TASOR epigenetic repressor cooperates with a CNOT1 RNA degradation pathway to repress HIV

The Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin recruits the histone methyl-transferase SETDB1 to spread H3K9me3 repressive marks across genes and transgenes in an integration site-dependent manner. The deposition of these repressive marks leads to heterochromatin formation and inhibits gene expression, but the underlying mechanism is not fully understood. Here, we show that TASOR silencing or HIV-2 Vpx expression, which induces TASOR degradation, increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. Furthermore, using a yeast 2-hybrid screen, we identify new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 synergistically repress HIV expression from its LTR. Similar to the RNA-induced transcriptional silencing complex found in fission yeast, we show that TASOR interacts with the RNA exosome and RNA Polymerase II, predominantly under its elongating state. Finally, we show that TASOR facilitates the association of RNA degradation proteins with RNA polymerase II and is detected at transcriptional centers. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral expression.

Relationship between the transcriptional expression of PIM1 and local control in patients with head and neck squamous cell carcinomas treated with radiotherapy

Purpose Proviral integration site for Moloney murine leukemia virus (PIMs) are proto-oncogenes encoding serine/threonine kinases that phosphorylate a variety of substrates involved in the regulation of cellular processes. Elevated expression of PIM-1 has been associated with poor prognosis in several types of cancer. There are no studies that have analyzed the response to radiotherapy in patients with head and neck squamous cell carcinoma (HNSCC) according to the expression of PIM-1. The aim of our study was to analyze the relationship between the transcriptional expression of PIM-1 and local response to radiotherapy in HNSCC patients. Methods We determined the transcriptional expression of PIM-1 in 135 HNSCC patients treated with radiotherapy, including patients treated with chemoradiotherapy (n = 65) and bioradiotherapy (n = 15). Results During the follow-up, 48 patients (35.6%) had a local recurrence of the tumor. Patients with local recurrence had a higher level of PIM-1 expression than those who achieved local control of the disease (P = 0.017). Five-year local recurrence-free survival for patients with a high expression of PIM-1 (n = 43) was 44.6% (95% CI 29.2–60.0%), and for patients with low expression (n = 92) it was 71.9% (95% CI 62.5–81.3%) (P = 0.007). According to the results of multivariate analysis, patients with a high PIM-1 expression had a 2.2-fold increased risk of local recurrence (95% CI 1.22–4.10, P = 0.009). Conclusion Patients with elevated transcriptional expression levels of PIM-1 had a significantly higher risk of local recurrence after radiotherapy.

Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs)-Derived miR-200c Regulates Wingless-Related Integration Site (Wnt)/β-Catenin Signaling in Prostate Cancer by Targeting Cortactin (CTTN)

Bone marrow-derived mesenchymal stem cells (BMSCs) are an integral part of cancer microenvironment. We intend to clarify BMSC-derived exosomes’ role in prostate cancer. The exosomes miR-200c secreted by BMSCs were identified by electron microscopy. The mice tumor model was used to explore the role of miR-200c’s in tumor mice. Cell invasion was assessed by transwell assay and Wnt/β-catenin expression was measured by western blot. Exosomes miR-200c derived from BMSCs promoted tumor cell invasion and activated Wnt/β-catenin signaling. miR-200c targets CTTN-mediated cell signal transduction, and blocking CTTN expression can suppression miR-200c-mediated Wnt/β-catenin signal transduction and inhibit cell invasion. In conclusion, miR-200c regulates CTTN, thereby inducing Wnt/β-catenin signaling to enhance tumor growth.

Immunohistochemical Expression of Wnt-4 Protein in Clear Cell Renal Carcinoma

Wingless binding integration site proteins (Wnt) have an important role in normal kidney development and in various kidney diseases. They are required for complete epithelial differentiation and normal nephron formation. Changes in these proteins could also have important role in carcinogenesis. This study included 185 patients with clear cell renal carcinoma (ccRCC) in whom immunohistochemical expression of Wnt-4 protein in healthy and tumorous tissue after surgery was investigated. There was higher expression of Wnt-4 in healthy than in tumor tissue. No difference between Fuhrman’s grade and Wnt-4 expression was found. A poor negative correlation between tumor size and Wnt-4 expression was found. Patients with suspected metastatic diseases had higher Wnt-4 expression. There was no difference in survival rates between Wnt-4 negative and positive groups. In our study we have shown that high Wnt-4 expression in healthy tissue decreases in low-grade tumors but then increases in high-grade tumors, suggesting that tumor progression requires Wnt-4 activation or reactivation.

