Expression In Primary Cells Research Articles

Potential Use of Extracellular Vesicles for the HER2 Status Assessment in Breast Cancer Patients.

Human epithelial growth factor receptor 2 (HER2)-targeted therapies are effective in patients with HER2-positive breast cancer. Recent advances have shown that HER2-targeted therapies can also be of benefit when treating tumors expressing low levels of HER2, highlighting the importance of identifying the HER2-low subgroup. This clinical trend has opened new therapeutic avenues for patients who were previously ineligible for HER2-targeted therapies. Thus, the development of new diagnostic methods for real-time HER2 profiling is crucial for accurately tailoring the treatment for these patients. We hypothesized that tumor-derived extracellular vesicles (TEVs) could reflect the HER2 profiles of primary tumors and potentially serve as diagnostic tools for HER2 status. This approach was validated using six breast cancer cell lines, which confirmed that the TEVs accurately reflected the HER2 profiles of the tumor cells. TEVs were isolated using an immunoaffinity method, and copy number variation (CNV) in the ERBB2/EIF2C ratio was assessed using droplet digital PCR of DNA from these vesicles. Clinical validation using plasma samples from 33 breast cancer patients further reinforced the diagnostic potential of our method. Pearson's correlation coefficient analysis of the flow cytometry results demonstrated that TEVs reflected HER2 expression in primary cells. To distinguish between HER2-negative and HER2-low patients, the area under the curve (AUC) of the ROC curve in our method was 0.796, with a sensitivity of 53.8% and a specificity of 100%. These findings suggest the clinical utility of extracellular vesicles derived from plasma and emphasize the need for further research to distinguish HER2-negative from HER2-low patients.

Multitranscript analysis reveals an effect of 2-deoxy-d-glucose on gene expression linked to unfolded protein response and integrated stress response in primary human monocytes and monocyte-derived macrophages

BackgroundGlycolytic inhibitor 2-deoxy-d-glucose (2-DG) binds to hexokinase in a non-competitive manner and phosphoglucose isomerase in a competitive manner, blocking the initial steps of the glycolytic pathway. Although 2-DG stimulates endoplasmic reticulum (ER) stress, activating the unfolded protein response to restore protein homeostasis, it is unclear which ER stress-related genes are modulated in response to 2-DG treatment in human primary cells. Here, we aimed to determine whether the treatment of monocytes and monocyte-derived macrophages (MDMs) with 2-DG leads to a transcriptional profile specific to ER stress. MethodsWe performed bioinformatics analysis to identify differentially expressed genes (DEGs) in previously reported RNA-seq datasets of 2-DG treated cells. RT-qPCR was performed to verify the sequencing data on cultured MDMs. ResultsA total of 95 common DEGs were found by transcriptional analysis of monocytes and MDMs treated with 2-DG. Among these, 74 were up-regulated and 21 were down-regulated. Multitranscript analysis showed that DEGs are linked to integrated stress response (GRP78/BiP, PERK, ATF4, CHOP, GADD34, IRE1α, XBP1, SESN2, ASNS, PHGDH), hexosamine biosynthetic pathway (GFAT1, GNA1, PGM3, UAP1), and mannose metabolism (GMPPA and GMPPB). ConclusionsResults reveal that 2-DG triggers a gene expression program that might be involved in restoring protein homeostasis in primary cells. General significance2-DG is known to inhibit glycolysis and induce ER stress; however, its effect on gene expression in primary cells is not well understood. This work shows that 2-DG is a stress inducer shifting the metabolic state of monocytes and macrophages.

Methods to Study NLR in Human Blood Cells.

