Lentiviral Expression Research Articles

Abstract MP38: Functional Investigation Of A Novel Adipose Long Intergenic Non-coding RNA, Linc- ADAIN, During Obesity

Background: Long intergenic non-coding RNAs (lincRNAs) are important emerging regulators of cellular functions. Dysfunctional adipose tissue, characterized by increased inflammation and insulin resistance, plays a central role in the development of type 2 diabetes (T2DM) and atherosclerotic cardiovascular diseases. Deep RNA-sequencing of human adipose during low-dose endotoxemia, identified novel lincRNAs shown to be regulated by inflammation and were validated in an independent human cohort, before and after bariatric surgery. LincADAIN was identified as a top candidate that is 1)abundantly expressed in adipose tissue 2)reduced in both obesity-induced chronic and LPS-induced acute inflammation (-77% and -53%, p<0.05). Methods: Knock down(KD) of lincRNAs was carried out via lentiviral expression of shRNA and antisense oligonucleotides (ASOs) in human adipose stromal cells (ASCs)-adipocytes. Subcellular location of LincADAIN was identified through qPCR after cellular fractionation. Biotinylated lincRNA pulldown assay was used to identify interacting proteins by Mass Spectrometry (MS). Results: Human LincADAIN expression negatively correlates to BMI in obese (p<0.001, r2-0.3042) but not lean individuals. PPARγ antagonist treatment showed reduced expression of LincADAIN and PPARγ ChIP-seq peaks indicate that PPARγ may be a transcriptional activator. KD via shRNA of LincADAIN in ASC-adipocytes, increased protein but not mRNA levels of inflammatory cytokines in adipocytes such as IL-8 and MCP-1. Cellular fractionation experiments indicate that LincADAIN is mainly located in the nucleus. MS analysis of biotinylated LincADAIN pulldown proteins revealed many central nuclear and ribosomal proteins as potential interactors, which may be involved in the post-transcriptional mechanism of LincADAIN regulating inflammatory cytokines. Conclusion: These results suggest that LincADAIN is a novel lincRNA with roles in regulating adipose inflammation through nuclear and cytoplasmic actions including modulation of RNA translation. Functional investigations of lincRNAs, such as these are warranted, as recent genomic studies suggest that lincRNAs are likely the causal element driving human disease at some GWAS loci.

Mutual regulation between chicken telomerase reverse transcriptase and the Wnt/\u03b2-catenin signalling pathway inhibits apoptosis and promotes the replication of ALV-J in LMH cells

This study aimed to explore the mutual regulation between chicken telomerase reverse transcriptase (chTERT) and the Wnt/β-catenin signalling pathway and its effects on cell growth and avian leukosis virus subgroup J (ALV-J) replication in LMH cells. First, LMH cells stably overexpressing the chTERT gene (LMH-chTERT cells) and corresponding control cells (LMH-NC cells) were successfully constructed with a lentiviral vector expression system. The results showed that chTERT upregulated the expression of β-catenin, Cyclin D1, TCF4 and c-Myc. chTERT expression level and telomerase activity were increased when cells were treated with LiCl. When the cells were treated with ICG001 or IWP-2, the activity of the Wnt/β-catenin signalling pathway was significantly inhibited, and chTERT expression and telomerase activity were also inhibited. However, when the β-catenin gene was knocked down by small interfering RNA (siRNA), the changes in chTERT expression and telomerase activity were consistent with those in cells treated with ICG001 or IWP-2. These results indicated that chTERT and the Wnt/β-catenin signalling pathway can be mutually regulated. Subsequently, we found that chTERT not only shortened the cell cycle to promote proliferation but also inhibited apoptosis by downregulating the expression of Caspase 3, Caspase 9 and BAX; upregulating BCL-2 and BCL-X expression; and promoting autophagy. Moreover, chTERT significantly enhanced the migration ability of LMH cells, upregulated the protein and mRNA expression of ALV-J and increased the virus titre. ALV-J replication promoted chTERT expression and telomerase activity.

Ribosomal L1 domain-containing protein 1 coordinates with HDM2 to negatively regulate p53 in human colorectal Cancer cells

