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1.
Cell ; 185(11): 1905-1923.e25, 2022 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-35523183

RESUMO

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.


Assuntos
Neoplasias , Animais , Genes ras , Camundongos , Neoplasias/genética , Filogenia , Sequenciamento do Exoma
2.
Nature ; 627(8003): 389-398, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38253266

RESUMO

The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived haematopoietic stem cells (HSCs)1. Perturbations to this process underlie diverse diseases, but the clonal contributions to human haematopoiesis and how this changes with age remain incompletely understood. Although recent insights have emerged from barcoding studies in model systems2-5, simultaneous detection of cell states and phylogenies from natural barcodes in humans remains challenging. Here we introduce an improved, single-cell lineage-tracing system based on deep detection of naturally occurring mitochondrial DNA mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs and map the physiological state and output of clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as both differences in total HSC output and biases towards the production of different mature cell types. We also find that the diversity of HSC clones decreases markedly with age, leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides a clonally resolved and cell-state-aware atlas of human haematopoiesis at single-cell resolution, showing an unappreciated functional diversity of human HSC clones and, more broadly, paving the way for refined studies of clonal dynamics across a range of tissues in human health and disease.


Assuntos
Linhagem da Célula , Hematopoese , Células-Tronco Hematopoéticas , Humanos , Cromatina/genética , Cromatina/metabolismo , Células Clonais/classificação , Células Clonais/citologia , Células Clonais/metabolismo , DNA Mitocondrial/genética , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Mutação , Análise de Célula Única , Transcrição Gênica , Envelhecimento
3.
Nature ; 607(7917): 149-155, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35705813

RESUMO

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1-5. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.


Assuntos
Antígenos de Neoplasias , Peptídeos , Proteômica , Células Epiteliais Alveolares/imunologia , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/química , Carcinoma Ductal Pancreático/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Neoplasias Pulmonares/química , Neoplasias Pulmonares/imunologia , Camundongos , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/imunologia , Peptídeos/análise , Peptídeos/química , Peptídeos/imunologia , RNA Mensageiro
4.
Proc Natl Acad Sci U S A ; 117(1): 513-521, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871154

RESUMO

Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified many recurrently mutated genes in human SCLC tumors. However, the functional roles of most of these genes remain to be validated. Here, we have adapted the CRISPR-Cas9 system to a well-established murine model of SCLC to rapidly model loss-of-function mutations in candidate genes identified from SCLC sequencing studies. We show that loss of the gene p107 significantly accelerates tumor progression. Notably, compared with loss of the closely related gene p130, loss of p107 results in fewer but larger tumors as well as earlier metastatic spread. In addition, we observe differences in proliferation and apoptosis as well as altered distribution of initiated tumors in the lung, resulting from loss of p107 or p130 Collectively, these data demonstrate the feasibility of using the CRISPR-Cas9 system to model loss of candidate tumor suppressor genes in SCLC, and we anticipate that this approach will facilitate efforts to investigate mechanisms driving tumor progression in this deadly disease.


Assuntos
Edição de Genes/métodos , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/genética , Animais , Apoptose/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Proliferação de Células/genética , Modelos Animais de Doenças , Progressão da Doença , Estudos de Viabilidade , Humanos , Mutação com Perda de Função , Pulmão/patologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Estadiamento de Neoplasias , Proteína p107 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Carga Tumoral/genética , Proteína Supressora de Tumor p53/genética
5.
Proc Natl Acad Sci U S A ; 112(11): E1288-96, 2015 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-25737542

RESUMO

BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.


