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The Drug-Gene Interaction Database (DGIdb, https://dgidb.org) is a publicly accessible resource that aggregates genes or gene products, drugs and drug-gene interaction records to drive hypothesis generation and discovery for clinicians and researchers. DGIdb 5.0 is the latest release and includes substantial architectural and functional updates to support integration into clinical and drug discovery pipelines. The DGIdb service architecture has been split into separate client and server applications, enabling consistent data access for users of both the application programming interface (API) and web interface. The new interface was developed in ReactJS, and includes dynamic visualizations and consistency in the display of user interface elements. A GraphQL API has been added to support customizable queries for all drugs, genes, annotations and associated data. Updated documentation provides users with example queries and detailed usage instructions for these new features. In addition, six sources have been added and many existing sources have been updated. Newly added sources include ChemIDplus, HemOnc, NCIt (National Cancer Institute Thesaurus), Drugs@FDA, HGNC (HUGO Gene Nomenclature Committee) and RxNorm. These new sources have been incorporated into DGIdb to provide additional records and enhance annotations of regulatory approval status for therapeutics. Methods for grouping drugs and genes have been expanded upon and developed as independent modular normalizers during import. The updates to these sources and grouping methods have resulted in an improvement in FAIR (findability, accessibility, interoperability and reusability) data representation in DGIdb.
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Medicina de Precisão , Humanos , Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas , Internet , Interface Usuário-Computador , Vocabulário ControladoRESUMO
HES3 is a basic helix-loop-helix transcription factor that regulates neural stem cell renewal during development. HES3 overexpression is predictive of reduced overall survival in patients with fusion-positive rhabdomyosarcoma, a pediatric cancer that resembles immature and undifferentiated skeletal muscle. However, the mechanisms of HES3 cooperation in fusion-positive rhabdomyosarcoma are unclear and are likely related to her3/HES3's role in neurogenesis. To investigate HES3's function during development, we generated a zebrafish CRISPR/Cas9 null mutation of her3, the zebrafish ortholog of HES3. Loss of her3 is not embryonic lethal and adults exhibit expected Mendelian ratios. Embryonic her3 zebrafish mutants exhibit dysregulated neurog1 expression, a her3 target gene, and the mutant her3 fails to bind the neurog1 promoter sequence. Further, her3 mutants are significantly smaller than wildtype and a subset present with lens defects as adults. Transcriptomic analysis of her3 mutant embryos indicates that genes involved in organ development, such as pctp and grinab, are significantly downregulated. Further, differentially expressed genes in her3 null mutant embryos are enriched for Hox and Sox10 motifs. Several cancer-related gene pathways are impacted, including the inhibition of matrix metalloproteinases. Altogether, this new model is a powerful system to study her3/HES3-mediated neural development and its misappropriation in cancer contexts.
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Rabdomiossarcoma , Peixe-Zebra , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Sistema Nervoso/metabolismo , Neurogênese , Rabdomiossarcoma/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Tumors are complex tissues containing collections of phenotypically diverse malignant and nonmalignant cells. We know little of the mechanisms that govern heterogeneity of tumor cells nor of the role heterogeneity plays in overcoming stresses, such as adaptation to different microenvironments. Osteosarcoma is an ideal model for studying these mechanisms-it exhibits widespread inter- and intra-tumoral heterogeneity, predictable patterns of metastasis, and a lack of clear targetable driver mutations. Understanding the processes that facilitate adaptation to primary and metastatic microenvironments could inform the development of therapeutic targeting strategies. RESULTS: We investigated single-cell RNA-sequencing profiles of 47,977 cells obtained from cell line and patient-derived xenograft models as cells adapted to growth within primary bone and metastatic lung environments. Tumor cells maintained phenotypic heterogeneity as they responded to the selective pressures imposed during bone and lung colonization. Heterogenous subsets of cells defined by distinct transcriptional profiles were maintained within bone- and lung-colonizing tumors, despite high-level selection. One prominent heterogenous feature involving glucose metabolism was clearly validated using immunofluorescence staining. Finally, using concurrent lineage tracing and single-cell transcriptomics, we found that lung colonization enriches for multiple clones with distinct transcriptional profiles that are preserved across cellular generations. CONCLUSIONS: Response to environmental stressors occurs through complex and dynamic phenotypic adaptations. Heterogeneity is maintained, even in conditions that enforce clonal selection. These findings likely reflect the influences of developmental processes promoting diversification of tumor cell subpopulations, which are retained, even in the face of selective pressures.
