Many viruses induce hepatitis in humans, highlighting the need to understand the underlying mechanisms of virus-induced liver pathology. The murine coronavirus, mouse hepatitis virus (MHV), causes ...acute hepatitis in its natural host and provides a useful model for understanding virus interaction with liver cells. The MHV accessory protein, ns2, antagonizes the type I interferon response and promotes hepatitis. We show that ns2 has 2′,5′-phosphodiesterase activity, which blocks the interferon inducible 2′,5′-oligoadenylate synthetase (OAS)-RNase L pathway to facilitate hepatitis development. Ns2 cleaves 2′,5′-oligoadenylate, the product of OAS, to prevent activation of the cellular endoribonuclease RNase L and consequently block viral RNA degradation. An ns2 mutant virus was unable to replicate in the liver or induce hepatitis in wild-type mice, but was highly pathogenic in RNase L deficient mice. Thus, RNase L is a critical cellular factor for protection against viral infection of the liver and the resulting hepatitis.
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► Mouse hepatitis virus ns2 protein inhibits the IFN-induced OAS-RNase L pathway ► ns2 reduces the intracellular level of 2′,5′-oligoadenylate (2-5A) ► ns2 has a 2′,5′-phosphodiesterase activity to directly cleave 2-5A ► ns2 mutant virus recovers wild-type virulence in RNase L-deficient mice
Loss of function TET2 mutation (TET2
) is one of the most frequently observed lesions in clonal hematopoiesis (CH). TET2 a member TET-dioxygenase family of enzymes that along with TET1 and TET3, ...progressively oxidize 5-methyl cytosine (mC) resulting in regulated demethylation of promoter, enhancer and silencer elements of the genome. This process is critical for efficient transcription that determine cell lineage fate, proliferation and survival and the maintenance of the genomic fidelity with aging of the organism. Partial or complete loss-of-function TET2 mutations create regional and contextual DNA hypermethylation leading to gene silencing or activation that result in skewed myeloid differentiation and clonal expansion. In addition to myeloid skewing, loss of TET2 creates differentiation block and provides proliferative advantage to hematopoietic stem and progenitor cells (HSPCs). TET2
is a prototypical lesion in CH, since the mutant clones dominate during stress hematopoiesis and often associates with evolution of myeloid malignancies. TET2
clones has unique privilege to create and persist in pro-inflammatory milieu. Despite extensive knowledge regarding biochemical mechanisms underlying distorted myeloid differentiation, and enhanced self-replication of TET2
HSPC, the mechanistic link of various pathogenesis associated with TET2 loss in CHIP is less understood. Here we review the recent development in TET2 biology and its probable mechanistic link in CH with aging and inflammation. We also explored the therapeutic strategies of targeting TET2
associated CHIP and the utility of targeting TET2 in normal hematopoiesis and somatic cell reprograming. We explore the biochemical mechanisms and candidate therapies that emerged in last decade of research.
Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by ...pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients' bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5-azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72-96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.
The GLI genes, GLI1 and GLI2, are transcription factors that regulate target genes at the distal end of the canonical Hedgehog (HH) signaling pathway (SHH->PTCH->SMO->GLI), tightly regulated in ...embryonic development, tissue patterning and differentiation. Both GLI1 and GLI2 are oncogenes, constitutively activated in many types of human cancers. In colon cancer cells oncogenic KRAS-GLI signaling circumvents the HH-SMO-GLI axis to channel through and activate GLI in the transcriptional regulation of target genes. We have observed extensive cell death in a panel of 7 human colon carcinoma cell lines using the small molecule GLI inhibitor GANT61. Using computational docking and experimental confirmation by Surface Plasmon Resonance, GANT61 binds to the 5-zinc finger GLI1 protein between zinc fingers 2 and 3 at sites E119 and E167, independent of the GLI-DNA binding region, and conserved between GLI1 and GLI2. GANT61 does not bind to other zinc finger transcription factors (KLF4, TFIIβ). Mutating the predicted GANT61 binding sites in GLI1 significantly inhibits GANT61-GLI binding and GLI-luciferase activity. Data establish the specificity of GANT61 for targeting GLI, and substantiate the critical role of GLI in cancer cell survival. Thus, targeting GLI in cancer therapeutics may be of high impact.
