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1.
Cell ; 170(6): 1079-1095.e20, 2017 Sep 07.
Article in English | MEDLINE | ID: mdl-28823558

ABSTRACT

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and α-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer. PAPERCLIP.


Subject(s)
Ascorbic Acid/pharmacology , DNA-Binding Proteins/metabolism , Leukemia, Myeloid, Acute/drug therapy , Myelodysplastic Syndromes/drug therapy , Proto-Oncogene Proteins/metabolism , Vitamins/pharmacology , Animals , Ascorbic Acid/administration & dosage , Cell Death , Cell Line, Tumor , DNA Methylation , DNA-Binding Proteins/genetics , Dioxygenases , Gene Knockdown Techniques , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Myelodysplastic Syndromes/genetics , Neoplasm Transplantation , Poly (ADP-Ribose) Polymerase-1/genetics , Proto-Oncogene Proteins/genetics , Transcription, Genetic , Transplantation, Heterologous , Vitamins/administration & dosage
2.
Immunity ; 50(1): 77-90.e5, 2019 01 15.
Article in English | MEDLINE | ID: mdl-30611612

ABSTRACT

Dendritic cells (DCs) are can be broadly divided into conventional (cDC) and plasmacytoid (pDC) subsets. Despite the importance of this lineage diversity, its genetic basis is not fully understood. We found that conditional ablation of the Ets-family transcription factor PU.1 in DC-restricted progenitors led to increased pDC production at the expense of cDCs. PU.1 controlled many of the cardinal functions of DCs, such as antigen presentation by cDCs and type I interferon production by pDCs. Conditional ablation of PU.1 de-repressed the pDC transcriptional signature in cDCs. The combination of genome-wide mapping of PU.1 binding and gene expression analysis revealed a key role for PU.1 in maintaining cDC identity through the induction of the transcriptional regulator DC-SCRIPT. PU.1 activated DC-SCRIPT expression, which in turn promoted cDC formation, particularly of cDC1s, and repressed pDC development. Thus, cDC identity is regulated by a transcriptional node requiring PU.1 and DC-SCRIPT.


Subject(s)
DNA-Binding Proteins/metabolism , Dendritic Cells/physiology , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Antigen Presentation , Cell Differentiation , Cell Lineage , DNA-Binding Proteins/genetics , Gene Expression Regulation , HEK293 Cells , Humans , Interferon Type I/metabolism , Mice , Mice, Transgenic , Nuclear Proteins/genetics , Proto-Oncogene Proteins/genetics , Signal Transduction , Trans-Activators/genetics , Transcription Factors/genetics , Transcriptome
3.
Nat Immunol ; 15(3): 283-93, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24509509

ABSTRACT

The transcription factor Ikaros is an essential regulator of lymphopoiesis. Here we studied its B cell-specific function by conditional inactivation of the gene encoding Ikaros (Ikzf1) in pro-B cells. B cell development was arrested at an aberrant 'pro-B cell' stage characterized by increased cell adhesion and loss of signaling via the pre-B cell signaling complex (pre-BCR). Ikaros activated genes encoding signal transducers of the pre-BCR and repressed genes involved in the downregulation of pre-BCR signaling and upregulation of the integrin signaling pathway. Unexpectedly, derepression of expression of the transcription factor Aiolos did not compensate for the loss of Ikaros in pro-B cells. Ikaros induced or suppressed active chromatin at regulatory elements of activated or repressed target genes. Notably, binding of Ikaros and expression of its target genes were dynamically regulated at distinct stages of early B lymphopoiesis.


