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1.
Stem Cell Reports ; 19(4): 529-544, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38552636

RESUMO

Alveolar type 2 (AT2) epithelial cells are tissue stem cells capable of differentiating into alveolar type 1 (AT1) cells for injury repair and maintenance of lung homeostasis. However, the factors involved in human AT2-to-AT1 cell differentiation are not fully understood. Here, we established SFTPCGFP and AGERmCherry-HiBiT dual-reporter induced pluripotent stem cells (iPSCs), which detected AT2-to-AT1 cell differentiation with high sensitivity and identified factors inducing AT1 cell differentiation from AT2 and their progenitor cells. We also established an "on-gel" alveolar epithelial spheroid culture suitable for medium-throughput screening. Among the 274 chemical compounds, several single compounds, including LATS-IN-1, converted AT1 cells from AT2 and their progenitor cells. Moreover, YAP/TAZ signaling activation and AKT signaling suppression synergistically recapitulated the induction of transcriptomic, morphological, and functionally mature AT1 cells. Our findings provide novel insights into human lung development and lung regenerative medicine.


Assuntos
Células Epiteliais Alveolares , Células-Tronco Pluripotentes Induzidas , Humanos , Células Cultivadas , Pulmão , Diferenciação Celular , Células Epiteliais
2.
Biochem Biophys Res Commun ; 681: 180-185, 2023 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-37783115

RESUMO

A bioluminescent immunoassay system was developed to determine serine/threonine protein kinase activity using an aequorin-labeled monoclonal antibody and a synthetic peptide as the substrate. A monoclonal antibody against the synthetic phosphorylated serine peptide (K9P peptide) of histone H3 (19 amino acid residues), referred to as the H3S10P antibody, was chemically conjugated to maleimide-activated aequorin to prepare aequorin-labeled H3S10P (AQ-S-H3S10P). For the serine/threonine kinase assay, a non-phosphorylated serine peptide (K9C peptide) coated on a microplate was incubated with serine/threonine protein kinase in the presence of ATP and Mg2+. The resulting phosphorylated K9C peptides (K9P peptide) were identified using AQ-S-H3S10P. Thus, after the removal of unbound AQ-S-H3S10P though washing, the serine/threonine kinase activity was determined by the luminescence activity of aequorin from AQ-S-H3S10P bound to the K9P peptide. This assay system, in combination with the K9C peptide and AQ-S-H3S10P, could be used to screen inhibitors of various serine/threonine protein kinases in general.


Assuntos
Equorina , Anticorpos Monoclonais , Equorina/metabolismo , Anticorpos Monoclonais/metabolismo , Imunoensaio/métodos , Peptídeos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases/metabolismo , Treonina/metabolismo , Especificidade por Substrato
3.
Cancer Rep (Hoboken) ; 6(2): e1701, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36806727

RESUMO

BACKGROUND: Several treatment strategies use upfront chemotherapy or androgen receptor axis-targeting therapies for metastatic prostate cancer. However, there are no useful biomarkers for selecting appropriate patients who urgently require these treatments. METHODS: Novel patient-derived xenograft (PDX) castration-sensitive and -resistant models were established and gene expression patterns were comprehensively compared. The function of a gene highly expressed in the castration-resistant models was evaluated by its overexpression in LNCaP prostate cancer cells. Protein expression in the tumors and serum of patients was examined by immunohistochemistry and ELISA, and correlations with castration resistance were analyzed. RESULTS: Expression of the α2 chain of interleukin-13 receptor (IL13Rα2) was higher in castration-resistant PDX tumors. LNCaP cells overexpressing IL13Rα2 acquired castration resistance in vitro and in vivo. In tissue samples, IL13Rα2 expression levels were significantly associated with castration-resistant progression (p < 0.05). In serum samples, IL13Rα2 levels could be measured in 5 of 28 (18%) castration-resistant prostate cancer patients. CONCLUSION: IL13Rα2 was highly expressed in castration-resistant prostate cancer PDX models and was associated with the castration resistance of prostate cancer cells. It might be a potential tissue and serum biomarker for predicting castration resistance in prostate cancer patients.


