RESUMEN

T cell development proceeds via discrete stages that require both gene induction and gene repression. Transcription factors direct gene repression by associating with corepressor complexes containing chromatin-remodeling enzymes; the corepressors NCOR1 and NCOR2 recruit histone deacetylases to these complexes to silence transcription of target genes. Earlier work identified the importance of NCOR1 in promoting the survival of positively-selected thymocytes. Here, we used flow cytometry and single-cell RNA sequencing to identify a broader role for NCOR1 and NCOR2 in regulating thymocyte development. Using Cd4-cre mice, we found that conditional deletion of NCOR2 had no effect on thymocyte development, whereas conditional deletion of NCOR1 had a modest effect. In contrast, Cd4-cre x Ncor1f/f x Ncor2f/f mice exhibited a significant block in thymocyte development at the DP to SP transition. Combined NCOR1/2 deletion resulted in increased signaling through the T cell receptor, ultimately resulting in elevated BIM expression and increased negative selection. The NF-κB, NUR77, and MAPK signaling pathways were also upregulated in the absence of NCOR1/2, contributing to altered CD4/CD8 lineage commitment, TCR rearrangement, and thymocyte emigration. Taken together, our data identify multiple critical roles for the combined action of NCOR1 and NCOR2 over the course of thymocyte development.

RESUMEN

Integration of external signals and B-lymphoid transcription factor activities organise B cell lineage commitment through alternating cycles of proliferation and differentiation, producing a diverse repertoire of mature B cells. We use single-cell transcriptomics/proteomics to identify differentially expressed gene networks across B cell development and correlate these networks with subtypes of B cell leukemia. Here we show unique transcriptional signatures that refine the pre-B cell expansion stages into pre-BCR-dependent and pre-BCR-independent proliferative phases. These changes correlate with reciprocal changes in expression of the transcription factor EBF1 and the RNA binding protein YBX3, that are defining features of the pre-BCR-dependent stage. Using pseudotime analysis, we further characterize the expression kinetics of different biological modalities across B cell development, including transcription factors, cytokines, chemokines, and their associated receptors. Our findings demonstrate the underlying heterogeneity of developing B cells and characterise developmental nodes linked to B cell transformation.

Asunto(s)

Linfocitos B/citología , Redes Reguladoras de Genes , Leucopoyesis/genética , Linfocitos B/metabolismo , Linfocitos B/patología , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proliferación Celular/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Pronóstico , Proteómica , Análisis de la Célula Individual , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

The transcription factors PAX5, IKZF1, and EBF1 are frequently mutated in B cell acute lymphoblastic leukemia (B-ALL). We demonstrate that compound heterozygous loss of multiple genes critical for B and T cell development drives transformation, including Pax5+/-xEbf1+/-, Pax5+/-xIkzf1+/-, and Ebf1+/-xIkzf1+/- mice for B-ALL, or Tcf7+/-xIkzf1+/- mice for T-ALL. To identify genetic defects that cooperate with Pax5 and Ebf1 compound heterozygosity to initiate leukemia, we performed a Sleeping Beauty (SB) transposon screen that identified cooperating partners including gain-of-function mutations in Stat5b (~65%) and Jak1 (~68%), or loss-of-function mutations in Cblb (61%) and Myb (32%). These findings underscore the role of JAK/STAT5B signaling in B cell transformation and demonstrate roles for loss-of-function mutations in Cblb and Myb in transformation. RNA-Seq studies demonstrated upregulation of a PDK1>SGK3>MYC pathway; treatment of Pax5+/-xEbf1+/- leukemia cells with PDK1 inhibitors blocked proliferation in vitro. In addition, we identified a conserved transcriptional gene signature between human and murine leukemias characterized by upregulation of myeloid genes, most notably involving the GM-CSF pathway, that resemble a B cell/myeloid mixed-lineage leukemia. Thus, our findings identify multiple mechanisms that cooperate with defects in B cell transcription factors to generate either progenitor B cell or mixed B/myeloid-like leukemias.