A common thread through malignant and nonmalignant diseases alerts us to a therapeutic opportunity to seize: disease may originate from genetic mutations, but resulting maladaptive/unhealthy cell ...fates and functions are mediated by epigenetic enzymes, that are druggable. Epigenetic enzymes modify DNA, and/or the histones around which DNA is organized, to regulate access to genes by the basal transcription factor machinery that transcribes genes. Epigenetic enzymes can be divided usefully into those that facilitate gene transcription ("on" enzymes or coactivators) and those that favor gene repression ("off" enzymes or corepressors). DNA-binding master transcription factors cooperate to recruit coactivators, and repulse corepressors, from thousands of genes, to thereby activate the gene expression programs that define cell fates and functions. In malignancy, this usual exchange of corepressors for coactivators fails, because of mutations to master transcription factors or the coactivators they recruit. Inhibiting corepressor enzymes using small molecules uses pharmacology to redress this coactivator/corepressor imbalance that originates from genetics, to in this way release cancer cells to the terminal lineage-fates intended by their master transcription factor content. Similarly, in nonmalignant β-hemoglobinopathies, inhibiting corepressors exploits transcription factor and lineage-context to activate unmutated fetal over mutated adult globin genes, to thereby treat these nonmalignant genetic diseases. Master transcription factors then are the "natural forces" in the Hippocratic dictum "Natural forces within us are the true healers of disease," and drugging epigenetic enzymes (corepressors) a way to harness these forces to heal.
Idiopathic aplastic anemia (IAA) is a rare autoimmune bone marrow failure (BMF) disorder initiated by a human leukocyte antigen (HLA)-restricted T-cell response to unknown antigens. As in other ...autoimmune disorders, the predilection for certain HLA profiles seems to represent an etiologic factor; however, the structure-function patterns involved in the self-presentation in this disease remain unclear. Herein, we analyzed the molecular landscape of HLA complexes of a cohort of 300 IAA patients and almost 3000 healthy and disease controls by deeply dissecting their genotypic configurations, functional divergence, self-antigen binding capabilities, and T-cell receptor (TCR) repertoire specificities. Specifically, analysis of the evolutionary divergence of HLA genotypes (HED) showed that IAA patients carried class II HLA molecules whose antigen-binding sites were characterized by a high level of structural homology, only partially explained by specific risk allele profiles. This pattern implies reduced HLA binding capabilities, confirmed by binding analysis of hematopoietic stem cell (HSC)-derived self-peptides. IAA phenotype was associated with the enrichment in a few amino acids at specific positions within the peptide-binding groove of DRB1 molecules, affecting the interface HLA-antigen-TCR β and potentially constituting the basis of T-cell dysfunction and autoreactivity. When analyzing associations with clinical outcomes, low HED was associated with risk of malignant progression and worse survival, underlying reduced tumor surveillance in clearing potential neoantigens derived from mechanisms of clonal hematopoiesis. Our data shed light on the immunogenetic risk associated with IAA etiology and clonal evolution and on general pathophysiological mechanisms potentially involved in other autoimmune disorders.
•Class II human leukocyte antigen (HLA) loci in idiopathic BMF disorders are characterized by low structural divergence.•This immunogenetic pattern contributes to a decreased T-cell receptor repertoire diversity, favoring crossreactivity and autoimmunity.
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Efficient and productive virus infection often requires viral countermeasures that block innate immunity. The IFN-inducible 2′,5′-oligoadenylate (2-5A) synthetases (OASs) and ribonuclease (RNase) L ...are components of a potent host antiviral pathway. We previously showed that murine coronavirus (MHV) accessory protein ns2, a 2H phosphoesterase superfamily member, is a phosphodiesterase (PDE) that cleaves 2-5A, thereby preventing activation of RNase L. The PDE activity of ns2 is required for MHV replication in macrophages and for hepatitis. Here, we show that group A rotavirus (RVA), an important cause of acute gastroenteritis in children worldwide, encodes a similar PDE. The RVA PDE forms the carboxy-terminal domain of the minor core protein VP3 (VP3-CTD) and shares sequence and predicted structural homology with ns2, including two catalytic HxT/S motifs. Bacterially expressed VP3-CTD exhibited 2′,5′-PDE activity, which cleaved 2-5A in vitro. In addition, VP3-CTD expressed transiently in mammalian cells depleted 2-5A levels induced by OAS activation with poly(rI):poly(rC), preventing RNase L activation. In the context of recombinant chimeric MHV expressing inactive ns2, VP3-CTD restored the ability of the virus to replicate efficiently in macrophages or in the livers of infected mice, whereas mutant viruses expressing inactive VP3-CTD (H718A or H798R) were attenuated. In addition, chimeric viruses expressing either active ns2 or VP3-CTD, but not nonfunctional equivalents, were able to protect ribosomal RNA from RNase L–mediated degradation. Thus, VP3-CTD is a 2′,5′-PDE able to functionally substitute for ns2 in MHV infection. Remarkably, therefore, two disparate RNA viruses encode proteins with homologous 2′,5′-PDEs that antagonize activation of innate immunity.
Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002. Like many ...coronaviruses, MERS-CoV carries genes that encode multiple accessory proteins that are not required for replication of the genome but are likely involved in pathogenesis. Evasion of host innate immunity through interferon (IFN) antagonism is a critical component of viral pathogenesis. The IFN-inducible oligoadenylate synthetase (OAS)-RNase L pathway activates upon sensing of viral double-stranded RNA (dsRNA). Activated RNase L cleaves viral and host single-stranded RNA (ssRNA), which leads to translational arrest and subsequent cell death, preventing viral replication and spread. Here we report that MERS-CoV, a lineage CBetacoronavirus, and related bat CoV NS4b accessory proteins have phosphodiesterase (PDE) activity and antagonize OAS-RNase L by enzymatically degrading 2',5'-oligoadenylate (2-5A), activators of RNase L. This is a novel function for NS4b, which has previously been reported to antagonize IFN signaling. NS4b proteins are distinct from lineage ABetacoronavirusPDEs and rotavirus gene-encoded PDEs, in having an amino-terminal nuclear localization signal (NLS) and are localized mostly to the nucleus. However, the expression level of cytoplasmic MERS-CoV NS4b protein is sufficient to prevent activation of RNase L. Finally, this is the first report of an RNase L antagonist expressed by a human or bat coronavirus and provides a specific mechanism by which this occurs. Our findings provide a potential mechanism for evasion of innate immunity by MERS-CoV while also identifying a potential target for therapeutic intervention.
Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV). MERS-CoV, like other coronaviruses, carries genes that encode accessory proteins that antagonize the host antiviral response, often the type I interferon response, and contribute to virulence. We found that MERS-CoV NS4b and homologs from related lineage C bat betacoronaviruses BtCoV-SC2013 (SC2013) and BtCoV-HKU5 (HKU5) are members of the 2H-phosphoesterase (2H-PE) enzyme family with phosphodiesterase (PDE) activity. Like murine coronavirus NS2, a previously characterized PDE, MERS NS4b, can antagonize activation of the OAS-RNase L pathway, an interferon-induced potent antiviral activity. Furthermore, MERS-CoV mutants with deletion of genes encoding accessory proteins NS3 to NS5 or NS4b alone or inactivation of the PDE can activate RNase L during infection of Calu-3 cells. Our report may offer a potential target for therapeutic intervention if NS4b proves to be critical to pathogenesis inin vivomodels of MERS-CoV infection.
Nucleophosmin (NPM1) is among the most frequently mutated genes in acute myeloid leukemia (AML). It is not known, however, how the resulting oncoprotein mutant NPM1 is leukemogenic. To reveal the ...cellular machinery in which NPM1 participates in myeloid cells, we analyzed the endogenous NPM1 protein interactome by mass spectrometry and discovered abundant amounts of the master transcription factor driver of monocyte lineage differentiation PU.1 (also known as SPI1). Mutant NPM1, which aberrantly accumulates in cytoplasm, dislocated PU.1 into cytoplasm with it. CEBPA and RUNX1, the master transcription factors that collaborate with PU.1 to activate granulomonocytic lineage fates, remained nuclear; but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, repressing instead of activating more than 500 granulocyte and monocyte terminal differentiation genes. An inhibitor of nuclear export, selinexor, by locking mutant NPM1/PU.1 in the nucleus, activated terminal monocytic fates. Direct depletion of the corepressor DNA methyltransferase 1 (DNMT1) from the CEBPA/RUNX1 protein interactome using the clinical drug decitabine activated terminal granulocytic fates. Together, these noncytotoxic treatments extended survival by more than 160 days versus vehicle in a patient-derived xenotransplant model of NPM1/FLT3-mutated AML. In sum, mutant NPM1 represses monocyte and granulocyte terminal differentiation by disrupting PU.1/CEBPA/RUNX1 collaboration, a transforming action that can be reversed by pharmacodynamically directed dosing of clinical small molecules.
Multiple myeloma (MM) is a complex hematologic malignancy characterized by the uncontrolled proliferation of clonal plasma cells in the bone marrow that secrete large amounts of immunoglobulins and ...other non-functional proteins. Despite decades of progress and several landmark therapeutic advancements, MM remains incurable in most cases. Standard of care frontline therapies have limited durable efficacy, with the majority of patients eventually relapsing, either early or later. Induced drug resistance via up-modulations of signaling cascades that circumvent the effect of drugs and the emergence of genetically heterogeneous sub-clones are the major causes of the relapsed-refractory state of MM. Cytopenias from cumulative treatment toxicity and disease refractoriness limit therapeutic options, hence creating an urgent need for innovative approaches effective against highly heterogeneous myeloma cell populations. Here, we present a comprehensive overview of the current and future treatment paradigm of MM, and highlight the gaps in therapeutic translations of recent advances in targeted therapy and immunotherapy. We also discuss the therapeutic potential of emerging preclinical research in multiple myeloma.
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