ABSTRACT
In the search for therapeutic combinations for the treatment of cancer, the pairing of targeted inhibitors of oncogenic driver pathways with immunotherapy has largely been overlooked. In Nature, Canon et al. (2019) describe how the novel KRAS-G12C inhibitor AMG 510 can potentiate immune rejection in combination with immune checkpoint blockade.
Subject(s)
Antineoplastic Agents , Neoplasms , Humans , Immunotherapy , Proto-Oncogene Proteins p21(ras) , Tumor EscapeABSTRACT
A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development1. Here we propose that environmental particulate matter measuring ≤2.5 µm (PM2.5), known to be associated with lung cancer risk, promotes lung cancer by acting on cells that harbour pre-existing oncogenic mutations in healthy lung tissue. Focusing on EGFR-driven lung cancer, which is more common in never-smokers or light smokers, we found a significant association between PM2.5 levels and the incidence of lung cancer for 32,957 EGFR-driven lung cancer cases in four within-country cohorts. Functional mouse models revealed that air pollutants cause an influx of macrophages into the lung and release of interleukin-1ß. This process results in a progenitor-like cell state within EGFR mutant lung alveolar type II epithelial cells that fuels tumorigenesis. Ultradeep mutational profiling of histologically normal lung tissue from 295 individuals across 3 clinical cohorts revealed oncogenic EGFR and KRAS driver mutations in 18% and 53% of healthy tissue samples, respectively. These findings collectively support a tumour-promoting role for PM2.5 air pollutants and provide impetus for public health policy initiatives to address air pollution to reduce disease burden.
Subject(s)
Adenocarcinoma of Lung , Air Pollutants , Air Pollution , Cell Transformation, Neoplastic , Lung Neoplasms , Animals , Mice , Adenocarcinoma of Lung/chemically induced , Adenocarcinoma of Lung/genetics , Air Pollutants/adverse effects , Air Pollutants/analysis , Air Pollution/adverse effects , Air Pollution/analysis , Cell Transformation, Neoplastic/chemically induced , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/genetics , Environmental Exposure , ErbB Receptors/genetics , Lung Neoplasms/chemically induced , Lung Neoplasms/genetics , Particulate Matter/adverse effects , Particulate Matter/analysis , Particle Size , Cohort Studies , Macrophages, Alveolar/drug effects , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/pathologyABSTRACT
In order to catalyse the splicing of messenger RNA, multiple proteins and RNA components associate and dissociate in a dynamic highly choreographed process. The Prp19 complex is a conserved essential part of the splicing machinery thought to facilitate the conformational changes the spliceosome undergoes during catalysis. Dynamic protein interactions often involve highly disordered regions that are difficult to study by structural methods. Using amine crosslinking and hydrogen-deuterium exchange coupled to mass spectrometry, we describe the architecture of the Prp19 sub-complex that contains CTNNBL1. Deficiency in CTNNBL1 leads to delayed initiation of cell division and embryonic lethality. Here we show that in vitro CTNNBL1 enhances the association of CWC15 and CDC5L, both core Prp19 complex proteins and identify an overlap in the region of CDC5L that binds either CTNNBL1 or CWC15 suggesting the two proteins might exchange places in the complex. Furthermore, in vivo, CTNNBL1 is required to maintain normal levels of the Prp19 complex and to facilitate the interaction of CWC15 with CDC5L. Our results identify a chaperone function for CTNNBL1 within the essential Prp19 complex, a function required to maintain the integrity of the complex and to support efficient splicing.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Cell Cycle Proteins/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Nuclear Proteins/metabolism , RNA-Binding Proteins/metabolism , Animals , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/physiology , Cell Cycle Proteins/chemistry , Cell Line, Tumor , HEK293 Cells , Humans , Mice , Models, Molecular , Nuclear Proteins/chemistry , Nuclear Proteins/physiology , RNA Splicing Factors , RNA-Binding Proteins/chemistry , Spliceosomes/metabolismABSTRACT
The growing scale and dimensionality of multiplexed imaging require reproducible and comprehensive yet user-friendly computational pipelines. TRACERx-PHLEX performs deep learning-based cell segmentation (deep-imcyto), automated cell-type annotation (TYPEx) and interpretable spatial analysis (Spatial-PHLEX) as three independent but interoperable modules. PHLEX generates single-cell identities, cell densities within tissue compartments, marker positivity calls and spatial metrics such as cellular barrier scores, along with summary graphs and spatial visualisations. PHLEX was developed using imaging mass cytometry (IMC) in the TRACERx study, validated using published Co-detection by indexing (CODEX), IMC and orthogonal data and benchmarked against state-of-the-art approaches. We evaluated its use on different tissue types, tissue fixation conditions, image sizes and antibody panels. As PHLEX is an automated and containerised Nextflow pipeline, manual assessment, programming skills or pathology expertise are not essential. PHLEX offers an end-to-end solution in a growing field of highly multiplexed data and provides clinically relevant insights.
