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1.
Cell ; 186(14): 3013-3032.e22, 2023 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-37352855

RESUMEN

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.


Asunto(s)
Cardiotoxicidad , ADN Mitocondrial , Animales , Ratones , ADN Mitocondrial/metabolismo , Inmunidad Innata , Interferones/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Fosforilación
2.
Cell ; 162(6): 1217-28, 2015 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-26321681

RESUMEN

Activated T cells engage aerobic glycolysis and anabolic metabolism for growth, proliferation, and effector functions. We propose that a glucose-poor tumor microenvironment limits aerobic glycolysis in tumor-infiltrating T cells, which suppresses tumoricidal effector functions. We discovered a new role for the glycolytic metabolite phosphoenolpyruvate (PEP) in sustaining T cell receptor-mediated Ca(2+)-NFAT signaling and effector functions by repressing sarco/ER Ca(2+)-ATPase (SERCA) activity. Tumor-specific CD4 and CD8 T cells could be metabolically reprogrammed by increasing PEP production through overexpression of phosphoenolpyruvate carboxykinase 1 (PCK1), which bolstered effector functions. Moreover, PCK1-overexpressing T cells restricted tumor growth and prolonged the survival of melanoma-bearing mice. This study uncovers new metabolic checkpoints for T cell activity and demonstrates that metabolic reprogramming of tumor-reactive T cells can enhance anti-tumor T cell responses, illuminating new forms of immunotherapy.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Melanoma/terapia , Monitorización Inmunológica , Fosfoenolpiruvato/metabolismo , Microambiente Tumoral , Animales , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Glucólisis , Hexoquinasa/metabolismo , Inmunoterapia , Ratones , Factores de Transcripción NFATC/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/inmunología
3.
Nature ; 598(7882): 682-687, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34671158

RESUMEN

Tumours use various strategies to evade immune surveillance1,2. Immunotherapies targeting tumour immune evasion such as immune checkpoint blockade have shown considerable efficacy on multiple cancers3,4 but are ineffective for most patients due to primary or acquired resistance5-7. Recent studies showed that some epigenetic regulators suppress anti-tumour immunity2,8-12, suggesting that epigenetic therapies could boost anti-tumour immune responses and overcome resistance to current immunotherapies. Here we show that, in mouse melanoma models, depletion of KDM5B-an H3K4 demethylase that is critical for melanoma maintenance and drug resistance13-15-induces robust adaptive immune responses and enhances responses to immune checkpoint blockade. Mechanistically, KDM5B recruits the H3K9 methyltransferase SETDB1 to repress endogenous retroelements such as MMVL30 in a demethylase-independent manner. Derepression of these retroelements activates cytosolic RNA-sensing and DNA-sensing pathways and the subsequent type-I interferon response, leading to tumour rejection and induction of immune memory. Our results demonstrate that KDM5B suppresses anti-tumour immunity by epigenetic silencing of retroelements. We therefore reveal roles of KDM5B in heterochromatin regulation and immune evasion in melanoma, opening new paths for the development of KDM5B-targeting and SETDB1-targeting therapies to enhance tumour immunogenicity and overcome immunotherapy resistance.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Silenciador del Gen , N-Metiltransferasa de Histona-Lisina/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Melanoma/inmunología , Retroelementos , Escape del Tumor , Animales , Línea Celular Tumoral , Epigénesis Genética , Heterocromatina , Humanos , Interferón Tipo I/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares , Proteínas Represoras
4.
Nature ; 583(7817): 609-614, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32581358

