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1.
Cell ; 186(7): 1295-1299, 2023 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-37001493

RESUMO

The transition to independence requires shared enthusiasm for one's research goals from broad audiences. In this commentary, we describe the use of "research vision workshopping" within peer mentoring networks. We contend that this approach is broadly useful for the development and refinement of research visions for the academic job search.


Assuntos
Tutoria , Humanos , Mentores , Grupo Associado
2.
Genes Dev ; 36(15-16): 936-949, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-36175034

RESUMO

Lung cancer is the leading cause of cancer-related death worldwide. Lung adenocarcinoma (LUAD), the most common histological subtype, accounts for 40% of all cases. While existing genetically engineered mouse models (GEMMs) recapitulate the histological progression and transcriptional evolution of human LUAD, they are time-consuming and technically demanding. In contrast, cell line transplant models are fast and flexible, but these models fail to capture the full spectrum of disease progression. Organoid technologies provide a means to create next-generation cancer models that integrate the most advantageous features of autochthonous and transplant-based systems. However, robust and faithful LUAD organoid platforms are currently lacking. Here, we describe optimized conditions to continuously expand murine alveolar type 2 (AT2) cells, a prominent cell of origin for LUAD, in organoid culture. These organoids display canonical features of AT2 cells, including marker gene expression, the presence of lamellar bodies, and an ability to differentiate into the AT1 lineage. We used this system to develop flexible and versatile immunocompetent organoid-based models of KRAS, BRAF, and ALK mutant LUAD. Notably, organoid-based tumors display extensive burden and complete penetrance and are histopathologically indistinguishable from their autochthonous counterparts. Altogether, this organoid platform is a powerful, versatile new model system to study LUAD.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Organoides , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo
3.
Cancer Discov ; 12(2): 562-585, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34561242

RESUMO

SMARCA4/BRG1 encodes for one of two mutually exclusive ATPases present in mammalian SWI/SNF chromatin remodeling complexes and is frequently mutated in human lung adenocarcinoma. However, the functional consequences of SMARCA4 mutation on tumor initiation, progression, and chromatin regulation in lung cancer remain poorly understood. Here, we demonstrate that loss of Smarca4 sensitizes club cell secretory protein-positive cells within the lung in a cell type-dependent fashion to malignant transformation and tumor progression, resulting in highly advanced dedifferentiated tumors and increased metastatic incidence. Consistent with these phenotypes, Smarca4-deficient primary tumors lack lung lineage transcription factor activities and resemble a metastatic cell state. Mechanistically, we show that Smarca4 loss impairs the function of all three classes of SWI/SNF complexes, resulting in decreased chromatin accessibility at lung lineage motifs and ultimately accelerating tumor progression. Thus, we propose that the SWI/SNF complex via Smarca4 acts as a gatekeeper for lineage-specific cellular transformation and metastasis during lung cancer evolution. SIGNIFICANCE: We demonstrate cell-type specificity in the tumor-suppressive functions of SMARCA4 in the lung, pointing toward a critical role of the cell-of-origin in driving SWI/SNF-mutant lung adenocarcinoma. We further show the direct effects of SMARCA4 loss on SWI/SNF function and chromatin regulation that cause aggressive malignancy during lung cancer evolution.This article is highlighted in the In This Issue feature, p. 275.


Assuntos
Adenocarcinoma de Pulmão/genética , Transformação Celular Neoplásica , DNA Helicases/genética , Neoplasias Pulmonares/genética , Metástase Neoplásica , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adenocarcinoma de Pulmão/secundário , Animais , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/patologia , Camundongos
4.
Nature ; 601(7891): 85-91, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34912115