Autoinflammatory diseases are a group of inherited and multifactorial disorders characterized by an over-activation of innate immune response. In most cases, the clinical manifestations are due to increased activity of the NLRP3 inflammasome resulting in increased IL-1β secretion. Investigating inflammatory cells from subjects affected by autoinflammatory diseases presents a number of technical difficulties related to the rarity of the diseases, to the young age of most patients, to the difficult modulation of gene expression in primary cells. However, since cell stress is involved in the pathophysiology of these diseases, the study of freshly drawn blood monocytes from patients affected by IL-1-mediated diseases strongly increases the chances that the observed phenomena is indeed pertinent to the pathogenesis of the disease and not influenced by the long-term cell culture conditions (e.g., the high O2 tension) or gene transfection in continuous cell lines that may lead to artifacts.

Krebs Memorial Fund 2022 awardees

Krebs Memorial Fund 2022 awardees

HEATR3 variants impair nuclear import of uL18 (RPL5) and drive Diamond-Blackfan anemia

AbstractThe congenital bone marrow failure syndrome Diamond-Blackfan anemia (DBA) is typically associated with variants in ribosomal protein (RP) genes impairing erythroid cell development. Here we report multiple individuals with biallelic HEATR3 variants exhibiting bone marrow failure, short stature, facial and acromelic dysmorphic features, and intellectual disability. These variants destabilize a protein whose yeast homolog is known to synchronize the nuclear import of RPs uL5 (RPL11) and uL18 (RPL5), which are both critical for producing ribosomal subunits and for stabilizing the p53 tumor suppressor when ribosome biogenesis is compromised. Expression of HEATR3 variants or repression of HEATR3 expression in primary cells, cell lines of various origins, and yeast models impairs growth, differentiation, pre–ribosomal RNA processing, and ribosomal subunit formation reminiscent of DBA models of large subunit RP gene variants. Consistent with a role of HEATR3 in RP import, HEATR3-depleted cells or patient-derived fibroblasts display reduced nuclear accumulation of uL18. Hematopoietic progenitor cells expressing HEATR3 variants or small-hairpin RNAs knocking down HEATR3 synthesis reveal abnormal acceleration of erythrocyte maturation coupled to severe proliferation defects that are independent of p53 activation. Our study uncovers a new pathophysiological mechanism leading to DBA driven by biallelic HEATR3 variants and the destabilization of a nuclear import protein important for ribosome biogenesis.

MOB3A Bypasses BRAF and RAS Oncogene-Induced Senescence by Engaging the Hippo Pathway.

These results suggest that MOB3 targeting to re-engage the Hippo pathway, or direct targeting of YAP/TAZ, may be viable therapeutic strategies potential for RAS-pathway driven tumours.

Abstract PO-043: Cytidine deaminase protects pancreatic cancer cells from replicative stress and drive response to DNA-targeting drugs

Abstract Cytidine deaminase (CDA) converts cytidine and deoxycytidine into uridine and deoxyuridine within the pyrimidine salvage pathway for DNA and RNA synthesis. In this regard, loss of CDA provokes genomic instability in Bloom Syndrome, and CDA overexpression is associated with tumor resistance to chemotherapy with pyrimidine analogs. However, the precise role of CDA per se in cancer has been totally underexplored so far. Patient cohort analysis demonstrate that CDA is overexpressed in PDAC, a disease with no cure with increasing incidence, and associated with a worse prognosis in patient. Functional studies demonstrate that CDA is essential to PDAC cell proliferation and tumor growth. We found that CDA expression is associated with gene set enrichment in DNA replication signature, in both PDAC tumors and experimental models. Using enforced expression, genetic or pharmacologic targeting, we demonstrate that CDA promotes DNA replication, localizes to the replication fork and increases replication fork fitness. These effects are strictly dependent on CDA deaminase activity. Hence, we found that CDA controls replication stress as CDA expression is inversely correlated with the level of DNA breaks during S-phase, and that CDA targeting is associated with gene set enrichment in replication stress signature and CHK1 protein activation. Next, we demonstrate that CDA preserves genomic stability of PDAC cells, as CDA expression is inversely correlated with micronuclei formation and DNA damage transfer to daughter cells. We next explored PDAC cell lines from the Cancer Cell Lines Encyclopedia and found that CDA expression is associated with resistance to drug targeting DNA synthesis. Functional studies demonstrate that overexpressing CDA, and not CDA catalytically inactive mutant, protects PDAC cell lines from camptothecin, a topoisomerase inhibitor, while targeting CDA sensitizes PDAC cells to treatment by camptothecin and oxaliplatin, that forms platinum-DNA adducts. To gain further insights into the clinical potential of such finding, we targeted CDA expression in primary cells from patient with PDAC and found that CDA is essential to primary cell growth, and that CDA targeting sensitizes primary cells to treatment by oxaliplatin. Taken together, our results reveal for the first time that CDA controls DNA replication, replication stress level and genomic stability of PDAC cells, and that this new role of CDA is involved in tumor resistance to drugs inducing DNA damages. Thus, our work stems for new strategies based on CDA targeting to defeat PDAC resistance to treatment. Citation Format: Audrey Lumeau, Nicolas Bery, Cyril Ribeyre, Samad Elkaoutari, Guillaume Labrousse, Miguel Madrid-Mencia, Vera Pancaldi, Marie-Jeanne Pillaire, Valérie Bergoglio, Nelson Dusseti, Jean-Sébastien Hoffmann, Louis Buscail, Malik Lutzmann, Pierre Cordelier. Cytidine deaminase protects pancreatic cancer cells from replicative stress and drive response to DNA-targeting drugs [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-043.