RESUMEN

Cytokines were the first modern immunotherapies to produce durable responses in patients with advanced cancer, but they have only modest efficacy and limited tolerability1,2. In an effort to identify alternative cytokine pathways for immunotherapy, we found that components of the interleukin-18 (IL-18) pathway are upregulated on tumour-infiltrating lymphocytes, suggesting that IL-18 therapy could enhance anti-tumour immunity. However, recombinant IL-18 previously did not demonstrate efficacy in clinical trials3. Here we show that IL-18BP, a high-affinity IL-18 decoy receptor, is frequently upregulated in diverse human and mouse tumours and limits the anti-tumour activity of IL-18 in mice. Using directed evolution, we engineered a 'decoy-resistant' IL-18 (DR-18) that maintains signalling potential but is impervious to inhibition by IL-18BP. Unlike wild-type IL-18, DR-18 exerted potent anti-tumour effects in mouse tumour models by promoting the development of poly-functional effector CD8+ T cells, decreasing the prevalence of exhausted CD8+ T cells that express the transcriptional regulator of exhaustion TOX, and expanding the pool of stem-like TCF1+ precursor CD8+ T cells. DR-18 also enhanced the activity and maturation of natural killer cells to effectively treat anti-PD-1 resistant tumours that have lost surface expression of major histocompatibility complex class I molecules. These results highlight the potential of the IL-18 pathway for immunotherapeutic intervention and implicate IL-18BP as a major therapeutic barrier.


Asunto(s)
Inmunoterapia , Péptidos y Proteínas de Señalización Intercelular/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interleucina-18/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Femenino , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Estimación de Kaplan-Meier , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones , Receptores de Interleucina-18/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología
5.
Proc Natl Acad Sci U S A ; 120(30): e2304319120, 2023 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-37459511

RESUMEN

Recurrence of advanced melanoma after therapy is a major risk factor for reduced survival, and treatment options are limited. Antitumor immune memory plays a critical role in preventing melanoma recurrence and memory T cells could be a potent cell-based therapy, but the identity, and functional properties of the required immune cells are incompletely understood. Here, we show that an IL-7Rhi tumor-specific CD8+ population is critical for antitumor memory and can be epigenetically augmented to drive powerful antitumor immune responses. Using a model of functional antimelanoma memory, we found that high IL-7R expression selectively marks a CD8+ population in lymphoid organs that plays critical roles in maintaining tumor remission after immunotherapy or surgical resection. This population has intrinsic cytotoxic activity, lacks markers of exhaustion and has superior antitumor efficacy. IL-7Rhi cells have a functionally poised epigenetic landscape regulated by DNA methylation, which can be augmented by hypomethylating agents to confer improved survival and complete melanoma clearance in naive mice. Importantly, greater than 95% of tumor-specific T cells in draining lymph nodes after therapy express high levels of IL-7R. This overlap between IL-7Rhi and antigen-specific T cells allows for enrichment of a potent functional CD8+ population without determining antigen-specificity, which we demonstrate in a melanoma model without a known antigen. We identify that IL-7R expression in human melanoma is an independent prognostic factor of improved survival. These findings advance our basic understanding of antitumor memory and suggest a cell-based therapy using high IL-7R expression to enrich for a lymph node population with superior antitumor activity that can be augmented by hypomethylating agents.


Asunto(s)
Linfocitos T CD8-positivos , Melanoma , Ratones , Humanos , Animales , Células T de Memoria , Melanoma/genética , Melanoma/terapia , Transducción de Señal , Antígenos , Concesión de Licencias , Memoria Inmunológica
6.
Immunity ; 44(5): 1190-203, 2016 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-27192578

RESUMEN

Chronic viruses and cancers thwart immune responses in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), that is upregulated on responding T cells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector T cells that had downregulated PD-1 as well as other inhibitory receptors. Notably, this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically, PSGL-1 ligation on exhausted CD8(+) T cells inhibited T cell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1, leading to diminished survival with TCR stimulation. In models of melanoma cancer in which T cell dysfunction occurs, PSGL-1 deficiency led to PD-1 downregulation, improved T cell responses, and tumor control. Thus, PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulate T cell responses in the tumor microenvironment.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Melanoma/inmunología , Glicoproteínas de Membrana/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Evasión Inmune , Interleucina-2/metabolismo , Activación de Linfocitos , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal
7.
Nature ; 565(7737): E3, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30523330

RESUMEN

In this Letter, 'released' should have been 'regulated' in the sentence starting: 'Deletion of Atg5 in the host similarly regulated circulating arginine and suppressed tumorigenesis...' This has been corrected online.

