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1.
Cell ; 159(7): 1524-37, 2014 Dec 18.
Article in English | MEDLINE | ID: mdl-25483777

ABSTRACT

The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA(+) enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.


Subject(s)
B-Lymphocytes/metabolism , Carcinogenesis , Cytidine Deaminase/genetics , Enhancer Elements, Genetic , Animals , DNA Damage , Humans , Lymphoma/metabolism , Mice
2.
Mol Cell ; 67(4): 566-578.e10, 2017 Aug 17.
Article in English | MEDLINE | ID: mdl-28803781

ABSTRACT

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.


Subject(s)
B-Lymphocytes/metabolism , Cell Cycle , Cell Nucleus/metabolism , Chromatin Assembly and Disassembly , Chromatin/metabolism , Histones/metabolism , Lymphocyte Activation , Proto-Oncogene Proteins c-myc/metabolism , Acetyl Coenzyme A/metabolism , Acetylation , Adenosine Triphosphate/metabolism , Animals , B-Lymphocytes/immunology , Cell Line , Chromatin/chemistry , Chromatin/genetics , DNA Methylation , Epigenesis, Genetic , Genotype , Histones/chemistry , Immunity, Humoral , Methylation , Mice, Inbred C57BL , Mice, Knockout , Nucleic Acid Conformation , Phenotype , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-myc/chemistry , Proto-Oncogene Proteins c-myc/genetics , Single Molecule Imaging , Structure-Activity Relationship , Time Factors , Transcription, Genetic
3.
PLoS Genet ; 18(6): e1010271, 2022 06.
Article in English | MEDLINE | ID: mdl-35727842

ABSTRACT

The TGF-ß-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-ß pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.


Subject(s)
Breast Neoplasms , Chloride Channels , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Chloride Channels/biosynthesis , Chloride Channels/genetics , Female , Humans , Mice , Neoplasm Metastasis , Proteomics , Transforming Growth Factor beta/metabolism , Tumor Microenvironment
4.
PLoS Genet ; 17(5): e1009553, 2021 05.
Article in English | MEDLINE | ID: mdl-33945523

ABSTRACT

The CBFB gene is frequently mutated in several types of solid tumors. Emerging evidence suggests that CBFB is a tumor suppressor in breast cancer. However, our understanding of the tumor suppressive function of CBFB remains incomplete. Here, we analyze genetic interactions between mutations of CBFB and other highly mutated genes in human breast cancer datasets and find that CBFB and TP53 mutations are mutually exclusive, suggesting a functional association between CBFB and p53. Integrated genomic studies reveal that TAp73 is a common transcriptional target of CBFB and p53. CBFB cooperates with p53 to maintain TAp73 expression, as either CBFB or p53 loss leads to TAp73 depletion. TAp73 re-expression abrogates the tumorigenic effect of CBFB deletion. Although TAp73 loss alone is insufficient for tumorigenesis, it enhances the tumorigenic effect of NOTCH3 overexpression, a downstream event of CBFB loss. Immunohistochemistry shows that p73 loss is coupled with higher proliferation in xenografts. Moreover, TAp73 loss-of-expression is a frequent event in human breast cancer tumors and cell lines. Together, our results significantly advance our understanding of the tumor suppressive functions of CBFB and reveal a mechanism underlying the communication between the two tumor suppressors CBFB and p53.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Core Binding Factor beta Subunit/genetics , Gene Expression Regulation, Neoplastic , Tumor Protein p73/genetics , Tumor Suppressor Protein p53/genetics , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Core Binding Factor beta Subunit/deficiency , Core Binding Factor beta Subunit/metabolism , Female , Genes, Tumor Suppressor , Humans , Immunohistochemistry , Mice , Mutation , Receptor, Notch3/genetics , Receptor, Notch3/metabolism , Transcription, Genetic , Tumor Protein p73/deficiency , Tumor Protein p73/metabolism , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays
5.
EMBO Rep ; 22(6): e52122, 2021 06 04.
Article in English | MEDLINE | ID: mdl-33950553

