Cell line-dependent variability in HIV activation employing DNMT inhibitors

Long-lived reservoirs of Human Immunodeficiency Virus (HIV) latently infected cells present the main barrier to a cure for HIV infection. Much interest has focused on identifying strategies to activate HIV, which would be used together with antiretrovirals to attack reservoirs. Several HIV activating agents, including Tumor Necrosis Factor alpha (TNFα) and other agents that activate via NF-kB are not fully effective in all latent infection models due to epigenetic restrictions, such as DNA methylation and the state of histone acetylation. DNA methyltransferases (DNMT) inhibitors like 5-aza-2'deoxycytidine (Aza-CdR) and histone deacetylase (HDAC) inhibitors like Trichostatin A (TSA) have been proposed as agents to enhance reactivation and have shown activity in model systems. However, it is not clear how the activities of DNMT and HDAC inhibitors range across different latently infected cell lines, potential models for the many different latently infected cells within an HIV patient. We determined HIV activation following treatment with TNFα, TSA and Aza-CdR across a range of well known latently infected cell lines. We assessed the activity of these compounds in four different Jurkat T cell-derived J-Lat cell lines (6.3, 8.4, 9.2 and 10.6), which have a latent HIV provirus in which GFP replaces Nef coding sequence, and ACH-2 and J1.1 (T cell-derived), and U1 (promonocyte-derived) cell lines with full-length provirus. We found that Aza-CdR plus TNFα activated HIV at least twice as well as TNFα alone for almost all J-Lat cells, as previously described, but not for J-Lat 10.6, in which TNFα plus Aza-CdR moderately decreased activation compared to TNFα alone. Surprisingly, a much greater reduction of TNFα-stimulated activation with Aza-CdR was detected for ACH-2, J1.1 and U1 cells. Reaching the highest reduction in U1 cells with a 75% reduction. Interestingly, Aza-CdR not only decreased TNFα induction of HIV expression in certain cell lines, but also decreased activation by TSA. Since DNMT inhibitors reduce the activity of provirus activators in some HIV latently infected cell lines the use of epigenetic modifying agents may need to be carefully optimized if they are to find clinical utility in therapies aimed at attacking latent HIV reservoirs

Epigenetic Regulation of HIV-1 Latency by Cytosine Methylation

Human immunodeficiency virus type 1 (HIV-1) persists in a latent state within resting CD4+ T cells of infected persons treated with highly active antiretroviral therapy (HAART). This reservoir must be eliminated for the clearance of infection. Using a cDNA library screen, we have identified methyl-CpG binding domain protein 2 (MBD2) as a regulator of HIV-1 latency. Two CpG islands flank the HIV-1 transcription start site and are methylated in latently infected Jurkat cells and primary CD4+ T cells. MBD2 and histone deacetylase 2 (HDAC2) are found at one of these CpG islands during latency. Inhibition of cytosine methylation with 5-aza-2′deoxycytidine (aza-CdR) abrogates recruitment of MBD2 and HDAC2. Furthermore, aza-CdR potently synergizes with the NF-κB activators prostratin or TNF-α to reactivate latent HIV-1. These observations confirm that cytosine methylation and MBD2 are epigenetic regulators of HIV-1 latency. Clearance of HIV-1 from infected persons may be enhanced by inclusion of DNA methylation inhibitors, such as aza-CdR, and NF-κB activators into current antiviral therapies

Cell-Dependent Role for the Poliovirus 3′ Noncoding Region in Positive-Strand RNA Synthesis

We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3′ noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3′ noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3′ noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3′ noncoding region is involved in positive-strand synthesis during genome replication

The SWI/SNF chromatin-remodeling complex is a cofactor for Tat transactivation of the HIV promoter

Tat is a critical viral transactivator essential for human immunodeficiency virus (HIV) gene expression. Activation involves binding to an RNA stem-loop structure and recruitment of the positive transcription elongation factor b. Tat also induces the remodeling of a single nucleosome in the HIV promoter. However, the mechanism of this remodeling has remained unclear. Knockdown of INI-1 and BRG-1, two components of the SWI/SNF chromatin-remodeling complex, suppressed Tat-mediated transactivation. Cells lacking INI-1 (G401 and MON) or BRG-1 (C33A) exhibited defective transactivation by Tat that was restored upon INI-1 and BRG-1 expression, respectively. Tat was co-immunoprecipitated with several SWI/SNF subunits, including INI-1, BRG-1, and β-actin. The SWI/SNF complex interacted with the integrated HIV promoter in a Tat-dependent manner. We also found that INI-1 and BRG-1 synergized with the p300 acetyltransferase to activate the HIV promoter. This synergism depended on the acetyltransferase activity of p300 and on Tat Lys 50 and Lys 51. In conclusion, Tat-mediated activation of the HIV promoter requires the SWI/SNF complex in synergy with the coactivator p300

ALX148 blocks CD47 and enhances innate and adaptive antitumor immunity with a favorable safety profile.