Asunto(s)

Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Factores de Transcripción/genética , Transposasas/genética , Animales , Mutación con Ganancia de Función , Pruebas Genéticas , Humanos , Mutación con Pérdida de Función , Ratones , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Análisis de Secuencia de ARN , Transducción de Señal , Transactivadores/genética

RESUMEN

While macrophages are among the most abundant immune cell type found within primary and metastatic mammary tumors, how their complexity and heterogeneity change with metastatic progression remains unknown. Here, macrophages were isolated from the lungs of mice bearing orthotopic mammary tumors for single-cell RNA sequencing (scRNA-seq). Seven distinct macrophage clusters were identified, including populations exhibiting enhanced differential expression of genes related to antigen presentation (H2-Aa, Cd74), cell cycle (Stmn1, Cdk1), and interferon signaling (Isg15, Ifitm3). Interestingly, one cluster demonstrated a profile concordant with lipid-associated macrophages (Lgals3, Trem2). Compared with nontumor-bearing controls, the number of these cells per gram of tissue was significantly increased in lungs from tumor-bearing mice, with the vast majority costaining positively with the alveolar macrophage marker Siglec-F. Enrichment of genes implicated in pathways related to lipid metabolism as well extracellular matrix remodeling and immunosuppression was observed. In addition, these cells displayed reduced capacity for phagocytosis. Collectively, these findings highlight the diversity of macrophages present within metastatic lesions and characterize a lipid-associated macrophage subset previously unidentified in lung metastases. SIGNIFICANCE: scRNA-seq of macrophages isolated from lung metastases reveals extensive macrophage heterogeneity and identifies a novel subpopulation enriched for genes involved in lipid metabolism, extracellular matrix remodeling, and immunosuppression.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Vesículas Extracelulares/patología , Regulación Neoplásica de la Expresión Génica , Lípidos/química , Neoplasias Pulmonares/secundario , Macrófagos/inmunología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Proliferación Celular , Vesículas Extracelulares/metabolismo , Femenino , Humanos , Terapia de Inmunosupresión , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Macrófagos/clasificación , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , RNA-Seq , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The authors wish to make the following corrections to this paper [...].

RESUMEN

Multiple studies have identified transcriptome subtypes of high-grade serous ovarian carcinoma (HGSOC), but their interpretation and translation are complicated by tumor evolution and polyclonality accompanied by extensive accumulation of somatic aberrations, varying cell type admixtures, and different tissues of origin. In this study, we examined the chronology of HGSOC subtype evolution in the context of these factors using a novel integrative analysis of absolute copy-number analysis and gene expression in The Cancer Genome Atlas complemented by single-cell analysis of six independent tumors. Tumor purity, ploidy, and subclonality were reliably inferred from different genomic platforms, and these characteristics displayed marked differences between subtypes. Genomic lesions associated with HGSOC subtypes tended to be subclonal, implying subtype divergence at later stages of tumor evolution. Subclonality of recurrent HGSOC alterations was evident for proliferative tumors, characterized by extreme genomic instability, absence of immune infiltration, and greater patient age. In contrast, differentiated tumors were characterized by largely intact genome integrity, high immune infiltration, and younger patient age. Single-cell sequencing of 42,000 tumor cells revealed widespread heterogeneity in tumor cell type composition that drove bulk subtypes but demonstrated a lack of intrinsic subtypes among tumor epithelial cells. Our findings prompt the dismissal of discrete transcriptome subtypes for HGSOC and replacement by a more realistic model of continuous tumor development that includes mixtures of subclones, accumulation of somatic aberrations, infiltration of immune and stromal cells in proportions correlated with tumor stage and tissue of origin, and evolution between properties previously associated with discrete subtypes. SIGNIFICANCE: This study infers whether transcriptome-based groupings of tumors differentiate early in carcinogenesis and are, therefore, appropriate targets for therapy and demonstrates that this is not the case for HGSOC.

