-1) restriction factor in the intrinsic antiviral immune system. Recent biochemical and biological studies have suggested that SAMHD1 restricts HIV-1 infection in non-cycling cells by limiting the pool of deoxyribonucleoside triphosphates, thereby interfering with HIV-1 reverse transcription. SAMHD1 also possesses single-stranded DNA and RNA binding activity, with reported nuclease activity, conferring additional HIV-1 restriction function. This review summarizes current knowledge regarding the structure of SAMHD1 and the regulation of its function in HIV-1 restriction.

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Wasik, Agata Magdalena, Elin Marin, Magali Merrien, Joana de Matos Rodrigues, Martin Lord, Ioanna Xagoraris, Birger Christensson et al. « SAMHD1 Is Variably Expressed in Mantle Cell Lymphoma and Correlated to SOX11 but Not to Survival ». Blood 132, Supplement 1 (29 novembre 2018) : 4136. http://dx.doi.org/10.1182/blood-2018-99-118087.

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Abstract Introduction Mantle cell lymphoma (MCL) is an incurable disease with a median survival of 3-5 years. Most cases express SOX11, a transcription factor associated with MCL pathobiology, which serves as a diagnostic marker. Current standard first-line therapy for younger MCL patients is rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and cytarabine (Ara-C), consolidated with high dose chemotherapy with autologous stem cell transplantation (ASCT), which is associated with prolonged survival (Eskelund et al. Br J Haematol 2016). Ara-C metabolizes into its active triphosphate form Ara-CTP which inhibits proliferation of the malignant cells. Recent reports suggest that the expression of the mammalian dNTPs hydrolase SAMHD1 determines response to Ara-C in acute myeloid leukemia (AML) by hydrolyzing Ara-CTP and thus by diminishing the anti-proliferative properties of Ara-C. Consequently, in vitro downregulation of SAMHD1 resulted in sensitization of AML cells to Ara-C (Herold et al., Nat Med 2017). SAMHD1 also exhibits anti-tumor properties via regulation of dNTP pool and is recurrently mutated in CLL (Clifford et al. Blood 2014) and T-PLL (Johansson et al. Blood Cancer J. 2018). In CLL a role in DNA-repair has been suggested (Clifford et al. Blood 2014). Thus, SAMHD1 may function as a tumor suppressor. In this study, we investigated for the first time the expression patterns of SAMHD1 in MCL and its association to clinical outcome, especially in patients receiving Ara-C as part of induction for ASCT. Methods SAMHD1 and SOX11 expression was investigated by qPCR, WB and IHC on whole tissue sections or tissue microarrays. MCL cell lines were treated with gene-specific siRNA for SAMHD1 or SOX11. Data on overall survival (OS) was retrieved from patient records. For patients included in the Nordic MCL2 and MCL3 trials data on progression free survival (PFS) and OS were retrieved. Results Initial IHC showed that expression of SAMHD1 is high in normal T-cells and macrophages and low in cells in the mantle zones of reactive tonsils. Co-staining with CD20 in a heterogeneously treated cohort of primary MCL (n=104) showed a large variation between cases (median 73.15%, range: 0.4%-99.6%) and the staining intensity was lower than in T-cells. Sixty two/104 samples were also evaluated for SOX11 expression and the percentage of SOX11 positive cells moderately correlated with SAMHD1 expression (Spearman correlation coefficient 0.27, p=0.036). Analysis of mRNA expression (normalized to mRNA levels of respective genes in Granta519 cell line) of SAMHD1 (median RFI: 2.09, range: 0.18-10.69) and SOX11 (median RFI: 2.00, range: 0.00-7.11) in flow cytometry sorted primary MCL cells (n=19) showed a trend for correlation (Spearman correlation coefficient 0.45, p=0.053). However, downregulation of SOX11 by siRNA did not alter the expression of SAMHD1 and neither did downregulation of SAMHD1 by siRNA change the expression of SOX11 suggesting that the observed correlation is not due to a mutual regulation. We investigated the relation of SAMHD1 expression to clinical and pathological parameters in this cohort and found no correlation to OS, blastoid morphology, proliferation (% Ki67+ tumor cells) nor p53 positivity (>20% positively stained cells). Further, a cohort of patients treated within the Nordic MCL2 and MCL3 protocols (n=51) were investigated for SAMHD1 expression by IHC. Interestingly the distribution of SAMHD1 expression was different in this cohort (median 34.9%, range: 2.2%-97.1%; Mann-Whitney p=0.0019). Survival analysis showed a trend for better PFS and OS in patients with high (>75% SAMHD1 positive cells) and low (<25% positive cells) SAMHD1, although the differences were not significant (Kaplan-Meier with log rank test, p=0.053). Conclusions In MCL the expression of SAMHD1 varies over a broad range and correlates to expression of SOX11. However, no significant difference in PFS or OS among patients receiving Ara-C containing induction chemotherapy is found in this study. Disclosures No relevant conflicts of interest to declare.