Subject(s)
B-Lymphocytes/cytology , Cell Differentiation/immunology , Ikaros Transcription Factor/immunology , Lymphopoiesis/immunology , Precursor Cells, B-Lymphoid/cytology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Chromatin Immunoprecipitation , Flow Cytometry , Gene Expression Regulation/immunology , Gene Knockdown Techniques , Ikaros Transcription Factor/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Precursor Cells, B-Lymphoid/immunology , Precursor Cells, B-Lymphoid/metabolism
4.
Cell ; 145(1): 145-58, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21458673

ABSTRACT

RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19(ARF) as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PAPERCLIP:


Subject(s)
Gene Knockdown Techniques/methods , RNA Interference , Adenocarcinoma/genetics , Adenocarcinoma/therapy , Animals , Embryonic Stem Cells/metabolism , Gene Knockdown Techniques/economics , Lung Neoplasms/genetics , Lung Neoplasms/therapy , Mice , Mice, Transgenic , MicroRNAs/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/therapy , RNA Processing, Post-Transcriptional , RNA, Small Interfering/genetics , Signal Transduction , Wnt Proteins/metabolism
5.
Nature ; 558(7711): E5, 2018 06.
Article in English | MEDLINE | ID: mdl-29849140

ABSTRACT

In Fig. 3c of this Letter, the the effects of CRISPR-Cas9-mediated deletion of NR3C1, TXNIP and CNR2 in patient-derived B-lineage leukaemia cells were shown. For curves depicting NR3C1 (left graph), data s for TXNIP (middle graph) were inadvertently plotted. This figure has been corrected online, and the original Fig. 3c is shown as Supplementary Information to this Amendment for transparency. The error does not affect the conclusions of the Letter. In addition, Source Data files have been added for the Figs. 1-4 and Extended Data Figs. 1-10 of the original Letter.

6.
Cell ; 134(4): 657-67, 2008 08 22.
Article in English | MEDLINE | ID: mdl-18724938

ABSTRACT

Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to noncancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell-cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix-degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage.


Subject(s)
Cellular Senescence , Liver Cirrhosis/immunology , Liver/cytology , Animals , Carbon Tetrachloride , Cells, Cultured , Female , Fibroblasts/metabolism , Humans , Killer Cells, Natural/immunology , Liver/physiology , Liver Cirrhosis/metabolism , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/immunology , Liver Cirrhosis, Experimental/metabolism , Mice
7.
Nature ; 542(7642): 479-483, 2017 02 23.
Article in English | MEDLINE | ID: mdl-28192788

ABSTRACT

B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development, yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL). The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK. Dominant-negative mutants of PAX5 and IKZF1, however, relieved this glucose and energy restriction. In a transgenic pre-B ALL mouse model, the heterozygous deletion of Pax5 increased glucose uptake and ATP levels by more than 25-fold. Reconstitution of PAX5 and IKZF1 in samples from patients with pre-B ALL restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5 and IKZF1 transcriptional targets identified the products of NR3C1 (encoding the glucocorticoid receptor), TXNIP (encoding a glucose-feedback sensor) and CNR2 (encoding a cannabinoid receptor) as central effectors of B-lymphoid restriction of glucose and energy supply. Notably, transport-independent lipophilic methyl-conjugates of pyruvate and tricarboxylic acid cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukaemic transformation. Conversely, pharmacological TXNIP and CNR2 agonists and a small-molecule AMPK inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapeutic targets. Furthermore, our results provide a mechanistic explanation for the empirical finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. Thus, B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation.


Subject(s)
B-Lymphocytes/metabolism , Energy Metabolism/genetics , Gene Expression Regulation, Neoplastic , Glucose/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Transcription Factors/metabolism , AMP-Activated Protein Kinase Kinases , AMP-Activated Protein Kinases/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Animals , B-Lymphocytes/drug effects , Carcinogenesis/genetics , Carrier Proteins/agonists , Carrier Proteins/metabolism , Cell Death , Chromatin Immunoprecipitation , Citric Acid Cycle , Disease Models, Animal , Female , Glucocorticoids/pharmacology , Glucocorticoids/therapeutic use , Humans , Ikaros Transcription Factor/metabolism , Mice , Mice, Transgenic , PAX5 Transcription Factor/deficiency , PAX5 Transcription Factor/genetics , PAX5 Transcription Factor/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Serine-Threonine Kinases/metabolism , Pyruvic Acid/metabolism , Receptor, Cannabinoid, CB2/agonists , Receptor, Cannabinoid, CB2/metabolism , Receptors, Glucocorticoid/metabolism , Sequence Analysis, RNA
8.
Blood ; 136(8): 957-973, 2020 08 20.
Article in English | MEDLINE | ID: mdl-32369597

ABSTRACT

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.