Asunto(s)

Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Femenino , Perfilación de la Expresión Génica , Inestabilidad Genómica , Humanos , Ploidias , Análisis de la Célula Individual

RESUMEN

Tissue-resident macrophages in the mammary gland are found in close association with epithelial structures and within the adipose stroma, and are important for mammary gland development and tissue homeostasis. Macrophages have been linked to ductal development in the virgin mammary gland, but less is known regarding the effects of macrophages on the adipose stroma. Using transcriptional profiling and single-cell RNA sequencing approaches, we identify a distinct resident stromal macrophage subpopulation within the mouse nulliparous mammary gland that is characterized by the expression of Lyve-1, a receptor for the extracellular matrix (ECM) component hyaluronan. This subpopulation is enriched in genes associated with ECM remodeling and is specifically associated with hyaluronan-rich regions within the adipose stroma and fibrous capsule of the virgin mammary gland. Furthermore, macrophage depletion leads to enhanced accumulation of hyaluronan-associated ECM in the adipose-associated stroma, indicating that resident macrophages are important for maintaining homeostasis within the nulliparous mammary gland stroma.

Asunto(s)

Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Glándulas Mamarias Animales/metabolismo , Tejido Adiposo/metabolismo , Animales , Matriz Extracelular/genética , Femenino , Homeostasis , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C

RESUMEN

Cancer has been conceptualized as a chronic wound with a predominance of tumor promoting inflammation. Given the accumulating evidence that the microenvironment supports tumor growth, we investigated hyaluronan (HA)-CD44 interactions within breast cancer cells, to determine whether this axis directly impacts the formation of an inflammatory microenvironment. Our results demonstrate that breast cancer cells synthesize and fragment HA and express CD44 on the cell surface. Using RNA sequencing approaches, we found that loss of CD44 in breast cancer cells altered the expression of cytokine-related genes. Specifically, we found that production of the chemokine CCL2 by breast cancer cells was significantly decreased after depletion of either CD44 or HA. In vivo, we found that CD44 deletion in breast cancer cells resulted in a delay in tumor formation and localized progression. This finding was accompanied by a decrease in infiltrating CD206+ macrophages, which are typically associated with tumor promoting functions. Importantly, our laboratory results were supported by human breast cancer patient data, where increased HAS2 expression was significantly associated with a tumor promoting inflammatory gene signature. Because high levels of HA deposition within many tumor types yields a poorer prognosis, our results emphasize that HA-CD44 interactions potentially have broad implications across multiple cancers.

RESUMEN

Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRASG12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSC-specific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg.

Asunto(s)

Proliferación Celular/genética , Autorrenovación de las Células/genética , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/citología , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Análisis de la Célula Individual/métodos , Animales , Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Células Madre Neoplásicas/metabolismo

RESUMEN

The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.

Asunto(s)

Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Células Progenitoras Linfoides/fisiología , Linfocitos T Reguladores/fisiología , Timo/crecimiento & desarrollo , Animales , Autoantígenos/inmunología , Colitis/inmunología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Adyuvante de Freund/administración & dosificación , Adyuvante de Freund/inmunología , Humanos , Tolerancia Inmunológica/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Células Progenitoras Linfoides/trasplante , Ratones , Ratones Transgénicos , Mycobacterium tuberculosis/inmunología , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Glicoproteína Mielina-Oligodendrócito/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Transducción de Señal , Organismos Libres de Patógenos Específicos , Timo/citología , Timo/inmunología