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Rentoft, Matilda, Kristoffer Lindell, Phong Tran, Anna Lena Chabes, Robert J. Buckland, Danielle L. Watt, Lisette Marjavaara et al. « Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance ». Proceedings of the National Academy of Sciences 113, n^o 17 (11 avril 2016) : 4723–28. http://dx.doi.org/10.1073/pnas.1519128113.

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Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that several SAMHD1 mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of one SAMHD1 allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associated SAMHD1 mutations increase mutation rates in cancer cells.

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Park, Kiwon, Jeongmin Ryoo, Heena Jeong, Minsu Kim, Sungwon Lee, Sung-Yeon Hwang, Jiyoung Ahn et al. « Aicardi-Goutières syndrome-associated gene SAMHD1 preserves genome integrity by preventing R-loop formation at transcription–replication conflict regions ». PLOS Genetics 17, n^o 4 (15 avril 2021) : e1009523. http://dx.doi.org/10.1371/journal.pgen.1009523.

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The comorbid association of autoimmune diseases with cancers has been a major obstacle to successful anti-cancer treatment. Cancer survival rate decreases significantly in patients with preexisting autoimmunity. However, to date, the molecular and cellular profiles of such comorbidities are poorly understood. We used Aicardi-Goutières syndrome (AGS) as a model autoimmune disease and explored the underlying mechanisms of genome instability in AGS-associated-gene-deficient patient cells. We found that R-loops are highly enriched at transcription-replication conflict regions of the genome in fibroblast of patients bearing SAMHD1 mutation, which is the AGS-associated-gene mutation most frequently reported with tumor and malignancies. In SAMHD1-depleted cells, R-loops accumulated with the concomitant activation of DNA damage responses. Removal of R-loops in SAMHD1 deficiency reduced cellular responses to genome instability. Furthermore, downregulation of SAMHD1 expression is associated with various types of cancer and poor survival rate. Our findings suggest that SAMHD1 functions as a tumor suppressor by resolving R-loops, and thus, SAMHD1 and R-loop may be novel diagnostic markers and targets for patient stratification in anti-cancer therapy.

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Ballana, Ester, Roger Badia, Gerard Terradas, Javier Torres-Torronteras, Alba Ruiz, Eduardo Pauls, Eva Riveira-Muñoz, Bonaventura Clotet, Ramon Martí et José A. Esté. « SAMHD1 Specifically Affects the Antiviral Potency of Thymidine Analog HIV Reverse Transcriptase Inhibitors ». Antimicrobial Agents and Chemotherapy 58, n^o 8 (9 juin 2014) : 4804–13. http://dx.doi.org/10.1128/aac.03145-14.

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ABSTRACTSterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase recently recognized as an antiviral factor that acts by depleting dNTP availability for viral reverse transcriptase (RT). SAMHD1 restriction is counteracted by the human immunodeficiency virus type 2 (HIV-2) accessory protein Vpx, which targets SAMHD1 for proteosomal degradation, resulting in an increased availability of dNTPs and consequently enhanced viral replication. Nucleoside reverse transcriptase inhibitors (NRTI), one of the most common agents used in antiretroviral therapy, compete with intracellular dNTPs as the substrate for viral RT. Consequently, SAMHD1 activity may be influencing NRTI efficacy in inhibiting viral replication. Here, a panel of different RT inhibitors was analyzed for their different antiviral efficacy depending on SAMHD1. Antiviral potency was measured for all the inhibitors in transformed cell lines and primary monocyte-derived macrophages and CD4+T cells infected with HIV-1 with or without Vpx. No changes in sensitivity to non-NRTI or the integrase inhibitor raltegravir were observed, but for NRTI, sensitivity significantly changed only in the case of the thymidine analogs (AZT and d4T). The addition of exogenous thymidine mimicked the change in viral sensitivity observed after Vpx-mediated SAMHD1 degradation, pointing toward a differential effect of SAMHD1 activity on thymidine. Accordingly, sensitivity to AZT was also reduced in CD4+T cells infected with HIV-2 compared to infection with the HIV-2ΔVpx strain. In conclusion, reduction of SAMHD1 levels significantly decreases HIV sensitivity to thymidine but not other nucleotide RT analog inhibitors in both macrophages and lymphocytes.