Assuntos
Subunidade alfa2 de Receptor de Interleucina-13 , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/patologia , Subunidade alfa2 de Receptor de Interleucina-13/uso terapêutico , Xenoenxertos , Orquiectomia , Biomarcadores
4.
BMC Cancer ; 23(1): 71, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36670405

RESUMO

Chronic infection with Kaposi's sarcoma-associated herpes virus (KSHV) in B lymphocytes causes primary effusion lymphoma (PEL), the most aggressive form of KSHV-related cancer, which is resistant to conventional chemotherapy. In this study, we report that the BCBL-1 KSHV+ PEL cell line does not harbor oncogenic mutations responsible for its aggressive malignancy. Assuming that KSHV viral oncogenes play crucial roles in PEL proliferation, we examined the effect of cyclin-dependent kinase 9 (CDK9) inhibitor FIT-039 on KSHV viral gene expression and KSHV+ PEL proliferation. We found that FIT-039 treatment impaired the proliferation of KSHV+ PEL cells and the expression of KSHV viral genes in vitro. The effects of FIT-039 treatment on PEL cells were further evaluated in the PEL xenograft model that retains a more physiological environment for the growth of PEL growth and KSHV propagation, and we confirmed that FIT-039 administration drastically inhibited PEL growth in vivo. Our current study indicates that FIT-039 is a potential new anticancer drug targeting KSHV for PEL patients.


Assuntos
Herpesvirus Humano 8 , Linfoma de Efusão Primária , Neoplasias , Sarcoma de Kaposi , Humanos , Sarcoma de Kaposi/tratamento farmacológico , Linfoma de Efusão Primária/patologia , Quinase 9 Dependente de Ciclina/metabolismo
5.
Hum Genet ; 142(1): 59-71, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36048237

RESUMO

Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal DMD transcripts were detected in 19 of 20 cases; Exonization of intronic sequences in 15 cases, exon skipping in one case, aberrantly spliced and polyadenylated transcripts in two cases and transcription termination in one case. Intronic single nucleotide variants, chromosomal rearrangements and nucleotide repeat expansion were identified in DMD gene as pathogenic causes of transcript alteration. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in DMD transcripts could inform the therapeutic options for patients with dystrophinopathy.


Assuntos
Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofina/genética , Splicing de RNA/genética , Íntrons/genética , Nucleotídeos , Análise de Sequência de RNA
6.
Sci Transl Med ; 14(673): eabn6056, 2022 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-36449604

RESUMO

Neoantigen production is a determinant of cancer immunotherapy. However, the expansion of neoantigen abundance for cancer therapeutics is technically challenging. Here, we report that the synthetic compound RECTAS can induce the production of splice-neoantigens that could be used to boost antitumor immune responses. RECTAS suppressed tumor growth in a CD8+ T cell- and tumor major histocompatibility complex class I-dependent manner and enhanced immune checkpoint blockade efficacy. Subsequent transcriptome analysis and validation for immunogenicity identified six splice-neoantigen candidates whose expression was induced by RECTAS treatment. Vaccination of the identified neoepitopes elicited T cell responses capable of killing cancer cells in vitro, in addition to suppression of tumor growth in vivo upon sensitization with RECTAS. Collectively, these results provide support for the further development of splice variant-inducing treatments for cancer immunotherapy.


Assuntos
Neoplasias Colorretais , Imunoterapia , Humanos , Mutação , Linfócitos T CD8-Positivos , Perfilação da Expressão Gênica , Neoplasias Colorretais/terapia
7.
JID Innov ; 1(3): 100026, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34909725

RESUMO

TRIAL DESIGN: Human papillomavirus infection causes verruca vulgaris. CDK9 inhibitor FIT039 inhibits DNA virus proliferation in animal models. We conducted a multicenter, single-blind, placebo-controlled, randomized phase I/II clinical trial evaluating the safety and efficacy of FIT039 against verruca vulgaris. METHODS: Target lesions were treated with liquid nitrogen once, and a FIT039 patch or placebo patch was applied for 14 days. The primary endpoint was lesion disappearance. The secondary endpoints were safety and changes in dimension, cross-sectional area, and the number of petechial lesions. RESULTS: A total of 24 participants were randomly allocated to the FIT039 (n = 13, median age, 54 years) and placebo (n = 11, median age, 62 years) groups. Verruca vulgaris did not disappear. FIT039 decreased the dimension to 76% of the initial value on day 29, followed by an increase to 98% on day 57. Placebo showed a monotonic increase to 107% on day 57. Changes in the cross-sectional area and petechiae number were comparable between the groups. CONCLUSIONS: No drug-related adverse reactions occurred. FIT039 efficacy was not determined in this study.

8.
Respir Res ; 22(1): 284, 2021 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-34736469

RESUMO

BACKGROUND: Somatic cells differentiated from patient-specific human induced pluripotent stem cells (iPSCs) could be a useful tool in human cell-based disease research. Hermansky-Pudlak syndrome (HPS) is an autosomal recessive genetic disorder characterized by oculocutaneous albinism and a platelet dysfunction. HPS patients often suffer from lethal HPS associated interstitial pneumonia (HPSIP). Lung transplantation has been the only treatment for HPSIP. Lysosome-related organelles are impaired in HPS, thereby disrupting alveolar type 2 (AT2) cells with lamellar bodies. HPSIP lungs are characterized by enlarged lamellar bodies. Despite species differences between human and mouse in HPSIP, most studies have been conducted in mice since culturing human AT2 cells is difficult. METHODS: We generated patient-specific iPSCs from patient-derived fibroblasts with the most common bi-allelic variant, c.1472_1487dup16, in HPS1 for modeling severe phenotypes of HPSIP. We then corrected the variant of patient-specific iPSCs using CRISPR-based microhomology-mediated end joining to obtain isogenic controls. The iPSCs were then differentiated into lung epithelial cells using two different lung organoid models, lung bud organoids (LBOs) and alveolar organoids (AOs), and explored the phenotypes contributing to the pathogenesis of HPSIP using transcriptomic and proteomic analyses. RESULTS: The LBOs derived from patient-specific iPSCs successfully recapitulated the abnormalities in morphology and size. Proteomic analysis of AOs involving iPSC-derived AT2 cells and primary lung fibroblasts revealed mitochondrial dysfunction in HPS1 patient-specific alveolar epithelial cells. Further, giant lamellar bodies were recapitulated in patient-specific AT2 cells. CONCLUSIONS: The HPS1 patient-specific iPSCs and their gene-corrected counterparts generated in this study could be a new research tool for understanding the pathogenesis of HPSIP caused by HPS1 deficiency in humans.


Assuntos
Células Epiteliais Alveolares/patologia , DNA/genética , Síndrome de Hermanski-Pudlak/genética , Proteínas de Membrana/genética , Mutação , Proteômica/métodos , Animais , Células Cultivadas , Análise Mutacional de DNA , Modelos Animais de Doenças , Síndrome de Hermanski-Pudlak/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Fenótipo
9.
Biomaterials ; 276: 121031, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34304138

RESUMO

Lung transplantation is the only treatment available for end-stage lung diseases; however, donor shortage is a global issue. The use of human pluripotent stem cells (hPSCs) for organ regeneration is a promising approach. Nevertheless, methods for the expansion of isolated hPSC-derived lung progenitors (hLPs) for transplantation purposes have not yet been reported. Herein, we established an expansion system of hLPs based on their three-dimensional culture in core-shell hydrogel microfibers, that ensures the maintenance of their bipotency for differentiation into alveolar and airway epithelial cells including alveolar type II (AT2) cells. Further, we developed an efficient in vivo transplantation method using an endoscope-assisted transtracheal administration system; the successful engraftment and in vivo differentiation of hLPs into alveolar epithelial cells (incorporated into the alveoli) was observed. Importantly, expanded hLPs in the context of microfibers were successfully transplanted into the murine lungs, opening avenues for cell-based therapies of lung diseases. Therefore, our novel method has potential regenerative medicine applications; additionally, the high-quality hLPs and AT2 cells generated via the microfiber-based technology are valuable for drug discovery purposes.