Assuntos
Compostos de Anilina/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Sulfonamidas/uso terapêutico , Compostos de Anilina/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Engenharia Genética , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Proteínas de Membrana/metabolismo , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Indução de Remissão , Carcinoma de Pequenas Células do Pulmão/patologia , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nat Biotechnol ; 42(3): 424-436, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37169967

RESUMO

Genetically engineered mouse models only capture a small fraction of the genetic lesions that drive human cancer. Current CRISPR-Cas9 models can expand this fraction but are limited by their reliance on error-prone DNA repair. Here we develop a system for in vivo prime editing by encoding a Cre-inducible prime editor in the mouse germline. This model allows rapid, precise engineering of a wide range of mutations in cell lines and organoids derived from primary tissues, including a clinically relevant Kras mutation associated with drug resistance and Trp53 hotspot mutations commonly observed in pancreatic cancer. With this system, we demonstrate somatic prime editing in vivo using lipid nanoparticles, and we model lung and pancreatic cancer through viral delivery of prime editing guide RNAs or orthotopic transplantation of prime-edited organoids. We believe that this approach will accelerate functional studies of cancer-associated mutations and complex genetic combinations that are challenging to construct with traditional models.


Assuntos
Neoplasias Pancreáticas , RNA Guia de Sistemas CRISPR-Cas , Camundongos , Humanos , Animais , Camundongos Transgênicos , Mutação/genética , Neoplasias Pancreáticas/genética , Linhagem Celular , Edição de Genes , Sistemas CRISPR-Cas/genética
7.
Cancer Cell ; 8(4): 275-85, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16226703

RESUMO

Loss of imprinting (LOI), commonly observed in human tumors, refers to loss of monoallelic gene regulation normally conferred by parent-of-origin-specific DNA methylation. To test the function of LOI in tumorigenesis, we developed a model by using transient demethylation to generate imprint-free mouse embryonic stem cells (IF-ES cells). Embryonic fibroblasts derived from IF-ES cells (IF-MEFs) display TGFbeta resistance and reduced p19 and p53 expression and form tumors in SCID mice. IF-MEFs exhibit spontaneous immortalization and cooperate with H-Ras in cellular transformation. Chimeric animals derived from IF-ES cells develop multiple tumors arising from the injected IF-ES cells within 12 months. These data demonstrate that LOI alone can predispose cells to tumorigenesis and identify a pathway through which immortality conferred by LOI lowers the threshold for transformation.


Assuntos
Impressão Genômica , Neoplasias Experimentais/patologia , Animais , Sequência de Bases , Linhagem Celular Transformada , Metilação de DNA , Primers do DNA , Mutação em Linhagem Germinativa , Camundongos , Neoplasias Experimentais/genética , Reação em Cadeia da Polimerase
8.
Nat Genet ; 55(10): 1686-1695, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37709863

RESUMO

DNA mismatch repair deficiency (MMRd) is associated with a high tumor mutational burden (TMB) and sensitivity to immune checkpoint blockade (ICB) therapy. Nevertheless, most MMRd tumors do not durably respond to ICB and critical questions remain about immunosurveillance and TMB in these tumors. In the present study, we developed autochthonous mouse models of MMRd lung and colon cancer. Surprisingly, these models did not display increased T cell infiltration or ICB response, which we showed to be the result of substantial intratumor heterogeneity of mutations. Furthermore, we found that immunosurveillance shapes the clonal architecture but not the overall burden of neoantigens, and T cell responses against subclonal neoantigens are blunted. Finally, we showed that clonal, but not subclonal, neoantigen burden predicts ICB response in clinical trials of MMRd gastric and colorectal cancer. These results provide important context for understanding immune evasion in cancers with a high TMB and have major implications for therapies aimed at increasing TMB.


Assuntos
Neoplasias Encefálicas , Neoplasias Colorretais , Síndromes Neoplásicas Hereditárias , Animais , Camundongos , Neoplasias Colorretais/genética , Antígenos de Neoplasias/genética , Mutação , Reparo de Erro de Pareamento de DNA/genética , Biomarcadores Tumorais/genética
9.
Cancer Discov ; 11(12): 3214-3229, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34344693

RESUMO

Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf-Mycl-driven mouse model of SCLC. RLF-MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF-MYCL genetically engineered mouse model displayed gene expression similarities with human RLF-MYCL SCLC. Together, our studies support RLF-MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. SIGNIFICANCE: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF-MYCL gene fusion by developing a Rlf-Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis. This article is highlighted in the In This Issue feature, p. 2945.


Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Fusão Gênica , Genes myc , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Proto-Oncogênicas c-myc , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Proteínas de Ligação a Telômeros
10.
Cancer Cell ; 39(10): 1342-1360.e14, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34358448

RESUMO

The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.


Assuntos
Evasão da Resposta Imune/imunologia , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Pancreáticas/imunologia , Receptores Virais/imunologia , Animais , Humanos , Camundongos , Neoplasias Pancreáticas
11.
Sci Rep ; 6: 16836, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26887506

RESUMO

Traditional cloning methods have limitations on the number of DNA fragments that can be simultaneously manipulated, which dramatically slows the pace of molecular assembly. Here we describe GMAP, a Gibson assembly-based modular assembly platform consisting of a collection of promoters and genes, which allows for one-step production of DNA constructs. GMAP facilitates rapid assembly of expression and viral constructs using modular genetic components, as well as increasingly complicated genetic tools using contextually relevant genomic elements. Our data demonstrate the applicability of GMAP toward the validation of synthetic promoters, identification of potent RNAi constructs, establishment of inducible lentiviral systems, tumor initiation in genetically engineered mouse models, and gene-targeting for the generation of knock-in mice. GMAP represents a recombinant DNA technology designed for widespread circulation and easy adaptation for other uses, such as synthetic biology, genetic screens, and CRISPR-Cas9.


Assuntos
DNA/genética , Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Engenharia Genética/métodos , Regiões Promotoras Genéticas , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Knockout
12.
Hematol J ; 5 Suppl 3: S114-7, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15190291

RESUMO

Nuclear transfer experiments in mammals have shown that the nucleus of an adult cell has the ability to direct the development of an entire organism, id est its genome is totipotent. However, these experiments did not conclusively demonstrate that the nuclei of terminally differentiated adult cells remain totipotent. It is possible that rare adult stem cells served as donors for the few surviving clones. To address this question, we have generated monoclonal mice from terminally differentiated lymphocytes that carry a single antigen receptor rearrangement in all tissues. Nuclear transfer technology may provide a powerful method for obtaining autologous cells for replacement therapy. We have demonstrated the feasibility of this concept by combining nuclear transfer with gene and cell therapy to treat the immune deficiency of Rag2 mutant mice, thus establishing a paradigm for 'therapeutic cloning'. Moreover, we will discuss the potential use of nuclear transfer to study the role of reversible genomic (epigenetic) modifications during tumorigenesis.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Técnicas de Transferência Nuclear , Transplante de Células-Tronco , Animais , Linfócitos B/citologia , Diferenciação Celular , Clonagem de Organismos , Proteínas de Ligação a DNA/deficiência , Epigênese Genética , Terapia Genética/métodos , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/terapia , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/terapia , Proteínas Nucleares , Linfócitos T/citologia , Transplante Autólogo
13.
Cancer Res ; 74(22): 6598-609, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25217525