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Ayinde, Diana, Timothée Bruel, Sylvain Cardinaud, Françoise Porrot, Julia G. Prado, Arnaud Moris et Olivier Schwartz. « SAMHD1 Limits HIV-1 Antigen Presentation by Monocyte-Derived Dendritic Cells ». Journal of Virology 89, n^o 14 (29 avril 2015) : 6994–7006. http://dx.doi.org/10.1128/jvi.00069-15.

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ABSTRACTMonocyte-derived dendritic cells (MDDC) stimulate CD8+cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses.IMPORTANCEUpon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early postentry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates the presentation of viral antigens derived from newly produced viral proteins, leading to the activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication but also impacts antigen presentation by DC. They highlight the link that exists between restriction factors and adaptive immune responses.

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Simon, Clara, Bastian Stielow, Andrea Nist, Iris Rohner, Lisa Marie Weber, Merle Geller, Sabrina Fischer, Thorsten Stiewe et Robert Liefke. « The CpG Island-Binding Protein SAMD1 Contributes to an Unfavorable Gene Signature in HepG2 Hepatocellular Carcinoma Cells ». Biology 11, n^o 4 (6 avril 2022) : 557. http://dx.doi.org/10.3390/biology11040557.

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The unmethylated CpG island-binding protein SAMD1 is upregulated in many human cancer types, but its cancer-related role has not yet been investigated. Here, we used the hepatocellular carcinoma cell line HepG2 as a cancer model and investigated the cellular and transcriptional roles of SAMD1 using ChIP-Seq and RNA-Seq. SAMD1 targets several thousand gene promoters, where it acts predominantly as a transcriptional repressor. HepG2 cells with SAMD1 deletion showed slightly reduced proliferation, but strongly impaired clonogenicity. This phenotype was accompanied by the decreased expression of pro-proliferative genes, including MYC target genes. Consistently, we observed a decrease in the active H3K4me2 histone mark at most promoters, irrespective of SAMD1 binding. Conversely, we noticed an increase in interferon response pathways and a gain of H3K4me2 at a subset of enhancers that were enriched for IFN-stimulated response elements (ISREs). We identified key transcription factor genes, such as IRF1, STAT2, and FOSL2, that were directly repressed by SAMD1. Moreover, SAMD1 deletion also led to the derepression of the PI3K-inhibitor PIK3IP1, contributing to diminished mTOR signaling and ribosome biogenesis pathways. Our work suggests that SAMD1 is involved in establishing a pro-proliferative setting in hepatocellular carcinoma cells. Inhibiting SAMD1’s function in liver cancer cells may therefore lead to a more favorable gene signature.

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Kim, Jin Young, Sunho An, Seungmee Lee, Byung Hoon Kim, Sojin Shin, Misun Choe, Eunyoung Ha et Ji Hae Seo. « SAMHD1-induced endosomal FAK signaling promotes human renal clear cell carcinoma metastasis through activating Rac1-mediated lamellipodia protrusion. » Journal of Clinical Oncology 41, n^o 16_suppl (1 juin 2023) : e16523-e16523. http://dx.doi.org/10.1200/jco.2023.41.16_suppl.e16523.