Subject(s)
Deep Learning , Humans , Image Processing, Computer-Assisted/methods , Animals , Software , Spatial Analysis , Single-Cell Analysis/methods , Phenotype , Mice , Image Cytometry/methodsABSTRACT
Understanding the role of the tumor microenvironment (TME) in lung cancer is critical to improving patient outcomes. We identified four histology-independent archetype TMEs in treatment-naïve early-stage lung cancer using imaging mass cytometry in the TRACERx study (n = 81 patients/198 samples/2.3 million cells). In immune-hot adenocarcinomas, spatial niches of T cells and macrophages increased with clonal neoantigen burden, whereas such an increase was observed for niches of plasma and B cells in immune-excluded squamous cell carcinomas (LUSC). Immune-low TMEs were associated with fibroblast barriers to immune infiltration. The fourth archetype, characterized by sparse lymphocytes and high tumor-associated neutrophil (TAN) infiltration, had tumor cells spatially separated from vasculature and exhibited low spatial intratumor heterogeneity. TAN-high LUSC had frequent PIK3CA mutations. TAN-high tumors harbored recently expanded and metastasis-seeding subclones and had a shorter disease-free survival independent of stage. These findings delineate genomic, immune, and physical barriers to immune surveillance and implicate neutrophil-rich TMEs in metastasis. SIGNIFICANCE: This study provides novel insights into the spatial organization of the lung cancer TME in the context of tumor immunogenicity, tumor heterogeneity, and cancer evolution. Pairing the tumor evolutionary history with the spatially resolved TME suggests mechanistic hypotheses for tumor progression and metastasis with implications for patient outcome and treatment. This article is featured in Selected Articles from This Issue, p. 897.
Subject(s)
Lung Neoplasms , Tumor Microenvironment , Humans , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Tumor Microenvironment/immunology , T-Lymphocytes/immunology , Myeloid Cells/immunology , Female , Male , Immune EvasionABSTRACT
Recently, conflicting results were reported on the hypermutation activity of activation-induced cytidine deaminase (AID) splice variants. With the generation of single point mutations, we studied the structure-function relationship of the amino acids that are commonly absent from all described splice variants. The results from this analysis pointed to several amino acids that are required for class switch recombination (CSR), without perturbing cellular localization or nucleocytoplasmic shuttling. A defect in deaminase activity was found to underlie this CSR deficiency. Interestingly, the most debilitating mutations concentrated on hydrophobic amino acids, suggesting a structural role for this part of the protein. Indeed, by generating homologous amino acid replacements, CSR activity could be restored. These results are in agreement with recent reports on the protein structure of the AID homolog APOBEC3G, suggesting a similar protein composition. In addition, the findings underscore that AID splice variants are unlikely to have preservation of catalytic activity.
Subject(s)
Alternative Splicing , Catalytic Domain/genetics , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Line , Cell Line, Tumor , Cells, Cultured , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunoglobulin Class Switching/genetics , Immunoglobulin Class Switching/immunology , Mice , Mice, Knockout , Microscopy, Fluorescence , Mutation, Missense , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombination, GeneticABSTRACT
Recently developed KRASG12C inhibitory drugs are beneficial to lung cancer patients harboring KRASG12C mutations, but drug resistance frequently develops. Because of the immunosuppressive nature of the signaling network controlled by oncogenic KRAS, these drugs can indirectly affect antitumor immunity, providing a rationale for their combination with immune checkpoint blockade. In this study, we have characterized how KRASG12C inhibition reverses immunosuppression driven by oncogenic KRAS in a number of preclinical lung cancer models with varying levels of immunogenicity. Mechanistically, KRASG12C inhibition up-regulates interferon signaling via Myc inhibition, leading to reduced tumor infiltration by immunosuppressive cells, enhanced infiltration and activation of cytotoxic T cells, and increased antigen presentation. However, the combination of KRASG12C inhibitors with immune checkpoint blockade only provides synergistic benefit in the most immunogenic tumor model. KRASG12C inhibition fails to sensitize cold tumors to immunotherapy, with implications for the design of clinical trials combining KRASG12C inhibitors with anti-PD1 drugs.