Characterization of NADPH Oxidase Expression and Activity in Acute Myeloid Leukemia Cell Lines: A Correlation with the Differentiation Status.

In acute myeloid leukemia (AML), a low level of reactive oxygen species (ROS) is associated with leukemic stem cell (LSC) quiescence, whereas a high level promotes blast proliferation. ROS homeostasis relies on a tightly-regulated balance between the antioxidant and oxidant systems. Among the oxidants, NADPH oxidases (NOX) generate ROS as a physiological function. Although it has been reported in AML initiation and development, the contribution of NOX to the ROS production in AML remains to be clarified. The aim of this study was to investigate the NOX expression and function in AML, and to examine the role of NOX in blast proliferation and differentiation. First, we interrogated the NOX expression in primary cells from public datasets, and investigated their association with prognostic markers. Next, we explored the NOX expression and activity in AML cell lines, and studied the impact of NOX knockdown on cell proliferation and differentiation. We found that NOX2 is ubiquitously expressed in AML blasts, and particularly in cells from the myelomonocytic (M4) and monocytic (M5) stages; however, it is less expressed in LSCs and in relapsed AML. This is consistent with an increased expression throughout normal hematopoietic differentiation, and is reflected in AML cell lines. Nevertheless, no endogenous NOX activity could be detected in the absence of PMA stimulation. Furthermore, CYBB knockdown, although hampering induced NOX2 activity, did not affect the proliferation and differentiation of THP-1 and HL-60 cells. In summary, our data suggest that NOX2 is a marker of AML blast differentiation, while AML cell lines lack any NOX2 endogenous activity.

Inhibition of miR-188-5p alleviates hepatic fibrosis by significantly reducing the activation and proliferation of HSCs through PTEN/PI3K/AKT pathway.

