A novel lectin receptor kinase gene, AtG-LecRK-I.2, enhances bacterial pathogen resistance through regulation of stomatal immunity in Arabidopsis.

The S-locus lectin receptor kinases (G-LecRKs) have been suggested as receptors for microbe/damage-associated molecular patterns (MAMPs/DAMPs) and to be involved in the pathogen defense responses, but the functions of most G-LecRKs in biotic stress response have not been characterized. Here, we identified a member of this family, G-LecRK-I.2, that positively regulates flg22- and Pseudomonas syringae pv. tomato (Pst) DC3000-induced stomatal closure. G-LecRK-I.2 was rapidly phosphorylated under flg22 treatment and could interact with the FLS2/BAK1 complex. Two T-DNA insertion lines, glecrk-i.2-1 and glecrk-i.2-2, had lower levels of reactive oxygen species (ROS) and nitric oxide (NO) production in guard cells, as compared with the wild-type Col-0, under Pst DC3000 infection. Also, the immunity marker genes CBP60g and PR1 were induced at lower levels under Pst DC3000 hrcC- infection in glecrk-i.2-1 and glecrk-i.2-2. The GUS reporter system also revealed that G-LecRK-I.2 was expressed only in guard cells. We also found that G-LecRK-I.2 could interact H+-ATPase AHA1 to regulate H+-ATPase activity in the guard cells. Taken together, our results show that G-LecRK-I.2 plays an important role in regulating stomatal closure under flg22 and Pst DC3000 treatments and in ROS and NO signaling specifically in guard cells.

Novel insights into the role of Discoidin domain receptor 2 (DDR2) in cancer progression: a new avenue of therapeutic intervention.

Discoidin domain receptors (DDRs), including DDR1 and DDR2, are a unique class of receptor tyrosine kinases (RTKs) activated by collagens at the cell-matrix boundary interface. The peculiar mode of activation makes DDRs as key cellular sensors of microenvironmental changes, with a critical role in all physiological and pathological processes governed by collagen remodeling. DDRs are widely expressed in fetal and adult tissues, and experimental and clinical evidence has shown that their expression is deregulated in cancer. Strong findings supporting the role of collagens in tumor progression and metastasis have led to renewed interest in DDRs.  However, despite an increasing number of studies, DDR biology remains poorly understood, particularly the less studied DDR2, whose involvement in cancer progression mechanisms is undoubted. Thus, the understanding of a wider range of DDR2 functions and related molecular mechanisms is expected. To date, several lines of evidence support DDR2 as a promising target in cancer therapy. Its involvement in key functions in the tumor microenvironment makes DDR2 inhibition particularly attractive to achieve simultaneous targeting of tumor and stromal cells, and tumor regression, which is beneficial for improving the response to different types of anti-cancer therapies, including chemo- and immunotherapy. This review summarizes current research on DDR2, focusing on its role in cancer progression through its involvement in tumor and stromal cell functions, and discusses findings that support the rationale for future development of direct clinical strategies targeting DDR2.

Exploring the Cellular and Molecular Mechanism of Discoidin Domain Receptors (DDR1 and DDR2) in Bone Formation, Regeneration, and Its Associated Disease Conditions.

The tyrosine kinase family receptor of discoidin domain receptors (DDR1 and DDR2) is known to be activated by extracellular matrix collagen catalytic binding protein receptors. They play a remarkable role in cell proliferation, differentiation, migration, and cell survival. DDR1 of the DDR family regulates matrix-metalloproteinase, which causes extracellular matrix (ECM) remodeling and reconstruction during unbalanced homeostasis. Collagenous-rich DDR1 triggers the ECM of cartilage to regenerate the cartilage tissue in osteoarthritis (OA) and temporomandibular disorder (TMD). Moreover, DDR2 is prominently present in the fibroblasts, smooth muscle cells, myofibroblasts, and chondrocytes. It is crucial in generating and breaking collagen vital cellular activities like proliferation, differentiation, and adhesion mechanisms. However, the deficiency of DDR1 rather than DDR2 was detrimental in cases of OA and TMDs. DDR1 stimulated the ECM cartilage and improved bone regeneration. Based on the above information, we made an effort to outline the advancement of the utmost promising DDR1 and DDR2 regulation in bone and cartilage, also summarizing their structural, biological activity, and selectivity.