New Approaches to Multi-Parametric HIV-1 Genetics Using Multiple Displacement Amplification: Determining the What, How, and Where of the HIV-1 Reservoir

Development of potential HIV-1 curative interventions requires accurate characterization of the proviral reservoir, defined as host-integrated viral DNA genomes that drive rebound of viremia upon halting ART (antiretroviral therapy). Evaluation of such interventions necessitates methods capable of pinpointing the rare, genetically intact, replication-competent proviruses within a background of defective proviruses. This evaluation can be achieved by identifying the distinct integration sites of intact proviruses within host genomes and monitoring the dynamics of these proviruses and host cell lineages over longitudinal sampling. Until recently, molecular genetic approaches at the single proviral level have been generally limited to one of a few metrics, such as proviral genome sequence/intactness, host-proviral integration site, or replication competency. New approaches, taking advantage of MDA (multiple displacement amplification) for WGA (whole genome amplification), have enabled multiparametric proviral characterization at the single-genome level, including proviral genome sequence, host-proviral integration site, and phenotypic characterization of the host cell lineage, such as CD4 memory subset and antigen specificity. In this review, we will examine the workflow of MDA-augmented molecular genetic approaches to study the HIV-1 reservoir, highlighting technical advantages and flexibility. We focus on a collection of recent studies in which investigators have used these approaches to comprehensively characterize intact and defective proviruses from donors on ART, investigate mechanisms of elite control, and define cell lineage identity and antigen specificity of infected CD4+ T cell clones. The highlighted studies exemplify how these approaches and their future iterations will be key in defining the targets and evaluating the impacts of HIV curative interventions.

Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

Stable expression of transgenes is essential in both therapeutic and research applications. Traditionally, transgene integration has been accomplished via viral vectors in a semi-random fashion, but with inherent integration site biases linked to the type of virus used. The randomly integrated transgenes may undergo silencing and more concerningly, can also lead to dysregulation of endogenous genes. Gene dysregulation can lead to malignant transformation of cells and has unfortunately given rise to cases of leukaemia in gene therapy trials. Genomic safe harbour (GSH) loci have been proposed as safe sites for transgene integration. To date, a number of sites in the human genome have been used for directed integration; however none of these pass scrutiny as bona fide GSH. Here, we conducted a computational analysis to identify 25 putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

A Pragmatic Pharmacophore Informatics Strategy to Discover New Potent Inhibitors Against Pim-3

Pim-3 (proviral integration site moloney murine leukemia virus-3) is an oncogene which encodes proteins belonging to serine/threonine kinase family, and PIM subfamily. It is generally over expressed in epithelial and hematological tumors. It is known to involve in numerous cellular functions such as cell growth, differentiation, survival, tumorigenesis and apoptosis. It also plays a crucial role in regulation of signal transduction cascades. Therefore it emerged as a hopefultherapeutic target for cancer treatment. In current study, indole derivatives having potent inhibitory activity against Pim 3 were taken and pharmacophore based virtual screening was carried out. A five point pharmacophore hypothesis with one hydrogen bond acceptor, one hydrogen bond donor and three aromatic rings i.e., ADRRR was developed with acceptable R2and Q2 values of 0.913 and 0.748 respectively. It was employed as a query and screening was conducted against Asinex and Otava lead library databases to screen out potent drug like candidates. The obtained compounds were subjected to SP, XP docking using 3D model of pim-3 which was constructed through comparative homology modelling and finally binding free energies were calculated for top hits. The docking and binding free energy studies revealed that six hit molecules showed higher binding energy in comparison to the best active molecule. Finally, MD simulations of the top hit with highest binding energy was carried out which indicated that the obtained hit N1 formed a stable complex with pim-3. We believe that these combined protocols will be helpful and cooperative to discover and design more potent pim-3 inhibitors in near future.

The retinoic acid receptor co-factor NRIP1 is uniquely upregulated and represents a therapeutic target in acute myeloid leukemia with chromosome 3q rearrangements

Aberrant expression of Ecotropic Viral Integration Site 1 (EVI1) is a hallmark of acute myeloid leukemia (AML) with inv(3) or t(3;3), which is a disease subtype with especially poor outcome. In studying transcriptomes from AML patients with chromosome 3q rearrangements, we identified a significant upregulation of the Nuclear Receptor Interacting Protein 1 (NRIP1) as well as its adjacent non-coding RNA LOC101927745. Utilizing transcriptomic and epigenomic data from over 900 primary patient samples as well as genetic and transcriptional engineering approaches, we have identified several mechanisms that can lead to upregulation of NRIP1 in AML. We hypothesize that the LOC101927745 transcription start site harbors a context-dependent enhancer that is bound by EVI1, causing upregulation of NRIP1 in AML with chr3 abnormalities. Furthermore, we show that NRIP1 knockdown negatively affects the proliferation and survival of 3q-rearranged AML cells and increases their sensitivity towards ATRA, suggesting that NRIP1 is relevant for the pathogenesis of inv(3)/t(3;3) AML and could serve as a novel therapeutic target in myeloid malignancies with 3q abnormalities.