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e16523 Background: Human sterile α motif and HD domain-containing protein 1 (SAMHD1) has deoxyribonucleoside triphosphohydrolase (dNTPase) activity that allows it to defend against human immunodeficiency virus type I (HIV-1) infections and regulate the cell cycle. Although SAMHD1 mutations have been identified in various cancer types, their role in cancer is unclear. Here, we aimed to investigate the oncogenic role of SAMHD1 in human clear cell renal cell carcinoma (ccRCC), particularly as a core molecule promoting cancer cell migration. Methods: HK-2 (#22190, a normal kidney cell line), Caki-1 (#30046), Caki-2 (#30047), SN12C (#80025), and ACHN (#21611) cells were obtained from the Korean Cell Line Bank (Seoul, Korea). SN12C and ACHN cells were transfected with siRNA targeting SAMHD1. From the Broad GDAC Firehose database ( https://gdac.broadinstitute.org/ ), we downloaded RNA-sequencing (RNA-seq) and clinical data from the pankidney cohort (KIPAN), clear cell renal cell carcinoma (KIRC, ccRCC in the present study), renal papillary cell carcinoma (KIRP, PRCC in the present study), and kidney chromophobe (KICH, chRCC in the present study) datasets. We analyzed 20 pairs of human tissues (tumor and normal specimens) from Dongsan Hospital. The results are expressed as the mean ± SD or SEM. We used a two-tailed Student’s t test for single comparisons or two-way ANOVA for multiple comparisons. Results: We found that SAMHD1 participated in endocytosis and lamellipodia formation. Mechanistically, SAMHD1 contributed to the formation of the endosomal complex by binding to cortactin. Thereafter, SAMHD1-stimulated endosomal focal adhesion kinase (FAK) signaling activated Rac1, which promoted lamellipodia formation on the plasma membrane and enhanced the motility of ccRCC cells. Finally, we observed a strong correlation between SAMHD1 expression and the activation of FAK and cortactin in tumor tissues obtained from patients with ccRCC. Conclusions: In brief, these findings reveal that SAMHD1 is an oncogene that plays a pivotal role in ccRCC cell migration through the endosomal FAK-Rac1 signaling pathway.

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Morris, Elizabeth R., et Ian A. Taylor. « The missing link : allostery and catalysis in the anti-viral protein SAMHD1 ». Biochemical Society Transactions 47, n^o 4 (11 juillet 2019) : 1013–27. http://dx.doi.org/10.1042/bst20180348.

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Abstract Vertebrate protein SAMHD1 (sterile-α-motif and HD domain containing protein 1) regulates the cellular dNTP (2′-deoxynucleoside-5′-triphosphate) pool by catalysing the hydrolysis of dNTP into 2′-deoxynucleoside and triphosphate products. As an important regulator of cell proliferation and a key player in dNTP homeostasis, mutations to SAMHD1 are implicated in hypermutated cancers, and germline mutations are associated with Chronic Lymphocytic Leukaemia and the inflammatory disorder Aicardi–Goutières Syndrome. By limiting the supply of dNTPs for viral DNA synthesis, SAMHD1 also restricts the replication of several retroviruses, such as HIV-1, and some DNA viruses in dendritic and myeloid lineage cells and resting T-cells. SAMHD1 activity is regulated throughout the cell cycle, both at the level of protein expression and post-translationally, through phosphorylation. In addition, allosteric regulation further fine-tunes the catalytic activity of SAMHD1, with a nucleotide-activated homotetramer as the catalytically active form of the protein. In cells, GTP and dATP are the likely physiological activators of two adjacent allosteric sites, AL1 (GTP) and AL2 (dATP), that bridge monomer–monomer interfaces to stabilise the protein homotetramer. This review summarises the extensive X-ray crystallographic, biophysical and molecular dynamics experiments that have elucidated important features of allosteric regulation in SAMHD1. We present a comprehensive mechanism detailing the structural and protein dynamics components of the allosteric coupling between nucleotide-induced tetramerization and the catalysis of dNTP hydrolysis by SAMHD1.

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Cai, Yiqing, Xiangxiang Zhou, Shunfeng Hu, Tiange Lu, Xiaomin Chen, Mengfei Ding et Xin Wang. « SAMHD1 Performs Tumor-Promoting Effects in Diffuse Large B-Cell Lymphoma By Inhibiting DNA Damage and Sting-Mediated Cell Pyroptosis ». Blood 138, Supplement 1 (5 novembre 2021) : 869. http://dx.doi.org/10.1182/blood-2021-147268.

