SAM Domain And HD Domain-containing Protein 1 Research Articles

Spatial resolution of HIV-1 post-entry steps in resting CD4 T cells

Resting CD4 Tcells resist productive HIV-1 infection. The HIV-2/simian immunodeficiency virus protein viral accessory protein X (Vpx) renders these cells permissive to infection, presumably by alleviating blocks at cytoplasmic reverse transcription and subsequent nuclear import of reverse-transcription/pre-integration complexes (RTC/PICs). Here, spatial analyses using quantitative virus imaging techniques reveal that HIV-1 capsids containing RTC/PICs are readily imported into the nucleus, recruit the host dependency factor CPSF6, and translocate to nuclear speckles in resting CD4 Tcells. Reverse transcription, however, remains incomplete, impeding proviral integration and viral gene expression. Vpx or pharmacological inhibition of the deoxynucleotide triphosphohydrolase (dNTPase) activity of the restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1) increases levels of nuclear reverse-transcribed cDNA and facilitates HIV-1 integration. Nuclear import and intranuclear transport of viral complexes therefore do not pose important blocks to HIV-1 in resting CD4 Tcells, and the limitation to reverse transcription by SAMHD1's dNTPase activity constitutes the main pre-integration block to infection.

Biochemical functions and structure of Caenorhabditis elegans ZK177.8 protein: Aicardi–Goutières syndrome SAMHD1 dNTPase ortholog

Mutations in SAM domain and HD domain containing protein 1 (SAMHD1) are found in a neuro-developmental disorder, Aicardi-Goutières Syndrome (AGS) and cancers, and SAMHD1, which is a dNTP tri-phosphorylase, was identified as a myeloid specific HIV-1 restriction factor. Here, we characterized the enzymology and structure of a SAMHD1 ortholog of C. elegans, ZK177.8, which also reportedly induces developmental defects upon gene knockdown. We found ZK177.8 protein is a dNTPase allosterically regulated by dGTP. The active site of ZK177.8 recognizes both 2' OH and tri-phosphate moieties of dNTPs, but not base moiety. The dGTP activator induces the formation of the enzymatically active ZK177.8 tetramers, and ZK177.8 protein lowers cellular dNTP levels in a human monocytic cell line. Finally, ZK177.8 tetramers display very similar X-ray crystal structure with human and mouse SAMHD1s except its lack of the canonical SAM domain. This striking conservation in structure, function and allosteric regulatory mechanism for the hydrolysis of the DNA building blocks supports their host developmental roles.

Vpx requires active cellular dNTP biosynthesis to effectively counteract the anti-lentivirus activity of SAMHD1 in macrophages

HIV-1 replication in primary monocyte-derived macrophages (MDMs) is kinetically restricted at the reverse transcription step due to the low deoxynucleoside triphosphates (dNTP) pools established by host dNTPase, SAM and HD domain containing protein 1 (SAMHD1). Lentiviruses such as HIV-2 and some Simian immunodeficiency virus counteract this restriction using viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates intracellular dNTP pools. However, how dNTP pools increase after Vpx degrades SAMHD1 in nondividing MDMs where no active dNTP biosynthesis is expected to exists remains unclear. In this study, we monitored known dNTP biosynthesis machinery during primary human monocyte differentiation to MDMs and unexpectedly found MDMs actively express dNTP biosynthesis enzymes such as ribonucleotide reductase, thymidine kinase 1, and nucleoside-diphosphate kinase. During differentiation from monocytes the expression levels of several biosynthesis enzymes are upregulated, while there is an increase in inactivating SAMHD1 phosphorylation. Correspondingly, we observed significantly lower levels of dNTPs in monocytes compared to MDMs. Without dNTP biosynthesis availability, Vpx failed to elevate dNTPs in monocytes, despite SAMHD1 degradation. These extremely low monocyte dNTP concentrations, which cannot be elevated by Vpx, impaired HIV-1 reverse transcription in a biochemical simulation. Furthermore, Vpx failed to rescue the transduction efficiency of a HIV-1 GFP vector in monocytes. Collectively, these data suggest that MDMs harbor active dNTP biosynthesis and Vpx requires this dNTP biosynthesis to elevate dNTP levels to effectively counteract SAMHD1 and relieve the kinetic block to HIV-1 reverse transcription in MDMs.

SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) infection by reducing the intracellular dNTP pool. We have shown that SAMHD1 suppresses nuclear factor kappa-B activation and type I interferon (IFN-I) induction by viral infection and inflammatory stimuli. However, the mechanism by which SAMHD1 inhibits IFN-I remains unclear. Here, we show that SAMHD1 inhibits IFN-I activation induced by the mitochondrial antiviral-signaling protein (MAVS). SAMHD1 interacted with MAVS and suppressed MAVS aggregation in response to Sendai virus infection in human monocytic THP-1cells. This resulted in increased phosphorylation of TANK binding kinase 1 (TBK1), inhibitor of nuclear factor kappa-B kinase epsilon (IKKε), and IFN regulatory factor 3 (IRF3). SAMHD1 suppressed IFN-I activation induced by IKKε and prevented IRF7 binding to the kinase domain of IKKε. We found that SAMHD1 interaction with the inhibitory domain (ID) of IRF7 (IRF7-ID) was necessary and sufficient for SAMHD1 suppression of IRF7-mediated IFN-I activation in HEK293T cells. Computational docking and molecular dynamics simulations revealed possible binding sites between IRF7-ID and full-length SAMHD1. Individual substitution of F411, E416, or V460 in IRF7-ID significantly reduced IRF7 transactivation activity and SAMHD1 binding. Furthermore, we investigated the role of SAMHD1 inhibition of IRF7-mediated IFN-I induction during HIV-1 infection. We found that THP-1cells lacking IRF7 expression had reduced HIV-1 infection and viral transcription compared to control cells, indicating a positive role of IRF7 in HIV-1 infection. Our findings suggest that SAMHD1 suppresses IFN-I induction through the MAVS, IKKε, and IRF7 signaling axis.

Recombinant Klotho attenuates IFNγ receptor signaling and SAMHD1 expression through blocking NF-κB translocation in glomerular mesangial cells.

Interferon gamma (IFNγ) is a cytokine implicated in the pathogenesis of autoimmune diseases. SAM and HD domain-containing protein 1 (SAMHD1) is an IFNγ-inducible protein that modulates cellular dNTP levels. Mutations in the human SAMHD1 gene cause Aicardi-Goutières (AG) syndrome, an autoimmune disease sharing similar clinical features with systemic lupus erythematosus (SLE). Klotho is an anti-inflammatory protein which suppresses aging through multiple mechanisms. Implication of Klotho in autoimmune response is identified in rheumatologic diseases such as SLE. Little information exists regarding the effect of Klotho in lupus nephritis, one of the prevalent symptoms of SLE. The present study verified the effect of IFNγ on SAMHD1 and Klotho expression in MES-13 glomerular mesangial cells, a special cell type in glomerulus that is critically involved in lupus nephritis. IFNγ upregulated SAMHD1 expression in MES-13 cells through the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT1) and the nuclear factor kappa B (NFκB) signaling pathways. IFNγ decreased Klotho protein expression in MES-13 cells. Treatment of MES-13 cells with recombinant Klotho protein inhibited SAMHD1 expression by blocking IFNγ-induced NFκB nuclear translocation, but showed no effect on JAK-STAT1 signaling. Collectively, our findings support the protective role of Klotho in attenuating lupus nephritis through the inhibition of IFNγ-induced SAMHD1 expression and IFNγ downstream signaling in MES-13 cells.

Activation of STING by SAMHD1 Deficiency Promotes PANoptosis and Enhances Efficacy of PD-L1 Blockade in Diffuse Large B-cell Lymphoma.