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Neoplasias Ósseas , Neoplasias Pulmonares , Osteossarcoma , Humanos , Osteossarcoma/genética , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Perfilação da Expressão Gênica , Neoplasias Ósseas/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Microambiente Tumoral/genéticaRESUMO
Plasmodium vivax and P. falciparum, the parasites responsible for most human malaria worldwide, exhibit striking biological differences, which have important clinical consequences. Unfortunately, P. vivax, unlike P. falciparum, cannot be cultivated continuously in vitro, which limits our understanding of its biology and, consequently, our ability to effectively control vivax malaria. Here, we describe single-cell gene expression profiles of 9,215 P. vivax parasites from bloodstream infections of Aotus and Saimiri monkeys. Our results show that transcription of most P. vivax genes occurs during short periods of the intraerythrocytic cycle and that this pattern of gene expression is conserved in other Plasmodium species. However, we also identify a strikingly high proportion of species-specific transcripts in late schizonts, possibly associated with the specificity of erythrocyte invasion. Our findings provide new and robust markers of blood-stage parasites, including some that are specific to the elusive P. vivax male gametocytes, and will be useful for analyzing gene expression data from laboratory and field samples.
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Plasmodium vivax/metabolismo , Transcriptoma , Animais , Aotidae , Cloroquina , Feminino , Expressão Gênica , Masculino , Família Multigênica , Plasmodium vivax/crescimento & desenvolvimento , Saimiri , Esquizontes/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Especificidade da EspécieRESUMO
Background: Plasmodium vivax resistance to chloroquine (CQ) has been reported worldwide, although the World Health Organization clinical drug efficacy studies protocol does not permit classification of patient outcomes. Methods: We enrolled 40 patients with P. vivax malaria in northeastern Cambodia, where >17% treatment failures were previously reported. Patients were treated with CQ (30 mg/kg) and followed for 2 months, with frequent clinical examination and capillary blood sample collection for microscopy, molecular parasite detection and genotyping, and drug concentration measurements. Reinfections were prevented by relocating patients to a transmission-free area. Results: P. vivax parasites were eliminated in all patients by day 3. Genomic analyses revealed that all clones in polyclonal infections were cleared at the same rate, indicating their equal susceptibility to CQ. CQ blood concentrations were below the therapeutic level in all recurrent infections (24 of 40 patients), which were efficiently cleared by a second course of CQ treatment. Genotyping (128 SNPs barcode) and sequences of entire parasite genome (Whole-Genome Sequencing, Illumina) indicated that two thirds (6 of 8) of the recurrent parasites resulted from heterologous relapses whose 50% are from by sibling/recombinant clones. Conclusions: No evidence of CQ resistance was observed. Our data suggest that P. vivax antimalarial drug resistance is likely overestimated and that the current guidelines for clinical drug studies of P. vivax malaria need to be revised.