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Abstract Sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a mammalian triphosphohydrolase, plays a critical role in regulating DNA replication and damage repair. Dysregulation of SAMHD1 facilitates DNA damage-mediated cell proliferation, anti-tumor immune response and chemoresistance of cancer cells. Stimulator of interferon genes (STING) is a critical regulator of the innate immune response through the perception of DNA damage. Here, we investigated the functional significance of SAMHD1 and its regulatory effect on STING signaling. We first elucidated the expression level of SAMHD1 in DLBCL. Upregulation of SAMHD1 mRNA were identified in DLBCL cells (Figure 1A). High protein levels of SAMHD1 was validated in a cohort of newly diagnosed DLBCL patients (n=80). To further identify the biological functions of SAMHD1, SAMHD1-knockdown model was constructed in vitro. Loss-of SAMHD1 resulted not only in impaired proliferation but also in increased cell apoptosis and G0/G1 blockage (Figure 1B). To explore the function of SAMHD1 in vivo, xenograft DLBCL mice model was established. Mice bearing tumors with silenced SAMHD1 revealed delayed tumor growth and diminished tumor activity (Figure 1C). To further explore the functional mechanism of SAMHD1 in DLBCL, we performed RNA-sequencing (RNA-seq) in LY1 cells with and without SAMHD1-knockdown. Gene set enrichment analysis (GSEA) revealed significant enrichment of the cytosolic DNA-sensing pathway, which included an elevation of STING (Figure 2A). Reactome analysis identified the enrichment of STING-mediated introduction of host immune response, suggesting the potential regulation of SAMHD1 on STING (Figure 2B). Nervetheless, co-immunoprecipitation analysis couldn't found the direct interactions between SAMHD1 and STING (Figure 2C). As a DNA damage sensor, STING activation responses to the presence of double stranded DNA (dsDNA). Our results then showed that SAMHD1-knockdown significantly induced nuclear DNA damage and cytosolic dsDNA accumulation (Figure 2D and E), thereby triggering STING activation in DLBCL cells (Figure 2F). Next, we investigated the biological roles of STING in DLBCL. Genetic activation of STING impaired cell viability and enhanced cell death (Figure 3A-3B). To identify the form of cell death, morphological observation was performed and revealed that cell pyroptosis, represented by cell swelling with large bubbles, could be induced by elevated STING (Figure 3C). High levels of LDH were detected in STING overexpressed DLBCL cells, further verifying the emergence of cell pyroptosis (Figure 3D). To validate the involvement of STING in pyroptosis, we deleted STING by CRISPR/Cas9 genomic-editing system (Figure 3E). STING deletion not only alleviated proliferation inhibition (Figure 3F) but also restrained pyroptosis-related morphological changes and LDH release (Figure 3G and H). Expression of pyroptotic effectors were then detected to investigated the molecular mechanisms of pyroptosis. It was worth noting that the Bak/Bax-Caspase-3 mediated gasdermin E (GSDME), rather than ASC/NLRP3/Caspase-1 mediated GSDMD, was activated in STING overexpressed DLBCL cells (Figure 4A and B). Furthermore, silencing of GSDME significantly inhibited pyroptosis-induced morphological changes and LDH release (Figure 4C). The anti-tumor effects of STING agonists in DLBCL were further explored. Treatment of DMXAA, a STING agonist, significantly promoted pyroptosis in DLBCL cells (Figure 5A-5B), further resulting in suppressed proliferation in time- and dose-dependent manners in vitro (Figure 5C). Besides, DMXAA also contributes to the upregulation of immune checkpoints, especially programmed cell death protein 1 (PD-1). We further detected the synergistic effect of DMXAA and PD-1 inhibitor (BMS1166). Of note, combination of DMXAA and BMS1166 remarkably produced an enhanced anti-tumor effect on DLBCL cells in vitro (Figure 5D). In summary, our present study firstly demonstrates that SAMHD1 functions as an oncogene in DLBCL by inhibiting DNA damage induced STING activation. Intrinsic STING induces cell pyroptosis in DLBCL cells via activating the Bak/Bax-Caspase3-GSDME pathway. Moreover, investigation of drug combination highlights the synergistic effect of STING agonist and PD-1 inhibitor, providing a novel option for improving therapeutic effects of anti-PD-1 treatment in DLBCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Clifford, Ruth, Tania Louis, Pauline Robbe, Sam Ackroyd, Adam Burns, Adele T. Timbs, Glen Wright Colopy et al. « SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage ». Blood 123, n^o 7 (13 février 2014) : 1021–31. http://dx.doi.org/10.1182/blood-2013-04-490847.

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Wing, Peter AC, Tamara Davenne, Jochen Wettengel, Alvina G. Lai, Xiaodong Zhuang, Anindita Chakraborty, Valentina D’Arienzo et al. « A dual role for SAMHD1 in regulating HBV cccDNA and RT-dependent particle genesis ». Life Science Alliance 2, n^o 2 (27 mars 2019) : e201900355. http://dx.doi.org/10.26508/lsa.201900355.