RESUMO

Cell-based drug screenings indicate that tumors displaying c-MET gene amplification are "addicted" to MET signaling and therefore are very sensitive to MET-targeted agents. However, these screenings were conducted in the absence of the MET ligand, hepatocyte growth factor (HGF), which is abundant in the tumor microenvironment. Sensitivity of six MET-addicted human tumor cells to three MET kinase inhibitors (JNJ-38877605, PHA-665752, crizotinib) and one antagonistic anti-MET antibody (DN30 Fab) was analyzed in the absence or presence of HGF, in a stroma-tumor coculture system, and by combining anti-MET drugs with an HGF neutralizing antibody (ficlatuzumab) in human HGF knock-in mice bearing c-MET-amplified tumors. In all models examined, HGF promoted resistance to MET-targeted agents, affecting both their potency and efficacy. HGF-induced resistance was due to restoration of physiologic GAB1-mediated PI3K activation that compensated for loss of aberrant HER3-dependent PI3K signaling. Ficlatuzumab restored sensitivity to MET-targeted agents in coculture systems and overcame resistance to JNJ-38877605, crizotinib, and DN30 Fab in human HGF knock-in mice. These data suggest that c-MET-amplified tumor cells-which normally exhibit ligand-independent, constitutive MET activation-become dependent on HGF for survival upon pharmacologic MET inhibition. Because HGF is frequently overexpressed in human cancer, this mechanism may represent a major cause of resistance to anti-MET therapies. The ability of ficlatuzumab to overcome HGF-mediated resistance generates proof of principle that vertical inhibition of both a tyrosine kinase receptor and its ligand can be therapeutically beneficial and opens new perspectives for the treatment of MET-dependent tumors.


Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Microambiente Tumoral , Animais , Anticorpos Monoclonais/farmacologia , Camundongos , Camundongos SCID , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-met/fisiologia , Receptor ErbB-3/fisiologia , Transdução de Sinais
14.
Cancer Res ; 74(6): 1857-69, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24448239

RESUMO

Metastatic colorectal cancer remains largely incurable, although in a subset of patients, survival is prolonged by new targeting agents such as anti-EGF receptor (anti-EGFR) antibodies. This disease is believed to be supported by a subpopulation of stem-like cells termed colon cancer-initiating cell (CCIC), which may also confer therapeutic resistance. However, how CCICs respond to EGFR inhibition has not been fully characterized. To explore this question, we systematically generated CCICs through spheroid cultures of patient-derived xenografts of metastatic colorectal cancer. These cultures, termed "xenospheres," were capable of long-term self-propagation in vitro and phenocopied the original patient tumors in vivo, thus operationally defining CCICs. Xenosphere CCICs retained the genetic determinants for EGFR therapeutic response in vitro and in xenografts; like the original tumors, xenospheres harboring a mutated KRAS gene were resistant to EGFR therapy, whereas those harboring wild-type RAS pathway genes (RAS(wt)) were sensitive. Notably, the effects of EGFR inhibition in sensitive CCICs could be counteracted by cytokines secreted by cancer-associated fibroblasts. In particular, we found that the MET receptor ligand hepatocyte growth factor (HGF) was especially active in supporting in vitro CCIC proliferation and resistance to EGFR inhibition. Ectopic production of human HGF in CCIC xenografts rendered the xenografts susceptible to MET inhibition, which sensitized the response to EGFR therapy. By showing that RAS(wt) CCICs rely on both EGFR and MET signaling, our results offer a strong preclinical proof-of-concept for concurrent targeting of these two pathways in the clinical setting.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias do Colo/enzimologia , Receptores ErbB/antagonistas & inibidores , Células-Tronco Neoplásicas/enzimologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Cetuximab , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Feminino , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Transdução de Sinais , Esferoides Celulares/enzimologia , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
15.
PLoS One ; 9(8): e105886, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25162504

RESUMO

Colon cancer is the second most common cause of cancer mortality in the Western world with metastasis commonly present at the time of diagnosis. Screening for propagation and metastatic behavior in a novel chimeric-mouse colon cancer model, driven by mutant p53 and ß-Catenin, led to the identification of a unique, invasive adenocarcinoma. Comparison of the genome of this tumor, CB42, with genomes from non-propagating tumors by array CGH and sequencing revealed an amplicon on chromosome five containing CDK6 and CDK14, and a KRAS mutation, respectively. Single agent small molecule inhibition of either CDK6 or MEK, a kinase downstream of KRAS, led to tumor growth inhibition in vivo whereas combination therapy not only led to regression of the subcutaneous tumors, but also near complete inhibition of lung metastasis; thus, genomic analysis of this tumor led to effective, individualized treatment.


