RESUMEN
BACKGROUND & AIMS: Patients with metastatic, treatment-refractory, and relapsed hepatoblastoma (HB) have survival rates of less than 50% due to limited treatment options. To develop new therapeutic strategies for these patients, our laboratory has developed a preclinical testing pipeline. Given that histone deacetylase (HDAC) inhibition has been proposed for HB, we hypothesized that we could find an effective combination treatment strategy utilizing HDAC inhibition. METHODS: RNA sequencing, microarray, NanoString, and immunohistochemistry data of patient HB samples were analyzed for HDAC class expression. Patient-derived spheroids (PDSp) were used to screen combination chemotherapy with an HDAC inhibitor, panobinostat. Patient-derived xenograft (PDX) mouse models were developed and treated with the combination therapy that showed the highest efficacy in the PDSp drug screen. RESULTS: HDAC RNA and protein expression were elevated in HB tumors compared to normal livers. Panobinostat (IC50 of 0.013-0.059 µM) showed strong in vitro effects and was associated with lower cell viability than other HDAC inhibitors. PDSp demonstrated the highest level of cell death with combination treatment of vincristine/irinotecan/panobinostat (VIP). All four models responded to VIP therapy with a decrease in tumor size compared to placebo. After 6 weeks of treatment, two models demonstrated necrotic cell death, with lower Ki67 expression, decreased serum alpha fetoprotein and reduced tumor burden compared to paired VI- and placebo-treated groups. CONCLUSIONS: Utilizing a preclinical HB pipeline, we demonstrate that panobinostat in combination with VI chemotherapy can induce an effective tumor response in models developed from patients with high-risk, relapsed, and treatment-refractory HB. IMPACT AND IMPLICATIONS: Patients with treatment-refractory hepatoblastoma have limited treatment options with survival rates of less than 50%. Our manuscript demonstrates that combination therapy with vincristine, irinotecan, and panobinostat reduces the size of high-risk, relapsed, and treatment-refractory tumors. With this work we provide preclinical evidence to support utilizing this combination therapy as an arm in future clinical trials.
Asunto(s)
Hepatoblastoma , Neoplasias Hepáticas , Humanos , Ratones , Animales , Panobinostat/farmacología , Panobinostat/uso terapéutico , Hepatoblastoma/tratamiento farmacológico , Irinotecán/uso terapéutico , Vincristina/uso terapéutico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/inducido químicamente , Inhibidores de Histona Desacetilasas/uso terapéutico , Neoplasias Hepáticas/patología , Ácidos Hidroxámicos/farmacologíaRESUMEN
Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with 5-year overall survival of less than 10% in patients over the age of 65. Limited progress has been made in the patient outcome because of the inability to selectively eradicate the leukemic stem cells (LSC) driving the refractory and relapsed disease. Herein, we investigated the role of the reprogramming factor KLF4 in AML because of its critical role in the self-renewal and stemness of embryonic and cancer stem cells. Using a conditional Cre-lox Klf4 deletion system and the MLL-AF9 retroviral mouse model, we demonstrated that loss-of-KLF4 does not significantly affect the induction of leukemia but markedly decreased the frequency of LSCs evaluated in limiting-dose transplantation studies. Loss of KLF4 in leukemic granulocyte-macrophage progenitors (L-GMP), a population enriched for AML LSCs, showed lessened clonogenicity and percentage in the G2/M phase of the cell cycle. RNAseq analysis of purified L-GMPs revealed decreased expression of stemness genes and MLL-target genes and upregulation of the RNA sensing helicase DDX58. However, silencing of DDX58 in KLF4 knockout leukemia indicated that DDX58 is not mediating this phenotype. CRISPR/Cas9 deletion of KLF4 in MOLM13 cell line and AML patient-derived xenograft cells showed impaired expansion in vitro and in vivo associated with a defective G2/M checkpoint. Collectively, our data suggest a mechanism in which KLF4 promotes leukemia progression by establishing a gene expression profile in AML LSCs supporting cell division and stemness.