Genomic instability is a significant driver of cancer. As the sensor of cytosolic DNA, the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a critical role in regulating anti-tumor immunity and cell death. However, the role and regulatory mechanisms of STING in diffuse large B-cell lymphoma (DLBCL) are still undefined. In this study, we reported that sterile alpha motif and HD domain-containing protein 1 (SAMHD1) deficiency induced STING expression and inhibited tumor growth in DLBCL. High level of SAMHD1 was associated with poor prognosis in DLBCL patients. Down-regulation of SAMHD1 inhibited DLBCL cell proliferation both in vitro and in vivo. Moreover, we found that SAMHD1 deficiency induced DNA damage and promoted the expression of DNA damage adaptor STING. STING overexpression promoted the formation of Caspase 8/RIPK3/ASC, further leading to MLKL phosphorylation, Caspase 3 cleavage, and GSDME cleavage. Up-regulation of necroptotic, apoptotic, and pyroptotic effectors indicated STING-mediated PANoptosis. Finally, we demonstrated that the STING agonist, DMXAA, enhanced the efficacy of a PD-L1 inhibitor in DLBCL. Our findings highlight the important role of STING-mediated PANoptosis in restricting DLBCL progression and provide a potential strategy for enhancing the efficacy of immune checkpoint inhibitor agents in DLBCL.

Porcine Endogenous Retrovirus Induces CXCL10 in Human Monocytes and Monocyte-Derived Primary Cells

Introduction: Pigs are suitable donor species for xenotransplantation and biological materials from these animals are used for this purpose for many years. A major risk of xenotransplantation is a zoonosis by transspecies transmission of animal viruses. In this regard, porcine endogenous retroviruses (PERVs) are of paramount importance because some of them are able to infect human cells and could induce innate immune responses. Methods: Using a replication-competent polytropic PERV-A/C strain, we have analysed the induction of innate immune responses by this virus in human monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells. Results: PERV-A/C elevates the expression of the C-X-C motif chemokine 10 (CXCL10) up to 1,000-fold in human monocytes and monocyte-derived primary cells. In comparison to CXCL10, the levels of interferon-β (IFN-β) and interferon-stimulated gene 54 (ISG54) were almost unchanged. Heat-inactivated virus did not induce CXCL10 expression. Neither treatment with the reverse transcriptase inhibitors azidothymidine (AZT) and stavudine (d4T) nor treatment with the integrase inhibitor raltegravir (RAL) reduced the activation levels. Furthermore, depletion of SAM domain and HD domain-containing protein 1 (SAMHD1), a restriction factor that blocks PERV-A/C infection at the level of reverse transcription in these myeloid cells, had no significant effect on the CXCL10 induction level. These results imply that innate immune sensing leading to the strong CXCL10 response occurs at an early step of the replication process and does not require products of reverse transcription. Inhibition of Janus kinases (JAKs) by AT9283 prevented the observed CXCL10 induction by the virus, providing evidence that the JAK-STAT signalling pathway is involved in the CXCL10 response in theses myeloid cells. Conclusion: Our findings highlight PERVs as inducers of the pro-inflammatory chemokine CXCL10 and other innate immune responses in human monocytes and derived cells with potential implications in the context of xenotransplantation.

SAMHD1 deacetylation by SIRT1 promotes DNA end resection by facilitating DNA binding at double-strand breaks

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) has a dNTPase-independent function in promoting DNA end resection to facilitate DNA double-strand break (DSB) repair by homologous recombination (HR); however, it is not known if upstream signaling events govern this activity. Here, we show that SAMHD1 is deacetylated by the SIRT1 sirtuin deacetylase, facilitating its binding with ssDNA at DSBs, to promote DNA end resection and HR. SIRT1 complexes with and deacetylates SAMHD1 at conserved lysine 354 (K354) specifically in response to DSBs. K354 deacetylation by SIRT1 promotes DNA end resection and HR but not SAMHD1 tetramerization or dNTPase activity. Mechanistically, K354 deacetylation by SIRT1 promotes SAMHD1 recruitment to DSBs and binding to ssDNA at DSBs, which in turn facilitates CtIP ssDNA binding, leading to promotion of genome integrity. These findings define a mechanism governing the dNTPase-independent resection function of SAMHD1 by SIRT1 deacetylation in promoting HR and genome stability.

SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA

Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here, we show that human SAM-domain- and HD-domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3'exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic-acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.

Identification of Biomarkers That Modulate Osteogenic Differentiation in Mesenchymal Stem Cells Related to Inflammation and Immunity: A Bioinformatics-Based Comprehensive Study.

Background: Inducing mesenchymal stem cells (MSCs) osteogenesis may be beneficial in a number of clinical applications. The aim of this study is to identify key novel biomarkers of this process and to analyze the possible regulatory effects on inflammation and immunity. Results: Seven datasets (GSE159137, GSE159138, GSE114117, GSE88865, GSE153829, GSE63754, GSE73087) were obtained from the Gene Expression Omnibus database and were assigned to either the training or the validation dataset. The least absolute shrinkage and selection operator (LASSO) logistic regression model was applied to the training data to select biomarkers of osteogenesis, which were then confirmed using the validation dataset. FK506 binding protein 5 (FKBP5), insulin-like growth factor binding protein (IGFBP2), prostaglandin E receptor 2 (PTGER2), SAM domain and HD domain-containing protein 1 (SAMHD1), and transmembrane tetratricopeptide 1 (TMTC1) were highlighted as potential biomarkers. In addition, the differential expressions of immunity and inflammation-related genes were examined and their correlations with the five identified biomarkers were analyzed. The results from performing RT-qPCR and Western blots confirmed that the levels of each of these biomarkers were all significantly increased following osteogenic differentiation of MSCs. Conclusions: Our results identify five biomarkers related to MSCs osteogenesis and allow us to identify their potential roles in immunoregulation and inflammation. Each biomarker was verified by in vitro experiments.

Abstract 3034: Purine nucleoside phosphorylase regulates metabolic and immune checkpoints

Abstract Purine nucleoside phosphorylase (PNP) functions as a central regulator of nucleotide metabolism by catalyzing the degradation of purine nucleosides guanosine and deoxyguanosine. PNP inactivation has been paradoxically associated with both immunodeficiency and autoimmunity in humans but the genetic and molecular determinants of these divergent outcomes are poorly defined and whether they reflect cell type specific mechanisms is currently unknown. Moreover, the mechanisms underlying the clinical efficacy of PNP inhibitors in a subset of patients with hematological malignancies are yet to be determined, thereby severely limiting the potential of this promising new class of metabolic targeted therapies. To address these gaps in knowledge we performed a systematic evaluation of the metabolic, immunological and cell-context phenotypic effects of targeting PNP using a clinical-stage inhibitor with picomolar affinity and excellent oral bioavailability. Here we show that across cancer types PNP inhibition is synthetically lethal with sterile alpha motif and HD domain-containing protein 1 (SAMHD1) deficiency by triggering massive dGTP pool expansion, and impairing DNA replication. These cytotoxic effects require the expression of deoxycytidine kinase (dCK) which mediates the intracellular trapping of deoxyguanosine and expansion of dGTP pools, and are opposed by environmental deoxycytidine produced by bone marrow stromal cells in a SAMHD1 and nucleoside transporter dependent manner. This resistance mechanism to PNP inhibition can be counteracted by the deoxycytidine catabolizing enzyme cytidine deaminase (CDA). We further show that in addition to, and independent of its dCK and CDA-dependent cytotoxic effects in SAMHD1 deficient cells, PNP inhibition elevates serum inflammatory cytokines and cytokine transcript levels in secondary lymphoid organs. Using scRNAseq we identify CD19+ B-cells and CSF1R+ macrophages, which express the pattern recognition receptor TLR7, as the drivers of these alterations. TLR7 activation in these cell types is promoted by guanosine accumulation which functions as a ligand for TLR7 alongside uridine-containing ssRNA. Importantly, TLR7 stimulation achieved by PNP inhibition initiates transcriptional alterations that are quantitatively and qualitatively unique from synthetic TLR7 agonists, such as the production of type I interferons. We additionally demonstrate that PNP inactivation stimulates germinal center responses in secondary lymphoid organs via the expansion of activated B-cells and T follicular helper cells. In summary, we demonstrate that by modulating multiple metabolic and immune checkpoints in a multigenic, cell type and environment dependent manner, pharmacological PNP inhibition elicits specific outcomes which can be leveraged for the development of novel therapeutic applications. Citation Format: Evan R. Abt, Khalid Rashid, Thuc M. Le, Hailey R. Lee, Amanda L. Creech, Ting-Ting Wu, Thomas Mehrling, Shanta Bantia, Caius G. Radu. Purine nucleoside phosphorylase regulates metabolic and immune checkpoints [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 3034.