BackgroundRibosomal L1 domain-containing protein 1 (RSL1D1) is a nucleolar protein that is essential in cell proliferation. In the current opinion, RSL1D1 translocates to the nucleoplasm under nucleolar stress and inhibits the E3 ligase activity of HDM2 via direct interaction, thereby leading to stabilization of p53.MethodsGene knockdown was achieved in HCT116p53+/+, HCT116p53−/−, and HCT-8 human colorectal cancer (CRC) cells by siRNA transfection. A lentiviral expression system was used to establish cell strains overexpressing genes of interest. The mRNA and protein levels in cells were evaluated by qRT-PCR and western blot analyses. Cell proliferation, cell cycle, and cell apoptosis were determined by MTT, PI staining, and Annexin V-FITC/PI double staining assays, respectively. The level of ubiquitinated p53 protein was assessed by IP. The protein-RNA interaction was investigated by RIP. The subcellular localization of proteins of interest was determined by IFA. Protein-protein interaction was investigated by GST-pulldown, BiFC, and co-IP assays. The therapeutic efficacy of RSL1D1 silencing on tumor growth was evaluated in HCT116 tumor-bearing nude mice.ResultsRSL1D1 distributed throughout the nucleus in human CRC cells. Silencing of RSL1D1 gene induced cell cycle arrest at G1/S and cell apoptosis in a p53-dependent manner. RSL1D1 directly interacted with and recruited p53 to HDM2 to form a ternary RSL1D1/HDM2/p53 protein complex and thereby enhanced p53 ubiquitination and degradation, leading to a decrease in the protein level of p53. Destruction of the ternary complex increased the level of p53 protein. RSL1D1 also indirectly decreased the protein level of p53 by stabilizing HDM2 mRNA. Consequently, the negative regulation of p53 by RSL1D1 facilitated cell proliferation and survival and downregulation of RSL1D1 remarkably inhibited the growth of HCT116p53+/+ tumors in a nude mouse model.ConclusionWe report, for the first time, that RSL1D1 is a novel negative regulator of p53 in human CRC cells and more importantly, a potential molecular target for anticancer drug development.

Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity.

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.

Lentiviral and AAV-mediated expression of palivizumab offer protection against Respiratory Syncytial Virus infection

Respiratory syncytial virus (RSV) infection is a common cause of hospitalisation in infants and the elderly. Palivizumab prophylaxis is the only approved treatment modality but is costly and only offered to select vulnerable populations. Here, we investigated gene delivery approaches via recombinant adeno-associated virus (rAAV2/8) and simian immunodeficiency virus (rSIV.F/HN) vectors to achieve sustained in vivo production of palivizumab in a murine model. Delivery of palivizumab-expressing vectors 28 days prior to RSV challenge resulted in complete protection from RSV-induced weight loss. This approach offers prophylaxis against RSV infection, allowing for wider use and reduction in treatment costs in vulnerable populations.

Functional analysis of a putative Bombyx mori cypovirus miRNA BmCPV-miR-10 and its effect on virus replication.

Bombyx mori cypovirus (BmCPV) is an important pathogen of silkworm (B. mori), the economically beneficial insect. The mechanism of its interaction with host immune defence system in the process of infection is still not yet completely clear. Researches have demonstrated that virus-encoded microRNAs (miRNA) play a crucial role in regulating host-pathogen interaction, but few reports are available so far on miRNAs encoded by insect viruses, especially the RNA viruses. In this study, a putative miRNA encoded by the 10th segment of BmCPV genomic RNA, BmCPV-miR-10, was identified and functionally analysed. The expression of the putative BmCPV-miR-10 could be detected via stem-loop RT-PCR (reverse transcription-Polymerase Chain Reaction) in the midgut of silkworm larvae infected with BmCPV. BmCSDE1 (B. mori cold shock domain E1 protein) gene was predicted to be a candidate target gene for BmCPV-miR-10 with the miRNA binding site located in 3' untranslated region of its mRNA. The regulation effect of the putative BmCPV-miR-10 on BmCSDE1 was verified in HEK293 cells by lentiviral expression system, in BmN cells by transfecting BmCPV-miR-10 mimics. The qRT-PCR (quantitative real-time PCR) results showed that the putative BmCPV-miR-10 could suppress the expression of BmCSDE1. By injection of BmCPV-miR-10 mimics into the silkworm larvae infected with BmCPV, it was further proved that the putative BmCPV-miR-10 could suppress the expression of BmCSDE1 in vivo, then inhibit the expression of BmApaf-1 (B. mori apoptotic protease activating factor 1), while enhance the replication of BmCPV genomic RNAs to a certain extent. These results implied that the putative BmCPV-miR-10 could down-regulate the expression of BmCSDE1, then suppress the expression of BmApaf-1, thereby created a favourable intracellular environment for virus replication and proliferation.

Pneumococcal surface adhesion A protein (PsaA) interacts with human Annexin A2 on airway epithelial cells

ABSTRACT Streptococcus pneumoniae (pneumococcus) is a normal colonizer of the human nasopharynx capable of causing serious invasive disease. Since colonization of the nasopharynx is a prerequisite for progression to invasive diseases, the development of future protein-based vaccines requires an understanding of the intimate interaction of bacterial adhesins with host receptors. In this study, we identified that pneumococcal surface adhesin A (PsaA), a highly conserved pneumococcal protein known to play an important role in colonization of pneumococcus, can interact with Annexin A2 (ANXA2) on Detroit 562 nasopharyngeal epithelial cells. Lentiviral expression of ANXA2 in HEK 293 T/17 cells, which normally express minimal ANXA2, significantly increased pneumococcal adhesion. Blocking of ANXA2 with recombinant PsaA negatively impacted pneumococcal adherence to ANXA2-transduced HEK cells. These results suggest that ANXA2 is an important host cellular receptor for pneumococcal colonization.