Asunto(s)
Factor 4 Similar a Kruppel , Leucemia Mieloide Aguda , Animales , Médula Ósea/patología , Modelos Animales de Enfermedad , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas de Fusión Oncogénica/metabolismoRESUMEN
BACKGROUND & AIMS: Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the predominant liver cancers in children, though their respective treatment options and associated outcomes differ dramatically. Risk stratification using a combination of clinical, histological, and molecular parameters can improve treatment selection, but it is particularly challenging for tumors with mixed histological features, including those in the recently created hepatocellular neoplasm not otherwise specified (HCN NOS) provisional category. We aimed to perform the first molecular characterization of clinically annotated cases of HCN NOS. METHODS: We tested whether these histological features are associated with genetic alterations, cancer gene dysregulation, and outcomes. Namely, we compared the molecular features of HCN NOS, including copy number alterations, mutations, and gene expression profiles, with those in other pediatric hepatocellular neoplasms, including HBs and HCCs, as well as HBs demonstrating focal atypia or pleomorphism (HB FPAs), and HBs diagnosed in older children (>8). RESULTS: Molecular profiles of HCN NOS and HB FPAs revealed common underlying biological features that were previously observed in HCCs. Consequently, we designated these tumor types collectively as HBs with HCC features (HBCs). These tumors were associated with high mutation rates (â¼3 somatic mutations/Mb) and were enriched with mutations and alterations in key cancer genes and pathways. In addition, recurrent large-scale chromosomal gains, including gains of chromosomal arms 2q (80%), 6p (70%), and 20p (70%), were observed. Overall, HBCs were associated with poor clinical outcomes. CONCLUSIONS: Our study indicates that histological features seen in HBCs are associated with combined molecular features of HB and HCC, that HBCs are associated with poor outcomes irrespective of patient age, and that transplanted patients are more likely to have good outcomes than those treated with chemotherapy and surgery alone. These findings highlight the importance of molecular testing and early therapeutic intervention for aggressive childhood hepatocellular neoplasms. LAY SUMMARY: We molecularly characterized a class of histologically aggressive childhood liver cancers and showed that these tumors are clinically aggressive and that their observed histological features are associated with underlying recurrent molecular features. We proposed a diagnostic algorithm to identify these cancers using a combination of histological and molecular features, and our analysis suggested that these cancers may benefit from specialized treatment strategies that may differ from treatment guidelines for other childhood liver cancers.
Asunto(s)
Carcinoma Hepatocelular , Hepatoblastoma , Neoplasias Hepáticas , Carcinoma Hepatocelular/patología , Niño , Aberraciones Cromosómicas , Hepatoblastoma/metabolismo , Humanos , Neoplasias Hepáticas/patología , Mutación , Adulto JovenRESUMEN
Despite being the most common liver cancer in children, hepatoblastoma (HB) is a rare neoplasm. Consequently, few pretreatment tumors have been molecularly profiled, and there are no validated prognostic or therapeutic biomarkers for HB patients. We report on the first large-scale effort to profile pretreatment HBs at diagnosis. Our analysis of 88 clinically annotated HBs revealed three risk-stratifying molecular subtypes that are characterized by differential activation of hepatic progenitor cell markers and metabolic pathways: high-risk tumors were characterized by up-regulated nuclear factor, erythroid 2-like 2 activity; high lin-28 homolog B, high mobility group AT-hook 2, spalt-like transcription factor 4, and alpha-fetoprotein expression; and high coordinated expression of oncofetal proteins and stem-cell markers, while low-risk tumors had low lin-28 homolog B and lethal-7 expression and high hepatic nuclear factor 1 alpha activity. CONCLUSION: Analysis of immunohistochemical assays using antibodies targeting these genes in a prospective study of 35 HBs suggested that these candidate biomarkers have the potential to improve risk stratification and guide treatment decisions for HB patients at diagnosis; our results pave the way for clinical collaborative studies to validate candidate biomarkers and test their potential to improve outcome for HB patients. (Hepatology 2017;65:104-121).