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Cloroquina/uso terapêutico , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Malária Vivax/tratamento farmacológico , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/genética , Adolescente , Adulto , Antimaláricos/uso terapêutico , Camboja , Cloroquina/sangue , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Recidiva , Análise de Sequência de DNA , Falha de Tratamento , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
N-glycosylation is a common posttranslational modification of secreted and membrane proteins, catalyzed by the two enzymatic isoforms of the oligosaccharyltransferase, STT3A and STT3B. Missense mutations are the most common mutations in inherited diseases; however, missense mutations that generate extra, non-native N-glycosylation sites have not been well characterized. Coagulation factor VIII (FVIII) contains five consensus N-glycosylation sites outside its functionally dispensable B domain. We developed a computer program that identified hemophilia A mutations in FVIII that can potentially create ectopic glycosylation sites. We determined that 18 of these ectopic sites indeed become N-glycosylated. These sites span the domains of FVIII and are primarily associated with a severe disease phenotype. Using STT3A and STT3B knockout cells, we determined that ectopic glycosylation exhibited different degrees of dependence on STT3A and STT3B. By separating the effects of ectopic N-glycosylation from those due to underlying amino acid changes, we showed that ectopic glycans promote the secretion of some mutants, but impair the secretion of others. However, ectopic glycans that enhanced secretion could not functionally replace a native N-glycan in the same domain. Secretion-deficient mutants, but not mutants with elevated secretion levels, show increased association with the endoplasmic reticulum chaperones BiP (immunoglobulin heavy chain-binding protein) and calreticulin. Though secreted to different extents, all studied mutants exhibited lower relative activity than wild-type FVIII. Our results reveal differential impacts of ectopic N-glycosylation on FVIII folding, trafficking and activity, which highlight complex disease-causing mechanisms of FVIII missense mutations. Our findings are relevant to other secreted and membrane proteins with mutations that generate ectopic N-glycans.
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Fator VIII/genética , Fator VIII/metabolismo , Mutação de Sentido Incorreto/fisiologia , Processamento de Proteína Pós-Traducional/genética , Sítios de Ligação/genética , Biologia Computacional , Fator VIII/química , Glicosilação , Humanos , Modelos Moleculares , Dobramento de Proteína , Transporte Proteico/genética , Análise de Sequência de DNA/métodosRESUMO
Gene amplification is a phenotype-causing form of chromosome instability and is initiated by DNA double-strand breaks (DSBs). Cells with mutant p53 lose G1/S checkpoint and are permissive to gene amplification. In this study we show that mammalian cells become proficient for spontaneous gene amplification when the function of the DSB repair protein complex MRN (Mre11/Rad50/Nbs1) is impaired. Cells with impaired MRN complex experienced severe replication stress and gained substrates for gene amplification during replication, as evidenced by the increase of replication-associated single-stranded breaks that were converted to DSBs most likely through replication fork reversal. Impaired MRN complex directly compromised ATM/ATR-mediated checkpoints and allowed cells to progress through cell cycle in the presence of DSBs. Such compromised intra-S phase checkpoints promoted gene amplification independently from mutant p53. Finally, cells adapted to endogenous replication stress by globally suppressing genes for DNA replication and cell cycle progression. Our results indicate that the MRN complex suppresses gene amplification by stabilizing replication forks and by securing DNA damage response to replication-associated DSBs.
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Reparo do DNA , Replicação do DNA/genética , Amplificação de Genes , Pontos de Checagem da Fase S do Ciclo Celular/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Western Blotting , Células CHO , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cricetinae , Cricetulus , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Targeting B-cell receptor (BCR) signaling in chronic lymphocytic leukemia (CLL) has been successful with durable remissions observed with several targeted therapeutics. Protein kinase C-ß (PKC-ß) is immediately downstream of BCR and has been shown to be essential to CLL cell survival and proliferation in vivo. We therefore evaluated sotrastaurin (AEB071), an orally administered potent PKC inhibitor, on CLL cell survival both in vitro and in vivo. AEB071 shows selective cytotoxicity against B-CLL cells in a dose-dependent manner. Additionally, AEB071 attenuates BCR-mediated survival pathways, inhibits CpG-induced survival and proliferation of CLL cells in vitro, and effectively blocks microenvironment-mediated survival signaling pathways in primary CLL cells. Furthermore, AEB071 alters ß-catenin expression, resulting in decreased downstream transcriptional genes as c-Myc, Cyclin D1, and CD44. Lastly, our preliminary in vivo studies indicate beneficial antitumor properties of AEB071 in CLL. Taken together, our results indicate that targeting PKC-ß has the potential to disrupt signaling from the microenvironment contributing to CLL cell survival and potentially drug resistance. Future efforts targeting PKC with the PKC inhibitor AEB071 as monotherapy in clinical trials of relapsed and refractory CLL patients are warranted.