RESUMEN

Follicular lymphoma and diffuse large B-cell lymphoma (DLBCL) are the most common non-Hodgkin lymphomas distinguishable by unique mutations, chromosomal rearrangements, and gene expression patterns. Here, it is demonstrated that early B-cell progenitors express 2',3'-cyclic-nucleotide 3' phosphodiesterase (CNP) and that when targeted with Sleeping Beauty (SB) mutagenesis, Trp53R270H mutation or Pten loss gave rise to highly penetrant lymphoid diseases, predominantly follicular lymphoma and DLBCL. In efforts to identify the genetic drivers and signaling pathways that are functionally important in lymphomagenesis, SB transposon insertions were analyzed from splenomegaly specimens of SB-mutagenized mice (n = 23) and SB-mutagenized mice on a Trp53R270H background (n = 7) and identified 48 and 12 sites with statistically recurrent transposon insertion events, respectively. Comparison with human data sets revealed novel and known driver genes for B-cell development, disease, and signaling pathways: PI3K-AKT-mTOR, MAPK, NFκB, and B-cell receptor (BCR). Finally, functional data indicate that modulating Ras-responsive element-binding protein 1 (RREB1) expression in human DLBCL cell lines in vitro alters KRAS expression, signaling, and proliferation; thus, suggesting that this proto-oncogene is a common mechanism of RAS/MAPK hyperactivation in human DLBCL. IMPLICATIONS: A forward genetic screen identified new genetic drivers of human B-cell lymphoma and uncovered a RAS/MAPK-activating mechanism not previously appreciated in human lymphoid disease. Overall, these data support targeting the RAS/MAPK pathway as a viable therapeutic target in a subset of human patients with DLBCL.

Asunto(s)

Proteínas de Unión al ADN/genética , Linfoma de Células B Grandes Difuso/genética , Factores de Transcripción/genética , Animales , Línea Celular Tumoral , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Transgénicos , Mutagénesis Insercional , Mutación , Proto-Oncogenes Mas

RESUMEN

Overall survival of patients with osteosarcoma (OS) has improved little in the past three decades, and better models for study are needed. OS is common in large dog breeds and is genetically inducible in mice, making the disease ideal for comparative genomic analyses across species. Understanding the level of conservation of intertumor transcriptional variation across species and how it is associated with progression to metastasis will enable us to more efficiently develop effective strategies to manage OS and to improve therapy. In this study, transcriptional profiles of OS tumors and cell lines derived from humans (n = 49), mice (n = 103), and dogs (n = 34) were generated using RNA sequencing. Conserved intertumor transcriptional variation was present in tumor sets from all three species and comprised gene clusters associated with cell cycle and mitosis and with the presence or absence of immune cells. Further, we developed a novel gene cluster expression summary score (GCESS) to quantify intertumor transcriptional variation and demonstrated that these GCESS values associated with patient outcome. Human OS tumors with GCESS values suggesting decreased immune cell presence were associated with metastasis and poor survival. We validated these results in an independent human OS tumor cohort and in 15 different tumor data sets obtained from The Cancer Genome Atlas. Our results suggest that quantification of immune cell absence and tumor cell proliferation may better inform therapeutic decisions and improve overall survival for OS patients.Significance: This study offers new tools to quantify tumor heterogeneity in osteosarcoma, identifying potentially useful prognostic biomarkers for metastatic progression and survival in patients. Cancer Res; 78(2); 326-37. ©2017 AACR.

Asunto(s)

Biomarcadores de Tumor/genética , Neoplasias Óseas/mortalidad , Regulación Neoplásica de la Expresión Génica , Inmunidad Celular/genética , Osteosarcoma/mortalidad , Transcriptoma , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/secundario , Estudios de Casos y Controles , Perros , Perfilación de la Expresión Génica , Humanos , Ratones , Metástasis de la Neoplasia , Osteosarcoma/genética , Osteosarcoma/secundario , Pronóstico , Tasa de Supervivencia