Asunto(s)
Hepatoblastoma/genética , Neoplasias Hepáticas/genética , Regulación Neoplásica de la Expresión Génica , Genómica , Hepatoblastoma/clasificación , Humanos , Neoplasias Hepáticas/clasificación , PronósticoRESUMEN
BACKGROUND & AIMS: Pediatric liver cancer is a rare but serious disease whose incidence is rising, and for which the therapeutic options are limited. Development of more targeted, less toxic therapies is hindered by the lack of an experimental animal model that captures the heterogeneity and metastatic capability of these tumors. METHODS: Here we established an orthotopic engraftment technique to model a series of patient-derived tumor xenograft (PDTX) from pediatric liver cancers of all major histologic subtypes: hepatoblastoma, hepatocellular cancer and hepatocellular malignant neoplasm. We utilized standard (immuno) staining methods for histological characterization, RNA sequencing for gene expression profiling and genome sequencing for identification of druggable targets. We also adapted stem cell culturing techniques to derive two new pediatric cancer cell lines from the xenografted mice. RESULTS: The patient-derived tumor xenografts recapitulated the histologic, genetic, and biological characteristics-including the metastatic behavior-of the corresponding primary tumors. Furthermore, the gene expression profiles of the two new liver cancer cell lines closely resemble those of the primary tumors. Targeted therapy of PDTX from an aggressive hepatocellular malignant neoplasm with the MEK1 inhibitor trametinib and pan-class I PI3 kinase inhibitor NVP-BKM120 resulted in significant growth inhibition, thus confirming this PDTX model as a valuable tool to study tumor biology and patient-specific therapeutic responses. CONCLUSIONS: The novel metastatic xenograft model and the isogenic xenograft-derived cell lines described in this study provide reliable tools for developing mutation- and patient-specific therapies for pediatric liver cancer. LAY SUMMARY: Pediatric liver cancer is a rare but serious disease and no experimental animal model currently captures the complexity and metastatic capability of these tumors. We have established a novel animal model using human tumor tissue that recapitulates the genetic and biological characteristics of this cancer. We demonstrate that our patient-derived animal model, as well as two new cell lines, are useful tools for experimental therapies.
Asunto(s)
Neoplasias Hepáticas , Animales , Carcinoma Hepatocelular , Línea Celular Tumoral , Niño , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Ratones , Trasplante de Neoplasias , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The stomach-derived hormone ghrelin regulates essential physiological functions. The ghrelin receptor (GHSR) has ligand-independent actions; therefore, GHSR gene deletion may be a reasonable approach to investigate the role of this system in feeding behaviors and diet-induced obesity (DIO). Here, we investigate the effects of a long-term (12-month) high-fat (HFD) versus regular diet on obesity-related measures in global GHSR-KO and wild-type (WT) Wistar male and female rats. Our main findings are that the GHSR gene deletion protects against DIO and decreases food intake during HFD in male but not in female rats. GHSR gene deletion increases thermogenesis and brain glucose uptake in male rats and modifies the effects of HFD on brain glucose metabolism in a sex-specific manner, as assessed with small animal positron emission tomography. We use RNA-sequencing to show that GHSR-KO rats have upregulated expression of genes responsible for fat oxidation in brown adipose tissue. Central administration of a novel GHSR inverse agonist, PF-5190457, attenuates ghrelin-induced food intake, but only in male, not in female mice. HFD-induced binge-like eating is reduced by inverse agonism in both sexes. Our results support GHSR as a promising target for new pharmacotherapies for obesity.
Asunto(s)
Dieta Alta en Grasa , Obesidad , Ratas Wistar , Receptores de Ghrelina , Caracteres Sexuales , Animales , Receptores de Ghrelina/genética , Receptores de Ghrelina/metabolismo , Dieta Alta en Grasa/efectos adversos , Masculino , Femenino , Ratas , Obesidad/metabolismo , Obesidad/genética , Ghrelina/metabolismo , Termogénesis/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/efectos de los fármacosRESUMEN
The stomach-derived hormone ghrelin regulates essential physiological functions. The ghrelin receptor (GHSR) has ligand-independent actions, therefore, GHSR gene deletion may be a reasonable approach to investigate the role of this system in feeding behaviors and diet-induced obesity (DIO). Here we investigated the effects of a long-term (12 month) high-fat (HFD) versus regular diet on obesity-related measures in global GHSR-KO and wild type (WT) Wistar male and female rats. Our main findings were that the GHSR gene deletion protects against DIO and decreases food intake during HFD in male but not in female rats. GHSR gene deletion increased thermogenesis and brain glucose uptake in male rats and modified the effects of HFD on brain glucose metabolism in a sex-specific manner, as assessed with small animal positron emission tomography. RNA-sequencing was also used to show that GHSR-KO rats had upregulated expression of genes responsible for fat oxidation in brown adipose tissue. Central administration of a novel GHSR inverse agonist, PF-5190457, attenuated ghrelin-induced food intake, but only in male, not in female mice. HFD-induced binge-like eating was reduced by inverse agonism in both sexes. Our results support GHSR as a promising target for new pharmacotherapies for obesity.