Disruption of dNTP homeostasis by ribonucleotide reductase hyperactivation overcomes AML differentiation blockade

AbstractDifferentiation blockade is a hallmark of acute myeloid leukemia (AML). A strategy to overcome such a blockade is a promising approach against the disease. The lack of understanding of the underlying mechanisms hampers development of such strategies. Dysregulated ribonucleotide reductase (RNR) is considered a druggable target in proliferative cancers susceptible to deoxynucleoside triphosphate (dNTP) depletion. Herein, we report an unanticipated discovery that hyperactivating RNR enables differentiation and decreases leukemia cell growth. We integrate pharmacogenomics and metabolomics analyses to identify that pharmacologically (eg, nelarabine) or genetically upregulating RNR subunit M2 (RRM2) creates a dNTP pool imbalance and overcomes differentiation arrest. Moreover, R-loop–mediated DNA replication stress signaling is responsible for RRM2 activation by nelarabine treatment. Further aggravating dNTP imbalance by depleting the dNTP hydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) enhances ablation of leukemia stem cells by RRM2 hyperactivation. Mechanistically, excessive activation of extracellular signal-regulated kinase (ERK) signaling downstream of the imbalance contributes to cellular outcomes of RNR hyperactivation. A CRISPR screen identifies a synthetic lethal interaction between loss of DUSP6, an ERK-negative regulator, and nelarabine treatment. These data demonstrate that dNTP homeostasis governs leukemia maintenance, and a combination of DUSP inhibition and nelarabine represents a therapeutic strategy.

Palbociclib in a patient with HR+/HER2- advanced breast cancer and HIV1 infection: a case report

The use of drugs that affect the cell cycle represents one of the common strategies for the control of some unrelated pathologies, such as chronic viral HIV infections or cancer. The authors report the case of a patient followed for a hormone receptor-positive (HR+)/HER2 negative (HER2-) advanced breast cancer, treated with hormone therapy and CDK 4/6 inhibitors, and a concomitant HIV infection under antiretroviral treatment. The authors consider the function of the sterile alpha motif and HD domain-containing protein-1 (SAMHD1) enzyme, its implications in the control of viral replication and the correlation between its activity and the mechanism of action of the CDK 4/6 inhibitor palbociclib.

SAMHD1 Performs Tumor-Promoting Effects in Diffuse Large B-Cell Lymphoma By Inhibiting DNA Damage and Sting-Mediated Cell Pyroptosis

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. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses.

The type I interferon (IFN) response is the major host arsenal against invading viruses. IRGM is a negative regulator of IFN responses under basal conditions. However, the role of human IRGM during viral infection has remained unclear. In this study, we show that IRGM expression is increased upon viral infection. IFN responses induced by viral PAMPs are negatively regulated by IRGM. Conversely, IRGM depletion results in a robust induction of key viral restriction factors including IFITMs, APOBECs, SAMHD1, tetherin, viperin, and HERC5/6. Additionally, antiviral processes such as MHC‐I antigen presentation and stress granule signaling are enhanced in IRGM‐deficient cells, indicating a robust cell‐intrinsic antiviral immune state. Consistently, IRGM‐depleted cells are resistant to the infection with seven viruses from five different families, including Togaviridae, Herpesviridae, Flaviviverdae, Rhabdoviridae, and Coronaviridae. Moreover, we show that Irgm1 knockout mice are highly resistant to chikungunya virus (CHIKV) infection. Altogether, our work highlights IRGM as a broad therapeutic target to promote defense against a large number of human viruses, including SARS‐CoV‐2, CHIKV, and Zika virus.