Persistent hepatic damage and chronic inflammation in liver activate the quiescent hepatic stellate cells (HSCs) and cause hepatic fibrosis (HF). Several microRNAs regulate the activation and proliferation of HSCs, thereby playing a critical role in HF progression. Previous studies have reported that miR‐188‐5p is dysregulated during the process of HF. However, the role of miR‐188‐5p in HF remains unclear. This study investigated the potential role of miR‐188‐5p in HSCs and HF. Firstly, we validated the miR‐188‐5p expression in primary cells isolated from liver of carbon tetrachloride (CCl4)‐induced mice, TGF‐β1‐induced LX‐2 cells, livers from 6‐month high‐fat diet (HFD)‐induced rat and 4‐month HFD‐induced mice NASH models, and human non‐alcoholic fatty liver disease (NAFLD) patients. Furthermore, we used miR‐188‐5p inhibitors to investigate the therapeutic effects of miR‐188‐5p inhibition in the HFD + CCl4 induced in vivo model and the potential role of miR‐188‐5p in the activation and proliferation of HSCs. This present study reported that miR‐188‐5p expression is significantly increased in the human NAFLD, HSCs isolated from liver of CCl4 induced mice, and in vitro and in vivo models of HF. Mimicking the miR‐188‐5p resulted in the up‐regulation of HSC activation and proliferation by directly targeting the phosphatase and tensin homolog (PTEN). Moreover, inhibition of miR‐188‐5p reduced the activation and proliferation markers of HSCs through PTEN/AKT pathway. Additionally, in vivo inhibition of miR‐188‐5p suppressed the HF parameters, pro‐fibrotic and pro‐inflammatory genes, and fibrosis. Collectively, our results uncover the pro‐fibrotic role of miR‐188‐5p. Furthermore, we demonstrated that miR‐188‐5p inhibition decreases the severity of HF by reducing the activation and proliferation of HSCs through PTEN/AKT pathway.

The epigenetic regulator RINF (CXXC5) maintains SMAD7 expression in human immature erythroid cells and sustains red blood cells expansion.

The gene CXXC5, encoding a retinoid-inducible nuclear factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome and adult acute myeloid leukemia. RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknown. Here, we evaluated the consequences of RINF silencing on cytokine-induced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells, without affecting cell viability. The phenotype induced by RINF-silencing was dependent on tumor growth factor b (TGFb) and mediated by SMAD7, a TGFb-signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and myelodysplastic syndrome patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdown-dependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a TET2-anchoring platform in mouse. Collectively, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFb superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.

ABCA1 plays an anti-inflammatory role by affecting TLR4 at the feto–maternal interface

AimsThis study aimed to evaluate the function and pathway of ATP-binding cassette transporter member A1 (ABCA1)-induced anti-inflammatory response in cells at the feto–maternal interface. Main methodsThe primary amniotic mesenchymal cells (AMCs), chorion cells and decidual cells were isolated from placental membranes of women with uncomplicated pregnancies at full-term (not in labor) using enzymatic digestion. Flow cytometry was used to measure the purity of isolated cells. Immunofluorescence assay was performed to detect the location of ABCA1 and toll-like receptor 4 (TLR4). Reverse transcription PCR and western blotting analyses were used to examine ABCA1, TLR4 and inflammatory factor expression in primary cells. ELISA was used to detect cytokine secretions from the primary cells. Key findingsABCA1 and TLR4 were mainly located in the cell nucleus and cytoplasm of feto-maternal interface cells. ABCA1 expression remained the highest in chorion cells, medium in decidual cells, and weakest in AMCs. Upregulated expression of ABCA1 decreased expression of TLR4 and the levels of pro-inflammatory factors, but increased cytoprotective factors in all cell types. In contrast, downregulated expression of ABCA1 increased the expression of TLR4 and pro-inflammatory factors, but decreased the levels of cytoprotective factors. Downregulated ABCA1 expression followed by decreased TLR4 expression using a small interference RNA (siRNA) induced reduction of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) in all cell types. SignificanceABCA1 at feto-maternal interface acts as an anti-inflammatory role by reducing the expression of TLR4 in uncomplicated pregnancies. ABCA1 might be a potential therapeutic target for preventing gestational diseases.

Epigenetic Regulation of Ferroportin in Primary Cultures of the Rat Blood-Brain Barrier.