Subject(s)
Cell Transformation, Neoplastic , Epithelial-Mesenchymal Transition/genetics , Histone Demethylases/metabolism , Leukemia, Myeloid, Acute/pathology , Snail Family Transcription Factors/physiology , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , HEK293 Cells , HL-60 Cells , Histone Demethylases/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice , Mice, Transgenic , Protein Binding , Snail Family Transcription Factors/genetics , Snail Family Transcription Factors/metabolism
9.
Genes Dev ; 28(12): 1337-50, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24939936

ABSTRACT

Loss-of-function mutations in hematopoietic transcription factors including PAX5 occur in most cases of B-progenitor acute lymphoblastic leukemia (B-ALL), a disease characterized by the accumulation of undifferentiated lymphoblasts. Although PAX5 mutation is a critical driver of B-ALL development in mice and humans, it remains unclear how its loss contributes to leukemogenesis and whether ongoing PAX5 deficiency is required for B-ALL maintenance. Here we used transgenic RNAi to reversibly suppress endogenous Pax5 expression in the hematopoietic compartment of mice, which cooperates with activated signal transducer and activator of transcription 5 (STAT5) to induce B-ALL. In this model, restoring endogenous Pax5 expression in established B-ALL triggers immunophenotypic maturation and durable disease remission by engaging a transcriptional program reminiscent of normal B-cell differentiation. Notably, even brief Pax5 restoration in B-ALL cells causes rapid cell cycle exit and disables their leukemia-initiating capacity. These and similar findings in human B-ALL cell lines establish that Pax5 hypomorphism promotes B-ALL self-renewal by impairing a differentiation program that can be re-engaged despite the presence of additional oncogenic lesions. Our results establish a causal relationship between the hallmark genetic and phenotypic features of B-ALL and suggest that engaging the latent differentiation potential of B-ALL cells may provide new therapeutic entry points.


Subject(s)
Cell Differentiation/genetics , PAX5 Transcription Factor/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cells, B-Lymphoid/cytology , Animals , Cell Line, Tumor , Cells, Cultured , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Genes, myc/genetics , Humans , Mice , Mice, Transgenic , PAX5 Transcription Factor/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction
10.
Mol Cell ; 41(6): 733-46, 2011 Mar 18.
Article in English | MEDLINE | ID: mdl-21353615

ABSTRACT

Short hairpin RNAs (shRNAs) provide powerful experimental tools by enabling stable and regulated gene silencing through programming of endogenous microRNA pathways. Since requirements for efficient shRNA biogenesis and target suppression are largely unknown, many predicted shRNAs fail to efficiently suppress their target. To overcome this barrier, we developed a "Sensor assay" that enables the biological identification of effective shRNAs at large scale. By constructing and evaluating 20,000 RNAi reporters covering every possible target site in nine mammalian transcripts, we show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. Our unbiased analyses reveal that potent shRNAs share various predicted and previously unknown features associated with specific microRNA processing steps, and suggest a model for competitive strand selection. Together, our study establishes a powerful tool for large-scale identification of highly potent shRNAs and provides insights into sequence requirements of effective RNAi.