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Chronic hepatitis B is one of the world’s unconquered diseases with more than 240 million infected subjects at risk of developing liver disease and hepatocellular carcinoma. Hepatitis B virus reverse transcribes pre-genomic RNA to relaxed circular DNA (rcDNA) that comprises the infectious particle. To establish infection of a naïve target cell, the newly imported rcDNA is repaired by host enzymes to generate covalently closed circular DNA (cccDNA), which forms the transcriptional template for viral replication. SAMHD1 is a component of the innate immune system that regulates deoxyribonucleoside triphosphate levels required for host and viral DNA synthesis. Here, we show a positive role for SAMHD1 in regulating cccDNA formation, where KO of SAMHD1 significantly reduces cccDNA levels that was reversed by expressing wild-type but not a mutated SAMHD1 lacking the nuclear localization signal. The limited pool of cccDNA in infectedSamhd1KO cells is transcriptionally active, and we observed a 10-fold increase in newly synthesized rcDNA-containing particles, demonstrating a dual role for SAMHD1 to both facilitate cccDNA genesis and to restrict reverse transcriptase-dependent particle genesis.

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Wichit, Sineewanlaya. « SAMHD1 Enhances Chikungunya and Zika Virus Replication in Human Skin Fibroblasts ». Proceedings 50, n^o 1 (17 juin 2020) : 81. http://dx.doi.org/10.3390/proceedings2020050081.

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Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis. We show that the expressions of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expressions of defense response proteins DDX58, STAT1, OAS3, EIF2AK2, and SAMHD1 were significantly upregulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx resulted in a strong increase or inhibition, respectively, in both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease in viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells.

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Wichit, Sineewanlaya, Rodolphe Hamel, Andreas Zanzoni, Fodé Diop, Alexandra Cribier, Loïc Talignani, Abibatou Diack et al. « SAMHD1 Enhances Chikungunya and Zika Virus Replication in Human Skin Fibroblasts ». International Journal of Molecular Sciences 20, n^o 7 (5 avril 2019) : 1695. http://dx.doi.org/10.3390/ijms20071695.

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Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using Stable Isotope Labelling by Amino acids in Cell culture (SILAC)-based mass-spectrometry analysis. We show that the expression of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expression of defense response proteins DDX58, STAT1, OAS3, EIF2AK2 and SAMHD1 was significantly up-regulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells, or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx, resulted in a strong increase or inhibition, respectively, of both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease of viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells.

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Baldauf, Hanna-Mari, Lena Stegmann, Sarah-Marie Schwarz, Ina Ambiel, Maud Trotard, Margarethe Martin, Manja Burggraf et al. « Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells ». Proceedings of the National Academy of Sciences 114, n^o 10 (22 février 2017) : 2729–34. http://dx.doi.org/10.1073/pnas.1613635114.

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Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2–like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.

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Martin, Michaël M., Roy Matkovic, Pauline Larrous, Marina Morel, Angélique Lasserre, Virginie Vauthier et Florence Margottin-Goguet. « Binding to DCAF1 distinguishes TASOR and SAMHD1 degradation by HIV-2 Vpx ». PLOS Pathogens 17, n^o 10 (26 octobre 2021) : e1009609. http://dx.doi.org/10.1371/journal.ppat.1009609.

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Human Immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) succeed to evade host immune defenses by using their viral auxiliary proteins to antagonize host restriction factors. HIV-2/SIVsmm Vpx is known for degrading SAMHD1, a factor impeding the reverse transcription. More recently, Vpx was also shown to counteract HUSH, a complex constituted of TASOR, MPP8 and periphilin, which blocks viral expression from the integrated viral DNA. In a classical ubiquitin ligase hijacking model, Vpx bridges the DCAF1 ubiquitin ligase substrate adaptor to SAMHD1, for subsequent ubiquitination and degradation. Here, we investigated whether the same mechanism is at stake for Vpx-mediated HUSH degradation. While we confirm that Vpx bridges SAMHD1 to DCAF1, we show that TASOR can interact with DCAF1 in the absence of Vpx. Nonetheless, this association was stabilized in the presence of Vpx, suggesting the existence of a ternary complex. The N-terminal PARP-like domain of TASOR is involved in DCAF1 binding, but not in Vpx binding. We also characterized a series of HIV-2 Vpx point mutants impaired in TASOR degradation, while still degrading SAMHD1. Vpx mutants ability to degrade TASOR correlated with their capacity to enhance HIV-1 minigenome expression as expected. Strikingly, several Vpx mutants impaired for TASOR degradation, but not for SAMHD1 degradation, had a reduced binding affinity for DCAF1, but not for TASOR. In macrophages, Vpx R34A-R42A and Vpx R42A-Q47A-V48A, strongly impaired in DCAF1, but not in TASOR binding, could not degrade TASOR, while being efficient in degrading SAMHD1. Altogether, our results highlight the central role of a robust Vpx-DCAF1 association to trigger TASOR degradation. We then propose a model in which Vpx interacts with both TASOR and DCAF1 to stabilize a TASOR-DCAF1 complex. Furthermore, our work identifies Vpx mutants enabling the study of HUSH restriction independently from SAMHD1 restriction in primary myeloid cells.