Subject(s)
Lung Neoplasms , Proto-Oncogene Proteins p21(ras) , Humans , Immune Checkpoint Inhibitors , Interferons , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mutation , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
In addition to the classical TH1 and TH2 cytokines, members of the recently identified IL-17 cytokine family play an important role in regulating cellular and humoral immune responses. At present nothing is known about the role of these cytokines in atherosclerosis. Expression of IL-17A, -E and -F was investigated in atherosclerotic tissue by rtPCR and immunohistochemistry. IL-17E and its receptor were further studied in cultured smooth muscle cells and endothelial cells, using rtPCR and western blot. rtPCR showed that IL-17A, -E and -F were expressed in the majority of plaques under investigation. IL-17A/F was expressed by mast cells in all stages of plaque development. IL-17A/F(+) neutrophils were always observed in complicated plaques, but hardly in intact lesions. IL-17A/F(+) Tcells ('TH17') were never observed. IL-17E was expressed by smooth muscle cells and endothelial cells in both normal and atherosclerotic arteries, and in advanced plaques also extensively by mature B cells. Cultured smooth muscle cells and endothelial cells were found to express both IL-17E and its functional receptor (IL-17RB). The constitutive expression of IL-17E by resident plaque cells, and the additional presence of IL-17E(+) B cells and IL-17A/F(+) neutrophils in advanced and complicated plaques indicates a complex contribution of IL-17 family cytokines in human atherosclerosis, depending on the stage and activity of the disease.
Subject(s)
Atherosclerosis/immunology , Interleukin-17/biosynthesis , Aged , Aged, 80 and over , Blotting, Western , Cells, Cultured , Endothelial Cells/immunology , Endothelium, Vascular/immunology , Gene Expression/immunology , Humans , Inflammation Mediators/metabolism , Interleukin-17/genetics , Middle Aged , Muscle, Smooth, Vascular/immunology , Plasma Cells/immunology , RNA, Messenger/genetics , Receptors, Interleukin/metabolism , Receptors, Interleukin-17 , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimise and validate image segmentation strategies and automate the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. With these methods we interrogate the remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, highlighting the infiltration and activation of antigen presenting cells and effector cells.
Subject(s)
Image Cytometry/methods , Oncogenes , Tumor Microenvironment/immunology , Animals , Antibodies , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/diagnostic imaging , Carcinoma, Non-Small-Cell Lung/immunology , Disease Models, Animal , Lung Neoplasms/diagnostic imaging , Lung Neoplasms/drug therapy , Macrophages , Mice , Mice, Inbred C57BL , Oncogenes/drug effects , T-Lymphocytes , Tumor Microenvironment/drug effectsABSTRACT
Extranodal marginal zone B-cell lymphomas (MZBCLs) arise on a background of chronic inflammation resulting from organ-specific autoimmunity, infection, or by unknown causes. Well-known examples are salivary gland MZBCL in Sjögren's sialadenitis and gastric MZBCL in Helicobacter pylori gastritis. MZBCLs express CXCR3, a receptor for interferon-gamma-induced chemokines highly expressed in the chronic inflammatory environment. The immunoglobulin (Ig) variable heavy/light chain (IgV(H)/IgV(L)) gene repertoire of salivary gland and gastric MZBCL appears restricted and frequently encodes B-cell receptors with rheumatoid factor reactivity. Primary cutaneous marginal zone B-cell lymphomas (PCMZLs) are regarded as the skin-involving counterparts of extranodal MZBCLs. Although PCMZLs have been associated with Borrelia burgdorferi dermatitis, PCMZLs generally arise because of unknown causes. We studied an extensive panel of PCMZLs and show that PCMZLs do not conform to the general profile of extranodal MZBCL. Whereas most noncutaneous MZBCLs express IgM, PCMZLs in majority express IgG, IgA, and IgE and do not show an obvious immunoglobulin repertoire bias. Furthermore, the isotype-switched PCMZLs lack CXCR3 and seem to arise in a different inflammatory environment, compared with other extranodal MZBCLs.