ABSTRACT

Metabolic regulation is critical for the maintenance of pluripotency and the survival of embryonic stem cells (ESCs). The transcription factor Tfcp2l1 has emerged as a key factor for the naïve pluripotency of ESCs. Here, we report an unexpected role of Tfcp2l1 in metabolic regulation in ESCs-promoting the survival of ESCs through regulating fatty acid oxidation (FAO) under metabolic stress. Tfcp2l1 directly activates many metabolic genes in ESCs. Deletion of Tfcp2l1 leads to an FAO defect associated with upregulation of glucose uptake, the TCA cycle, and glutamine catabolism. Mechanistically, Tfcp2l1 activates FAO by inducing Cpt1a, a rate-limiting enzyme transporting free fatty acids into the mitochondria. ESCs with defective FAO are sensitive to cell death induced by glycolysis inhibition and glutamine deprivation. Moreover, the Tfcp2l1-Cpt1a-FAO axis promotes the survival of quiescent ESCs and diapause-like blastocysts induced by mTOR inhibition. Thus, our results reveal how ESCs orchestrate pluripotent and metabolic programs to ensure their survival in response to metabolic stress.


Subject(s)
Embryonic Stem Cells , Lipid Metabolism , Fatty Acids , Oxidation-Reduction , Stress, Physiological
6.
Mol Carcinog ; 61(4): 408-416, 2022 04.
Article in English | MEDLINE | ID: mdl-34964999

ABSTRACT

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase and central regulator of cell growth, differentiation, and survival. mTOR is commonly hyperactivated in a diverse number of cancers and critical roles for mTOR in regulating immune cell differentiation and function have been demonstrated. However, there is little work investigating the roles of mTOR in early B-cell development. Here we demonstrate that conditional disruption of mTOR in developing mouse B cells results in reduced pre-B-cell proliferation and survival, as well as a developmental block at the pre-B-cell stage, with a corresponding lack of peripheral B cells. Upon immunization with NP-CGG antigen, mice with Mtor conditional disruption in early B cells lost their ability to form germinal centers and produce specific antibodies. In competitive BM repopulation assays, donor BM cells from conditional knock-out mice were completely impaired in their ability to reconstitute B cells. Our data reveal the essential role of mTOR in early pre-B-cell development and survival.


Subject(s)
Signal Transduction , Sirolimus , Animals , B-Lymphocytes/metabolism , Cell Differentiation , Lymphocyte Activation , Mice , Mice, Knockout , TOR Serine-Threonine Kinases/metabolism
7.
J Biol Chem ; 294(45): 16756-16764, 2019 11 08.
Article in English | MEDLINE | ID: mdl-31548308

ABSTRACT

Mechanistic target of rapamycin (MTOR) is a highly conserved serine/threonine kinase that critically regulates cell growth, proliferation, differentiation, and survival. Previously, we have implicated Mtor as a plasmacytoma-resistance locus, Pctr2, in mice. Here, we report that administration of the tumor-inducing agent pristane decreases Mtor gene expression to a greater extent in mesenteric lymph nodes of BALB/cAnPt mice than of DBA/2N mice. We identified six allelic variants in the Mtor promoter region in BALB/cAnPt and DBA/2N mice. To determine the effects of these variants on Mtor transcription, we constructed a series of luciferase reporters containing these promoter variants and transfected them into mouse plasmacytoma cells. We could attribute the differences in Mtor promoter activity between the two mouse strains to a C → T change at the -6 position relative to the transcriptional start site Tssr 40273; a T at this position in the BALB promoter creates a consensus binding site for the transcription factor MZF1 (myeloid zinc finger 1). Results from electrophoretic mobility shift assays and DNA pulldown assays with ChIP-PCR confirmed that MZF1 binds to the cis-element TGGGGA located in the -6/-1 Mtor promoter region. Of note, MZF1 significantly and differentially down-regulated Mtor promoter activity, with MZF1 overexpression reducing Mtor expression more strongly in BALB mice than in DBA mice. Moreover, MZF1 overexpression reduced Mtor expression in both fibroblasts and mouse plasmacytoma cells, and Mzf1 knockdown increased Mtor expression in BALB3T3 and NIH3T3 fibroblast cells. Our results provide evidence that MZF1 down-regulates Mtor expression in pristane-induced plasmacytomas in mice.