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Kim, Yong Chan, Kee Kwang Kim, Jeong Heon Yoon, Ethan M. Shevach et David W. Scott. « Stability of Foxp3 mRNA is controlled by SAMHD1 in human T regulatory cells ». Journal of Immunology 196, n^o 1_Supplement (1 mai 2016) : 48.10. http://dx.doi.org/10.4049/jimmunol.196.supp.48.10.

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Abstract Clinical application of antigen-specific T regulatory cells (Tregs) offers promise for the treatment of undesirable immune diseases. To achieve this goal, long-term expansion of Tregs is required to obtain sufficient numbers of cells. However, human Tregs are not stable ex vivo. Therefore, we previously developed an innovative Treg expansion protocol using phosphorothioated random oligonucleotides (ODNps25). The addition of ODNps25 successfully resulted in the stabilization of engineered antigen-specific Tregs, however, the mechanism is not fully characterized. We first identified SAMHD1 (sterile alpha motif and HD domain 1) as an ODNps25 binding protein using a UV-crosslinking pull-down strategy. SAMHD1 physically interacted with the 3′ untranslated region (UTR) of Foxp3 mRNA and was translocated from nucleus to cytoplasm after ODNps25 treatment. Importantly, addition of ODNps25 enhanced the interaction of SAMHD1 and Foxp3 mRNA significantly, and this interaction was increased by TCR stimulation. Since ODNps25 binds to the nuclease (HD) domain of SAMHD1, we then established that overexpression of a nuclease-deficient mutant (D137N) in Tregs significantly stabilized the expression level of Foxp3 protein. This mutant also enhanced stability of a luciferase reporter, followed by the 3′ UTR of Foxp3 mRNA. Additionally, we also found that phosphorylation of the threonine residue (Thr592), which is a regulatory site to control SAMHD1 activity, was clearly downregulated in Tregs. Taken together, we suggest that ODNs interact with the HD of SAMHD1 to interfere with its nuclease activity, thereby stabilizing 3′ UTR of FoxP3 mRNA in long-term culture.

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Huber, Andrew D., Eleftherios Michailidis, Megan L. Schultz, Yee T. Ong, Nicolin Bloch, Maritza N. Puray-Chavez, Maxwell D. Leslie et al. « SAMHD1 Has Differential Impact on the Efficacies of HIV Nucleoside Reverse Transcriptase Inhibitors ». Antimicrobial Agents and Chemotherapy 58, n^o 8 (27 mai 2014) : 4915–19. http://dx.doi.org/10.1128/aac.02745-14.

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ABSTRACTSterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways.

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Banchenko, Sofia, Ferdinand Krupp, Christine Gotthold, Jörg Bürger, Andrea Graziadei, Francis J. O’Reilly, Ludwig Sinn et al. « Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses ». PLOS Pathogens 17, n^o 8 (2 août 2021) : e1009775. http://dx.doi.org/10.1371/journal.ppat.1009775.

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Viruses have evolved means to manipulate the host’s ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIVmus). X-ray crystallography reveals commonalities between SIVmus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIVmus Vpr exploits the dynamic architecture of the multi-subunit CRL4DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins.

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Seamon, Kyle J., et James T. Stivers. « A High-Throughput Enzyme-Coupled Assay for SAMHD1 dNTPase ». Journal of Biomolecular Screening 20, n^o 6 (9 mars 2015) : 801–9. http://dx.doi.org/10.1177/1087057115575150.