Subject(s)
Immunoglobulins/metabolism , Lymphoma, B-Cell, Marginal Zone/immunology , Lymphoma, B-Cell, Marginal Zone/metabolism , Th2 Cells/metabolism , Antigens, CD20/biosynthesis , Complementarity Determining Regions/metabolism , Humans , Immunoglobulin A/chemistry , Immunoglobulin Class Switching , Immunoglobulin E/chemistry , Immunoglobulin G/chemistry , Inflammation , Interferon-gamma/metabolism , Lymphoma, B-Cell, Marginal Zone/pathology , Models, Biological , Receptors, CXCR3/metabolism , Rheumatoid Factor/metabolismABSTRACT
Chronic autoimmune or pathogen-induced immune reactions resulting in lymphoid neogenesis are associated with development of malignant lymphomas, mostly extranodal marginal zone B-cell lymphomas (MZBCLs). In this review we address (i) chemokines and adhesion molecules involved in lymphoid neogenesis; (ii) the autoimmune diseases and pathogens which are associated with development of B-cell lymphomas; (iii) the molecular mechanisms involved in the initiation and progression of MZBCL; and (iv) 'potential' mouse models for MZBCL.
Subject(s)
Autoimmune Diseases/immunology , Lymphoid Tissue/immunology , Lymphoma, B-Cell/immunology , Animals , Autoimmune Diseases/genetics , Autoimmune Diseases/metabolism , Cell Adhesion Molecules/metabolism , Chemokines/metabolism , Chronic Disease , Humans , Lymphoid Tissue/metabolism , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/metabolism , Models, Biological , Translocation, GeneticABSTRACT
KRAS represents an excellent therapeutic target in lung cancer, the most commonly mutated form of which can now be blocked using KRAS-G12C mutant-specific inhibitory trial drugs. Lung adenocarcinoma cells harboring KRAS mutations have been shown previously to be selectively sensitive to inhibition of mitogen-activated protein kinase kinase (MEK) and insulin-like growth factor 1 receptor (IGF1R) signaling. Here, we show that this effect is markedly enhanced by simultaneous inhibition of mammalian target of rapamycin (mTOR) while maintaining selectivity for the KRAS-mutant genotype. Combined mTOR, IGF1R, and MEK inhibition inhibits the principal signaling pathways required for the survival of KRAS-mutant cells and produces marked tumor regression in three different KRAS-driven lung cancer mouse models. Replacing the MEK inhibitor with the mutant-specific KRAS-G12C inhibitor ARS-1620 in these combinations is associated with greater efficacy, specificity, and tolerability. Adding mTOR and IGF1R inhibitors to ARS-1620 greatly improves its effectiveness on KRAS-G12C mutant lung cancer cells in vitro and in mouse models. This provides a rationale for the design of combination treatments to enhance the impact of the KRAS-G12C inhibitors, which are now entering clinical trials.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation/genetics , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Cell Survival/drug effects , Imidazoles/pharmacology , Imidazoles/therapeutic use , Mice, Nude , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-akt/metabolism , Pyrazines/pharmacology , Pyrazines/therapeutic use , Pyridones/pharmacology , Pyridones/therapeutic use , Pyrimidinones/pharmacology , Pyrimidinones/therapeutic use , RNA, Small Interfering/metabolism , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolismABSTRACT
Contemporary pathology involves an emerging role for molecular diagnostics. Current tissue handling procedures [ie, formalin fixation and paraffin embedment (FFPE)] have their origin in the aim to obtain good tissue morphology and optimal results within immunohistochemistry. Unfortunately, FFPE is notorious for its poor RNA conservation capacities. In this study, we have examined the impact of the individual steps in tissue handling processes on the RNA extractability, quality, and usability for reverse-transcription polymerase chain reaction. It was found that a prolonged prefixation time (ie, the time between tissue dissection and fixation) has a measurable impact on RNA integrity when analyzed with the Agilent Bioanalyzer. Surprisingly, however, the deteriorated RNA quality hardly had any consequences for reverse-transcription polymerase chain reaction yields. Furthermore, we assessed the optimal fixation time for RNA preservation, and we found that an RNA heating step, preceding copy DNA synthesis, significantly increases the RNA template length. Finally, we provide a protocol for RNA isolation from immunohistochemically stained FFPE tissue sections. Thus, by applying alterations to tissue handling procedures, archival FFPE tissues become well suitable for RNA-based molecular diagnostics.