RESUMO

The state and behaviour of a cell can be influenced by both genetic and environmental factors. In particular, tumour progression is determined by underlying genetic aberrations1-4 as well as the makeup of the tumour microenvironment5,6. Quantifying the contributions of these factors requires new technologies that can accurately measure the spatial location of genomic sequence together with phenotypic readouts. Here we developed slide-DNA-seq, a method for capturing spatially resolved DNA sequences from intact tissue sections. We demonstrate that this method accurately preserves local tumour architecture and enables the de novo discovery of distinct tumour clones and their copy number alterations. We then apply slide-DNA-seq to a mouse model of metastasis and a primary human cancer, revealing that clonal populations are confined to distinct spatial regions. Moreover, through integration with spatial transcriptomics, we uncover distinct sets of genes that are associated with clone-specific genetic aberrations, the local tumour microenvironment, or both. Together, this multi-modal spatial genomics approach provides a versatile platform for quantifying how cell-intrinsic and cell-extrinsic factors contribute to gene expression, protein abundance and other cellular phenotypes.


Assuntos
Células Clonais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Genômica/métodos , Animais , Células Clonais/patologia , Variações do Número de Cópias de DNA/genética , Humanos , Camundongos , Fenótipo , RNA-Seq , Análise de Sequência de DNA , Transcrição Gênica , Transcriptoma
5.
STAR Protoc ; 2(2): 100583, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34142101

RESUMO

Single-cell ATAC sequencing using combinatorial indexing (sciATAC-seq) enables the identification of chromatin accessibility profiles at single-cell resolution with a dual-barcoding approach during transposition and library construction. Unlike commercial droplet-based approaches, sciATAC-seq is a cost-effective, extensible strategy that permits flexibility in the experimental scale via multiplexed barcoding across samples or perturbations. In contrast, droplet-based approaches have higher cell recovery and may be advantageous when cell input is limited. The step-by-step sciATAC-seq protocol described here is amenable to diverse cell types and fixed samples. For complete details on the use and execution of this protocol, please refer to LaFave et al. (2020).


Assuntos
Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/genética , Análise de Célula Única/métodos , Animais , Biologia Computacional/métodos , Epigênese Genética , Camundongos
7.
Cell ; 183(4): 1103-1116.e20, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33098772

RESUMO

Cell differentiation and function are regulated across multiple layers of gene regulation, including modulation of gene expression by changes in chromatin accessibility. However, differentiation is an asynchronous process precluding a temporal understanding of regulatory events leading to cell fate commitment. Here we developed simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq), a highly scalable approach for measurement of chromatin accessibility and gene expression in the same single cell, applicable to different tissues. Using 34,774 joint profiles from mouse skin, we develop a computational strategy to identify cis-regulatory interactions and define domains of regulatory chromatin (DORCs) that significantly overlap with super-enhancers. During lineage commitment, chromatin accessibility at DORCs precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for lineage commitment. We computationally infer chromatin potential as a quantitative measure of chromatin lineage-priming and use it to predict cell fate outcomes. SHARE-seq is an extensible platform to study regulatory circuitry across diverse cells in tissues.


Assuntos
Cromatina/metabolismo , Perfilação da Expressão Gênica , RNA/genética , Análise de Célula Única , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Elementos Facilitadores Genéticos/genética , Feminino , Regulação da Expressão Gênica , Histonas/metabolismo , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , RNA/metabolismo
8.
Cancer Cell ; 38(2): 212-228.e13, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32707078

RESUMO

Regulatory networks that maintain functional, differentiated cell states are often dysregulated in tumor development. Here, we use single-cell epigenomics to profile chromatin state transitions in a mouse model of lung adenocarcinoma (LUAD). We identify an epigenomic continuum representing loss of cellular identity and progression toward a metastatic state. We define co-accessible regulatory programs and infer key activating and repressive chromatin regulators of these cell states. Among these co-accessibility programs, we identify a pre-metastatic transition, characterized by activation of RUNX transcription factors, which mediates extracellular matrix remodeling to promote metastasis and is predictive of survival across human LUAD patients. Together, these results demonstrate the power of single-cell epigenomics to identify regulatory programs to uncover mechanisms and key biomarkers of tumor progression.