8.
Nature ; 563(7732): 569-573, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30429607

RESUMEN

Autophagy captures intracellular components and delivers them to lysosomes, where they are degraded and recycled to sustain metabolism and to enable survival during starvation1-5. Acute, whole-body deletion of the essential autophagy gene Atg7 in adult mice causes a systemic metabolic defect that manifests as starvation intolerance and gradual loss of white adipose tissue, liver glycogen and muscle mass1. Cancer cells also benefit from autophagy. Deletion of essential autophagy genes impairs the metabolism, proliferation, survival and malignancy of spontaneous tumours in models of autochthonous cancer6,7. Acute, systemic deletion of Atg7 or acute, systemic expression of a dominant-negative ATG4b in mice induces greater regression of KRAS-driven cancers than does tumour-specific autophagy deletion, which suggests that host autophagy promotes tumour growth1,8. Here we show that host-specific deletion of Atg7 impairs the growth of multiple allografted tumours, although not all tumour lines were sensitive to host autophagy status. Loss of autophagy in the host was associated with a reduction in circulating arginine, and the sensitive tumour cell lines were arginine auxotrophs owing to the lack of expression of the enzyme argininosuccinate synthase 1. Serum proteomic analysis identified the arginine-degrading enzyme arginase I (ARG1) in the circulation of Atg7-deficient hosts, and in vivo arginine metabolic tracing demonstrated that serum arginine was degraded to ornithine. ARG1 is predominantly expressed in the liver and can be released from hepatocytes into the circulation. Liver-specific deletion of Atg7 produced circulating ARG1, and reduced both serum arginine and tumour growth. Deletion of Atg5 in the host similarly regulated [corrected] circulating arginine and suppressed tumorigenesis, which demonstrates that this phenotype is specific to autophagy function rather than to deletion of Atg7. Dietary supplementation of Atg7-deficient hosts with arginine partially restored levels of circulating arginine and tumour growth. Thus, defective autophagy in the host leads to the release of ARG1 from the liver and the degradation of circulating arginine, which is essential for tumour growth; this identifies a metabolic vulnerability of cancer.


Asunto(s)
Arginina/sangre , Autofagia , Neoplasias/sangre , Neoplasias/patología , Aloinjertos , Animales , Arginasa/sangre , Arginasa/metabolismo , Arginina/administración & dosificación , Arginina/farmacología , Autofagia/genética , Proteína 5 Relacionada con la Autofagia/deficiencia , Proteína 5 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/deficiencia , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Suplementos Dietéticos , Hepatocitos/enzimología , Hepatocitos/metabolismo , Hígado/enzimología , Masculino , Ratones , Trasplante de Neoplasias , Neoplasias/genética , Ornitina/metabolismo
9.
Cell Mol Life Sci ; 79(7): 377, 2022 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-35737114

RESUMEN

We aimed to study mechanisms controlling metastatic outgrowth of melanoma into clinically relevant lesions, a critical process responsible for the majority of melanoma deaths. To this end, we developed novel in vivo models and identified molecular events that can be ascribed to their distinct phenotypes, indolent or highly metastatic. Induction of a proliferative state at distant sites was associated with high levels of the stem-like/progenitor marker, SOX2, and required the upregulation of FMOD, an extracellular matrix component, which modulates tumor-stroma interactions. Functional studies revealed a possible link between FMOD and SOX2; dual FMOD and SOX2 silencing nearly abolished brain metastasis and had a similar effect on distant metastasis to other sites. Our in vitro data suggests that FMOD and SOX2 cooperation plays an important role in tumor vasculogenic mimicry. Furthermore, we found that FMOD and SOX2 functional roles might converge at the activation of transcriptional co-factors YAP and TAZ, possibly via crosstalk with the tumor suppressor Hippo pathway. Finally, high expression of both genes in patient specimens predicted early development of brain metastasis. Thus, our study identifies FMOD and SOX2 cooperation as a novel regulatory mechanism that might be linked functionally to melanoma metastatic competence.