Subject(s)
Genetic Predisposition to Disease/genetics , Kruppel-Like Transcription Factors/metabolism , Mutation , Plasmacytoma/genetics , Promoter Regions, Genetic/genetics , TOR Serine-Threonine Kinases/genetics , Alleles , Animals , Base Sequence , Cell Line, Tumor , Down-Regulation , Mice , Plasmacytoma/pathology
8.
Mol Carcinog ; 59(2): 237-245, 2020 02.
Article in English | MEDLINE | ID: mdl-31898340

ABSTRACT

In humans, bone marrow (BM) failure syndromes, both constitutional and acquired, predispose to myeloid malignancies. We have modeled acquired immune aplastic anemia, the paradigmatic disease of these syndromes, in the mouse by infusing lymph node cells from specific pathogen-free (SPF) CD45.1 congenic C57BL/6 (B6) donors into hybrid CByB6F1 recipients housed either in conventional (CVB) or SPF facilities. The severity of BM damage was reduced in CVB recipients; they also had reduced levels of CD44+ CD62L- effector memory T cells, reduced numbers of donor-type CD44+ T cells, and reduced expansion of donor-type CD8 T cells carrying T-cell receptor ß-variable regions 07, 11, and 17. Analyses of fecal samples through 16S ribosomal RNA amplicon sequencing revealed greater gut microbial alpha diversity in CVB mice relative to that of SPF mice. Thus, the presence of a broader spectrum of gut microorganisms in CVB-housed CByB6F1 could have primed recipient animal's immune system leading to suppression of allogeneic donor T-cell activation and expansion and attenuation of host BM destruction. These results suggest the potential benefit of diverse gut microbiota in patients receiving BM transplants.


Subject(s)
Anemia, Aplastic/therapy , Bone Marrow Transplantation/methods , Bone Marrow/immunology , Gastrointestinal Microbiome/immunology , T-Lymphocytes/immunology , Anemia, Aplastic/immunology , Anemia, Aplastic/pathology , Animals , Bone Marrow/pathology , Feces/microbiology , Hyaluronan Receptors/immunology , Hyaluronan Receptors/metabolism , Immunologic Memory/immunology , Lymphocyte Activation/immunology , Mice, Inbred BALB C , Mice, Inbred C57BL , Specific Pathogen-Free Organisms , T-Lymphocytes/metabolism , T-Lymphocytes/transplantation , Transplantation Immunology , Transplantation, Homologous
9.
Mol Cell ; 46(1): 30-42, 2012 Apr 13.
Article in English | MEDLINE | ID: mdl-22387025

ABSTRACT

p53 is critical in regulating the differentiation of ES and induced pluripotent stem (iPS) cells. Here, we report a whole-genome study of p53-mediated DNA damage signaling in mouse ES cells. Systems analyses reveal that binding of p53 at the promoter region significantly correlates with gene activation but not with repression. Unexpectedly, we identify a regulatory mode for p53-mediated repression through interfering with distal enhancer activity. Importantly, many ES cell-enriched core transcription factors are p53-repressed genes. Further analyses demonstrate that p53-repressed genes are functionally associated with ES/iPS cell status while p53-activated genes are linked to differentiation. p53-activated genes and -repressed genes also display distinguishable features of expression levels and epigenetic markers. Upon DNA damage, p53 regulates the self-renewal and pluripotency of ES cells. Together, these results support a model where, in response to DNA damage, p53 affects the status of ES cells through activating differentiation-associated genes and repressing ES cell-enriched genes.