The Arabidopsis thaliana onset of leaf death 12 mutation in the lectin receptor kinase P2K2 results in an autoimmune phenotype.

BACKGROUND: Plant immunity relies on the perception of immunogenic signals by cell-surface and intracellular receptors and subsequent activation of defense responses like programmed cell death. Under certain circumstances, the fine-tuned innate immune system of plants results in the activation of autoimmune responses that cause constitutive defense responses and spontaneous cell death in the absence of pathogens. RESULTS: Here, we characterized the onset of leaf death 12 (old12) mutant that was identified in the Arabidopsis accession Landsberg erecta. The old12 mutant is characterized by a growth defect, spontaneous cell death, plant-defense gene activation, and early senescence. In addition, the old12 phenotype is temperature reversible, thereby exhibiting all characteristics of an autoimmune mutant. Mapping the mutated locus revealed that the old12 phenotype is caused by a mutation in the Lectin Receptor Kinase P2-TYPE PURINERGIC RECEPTOR 2 (P2K2) gene. Interestingly, the P2K2 allele from Landsberg erecta is conserved among Brassicaceae. P2K2 has been implicated in pathogen tolerance and sensing extracellular ATP. The constitutive activation of defense responses in old12 results in improved resistance against Pseudomonas syringae pv. tomato DC3000. CONCLUSION: We demonstrate that old12 is an auto-immune mutant and that allelic variation of P2K2 contributes to diversity in Arabidopsis immune responses.

Behçet syndrome: The disturbed balance between anti- (CLEC12A, CLC) and proinflammatory (IFI27) gene expressions.

INTRODUCTION: Behçet syndrome (BS) is a chronic, multisystemic inflammatory condition with unanswered questions regarding its pathogenesis and rational therapeutics. A microarray-based comparative transcriptomic analysis was performed to elucidate the molecular mechanisms of BS and identify any potential therapeutic targets. METHODS: Twenty-nine BS patients (B) and 15 age and sex-matched control subjects (C) were recruited. Patients were grouped as mucocutaneous (M), ocular (O), and vascular (V) according to their clinical phenotypes. GeneChip Human Genome U133 Plus 2.0 arrays were used for expression profiling on peripheral blood samples of the patients and the control subjects. Following documentation of the differentially expressed gene (DEG) sets, the data were further evaluated with bioinformatics analysis, visualization, and enrichment tools. Validation of the microarray data was performed using quantitative reverse transcriptase polymerase chain reaction. RESULTS: When p ≤ 0.05 and fold change ≥2.0 were chosen, the following numbers of DEGs were obtained; B versus C: 28, M versus C: 20, O versus C: 8, V versus C: 555, M versus O: 6, M versus V: 324, O versus V: 142. Venn diagram analysis indicated only two genes, CLEC12A and IFI27, in the intersection of M versus C ∩ O versus C ∩ V versus C. Another noteworthy gene appeared as CLC in the DEG sets. Cluster analyses successfully clustered distinct clinical phenotypes of BS. While innate immunity-related processes were enriched in the M group, adaptive immunity-specific processes were significantly enriched in the O and V groups. CONCLUSIONS: Distinct clinical phenotypes of BS patients displayed distinct expression profiles. In Turkish BS patients, expression differences regarding the genes CLEC12A, IFI27, and CLC seemed to be operative in the disease pathogenesis. Based on these findings, future research should consider the immunogenetic heterogeneity of BS clinical phenotypes. Two anti-inflammatory genes, namely CLEC12A and CLC, may be valuable as therapeutic targets and may also help design an experimental model in BS.