RESUMEN

Pain is a hallmark feature of sickle cell disease (SCD). Recurrent and unpredictable acute pain due to vaso-oclussive crises (VOC) is unique to SCD; and can be superimposed on chronic pain. To examine the mechanisms underlying pain in SCD, we performed RNA sequencing of dorsal root ganglion (DRG) of transgenic sickle mice and their age-matched control mice expressing normal human hemoglobin A, at 2 and 5 months of age. Sickle and control mice of both ages were equally divided into hypoxia/reoxygenation (to simulate VOC) and normoxia treatment, resulting in eight groups of mice. Each group had at least six mice. RNA isolated from the DRG was sequenced and paired-end 50 bp sequencing data were generated using Illumina's HiSeq 2000. This large dataset can serve as a resource for examining transcriptional changes in the DRG that are associated with age and hypoxia/reoxygenation associated signatures of nociceptive mechanisms underlying chronic and acute pain, respectively.

Asunto(s)

Anemia de Células Falciformes/fisiopatología , Ganglios Espinales , Perfilación de la Expresión Génica , Dolor/genética , Anemia de Células Falciformes/genética , Animales , Humanos , Ratones , Ratones Transgénicos , ARN/genética

RESUMEN

BACKGROUND: Mutation studies of pancreatic ductal adenocarcinoma (PDA) have revealed complicated heterogeneous genomic landscapes of the disease. These studies cataloged a number of genes mutated at high frequencies, but also report a very large number of genes mutated in lower percentages of tumors. Taking advantage of a well-established forward genetic screening technique, with the Sleeping Beauty (SB) transposon, several studies produced PDA and discovered a number of common insertion sites (CIS) and associated genes that are recurrently mutated at high frequencies. As with human mutation studies, a very large number of genes were found to be altered by transposon insertion at low frequencies. These low frequency CIS associated genes may be very valuable to consider for their roles in cancer, since collectively they might emerge from a core group of genetic pathways. RESULT: In this paper, we determined whether the genetic mutations in SB-accelerated PDA occur within a collated group of biological processes defined as gene sets. The approach considered both genes mutated in high and lower frequencies. We implemented a case-oriented, gene set enrichment analysis (CO-GSEA) on SB altered genes in PDA. Compared to traditional GSEA, CO-GSEA enables us to consider individual characteristics of mutation profiles of each PDA tumor. We identified genetic pathways with higher numbers of genetic mutations than expected by chance. We also present the correlations between these significant enriched genetic pathways, and their associations with CIS genes. CONCLUSION: These data suggest that certain pathway alterations cooperate in PDA development.

Asunto(s)

Elementos Transponibles de ADN/genética , Pruebas Genéticas , Mutagénesis Insercional/genética , Neoplasias Pancreáticas/genética , Humanos , Mutación/genética , Proteolisis , Transducción de Señal/genética , Ubiquitina/metabolismo , Neoplasias Pancreáticas

RESUMEN

Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma.

Asunto(s)

Neoplasias Óseas/genética , Osteosarcoma/genética , Animales , Neoplasias Óseas/patología , Carcinogénesis/genética , Línea Celular Tumoral , Elementos Transponibles de ADN , Perros , Predisposición Genética a la Enfermedad , Inestabilidad Genómica , Humanos , Ratones Transgénicos , Mutagénesis Insercional , Osteosarcoma/secundario , Fosfohidrolasa PTEN/genética , Semaforinas/genética , Semaforinas/metabolismo , Proteína p53 Supresora de Tumor/genética

RESUMEN

Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRAS(G12V)). Using computational approaches to explore our gene-expression data sets, we found that NRAS(G12V) enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9- and Myb-dependent leukemia self-renewal gene-expression program. NRAS(G12V) was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRAS(G12V)-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell-enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1(Low) cells, which harbor leukemia stem cells, were preferentially sensitive to NRAS(G12V) withdrawal. NRAS(G12V) maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction.