Ferroportin plays an essential role for iron transport through the blood-brain barrier (BBB), which is formed by brain capillary endothelial cells (BCECs). To maintain the integrity of the BBB, the BCECs gain support from pericytes and astrocytes, which together with neurons form the neurovascular unit (NVU). The objectives of the present study were to investigate ferroportin expression in primary cells of the NVU and to determine if ferroportin mRNA (Fpn) expression is epigenetically regulated. Primary rat BCECs, pericytes, astrocytes, and neurons all expressed ferroportin mRNA at varying levels, with BCECs exhibiting the highest expression of Fpn, peaking when co-cultured but examined separately from astrocytes. Conversely, Fpn expression was lowest in isolated astrocytes, which correlated with high DNA methylation in their Slc40a1 promoter. To provide further evidence for epigenetic regulation, mono-cultured BCECs, pericytes, and astrocytes were treated with the histone deacetylase inhibitors valproic acid (VPA) and sodium butyrate (SB), which significantly increased Fpn and ferroportin protein in BCECs and pericytes. Furthermore, 59Fe export from BCECs was elevated after treatment with VPA. In conclusion, we present first time evidence stating that Fpn expression is epigenetically regulated in BCECs, which may have implications for pharmacological induction of iron transport through the BBB.

Regulation of Erythroblast Protein Expression By an RNA Splicing Mechanism, Decoy Exon-Mediated Intron Retention

The erythroid transcriptome is extensively remodeled during terminal erythropoiesis by dynamic changes in RNA splicing of cassette exons and retained introns. Mechanistic studies of these RNA processing networks will provide new insight into pathways that impact structure and function of the erythroid proteome during erythroblast differentiation. We previously showed that up-regulation of EPB41 exon 16 splicing imparts new functionality to the encoded protein, enhancing protein-protein interactions that mechanically strengthen the red cell membrane. More recently, RNA-seq analysis revealed that numerous erythroid transcripts exhibit up-regulation of intron retention (IR) events, some of which are controlled by a decoy exon-mediated mechanism that can reduce the output of translated mRNA so as to limit protein expression. Here we demonstrate that modulation of decoy-mediated IR quantitatively affects protein expression in primary human erythroid cells. We first studied the OGT gene (O-GlcNAc transferase), a key regulator of O-GlcNAC homeostasis. OGT expression responds to pharmacological inhibitors by regulating intron 4 retention, by a mechanism requiring a intronic splicing silencer (1) that functions as a decoy exon (2) to nonproductively engage annotated splice sites at the ends of the intron, thereby blocking excision and enforcing its retention. In erythroid CD34+ progenitors at day 7 of culture, we found that treatment with an OGT inhibitor (OSMI-1) reduced OGT IR and increased spliced OGT RNA and OGT protein. Conversely, an inhibitor of the antagonistic OGA enzyme (thiamet-G) induced greater OGT IR and reduced OGT protein expression. These results are similar to what was reported previously in established cell lines (1), and suggest that modulation of IR can vary mRNA and protein expression in primary cells >5-fold. To further explore the model, we independently blocked OGT IR in erythroid cultures by electroporation of an antisense morpholino directed against the OGT decoy exon 5' splice site. RT-qPCR and western blot analysis confirmed substantial reduction in IR, coupled with an increase in spliced RNA and elevated OGT protein expression, compared to control cells or cells treated with an irrelevant morpholino. The OGT-specific MO also substantially blocked IR induced by thiamet-G. These results show that pharmacological- or antisense-mediated alteration in IR can significantly change protein expression in primary erythroblast cultures. We propose that the abundance of IR transcripts in late erythropoiesis represents a widespread modulation of protein output by post-transcriptional pathways operating at the level of intron retention. Park SK, et al. (2017) Cell Rep. 20: 1088-99.Parra M et al. (2018) RNA 24: 1255-65. Disclosures No relevant conflicts of interest to declare.