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

Dual roles of SAMHD1 in tumor development and chemoresistance to anticancer drugs.

Human sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) has been identified as a GTP or dGTP-dependent deoxynucleotide triphosphohydrolase (dNTPase) and acts as an antiviral factor against certain retroviruses and DNA viruses. Genetic mutation in SAMHD1 causes the inflammatory Aicardi-Goutières Syndrome and abnormal intracellular deoxyribonucleoside triphosphates (dNTPs) pool. At present, the role of SAMHD1 in numerous types of cancer, such as chronic lymphocytic leukemia, lung cancer and colorectal cancer, is highly studied. Furthermore, it has been found that methylation, acetylation and phosphorylation are involved in the regulation of SAMHD1 expression, and that genetic mutations can cause changes in its activities, including dNTPase activity, long interspersed element type 1 (LINE-1) suppression and DNA damage repair, which could lead to uncontrolled cell cycle progression and cancer development. In addition, SAMHD1 has been reported to have a negative regulatory role in the chemosensitivity to anticancer drugs through its dNTPase activity. The present review aimed to summarize the regulation of SAMHD1 expression in cancer and its function in tumor growth and chemotherapy sensitivity, and discussed controversial points and future directions.

SAMHD1 … and Viral Ways around It.

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.

Expression of the novel tumour suppressor sterile alpha motif and HD domain‐containing protein 1 is an independent adverse prognostic factor in classical Hodgkin lymphoma

The expression patterns and prognostic significance of sterile alpha motif and HD domain-containing protein 1 (SAMHD1) protein in the neoplastic Hodgkin and Reed Sternberg (HRS) cells of Hodgkin lymphoma (HL) were investigated in a cohort of 154 patients with HL treated with standard regimens. SAMHD1 expression was assessed by immunohistochemistry using diagnostic lymph node biopsies obtained prior to treatment. Using an arbitrary 20% cut-off, SAMHD1 was positive in HRS cells of 48/154 (31·2%) patients. SAMHD1 expression was not associated with clinicopathologic parameters, such as age, gender, stage or histologic subtype. In 125 patients with a median follow-up of 90months (7-401months), SAMHD1 expression in HRS cells significantly correlated with inferior freedom from progression (FFP) (P=0·025), disease-specific survival (DSS) (P=0·013) and overall survival (OS) (P=0·01). Importantly, in multivariate models together with disease stage, histology subtype and type of treatment as covariates, SAMHD1 expression retained an independent significant association with unfavourable FFP (P=0·005) as well as DSS (P=0·022) and OS (P=0·018). These findings uncover the significance of a novel, adverse prognostic factor in HL that may have therapeutic implications since SAMHD1 inhibitors are now available for clinical use.