MAFA Expression Promotes Proliferation of Myeloma Cells and Its Protein Stability Is Regulated By Proteasome Inhibitors

IntroductionMultiple myeloma (MM) patients with a t(14;16) or t(14;20) which result in high expression of MAF and MAFB, respectively, have a poor prognosis, and have not benefitted from the recent advances in MM therapy. Our previous work showed that post-translational modifications (PTM) further up-regulated MAF (Blood, 2016) and MAFB proteins (ASH Abstract #2091, 2014) conferring innate resistance to proteasome inhibitors (PI). The large MAF family of proteins contains a third member, MAFA. Little is known about its biological role in MM pathogenesis, as a probeset for MAFA is not present in gene expression profiling (GEP) analysis of molecular subgroups, and MAFA probes are not routinely used in fluorescence in situ hybridization (FISH) analysis. In the present study we therefore investigated the biological role of MAFA in MM and its effect on PI resistance.MethodsThe MAFA translocation in primary MM cells was determined by FISH analysis in 19 newly diagnosed MM patients. MAFA protein expression and protein level in response to PIs were assessed by immunoblotting and immunofluorescence staining (IFS). The effect of MAFA on proliferation of MM cells was measured by MTT assay. Annexin V staining and BrdU incorporation, analyzed by FACS analysis, was performed to determine the molecular action of MAFA on the survival of MM cells. MAFA expression was down-regulated in human MM cell lines (HMCLs). HMCLs using a lentiviral shRNA expression system. qRT-PCR analysis was used to determine MAFA mRNA expression in 32 HMCL and primary CD138+ plasma cells from 14 MM patients, and MAFA levels after shRNA treatment.ResultsFour levels of MAFA mRNA expression (relative to GAPDH) by RT-PCR analysis were determined: high levels were detected in 5 HMCLs, intermediate levels (100 to 500 ) in 10, low levels (10<100) in 7 and negative (<10) in 10 HMCLs. Among primary CD138+ patient cells (n=14), high levels were found in one patient, intermediate levels in 3, low levels in 4 and negative in 6 patients. FISH analysis found MAFA /t(8;14)(q24;q32) translocation in one out of 19 patients, which was also in the MF group by GEP analysis suggesting common downstream targets of MAFA with MAF and MAFB . MAFA protein was detected in the 6 HMCLs which expressed the highest MAFA mRNA levels by RT-PCR but not in the others.To determine if MAFA is required for MM cell survival, we knocked-down MAFA expression in three HMCLs that expressed high levels of MAFA . sh MAFA lentiviral supernatant infected HMCLs had 75% lower levels of MAFA mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, bioinformatically defined MAFA target genes were significantly decreased, including TLR4 (2.4-fold, P<0.01), NUAK1 (2.3-fold, p<0.01) and SPP1 (osteopontin, 2.2-fold, P<0.01), compared with HMCLs infected with control viral vector. Silencing MAFA expression significantly decreased the proliferation of HMCLs (62.5% decrease, P=2.1x10-4). Among 6 high MAFA expressing HMCLs, 3 co-expressed MAF and 2 co-expressed MAFB . To determine if MAFA synergized with MAF in promoting MM proliferation, we silenced MAFA and MAFB genes in cells co-expressing these genes. Simultaneously silencing MAFA and MAFB did not further decrease proliferation compared to either silencing MAFA or MAFB alone. These results indicate that MAFA promotes MM cell proliferation and that there is no synergetic effect between MAFA and MAFB in regulating MM proliferation.To determine if MAFA protein is regulated by PTM, we treated MM cells with PIs including bortezomib (Bzb) and carfilzomib (CFZ). Bzb induced stabilization of MAFA protein in a dose-dependent manner. Similar effects were seen with CFZ indicating that MAFA protein may be degraded by the ubiquitin proteasome system.ConclusionTaken together, our results indicate that increased levels of MAFA are seen in MM including the MAFA /t(8;14) translocation. High expression of MAFA mRNA and protein occurs in 18.7 % of MM cell lines. MAFA promotes myeloma proliferation and regulates TLR4, NUAK1 and SPP1 gene expression. The stability of MAF protein is regulated by PIs. These results provide new insights in understanding the role of the MAF family and, in particular, MAFA in MM pathogenesis. Experiments are ongoing to determine the function of MAFA in PI-resistance and to confirm MAFA targets in order to identify potential alternate approaches for the treatment of this poor performing molecular subgroup. DisclosuresMorgan:Bristol Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Davies:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria.