Subject(s)
Cell Differentiation , DNA Damage , Embryonic Stem Cells/metabolism , Models, Biological , Pluripotent Stem Cells/metabolism , Repressor Proteins/metabolism , Response Elements , Tumor Suppressor Protein p53/metabolism , Animals , Cells, Cultured , Embryonic Stem Cells/cytology , Gene Expression Regulation , Genome , Mice , Repressor Proteins/genetics
10.
Int J Mol Sci ; 21(17)2020 Aug 26.
Article in English | MEDLINE | ID: mdl-32858886

ABSTRACT

Specific-pathogen-free (SPF) mice have improved hematopoietic characteristics relative to germ-free mice, however, it is not clear whether improvements in hematopoietic traits will continue when the level of microorganism exposure is further increased. We co-housed SPF C57BL/6 mice in a conventional facility (CVT) and found a significant increase in gut microbiota diversity along with increased levels of myeloid cells and T cells, especially effector memory T cells. Through single cell RNA sequencing of sorted KL (c-Kit+Lin-) cells, we imputed a decline in long-term hematopoietic stem cells and an increase in granulocyte-monocyte progenitors in CVT mice with up-regulation of genes associated with cell survival. Bone marrow transplantation through competitive repopulation revealed a significant increase in KSL (c-Kit+Sca-1+Lin-) cell reconstitution in recipients of CVT donor cells which occurred when donors were co-housed for both one and twelve months. However, there was minimal to no gain in mature blood cell engraftment in recipients of CVT donor cells relative to those receiving SPF donor cells. We conclude that co-housing SPF mice with mice born in a conventional facility increased gut microbiota diversity, augmented myeloid cell production and T cell activation, stimulated KSL cell reconstitution, and altered hematopoietic gene expression.


Subject(s)
Bacteria/classification , Gene Expression Profiling/methods , Hematopoiesis , Myeloid Cells/metabolism , Sequence Analysis, RNA/methods , T-Lymphocytes/metabolism , Animals , Bacteria/genetics , Bacteria/isolation & purification , Bone Marrow Transplantation , Gastrointestinal Microbiome , Gene Expression Regulation , Housing, Animal , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Phylogeny , Single-Cell Analysis , Specific Pathogen-Free Organisms
11.
Immunity ; 32(6): 828-39, 2010 Jun 25.
Article in English | MEDLINE | ID: mdl-20605486

ABSTRACT

Although the cellular concentration of miRNAs is critical to their function, how miRNA expression and abundance are regulated during ontogeny is unclear. We applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. We show that lymphocyte-specific miRNAs are either tightly controlled by polycomb group-mediated H3K27me3 or maintained in a semi-activated epigenetic state prior to full expression. Because of miRNA biogenesis, the cellular concentration of mature miRNAs does not typically reflect transcriptional changes. However, we uncover a subset of miRNAs for which abundance is dictated by miRNA gene expression. We confirm that concentration of 5p and 3p miRNA strands depends largely on free energy properties of miRNA duplexes. Unexpectedly, we also find that miRNA strand accumulation can be developmentally regulated. Our data provide a comprehensive map of immunity's microRNome and reveal the underlying epigenetic and transcriptional forces that shape miRNA homeostasis.


Subject(s)
Epigenesis, Genetic , Gene Expression Regulation/genetics , Lymphocytes , Lymphopoiesis/genetics , MicroRNAs/genetics , Animals , Gene Expression , Humans , Mice , Reverse Transcriptase Polymerase Chain Reaction
12.
Stem Cells ; 33(4): 1304-19, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25524638