Assuntos
Adenocarcinoma , Neoplasias do Colo , Neoplasias Pulmonares , Mutação , Proteínas de Neoplasias , Neoplasias Experimentais , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia
16.
Nat Biotechnol ; 28(1): 71-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20023657

RESUMO

To recapitulate the stochastic nature of human cancer development, we have devised a strategy for generating mouse tumor models that involves stepwise genetic manipulation of embryonic stem (ES) cells and chimera generation. Tumors in the chimeric animals develop from engineered cells in the context of normal tissue. Adenocarcinomas arising in an allelic series of lung cancer models containing HER2 (also known as ERBB2), KRAS or EGFR oncogenes exhibit features of advanced malignancies. Treatment of EGFR(L858R) and KRAS(G12V) chimeric models with an EGFR inhibitor resulted in near complete tumor regression and no response to the treatment, respectively, accurately reflecting previous clinical observations. Transcriptome and immunohistochemical analyses reveal that PI3K pathway activation is unique to ERBB family tumors whereas KRAS-driven tumors show activation of the JNK/SAP pathway, suggesting points of therapeutic intervention for this difficult-to-treat tumor category.


Assuntos
Adenocarcinoma/metabolismo , Quimera/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Adenocarcinoma/patologia , Animais , Modelos Animais de Doenças , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Fenótipo , Piperazinas/farmacologia , Quinazolinas/farmacologia , Insuficiência Respiratória/metabolismo , Insuficiência Respiratória/patologia , Transdução de Sinais/efeitos dos fármacos
17.
Cell ; 109(1): 17-27, 2002 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-11955443

RESUMO

Immune-deficient Rag2(-/-) mice were used as nuclear donors for transfer into enucleated oocytes, and the resulting blastocysts were cultured to isolate an isogenic embryonic stem cell line. One of the mutated alleles in the Rag2(-/-) ES cells was repaired by homologous recombination, thereby restoring normal Rag2 gene structure. Mutant mice were treated with the repaired ES cells in two ways. (1) Immune-competent mice were generated from the repaired ES cells by tetraploid embryo complementation and were used as bone marrow donors for transplantation. (2) Hematopoietic precursors were derived by in vitro differentiation from the repaired ES cells and engrafted into mutant mice. Mature myeloid and lymphoid cells as well as immunoglobulins became detectable 3-4 weeks after transplantation. Our results establish a paradigm for the treatment of a genetic disorder by combining therapeutic cloning with gene therapy.


Assuntos
Clonagem de Organismos/métodos , Proteínas de Ligação a DNA/deficiência , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Técnicas de Transferência Nuclear , Imunodeficiência Combinada Severa/terapia , Animais , Blastocisto/citologia , Blastocisto/imunologia , Blastocisto/metabolismo , Transplante de Medula Óssea , Diferenciação Celular/genética , Núcleo Celular/genética , Núcleo Celular/imunologia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Transferência Embrionária , Feminino , Sobrevivência de Enxerto/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Mutação/genética , Oócitos/citologia , Oócitos/imunologia , Oócitos/metabolismo , Poliploidia , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia
18.
Cell ; 114(4): 431-43, 2003 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-12941272

RESUMO

E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.


Assuntos
Ciclina E/genética , Ciclina E/metabolismo , Embrião de Mamíferos/fisiologia , Animais , Anormalidades Cardiovasculares , Ciclo Celular/fisiologia , Transformação Celular Neoplásica , Replicação do DNA , Feminino , Marcação de Genes , Masculino , Megacariócitos/fisiologia , Camundongos , Camundongos Knockout , Placenta/citologia , Placenta/metabolismo , Gravidez , Espermatogênese/fisiologia , Trofoblastos/citologia , Trofoblastos/metabolismo
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