The Transcription Factor TAp63 Exerts Pro-Survival Effects in Chronic Lymphocytic Leukemia Acting through the BCL2 Pathway

Recent evidence indicates that TAp63, the prevalent isoform of TP63 in Chronic Lymphocytic Leukemia (CLL), is implicated in disease pathogenesis. In CLL, TAp63 expression, modulated by both immune signaling and epigenetic modifications, promotes leukemic cell survival and homing to the bone marrow. In activated normal B cells, the TAp63 transcription factor binds the BCL2 gene, participating in an anti-apoptotic pathway (axis NF-κB/TAp63/BCL2) augmenting cell survival. In this study, we investigated the expression of TAp63 in a large cohort of CLL cases and its potential fluctuation during disease progression. Additionally, in order to further understand the pro-survival role of TAp63 in CLL, we interrogated at the molecular level the interplay betweenΤAp63 and BCL2.Initially, using RT-qPCR we quantified TAp63 mRNA expression in 166 CLL patients, consisting of 89 with unmutated IGHV genes (U-CLL) and 77 with mutated IGHV genes (M-CLL), prior to administration of treatment. Significantly higher TAp63 mRNA levels were observed in U-CLL vs M-CLL (FD=13.83, p<0.0001). However, outliers were identified in both subgroups, prompting us to re-classify all cases into TAp63high and TAp63low subgroups using ROC curve and Youden index statistical procedures. TAp63high patients displayed significantly shorter time-to-first-treatment (TTFT) (TAp63highmedian TTFT: 1.58 years; TAp63lowmedian TTFT: 4.07 years; p=0.03) and shorter overall survival (OS) (TAp63highmedian OS: 7.825 years; TAp63lowmedian OS: not yet reached; p=0.046). Next, we analyzed TAp63 mRNA expression in longitudinal samples of 25 U-CLL cases treated with either FCR or rituximab-chlorambucil. In each case, samples were collected at three ‘landmarks’; diagnosis, first progressionand first relapse. Expression analysis by RT-qPCR showed that TAp63 levels significantly increased at disease relapse compared to diagnosis (FD=3.47, p=0.02).We subsequently investigated links between TAp63 and BCL2 by measuring BCL2 mRNA levels in 56 U-CLL cases from the present cohort and found statistically significant correlation with the corresponding TAp63 mRNA levels (spearman rho=0.31, p=0.01). To validate this observation, we undertook functional studies in the MEC1 CLL cell line. Considering that MEC1 cells express high TAp63 mRNA levels, we generated a stable MEC1 cell line to inducibly downregulate TAp63, using CRISPR/dCas9-KRAB upon treatment with doxycycline (Dox). We used 2 different guide RNAs (sgRNAs; sgRNA1, sgRNA2) targeting 2 distinct regions of the endogenous TAp63 promoter. After 5 days of induction, the expression levels of both TAp63 and BCL2 were quantified by one step RT-qPCR in Tet-on-dCas9-KRAB-sgRNA-TAp63 MEC1 cells. Inducible downregulation of TAp63 expression (gRNA1: FD=1.7, gRNA2: FD=1.53) resulted in downregulation of BCL2 expression (gRNA1: FD=1.34, gRNA2: FD=1.12) with strong correlation (rho=0.97, p<0.0001) between TP63 and BCL2 mRNA levels. Furthermore, we also observed correlation between TAp63 and BCL2 protein expression in primary cells of one representative TP63high CLL case (rho=0.94, p=0.01), in which TAp63 was silenced by RNA interference (RNAi) with 3 different siRNAs. Prompted by these results, we additionally assessed ex vivo the effect of the BCL2 inhibitor Venetoclax in primary CLL cells of both TAp63high (n=8) and TAp63low (n=6) cases. Cell viability was measured by flow cytometry at 24 and 48 hours after treatment. TAp63high cases were more resistant to treatment with Venetoclax as they showed no statistically significant reduction in cell viability compared to the respective (DMSO-treated) controls, in contrast to TAp63low cases (24h: FD=3.63, p=0.004; 48h: FD=7.17, p=0.005).In conclusion, we provide evidence suggesting that up-regulated TAp63 expression represents a novel resistance mechanism to chemoimmunotherapy in CLL. The pro-survival role of TAp63 is supported by its strong association with BCL2. Indeed, based on the present findings, TAp63 appears to act as a positive modulator of BCL2 in CLL cells, rendering them less responsive to apoptosis induction with the BCL2 inhibitor Venetoclax. DisclosuresHadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

