RESUMEN
Multiple myeloma is a treatable, but currently incurable, hematological malignancy of plasma cells characterized by diverse and complex tumor genetics for which precision medicine approaches to treatment are lacking. The Multiple Myeloma Research Foundation's Relating Clinical Outcomes in Multiple Myeloma to Personal Assessment of Genetic Profile study ( NCT01454297 ) is a longitudinal, observational clinical study of newly diagnosed patients with multiple myeloma (n = 1,143) where tumor samples are characterized using whole-genome sequencing, whole-exome sequencing and RNA sequencing at diagnosis and progression, and clinical data are collected every 3 months. Analyses of the baseline cohort identified genes that are the target of recurrent gain-of-function and loss-of-function events. Consensus clustering identified 8 and 12 unique copy number and expression subtypes of myeloma, respectively, identifying high-risk genetic subtypes and elucidating many of the molecular underpinnings of these unique biological groups. Analysis of serial samples showed that 25.5% of patients transition to a high-risk expression subtype at progression. We observed robust expression of immunotherapy targets in this subtype, suggesting a potential therapeutic option.
RESUMEN
Osteogenesis imperfecta (OI) type V is the second most common form of OI, distinguished by hyperplastic callus formation and calcification of the interosseous membranes, in addition to the bone fragility. It is caused by a recurrent, dominant pathogenic variant (c.-14C>T) in interferon-induced transmembrane protein 5 (IFITM5). Here, we generated a conditional Rosa26-knockin mouse model to study the mechanistic consequences of the recurrent mutation. Expression of the mutant Ifitm5 in osteo-chondroprogenitor or chondrogenic cells resulted in low bone mass and growth retardation. Mutant limbs showed impaired endochondral ossification, cartilage overgrowth, and abnormal growth plate architecture. The cartilage phenotype correlates with the pathology reported in patients with OI type V. Surprisingly, expression of mutant Ifitm5 in mature osteoblasts caused no obvious skeletal abnormalities. In contrast, earlier expression in osteo-chondroprogenitors was associated with an increase in the skeletal progenitor cell population within the periosteum. Lineage tracing showed that chondrogenic cells expressing the mutant Ifitm5 had decreased differentiation into osteoblastic cells in diaphyseal bone. Moreover, mutant IFITM5 disrupted early skeletal homeostasis in part by activating ERK signaling and downstream SOX9 protein, and inhibition of these pathways partially rescued the phenotype in mutant animals. These data identify the contribution of a signaling defect altering osteo-chondroprogenitor differentiation as a driver in the pathogenesis of OI type V.
Asunto(s)
Diferenciación Celular , Sistema de Señalización de MAP Quinasas , Osteoblastos , Osteogénesis Imperfecta , Factor de Transcripción SOX9 , Animales , Femenino , Masculino , Ratones , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Mutación , Osteoblastos/metabolismo , Osteoblastos/patología , Osteogénesis/genética , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/patología , Osteogénesis Imperfecta/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Células Madre/metabolismo , Células Madre/patología , Quinasas MAP Reguladas por Señal ExtracelularRESUMEN
The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) remains a public health concern and a subject of active research effort. Development of pre-clinical animal models is critical to study viral-host interaction, tissue tropism, disease mechanisms, therapeutic approaches, and long-term sequelae of infection. Here, we report two mouse models for studying SARS-CoV-2: A knock-in mAce2F83Y,H353K mouse that expresses a mouse-human hybrid form of the angiotensin-converting enzyme 2 (ACE2) receptor under the endogenous mouse Ace2 promoter, and a Rosa26 conditional knock-in mouse carrying the human ACE2 allele (Rosa26hACE2). Although the mAce2F83Y,H353K mice were susceptible to intranasal inoculation with SARS-CoV-2, they did not show gross phenotypic abnormalities. Next, we generated a Rosa26hACE2;CMV-Cre mouse line that ubiquitously expresses the human ACE2 receptor. By day 3 post infection with SARS-CoV-2, Rosa26hACE2;CMV-Cre mice showed significant weight loss, a variable degree of alveolar wall thickening and reduced survival rates. Viral load measurements confirmed inoculation in lung and brain tissues of infected Rosa26hACE2;CMV-Cre mice. The phenotypic spectrum displayed by our different mouse models translates to the broad range of clinical symptoms seen in the human patients and can serve as a resource for the community to model and explore both treatment strategies and long-term consequences of SARS-CoV-2 infection.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Modelos Animales de Enfermedad , SARS-CoV-2 , Animales , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/genética , COVID-19/patología , COVID-19/virología , Ratones , Humanos , SARS-CoV-2/genética , Ratones Transgénicos , Pulmón/virología , Pulmón/patología , Pulmón/metabolismo , Técnicas de Sustitución del GenRESUMEN
BACKGROUND: Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. METHODS: To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. RESULTS: C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. CONCLUSION: We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.
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Cinesinas , Osteogénesis Imperfecta , Animales , Humanos , Ratones , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/genética , Regulación hacia Abajo , Cinesinas/genética , Cinesinas/metabolismo , Células 3T3 NIH , Proteómica , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.