CD47 is a widely expressed cell surface protein that functions as an immune checkpoint in cancer. When expressed by tumor cells, CD47 can bind SIRPα on myeloid cells, leading to suppression of tumor cell phagocytosis and other innate immune functions. CD47-SIRPα signaling has also been implicated in the suppression of adaptive antitumor responses, but the relevant cellular functions have yet to be elucidated. Therapeutic blockade of the CD47 pathway may stimulate antitumor immunity and improve cancer therapy. To this end, a novel CD47-blocking molecule, ALX148, was generated by fusing a modified SIRPα D1 domain to an inactive human IgG1 Fc. ALX148 binds CD47 from multiple species with high affinity, inhibits wild type SIRPα binding, and enhances phagocytosis of tumor cells by macrophages. ALX148 has no effect on normal human blood cells in vitro or on blood cell parameters in rodent and non-human primate studies. Across several murine tumor xenograft models, ALX148 enhanced the antitumor activity of different targeted antitumor antibodies. Additionally, ALX148 enhanced the antitumor activity of multiple immunotherapeutic antibodies in syngeneic tumor models. These studies revealed that CD47 blockade with ALX148 induces multiple responses that bridge innate and adaptive immunity. ALX148 stimulates antitumor properties of innate immune cells by promoting dendritic cell activation, macrophage phagocytosis, and a shift of tumor-associated macrophages toward an inflammatory phenotype. ALX148 also stimulated the antitumor properties of adaptive immune cells, causing increased T cell effector function, pro-inflammatory cytokine production, and a reduction in the number of suppressive cells within the tumor microenvironment. Taken together, these results show that ALX148 binds and blocks CD47 with high affinity, induces a broad antitumor immune response, and has a favorable safety profile

Poliovirus tropism and attenuation are determined after internal ribosome entry

Poliovirus replication is limited to a few organs, including the brain and spinal cord. This restricted tropism may be a consequence of organ-specific differences in translation initiation by the poliovirus internal ribosome entry site (IRES). A C-to-U mutation at base 472 in the IRES of the Sabin type 3 poliovirus vaccine strain, known to attenuate neurovirulence, may further restrict tropism by eliminating viral replication in the CNS. To determine the relationship between IRES-mediated translation and poliovirus tropism, recombinant human adenoviruses were used to express bicistronic mRNAs in murine organs. The IRESs of poliovirus, the cardiotropic coxsackievirus B3 (CVB3), and the hepatotropic hepatitis C virus (HCV) mediate translation in many organs, including those that do not support viral replication. A translation defect associated with the Sabin type 3 IRES was observed in all organs examined. Poliovirus type 1 and recombinant polioviruses dependent on the IRES of CVB3 or HCV replicate in the CNS of mice and cause paralysis. Although the type 3 Sabin strain is an effective vaccine, polioviruses with a U at base 472 of the IRES cause paralysis in newborn mice. Tropism of wild-type and vaccine strains of poliovirus is therefore determined after internal ribosome entry

Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis.

BackgroundThere is microscopic spatial and temporal heterogeneity of pathological changes in idiopathic pulmonary fibrosis (IPF) lung tissue, which may relate to heterogeneity in pathophysiological mediators of disease and clinical progression. We assessed relationships between gene expression patterns, pathological features, and systemic biomarkers to identify biomarkers that reflect the aggregate disease burden in patients with IPF.MethodsGene expression microarrays (N=40 IPF; 8 controls) and immunohistochemical analyses (N=22 IPF; 8 controls) of lung biopsies. Clinical characterisation and blood biomarker levels of MMP3 and CXCL13 in a separate cohort of patients with IPF (N=80).Results2940 genes were significantly differentially expressed between IPF and control samples (|fold change| >1.5, p<0.05). Two clusters of co-regulated genes related to bronchiolar epithelium or lymphoid aggregates exhibited substantial heterogeneity within the IPF population. Gene expression in bronchiolar and lymphoid clusters corresponded to the extent of bronchiolisation and lymphoid aggregates determined by immunohistochemistry in adjacent tissue sections. Elevated serum levels of MMP3, encoded in the bronchiolar cluster, and CXCL13, encoded in the lymphoid cluster, corresponded to disease severity and shortened survival time (p<10(-7) for MMP3 and p<10(-5) for CXCL13; Cox proportional hazards model).ConclusionsMicroscopic pathological heterogeneity in IPF lung tissue corresponds to specific gene expression patterns related to bronchiolisation and lymphoid aggregates. MMP3 and CXCL13 are systemic biomarkers that reflect the aggregate burden of these pathological features across total lung tissue. These biomarkers may have clinical utility as prognostic and/or surrogate biomarkers of disease activity in interventional studies in IPF

Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis.

BackgroundThere is microscopic spatial and temporal heterogeneity of pathological changes in idiopathic pulmonary fibrosis (IPF) lung tissue, which may relate to heterogeneity in pathophysiological mediators of disease and clinical progression. We assessed relationships between gene expression patterns, pathological features, and systemic biomarkers to identify biomarkers that reflect the aggregate disease burden in patients with IPF.MethodsGene expression microarrays (N=40 IPF; 8 controls) and immunohistochemical analyses (N=22 IPF; 8 controls) of lung biopsies. Clinical characterisation and blood biomarker levels of MMP3 and CXCL13 in a separate cohort of patients with IPF (N=80).Results2940 genes were significantly differentially expressed between IPF and control samples (|fold change| >1.5, p<0.05). Two clusters of co-regulated genes related to bronchiolar epithelium or lymphoid aggregates exhibited substantial heterogeneity within the IPF population. Gene expression in bronchiolar and lymphoid clusters corresponded to the extent of bronchiolisation and lymphoid aggregates determined by immunohistochemistry in adjacent tissue sections. Elevated serum levels of MMP3, encoded in the bronchiolar cluster, and CXCL13, encoded in the lymphoid cluster, corresponded to disease severity and shortened survival time (p<10(-7) for MMP3 and p<10(-5) for CXCL13; Cox proportional hazards model).ConclusionsMicroscopic pathological heterogeneity in IPF lung tissue corresponds to specific gene expression patterns related to bronchiolisation and lymphoid aggregates. MMP3 and CXCL13 are systemic biomarkers that reflect the aggregate burden of these pathological features across total lung tissue. These biomarkers may have clinical utility as prognostic and/or surrogate biomarkers of disease activity in interventional studies in IPF