Assuntos
Adenocarcinoma/genética , Modelos Animais de Doenças , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Progressão da Doença , Perfilação da Expressão Gênica/métodos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais/genética , Análise de Célula Única/métodos
11.
Nat Med ; 21(11): 1344-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26437366

RESUMO

The tumor suppressors BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). However, BAP1 and ASXL1 are mutated in distinct cancer types, consistent with independent roles in regulating epigenetic state and malignant transformation. Here we demonstrate that Bap1 loss in mice results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) expression, and enhanced repression of polycomb repressive complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 levels seen with Asxl1 loss. Conditional deletion of Bap1 and Ezh2 in vivo abrogates the myeloid progenitor expansion induced by Bap1 loss alone. Loss of BAP1 results in a marked decrease in H4K20 monomethylation (H4K20me1). Consistent with a role for H4K20me1 in the transcriptional regulation of EZH2, expression of SETD8-the H4K20me1 methyltransferase-reduces EZH2 expression and abrogates the proliferation of BAP1-mutant cells. Furthermore, mesothelioma cells that lack BAP1 are sensitive to EZH2 pharmacologic inhibition, suggesting a novel therapeutic approach for BAP1-mutant malignancies.


Assuntos
Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética , Técnicas de Inativação de Genes , Células HEK293 , Código das Histonas , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imunoprecipitação , Leucemia/genética , Mesotelioma/genética , Metilação , Camundongos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo
12.
Blood ; 124(14): 2280-4, 2014 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-25115888

RESUMO

JAK inhibitor treatment is limited by the variable development of anemia and thrombocytopenia thought to be due to on-target JAK2 inhibition. We evaluated the impact of Jak2 deletion in platelets (PLTs) and megakaryocytes (MKs) on blood counts, stem/progenitor cells, and Jak-Stat signaling. Pf4-Cre-mediated Jak2 deletion in PLTs and MKs did not compromise PLT formation but caused thrombocytosis, and resulted in expansion of MK progenitors and Lin(-)Sca1(+)Kit+ cells. Serum thrombopoietin (TPO) was maintained at normal levels in Pf4-Cre-positive Jak2(f/f) mice, consistent with reduced internalization/turnover by Jak2-deficient PLTs. These data demonstrate that Jak2 in terminal megakaryopoiesis is not required for PLT production, and that Jak2 loss in PLTs and MKs results in non-autonomous expansion of stem/progenitors and of MKs and PLTs via dysregulated TPO turnover. This suggests that the thrombocytopenia frequently seen with JAK inhibitor treatment is not due to JAK2 inhibition in PLTs and MKs, but rather due to JAK2 inhibition in stem/progenitor cells.


Assuntos
Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Trombocitose/metabolismo , Trombopoese/fisiologia , Animais , Plaquetas/citologia , Cruzamentos Genéticos , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Camundongos , Transdução de Sinais , Células-Tronco/citologia , Trombopoetina/sangue , Trombopoetina/metabolismo
13.
J Exp Med ; 210(12): 2641-59, 2013 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-24218140

RESUMO

Somatic Addition of Sex Combs Like 1 (ASXL1) mutations occur in 10-30% of patients with myeloid malignancies, most commonly in myelodysplastic syndromes (MDSs), and are associated with adverse outcome. Germline ASXL1 mutations occur in patients with Bohring-Opitz syndrome. Here, we show that constitutive loss of Asxl1 results in developmental abnormalities, including anophthalmia, microcephaly, cleft palates, and mandibular malformations. In contrast, hematopoietic-specific deletion of Asxl1 results in progressive, multilineage cytopenias and dysplasia in the context of increased numbers of hematopoietic stem/progenitor cells, characteristic features of human MDS. Serial transplantation of Asxl1-null hematopoietic cells results in a lethal myeloid disorder at a shorter latency than primary Asxl1 knockout (KO) mice. Asxl1 deletion reduces hematopoietic stem cell self-renewal, which is restored by concomitant deletion of Tet2, a gene commonly co-mutated with ASXL1 in MDS patients. Moreover, compound Asxl1/Tet2 deletion results in an MDS phenotype with hastened death compared with single-gene KO mice. Asxl1 loss results in a global reduction of H3K27 trimethylation and dysregulated expression of known regulators of hematopoiesis. RNA-Seq/ChIP-Seq analyses of Asxl1 in hematopoietic cells identify a subset of differentially expressed genes as direct targets of Asxl1. These findings underscore the importance of Asxl1 in Polycomb group function, development, and hematopoiesis.