MAF and NF-κB Signaling Pathways Regulate Expression of APOBEC3B in Multiple Myeloma

IntroductionAPOBEC mutagenesis is a source of mutations in ~50% of human cancers, resulting from the over-expression of APOBEC proteins, including APOBEC3B (A3B). High expression of A3B is associated with poor prognosis and drug resistance in many cancers. In multiple myeloma, the chromosomal translocation t(14;16) leads to the over-expression of the transcription factor MAF through juxtaposition with the IGH super-enhancer. We have shown that in this poor prognosis sub-group A3B is also over-expressed and there is an enrichment for APOBEC signature mutations and a higher mutational load. It is not clear whether over-expression of A3B results from direct activation by MAF or indirectly through downstream pathways. In other cancers, mitosis, cell-cycle related gene expression, and the PKC/NF-κB signaling pathway may also affect expression of A3B.MethodsGene expression profile (GEP, U133 Plus 2.0) of 50 human myeloma cell lines (HMCLs) and 1310 myeloma patients was performed. mRNA levels of MAF and the AID /APOBEC family was investigated by qPCR in HMCLs. Western blotting and a DNA cytosine deaminase activity assay were used to detect A3B protein and analyze A3B enzymatic activity which results in mutagenesis, respectively. The effect of MAF on expression of A3B was investigated in inducible TRIPZ lentiviral shRNA c-MAF knockdown in HMCLs with high expression of MAF. Bortezomib, a proteasome inhibitor, and phorbol 12-myristate 13-acetate (PMA), a PKC dependent activator of the NF-κB signaling pathway, was used to investigate A3B expression in TRIPZ lentiviral shRNA MAF (MF KO) and A3B (A3B KO) knockdown HMCLs. Localization of A3B in regular HMCLs and cell lines with over-expression of A3B (generated using ViraPower T-Rex lentiviral expression system) was investigated by immunofluorescent analysis (IFA) using laser confocal and high resolution microscopy.ResultsBy microarray, high levels (log2>10) of A3B mRNA were found in approximately half of multiple myeloma patients. The majority of t(14;16) MAF patients had high levels of A3B. Increased expression of A3B was also detected in 72% of HMCLs. In t(14;16), MAF, and t(14;20), MAFB, HMCLs there was a strong positive correlation between MAF and A3B mRNA expression (r2= 0.51 and r2=0.43, respectively). On the basis of levels of MAF expression two groups of HMCLs were selected. Group 1 with high expression of MAF (MF) with the presence of a t(14;16) ARP1, CAG, JJN3, OCMY5, MM1S, and RPMI8226. Group 2 had low expression of MAF (non-MF) and was represented by cell lines with t(11;14) - U266, XG1 and KMS12BM; t(4;14) - KMS28PE and H929; or t(14;20) SACHI. We found a strong positive correlation (r2=0.68) between MAF and A3B mRNA expression in MF cell lines and no such correlation in the non-MF group (r2=0.09). Upon down-regulation of MAF by shRNA, JJN3 and RPMI8226 showed reduced expression of A3B protein (-33% and -29%, respectively) by 72 hours with accompanying reduction in cytosine deaminase activity (-23% and -26%, respectively). There was a positive correlation between expression of MAF protein and A3B protein in JJN3 MAF KO r2= 0.21, in RPMI8226 MAF KO r2=0.50; and a positive correlation between MAF protein and DNA cytosine deaminase activity in JJN3 MAF KO r2=0.21 and in RPMI8226 MAF KO r2=0.34. Bortezomib reduced expression of MAF and A3B and DNA cytosine deaminase activity in JJN3 and RPMI8226 cell lines. PMA increased the expression of A3B mRNA in HMCLs within 6 hours of treatment and lasted for 7 days after treatment. The highest sensitivity of A3B to PMA was detected in JJN3 and U266 cell lines, with increases of 4.4 and 8.2-fold, respectively, after 6 hours of treatment. Protein levels of A3B were increased 2.4-fold in both cell lines but DNA cytosine deaminase activity was increased (27%) only in JJN3 cells after PMA treatment.ConclusionsKnock down of MAF results in decreased expression and activity of A3B, indicating that MAF is a regulator of A3B expression in multiple myeloma. Expression of A3B in t(14;16) cases is dependent upon MAF expression but can also be increased through activation of the PKC/NF-κB signaling pathway, resulting in increased DNA damage. Understanding the mechanisms responsible for mutagenesis in the t(14;16) subgroup through A3B, which results in new clone formation and drug resistance in multiple myeloma will be highly beneficial in designing new therapeutic strategies. DisclosuresDavies:Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Morgan:Bristol Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria.