ABSTRACT

The tumor suppressor, p53, plays a critical role in suppressing osteosarcoma. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested to give rise to osteosarcomas. However, the role of p53 in BMSCs has not been extensively explored. Here, we report that p53 regulates the lineage choice of mouse BMSCs (mBMSCs). Compared to mBMSCs with wild-type p53, mBMSCs deficient in p53 have enhanced osteogenic differentiation, but with similar adipogenic and chondrogenic differentiation. The role of p53 in inhibiting osteogenic lineage differentiation is mainly through the action of Runx2, a master transcription factor required for the osteogenic differentiation of mBMSCs. We find that p53 indirectly represses the expression of Runx2 by activating the microRNA-34 family, which suppresses the translation of Runx2. Since osteosarcoma may derive from BMSCs, we examined whether p53 has a role in the osteogenic differentiation of osteosarcoma cells and found that osteosarcoma cells with p53 deletion have higher levels of Runx2 and faster osteogenic differentiation than those with wild-type p53. A systems biology approach reveals that p53-deficient mBMSCs are more closely related to human osteosarcoma while mBMSCs with wild-type p53 are similar to normal human BMSCs. In summary, our results indicate that p53 activity can influence cell fate specification of mBMSCs, and provide molecular and cellular insights into the observation that p53 loss is associated with increased osteosarcoma incidence.


Subject(s)
Cell Differentiation/physiology , Mesenchymal Stem Cells/metabolism , Osteogenesis/physiology , Tumor Suppressor Protein p53/deficiency , Animals , Cell Line, Tumor , Cells, Cultured , Humans , Mice , Mice, Knockout
13.
Proc Natl Acad Sci U S A ; 109(34): 13728-32, 2012 Aug 21.
Article in English | MEDLINE | ID: mdl-22869734

ABSTRACT

Many tumors are characterized by recurrent translocations between a tissue-specific gene and a proto-oncogene. The juxtaposition of the Ig heavy chain gene and Myc in Burkitt's lymphoma and in murine plasmacytoma is a classic example. Regulatory elements within the heavy chain constant region locus are required for Myc translocation and/or deregulation. However, many genes are regulated by cis-acting elements at distances up to 1,000 kb outside the locus. Such putative distal elements have not been examined for the heavy chain locus, particularly in the context of Myc translocations. We demonstrate that a transgene containing the Ig heavy chain constant region locus, inserted into five different chromosomal locations, can undergo translocations involving Myc. Furthermore, these translocations are able to generate plasmacytomas in each transgenic line. We conclude that the heavy chain constant region locus itself includes all of the elements necessary for both the translocation and the deregulation of the proto-oncogene.


Subject(s)
Genes, Immunoglobulin Heavy Chain , Proto-Oncogene Proteins c-myc/genetics , Translocation, Genetic , Animals , Cell Line, Tumor , Chromosome Mapping , Gene Expression Regulation, Neoplastic , Genome , Humans , Lymphoma, B-Cell/genetics , Mice , Mice, Inbred BALB C , Models, Genetic , Molecular Sequence Data , Proto-Oncogene Mas , Transgenes
14.
Proc Natl Acad Sci U S A ; 109(27): 10972-7, 2012 Jul 03.
Article in English | MEDLINE | ID: mdl-22711821

ABSTRACT

Human Burkitt lymphomas are divided into two main clinical variants: the endemic form, affecting African children infected with malaria and the Epstein-Barr virus, and the sporadic form, distributed across the rest of the world. However, whereas sporadic translocations decapitate Myc from 5' proximal regulatory elements, most endemic events occur hundreds of kilobases away from Myc. The origin of these rearrangements and how they deregulate oncogenes at such distances remain unclear. We here recapitulate endemic Burkitt lymphoma-like translocations in plasmacytomas from uracil N-glycosylase and activation-induced cytidine deaminase-deficient mice. Mapping of translocation breakpoints using an acetylated histone H3 lysine 9 chromatin immunoprecipitation sequencing approach reveals Igh fusions up to ∼350 kb upstream of Myc or the related oncogene Mycn. A comprehensive analysis of epigenetic marks, PolII recruitment, and transcription in tumor cells demonstrates that the 3' Igh enhancer (Eα) vastly remodels ∼450 kb of chromatin into translocated sequences, leading to significant polymerase occupancy and constitutive oncogene expression. We show that this long-range epigenetic reprogramming is directly proportional to the physical interaction of Eα with translocated sites. Our studies thus uncover the extent of epigenetic remodeling by Ig 3' enhancers and provide a rationale for the long-range deregulation of translocated oncogenes in endemic Burkitt lymphomas. The data also shed light on the origin of endemic-like chromosomal rearrangements.