RESUMEN
Children with Down syndrome (DS) are 10-fold more likely to develop B-cell acute lymphoblastic leukemia (B-ALL), with a higher frequency of rearrangements resulting in overexpression of cytokine receptor-like factor 2 (CRLF2). Here, we investigated the impact of CRLF2 overexpression on B-cell progenitor proliferation, immunophenotype, and gene expression profile in the Dp(16)1Yey (Dp16) mouse model of DS compared with wild-type (WT) mice. CRLF2 overexpression enhanced immature B-lymphoid colony development and increased the proportion of less differentiated pre-pro-B cells, with a greater effect in Dp16 versus WT. In CRLF2-rearranged (CRLF2-R) B-ALL patient samples, cells with higher CRLF2 expression exhibited a less differentiated B-cell immunophenotype. CRLF2 overexpression resulted in a gene expression signature associated with E2F signaling both in Dp16 B-progenitors and in DS-ALL patient samples, and PI3K/mTOR and pan-CDK inhibitors, which reduce E2F-mediated signaling, exhibited cytotoxicity in CRLF2-R B-ALL cell lines and patient samples. CRLF2 overexpression alone in Dp16 stem and progenitor cells did not result in leukemic transformation in recipient mice. Thus, CRLF2 overexpression results in reduced B-cell differentiation and enhanced E2F signaling in Dp16 B-progenitor cells and DS-ALL patient samples. These findings suggest a functional basis for the high frequency of CRLF2-R in DS-ALL as well as a potential therapeutically targetable pathway.
Asunto(s)
Síndrome de Down , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Síndrome de Down/complicaciones , Síndrome de Down/genética , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Citocinas/genética , Transducción de SeñalRESUMEN
Substance use disorder is associated with accelerated disease progression in people with human immunodeficiency virus (HIV; PWH). Problem opioid use, including high-dose opioid therapy, prescription drug misuse, and opioid abuse, is high and increasing in the PWH population. Oxycodone is a broadly prescribed opioid in both the general population and PWH. Here, we allowed HIV transgenic (Tg) rats and wildtype (WT) littermates to intravenously self-administer oxycodone under short-access (ShA) conditions, which led to moderate, stable, "recreational"-like levels of drug intake, or under long-access (LgA) conditions, which led to escalated (dependent) drug intake. HIV Tg rats with histories of oxycodone self-administration under LgA conditions exhibited significant impairment in memory performance in the novel object recognition (NOR) paradigm. RNA-sequencing expression profiling of the medial prefrontal cortex (mPFC) in HIV Tg rats that self-administered oxycodone under ShA conditions exhibited greater transcriptional evidence of inflammation than WT rats that self-administered oxycodone under the same conditions. HIV Tg rats that self-administered oxycodone under LgA conditions exhibited transcriptional evidence of an increase in neuronal injury and neurodegeneration compared with WT rats under the same conditions. Gene expression analysis indicated that glucocorticoid-dependent adaptations contributed to the gene expression effects of oxycodone self-administration. Overall, the present results indicate that a history of opioid intake promotes neuroinflammation and glucocorticoid dysregulation, and excessive opioid intake is associated with neurotoxicity and cognitive impairment in HIV Tg rats.
Asunto(s)
Disfunción Cognitiva , Infecciones por VIH , Analgésicos Opioides/efectos adversos , Animales , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/complicaciones , Glucocorticoides , VIH , Infecciones por VIH/complicaciones , Humanos , Oxicodona/efectos adversos , Ratas , Ratas TransgénicasRESUMEN
Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. MYCN amplification is found in half of high-risk NB patients; however, no available therapies directly target MYCN. Using multi-dimensional metabolic profiling in MYCN expression systems and primary patient tumors, we comprehensively characterized the metabolic landscape driven by MYCN in NB. MYCN amplification leads to glycerolipid accumulation by promoting fatty acid (FA) uptake and biosynthesis. We found that cells expressing amplified MYCN depend highly on FA uptake for survival. Mechanistically, MYCN directly upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic depletion of SLC27A2 impairs NB survival, and pharmacological SLC27A2 inhibition selectively suppresses tumor growth, prolongs animal survival, and exerts synergistic anti-tumor effects when combined with conventional chemotherapies in multiple preclinical NB models. This study identifies FA uptake as a critical metabolic dependency for MYCN-amplified tumors. Inhibiting FA uptake is an effective approach for improving current treatment regimens.
Asunto(s)
Ácidos Grasos , Neuroblastoma , Animales , Línea Celular Tumoral , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/metabolismoRESUMEN
Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence-activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Hepatoblastoma , Neoplasias Hepáticas , Células Neoplásicas Circulantes , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Hepatoblastoma/genética , Hepatoblastoma/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , RatonesRESUMEN
Membrane proteins comprise a significant fraction of the proteomes of sequenced organisms and are the targets of approximately half of marketed drugs. However, in spite of their prevalence and biomedical importance, relatively few experimental structures are available due to technical challenges. Computational simulations can potentially address this deficit by providing structural models of membrane proteins. Solvation within the spatially heterogeneous membrane/solvent environment provides a major component of the energetics driving protein folding and association within the membrane. We have developed an implicit solvation model for membranes that is both computationally efficient and accurate enough to enable molecular mechanics predictions for the folding and association of peptides within the membrane. We derived the new atomic solvation model parameters using an unbiased fitting procedure to experimental data and have applied it to diverse problems in order to test its accuracy and to gain insight into membrane protein folding. First, we predicted the positions and orientations of peptides and complexes within the lipid bilayer and compared the simulation results with solid-state NMR structures. Additionally, we performed folding simulations for a series of host-guest peptides with varying propensities to form alpha helices in a hydrophobic environment and compared the structures with experimental measurements. We were also able to successfully predict the structures of amphipathic peptides as well as the structures for dimeric complexes of short hexapeptides that have experimentally characterized propensities to form beta sheets within the membrane. Finally, we compared calculated relative transfer energies with data from experiments measuring the effects of mutations on the free energies of translocon-mediated insertion of proteins into lipid bilayers and of combined folding and membrane insertion of a beta barrel protein.
Asunto(s)
Simulación por Computador , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Modelos Moleculares , Transferencia de Energía , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/metabolismo , Simulación de Dinámica Molecular , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Soluciones/química , TermodinámicaRESUMEN
BACKGROUND & AIMS: The accumulation of neutral lipids within hepatocytes underlies non-alcoholic fatty liver disease (NAFLD), which affects a quarter of the world's population and is associated with hepatitis, cirrhosis, and hepatocellular carcinoma. Despite insights gained from both human and animal studies, our understanding of NAFLD pathogenesis remains limited. To better study the molecular changes driving the condition we aimed to generate a humanised NAFLD mouse model. METHODS: We generated TIRF (transgene-free Il2rg -/-/Rag2 -/-/Fah -/-) mice, populated their livers with human hepatocytes, and fed them a Western-type diet for 12 weeks. RESULTS: Within the same chimeric liver, human hepatocytes developed pronounced steatosis whereas murine hepatocytes remained normal. Unbiased metabolomics and lipidomics revealed signatures of clinical NAFLD. Transcriptomic analyses showed that molecular responses diverged sharply between murine and human hepatocytes, demonstrating stark species differences in liver function. Regulatory network analysis indicated close agreement between our model and clinical NAFLD with respect to transcriptional control of cholesterol biosynthesis. CONCLUSIONS: These NAFLD xenograft mice reveal an unexpected degree of evolutionary divergence in food metabolism and offer a physiologically relevant, experimentally tractable model for studying the pathogenic changes invoked by steatosis. LAY SUMMARY: Fatty liver disease is an emerging health problem, and as there are no good experimental animal models, our understanding of the condition is poor. We here describe a novel humanised mouse system and compare it with clinical data. The results reveal that the human cells in the mouse liver develop fatty liver disease upon a Western-style fatty diet, whereas the mouse cells appear normal. The molecular signature (expression profiles) of the human cells are distinct from the mouse cells and metabolic analysis of the humanised livers mimic the ones observed in humans with fatty liver. This novel humanised mouse system can be used to study human fatty liver disease.