ZNF598 Plays Distinct Roles in Interferon-Stimulated Gene Expression and Poxvirus Protein Synthesis

SUMMARYPost-translational modification of ribosomal subunit proteins (RPs) is emerging as an important means of regulating gene expression. Recently, regulatory ubiquitination of small RPs RPS10 and RPS20 by the ubiquitin ligase ZNF598 was found to function in ribosome sensing and stalling on internally polyadenylated mRNAs during ribosome quality control (RQC). Here, we reveal that ZNF598 and RPS10 negatively regulate interferon-stimulated gene (ISG) expression in primary cells, depletion of which induced ISG expression and a broad antiviral state. However, cell lines lacking interferon responses revealed that ZNF598 E3 ligase activity and ubiquitination of RPS20, but not RPS10, were specifically required for poxvirus replication and synthesis of poxvirus proteins whose encoding mRNAs contain unusual 5′ poly(A) leaders. Our findings reveal distinct functions for ZNF598 and its downstream RPS targets, one that negatively regulates ISG expression and infection by a range of viruses while the other is positively exploited by poxviruses.

Analysis of Bruton’s Tyrosine Kinase Deficiency in Patients with Presumed X-Linked Agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a fully penetrant X-linked recessive disorder characterized by the early onset of recurrent bacterial infections, profound hypogammaglobulinemia and a marked decrease in the number of B-lymphocytes [1]. The gene defective in XLA has been identified as a non-receptor proteine tyrosine kinase, BTK (Bruton´s tyrosine kinase) [2,3]. Over 600 mutations have been described in the BTK gene and they are spreaded throughout the gene [4]. Mutational analysis has revealed that there is considerable heterogeneity in the clinical spectrum of XLA, with atypical individuals having small numbers of mature B lymphocytes and some immnoglobulin production [5]. Analysis of Btk protein expression in primary cells of XLA patients has shown that the large majority of individuals do not express protein regardless of their mutation [6]. As it is yet known Btk is expressed not only in B cells but also in granulocytes and monocytes. In the present work we applied a flow cytometric test employing whole blood to detect Btk expression in monocytes to identify XLA patients.

Aberrant expression of p16INK4a in human cancers – a new biomarker?

The ARF and INK4a genes are located in the same CDKN2a locus, both showing its tumor suppressive activity. ARF has been shown to detect potentially harmful oncogenic signals, making incipient cancer cells undergo senescence or apoptosis. INK4a, on the other hand, responds to signals from aging in a variety of tissues including islets of Langerhans, neuronal cells, and cancer stem cells in general. It also detects oncogenic signals from incipient cancer cells to induce them senescent to prevent neoplastic transformation. Both of these genes are inactivated by gene deletion, promoter methylation, frame shift, and aberrant splicing although mutations changing the amino acid sequences affect only the latter. Recent studies indicated that polycomb gene products EZH2 and BMI1 repressed p16INK4a expression in primary cells, but not in cells deficient for pRB protein function. It was also reported that that p14ARF inhibits the stability of the p16INK4a protein in human cancer cell lines and mouse embryonic fibroblasts through its interaction with regenerating islet-derived protein 3γ. Overexpression of INK4a is associated with better prognosis of cancer when it is associated with human papilloma virus infection. However, it has a worse prognostic value in other tumors since it is an indicator of pRB loss. The p16INK4a tumor suppressive protein can thus be used as a biomarker to detect early stage cancer cells as well as advanced tumor cells with pRB inactivation since it is not expressed in normal cells.