Subject(s)
Burkitt Lymphoma/genetics , Gene Expression Regulation, Neoplastic/genetics , Genes, Immunoglobulin Heavy Chain/genetics , Genes, myc/genetics , Immunoglobulin Class Switching/genetics , Translocation, Genetic/genetics , Animals , B-Lymphocytes/cytology , B-Lymphocytes/physiology , Burkitt Lymphoma/epidemiology , Cells, Cultured , Cytidine/genetics , Disease Models, Animal , Endemic Diseases , Enhancer Elements, Genetic/genetics , Epigenesis, Genetic/genetics , Gene Rearrangement, B-Lymphocyte/genetics , Humans , Mice , Oncogene Proteins, Fusion/genetics , Plasma Cells/cytology , Plasma Cells/physiology , Transcription Initiation Site/physiology , Uracil-DNA Glycosidase/genetics
15.
Blood ; 119(4): 1018-28, 2012 Jan 26.
Article in English | MEDLINE | ID: mdl-22147894

ABSTRACT

Tumor progression usually proceeds through several sequential stages, any of which could be targets for interrupting the progression process if one understood these steps at the molecular level. We extracted nascent plasma cell tumor (PCT) cells from within inflammatory oil granulomas (OG) isolated from IP pristane-injected BALB/c.iMyc(Eµ) mice at 5 different time points during tumor progression. We used laser capture microdissection to collect incipient PCT cells and analyzed their global gene expression on Affymetrix Mouse Genome 430A microarrays. Two independent studies were performed with different sets of mice. Analysis of the expression data used ANOVA and Bayesian estimation of temporal regulation. Genetic pathway analysis was performed using MetaCore (GeneGo) and IPA (Ingenuity). The gene expression profiles of PCT samples and those of undissected OG samples from adjacent sections showed that different genes and pathways were mobilized in the tumor cells during tumor progression, compared with their stroma. Our analysis implicated several genetic pathways in PCT progression, including biphasic (up- and then down-regulation) of the Spp1/osteopontin-dependent network and up-regulation of mRNA translation/protein synthesis. The latter led to a biologic validation study that showed that the AMPK-activating diabetes drug, metformin, was a potent specific PCT inhibitor in vitro.


Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Neoplasms, Plasma Cell/drug therapy , Stromal Cells/drug effects , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Gene Expression Profiling , Granuloma, Plasma Cell/drug therapy , Granuloma, Plasma Cell/metabolism , Granuloma, Plasma Cell/pathology , Metformin/pharmacology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Molecular Targeted Therapy , Neoplasms, Plasma Cell/metabolism , Neoplasms, Plasma Cell/pathology , Oligonucleotide Array Sequence Analysis , Osteopontin/genetics , Osteopontin/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Messenger/metabolism , Stromal Cells/cytology , Stromal Cells/metabolism , Tumor Cells, Cultured
16.
Sci Adv ; 10(32): eadl4893, 2024 Aug 09.
Article in English | MEDLINE | ID: mdl-39121214

ABSTRACT

Discontinuous transcription is evolutionarily conserved and a fundamental feature of gene regulation; yet, the exact mechanisms underlying transcriptional bursting are unresolved. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements, but other factors that regulate this process remain elusive. We applied mathematical modeling to single-cell RNA sequencing data to infer bursting dynamics transcriptome-wide under multiple conditions to identify possible molecular mechanisms. We found that Mediator complex subunit 26 (MED26) primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. Despite comparable effects on RNA levels among these perturbations, acute depletion of MED26 had the most profound impact on the entire gene regulatory network, acting downstream of chromatin spatial architecture and without affecting TATA box-binding protein (TBP) recruitment. These results indicate that later steps in the initiation of transcriptional bursts are primary nodes for integrating gene networks in single cells.