RESUMEN
Neuroblastoma (NB) arises from oncogenic disruption of neural crest (NC) differentiation. Treatment with retinoic acid (RA) to induce differentiation has improved survival in some NB patients, but not all patients respond, and most NBs eventually develop resistance to RA. Loss of the chromatin modifier chromatin assembly factor 1 subunit p150 (CHAF1A) promotes NB cell differentiation; however, the mechanism by which CHAF1A drives NB oncogenesis has remained unexplored. This study shows that CHAF1A gain-of-function supports cell malignancy, blocks neuronal differentiation in three models (zebrafish NC, human NC, and human NB), and promotes NB oncogenesis. Mechanistically, CHAF1A upregulates polyamine metabolism, which blocks neuronal differentiation and promotes cell cycle progression. Targeting polyamine synthesis promotes NB differentiation and enhances the anti-tumor activity of RA. The authors' results provide insight into the mechanisms that drive NB oncogenesis and suggest a rapidly translatable therapeutic approach (DFMO plus RA) to enhance the clinical efficacy of differentiation therapy in NB patients.
Asunto(s)
Carcinogénesis/metabolismo , Diferenciación Celular/genética , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Neuroblastoma/metabolismo , Neuronas/metabolismo , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Factor 1 de Ensamblaje de la Cromatina/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Neuroblastoma/genética , Pez CebraRESUMEN
In October 2020, 62 scientists from nine nations worked together remotely in the Second Baylor College of Medicine & DNAnexus hackathon, focusing on different related topics on Structural Variation, Pan-genomes, and SARS-CoV-2 related research. The overarching focus was to assess the current status of the field and identify the remaining challenges. Furthermore, how to combine the strengths of the different interests to drive research and method development forward. Over the four days, eight groups each designed and developed new open-source methods to improve the identification and analysis of variations among species, including humans and SARS-CoV-2. These included improvements in SV calling, genotyping, annotations and filtering. Together with advancements in benchmarking existing methods. Furthermore, groups focused on the diversity of SARS-CoV-2. Daily discussion summary and methods are available publicly at https://github.com/collaborativebioinformatics provides valuable insights for both participants and the research community.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Genoma Viral , Humanos , VertebradosRESUMEN
The abuse of stimulants, such as methamphetamine (METH), is associated with treatment non-compliance, a greater risk of viral transmission, and the more rapid clinical progression of immunological and central nervous system human immunodeficiency virus (HIV) disease. The behavioral effects of METH in the setting of HIV remain largely uncharacterized. We used a state-of-the-art paradigm of the escalation of voluntary intravenous drug self-administration in HIV transgenic (Tg) and wildtype rats. The rats were first allowed to self-administer METH under short-access (ShA) conditions, which is characterized by a nondependent and more "recreational" pattern of METH use, and then allowed to self-administer METH under long-access (LgA) conditions, which leads to compulsive (dependent) METH intake. HIV Tg and wildtype rats self-administered equal amounts of METH under ShA conditions. HIV Tg rats self-administered METH under LgA conditions following a 4-week enforced abstinence period to model the intermittent pattern of stimulant abuse in humans. These HIV Tg rats developed greater motivation to self-administer METH and self-administered larger amounts of METH. Impairments in function of the medial prefrontal cortex (mPFC) contribute to compulsive drug and alcohol intake. Gene expression profiling of the mPFC in HIV Tg rats with a history of escalated METH self-administration under LgA conditions showed transcriptional evidence of increased inflammation, greater neural injury, and impaired aerobic glucose metabolism than wildtype rats that self-administered METH under LgA conditions. The detrimental effects of the interaction between neuroHIV and escalated METH intake on the mPFC are likely key factors in the greater vulnerability to excessive drug intake in the setting of HIV.