Repurposing Nelarabine to Induce Differentiation of Acute Myeloid Leukemia

The success of all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL) pioneered the concept of differentiation therapy. However, comparable approaches to overcome differentiation blockage for non-APL acute myeloid leukemia (AML) are hampered by lack of an effective drug discovery platform. Recently, we analyzed gene signatures of compounds (ATRA, arsenic trioxide, zalcitabine, and sodium butyrate) that trigger myeloid differentiation in the NCI-60 collection datasets and identified CD38 as the top gene upregulated by differentiation induction. We next initiated an in silico screen in the DTP database of >20,000 compounds to identify compounds that increase CD38 levels. Among those retrieved from "CellMiner" with CD38 as input, we assessed the top 193 available from NCI (r>0.6, p=0) for effects on differentiation utilizing a conditional murine myeloid differentiation-arrest model overexpressing estrogen receptor-HoxA9 (ER-HoxA9) fusion proteins (Cell, 2016). We identified NSC755985 (Nelarabine, NEL) in that screen. NEL is an orphan drug approved to treat relapsed or refractory T-cell acute lymphoblastic leukemia (ALL). NEL at clinically achievable doses (Cmax: 6.73 μM~ 26.91 μM, at a proposed adult dosing schedule of 1,500 mg/m2/day) markedly induced primary AML cell differentiation and death while sparing normal hematopoietic cells (AML vs normal, IC50: 14.7 ± 4.3 μM, n=7, vs 45.3 ± 1.3 μM, n=3), suggesting a therapeutic window in AML. Ex vivo NEL treatment compromised BM engraftment of CD34+ cells from one primary AML specimen in immunodeficient NSG mice at 12 weeks post-transplant (human CD45+ cells in BM: NEL 0.73% vs vehicle 33.42%, n=6/group, p<0.01). NEL administration in vivo (130 mg/kg/day, i.v. for 5 consecutive days) reduced leukemic burden of NSG mice xenografted with luciferase-expressing U937 cells (Radiance: NEL 2.83×107 vs vehicle 1.65×108 photons/s/cm2, n=9/group,p<0.01) and extended mouse survival. Transcriptome analyses in U397 cells and primary AML specimens revealed that NEL treatment upregulated RAS-related pathways. NEL-elicited RAS activation was confirmed by pull-down assay using a GST-Raf1-RBD affinity probe, followed by blotting with a pan-RAS antibody. We performed functional analysis by infecting ER-HoxA9 cells with lentiviral vector expressing oncogenic RAS and observed enhanced myeloid differentiation, as evidenced by increased CD11b/GFP levels relative to MOCK-infected controls. Given that NEL's active metabolite Ara-GTP perturbs guanine nucleotide metabolism, we asked if NEL-evoked RAS activation was associated with accrual of intracellular GTP. HPLC/MS analyses of U937 cells showed that NEL treatment resulted in a marked increase in GTP (approximately 5-fold higher than baseline at sub-millimolar levels) which was secondary to Ara-GTP. Importantly, either electroporation of GTP into U937 cells or indirect introduction of GTP by addition of guanine utilizing purine salvage pathways activated RAS and recapitulated differentiation induction phenotypes. To determine whether AML cells with higher RAS activity exhibited greater NEL sensitivity, we pretreated U937 cells with a RAS agonist KRA-553 or ectopically expressed RAS mutants and observed enhanced NEL inhibitory effects in both cases. We also observed enhanced vulnerability to NEL treatment in MLL-AF9 transformed murine hematopoietic cells from KrasLox-Stop-Lox (LSL) G12D/+/Vav-Cre mice (Blood, 2009) versus Cre+ counterparts. Relevant to AML line THP-1 which is poorly responsive to NEL (IC50>100 µM), we observed extremely low levels of Ara-GTP, no GTP increase or RAS hyperactivation after NEL treatment; Ara-GTP is inactivated by SAM domain and HD domain-containing protein 1 (SAMHD1), a dNTP hydrolase, whose high expression reportedly underlies NEL resistance in T-ALL. Indeed, SAMHD1 deletion remarkably increased RAS activity in THP-1 cells treated with NEL, thereby fully reversing NEL resistance. Our study provides a preclinical basis for testing NEL efficacy in a large cohort of AML patients, given that RAS activity is generally high in AML, or even against other malignancies harboring RAS mutations, which are considered "undruggable". Additionally, further study to test whether SAMHD1 inhibition enhances NEL efficacy against RAS active cancers is warranted. Disclosures Marcucci: Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Novartis: Speakers Bureau; Takeda: Other: Research Support (Investigation Initiated Clinical Trial); Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Merck: Other: Research Support (Investigation Initiated Clinical Trial); Abbvie: Speakers Bureau. Sykes:Clear Creek Bio: Current equity holder in private company, Other: co-founder.