MBD2-NuRD but Not MBD3-NuRD Mediates Strong Silencing of Fetal γ-Globin Gene Expression in Human Adult Erythroid Cells

Elevated fetal hemoglobin (HbF) levels are effective in ameliorating the underlying pathophysiologic defects in β-thalassemia and sickle cell anemia. Studies aimed at a full understanding of the mechanisms that enforce silencing of HbF expression in adult erythroid cells offer the promise of targeted molecular therapy. Here we show that depletion of Methyl CpG Binding Domain Protein 2 (MBD2) or disruption of MBD2-Nucleosome Remodeling and Deacetylose Complex (NuRD) interactions results in high levels of HbF in HUDEP-2 cell progenitor derived primary erythroblasts and CD34 + progenitor derived primary adult erythroid cells.We have previously shown that depletion of MBD2 causes a 10-20 fold increase in γ-globin gene expression in adult erythroblasts of β-YAC transgenic mice. To determine the effect of total depletion of MBD2 in human erythroid cells, CRISPR/Cas9 mediated knockout was carried out in HUDEP-2 cells. The absolute level of γ-globin gene expression increased by > 100 fold and the γ/ γ+β mRNA ratio increased from 1-3% in CRISPR/Cas9 scrambled guide control cells to 50-60% in MBD2 knockout HUDEP-2 cells after erythroid differentiation, equivalent to the highest levels reported with knockout of other silencing factors. In contrast CRISPR/Cas9 mediated knockout of Methyl CpG Binding Domain Protein 3 (MBD3) had no significant effect on γ-globin gene expression levels in HUDEP-2 cells after erythroid differentiation.Partial depletion of several strong γ-globin gene silencers, including BCL11A, LRF and KLF-1 has been shown to relieve silencing in CD34+ progenitor derived primary adult erythroid cells, but differences in the differentiation states reported in individual studies make comparisons of the relative effects of these silencer factors difficult. We show here that knockdown of MBD2 by shRNA in CD34+ progenitor derived primary adult erythroid cells consistently increased γ/ γ+β mRNA rations by 7-10 fold across different stages of differentiation without affecting the level of differentiation as compared to shRNA scrambled control cells. MBD2 knockdown led to ~30-40% γ-globin mRNA levels compared to 3%-4% in scrambled shRNA control cells, a level comparable to that typically observed in sickle cell patient reticulocytes. A comparable increase in HbF levels was observed in the MBD2 shRNA knockdown CD34+ progenitor derived erythroid cells compared to scrambled shRNA controls.We have previously shown that MBD2 interacts with the CHD4 chromatin remodeling component of the NuRD complex through a coiled-coil domain, and with the histone deacetylase component of the complex via an intrinsically disordered region (IDR) within MBD2. To test the effects of disrupting these domains in a human adult erythroid cell model, MBD2 knockout HUDEP-2 cells were infected with lentiviral expression vectors containing either wild type MBD2, coiled-coil mutant MBD2, or IDR mutant MBD2. Wild type MBD2 expression resulted in significant silencing of γ-globin gene expression while neither the coiled-coil mutant nor the IDR mutant MBD2 had any silencing effect.These results establish MBD2-NuRD as a major silencer of fetal hemoglobin expression in adult human erythroid cells. Given the minimal phenotype of MBD2 knockout mice, these results suggest that the coiled-coil and IDR domains of the MBD2-NuRD complex are potential targets for therapeutic agents aimed at increasing HbF levels in patients with β-hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

APOC2 Is Upregulated in Acute Myeloid Leukemia and Presents a Novel Therapeutic Target