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Huesos/metabolismo , Proteína Coat de Complejo I/genética , Proteína Coatómero/genética , Discapacidades del Desarrollo/genética , Discapacidad Intelectual/genética , Osteoporosis/genética , Animales , Ácido Ascórbico/farmacología , Huesos/efectos de los fármacos , Huesos/patología , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Niño , Preescolar , Proteína Coat de Complejo I/deficiencia , Proteína Coatómero/química , Proteína Coatómero/deficiencia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Embrión no Mamífero , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación del Desarrollo de la Expresión Génica , Aparato de Golgi , Haploinsuficiencia , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Masculino , Ratones , Osteoporosis/tratamiento farmacológico , Osteoporosis/metabolismo , Osteoporosis/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Índice de Severidad de la Enfermedad , Pez CebraRESUMEN
Mosquito-borne pathogens contribute significantly to the global burden of infectious diseases and are a continuing public health concern in the United States. Blood feeding by vector mosquitoes is a critical step in the transmission of human pathogens. Continuous surveillance of mosquito feeding patterns, especially in major population centers, is necessary for sustainable, effective control strategies. To better understand female feeding habits in Harris County, TX, we trapped mosquitoes from various locations, distributed among urban and semi-urban environments. Bloodmeal hosts were determined using a cytochrome C oxidase I DNA barcoding strategy. We identified a diverse array of vertebrate hosts with a high degree of avian host utilization, most surprisingly from anthropophilic species like Aedes aegypti (L.). We also detected sequences from two different vertebrate hosts in about half of specimens examined, suggesting that multiple bloodmeals had been acquired in the same feeding cycle by a sizable fraction of females in both urban and semi-urban locations. The high proportion of feeding on domestic chickens may indicate that a significant number of homeowners are rearing chickens within close proximity to study trap sites. As non-amplifying hosts, chickens may have a diluting effect on West Nile virus, as well as a zooprophylactic effect in their immediate vicinities. Ultimately, spatial and temporal host utilization patterns add insight into potential disease transmission dynamics, thereby informing vector control strategies in Harris County and other metropolitan areas.
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Culicidae/fisiología , Mosquitos Vectores/fisiología , Aedes/fisiología , Animales , Culex/fisiología , Conducta Alimentaria , Femenino , TexasRESUMEN
Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.
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Melanoma/genética , Melanoma/veterinaria , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/veterinaria , Animales , Ciclo Celular/genética , Proliferación Celular/genética , Hibridación Genómica Comparativa , Análisis Mutacional de ADN , Enfermedades de los Perros/genética , Perros , Femenino , Masculino , Melanoma/sangre , Melanoma/patología , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Transducción de Señal/genética , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/patología , Análisis de Matrices TisularesRESUMEN
Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT.Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT.Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%.Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932-43. ©2018 AACR.
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Antineoplásicos/farmacología , Carcinoma de Células Pequeñas/metabolismo , Carcinoma de Células Pequeñas/patología , Imidazoles/farmacología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/farmacología , Animales , Carcinoma de Células Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Biología Computacional/métodos , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Neoplasias Ováricas/tratamiento farmacológico , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , ARN Interferente Pequeño/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Multiple Myeloma (MM) is a plasma cell malignancy with significantly greater incidence and mortality rates among African Americans (AA) compared to Caucasians (CA). The overall goal of this study is to elucidate differences in molecular alterations in MM as a function of self-reported race and genetic ancestry. Our study utilized somatic whole exome, RNA-sequencing, and correlated clinical data from 718 MM patients from the Multiple Myeloma Research Foundation CoMMpass study Interim Analysis 9. Somatic mutational analyses based upon self-reported race corrected for ancestry revealed significant differences in mutation frequency between groups. Of interest, BCL7A, BRWD3, and AUTS2 demonstrate significantly higher mutation frequencies among AA cases. These genes are all involved in translocations in B-cell malignancies. Moreover, we detected a significant difference in mutation frequency of TP53 and IRF4 with frequencies higher among CA cases. Our study provides rationale for interrogating diverse tumor cohorts to best understand tumor genomics across populations.
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Factores Reguladores del Interferón/genética , Proteínas de Microfilamentos/genética , Mieloma Múltiple/genética , Proteínas Oncogénicas/genética , Proteínas/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Población Negra/genética , Proteínas del Citoesqueleto , Exoma/genética , Femenino , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/epidemiología , Mieloma Múltiple/patología , Mutación , Tasa de Mutación , Grupos Raciales , Población Blanca/genéticaRESUMEN
Early results of a prospective phase 2 clinical trial of carfilzomib, lenalidomide, and dexamethasone followed by lenalidomide maintenance in high-risk smoldering myeloma showed promising results that were previously published. Here, we provide novel insights into the genetic landscape of high-risk smoldering myeloma and information on sustained minimal residual disease (MRD) negativity with an expanded cohort of patients. Eighteen patients with high-risk smoldering myeloma were enrolled between 29 May 2012, and 14 January 2014. We included patients with newly diagnosed multiple myeloma enrolled in a parallel trial who received the same therapy (reference group). The overall response rate was 100%. With median potential follow-up of 43.3 months, 10 (63%) remain in MRD negativity, and the estimated 4-year progression-free and overall survival rates are 71% and 100%, respectively. Importantly, we report differences in mutational patterns in patients with high-risk smoldering myeloma and newly diagnosed multiple myeloma, reflected in a lower frequency of mutations in significant myeloma genes (6.6% vs 45%) and NFKB pathway genes (6.6% vs 25%). Treatment with carfilzomib, lenalidomide, and dexamethasone followed by lenalidomide maintenance was associated with a 100% response rate and 63% MRD negativity with a safety profile consistent with previous reports for this regimen. This study had a small numbers of participants, but there seemed to be important differences in the genetic landscape of patients with high-risk smoldering myeloma and those with newly diagnosed multiple myeloma, suggestive of a more treatment-responsive biology in early disease.