Asunto(s)
Melanoma , Neoplasias Encefálicas/secundario , Fibromodulina/genética , Fibromodulina/metabolismo , Humanos , Melanoma/genética , Metástasis de la Neoplasia , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/genética
10.
Cancer ; 126(5): 1016-1030, 2020 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-31769872

RESUMEN

BACKGROUND: PLEKHA5 has previously been identified as a novel molecule implicated in melanoma brain metastasis, a disease that continues to portend a poor prognosis. The aim of this study was to further investigate the functional role of PLEKHA5 in disseminated melanoma. METHODS: The impact of PLEKHA5 on proliferation and tumor growth was examined in vitro and in melanoma xenograft models, including brain-tropic melanomas (melanomas tending to disseminate to the brain). In vitro loss- and gain-of-function studies were used to explore the underlying mechanisms of PLEKHA5-mediated tumor growth and the crosstalk between PLEKHA5 and PI3K/AKT/mTOR or MAPK/ERK signaling. The clinical relevance of PLEKHA5 dysregulation was further investigated in a cohort of matched cranial and extracranial melanoma metastases. RESULTS: PLEKHA5 stable knockdown negatively regulated cell proliferation by inhibiting the G1 -to-S cell cycle transition, which coincided with upregulation of the cell cycle regulator PDCD4. Conversely, ectopic PLEKHA5 expression exhibited the inverse effect. PLEKHA5 knockdown significantly inhibited tumor growth, whereas its overexpression upregulated the growth of tumors, which was induced by cranial and subcutaneous inoculation of cells in nude mice. PLEKHA5 modulation affected PDCD4 protein stability and was coupled with changes in PI3K/AKT/mTOR pathway signaling. High PDCD4 expression in cerebral specimens was associated with better overall survival. CONCLUSIONS: This study further supports the role of PLEKHA5 as a regulator of melanoma growth at distant sites, including the brain. Furthermore, the results highlight the significance of PDCD4 dysregulation in disseminated melanoma and implicate PDCD4 as a possible causal link between PLEKHA5 and cell proliferation and growth.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/secundario , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Melanoma/patología , Adulto , Anciano , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Desnudos , Persona de Mediana Edad , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto Joven
12.
Proc Natl Acad Sci U S A ; 114(30): E6157-E6165, 2017 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-28710334

RESUMEN

The recent Food and Drug Administration approval of immunogenic oncolytic virus (OV) has opened a new era in the treatment of advanced melanoma; however, approximately 50% of patients with melanoma develop brain metastasis, and currently there are no beneficial treatment options for such patients. To model the progression of metastases seen in patients and to overcome the hurdles of systemic delivery of OV, we developed melanoma brain metastasis models in immunocompromised and immunocompetent mice, and tested the fate and efficacy of oncolytic herpes simplex virus (oHSV)-armed mesenchymal stem cells (MSCs). Using brain-seeking patient-derived melanoma cells and real-time in vivo imaging, we show a widespread distribution of micrometastases and macrometastases in the brain, recapitulating the progression of multifoci metastases seen in patients. We armed MSCs with different oHSV variants (MSC-oHSV) and found that intracarotid administration of MSC-oHSV, but not of purified oHSV alone, effectively tracks metastatic tumor lesions and significantly prolongs the survival of brain tumor-bearing mice. In a syngeneic model of melanoma brain metastasis, a combination of MSC-oHSV and PD-L1 blockade increases IFNγ-producing CD8+ tumor-infiltrating T lymphocytes and results in a profound extension of the median survival of treated animals. This study thus demonstrates the utility of MSCs as OV carriers to disseminated brain lesions, and provides a clinically applicable therapeutic platform to target melanoma brain metastasis.


Asunto(s)
Neoplasias Encefálicas/terapia , Melanoma Experimental/terapia , Células Madre Mesenquimatosas , Viroterapia Oncolítica/métodos , Animales , Neoplasias Encefálicas/patología , Línea Celular , Humanos , Ratones , Metástasis de la Neoplasia , Virus Oncolíticos/genética , Simplexvirus/genética , Células Tumorales Cultivadas
13.
Cancer Immunol Immunother ; 68(7): 1121-1132, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31134297