Acute Myeloid Leukemia (AML), is a devastating hematologic malignancy that affects the hematopoietic stem cells. Certain cytogenetic and molecular abnormalities have been implicated in disease pathogenesis, which result in abnormal proliferation and differentiation of myeloid blasts. Current therapy provides five years' overall survival (OS) in 35%-40% of young patients and 5-15% of old patients. Thus, there is an urgent need to investigate new therapeutic targets.To identify therapeutic targets in AML, we analyzed gene expression datasets (GSE1159, GSE7186, GSE13159 and GSE13164) for genes that are differentially overexpressed in AML compared with healthy donor cells. We found that Apolipoprotein CII (APOC2) was consistently expressed at a significantly higher level in AML than in control samples (P<0.0001). APOC2 is component of low density lipoprotein which activates lipase and thus contributes to lipoproteins metabolism. However, the role of APOC2 in cancer is unknown.Here, we report that APOC2 mRNA is significantly overexpressed in 542 AML patients compared with 74 healthy peripheral blood mononuclear cells (PBMC) samples (GSE13159, 2.9 fold, P<0.0001). In GSE13164, APOC2 median mRNA level was higher in 257 AML samples compared with 58 healthy PBMC samples (1.7 fold, P<0.0001). Consistently, APOC2 median mRNA level was upregulated in 285 AML samples compared with 8 control samples in the GSE1159 dataset (6 fold, P<0.001). In the GSE7186, APOC2 showed a higher median mRNA level in 23 AML patients compared with 6 normal bone marrow samples (32 fold, P<0.0001).To understand the role of APOC2 and its deregulation in AML, we analyzed its expression according to cytogenetic and mutational status in patients with AML. APOC2 is significantly upregulated in t(11q23)/MLL-rearranged AML. APOC2 median mRNA level was higher in 38 patients with MLL rearrangement than that in 504 patients without MLL rearrangement (GSE13159, 9 fold, P<0.0001). APOC2 median mRNA level was also higher in 47 patients with MLL rearrangement than that in 190 patients without MLL rearrangement (GSE17855, 6.6 fold, P<0.0001). Similar findings were observed in three other data sets (GSE1159, 19-fold, P<0.0001; TCGA, 6 fold, P=0.0003; GSE13164, 6.7 fold, P<0.0001). In addition, patients with FLT3 mutation had 5-7 fold higher APOC2 median mRNA expression compared with patients carrying the wild type FLT3 (GSE1159, 5.4 fold, P=0.0006; TCGA data, 6.9 fold, P=0.0007). APOC2 levels were also higher in patients with t(15;17) translocation than that in patients without t(15;17) (GSE1159, 4.9 fold, P=0.0024; TCGA, 36.5 fold, P=0.0098).We also examined the association between APOC2 overexpression and clinical outcome of patients with AML excluding t(15;17) patients. Patients in the TCGA data were dichotomized based on their APOC2 mRNA expression Z-score (RNA Seq V2 RSEM) into high (Z-score ≥1) and low (Z-Score <1). Patients with high APOC2 levels had significantly shorter OS than patients with low APOC2 expression (median OS 4.05 vs 10.3 months, P=0.002).To assess the function of APOC2 in AML, we established an ectopic lentiviral expression system to generate stable cells overexpressing APOC2 (APOC2-OE). In three different AML cell lines (U937, MOLM13 and THP-1), APOC2 overexpression caused significant increase in cell proliferation compared with empty vector control (2 fold, P<0.0001). Similarly, in primary blasts from three patients with AML, overexpression of APOC2 increased cell number of primary blasts (3 fold, P=0.0364). Given that APOC2 is a secreted protein, we examined the effect of secreted APOC2 on the growth of AML cell lines. Naïve cell lines (U937, MOLM13 and THP-1) were treated with supernatant collected from their respective APOC2-OE cells or empty vector cells. Naïve cells treated with supernatant from APOC2-OE cells showed a significant increase of cell proliferation compared with cells treated with supernatant from empty vector cells (30 to 70% increase, P<0.01).In conclusion, APOC2 is highly expressed in AML, specifically in patients with MLL-rearrangement and in patients with FLT3 -ITD mutation. We also demonstrated that secreted APOC2 promotes cell proliferation in AML cells. More studies are ongoing to characterize the functional role of APOC2 overexpression in leukemogenesis and to investigate the mechanisms by which this gene is deregulated in MLL-rearrangements AML. DisclosuresNo relevant conflicts of interest to declare.

In vitro functional analysis of four variants of human asparagine synthetase.

The loss-of-function variants of the human asparagine synthetase (ASNS) gene cause asparagine synthetase deficiency (ASNSD). Diagnosis of ASNSD requires genetic tests because a specific biochemical diagnostic for ASNSD is not available. There are a few reports describing the functional evaluation of ASNS variants. Therefore, in vitro methods are needed to evaluate the detected variants in patients. In this report, five types of human ASNS proteins (wild-type and our reported four variants: p.Leu145Ser, p.Leu247Trp, p.Val489Asp, and p.Trp541Cysfs*5) were expressed in silkworm using a baculoviral expression system. An enzymatic activity assay of ASNS was performed, and the concentration of asparagine by ninhydrin and High Performance Liquid Chromatography methods using the purified recombinant proteins was measured. We established ASNS deficient HEK293 cells using the CRISPR/Cas9 method and evaluated the growth of cells without asparagine after transduction of ASNS variants with a lentiviral expression system. The four ASNS variants displayed significantly low enzymatic activity. The ASNS deficient HEK293 cells transduced with wild-type ASNS grew without asparagine, whereas cells transduced with the variants did not grow or showed significantly slower growth than cells transduced with wild-type ASNS. Herein, we established a method for evaluating the enzymatic activity of the recombinant human ASNS variants. The results of the cell-based assay corroborated the results of the enzymatic activity. These methods should enable the evaluation of the pathogenicity of ASNS variants.

Immunoprecipitation Strategies to Isolate RIPK1/RIPK3 Complexes in Mouse Macrophages.

A large protein complex, containing RIPK1, RIPK3, and caspase-8 and known as Complex II, has emerged as one of the key mediators of cell death downstream from a range of innate immune triggers. This regulatory mechanism plays a prominent role in macrophages, where Complex II has been linked to apoptosis, pyroptosis, and necroptosis as well as the enhancement of inflammatory gene expression. Although core components of this complex are fairly well understood, more subtle proteomic changes that determine the direction of a response once the complex is assembled remain much less clear. In addition, Complex II components undergo a wealth of post-translational changes that modify the functions of the complex components. This necessitates development of robust and efficient methods of isolating Complex II for further interrogation of its composition and the post-translational modifications of its components. This article describes several methods that we have developed for Complex II isolation, which can be used to obtain complementary information about this signaling mechanism. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of Complex II in necroptotic and pyroptotic macrophages using FADD immunoprecipitation Basic Protocol 2: Isolation of the complexes formed by the conditionally expressed 3XFLAG-RIPK1 protein Alternate Protocol: Alternative methods of immunoprecipitation of RIPK1 and other Complex-II-related factors Support Protocol: Generation of stable macrophage cell lines using lentiviral expression Basic Protocol 3: Use of proximity labeling to identify necrosome components in the detergent-insoluble fraction of the cell lysates.

