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1.
Nat Commun ; 10(1): 4730, 2019 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-31628330

RESUMEN

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.


Asunto(s)
Sistemas CRISPR-Cas , Diferenciación Celular/genética , Proliferación Celular/genética , Edición Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Animales , Electroporación/métodos , Femenino , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Células Madre Hematopoyéticas/citología , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Trasplante Heterólogo
2.
Cell Stem Cell ; 25(5): 639-653.e7, 2019 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-31631013

RESUMEN

Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics.

3.
Blood ; 133(20): 2198-2211, 2019 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-30796022

RESUMEN

There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.


Asunto(s)
Regulación Leucémica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mieloide Aguda/genética , Células Madre Neoplásicas/metabolismo , Animales , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Ratones Endogámicos NOD , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/patología , Regulación hacia Arriba , Quinasas p21 Activadas/análisis
4.
Cell Rep ; 25(5): 1109-1117.e5, 2018 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-30380403

RESUMEN

Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Severe perturbations of cellular homeostasis result in rapid HSC loss to maintain clonal purity. However, normal homeostatic processes can also generate lower-level stress; how HSCs survive these conditions remains unknown. Here we show that the integrated stress response (ISR) is uniquely active in HSCs and facilitates their persistence. Activating transcription factor 4 (ATF4) mediates the ISR and is highly expressed in HSCs due to scarcity of the eIF2 translation initiation complex. Amino acid deprivation results in eIF2α phosphorylation-dependent upregulation of ATF4, promoting HSC survival. Primitive acute myeloid leukemia (AML) cells also display eIF2 scarcity and ISR activity marks leukemia stem cells (LSCs) in primary AML samples. These findings identify a link between the ISR and stem cell survival in the normal and leukemic contexts.


Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Leucemia/metabolismo , Estrés Fisiológico , Factor de Transcripción Activador 4/metabolismo , Animales , Supervivencia Celular , Citoprotección , Factor 2 Eucariótico de Iniciación/metabolismo , Sangre Fetal/citología , Genes Reporteros , Humanos , Masculino , Ratones Endogámicos NOD , Ratones SCID , Células Madre Multipotentes/metabolismo , Fosforilación , Regulación hacia Arriba , Valina/deficiencia
5.
PLoS Biol ; 16(6): e2004049, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29924804

RESUMEN

The contribution of basal and luminal cells to cancer progression and metastasis is poorly understood. We report generation of reporter systems driven by either keratin-14 (K14) or keratin-8 (K8) promoter that not only express a fluorescent protein but also an inducible suicide gene. Transgenic mice express the reporter genes in the right cell compartments of mammary gland epithelia and respond to treatment with toxins. In addition, we engineered the reporters into 4T1 metastatic mouse tumor cell line and demonstrate that K14+ cells, but not K14- or K8+, are both highly invasive in three-dimensional (3D) culture and metastatic in vivo. Treatment of cells in culture, or tumors in mice, with reporter-targeting toxin inhibited both invasive behavior and metastasis in vivo. RNA sequencing (RNA-seq), secretome, and epigenome analysis of K14+ and K14- cells led to the identification of amphoterin-induced protein 2 (Amigo2) as a new cell invasion driver whose expression correlated with decreased relapse-free survival in patients with TP53 wild-type (WT) breast cancer.


Asunto(s)
Genes Reporteros/genética , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Animales , División Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Células Epiteliales/patología , Femenino , Proteínas Fluorescentes Verdes/genética , Queratina-14/genética , Queratina-8/genética , Glándulas Mamarias Animales/citología , Neoplasias Mamarias Animales/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Metástasis de la Neoplasia/patología , Regiones Promotoras Genéticas/genética
7.
Nature ; 554(7692): 378-381, 2018 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-29414946

RESUMEN

Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites. A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient's primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l-asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.