RESUMEN

Immune-cell infiltration is associated with improved survival in melanoma. Human melanoma metastases may be grouped into immunotypes representing patterns of immune-cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse). Immunotypes have not been defined for murine melanomas, but may provide opportunities to understand mechanism-driving immunotype differences. We performed immunohistochemistry with immune-cell enumeration, immunotyping, and vascular density scoring in genetically engineered (Braf/Pten and Braf/Pten/ß-catenin) and transplantable (B16-F1, B16-OVA, and B16-AAD) murine melanomas. The transplantable tumors were grown in subcutaneous (s.c.) or intraperitoneal (i.p.) locations. Braf/Pten and Braf/Pten/ß-catenin tumors had low immune-cell densities, defining them as Immunotype A, as did B16-F1 tumors. B16-OVA (s.c. and i.p.) and B16-AAD s.c. tumors were Immunotype B, while B16-AAD i.p. tumors were primarily Immunotype C. Interestingly, the i.p. location was characterized by higher immune-cell counts in B16-OVA tumors, with counts that trended higher for B16-F1 and B16-AAD. The i.p. location was also characterized by higher vascularity in B16-F1 and B16-AAD tumors. These findings demonstrate that spontaneously mutated neoantigens in B16 melanomas were insufficient to induce robust intratumoral immune-cell infiltrates, but instead were Immunotype A tumors. The addition of model neoantigens (OVA or AAD) to B16 enhanced infiltration, but this most often resulted in Immunotype B. We find that tumor location may be an important element in enabling Immunotype C tumors. In aggregate, these data suggest important roles both for the antigen type and for the tumor location in defining immunotypes.


Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunofenotipificación , Melanoma Experimental/inmunología , Neoplasias Cutáneas/inmunología , Animales , Línea Celular Tumoral/trasplante , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas B-raf/genética , Piel/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Análisis de Matrices Tisulares , beta Catenina/genética
14.
Cytometry A ; 95(4): 399-410, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30468565

RESUMEN

Phenotyping immune cells and cell clusters in situ, including their activation state and function, can aid in interpretation of spatial relationships within the tissue microenvironment. Immune cell phenotypes require multiple biomarkers. However, conventional microscopy setups can only image up to four biomarkers at one time. In this report, we describe and give an example of a workflow to phenotype, quantitate, and visualize greater than four biomarkers in silico utilizing multiplexed fluorescence histology and the TissueFAXS quantitative imaging system with a conventional microscopy setup. Biomarkers were conjugated to Cy3 or Cy5. Multiplexed staining was performed on formalin-fixed paraffin-embedded tissue sections. We imaged the slides, inactivated the dyes, and repeated the process until all biomarkers were stained. Phenotype profiles were built based on in silico combinations of the biomarkers. We used algorithms that aligned all images to create a composite image, isolated each cell in the image, and identified biomarker positive cells in the image. The in silico phenotypes were quantitated and displayed through flow cytometry-like histograms and dot scatterplots in addition to backgating into the tissue images. The advantage of our workflow is that it provides visual verification of cell isolation and identification as well as highlight characteristics of cells and cell clusters. © 2018 International Society for Advancement of Cytometry.


Asunto(s)
Simulación por Computador , Citometría de Imagen/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Inmunofenotipificación/métodos , Linfocitos/citología , Animales , Recuento de Células/métodos , Línea Celular Tumoral , Citometría de Flujo/métodos , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente/métodos , Esferoides Celulares/citología , Coloración y Etiquetado/métodos , Fijación del Tejido/métodos
15.
PLoS Comput Biol ; 13(1): e1005308, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-28085880

RESUMEN

A promising alternative to address the problem of acquired drug resistance is to rely on combination therapies. Identification of the right combinations is often accomplished through trial and error, a labor and resource intensive process whose scale quickly escalates as more drugs can be combined. To address this problem, we present a broad computational approach for predicting synergistic combinations using easily obtainable single drug efficacy, no detailed mechanistic understanding of drug function, and limited drug combination testing. When applied to mutant BRAF melanoma, we found that our approach exhibited significant predictive power. Additionally, we validated previously untested synergy predictions involving anticancer molecules. As additional large combinatorial screens become available, this methodology could prove to be impactful for identification of drug synergy in context of other types of cancers.