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Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a recently discovered enzyme that plays a central role in nucleotide metabolism and innate immunity. SAMHD1 has deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase activity that depletes the dNTP substrates required for DNA synthesis in cells. The involvement of SAMHD1 in biological processes as varied as viral restriction, endogenous retroelement control, cancer, and modulation of anticancer/antiviral nucleoside drug efficacy makes it a valuable target for the development of small-molecule inhibitors. We report a high-throughput colorimetric assay for SAMHD1 dNTP hydrolase activity that takes advantage of Escherichia coli inorganic pyrophosphatase to convert PPPi to 3 Pi. The assay was validated by screening a library of 2653 clinically used compounds. Fifteen primary hits were obtained (0.57% hit rate); 80% of these were confirmed in a direct secondary assay for dNTP hydrolysis. The zinc salt of the antibiotic cephalosporin C was a potent inhibitor of SAMHD1 with an IC50 of 1.1 ± 0.1 µM, and this inhibition was largely attributable to the presence of zinc. The assay also screened a targeted library of nucleosides and their analogs, revealing that the antiviral drug acycloguanosine (acyclovir) is an inhibitor possessing excellent properties for future fragment-based drug development efforts.

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Akimova, Ekaterina, Franz Josef Gassner, Maria Schubert, Stefan Rebhandl, Claudia Arzt, Stefanie Rauscher, Vanessa Tober, Nadja Zaborsky, Richard Greil et Roland Geisberger. « SAMHD1 restrains aberrant nucleotide insertions at repair junctions generated by DNA end joining ». Nucleic Acids Research 49, n^o 5 (16 février 2021) : 2598–608. http://dx.doi.org/10.1093/nar/gkab051.

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Abstract Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.

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Jáuregui, Paula, Eric C. Logue, Megan L. Schultz, Stephanie Fung et Nathaniel R. Landau. « Degradation of SAMHD1 by Vpx Is Independent of Uncoating ». Journal of Virology 89, n^o 10 (11 mars 2015) : 5701–13. http://dx.doi.org/10.1128/jvi.03575-14.

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ABSTRACTSterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid and resting T cells. Lentiviruses such as HIV-2 and some simian immunodeficiency viruses (SIVs) counteract the restriction by encoding Vpx or Vpr, accessory proteins that are packaged in virions and which, upon entry of the virus into the cytoplasm, induce the proteasomal degradation of SAMHD1. As a tool to study these mechanisms, we generated HeLa cell lines that express a fusion protein termed NLS.GFP.SAM595 in which the Vpx binding domain of SAMHD1 is fused to the carboxy terminus of green fluorescent protein (GFP) and a nuclear localization signal is fused to the amino terminus of GFP. Upon incubation of Vpx-containing virions with the cells, the NLS.GFP.SAM595 fusion protein was degraded over several hours and the levels remained low over 5 days as the result of continued targeting of the CRL4 E3 ubiquitin ligase. Degradation of the fusion protein required that it contain a nuclear localization sequence. Fusion to the cytoplasmic protein muNS rendered the protein resistant to Vpx-mediated degradation, confirming that SAMHD1 is targeted in the nucleus. Virions treated with protease inhibitors failed to release Vpx, indicating that Gag processing was required for Vpx release from the virion. Mutations in the capsid protein that altered the kinetics of virus uncoating and the Gag binding drug PF74 had no effect on the Vpx-mediated degradation. These results suggest that Vpx is released from virions without a need for uncoating of the capsid, allowing Vpx to transit to the nucleus rapidly upon entry into the cytoplasm.IMPORTANCESAMHD1 restricts lentiviral replication in myeloid cells and resting T cells. Its importance is highlighted by the fact that viruses such as HIV-2 encode an accessory protein that is packaged in the virion and is dedicated to inducing SAMHD1 degradation. Vpx needs to act rapidly upon infection to allow reverse transcription to proceed. The limited number of Vpx molecules in a virion also needs to clear the cell of SAMHD1 over a prolonged period of time. Using an engineered HeLa cell line that expresses a green fluorescent protein (GFP)-SAMHD1 fusion protein, we showed that the Vpx-dependent degradation occurs without a need for viral capsid uncoating. In addition, the fusion protein was degraded only when it was localized to the nucleus, confirming that SAMHD1 is targeted in the nucleus and thus explaining why Vpx also localizes to the nucleus.

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