MicroRNA-155 induction via TNF-α and IFN-γ suppresses expression of programmed death ligand-1 (PD-L1) in human primary cells

Programmed death ligand-1 (PD-L1) is a critical regulator of T cell function contributing to peripheral immune tolerance. Although it has been shown that posttranscriptional regulatory mechanisms control PD-L1 expression in cancer, it remains unknown whether such regulatory loops operate also in non-transformed cells. Here we studied PD-L1 expression in human dermal lymphatic endothelial cells (HDLECs), which play key roles in immunity and cancer. Treatment of HDLECs with the pro-inflammatory cytokines IFN-γ and TNF-α synergistically up-regulated PD-L1 expression. IFN-γ and TNF-α also affected expression of several microRNAs (miRNAs) that have the potential to suppress PD-L1 expression. The most highly up-regulated miRNA following IFN-γ and TNF-α treatment in HDLECs was miR-155, which has a central role in the immune system and cancer. Induction of miR-155 was driven by TNF-α, the effect of which was significantly enhanced by IFN-γ. The PD-L1 3′-UTR contains two functional miR-155-binding sites. Endogenous miR-155 controlled the kinetics and maximal levels of PD-L1 induction upon IFN-γ and TNF-α treatments. We obtained similar findings in dermal fibroblasts, demonstrating that the IFN-γ/TNF-α/miR-155/PD-L1 pathway is not restricted to HDLECs. These results reveal miR-155 as a critical component of an inflammation-induced regulatory loop controlling PD-L1 expression in primary cells.

OTX2 Activity at Distal Regulatory Elements Shapes the Chromatin Landscape of Group 3 Medulloblastoma.

Medulloblastoma is the most frequent malignant pediatric brain tumor and is divided into at least four subgroups known as WNT, SHH, Group 3, and Group 4. Here, we characterized gene regulation mechanisms in the most aggressive subtype, Group 3 tumors, through genome-wide chromatin and expression profiling. Our results show that most active distal sites in these tumors are occupied by the transcription factor OTX2. Highly active OTX2-bound enhancers are often arranged as clusters of adjacent peaks and are also bound by the transcription factor NEUROD1. These sites are responsive to OTX2 and NEUROD1 knockdown and could also be generated de novo upon ectopic OTX2 expression in primary cells, showing that OTX2 cooperates with NEUROD1 and plays a major role in maintaining and possibly establishing regulatory elements as a pioneer factor. Among OTX2 target genes, we identified the kinase NEK2, whose knockdown and pharmacologic inhibition decreased cell viability. Our studies thus show that OTX2 controls the regulatory landscape of Group 3 medulloblastoma through cooperative activity at enhancer elements and contributes to the expression of critical target genes.Significance: The gene regulation mechanisms that drive medulloblastoma are not well understood. Using chromatin profiling, we find that the transcription factor OTX2 acts as a pioneer factor and, in cooperation with NEUROD1, controls the Group 3 medulloblastoma active enhancer landscape. OTX2 itself or its target genes, including the mitotic kinase NEK2, represent attractive targets for future therapies. Cancer Discov; 7(3); 288-301. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

Comprehensive mapping of the effects of azacitidine on DNA methylation, repressive/permissive histone marks and gene expression in primary cells from patients with MDS and MDS-related disease.

Azacitidine (Aza) is first-line treatment for patients with high-risk myelodysplastic syndromes (MDS), although its precise mechanism of action is unknown. We performed the first study to globally evaluate the epigenetic effects of Aza on MDS bone marrow progenitor cells assessing gene expression (RNA seq), DNA methylation (Illumina 450k) and the histone modifications H3K18ac and H3K9me3 (ChIP seq). Aza induced a general increase in gene expression with 924 significantly upregulated genes but this increase showed no correlation with changes in DNA methylation or H3K18ac, and only a weak association with changes in H3K9me3. Interestingly, we observed activation of transcripts containing 15 endogenous retroviruses (ERVs) confirming previous cell line studies. DNA methylation decreased moderately in 99% of all genes, with a median β-value reduction of 0.018; the most pronounced effects seen in heterochromatin. Aza-induced hypomethylation correlated significantly with change in H3K9me3. The pattern of H3K18ac and H3K9me3 displayed large differences between patients and healthy controls without any consistent pattern induced by Aza. We conclude that the marked induction of gene expression only partly could be explained by epigenetic changes, and propose that activation of ERVs may contribute to the clinical effects of Aza in MDS.