Subject(s)
Formaldehyde/pharmacology , Immunohistochemistry , Paraffin Embedding , RNA/isolation & purification , Specimen Handling , Algorithms , DNA, Complementary/analysis , DNA, Complementary/chemistry , Efficiency , Female , Frozen Sections , Humans , Quality Control , Reverse Transcriptase Polymerase Chain Reaction , Temperature , Time Factors , Tissue Fixation/methodsABSTRACT
BACKGROUND AND OBJECTIVES: Activation-induced cytidine deaminase is essential for somatic hypermutation and class switch recombination of the immunoglobulin genes in B cells. It has been proposed that aberrant targeting of the somatic hypermutation machinery is instrumental in initiation and progression of B-cell non Hodgkin's lymphomas. In this study, we investigated the B-cell receptor and the role of the somatic hypermutation machinery in B-cell chronic lymphocytic leukemias (B-CLL) prior to and after transformation to a lymphoma of a higher malignancy grade (Richter's transformation). DESIGN AND METHODS: We investigated the activity of the somatic hypermutation machinery in nine B-CLL and secondary diffuse large B-cell lymphomas by measuring the expression of activation-induced cytidine deaminase, in combination with mutation analysis of immunoglobulin (Ig) and non-Ig genes. Furthermore, the structure of the antigen receptors of B-CLL known to have developed a Richter's syndrome (RS B-CLL) was analyzed by comparing the most variable region of the Ig, the CDR3 region, to CDR3 sequences present in GenBank. RESULTS: Ig variable heavy chain (IgV(H)) gene studies revealed that Richter's transformation occurs almost exclusively in unmutated B-CLL. Furthermore, activated-induced cytidine deaminase expression and somatic hypermutation activity of most RS B-CLL were found to be higher than those of control (non-transforming) B-CLL. Finally, comparison of the IgVH-CDR3 regions showed a remarkable amino acid sequence homology between two RS B-CLL of our panel and two RS B-CLL described in the literature. INTERPRETATIONS AND CONCLUSIONS: The combined findings suggest a role for the Ig gene diversification apparatus during Richter's transformation and show that distinct RS-B-CLL may recognize recurrent antigenic epitopes.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Receptors, Antigen/immunology , Somatic Hypermutation, Immunoglobulin , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Neoplasms, Second Primary , PrognosisABSTRACT
Matrix metalloproteinases (MMPs) such as gelatinases are believed to play an important role in invasion and metastasis of cancer. In this study we investigated the possible role of MMP-2 and MMP-9 in an experimental model of colon cancer metastasis in rat liver. We demonstrated with gelatin zymography that the tumors contained MMP-2 and MMP-9, but only MMP-2 was present in the active form. Immunolocalization of MMP-2 showed that the protein was localized at basement membranes of colon cancer cells and in intratumor stroma, associated with extracellular matrix (ECM) components. However, zymography and immunohistochemistry (IHC) do not provide information on the localization of MMP activity. Therefore, we developed an in situ zymography technique using the quenched fluorogenic substrate DQ-gelatin in unfixed cryostat sections. The application of DQ-gelatin in combination with a gelled medium allows precise localization of gelatinolytic activity. Fluorescence due to gelatinolytic activity was found in the ECM of tumors and was localized similarly to both MMP-2 protein and collagen type IV, its natural substrate. The localization of MMP-2 activity and collagen type IV at similar sites suggests a role of MMP-2 in remodeling of ECM of stroma in colon cancer metastases in rat liver.
Subject(s)
Colonic Neoplasms/pathology , Extracellular Matrix/enzymology , Gelatin/metabolism , Liver Neoplasms/enzymology , Liver/enzymology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Animals , Fluorescent Dyes , Immunohistochemistry , Liver/ultrastructure , Liver Neoplasms/secondary , Liver Neoplasms/ultrastructure , Male , Neoplasm Transplantation , RatsABSTRACT
CTNNBL1 is a spliceosome-associated protein that binds nuclear localization signals (NLSs) in splice factors CDC5L and Prp31 as well as the antibody diversifying enzyme AID. Here, crystal structures of human CTNNBL1 reveal a distinct structure from its closest homologue karyopherin-α. CTNNBL1 comprises a HEAT-like domain (including a nuclear export signal), a central armadillo domain, and a coiled-coil C-terminal domain. Structure-guided mutations of the region homologous to the karyopherin-α NLS-binding site fail to disrupt CTNNBL1-NLS interactions. Our results identify CTNNBL1 as a unique selective NLS-binding protein with striking differences from karyopherin-αs.
Subject(s)
Apoptosis Regulatory Proteins/chemistry , Nuclear Localization Signals/chemistry , Nuclear Proteins/chemistry , alpha Karyopherins/chemistry , Amino Acid Sequence , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Armadillo Domain Proteins/chemistry , Armadillo Domain Proteins/genetics , Armadillo Domain Proteins/metabolism , Binding Sites/genetics , Crystallography, X-Ray , DNA Mutational Analysis , Electrophoresis, Polyacrylamide Gel , HEK293 Cells , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Nuclear Localization Signals/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , alpha Karyopherins/genetics , alpha Karyopherins/metabolismABSTRACT
CTNNBL1 is an armadillo-repeat protein that associates with the CDC5L/Prp19 complex of the spliceosome. Unlike the majority of spliceosomal proteins (and despite having no obvious homologs), CTNNBL1 is inessential for cell viability as revealed by studies in both vertebrate B cell lines and in fission yeast. Here, however, we show that ablation of CTNNBL1 in the mouse germline results in mid-gestation embryonic lethality but that lineage-specific CTNNBL1 ablation in early B cell precursors does not affect the production and abundance of mature B lymphocytes. However, CTNNBL1-deficient resting B lymphocytes show sluggish exit from quiescence on cell activation, although once entry into cycle has initiated, proliferation and differentiation in response to mitogenic stimuli continue largely unaffected. A similar sluggish exit from quiescence is also observed on reprovision of nutrients to nitrogen-starved CTNNBL1-deficient yeast. The results indicate that, whereas other RNA splicing-associated factors have been connected to cell cycle progression, CTNNBL1 plays no essential role in cycling cells but does fulfill an evolutionarily conserved function in helping cells to undergo efficient exit from quiescence following activation.
Subject(s)
Apoptosis Regulatory Proteins/deficiency , Cell Cycle , Embryo Loss/pathology , Nuclear Proteins/deficiency , Spliceosomes/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Biomarkers/metabolism , Cell Cycle/drug effects , Cell Lineage/drug effects , Cell Lineage/genetics , Embryo Loss/genetics , Gene Targeting , Genes, Immediate-Early/genetics , Germ-Line Mutation/genetics , Immunoglobulin Class Switching/drug effects , Immunoglobulin Class Switching/genetics , Lipopolysaccharides/pharmacology , Mice , Nuclear Proteins/metabolism , RNA Splicing/drug effects , RNA Splicing/genetics , S Phase/drug effects , Schizosaccharomyces/cytology , Schizosaccharomyces/drug effects , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Spliceosomes/drug effects , Transcriptional Activation/drug effects , Up-Regulation/drug effectsABSTRACT
To reveal migration trails of antigen-responsive B cells in lymphoid tissue, we analyzed immunoglobulin (Ig)M-V(H) and IgG-V(H) transcripts of germinal center (GC) samples microdissected from three reactive human lymph nodes. Single B cell clones were found in multiple GCs, one clone even in as many as 19 GCs. In several GCs, IgM and IgG variants of the same clonal origin were identified. The offspring of individual hypermutated IgG memory clones were traced in multiple GCs, indicating repeated engagement of memory B cells in GC reactions. These findings imply that recurring somatic hypermutation progressively drives the Ig repertoire of memory B cells to higher affinities and infer that transforming genetic hits in non-Ig genes during lymphomagenesis do not have to arise during a single GC passage, but can be collected during successive recall responses.