Generating An Exosome Packaged p38 Inhibitory Peptide To Block GPCR Induced Vascular Inflammation

A key mediator of angiogenesis and inflammatory response in chronic lung disease is the mitogen-activated protein kinase (MAPK), p38α. The typical activation pathway for p38α uses MAP kinase kinase 3 (MKK3), and MKK6 to phosphorylate and activate p38α. However, an atypical p38α signaling pathway activated by a family of pro-inflammatory G-protein coupled receptors (GPCRs) induces a direct interaction between transforming growth factor β activated kinase 1 binding protein-1 (TAB1) and p38α, bypassing MKK3/6-dependent p38α activation. The binding of TAB1 to p38α induces the autophosphorylation of p38α, triggering proinflammatory signaling in the vasculature. Recent studies have highlighted the use of cell-penetrating inhibitors that specifically block atypical p38α signaling, leaving MKK3/6-dependent signaling intact. Using the Xpack Lentiviral expression system, we have generated a stable cell line that expresses red fluorescent protein (RFP) labeled peptide inhibitors in endothelial cells. The Xpack system contains an exosomal packaging motif enabling RFP-linked inhibitor peptides to be targeted for exosomes. Using filter centrifugation and ultracentrifugation we have isolated RFP peptide loaded exosomes/extracellular vesicles. These inhibitor loaded extracellular vesicles can successfully deliver inhibitors to vascular endothelial cells in vitro. Our current studies focus on assessing whether cellular uptake of peptide loaded exosomes can efficiently attenuate GPCR-induced proinflammatory signaling. To investigate this we are using a combination of immunoblotting and live-cell Forster's Resonance Energy Transfer (FRET) assays. We predict that exosomally delivered peptides will inhibit p38 signaling by GPCR ligands in endothelial cells providing a tool to produce large quantities of bioavailable inhibitors that can be developed into therapeutics. These studies demonstrate the potential for exosomal nanocarriers to deliver peptide inhibitors to block GPCR inflammation.

Breaching the Delivery Barrier: Chemical and Physical Airway Epithelium Disruption Strategies for Enhancing Lentiviral-Mediated Gene Therapy

The lungs have evolved complex physical, biological and immunological defences to prevent foreign material from entering the airway epithelial cells. These mechanisms can also affect both viral and non-viral gene transfer agents, and significantly diminish the effectiveness of airway gene-addition therapies. One strategy to overcome the physical barrier properties of the airway is to transiently disturb the integrity of the epithelium prior to delivery of the gene transfer vector. In this study, chemical (lysophosphatidylcholine, LPC) and physical epithelium disruption using wire abrasion were compared for their ability to improve airway-based lentiviral (LV) vector mediated transduction and reporter gene expression in rats. When luciferase expression was assessed at 1-week post LV delivery, LPC airway conditioning significantly enhanced gene expression levels in rat lungs, while a long-term assessment in a separate cohort of rats at 12 months revealed that LPC conditioning did not improve gene expression longevity. In rats receiving physical perturbation to the trachea prior to gene delivery, significantly higher LacZ gene expression levels were found when compared to LPC-conditioned or LV-only control rats when evaluated 1-week post gene transfer. This proof-of-principle study has shown that airway epithelial disruption strategies based on physical perturbation substantially enhanced LV-mediated airway gene transfer in the trachea.

Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate de novo T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent “induced DCs” (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14+ monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34+ cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.

CHI3L1 promotes Staphylococcus aureus-induced osteomyelitis by activating p38/MAPK and Smad signaling pathways