Asunto(s)
Combinación de Medicamentos , Descubrimiento de Drogas/métodos , Sinergismo Farmacológico , Antineoplásicos , Línea Celular Tumoral , Biología Computacional , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Modelos Teóricos , Proteínas Proto-Oncogénicas B-raf/genética
16.
Nature ; 491(7424): 449-53, 2012 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-23123854

RESUMEN

People with pale skin, red hair, freckles and an inability to tan--the 'red hair/fair skin' phenotype--are at highest risk of developing melanoma, compared to all other pigmentation types. Genetically, this phenotype is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene. MC1R encodes a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Minimal receptor activity, as in red hair/fair skin polymorphisms, produces the red/yellow pheomelanin pigment, whereas increasing MC1R activity stimulates the production of black/brown eumelanin. Pheomelanin has weak shielding capacity against ultraviolet radiation relative to eumelanin, and has been shown to amplify ultraviolet-A-induced reactive oxygen species. Several observations, however, complicate the assumption that melanoma risk is completely ultraviolet-radiation-dependent. For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and ultraviolet radiation signature mutations are infrequently oncogenic drivers. Although linkage of melanoma risk to ultraviolet radiation exposure is beyond doubt, ultraviolet-radiation-independent events are likely to have a significant role. Here we introduce a conditional, melanocyte-targeted allele of the most common melanoma oncoprotein, BRAF(V600E), into mice carrying an inactivating mutation in the Mc1r gene (these mice have a phenotype analogous to red hair/fair skin humans). We observed a high incidence of invasive melanomas without providing additional gene aberrations or ultraviolet radiation exposure. To investigate the mechanism of ultraviolet-radiation-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1r(e/e) background. Selective absence of pheomelanin synthesis was protective against melanoma development. In addition, normal Mc1r(e/e) mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1r(e/e) mouse skin. These data suggest that the pheomelanin pigment pathway produces ultraviolet-radiation-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage. Although protection from ultraviolet radiation remains important, additional strategies may be required for optimal melanoma prevention.


Asunto(s)
Color del Cabello/genética , Melanoma/genética , Pigmentación de la Piel/genética , Rayos Ultravioleta , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Melaninas/metabolismo , Ratones , Ratones Endogámicos C57BL , Monofenol Monooxigenasa/genética , Peroxidasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Receptor de Melanocortina Tipo 1/genética , Sulfonamidas/farmacología , Análisis de Supervivencia , Células Tumorales Cultivadas
17.
Proc Natl Acad Sci U S A ; 112(6): E536-45, 2015 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-25624498

RESUMEN

BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in ∼ 80% of BRAF(V600)-mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of Braf(V618E) (analogous to the human BRAF(V600E) mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of Braf(V618E) transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes.


Asunto(s)
Resistencia a Antineoplásicos/fisiología , Melanoma/tratamiento farmacológico , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Animales , Animales Modificados Genéticamente , Southern Blotting , Western Blotting , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Células Madre Embrionarias/metabolismo , Exoma/genética , Estudios de Asociación Genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Inmunohistoquímica , Indoles/farmacología , Melanoma/metabolismo , Ratones , Mutagénesis , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transducción de Señal/genética , Sulfonamidas/farmacología , Transposasas/metabolismo , Proteína Letal Asociada a bcl/metabolismo
18.
Proc Natl Acad Sci U S A ; 110(38): E3640-9, 2013 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-24003131

RESUMEN

Despite considerable efforts to sequence hypermutated cancers such as melanoma, distinguishing cancer-driving genes from thousands of recurrently mutated genes remains a significant challenge. To circumvent the problematic background mutation rates and identify new melanoma driver genes, we carried out a low-copy piggyBac transposon mutagenesis screen in mice. We induced eleven melanomas with mutation burdens that were 100-fold lower relative to human melanomas. Thirty-eight implicated genes, including two known drivers of human melanoma, were classified into three groups based on high, low, or background-level mutation frequencies in human melanomas, and we further explored the functional significance of genes in each group. For two genes overlooked by prevailing discovery methods, we found that loss of membrane associated guanylate kinase, WW and PDZ domain containing 2 and protein tyrosine phosphatase, receptor type, O cooperated with the v-raf murine sarcoma viral oncogene homolog B (BRAF) recurrent V600E mutation to promote cellular transformation. Moreover, for infrequently mutated genes often disregarded by current methods, we discovered recurrent mitogen-activated protein kinase kinase kinase 1 (Map3k1)-activating insertions in our screen, mirroring recurrent MAP3K1 up-regulation in human melanomas. Aberrant expression of Map3k1 enabled growth factor-autonomous proliferation and drove BRAF-independent ERK signaling, thus shedding light on alternative means of activating this prominent signaling pathway in melanoma. In summary, our study contributes several previously undescribed genes involved in melanoma and establishes an important proof-of-principle for the utility of the low-copy transposon mutagenesis approach for identifying cancer-driving genes, especially those masked by hypermutation.


Asunto(s)
Elementos Transponibles de ADN/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Melanoma/genética , Mutagénesis Insercional/genética , Transducción de Señal/fisiología , Animales , Western Blotting , Cartilla de ADN/genética , Regulación Neoplásica de la Expresión Génica/genética , Pruebas Genéticas , Células HEK293 , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Especificidad de la Especie
19.
Proc Natl Acad Sci U S A ; 110(45): 18226-31, 2013 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-24145418

RESUMEN

Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. Based on our discovery of cross-talk between the AMPK and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) signaling pathways, we investigated the antitumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF-mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Cotreatment of BRAF-mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared with the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells, whereas PLX4720 selectively targeted JARID1B-negative cells. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAF(V600E)/PTEN(null)-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma.


Asunto(s)
Indoles/farmacología , Melanoma/tratamiento farmacológico , Fenformina/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/farmacología , Análisis de Varianza , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Inmunohistoquímica , Indoles/administración & dosificación , Histona Demetilasas con Dominio de Jumonji/metabolismo , Melanoma/genética , Ratones , Mutación Missense/genética , Fenformina/administración & dosificación , Proteínas Proto-Oncogénicas B-raf/genética , Sulfonamidas/administración & dosificación
20.
J Virol ; 87(12): 6644-59, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23552414

RESUMEN

Metastatic malignant melanoma remains one of the most therapeutically challenging forms of cancer. Here we test replication-competent vesicular stomatitis viruses (VSV) on 19 primary human melanoma samples and compare these infections with those of normal human melanocyte control cells. Even at a low viral concentration, we found a strong susceptibility to viral oncolysis in over 70% of melanomas. In contrast, melanocytes displayed strong resistance to virus infection and showed complete protection by interferon. Several recombinant VSVs were compared, and all infected and killed most melanomas with differences in the time course with increasing rates of melanoma infection, as follows: VSV-CT9-M51 < VSV-M51 < VSV-G/GFP < VSV-rp30. VSV-rp30 sequencing revealed 2 nonsynonymous mutations at codon positions P126 and L223, both of which appear to be required for the enhanced phenotype. VSV-rp30 showed effective targeting and infection of multiple subcutaneous and intracranial melanoma xenografts in SCID mice after tail vein virus application. Sequence analysis of mutations in the melanomas used revealed that BRAF but not NRAS gene mutation status was predictive for enhanced susceptibility to infection. In mouse melanoma models with specific induced gene mutations including mutations of the Braf, Pten, and Cdkn2a genes, viral infection correlated with the extent of malignant transformation. Similar to human melanocytes, mouse melanocytes resisted VSV-rp30 infection. This study confirms the general susceptibility of the majority of human melanoma types for VSV-mediated oncolysis.


Asunto(s)
Melanocitos/virología , Melanoma/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/patogenicidad , Virus de la Estomatitis Vesicular Indiana/patogenicidad , Animales , Línea Celular Tumoral , Humanos , Melanoma/virología , Ratones , Ratones SCID , Mutación , Virus Oncolíticos/genética , Proteínas Proto-Oncogénicas B-raf/genética , Trasplante Heterólogo/fisiología , Virus de la Estomatitis Vesicular Indiana/genética , Replicación Viral , Ensayos Antitumor por Modelo de Xenoinjerto
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