Asunto(s)
Estimulantes del Sistema Nervioso Central/administración & dosificación , Conducta Compulsiva/complicaciones , Encefalitis/complicaciones , Infecciones por VIH/complicaciones , Infecciones por VIH/metabolismo , Metanfetamina/administración & dosificación , Animales , Conducta Compulsiva/virología , Encefalitis/metabolismo , Encefalitis/virología , Expresión Génica/efectos de los fármacos , Masculino , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/virología , Ratas TransgénicasRESUMEN
Internal RNA modifications have been known for decades, however their roles in mRNA regulation have only recently started to be elucidated. Here we investigated the most abundant mRNA modification, N6-methyladenosine (m6A) in transcripts from the hippocampus of HIV transgenic (Tg) rats. The distribution of m6A peaks within HIV transcripts in HIV Tg rats largely corresponded to the ones observed for HIV transcripts in cell lines and T cells. Host transcripts were found to be differentially m6A methylated in HIV Tg rats. The functional roles of the differentially m6A methylated pathways in HIV Tg rats is consistent with a key role of RNA methylation in the regulation of the brain transcriptome in chronic HIV disease. In particular, host transcripts show significant differential m6A methylation of genes involved in several pathways related to neural function, suggestive of synaptodendritic injury and neurodegeneration, inflammation and immune response, as well as RNA processing and metabolism, such as splicing. Changes in m6A methylation were usually positively correlated with differential expression, while differential m6A methylation of pathways involved in RNA processing were more likely to be negatively correlated with gene expression changes. Thus, sets of differentially m6A methylated, functionally-related transcripts appear to be involved in coordinated transcriptional responses in the context of chronic HIV. Altogether, our results support that m6A methylation represents an additional layer of regulation of HIV and host gene expression in vivo that contributes significantly to the transcriptional effects of chronic HIV.
Asunto(s)
Complejo SIDA Demencia/genética , VIH-1/genética , Hipocampo/patología , ARN Mensajero/genética , Transcriptoma/genética , Complejo SIDA Demencia/inmunología , Complejo SIDA Demencia/patología , Complejo SIDA Demencia/virología , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Modelos Animales de Enfermedad , Epigénesis Genética/genética , Epigénesis Genética/inmunología , Regulación de la Expresión Génica/inmunología , VIH-1/inmunología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Masculino , Metilación , Empalme del ARN/genética , ARN Mensajero/aislamiento & purificación , ARN Viral/genética , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Análisis de Secuencia de ARN , Transcriptoma/inmunologíaRESUMEN
Long noncoding RNAs (lncRNAs) are commonly dysregulated in tumors, but only a handful are known to play pathophysiological roles in cancer. We inferred lncRNAs that dysregulate cancer pathways, oncogenes, and tumor suppressors (cancer genes) by modeling their effects on the activity of transcription factors, RNA-binding proteins, and microRNAs in 5,185 TCGA tumors and 1,019 ENCODE assays. Our predictions included hundreds of candidate onco- and tumor-suppressor lncRNAs (cancer lncRNAs) whose somatic alterations account for the dysregulation of dozens of cancer genes and pathways in each of 14 tumor contexts. To demonstrate proof of concept, we showed that perturbations targeting OIP5-AS1 (an inferred tumor suppressor) and TUG1 and WT1-AS (inferred onco-lncRNAs) dysregulated cancer genes and altered proliferation of breast and gynecologic cancer cells. Our analysis indicates that, although most lncRNAs are dysregulated in a tumor-specific manner, some, including OIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergistically dysregulate cancer pathways in multiple tumor contexts.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , ARN Largo no Codificante/genética , Línea Celular , Línea Celular Tumoral , Redes Reguladoras de Genes , Genes Supresores de Tumor , Humanos , OncogenesRESUMEN
Only one out of 10 drugs in development passes clinical trials. Many fail because experimental animal models poorly predict human xenobiotic metabolism. Human liver chimeric mice are a step forward in this regard, as the human hepatocytes in chimeric livers generate human metabolites, but the remaining murine hepatocytes contain an expanded set of P450 cytochromes that form the major class of drug-metabolizing enzymes. We therefore generated a conditional knock-out of the NADPH-P450 oxidoreductase (Por) gene combined with Il2rg - /- /Rag2 - /- /Fah - /- (PIRF) mice. Here we show that homozygous PIRF mouse livers are readily repopulated with human hepatocytes, and when the murine Por gene is deleted (<5%), they predominantly use human cytochrome metabolism. When given the anticancer drug gefitinib or the retroviral drug atazanavir, the Por-deleted humanized PIRF mice develop higher levels of the major human metabolites than current models. Humanized, murine Por-deficient PIRF mice can thus predict human drug metabolism and should be useful for preclinical drug development.Human liver chimeric mice are increasingly used for drug testing in preclinical development, but express residual murine p450 cytochromes. Here the authors generate mice lacking the Por gene in the liver, and show that human cytochrome metabolism is used following repopulation with human hepatocytes.