Subject(s)
Cell Cycle Proteins , Chromatin , Gene Regulatory Networks , Transcription Factors , Transcription, Genetic , Chromatin/metabolism , Chromatin/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Humans , Gene Expression Regulation , Mediator Complex/metabolism , Mediator Complex/genetics , Single-Cell Analysis , Transcriptome , Cohesins , Bromodomain Containing Proteins
17.
Blood ; 117(4): 1228-38, 2011 Jan 27.
Article in English | MEDLINE | ID: mdl-21079150

ABSTRACT

Mammalian TOR (mTOR) regulates cell growth, proliferation, and migration. Because mTOR knock-outs are embryonic lethal, we generated a viable hypomorphic mouse by neo-insertion that partially disrupts mTOR transcription and creates a potential physiologic model of mTORC1/TORC2 inhibition. Homozygous knock-in mice exhibited reductions in body, organ, and cell size. Although reductions in most organ sizes were proportional to decreased body weight, spleens were disproportionately smaller. Decreases in the total number of T cells, particularly memory cells, and reduced responses to chemokines suggested alterations in T-cell homing/homeostasis. T-cell receptor-stimulated T cells proliferated less, produced lower cytokine levels, and expressed FoxP3. Decreased neutrophil numbers were also observed in the spleen, despite normal development and migration in the bone marrow. However, B-cell effects were most pronounced, with a partial block in B-cell development in the bone marrow, altered splenic populations, and decreases in proliferation, antibody production, and migration to chemokines. Moreover, increased AKT(Ser473) phosphorylation was observed in activated B cells, reminiscent of cancers treated with rapamycin, and was reduced by a DNA-pk inhibitor. Thus, mTOR is required for the maturation and differentiation of multiple immune cell lineages. These mice provide a novel platform for studying the consequences of constitutively reduced mTORC1/TORC2 activity.


Subject(s)
Antibody Formation/genetics , B-Lymphocytes/cytology , B-Lymphocytes/physiology , TOR Serine-Threonine Kinases/genetics , Animals , B-Lymphocytes/metabolism , Body Size/genetics , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Lineage/genetics , Cell Lineage/immunology , Cell Size , Down-Regulation/immunology , Down-Regulation/physiology , Gene Knockdown Techniques , Mice , Mice, Inbred BALB C , Mice, Transgenic , Organ Size/genetics , Spleen/anatomy & histology , Spleen/metabolism
18.
Blood ; 118(26): 6803-13, 2011 Dec 22.
Article in English | MEDLINE | ID: mdl-22053105

ABSTRACT

Increased mast cell burden is observed in the inflamed tissues and affected organs and tissues of patients with mast cell proliferative disorders. However, normal mast cells participate in host defense, so approaches to preferentially target clonally expanding mast cells are needed. We found that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) are up-regulated in neoplastic and developing immature mast cells compared with their terminally differentiated counterparts. Elevated mTOR mRNA was also observed in bone marrow mononuclear cells of patients exhibiting mast-cell hyperplasia. Selective inhibition of mTORC1 and mTORC2 through genetic and pharmacologic manipulation revealed that, whereas mTORC1 may contribute to mast-cell survival, mTORC2 was only critical for homeostasis of neoplastic and dividing immature mast cells. The cytostatic effect of mTORC2 down-regulation in proliferating mast cells was determined to be via inhibition of cell-cycle progression. Because mTORC2 was observed to play little role in the homeostasis of differentiated, nonproliferating, mature mast cells, these data provide a rationale for adopting a targeted approaching selectively inhibiting mTORC2 to effectively reduce the proliferation of mast cells associated with inflammation and disorders of mast cell proliferation while leaving normal differentiated mast cells largely unaffected.