Asunto(s)

Proliferación Celular/genética , GTP Fosfohidrolasas/fisiología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteínas de la Membrana/fisiología , Sustitución de Aminoácidos , Animales , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , GTP Fosfohidrolasas/genética , Regulación Leucémica de la Expresión Génica , Glicina/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones SCID , Oncogenes/fisiología , Transcriptoma , Células Tumorales Cultivadas , Valina/genética

RESUMEN

Cytotoxic T cells typically are expanded ex vivo in culture with IL2 for adoptive immunotherapy. This culture period leads to a differentiated phenotype and acquisition of effector function, as well as a loss of in vivo proliferative capability and antitumor efficacy. Here, we report antigen-specific and polyclonal expansion of cytotoxic T cells in a cocktail of cytokines and small molecules that leads to a memory-like phenotype in mouse and human cells even during extended culture, leading to enhanced in vivo expansion and tumor control in mice.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Memoria Inmunológica , Inmunoterapia Adoptiva , Linfocitos Infiltrantes de Tumor/inmunología , Animales , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Citocinas/inmunología , Epítopos , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias/terapia

RESUMEN

HIV-1 requires the cellular transcription factor CBFß to stabilize its accessory protein Vif and promote APOBEC3G degradation. Here, we demonstrate that both isoforms of CBFß allow for increased steady-state levels of Vif, enhanced APOBEC3G degradation, and increased viral infectivity. This conserved functional interaction enhances the steady-state levels of Vif proteins from multiple HIV-1 subtypes and is required for the degradation of all human and rhesus Vif-sensitive APOBEC3 proteins by their respective lentiviral Vif proteins.

Asunto(s)

Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Citidina Desaminasa/metabolismo , Productos del Gen vif/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Animales , Subunidad beta del Factor de Unión al Sitio Principal/genética , Citidina Desaminasa/genética , Productos del Gen vif/química , Productos del Gen vif/genética , Infecciones por VIH/enzimología , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , Humanos , Macaca mulatta , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad Proteica , Síndrome de Inmunodeficiencia Adquirida del Simio/enzimología , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/química , Virus de la Inmunodeficiencia de los Simios/genética

RESUMEN

Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication. The AIDS virus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host's antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation. Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-ß to this ubiquitin ligase complex. CBF-ß, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-ß is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-ß to degrade rhesus macaque APOBEC3G, indicating functional conservation. Methods of disrupting the CBF-ß-Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins.

Asunto(s)

Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Citidina Desaminasa/metabolismo , Productos del Gen vif/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Desaminasa APOBEC-3G , Marcadores de Afinidad , Animales , Proteínas Cullin/metabolismo , Técnicas de Silenciamiento del Gen , Prueba de Complementación Genética , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Células Jurkat , Macaca mulatta/metabolismo , Macaca mulatta/virología , Espectrometría de Masas , Modelos Biológicos , Unión Proteica , Proteolisis , Virus de la Inmunodeficiencia de los Simios/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Replicación Viral

RESUMEN

Successful intracellular pathogens must evade or neutralize the innate immune defenses of their host cells and render the cellular environment permissive for replication. For example, to replicate efficiently in CD4(+) T lymphocytes, human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral infectivity factor (Vif) that promotes pathogenesis by triggering the degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3 proteins have been implicated in HIV-1 restriction, but the relevant repertoire remains ambiguous. Here we present the first comprehensive analysis of the complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction. In addition to APOBEC3G, we find that three other human APOBEC3 proteins, APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors. These four proteins are expressed in CD4(+) T lymphocytes, are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, and they are all neutralized by the simian immunodeficiency virus Vif protein. On the other hand, neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant impact on HIV-1 replication. These data strongly implicate a combination of four APOBEC3 proteins--APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H--in HIV-1 restriction.

Asunto(s)

Citosina Desaminasa/inmunología , VIH-1/inmunología , VIH-1/patogenicidad , Factores de Virulencia/deficiencia , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/deficiencia , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Citosina Desaminasa/metabolismo , Humanos , Macaca mulatta , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad