RESUMO
Intrinsic ureteropelvic junction obstruction is the most common cause of congenital hydronephrosis, yet the underlying pathogenesis is undefined. Hedgehog proteins control morphogenesis by promoting GLI-dependent transcriptional activation and inhibiting the formation of the GLI3 transcriptional repressor. Hedgehog regulates differentiation and proliferation of ureteric smooth muscle progenitor cells during murine kidney-ureter development. Histopathologic findings of smooth muscle cell hypertrophy and stroma-like cells, consistently observed in obstructing tissue at the time of surgical correction, suggest that Hedgehog signaling is abnormally regulated during the genesis of congenital intrinsic ureteropelvic junction obstruction. Here, we demonstrate that constitutively active Hedgehog signaling in murine intermediate mesoderm-derived renal progenitors results in hydronephrosis and failure to develop a patent pelvic-ureteric junction. Tissue obstructing the ureteropelvic junction was marked as early as E13.5 by an ectopic population of cells expressing Ptch2, a Hedgehog signaling target. Constitutive expression of GLI3 repressor in Ptch1-deficient mice rescued ectopic Ptch2 expression and obstructive hydronephrosis. Whole transcriptome analysis of isolated Ptch2+ cells revealed coexpression of genes characteristic of stromal progenitor cells. Genetic lineage tracing indicated that stromal cells blocking the ureteropelvic junction were derived from intermediate mesoderm-derived renal progenitors and were distinct from the smooth muscle or epithelial lineages. Analysis of obstructive ureteric tissue resected from children with congenital intrinsic ureteropelvic junction obstruction revealed a molecular signature similar to that observed in Ptch1-deficient mice. Together, these results demonstrate a Hedgehog-dependent mechanism underlying mammalian intrinsic ureteropelvic junction obstruction.
Assuntos
Proteínas Hedgehog/genética , Hidronefrose/genética , Proteínas do Tecido Nervoso/genética , Receptor Patched-1/genética , Receptor Patched-2/genética , Transdução de Sinais , Obstrução Ureteral/genética , Proteína Gli3 com Dedos de Zinco/genética , Aldeído Oxirredutases/genética , Animais , Linhagem da Célula , Criança , Feminino , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Hidronefrose/congênito , Hidronefrose/patologia , Hibridização In Situ , Pelve Renal/embriologia , Pelve Renal/metabolismo , Masculino , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Transcriptoma , Regulação para Cima , Ureter/embriologia , Ureter/metabolismo , Obstrução Ureteral/congênito , Obstrução Ureteral/patologia , Proteína Gli3 com Dedos de Zinco/metabolismoRESUMO
Pallister-Hall syndrome (PHS) is a rare disorder caused by mutations in GLI3 that produce a transcriptional repressor (GLI3R). Individuals with PHS present with a variably penetrant variety of urogenital system malformations, including renal aplasia or hypoplasia, hydroureter, hydronephrosis or a common urogenital sinus. The embryologic mechanisms controlled by GLI3R that result in these pathologic phenotypes are undefined. We demonstrate that germline expression of GLI3R causes renal hypoplasia, associated with decreased nephron number, and hydroureter and hydronephrosis, caused by blind-ending ureters. Mice with obligate GLI3R expression also displayed duplication of the ureters that was caused by aberrant common nephric duct patterning and ureteric stalk outgrowth. These developmental abnormalities are associated with suppressed Hedgehog signaling activity in the cloaca and adjacent vesicular mesenchyme. Mice with conditional expression of GLI3R were utilized to identify lineage-specific effects of GLI3R. In the ureteric bud, GLI3R expression decreased branching morphogenesis. In Six2-positive nephrogenic progenitors, GLI3R decreased progenitor cell proliferation reducing the number of nephrogenic precursor structures. Using mutant mice with Gli3R and Gli3 null alleles, we demonstrate that urogenital system patterning and development is controlled by the levels of GLI3R and not by an absence of full-length GLI3. We conclude that the urogenital system phenotypes observed in PHS are caused by GLI3R-dependent perturbations in nephric duct patterning, renal branching morphogenesis and nephrogenic progenitor self-renewal.
Assuntos
Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Hidronefrose/genética , Rim/anormalidades , Fatores de Transcrição Kruppel-Like/genética , Proteínas do Tecido Nervoso/genética , Síndrome de Pallister-Hall/genética , Anormalidades Urogenitais/genética , Animais , Padronização Corporal/genética , Proliferação de Células , Modelos Animais de Doenças , Embrião de Mamíferos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hidronefrose/metabolismo , Hidronefrose/patologia , Rim/metabolismo , Rim/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , Mutação , Néfrons/anormalidades , Néfrons/embriologia , Néfrons/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Síndrome de Pallister-Hall/metabolismo , Síndrome de Pallister-Hall/patologia , Fenótipo , Transdução de Sinais , Células-Tronco/metabolismo , Células-Tronco/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ureter/anormalidades , Ureter/embriologia , Ureter/metabolismo , Anormalidades Urogenitais/metabolismo , Anormalidades Urogenitais/patologia , Proteína Gli3 com Dedos de ZincoRESUMO
Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies.
Assuntos
Recombinação Homóloga , Imunidade Inata , Integrases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Integrases/genética , Macrófagos/metabolismo , CamundongosRESUMO
STUDY HYPOTHESIS: The mouse endometrium harbours stem/progenitor cells that express the stem cell marker mouse telomerase reverse transcriptase (mTert). STUDY FINDING: We used a mouse carrying a transgenic reporter for mTert promoter activity to identify rare endometrial populations of epithelial and endothelial cells that express mTert. WHAT IS KNOWN ALREADY: Stem/progenitor cells are hypothesized to be responsible for the remarkable regenerative capacity of the endometrium, but the lack of convenient endometrial stem/progenitor markers in the mouse has hampered investigations into the identity of these cells. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A mouse containing a green fluorescent protein (GFP) reporter under the control of the telomerase reverse transcriptase promoter (mTert-GFP) was used to identify potential stem/progenitor cells in the endometrium. mTert promoter activity was determined using fluorescence microscopy and flow cytometry to identify GFP(+) cells. GFP(+) cells were examined for epithelial, stromal, endothelial, leucocyte and proliferation markers and bromodeoxyuridine retention to determine their identity. The endometrium of ovariectomized mice was compared to that of intact cycling mice to establish the role of ovarian hormones in maintaining mTert-expressing cells. MAIN RESULTS AND THE ROLE OF CHANCE: We found that mTert-GFP is expressed by rare luminal and glandular epithelial cells (0.3% of epithelial cells by flow cytometry), rare CD45(-) cells in the stromal compartment (0.028 ± 0.010% of stromal cells by microscopy) and many CD45(+) leucocytes. Ovariectomy resulted in significant decrease of mTert-GFP(+) epithelial cells (P = 0.029 for luminal epithelium; P = 0.034 for glandular epithelium) and a decrease in the percentage of mTert-GFP(+) CD45(+) leucocytes in the stromal compartment (P = 0.015). However, CD45(-) mTert-GFP(+) cells in the stromal compartment were maintained in ovariectomized mice. This population is enriched for cells bearing the endothelial marker CD31 (10.3% of CD90(-) CD45(-) and 97.8% CD90(+) CD45(-) by flow cytometry). CD45(-) mTert-GFP(+) cells also immunostained for the endothelial marker von Willebrand factor. These results suggest that the endometrial epithelium and vasculature are foci of stem/progenitor activity and provide a system to investigate molecular mechanisms involved in endometrial regeneration and repair. LIMITATIONS, REASONS FOR CAUTION: The stem/progenitor activity of endometrial mTert-GFP(+) cells needs to be experimentally verified. WIDER IMPLICATIONS OF THE FINDINGS: The identification and characterization of mTert-expressing progenitor cells in the mouse will facilitate the identification of equivalent populations in the human endometrium that are likely to be involved in endometrial function, fertility and disease. LARGE-SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This study was funded by National Health and Medical Research Council (NHMRC) of Australia grants (1085435, C.E.G., J.A.D.), 1021127 (C.E.G.), NHMRC Senior Research Fellowship (1042298, C.E.G.), the Victorian Infrastructure Support Program, U.S. National Institutes of Health grant R01 DK084056 (D.T.B.) and the Harvard Stem Cell Institute (D.T.B.). The authors have no conflicts of interest to declare.
Assuntos
Endométrio/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Células-Tronco/metabolismo , Telomerase/genética , Animais , Biomarcadores/metabolismo , Proliferação de Células , Endométrio/citologia , Células Endoteliais/citologia , Células Epiteliais/citologia , Feminino , Citometria de Fluxo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/citologia , Leucócitos/metabolismo , Camundongos , Ovariectomia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Regiões Promotoras Genéticas , Células-Tronco/citologia , Telomerase/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
Here we report that ILK localizes in the mouse primary cilium, a sensory organelle required for signalling by the Hedgehog (Hh) pathway. Genetic or pharmacological inhibition of ILK blocks ciliary accumulation of the Hh pathway effector smoothened (Smo) and suppresses the induction of Gli transcription factor mRNAs by SHh. Conditional deletion of ILK or Smo also inhibits SHh-driven activation of Gli2 in the embryonic mouse cerebellum. ILK regulation of Hh signalling probably requires the physical interaction of ILK and Smo in the cilium, and we also show selective cilia-associated interaction of ILK with ß-arrestin, a known mediator of Smo-dependent signalling.
Assuntos
Cerebelo/metabolismo , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Arrestinas/metabolismo , Linhagem Celular , Cerebelo/embriologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened , Proteína Gli2 com Dedos de ZincoRESUMO
BACKGROUND: The contribution of copy-number variation (CNV) to disease has been highlighted with the widespread adoption of array-based comparative genomic hybridisation (aCGH) and microarray technology. Contiguous gene deletions involving ANKRD11 in 16q24.3 are associated with autism spectrum disorder (ASD) and intellectual disability (ID), while 16q24.1 deletions affecting FOXF1 are associated with congenital renal malformations, alveolar capillary dysplasia, and various other abnormalities. The disease associations of deletions in the intervening region, 16q24.2, have only been defined to a limited extent. AIM: To determine whether deletions affecting 16q24.2 are correlated with congenital anomalies. METHODS: 35 individuals, each having a deletion in 16q24.2, were characterised clinically and by aCGH and/or SNP-genotyping microarray. RESULTS: Several of the 35 16q24.2 deletions identified here closely abut or overlap the coding regions of FOXF1 and ANKRD11, two genes that have been previously associated with the disease. 25 patients were reported to have ASD/ID, and three were found to have bilateral hydronephrosis. 14 of the deletions associated with ASD/ID overlap the coding regions of FBXO31 and MAP1LC3B. These same genes and two others, C16orf95 and ZCCHC14, are also included in the area of minimal overlap of the three deletions associated with hydronephrosis. CONCLUSIONS: Our data highlight 16q24.2 as a region of interest for ASD, ID and congenital renal malformations. These conditions are associated, albeit without complete penetrance, with deletions affecting C16orf95, ZCCHC14, MAP1LC3B and FBXO31. The function of each gene in development and disease warrants further investigation.
Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Cromossomos Humanos Par 16 , Deleção de Genes , Deficiência Intelectual/genética , Rim/anormalidades , Adolescente , Criança , Pré-Escolar , Mapeamento Cromossômico , Variações do Número de Cópias de DNA/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas Repressoras/genética , Adulto JovemRESUMO
Genomic profiling has identified therapeutic targets for precision treatment of certain cancers, but many patients lack actionable mutations. Additional omics approaches, like proteomics and phosphoproteomics, are essential for comprehensive mapping of cancer-associated molecular phenotypes. In vivo models, such as cell line and patient-derived xenografts (PDX), offer valuable insights into cancer biology and treatment strategies.This chapter presents a semiautomated high-throughput workflow for integrated proteomics and phosphoproteomics analysis on the Kingfish platform coupled with MagReSyn® Zr-IMAC HP. It enhances protein extraction from in vivo xenograft samples and provides better insights into cancers with poor prognosis. The approach successfully identified over 11,000 unique phosphosites and ~6000 proteins in SJSA-1 pediatric osteosarcoma xenografts, demonstrating its efficacy. This workflow is a valuable tool for studying tumor biology and developing precision oncology strategies.
Assuntos
Biomarcadores Tumorais , Fosfoproteínas , Proteômica , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Proteômica/métodos , Biomarcadores Tumorais/metabolismo , Camundongos , Fosfoproteínas/metabolismo , Linhagem Celular Tumoral , Neoplasias/metabolismo , Neoplasias/patologia , Osteossarcoma/metabolismo , Osteossarcoma/patologia , CriançaRESUMO
Renal primary cilia are microscopic sensory organelles found on the apical surface of epithelial cells of the nephron and collecting duct. They are based upon a microtubular cytoskeleton, bounded by a specialized membrane, and contain an array of proteins that facilitate their assembly, maintenance and function. Cilium-based signalling is important for the control of epithelial differentiation and has been implicated in the pathogenesis of various cystic kidney diseases and in renal repair. As such, visualizing renal primary cilia and understanding their composition has become an essential component of many studies of inherited kidney disease and mechanisms of epithelial regeneration. Primary cilia were initially identified in the kidney using electron microscopy and this remains a useful technique for the high resolution examination of these organelles. New reagents and techniques now also allow the structure and composition of primary cilia to be analysed in detail using fluorescence microscopy. Primary cilia can be imaged in situ in sections of kidney, and many renal-derived cell lines produce primary cilia in culture providing a simplified and accessible system in which to investigate these organelles. Here we outline microscopy-based techniques commonly used for studying renal primary cilia.
Assuntos
Células Epiteliais/ultraestrutura , Rim/ultraestrutura , Microscopia , Animais , Biomarcadores/análise , Cílios , Células Epiteliais/química , Imunofluorescência , Humanos , Processamento de Imagem Assistida por Computador , Rim/química , Microscopia/métodos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de FluorescênciaRESUMO
BACKGROUND: Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer with an appalling overall survival of less than 5% (Zimmerman et al. J Thor Oncol 14:768-83, 2019). Patients typically respond to front line platinum-based doublet chemotherapy, but almost universally relapse with drug resistant disease. Elevated MYC expression is common in SCLC and has been associated with platinum resistance. This study evaluates the capacity of MYC to drive platinum resistance and through screening identifies a drug capable of reducing MYC expression and overcoming resistance. METHODS: Elevated MYC expression following the acquisition of platinum resistance in vitro and in vivo was assessed. Moreover, the capacity of enforced MYC expression to drive platinum resistance was defined in SCLC cell lines and in a genetically engineered mouse model that expresses MYC specifically in lung tumors. High throughput drug screening was used to identify drugs able to kill MYC-expressing, platinum resistant cell lines. The capacity of this drug to treat SCLC was defined in vivo in both transplant models using cell lines and patient derived xenografts and in combination with platinum and etoposide chemotherapy in an autochthonous mouse model of platinum resistant SCLC. RESULTS: MYC expression is elevated following the acquisition of platinum resistance and constitutively high MYC expression drives platinum resistance in vitro and in vivo. We show that fimepinostat decreases MYC expression and that it is an effective single agent treatment for SCLC in vitro and in vivo. Indeed, fimepinostat is as effective as platinum-etoposide treatment in vivo. Importantly, when combined with platinum and etoposide, fimepinostat achieves a significant increase in survival. CONCLUSIONS: MYC is a potent driver of platinum resistance in SCLC that is effectively treated with fimepinostat.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Animais , Humanos , Camundongos , Etoposídeo/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Recidiva Local de Neoplasia , Fosfatidilinositol 3-Quinases , Platina/farmacologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
TP53 and RB1 loss-of-function mutations are common in osteosarcoma. During development, combined loss of TP53 and RB1 function leads to downregulation of autophagy and the aberrant formation of primary cilia, cellular organelles essential for the transmission of canonical Hedgehog (Hh) signaling. Excess cilia formation then leads to hypersensitivity to Hedgehog (Hh) ligand signaling. In mouse and human models, we now show that osteosarcomas with mutations in TP53 and RB1 exhibit enhanced ligand-dependent Hh pathway activation through Smoothened (SMO), a transmembrane signaling molecule required for activation of the canonical Hh pathway. This dependence is mediated by hypersensitivity to Hh ligand and is accompanied by impaired autophagy and increased primary cilia formation and expression of Hh ligand in vivo. Using a conditional genetic mouse model of Trp53 and Rb1 inactivation in osteoblast progenitors, we further show that deletion of Smo converts the highly malignant osteosarcoma phenotype to benign, well differentiated bone tumors. Conversely, conditional overexpression of SHH ligand, or a gain-of-function SMO mutant in committed osteoblast progenitors during development blocks terminal bone differentiation. Finally, we demonstrate that the SMO antagonist sonidegib (LDE225) induces growth arrest and terminal differentiation in vivo in osteosarcomas that express primary cilia and Hh ligand combined with mutations in TP53. These results provide a mechanistic framework for aberrant Hh signaling in osteosarcoma based on defining mutations in the tumor suppressor, TP53.
Assuntos
Antineoplásicos , Osteossarcoma , Humanos , Animais , Camundongos , Proteínas Hedgehog/metabolismo , Ligantes , Transdução de Sinais , Antineoplásicos/farmacologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Cílios/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
DNA methylation array profiling for classifying pediatric central nervous system (CNS) tumors is a valuable adjunct to histopathology. However, unbiased prospective and interlaboratory validation studies have been lacking. The AIM BRAIN diagnostic trial involving 11 pediatric cancer centers in Australia and New Zealand was designed to test the feasibility of routine clinical testing and ran in parallel with the Molecular Neuropathology 2.0 (MNP2.0) study at Deutsches Krebsforschungszentrum (German Cancer Research Center). CNS tumors from 269 pediatric patients were prospectively tested on Illumina EPIC arrays, including 104 cases co-enrolled on MNP2.0. Using MNP classifier versions 11b4 and 12.5, we report classifications with a probability score ≥0.90 in 176 of 265 (66.4%) and 213 of 269 (79.2%) cases, respectively. Significant diagnostic information was obtained in 130 of 176 (74%) for 11b4, and 12 of 174 (7%) classifications were discordant with histopathology. Cases prospectively co-enrolled on MNP2.0 gave concordant classifications (99%) and score thresholds (93%), demonstrating excellent test reproducibility and sensitivity. Overall, DNA methylation profiling is a robust single workflow technique with an acceptable diagnostic yield that is considerably enhanced by the extensive subgroup and copy number profile information generated by the platform. The platform has excellent test reproducibility and sensitivity and contributes significantly to CNS tumor diagnosis.
Assuntos
Neoplasias do Sistema Nervoso Central , Metilação de DNA , Criança , Humanos , Austrália , Neoplasias do Sistema Nervoso Central/diagnóstico , Neoplasias do Sistema Nervoso Central/genética , Metilação de DNA/genética , Nova Zelândia , Estudos Prospectivos , Reprodutibilidade dos TestesRESUMO
Mutations in the type III receptor tyrosine kinase FLT3 are frequent in patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. AML is characterized by the overproduction of reactive oxygen species (ROS), which can induce cysteine oxidation in redox-sensitive signaling proteins. Here, we sought to characterize the specific pathways affected by ROS in AML by assessing oncogenic signaling in primary AML samples. The oxidation or phosphorylation of signaling proteins that mediate growth and proliferation was increased in samples from patient subtypes with FLT3 mutations. These samples also showed increases in the oxidation of proteins in the ROS-producing Rac/NADPH oxidase-2 (NOX2) complex. Inhibition of NOX2 increased the apoptosis of FLT3-mutant AML cells in response to FLT3 inhibitors. NOX2 inhibition also reduced the phosphorylation and cysteine oxidation of FLT3 in patient-derived xenograft mouse models, suggesting that decreased oxidative stress reduces the oncogenic signaling of FLT3. In mice grafted with FLT3 mutant AML cells, treatment with a NOX2 inhibitor reduced the number of circulating cancer cells, and combining FLT3 and NOX2 inhibitors increased survival to a greater extent than either treatment alone. Together, these data raise the possibility that combining NOX2 and FLT3 inhibitors could improve the treatment of FLT3 mutant AML.
Assuntos
Cisteína , Leucemia Mieloide Aguda , Humanos , Animais , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Cisteína/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Modelos Animais de Doenças , Linhagem Celular Tumoral , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9-11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA-mutations showed increased sensitivity to ONC201, while those harboring TP53-mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992.
RESUMO
Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. SIGNIFICANCE: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.
RESUMO
Renal dysplasia, defined by defective ureteric branching morphogenesis and nephrogenesis, is the major cause of renal failure in infants and children. Here, we define a pathogenic role for a ß-catenin-activated genetic pathway in murine renal dysplasia. Stabilization of ß-catenin in the ureteric cell lineage before the onset of kidney development increased ß-catenin levels and caused renal aplasia or severe hypodysplasia. Analysis of gene expression in the dysplastic tissue identified downregulation of genes required for ureteric branching and upregulation of Tgfß2 and Dkk1. Treatment of wild-type kidney explants with TGFß2 or DKK1 generated morphogenetic phenotypes strikingly similar to those observed in mutant kidney tissue. Stabilization of ß-catenin after the onset of kidney development also caused dysplasia and upregulation of Tgfß2 and Dkk1 in the epithelium. Together, these results demonstrate that elevation of ß-catenin levels during kidney development causes dysplasia.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Rim/anormalidades , Rim/embriologia , Fator de Crescimento Transformador beta2/fisiologia , Regulação para Cima/fisiologia , beta Catenina/fisiologia , Animais , Apoptose/fisiologia , Proliferação de Células , Modelos Animais de Doenças , Feminino , Rim/fisiopatologia , Camundongos , Camundongos Mutantes , Morfogênese/fisiologia , Gravidez , Transdução de Sinais/fisiologia , Ureter/anormalidades , Ureter/embriologia , Ureter/fisiopatologia , Proteínas Wnt/fisiologiaRESUMO
Diffuse midline glioma (DMG) is a deadly pediatric and adolescent central nervous system (CNS) tumor localized along the midline structures of the brain atop the spinal cord. With a median overall survival (OS) of just 9-11-months, DMG is characterized by global hypomethylation of histone H3 at lysine 27 (H3K27me3), driven by recurring somatic mutations in H3 genes including, HIST1H3B/C (H3.1K27M) or H3F3A (H3.3K27M), or through overexpression of EZHIP in patients harboring wildtype H3. The recent World Health Organization's 5th Classification of CNS Tumors now designates DMG as, 'H3 K27-altered', suggesting that global H3K27me3 hypomethylation is a ubiquitous feature of DMG and drives devastating transcriptional programs for which there are no treatments. H3-alterations co-segregate with various other somatic driver mutations, highlighting the high-level of intertumoral heterogeneity of DMG. Furthermore, DMG is also characterized by very high-level intratumoral diversity with tumors harboring multiple subclones within each primary tumor. Each subclone contains their own combinations of driver and passenger lesions that continually evolve, making precision-based medicine challenging to successful execute. Whilst the intertumoral heterogeneity of DMG has been extensively investigated, this is yet to translate to an increase in patient survival. Conversely, our understanding of the non-genomic factors that drive the rapid growth and fatal nature of DMG, including endogenous and exogenous microenvironmental influences, neurological cues, and the posttranscriptional and posttranslational architecture of DMG remains enigmatic or at best, immature. However, these factors are likely to play a significant role in the complex biological sequelae that drives the disease. Here we summarize the heterogeneity of DMG and emphasize how analysis of the posttranslational architecture may improve treatment paradigms. We describe factors that contribute to treatment response and disease progression, as well as highlight the potential for pharmaco-proteogenomics (i.e., the integration of genomics, proteomics and pharmacology) in the management of this uniformly fatal cancer.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Glioma/tratamento farmacológico , Glioma/genética , Proteogenômica/métodos , Animais , Neoplasias Encefálicas/mortalidade , Criança , Pré-Escolar , Feminino , Glioma/mortalidade , Humanos , Masculino , Camundongos , Análise de Sobrevida , Microambiente TumoralRESUMO
Maintenance of male fertility requires spermatogonial stem cells (SSCs) that self-renew and generate differentiating germ cells for production of spermatozoa. Germline cells are sensitive to genotoxic drugs and patients receiving chemotherapy can become infertile. SSCs surviving treatment mediate germline recovery but pathways driving SSC regenerative responses remain poorly understood. Using models of chemotherapy-induced germline damage and recovery, here we identify unique molecular features of regenerative SSCs and characterise changes in composition of the undifferentiated spermatogonial pool during germline recovery by single-cell analysis. Increased mitotic activity of SSCs mediating regeneration is accompanied by alterations in growth factor signalling including PI3K/AKT and mTORC1 pathways. While sustained mTORC1 signalling is detrimental for SSC maintenance, transient mTORC1 activation is critical for the regenerative response. Concerted inhibition of growth factor signalling disrupts core features of the regenerative state and limits germline recovery. We also demonstrate that the FOXM1 transcription factor is a target of growth factor signalling in undifferentiated spermatogonia and provide evidence for a role in regeneration. Our data confirm dynamic changes in SSC functional properties following damage and support an essential role for microenvironmental growth factors in promoting a regenerative state.
Assuntos
Fosfatidilinositol 3-Quinases , Espermatogênese , Diferenciação Celular/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espermatogênese/genética , Espermatogônias , Células-Tronco/metabolismo , Testículo/metabolismoRESUMO
Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.
Assuntos
Neoplasias Pulmonares/genética , Oncogenes/fisiologia , Proteína do Retinoblastoma/metabolismo , Carcinoma de Pequenas Células do Pulmão/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Carcinoma de Pequenas Células do Pulmão/patologiaRESUMO
BACKGROUND: Pediatric diffuse midline gliomas (DMGs) are incurable childhood cancers. The imipridone ONC201 has shown early clinical efficacy in a subset of DMGs. However, the anticancer mechanisms of ONC201 and its derivative ONC206 have not been fully described in DMGs. METHODS: DMG models including primary human in vitro (n = 18) and in vivo (murine and zebrafish) models, and patient (n = 20) frozen and FFPE specimens were used. Drug-target engagement was evaluated using in silico ChemPLP and in vitro thermal shift assay. Drug toxicity and neurotoxicity were assessed in zebrafish models. Seahorse XF Cell Mito Stress Test, MitoSOX and TMRM assays, and electron microscopy imaging were used to assess metabolic signatures. Cell lineage differentiation and drug-altered pathways were defined using bulk and single-cell RNA-seq. RESULTS: ONC201 and ONC206 reduce viability of DMG cells in nM concentrations and extend survival of DMG PDX models (ONC201: 117 days, P = .01; ONC206: 113 days, P = .001). ONC206 is 10X more potent than ONC201 in vitro and combination treatment was the most efficacious at prolonging survival in vivo (125 days, P = .02). Thermal shift assay confirmed that both drugs bind to ClpP, with ONC206 exhibiting a higher binding affinity as assessed by in silico ChemPLP. ClpP activation by both drugs results in impaired tumor cell metabolism, mitochondrial damage, ROS production, activation of integrative stress response (ISR), and apoptosis in vitro and in vivo. Strikingly, imipridone treatment triggered a lineage shift from a proliferative, oligodendrocyte precursor-like state to a mature, astrocyte-like state. CONCLUSION: Targeting mitochondrial metabolism and ISR activation effectively impairs DMG tumorigenicity. These results supported the initiation of two pediatric clinical trials (NCT05009992, NCT04732065).
Assuntos
Antineoplásicos , Glioma , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Linhagem da Célula , Criança , Metabolismo Energético , Glioma/tratamento farmacológico , Glioma/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Camundongos , Peixe-ZebraRESUMO
The kidney is the most common site of congenital malformations that result in impaired renal function. Yet, the molecular mechanisms that control renal malformations are poorly understood. The Hedgehog signaling pathway plays critical roles during mammalian organogenesis. Aberrant Hedgehog signaling results in severe congenital abnormalities, including renal malformations. Here, we review the current body of knowledge on Hedgehog signaling during renal morphogenesis and highlight the gaps in our understanding. Furthermore, we propose mechanisms by which Hedgehog signaling contributes to both normal and abnormal renal development.