Subject(s)
Biosensing Techniques , High-Throughput Screening Assays/methods , RNA Interference , RNA, Small Interfering/genetics , Algorithms , Animals , Fibroblasts/cytology , Fibroblasts/physiology , Gene Silencing , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Mice , MicroRNAs/genetics , NIH 3T3 Cells
11.
Genesis ; 55(4)2017 04.
Article in English | MEDLINE | ID: mdl-28170160

ABSTRACT

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that has many essential roles during inflammation, development and cancer. Stat3 is therefore an attractive therapeutic target in many diseases. While current Stat3 knockout mouse models led to a better understanding of the role of Stat3, the irreversible nature of Stat3 ablation does not model the effects of transient Stat3 therapeutic inhibition, and does not inform on potential dosage effects of Stat3. Using RNAi technology, we have generated a new mouse model allowing the inducible and reversible silencing of Stat3 in vivo, which mirrors the effects of specific Stat3 therapeutic interference. We showed that upon Doxycycline-mediated activation of the Stat3 short-hairpin RNA, Stat3 expression was efficiently reduced by about 80% in multiple organs and cell types. Moreover, Stat3 reduction was sufficient to reduce tumor burden in a clinically-validated mouse model of gastric cancer. Finally, we demonstrated that Stat3 silencing during embryonic development led to reduced birth rate without leading to complete embryonic lethality, in contrast to full Stat3 ablation. In conclusion, this new mouse model will be invaluable to understand the effects of Stat3 therapeutic interference and Stat3 dosage effects.


Subject(s)
Gene Silencing , Gene Targeting/methods , STAT3 Transcription Factor/genetics , Animals , Cell Line , Doxycycline/pharmacology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Gene Dosage , Mice , Mice, Inbred C57BL , Mice, Knockout , STAT3 Transcription Factor/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Transcription, Genetic/drug effects
13.
Nat Genet ; 39(7): 914-21, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17572676

ABSTRACT

Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.


Subject(s)
Organ Specificity/genetics , Animals , Drug Delivery Systems , Mice , Mice, Transgenic , Promoter Regions, Genetic , RNA Interference/physiology , Tetracycline
14.
BMC Cancer ; 15: 221, 2015 Apr 03.
Article in English | MEDLINE | ID: mdl-25879659

ABSTRACT

BACKGROUND: The molecular regulators that orchestrate stem cell renewal, proliferation and differentiation along the mammary epithelial hierarchy remain poorly understood. Here we have performed a large-scale pooled RNAi screen in primary mouse mammary stem cell (MaSC)-enriched basal cells using 1295 shRNAs against genes principally involved in transcriptional regulation. METHODS: MaSC-enriched basal cells transduced with lentivirus pools carrying shRNAs were maintained as non-adherent mammospheres, a system known to support stem and progenitor cells. Integrated shRNAs that altered culture kinetics were identified by next generation sequencing as relative frequency changes over time. RNA-seq-based expression profiling coupled with in vitro progenitor and in vivo transplantation assays was used to confirm a role for candidate genes in mammary stem and/or progenitor cells. RESULTS: Utilizing a mammosphere-based assay, the screen identified several candidate regulators. Although some genes had been previously implicated in mammary gland development, the vast majority of genes uncovered have no known function within the mammary gland. RNA-seq analysis of freshly purified primary mammary epithelial populations and short-term cultured mammospheres was used to confirm the expression of candidate regulators. Two genes, Asap1 and Prox1, respectively implicated in breast cancer metastasis and progenitor cell function in other systems, were selected for further analysis as their roles in the normal mammary gland were unknown. Both Prox1 and Asap1 were shown to act as negative regulators of progenitor activity in vitro, and Asap1 knock-down led to a marked increase in repopulating activity in vivo, implying a role in stem cell activity. CONCLUSIONS: This study has revealed a number of novel genes that influence the activity or survival of mammary stem and/or progenitor cells. Amongst these, we demonstrate that Prox1 and Asap1 behave as negative regulators of mammary stem/progenitor function. Both of these genes have also been implicated in oncogenesis. Our findings provide proof of principle for the use of short-term cultured primary MaSC/basal cells in functional RNAi screens.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Homeodomain Proteins/genetics , Mammary Glands, Animal/metabolism , RNA, Small Interfering/genetics , Stem Cells/metabolism , Tumor Suppressor Proteins/genetics , Animals , Cell Count , Cell Differentiation/genetics , Epithelial Cells/metabolism , Female , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Immunophenotyping , Mice , Reproducibility of Results
15.
Nat Genet ; 37(11): 1289-95, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16200064

ABSTRACT

RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA-based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA-based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.


Subject(s)
Genetic Vectors , MicroRNAs/genetics , Neoplasms/metabolism , RNA Interference , RNA Polymerase II/genetics , RNA, Small Interfering/pharmacology , Animals , Cell Proliferation , Cellular Senescence , Fibroblasts/metabolism , Mice , Oncogenes , Phenotype , Promoter Regions, Genetic/genetics , Protein Synthesis Inhibitors/pharmacology , Tetracycline/pharmacology , Transcription, Genetic , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
16.
J Immunol ; 186(6): 3666-73, 2011 Mar 15.
Article in English | MEDLINE | ID: mdl-21300820

ABSTRACT

Cystatin C (CstC) is a cysteine protease inhibitor of major clinical importance. Low concentration of serum CstC is linked to atherosclerosis. CstC can prevent formation of amyloid ß associated with Alzheimer's disease and can itself form toxic aggregates. CstC regulates NO secretion by macrophages and is a TGF-ß antagonist. Finally, the serum concentration of CstC is an indicator of kidney function. Yet, little is known about the regulation of CstC expression in vivo. In this study, we demonstrate that the transcription factor IFN regulatory factor 8 (IRF-8) is critical for CstC expression in primary dendritic cells. Only those cells with IRF-8 bound to the CstC gene promoter expressed high levels of the inhibitor. Secretion of IL-10 in response to inflammatory stimuli downregulated IRF-8 expression and consequently CstC synthesis in vivo. Furthermore, the serum concentration of CstC decreased in an IL-10-dependent manner in mice treated with the TLR9 agonist CpG. CstC synthesis is therefore more tightly regulated than hitherto recognized. The mechanisms involved in this regulation might be targeted to alter CstC production, with potential therapeutic value. Our results also indicate that caution should be exerted when using the concentration of serum CstC as an indicator of kidney function in conditions in which inflammation may alter CstC production.


Subject(s)
Cystatin C/biosynthesis , Cystatin C/blood , Down-Regulation/immunology , Inflammation Mediators/physiology , Interferon Regulatory Factors/biosynthesis , Interleukin-10/physiology , Animals , Bone Marrow Transplantation/immunology , Bone Marrow Transplantation/pathology , Cell Line, Tumor , Cells, Cultured , Coculture Techniques , Cystatin C/deficiency , Dendritic Cells/classification , Dendritic Cells/immunology , Dendritic Cells/pathology , Down-Regulation/genetics , Inflammation Mediators/antagonists & inhibitors , Interferon Regulatory Factors/deficiency , Interferon Regulatory Factors/physiology , Interleukin-10/metabolism , Melanoma, Experimental , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic
17.
Nature ; 445(7128): 656-60, 2007 Feb 08.
Article in English | MEDLINE | ID: mdl-17251933

ABSTRACT

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.


Subject(s)
Cellular Senescence/physiology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/metabolism , Animals , Apoptosis , Cell Differentiation , Immunity, Innate/immunology , Liver Neoplasms/immunology , Mice , RNA Interference , Tumor Suppressor Protein p53/genetics
18.
Cancers (Basel) ; 15(18)2023 Sep 20.
Article in English | MEDLINE | ID: mdl-37760615

ABSTRACT

Tumour progression relies on interactions with untransformed cells in the tumour microenvironment (TME), including cancer-associated fibroblasts (CAFs), which promote blood supply, tumour progression, and immune evasion. Eph receptor tyrosine kinases are cell guidance receptors that are most active during development but re-emerge in cancer and are recognised drug targets. EphA3 is overexpressed in a wide range of tumour types, and we previously found expression particularly in stromal and vascular tissues of the TME. To investigate its role in the TME, we generated transgenic mice with inducible shRNA-mediated knockdown of EphA3 expression. EphA3 knockdown was confirmed in aortic mesenchymal stem cells (MSCs), which displayed reduced angiogenic capacity. In mice with syngeneic lung tumours, EphA3 knockdown reduced vasculature and CAF/MSC-like cells in tumours, and inhibited tumour growth, which was confirmed also in a melanoma model. Single cell RNA sequencing analysis of multiple human tumour types confirmed EphA3 expression in CAFs, including in breast cancer, where EphA3 was particularly prominent in perivascular- and myofibroblast-like CAFs. Our results thus indicate expression of the cell guidance receptor EphA3 in distinct CAF subpopulations is important in supporting tumour angiogenesis and tumour growth, highlighting its potential as a therapeutic target.

19.
Nature ; 440(7080): 105-9, 2006 Mar 02.
Article in English | MEDLINE | ID: mdl-16400328

ABSTRACT

Chromosomal translocations involving the immunoglobulin switch region are a hallmark feature of B-cell malignancies. However, little is known about the molecular mechanism by which primary B cells acquire or guard against these lesions. Here we find that translocations between c-myc and the IgH locus (Igh) are induced in primary B cells within hours of expression of the catalytically active form of activation-induced cytidine deaminase (AID), an enzyme that deaminates cytosine to produce uracil in DNA. Translocation also requires uracil DNA glycosylase (UNG), which removes uracil from DNA to create abasic sites that are then processed to double-strand breaks. The pathway that mediates aberrant joining of c-myc and Igh differs from intrachromosomal repair during immunoglobulin class switch recombination in that it does not require histone H2AX, p53 binding protein 1 (53BP1) or the non-homologous end-joining protein Ku80. In addition, translocations are inhibited by the tumour suppressors ATM, Nbs1, p19 (Arf) and p53, which is consistent with activation of DNA damage- and oncogenic stress-induced checkpoints during physiological class switching. Finally, we demonstrate that accumulation of AID-dependent, IgH-associated chromosomal lesions is not sufficient to enhance c-myc-Igh translocations. Our findings reveal a pathway for surveillance and protection against AID-dependent DNA damage, leading to chromosomal translocations.


Subject(s)
Cytidine Deaminase/metabolism , Genes, Immunoglobulin Heavy Chain/genetics , Genes, myc/genetics , Genomic Instability/genetics , Models, Genetic , Translocation, Genetic/genetics , Tumor Suppressor Protein p53/metabolism , Animals , B-Lymphocytes/metabolism , Cell Line , Cytidine Deaminase/genetics , DNA Damage/genetics , Gene Expression , Immunoglobulin Class Switching/genetics , Mice , Mutation/genetics , Tumor Suppressor Protein p53/genetics
20.
Nat Commun ; 12(1): 6546, 2021 11 11.
Article in English | MEDLINE | ID: mdl-34764270

ABSTRACT

Acute myeloid leukemia (AML) is a malignancy of immature progenitor cells. AML differentiation therapies trigger leukemia maturation and can induce remission, but relapse is prevalent and its cellular origin is unclear. Here we describe high resolution analysis of differentiation therapy response and relapse in a mouse AML model. Triggering leukemia differentiation in this model invariably produces two phenotypically distinct mature myeloid lineages in vivo. Leukemia-derived neutrophils dominate the initial wave of leukemia differentiation but clear rapidly and do not contribute to residual disease. In contrast, a therapy-induced population of mature AML-derived eosinophil-like cells persists during remission, often in extramedullary organs. Using genetic approaches we show that restricting therapy-induced leukemia maturation to the short-lived neutrophil lineage markedly reduces relapse rates and can yield cure. These results indicate that relapse can originate from therapy-resistant mature AML cells, and suggest differentiation therapy combined with targeted eradication of mature leukemia-derived lineages may improve disease outcome.


Subject(s)
Leukemia, Myeloid, Acute/metabolism , Neoplasm, Residual/metabolism , Cell Differentiation , Humans , Leukemia, Myeloid, Acute/genetics , Neoplasm, Residual/genetics
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