Assuntos
Anormalidades Múltiplas/etiologia , Síndromes Mielodisplásicas/etiologia , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Anormalidades Múltiplas/genética , Animais , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Dioxigenases , Modelos Animais de Doenças , Epigênese Genética , Feminino , Deleção de Genes , Mutação em Linhagem Germinativa , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Fenótipo , Gravidez , Ligação Proteica , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/metabolismo
14.
Nat Genet ; 45(11): 1269-70, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24165727

RESUMO

The significance of epigenomic aberrations in cancer development has been underscored by the discovery of mutations in key chromatin modifiers, most notably in hematological malignancies. A new study of pediatric acute lymphoblastic leukemia (ALL) demonstrates the usefulness of mapping global epigenetic signatures and applying these data in a framework to identify and characterize underlying somatic genetic alterations in human cancers.


Assuntos
Cromatina/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Repressoras/genética , Animais , Feminino , Humanos
15.
Trends Pharmacol Sci ; 33(11): 574-82, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22995223

RESUMO

The Janus kinase (JAK) proteins are a family of intracellular nonreceptor tyrosine kinases involved in cytokine signaling via the JAK-STAT (signal transducers and activators of transcription) pathway. Genetic studies have identified somatic JAK2(V617F) mutations and other mutant alleles that activate JAK-STAT signaling in most patients with myeloproliferative neoplasms (MPNs). As a result, JAK inhibitors have been developed to treat various malignancies and have been shown to be efficacious in both preclinical and clinical settings. However, available ATP-competitive JAK (type I) inhibitors are associated with dose-dependent toxicities, and do not yet reduce disease burden in MPN patients. Recent studies suggest that genetic and epigenetic mechanisms can cause insensitivity to type I JAK inhibitors. Novel therapies include the development of type II JAK inhibitors and the use of alternative strategies to abrogate JAK-STAT signaling, perhaps with histone deacetylase (HDAC) and heat shock protein 90 (HSP90) inhibitors. These innovative therapies may translate to treatment of other diseases that are dependent on JAK signaling, including B-precursor acute lymphoblastic leukemia (B-ALL).


Assuntos
Janus Quinases/antagonistas & inibidores , Transtornos Mieloproliferativos/tratamento farmacológico , Animais , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais
16.
Science ; 337(6101): 1541-6, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22878500

RESUMO

De-ubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knockin mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with host cell factor-1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mice and humans.


Assuntos
Transformação Celular Neoplásica , Genes Supressores de Tumor , Leucemia Mielomonocítica Crônica/genética , Síndromes Mielodisplásicas/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Animais , Transplante de Medula Óssea , Imunoprecipitação da Cromatina , Desenvolvimento Embrionário , Deleção de Genes , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Hematopoese , Fator C1 de Célula Hospedeira/metabolismo , Humanos , Leucemia Mielomonocítica Crônica/metabolismo , Leucemia Mielomonocítica Crônica/patologia , Camundongos , Camundongos Knockout , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Células Mieloides/citologia , Células Mieloides/fisiologia , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/fisiologia , N-Acetilglucosaminiltransferases/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo
17.
Cancer Cell ; 22(2): 180-93, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22897849

RESUMO

Recurrent somatic ASXL1 mutations occur in patients with myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia, and are associated with adverse outcome. Despite the genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here, we identify that ASXL1 mutations result in loss of polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data, we identify targets of ASXL1 repression, including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the PRC2, and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis.


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Mutação/genética , Células Mieloides/patologia , Proteínas Repressoras/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Sistema Hematopoético/metabolismo , Sistema Hematopoético/patologia , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Metilação , Camundongos , Células Mieloides/metabolismo , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Ligação Proteica , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Regulação para Cima/genética , Proteínas ras/metabolismo
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