Subject(s)
Homeostasis , Mast Cells/metabolism , Mastocytosis, Systemic/metabolism , Proteins/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antibiotics, Antineoplastic/pharmacology , Blotting, Western , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Gene Expression Profiling , Humans , Mast Cells/drug effects , Mastocytosis, Systemic/genetics , Mastocytosis, Systemic/pathology , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Transgenic , Multiprotein Complexes , Naphthyridines/pharmacology , Proteins/antagonists & inhibitors , Proteins/genetics , RNA Interference , Rapamycin-Insensitive Companion of mTOR Protein , Regulatory-Associated Protein of mTOR , Reverse Transcriptase Polymerase Chain Reaction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Tumor Cells, Cultured
19.
Cancer Lett ; 568: 216284, 2023 08 01.
Article in English | MEDLINE | ID: mdl-37356470

ABSTRACT

Drug resistance and disease progression are common in multiple myeloma (MM) patients, underscoring the need for new therapeutic combinations. A high-throughput drug screen in 47 MM cell lines and in silico Huber robust regression analysis of drug responses revealed 43 potentially synergistic combinations. We hypothesized that effective combinations would reduce MYC expression and enhance p16 activity. Six combinations cooperatively reduced MYC protein, frequently over-expressed in MM and also cooperatively increased p16 expression, frequently downregulated in MM. Synergistic reductions in viability were observed with top combinations in proteasome inhibitor-resistant and sensitive MM cell lines, while sparing fibroblasts. Three combinations significantly prolonged survival in a transplantable Ras-driven allograft model of advanced MM closely recapitulating high-risk/refractory myeloma in humans and reduced viability of ex vivo treated patient cells. Common genetic pathways similarly downregulated by these combinations promoted cell cycle transition, whereas pathways most upregulated were involved in TGFß/SMAD signaling. These preclinical data identify potentially useful drug combinations for evaluation in drug-resistant MM and reveal potential mechanisms of combined drug sensitivity.


Subject(s)
Multiple Myeloma , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , High-Throughput Screening Assays , Drug Synergism , Cell Cycle , Drug Combinations , Cell Line, Tumor , Drug Resistance, Neoplasm
20.
Blood ; 115(9): 1746-54, 2010 Mar 04.
Article in English | MEDLINE | ID: mdl-20018915

ABSTRACT

Interleukin-6 (IL-6) plays a critical role in the natural history of human plasma cell neoplasms (PCNs), such as plasma cell myeloma and plasmacytoma (PCT). IL-6 is also at the center of neoplastic plasma cell transformation in BALB/c (C) mice carrying a transgene, H2-L(d)-IL6, that encodes human IL-6 under control of the major histocompatibility complex H2-L(d) promoter: strain C.H2-L(d)-IL6. These mice are prone to PCT, but tumor development is incomplete with long latencies ( approximately 40% PCT at 12 months of age). To generate a more robust mouse model of IL-6-dependent PCN, we intercrossed strain C.H2-L(d)-IL6 with strains C.iMyc(Emu) or C.iMyc(Calpha), 2 interrelated gene-insertion models of the chromosomal T(12;15) translocation causing deregulated expression of Myc in mouse PCT. Deregulation of MYC is also a prominent feature of human PCN. We found that double-transgenic C.H2-L(d)-IL6/iMyc(Emu) and C.H2-L(d)-IL6/iMyc(Calpha) mice develop PCT with full penetrance (100% tumor incidence) and short latencies (3-6 months). The mouse tumors mimic molecular hallmarks of their human tumor counterparts, including elevated IL-6/Stat3/Bcl-X(L) signaling. The newly developed mouse strains may provide a good preclinical research tool for the design and testing of new approaches to target IL-6 in treatment and prevention of human PCNs.


Subject(s)
Genes, myc , Interleukin-6/genetics , Plasmacytoma/etiology , Animals , Antibodies, Monoclonal/biosynthesis , Disease Models, Animal , Gene Expression Profiling , Humans , Hyperplasia , Mice , Mice, Transgenic , Neoplasm Transplantation , Plasma Cells/pathology , Plasmacytoma/genetics , Plasmacytoma/immunology , Plasmacytoma/pathology , Signal Transduction , Translocation, Genetic
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