Asunto(s)
Asparagina/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Metástasis de la Neoplasia/patología , Animales , Asparaginasa/metabolismo , Asparaginasa/uso terapéutico , Asparagina/deficiencia , Aspartatoamoníaco Ligasa/genética , Aspartatoamoníaco Ligasa/metabolismo , Disponibilidad Biológica , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Masculino , Ratones , Invasividad Neoplásica/patología , Pronóstico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Interferencia de ARN , Reproducibilidad de los Resultados
8.
Nature ; 520(7547): 358-62, 2015 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-25855289

RESUMEN

Cancer metastasis requires that primary tumour cells evolve the capacity to intravasate into the lymphatic system or vasculature, and extravasate into and colonize secondary sites. Others have demonstrated that individual cells within complex populations show heterogeneity in their capacity to form secondary lesions. Here we develop a polyclonal mouse model of breast tumour heterogeneity, and show that distinct clones within a mixed population display specialization, for example, dominating the primary tumour, contributing to metastatic populations, or showing tropism for entering the lymphatic or vasculature systems. We correlate these stable properties to distinct gene expression profiles. Those clones that efficiently enter the vasculature express two secreted proteins, Serpine2 and Slpi, which were necessary and sufficient to program these cells for vascular mimicry. Our data indicate that these proteins not only drive the formation of extravascular networks but also ensure their perfusion by acting as anticoagulants. We propose that vascular mimicry drives the ability of some breast tumour cells to contribute to distant metastases while simultaneously satisfying a critical need of the primary tumour to be fed by the vasculature. Enforced expression of SERPINE2 and SLPI in human breast cancer cell lines also programmed them for vascular mimicry, and SERPINE2 and SLPI were overexpressed preferentially in human patients that had lung-metastatic relapse. Thus, these two secreted proteins, and the phenotype they promote, may be broadly relevant as drivers of metastatic progression in human cancer.


Asunto(s)
Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/patología , Endotelio Vascular/patología , Metástasis de la Neoplasia/patología , Animales , Anticoagulantes/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Células Clonales/metabolismo , Células Clonales/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Metástasis de la Neoplasia/genética , Recurrencia , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Análisis de Secuencia de ADN , Serpina E2/metabolismo
9.
Mol Cell ; 56(6): 796-807, 2014 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-25435137

RESUMEN

The strength of conclusions drawn from RNAi-based studies is heavily influenced by the quality of tools used to elicit knockdown. Prior studies have developed algorithms to design siRNAs. However, to date, no established method has emerged to identify effective shRNAs, which have lower intracellular abundance than transfected siRNAs and undergo additional processing steps. We recently developed a multiplexed assay for identifying potent shRNAs and used this method to generate ∼250,000 shRNA efficacy data points. Using these data, we developed shERWOOD, an algorithm capable of predicting, for any shRNA, the likelihood that it will elicit potent target knockdown. Combined with additional shRNA design strategies, shERWOOD allows the ab initio identification of potent shRNAs that specifically target the majority of each gene's multiple transcripts. We validated the performance of our shRNA designs using several orthogonal strategies and constructed genome-wide collections of shRNAs for humans and mice based on our approach.


Asunto(s)
ARN Interferente Pequeño/genética , Programas Informáticos , Algoritmos , Secuencia de Bases , Línea Celular Tumoral , Simulación por Computador , Secuencia de Consenso , Técnicas de Silenciamiento del Gen , Humanos , MicroARNs/genética , Modelos Genéticos , Datos de Secuencia Molecular
10.
Cell ; 157(2): 382-394, 2014 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-24725405

RESUMEN

Missense mutations in the p53 tumor suppressor inactivate its antiproliferative properties but can also promote metastasis through a gain-of-function activity. We show that sustained expression of mutant p53 is required to maintain the prometastatic phenotype of a murine model of pancreatic cancer, a highly metastatic disease that frequently displays p53 mutations. Transcriptional profiling and functional screening identified the platelet-derived growth factor receptor b (PDGFRb) as both necessary and sufficient to mediate these effects. Mutant p53 induced PDGFRb through a cell-autonomous mechanism involving inhibition of a p73/NF-Y complex that represses PDGFRb expression in p53-deficient, noninvasive cells. Blocking PDGFRb signaling by RNA interference or by small molecule inhibitors prevented pancreatic cancer cell invasion in vitro and metastasis formation in vivo. Finally, high PDGFRb expression correlates with poor disease-free survival in pancreatic, colon, and ovarian cancer patients, implicating PDGFRb as a prognostic marker and possible target for attenuating metastasis in p53 mutant tumors.


Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Metástasis de la Neoplasia , Neoplasias Pancreáticas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Carcinoma Ductal Pancreático/patología , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteína p53 Supresora de Tumor/genética
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