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Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/enzimologia , Proteína Quinase C beta/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Pirróis/uso terapêutico , Quinazolinas/uso terapêutico , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Receptores de Antígenos de Linfócitos B/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
The import of nuclear transcribed RNAs into mitochondria is an emerging area that presents a tremendous opportunity to develop human metabolic therapeutics. However, our knowledge base is quite limited. Much remains to be discovered regarding specific RNA localization and mechanisms of import. To identify novel RNAs imported into mitochondria, all RNAs within the mitochondria were characterized using next generation sequencing technology. Several nuclear transcribed RNAs were found within mitochondrial RNA (mtRNA) samples, including nuclear ribosomal RNAs, gamma satellite RNA and VL30 retroelement RNA. The presence of these RNAs within mitochondria coupled with RNA sequencing data from other laboratories investigating mtRNA processing, lead us to hypothesize that nuclease treatment of mitoplasts is insufficient for removing contaminating cytoplasmic RNAs. In contrast to traditional methodology, mitochondrial import was evaluated by qRT-PCR after stepwise removal of the outer mitochondrial membrane and subsequent lysis of mitochondria. This allowed identification of RNAs lost from the mitochondria with the same kinetics as mitochondrial DNA-transcribed RNAs. This approach provided an improved evaluation of nuclear RNA enrichment within mitochondrial membranes to characterize nuclease protection and mitochondrial import and identify false-positive detection errors. qRT-PCR results confirmed the presence of VL30 retroelement RNA within mitochondria and question the hypothesis that the RNA component of RNase P is imported. These results illustrate a reliable approach for evaluating the presence of RNAs within mitochondria and open new avenues of investigation relating to mtRNA biology and in targeting mitochondrial based therapeutics.
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Mitocôndrias/genética , Transporte de RNA , RNA/metabolismo , Animais , Núcleo Celular/genética , Descoberta de Drogas , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Mitocôndrias/fisiologia , RNA/análise , RNA/isolamento & purificação , RNA Mitocondrial , RNA Ribossômico/metabolismo , RNA Satélite/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Retroelementos , Ribonucleases/metabolismo , Análise de Sequência de RNARESUMO
Purpose: Lung metastasis is responsible for nearly all deaths caused by osteosarcoma, the most common pediatric bone tumor. How malignant bone cells coerce the lung microenvironment to support metastatic growth is unclear. This study delineates how osteosarcoma cells educate the lung microenvironment during metastatic progression. Experimental design: Using single-cell transcriptomics (scRNA-seq), we characterized genome- and tissue-wide molecular changes induced within lung tissues by disseminated osteosarcoma cells in both immunocompetent murine models of metastasis and patient samples. We confirmed transcriptomic findings at the protein level and determined spatial relationships with multi-parameter immunofluorescence. We evaluated the ability of nintedanib to impair metastatic colonization and prevent osteosarcoma-induced education of the lung microenvironment in both immunocompetent murine osteosarcoma and immunodeficient human xenograft models. Results: Osteosarcoma cells induced acute alveolar epithelial injury upon lung dissemination. scRNA-seq demonstrated that the surrounding lung stroma adopts a chronic, non-resolving wound-healing phenotype similar to that seen in other models of lung injury. Accordingly, metastasis-associated lung demonstrated marked fibrosis, likely due to the accumulation of pathogenic, pro-fibrotic, partially-differentiated epithelial intermediates. Inhibition of fibrotic pathways with nintedanib prevented metastatic progression in multiple murine and human xenograft models. Conclusions: Our work demonstrates that osteosarcoma cells co-opt fibrosis to promote metastatic outgrowth. When harmonized with data from adult epithelial cancers, our results support a generalized model wherein aberrant mesenchymal-epithelial interactions are critical for promoting lung metastasis. Adult epithelial carcinomas induce fibrotic pathways in normal lung fibroblasts, whereas osteosarcoma, a pediatric mesenchymal tumor, exhibits fibrotic reprogramming in response to the aberrant wound-healing behaviors of an otherwise normal lung epithelium, which are induced by tumor cell interactions. Statement of translational relevance: Therapies that block metastasis have the potential to save the majority of lives lost due to solid tumors. Disseminated tumor cells must educate the foreign, inhospitable microenvironments they encounter within secondary organs to facilitate metastatic colonization. Our study elucidated that disseminated osteosarcoma cells survive within the lung by co-opting and amplifying the lung's endogenous wound healing response program. More broadly, our results support a model wherein mesenchymal-epithelial cooperation is a key driver of lung metastasis. Osteosarcoma, a pediatric mesenchymal tumor, undergoes lung epithelial induced fibrotic activation while also transforming normal lung epithelial cells towards a fibrosis promoting phenotype. Conversely, adult epithelial carcinomas activate fibrotic signaling in normal lung mesenchymal fibroblasts. Our data implicates fibrosis and abnormal wound healing as key drivers of lung metastasis across multiple tumor types that can be targeted therapeutically to disrupt metastasis progression.
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Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer molecularly characterized by arrested myogenesis. The defining genetic driver, PAX3::FOXO1, functions as a chimeric gain-of-function transcription factor. An incomplete understanding of PAX3::FOXO1's in vivo epigenetic mechanisms has hindered therapeutic development. Here, we establish a PAX3::FOXO1 zebrafish injection model and semi-automated ChIP-seq normalization strategy to evaluate how PAX3::FOXO1 initially interfaces with chromatin in a developmental context. We investigated PAX3::FOXO1's recognition of chromatin and subsequent transcriptional consequences. We find that PAX3::FOXO1 interacts with inaccessible chromatin through partial/homeobox motif recognition consistent with pioneering activity. However, PAX3::FOXO1-genome binding through a composite paired-box/homeobox motif alters chromatin accessibility and redistributes H3K27ac to activate neural transcriptional programs. We uncover neural signatures that are highly representative of clinical rhabdomyosarcoma gene expression programs that are enriched following chemotherapy. Overall, we identify partial/homeobox motif recognition as a new mode for PAX3::FOXO1 pioneer function and identify neural signatures as a potentially critical PAX3::FOXO1 tumor initiation event.
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Genes encoding the RNA-binding proteins FUS, EWSR1, and TAF15 (FET proteins) are involved in chromosomal translocations in rare sarcomas. FET-rearranged sarcomas are often aggressive malignancies affecting patients of all ages. New therapies are needed. These translocations fuse the 5' portion of the FET gene with a 3' partner gene encoding a transcription factor (TF). The resulting fusion proteins are oncogenic TFs with a FET protein low complexity domain (LCD) and a DNA binding domain. FET fusion proteins have proven stubbornly difficult to target directly and promising strategies target critical co-regulators. One candidate is lysine specific demethylase 1 (LSD1). LSD1 is recruited by multiple FET fusions, including EWSR1::FLI1. LSD1 promotes EWSR1::FLI1 activity and treatment with the noncompetitive inhibitor SP-2509 blocks EWSR1::FLI1 transcriptional function. A similar molecule, seclidemstat (SP-2577), is currently in clinical trials for FET-rearranged sarcomas (NCT03600649). However, whether seclidemstat has pharmacological activity against FET fusions has not been demonstrated. Here, we evaluate the in vitro potency of seclidemstat against multiple FET-rearranged sarcoma cell lines, including Ewing sarcoma, desmoplastic small round cell tumor, clear cell sarcoma, and myxoid liposarcoma. We also define the transcriptomic effects of seclidemstat treatment and evaluated the activity of seclidemstat against FET fusion transcriptional regulation. Seclidemstat showed potent activity in cell viability assays across FET-rearranged sarcomas and disrupted the transcriptional function of all tested fusions. Though epigenetic and targeted inhibitors are unlikely to be effective as a single agents in the clinic, these data suggest seclidemstat remains a promising new treatment strategy for patients with FET-rearranged sarcomas.
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PURPOSE: For patients with osteosarcoma, disease-related mortality most often results from lung metastasis-a phenomenon shared with many solid tumors. While established metastatic lesions behave aggressively, very few of the tumor cells that reach the lung will survive. By identifying mechanisms that facilitate survival of disseminated tumor cells, we can develop therapeutic strategies that prevent and treat metastasis. METHODS: We analyzed single cell RNA-sequencing (scRNAseq) data from murine metastasis-bearing lungs to interrogate changes in both host and tumor cells during colonization. We used these data to elucidate pathways that become activated in cells that survive dissemination and identify candidate host-derived signals that drive activation. We validated these findings through live cell reporter systems, immunocytochemistry, and fluorescent immunohistochemistry. We then validated the functional relevance of key candidates using pharmacologic inhibition in models of metastatic osteosarcoma. RESULTS: Expression patterns suggest that the MAPK pathway is significantly elevated in early and established metastases. MAPK activity correlates with expression of anti-apoptotic genes, especially MCL1. Niche cells produce growth factors that increase ERK phosphorylation and MCL1 expression in tumor cells. Both early and established metastases are vulnerable to MCL1 inhibition, but not MEK inhibition in vivo. Combining MCL1 inhibition with chemotherapy both prevented colonization and eliminated established metastases in murine models of osteosarcoma. CONCLUSION: Niche-derived growth factors drive MAPK activity and MCL1 expression in osteosarcoma, promoting metastatic colonization. Although later metastases produce less MCL1, they remain dependent on it. MCL1 is a promising target for clinical trials in both human and canine patients.
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Neoplasias Ósseas , Neoplasias Pulmonares , Proteína de Sequência 1 de Leucemia de Células Mieloides , Osteossarcoma , Animais , Cães , Humanos , Camundongos , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Pulmão/metabolismo , Neoplasias Pulmonares/secundário , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Osteossarcoma/patologia , FosforilaçãoRESUMO
We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript spread throughout the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing immunosuppressive macrophages and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.
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BACKGROUND: Mutations in mitochondrial DNA (mtDNA) cause a variety of pathologic states in human patients. Development of animal models harboring mtDNA mutations is crucial to elucidating pathways of disease and as models for preclinical assessment of therapeutic interventions. SCOPE OF REVIEW: This review covers the knowledge gained through animal models of mtDNA mutations and the strategies used to produce them. Animals derived from spontaneous mtDNA mutations, somatic cell nuclear transfer (SCNT), nuclear translocation of mitochondrial genes followed by mitochondrial protein targeting (allotopic expression), mutations in mitochondrial DNA polymerase gamma, direct microinjection of exogenous mitochondria, and cytoplasmic hybrid (cybrid) embryonic stem cells (ES cells) containing exogenous mitochondria (transmitochondrial cells) are considered. MAJOR CONCLUSIONS: A wide range of strategies have been developed and utilized in attempts to mimic human mtDNA mutation in animal models. Use of these animals in research studies has shed light on mechanisms of pathogenesis in mitochondrial disorders, yet methods for engineering specific mtDNA sequences are still in development. GENERAL SIGNIFICANCE: Research animals containing mtDNA mutations are important for studies of the mechanisms of mitochondrial disease and are useful for the development of clinical therapies. This article is part of a Special Issue entitled Biochemistry of Mitochondria.
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DNA Mitocondrial/genética , Modelos Animais de Doenças , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Mutação/genética , Animais , HumanosRESUMO
Bronchiectasis is an irreversible dilatation of the bronchi resulting from chronic airway inflammation. In people, computed tomography (CT) has been described as the noninvasive gold standard for diagnosing bronchiectasis. In dogs, normal CT bronchoarterial ratios have been described as <2.0. The purpose of this retrospective study was to describe quantitative and qualitative CT characteristics of bronchiectasis in a cohort of dogs with confirmed disease. Inclusion criteria for the study were thoracic radiography, thoracic CT, and a diagnosis of bronchiectasis based on bronchoscopy and/or histopathology. For each included dog, a single observer measured CT bronchoarterial ratios at 6 lobar locations. Qualitative thoracic radiography and CT characteristics were recorded by consensus opinion of two board-certified veterinary radiologists. Twelve dogs met inclusion criteria. The mean bronchoarterial ratio from 28 bronchiectatic lung lobes was 2.71 ± 0.80 (range 1.4 to 4.33), and 23/28 measurements were >2.0. Averaged bronchoarterial ratios from bronchiectatic lung lobes were significantly larger (P < 0.01) than averaged ratios from nonbronchiectatic lung lobes. Qualitative CT characteristics of bronchiectasis included lack of peripheral airway tapering (12/12), lobar consolidation (11/12), bronchial wall thickening (7/12), and bronchial lumen occlusion (4/12). Radiographs detected lack of airway tapering in 7/12 dogs. In conclusion, the most common CT characteristics of bronchiectasis were dilatation, a lack of peripheral airway tapering, and lobar consolidation. Lack of peripheral airway tapering was not visible in thoracic radiographs for some dogs. For some affected dogs, bronchoarterial ratios were less than published normal values.
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Brônquios/patologia , Bronquiectasia/veterinária , Doenças do Cão/patologia , Animais , Bronquiectasia/diagnóstico por imagem , Bronquiectasia/patologia , Broncoscopia/veterinária , Doenças do Cão/diagnóstico por imagem , Cães , Feminino , Masculino , Estudos Retrospectivos , Tomografia Computadorizada por Raios X/veterináriaRESUMO
Osteosarcoma is an aggressive malignancy characterized by high genomic complexity. Identification of few recurrent mutations in protein coding genes suggests that somatic copy-number aberrations (SCNA) are the genetic drivers of disease. Models around genomic instability conflict-it is unclear whether osteosarcomas result from pervasive ongoing clonal evolution with continuous optimization of the fitness landscape or an early catastrophic event followed by stable maintenance of an abnormal genome. We address this question by investigating SCNAs in >12,000 tumor cells obtained from human osteosarcomas using single-cell DNA sequencing, with a degree of precision and accuracy not possible when inferring single-cell states using bulk sequencing. Using the CHISEL algorithm, we inferred allele- and haplotype-specific SCNAs from this whole-genome single-cell DNA sequencing data. Surprisingly, despite extensive structural complexity, these tumors exhibit a high degree of cell-cell homogeneity with little subclonal diversification. Longitudinal analysis of patient samples obtained at distant therapeutic timepoints (diagnosis, relapse) demonstrated remarkable conservation of SCNA profiles over tumor evolution. Phylogenetic analysis suggests that the majority of SCNAs were acquired early in the oncogenic process, with relatively few structure-altering events arising in response to therapy or during adaptation to growth in metastatic tissues. These data further support the emerging hypothesis that early catastrophic events, rather than sustained genomic instability, give rise to structural complexity, which is then preserved over long periods of tumor developmental time. Significance: Chromosomally complex tumors are often described as genomically unstable. However, determining whether complexity arises from remote time-limited events that give rise to structural alterations or a progressive accumulation of structural events in persistently unstable tumors has implications for diagnosis, biomarker assessment, mechanisms of treatment resistance, and represents a conceptual advance in our understanding of intratumoral heterogeneity and tumor evolution.
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Neoplasias Ósseas , Osteossarcoma , Humanos , Filogenia , Variações do Número de Cópias de DNA/genética , Recidiva Local de Neoplasia , Osteossarcoma/genética , Instabilidade Genômica/genética , Neoplasias Ósseas/genéticaRESUMO
Objective: The diversity of nomenclature and naming strategies makes therapeutic terminology difficult to manage and harmonize. As the number and complexity of available therapeutic ontologies continues to increase, the need for harmonized cross-resource mappings is becoming increasingly apparent. This study creates harmonized concept mappings that enable the linking together of like-concepts despite source-dependent differences in data structure or semantic representation. Materials and Methods: For this study, we created Thera-Py, a Python package and web API that constructs searchable concepts for drugs and therapeutic terminologies using 9 public resources and thesauri. By using a directed graph approach, Thera-Py captures commonly used aliases, trade names, annotations, and associations for any given therapeutic and combines them under a single concept record. Results: We highlight the creation of 16 069 unique merged therapeutic concepts from 9 distinct sources using Thera-Py and observe an increase in overlap of therapeutic concepts in 2 or more knowledge bases after harmonization using Thera-Py (9.8%-41.8%). Conclusion: We observe that Thera-Py tends to normalize therapeutic concepts to their underlying active ingredients (excluding nondrug therapeutics, eg, radiation therapy, biologics), and unifies all available descriptors regardless of ontological origin.
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Background: To address the lack of non-cytotoxic, non-surgical options to treat undesirable focal adiposity of the face, we propose use of the anti-glaucoma medication and prostaglandin F2α analogue latanoprost, which has a well-described side effect of periorbital adipose shrinkage. Objective: To evaluate the safety and efficacy of soluble and liposomal latanoprost for focal fat reduction. Approach: To compare efficacy, single administrations of either the FDA-approved cytolytic drug deoxycholic acid (DOCA), latanoprost, or liposomal latanoprost were injected into ob/ob mouse inguinal fat pads. Study outcomes included mouse weight, inguinal fat pad volume, architecture, and cytotoxicity. Results: Both DOCA and soluble latanoprost significantly reduced inguinal fat pad volume whereas liposome encapsulation reduced inguinal fat pad volume insignificantly over the 14-day study period. Hematoxylin and eosin demonstrated effective reduction in adipocyte volume without histologic evidence of cytolysis or inflammation whereas DOCA caused dermal ulcerations, adipocyte lysis, and increased tissue inflammation. Conclusion: Latanoprost reduced fat volume without inducing cell lysis or inflammation.
Assuntos
Acetato de Desoxicorticosterona , Lipossomos , Humanos , Animais , Camundongos , Latanoprosta/uso terapêutico , Preparações de Ação Retardada , Adiposidade , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêuticoRESUMO
Clinical sequencing efforts are rapidly identifying sarcoma gene fusions that lack functional validation. An example is the fusion of transcriptional coactivators, VGLL2-NCOA2, found in infantile rhabdomyosarcoma. To delineate VGLL2-NCOA2 tumorigenic mechanisms and identify therapeutic vulnerabilities, we implement a cross-species comparative oncology approach with zebrafish, mouse allograft, and patient samples. We find that VGLL2-NCOA2 is sufficient to generate mesenchymal tumors that display features of immature skeletal muscle and recapitulate the human disease. A subset of VGLL2-NCOA2 zebrafish tumors transcriptionally cluster with embryonic somitogenesis and identify VGLL2-NCOA2 developmental programs, including a RAS family GTPase, ARF6. In VGLL2-NCOA2 zebrafish, mouse, and patient tumors, ARF6 is highly expressed. ARF6 knockout suppresses VGLL2-NCOA2 oncogenic activity in cell culture, and, more broadly, ARF6 is overexpressed in adult and pediatric sarcomas. Our data indicate that VGLL2-NCOA2 is an oncogene that leverages developmental programs for tumorigenesis and that reactivation or persistence of ARF6 could represent a therapeutic opportunity.