Ablation of myeloid discoidin domain receptor 2 exacerbates arthritis and high fat diet induced inflammation.

Chronic systemic inflammation leads to sever disorders and diseases. It is of great importance to explore novel target for effective treatment. Discoidin domain receptor 2 (Ddr2) is a member of receptor tyrosine kinase (RTK) family and is implicated in skeletal and fat hemostasis. However, the role of Ddr2 in myeloid cells remains obscure. In this study, we conditionally deleted Ddr2 in myeloid lineage cells to generate cKO mice to investigate the role of Ddr2 in myeloid lineage cells. We found that cKO mice exhibited more severe inflammation both in collagen antibody-induced arthritis (CAIA) and high-fat diet (HFD)-induced obesity, indicating the protective role of Ddr2 against inflammation. Mechanistically, Ddr2 promotes macrophage repolarization from the M1 to M2 phenotype, and protect against systemic inflammation. Our study reveals for the first time that Ddr2 modulates macrophage repolarization and plays critical roles in macrophage-mediated inflammation, providing potential target for the intervention of inflammation and related diseases.

Discoidin Domain Receptor 1 Inhibitors: Advances and Future Directions for Novel Therapeutics with Aid of DNA Encoded Library Screens and Artificial Intelligence.

Discoidin domain receptor (DDR) 1, a collagen binding receptor kinase, is an intensively researched therapeutic target for cancer, fibrosis and other diseases. The majority of early known DDR1 inhibitors targeted the ATP binding pocket of this enzyme that shares structural similarities with other kinase pockets across the biological system. This structural similarity of DDR1 kinase with other protein kinases often leads to "off target "toxicity issues. Understanding of uniqueness in DDR:ATP-phosphate-binding loop (P-loop), DNA encoded library screen, structure-guided optimization studies, and machine learning drug design platforms that come under the umbrella of artificial intelligence has led to the discovery of a new array of inhibitors that are highly selective for DDR1 over DDR2 and other similar kinases. Most of the drug discovery platforms concentrated on the ATP binding region of DDR1 kinase and never looked beyond this region for novel therapeutic options. Recent findings have disclosed the kinase-independent functions of DDR1 in immune exclusion, which resides in the extracellular collagen-binding domain, thus opening avenues for the development of inhibitors that veer away from targeting ATP binding pockets. This recent understanding of the functional modalities of DDR1 opens the complexity of targeting this transmembrane protein as per its functional prominence in the respective disease and thus demands the development of specific novel therapeutics. The perspective gives a short overview of recent developments of DDR1 inhibitors with the aid of the latest technologies, future directions for therapeutic development, and possibility of combinational therapeutic treatments to completely disengage functions of DDR1.

Identification of Potential Differentially-Methylated/Expressed Genes in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease that causes obstructed airflow from the lungs. DNA methylation can regulate gene expression. Understanding the potential molecular mechanism of COPD is of great importance. The aim of this study was to find differentially methylated/expressed genes in COPD. DNA methylation and gene expression profiles in COPD were downloaded from the dataset, followed by functional analysis of differentially-methylated/expressed genes. The potential diagnostic value of these differentially-methylated/expressed genes was determined by receiver operating characteristic (ROC) analysis. Expression validation of differentially-methylated/expressed genes was performed by in vitro experiment and extra online datasets. Totally, 81 hypermethylated-low expression genes and 121 hypomethylated-high expression genes were found in COPD. Among which, 9 core hypermethylated-low expression genes (CD247, CCR7, CD5, IKZF1, SLAMF1, IL2RB, CD3E, CD7 and IL7R) and 8 core hypomethylated-high expression genes (TREM1, AQP9, CD300LF, CLEC12A, NOD2, IRAK3, NLRP3 and LYZ) were identified in the protein-protein interaction (PPI) network. Moreover, these genes had a potential diagnostic utility for COPD. Some signaling pathways were identified in COPD, including T cell receptor signaling pathway, cytokine-cytokine receptor interaction, hematopoietic cell lineage, HTLV-I infection, endocytosis and Jak-STAT signaling pathway. In conclusion, differentially-methylated/expressed genes and involved signaling pathways are likely to be associated with the process of COPD.

Discoidin Domain Receptor-Driven Gene Signatures as Markers of Patient Response to Anti-PD-L1 Immune Checkpoint Therapy.

BACKGROUND: Anti-programmed cell death 1 (anti-PD-1) and PD ligand 1 (PD-L1) immune checkpoint therapies (ICTs) provided durable responses only in a subset of cancer patients. Thus, biomarkers are needed to predict nonresponders and offer them alternative treatments. We recently implicated discoidin domain receptor tyrosine kinase 2 (DDR2) as a contributor to anti-PD-1 resistance in animal models; therefore, we sought to investigate whether this gene family may provide ICT response prediction. METHODS: We assessed mRNA expression of DDR2 and its family member DDR1. Transcriptome analysis of bladder cancer (BCa) models in which DDR1 and 2 were perturbed was used to derive DDR1- and DDR2-driven signature scores. DDR mRNA expression and gene signature scores were evaluated using BCa-The Cancer Genome Atlas (n = 259) and IMvigor210 (n = 298) datasets, and their relationship to BCa subtypes, pathway enrichment, and immune deconvolution analyses was performed. The potential of DDR-driven signatures to predict ICT response was evaluated and independently validated through a statistical framework in bladder and lung cancer cohorts. All statistical tests were 2-sided. RESULTS: DDR1 and DDR2 showed mutually exclusive gene expression patterns in human tumors. DDR2high BCa exhibited activation of immune pathways and a high immune score, indicative of a T-cell-inflamed phenotype, whereas DDR1high BCa exhibited a non-T-cell-inflamed phenotype. In IMvigor210 cohort, tumors with high DDR1 (hazard ratio [HR] = 1.53, 95% confidence interval [CI] = 1.16 to 2.06; P = .003) or DDR2 (HR = 1.42, 95% CI = 1.01 to 1.92; P = .04) scores had poor overall survival. Of note, DDR2high tumors from IMvigor210 and CheckMate 275 (n = 73) cohorts exhibited poorer overall survival (HR = 1.56, 95% CI = 1.20 to 2.06; P < .001) and progression-free survival (HR = 1.77 95%, CI = 1.05 to 3.00; P = .047), respectively. This result was validated in independent cancer datasets. CONCLUSIONS: These findings implicate DDR1 and DDR2 driven signature scores in predicting ICT response.

Increased expression of Clec9A on cDC1s associated with cytotoxic CD8+ T cell response in COPD.

Although C-type lectin domain family 9A (Clec9A) on conventional type 1 dendritic cells (cDC1s) plays a critical role in cytotoxic CD8+ T cell response in cancers and viral infections, its role in chronic obstructive pulmonary disease (COPD) is unknown. We measured the expression of Clec9A in sera, bronchoalveolar lavage fluid (BALF), and peripheral blood mononuclear cells (PBMCs) from controls and COPD patients. The percentages of Clec9A+ DC and cytotoxic CD8+ T cell in the BALF were determined by flow cytometry between patients with COPD and non-obstructive chronic bronchitis (NOCB). Compared with healthy individuals, the serum levels of Clec9A were increased at different stages of COPD patients, and the mRNA and protein levels of Clec9A were both increased in COPD patients at GOLD stages III-IV. The percentage of Clec9A+ DCs was also increased in the BALF of COPD patients compared with NOCB patients. Moreover, enhanced Clec9A+ DCs recruitment was positively correlated with cytotoxic CD8+ T cell response in the BALF of COPD patients. This study suggests that Clec9A+ DCs participate in the CD8+ T cell-mediated chronic airway inflammation in COPD.

Single-cell RNA-sequencing of peripheral blood mononuclear cells reveals widespread, context-specific gene expression regulation upon pathogenic exposure.

The host's gene expression and gene regulatory response to pathogen exposure can be influenced by a combination of the host's genetic background, the type of and exposure time to pathogens. Here we provide a detailed dissection of this using single-cell RNA-sequencing of 1.3M peripheral blood mononuclear cells from 120 individuals, longitudinally exposed to three different pathogens. These analyses indicate that cell-type-specificity is a more prominent factor than pathogen-specificity regarding contexts that affect how genetics influences gene expression (i.e., eQTL) and co-expression (i.e., co-expression QTL). In monocytes, the strongest responder to pathogen stimulations, 71.4% of the genetic variants whose effect on gene expression is influenced by pathogen exposure (i.e., response QTL) also affect the co-expression between genes. This indicates widespread, context-specific changes in gene expression level and its regulation that are driven by genetics. Pathway analysis on the CLEC12A gene that exemplifies cell-type-, exposure-time- and genetic-background-dependent co-expression interactions, shows enrichment of the interferon (IFN) pathway specifically at 3-h post-exposure in monocytes. Similar genetic background-dependent association between IFN activity and CLEC12A co-expression patterns is confirmed in systemic lupus erythematosus by in silico analysis, which implies that CLEC12A might be an IFN-regulated gene. Altogether, this study highlights the importance of context for gaining a better understanding of the mechanisms of gene regulation in health and disease.

Recognition of pathogen-derived sphingolipids in Arabidopsis.

In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors, which induce defense responses. Here, we show that the ceramide Phytophthora infestans-ceramide D (Pi-Cer D) from the plant pathogenic oomycete P. infestans triggers defense responses in Arabidopsis. Pi-Cer D is cleaved by an Arabidopsis apoplastic ceramidase, NEUTRAL CERAMIDASE 2 (NCER2), and the resulting 9-methyl-branched sphingoid base is recognized by a plasma membrane lectin receptor-like kinase, RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING 2 (RDA2). 9-Methyl-branched sphingoid base is specific to microbes and induces plant immune responses by physically interacting with RDA2. Loss of RDA2 or NCER2 function compromised Arabidopsis resistance against an oomycete pathogen. Thus, we elucidated the recognition mechanisms of pathogen-derived lipid molecules in plants.

CLEC12A plays an important role in immunomodulatory function and prognostic significance of patients with acute myeloid leukemia.

The physiological function and prognostic significance of C-type lectin domain family 12 member A (CLEC12A) in acute myeloid leukemia (AML) patients are unclear. CLEC12A transcriptional expression in a variety of tumors from several public databases was collected and compared. We found that CLEC12A was highly expressed in AML cell lines and in tissues from AML patients and a higher CLEC12A expression in leukemia stem cells. CLEC12A low expression was associated with poor prognosis in the chemotherapy-only group and high CLEC12A expression may benefit from autologous or allogeneic hematopoietic stem cell transplantation (HSCT). CLEC12A expression was positively correlated with infiltrating levels of type 2 macrophages and monocytes and negatively associated with NK cells and regulatory T cells in AML. CLEC12A high was positively associated with immune checkpoint genes as well as macrophage associated genes. CLEC12A is an ideal chimeric antigen receptor T-cell (CAR-T) therapy target for AML and its expression level was closely linked to treatment response and patients' survival outcome. CLEC12A plays an important immunomodulatory role in AML.

WHO grading system for invasive pulmonary lung adenocarcinoma reveals distinct molecular signature: An analysis from the cancer genome atlas database.

Lung adenocarcinoma grading has gained interest in the past years. Recently a three-tier tumor grading was proposed showing that it is related to patients' prognosis. Nevertheless, the underlying molecular basis of this morphological grading remains partly unknown. The aim of our work is to take advantage of The Cancer Genome Atlas lung adenocarcinoma (TCGA_LUAD) cohort to describe the molecular data associated to tumor grading. We performed a study on publicly available data of the TCGA database first by assessing a tumor grade on downloadable tumor slides. Secondly we analyzed the molecular features of each tumor grade group. Our work was performed on a study group of 449 patients. We show that aneuploidy score was significantly different between grade 2 and grade 3 groups with different chromosomal imbalance (p < 0.001). SCGB1A1 mRNA expression was higher in grade 2 (p = 0.0179) whereas NUP155, CHFR, POLQ and CDC7 have a higher expression in grade 3 (p = 0.0189, 0.0427, 0.0427 and 0.427 respectively). GZMB and KRT80 have a higher methylation of DNA in grade 2 (p = 0.0201 and 0.0359 respectively). MT1G, CLEC12B and NDUFA7 have a higher methylation of DNA in grade 3 (p < 0.001, 0.0246 and 0.0359 respectively). We showed that the number of activated pathways is different between grade 2 and grade 3 patients (p = 0.004). We showed that differentially expressed genes by mRNA analysis and DNA methylation analysis involve several genes implied in chemoresistance. This could suggest that grade 3 lung adenocarcinoma might be more resistant to chemotherapy.

Immunophenotypically defined stem cell subsets in paediatric AML are highly heterogeneous and demonstrate differences in BCL-2 expression by cytogenetic subgroups.

In adult acute myeloid leukaemia (AML), immunophenotypic differences enable discrimination of leukaemic stem cells (LSCs) from healthy haematopoietic stem cells (HSCs). However, immunophenotypic stem cell characteristics are less explored in paediatric AML. Employing a 15-colour flow cytometry assay, we analysed the expression of eight aberrant surface markers together with BCL-2 on CD34+ CD38- bone marrow stem cells from 38 paediatric AML patients and seven non-leukaemic, age-matched controls. Furthermore, clonality was investigated by genetic analyses of sorted immunophenotypically abnormal stem cells from six patients. A total of 50 aberrant marker positive (non-HSC-like) subsets with 41 different immunophenotypic profiles were detected. CD123, CLEC12A, and IL1RAP were the most frequently expressed markers. IL1RAP, CD93, and CD25 expression were not restricted to stem cells harbouring leukaemia-associated mutations. Differential BCL-2 expression was found among defined cytogenetic subgroups. Interestingly, only immunophenotypically abnormal non-HSC-like subsets demonstrated BCL-2 overexpression. Collectively, we observed pronounced immunophenotypic heterogeneity within the stem cell compartment of paediatric AML patients. Additionally, certain aberrant markers used in adults seemed to be ineligible for detection of leukaemia-representing stem cells in paediatric patients implying that inference from adult studies must be done with caution.

Identification of potential blood biomarkers for early diagnosis of schizophrenia through RNA sequencing analysis.

Schizophrenia (SCZ) is a highly heritable, polygenic complex mental disorder with imprecise diagnostic boundaries. Finding sensitive and specific novel biomarkers to improve the biological homogeneity of SCZ diagnosis is still one of the research hotspots. To identify the blood specific diagnostic biomarkers of SCZ, we performed RNA sequencing (RNA-seq) on 30 peripheral blood samples from 15 first-episode drug-naïve SCZ patients and 15 healthy controls (CTL). By performing multiple bioinformatics analysis algorithms based on RNA-seq data and microarray datasets, including differential expression genes (DEGs) analysis, WGCNA and CIBERSORT, we first identified 6 specific key genes (TOMM7, SNRPG, KRT1, AQP10, TMEM14B and CLEC12A) in SCZ. Moreover, we found that the proportions of lymphocyte, monocyte and neutrophils were significantly distinct in SCZ patients with CTL samples. Therefore, combining various features including age, sex and the novel blood biomarkers, we constructed the risk prediction model with three classifiers (RF: Random Forest; SVM: support vector machine; DT: decision tree) through repeated k-fold cross validation ensuring better generalizability. Finest result of Area under Receiver Operating Characteristic (AUROC) score of 0.91 was achieved by RF classifier and with a comparable good performance of AUROC 0.77 in external validation dataset. A lower AUROC of 0.63 was demonstrated when it was further applied to a Bipolar disorder (BPD) cohort. In conclusion, the study identified three peripheral core immunocytes and six key genes associated with the occurrence of SCZ, and further studies are required to test and validate these novel biomarkers for early diagnosis and treatment of SCZ.

The Collagen Receptor uPARAP in Malignant Mesothelioma: A Potential Diagnostic Marker and Therapeutic Target.

Malignant mesothelioma (MM) is a highly aggressive cancer with limited therapeutic options. We have previously shown that the endocytic collagen receptor, uPARAP, is upregulated in certain cancers and can be therapeutically targeted. Public RNA expression data display uPARAP overexpression in MM. Thus, to evaluate its potential use in diagnostics and therapy, we quantified uPARAP expression by immunohistochemical H-score in formalin-fixed paraffin-embedded bioptic/surgical human tissue samples and tissue microarrays. We detected pronounced upregulation of uPARAP in the three main MM subtypes compared to non-malignant reactive mesothelial proliferations, with higher expression in sarcomatoid and biphasic than in epithelioid MM. The upregulation appeared to be independent of patients' asbestos exposure and unaffected after chemotherapy. Using immunoblotting, we demonstrated high expression of uPARAP in MM cell lines and no expression in a non-malignant mesothelial cell line. Moreover, we showed the specific internalization of an anti-uPARAP monoclonal antibody by the MM cell lines using flow cytometry-based assays and confocal microscopy. Finally, we demonstrated the sensitivity of these cells towards sub-nanomolar concentrations of an antibody-drug conjugate formed with the uPARAP-directed antibody and a potent cytotoxin that led to efficient, uPARAP-specific eradication of the MM cells. Further studies on patient cohorts and functional preclinical models will fully reveal whether uPARAP could be exploited in diagnostics and therapeutic targeting of MM.

The Fusion of CLEC12A and MIR223HG Arises from a trans-Splicing Event in Normal and Transformed Human Cells.

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein-protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs.

CLEC12B suppresses lung cancer progression by inducing SHP-1 expression and inactivating the PI3K/AKT signaling pathway.

Lung cancer is the leading cause of cancer mortality worldwide. CLEC12B, a C-type lectin-like receptor, is low-expressed in lung cancer tissues. However, the function of CLEC12B in lung cancer and its underlying mechanism remain unclear. Here, an obvious down-regulation of CLEC12B was observed in lung cancer cells compared with the normal lung epithelial cells. CLEC12B over-expression suppressed cell viability and cell cycle entry in lung cancer, along with the reduction of PCNA and cyclin D1 expressions, while silencing CLEC12B possessed the opposite effects. Over-expression of CLEC12B promoted lung cancer cell apoptosis, accompanied by decreased Bcl-2 and increased Bax, cleaved caspase-3 and cleaved caspase-9. Moreover, CLEC12B decreased phosphorylation of PI3K-p85 and AKT proteins. By contrast, CLEC12B knockdown activated the PI3K/AKT pathway. In vivo, CLEC12B inhibited tumor growth in lung cancer, which can be reversed by CLEC12B inhibition. Co-IP and immunofluorescence assays confirmed the interaction between CLEC12B and SHP-1, and CLEC12B over-expression increased SHP-1 level. Furthermore, knocking down SHP-1 abrogated the above biological phenotypes caused by CLEC12B elevation. Taken together, our findings demonstrate that CLEC12B serves as a tumor-suppressing gene in lung cancer through positively regulating SHP-1 expression, which may be mediated by the PI3K/AKT signaling pathway.

Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region.

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor's expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.