AimsStaphylococcus aureus (S. aureus) is the most common causative bacterial pathogen involved in promoting infection-induced osteomyelitis, a disease resulting in severe bone degradation. In this study, we aimed to identify the mechanism behind inhibition of osteoclast survival and differentiation by CHI3L1, a lectin previously reported to regulate S. aureus-induced osteomyelitis. Main methodsThe role of CHI3L1 in osteoclast survival, proliferation, and differentiation was studied ex vivo using primary human bone marrow derived stem cells (HBMSCs) and transducing them with lentiviral expression vectors or shRNA knockdown constructs. Cell apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate/propidium iodide staining. Cell proliferation was assessed using alkaline phosphatase, Alcian Blue, and TRAP staining. The qRT-PCR was used to measure mRNA levels of osteoclast maturation markers, and western blotting was used to analyze protein expression. An in vivo murine model for osteomyelitis and microcomputed tomography analyses of infected femurs were used to study the effects of CHI3L1 on bone erosion. Key findingsOverexpression of CHI3L1 significantly reduced HBMSC cell viability, proliferation, and differentiation, and knockdown improved these effects in the presence of S. aureus infection. More specifically, CHI3L1 constitutively activated the p38/MAPK pathway to promote apoptosis. Furthermore, CHI3L1 induced activation of the Smad pathway by promoting phosphorylation of Smad-1/5 proteins. Finally, overexpression of CHI3L1 significantly induced bone erosion upon S. aureus infection in a murine osteomyelitis model, and knockdown of CHI3L1 significantly alleviated this effect. SignificanceCHI3L1 played a vital role in osteoblast differentiation and proliferation by regulating the p38/MAPK and Smad signaling pathways to promote S. aureus-induced osteomyelitis.

EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells

BackgroundChromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells.MethodsHere we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes.ResultsThe lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated β-galactosidase (SA-β-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice.ConclusionsOur data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

Lentiviral vector-mediated expression of C3 transferase attenuates retinal ischemia and reperfusion injury in rats

AimsOur previous study showed that intravitreal delivery of self-complementary AAV2 (scAAV2)-mediated exoenzyme C3 transferase (C3) can attenuate retinal ischemia/reperfusion (I/R) injury. The current study investigated the neuroprotective effects of lentivirus (LV)-mediated C3 transgene expression on rat retinal I/R injury. Main methodsThe LV encoding C3 and green fluorescent protein (GFP) together (LV-C3-GFP) or GFP only (LV-GFP) was intravitreally injected to SPRAGUE-DAWLEY rats. On day 5 post-intravitreal injection, eyes were evaluated by slit-lamp examination. The GFP expression on retina was confirmed by in vivo and ex vivo assessments. RhoA GTPase expression in retina was examined by western blot. Retinal I/R injury was generated by transiently increasing intraocular pressure (110 mmHg, 90 min). Eyes were then enucleated, and retinas processed for morphological analysis and TdT-dUTP terminal nick-end labeling (TUNEL) assay. Key findingsNo obvious inflammatory reactions or surgical complications were observed after intravitreal injection of LV vectors. There was a significant decrease of total RhoA GTPase level in the retina treated with LV-C3-GFP. Compared to the blank control group, LV-C3-GFP and LV-GFP did not affect the retinal thickness, cell density in ganglion cell layer (GCL), or numbers of apoptotic cells in retinal flat-mounts. In the LV-GFP-treated retinas, I/R decreased the retinal thickness and GCL cell density and increased apoptotic retinal cell numbers. LV-C3-GFP significantly protected against all these degenerative effects of I/R. SignificanceThis study indicated that LV-mediated C3 transgene expression exhibits neuroprotective effects on the retinal I/R injury and holds potential as a novel neuroprotective approach targeting certain retinopathies.

Somatic Epigenetic Silencing of RIPK3 Inactivates Necroptosis and Contributes to Chemoresistance in Malignant Mesothelioma.

Receptor-interacting protein kinase 3 (RIPK3) phosphorylates effector molecule MLKL to trigger necroptosis. Although RIPK3 loss is seen in several human cancers, its role in malignant mesothelioma is unknown. This study aimed to determine whether RIPK3 functions as a potential tumor suppressor to limit development of malignant mesothelioma. RIPK3 expression was examined in 66 malignant mesothelioma tumors and cell lines. Promoter methylation and DNMT1 siRNA studies were performed to assess the mode of RIPK3 silencing in RIPK3-deficient malignant mesothelioma cells. Restoration of RIPK3 expression in RIPK3-negative malignant mesothelioma cells, either by treatment with 5-aza-2'-deoxycytidine or lentiviral expression of cDNA, was performed to assess effects on cell viability, necrosis, and chemosensitization. Loss of RIPK3 expression was observed in 42/66 (63%) primary malignant mesotheliomas and malignant mesothelioma cell lines, and RT-PCR analysis demonstrated that downregulation occurs at the transcriptional level, consistent with epigenetic silencing. RIPK3-negative malignant mesothelioma cells treated with 5-aza-2'-deoxycytidine resulted in reexpression of RIPK3 and chemosensitization. Ectopic expression of RIPK3 also resulted in chemosensitization and led to necroptosis, the latter demonstrated by phosphorylation of downstream target MLKL and confirmed by rescue experiments. Mining of RIPK3 expression and survival outcomes among patients with malignant mesothelioma available from The Cancer Genome Atlas repository revealed that promoter methylation of RIPK3 is associated with reduced RIPK3 expression and poor prognosis. These data suggest that RIPK3 acts as a tumor suppressor in malignant mesothelioma by triggering necroptosis and that epigenetic silencing of RIPK3 by DNA methylation impairs necroptosis and contributes to chemoresistance and poor survival in this incurable disease.