Assuntos
Hidrogéis , Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Humanos , Pulmão , Camundongos , Medicina Regenerativa
10.
Sci Rep ; 11(1): 7963, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846420

RESUMO

Dysregulation of alternative splicing is a feature of cancer, both in aetiology and progression. It occurs because of mutations in splice sites or sites that regulate splicing, or because of the altered expression and activity of splice factors and of splice factor kinases that regulate splice factor activity. Recently the CDC2-like kinases (CLKs) have attracted attention due to their increasing involvement in cancer. We measured the effect of the CLK inhibitor, the benzothiazole TG003, on two prostate cancer cell lines. TG003 reduced cell proliferation and increased apoptosis in PC3 and DU145 cells. Conversely, the overexpression of CLK1 in PC3 cells prevented TG003 from reducing cell proliferation. TG003 slowed scratch closure and reduced cell migration and invasion in a transwell assay. TG003 decisively inhibited the growth of a PC3 cell line xenograft in nude mice. We performed a transcriptomic analysis of cells treated with TG003. We report widespread and consistent changes in alternative splicing of cancer-associated genes including CENPE, ESCO2, CKAP2, MELK, ASPH and CD164 in both HeLa and PC3 cells. Together these findings suggest that targeting CLKs will provide novel therapeutic opportunities in prostate cancer.


Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Terapia de Alvo Molecular , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Processamento Alternativo/genética , Animais , Apoptose/efeitos dos fármacos , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos Nus , Invasividade Neoplásica , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , RNA-Seq , Tiazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cell Chem Biol ; 28(8): 1132-1144.e9, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-33561428

RESUMO

Metabolic activities are altered in cancer cells compared with those in normal cells, and the cancer-specific pathway becomes a potential therapeutic target. Higher cellular glucose consumption, which leads to lower glucose levels, is a hallmark of cancer cells. In an objective screening for chemicals that induce cell death under low-glucose conditions, we discovered a compound, denoted as ALESIA (Anticancer Ligand Enhancing Starvation-induced Apoptosis). By our shedding assay of transforming growth factor α in HEK293A cells, ALESIA was determined to act as a sphingosine-1-phosphate receptor 3-G12-biased agonist that promotes nitric oxide production and oxidative stress. The oxidative stress triggered by ALESIA resulted in the exhaustion of glucose, cellular NADPH deficiency, and then cancer cell death. Intraperitoneal administration of ALESIA improved the survival of mice with peritoneally disseminated rhabdomyosarcoma, indicating its potential as a new type of anticancer drug for glucose starvation therapy.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Glucose/metabolismo , Neoplasias/tratamento farmacológico , Receptores de Esfingosina-1-Fosfato/agonistas , Animais , Antineoplásicos/química , Linhagem Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Receptores de Esfingosina-1-Fosfato/metabolismo
12.
Am J Respir Cell Mol Biol ; 64(4): 504-514, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33493427

RESUMO

Alveolar epithelial type II (AT2) cells secrete pulmonary surfactant via lamellar bodies (LBs). Abnormalities in LBs are associated with pulmonary disorders, including fibrosis. However, high-content screening (HCS) for LB abnormalities is limited by the lack of understanding of AT2 cell functions. In the present study, we have developed LB cells harboring LB-like organelles that secrete surfactant proteins. These cells were more similar to AT2 cells than to parental A549 cells. LB cells recapitulated amiodarone (AMD)-induced LB enlargement, similar to AT2 cells of patients exposed to AMD. To reverse AMD-induced LB abnormalities, we performed HCS of approved drugs and identified 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a cyclic oligosaccharide, as a potential therapeutic agent. A transcriptome analysis revealed that HPßCD modulates lipid homeostasis. In addition, HPßCD inhibited AMD-induced LB abnormalities in human induced pluripotent stem cell-derived AT2 cells. Our results demonstrate that LB cells are useful for HCS and suggest that HPßCD is a candidate therapeutic agent for AMD-induced interstitial pneumonia.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Células Epiteliais Alveolares/efeitos dos fármacos , Amiodarona/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Células A549 , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Ensaios de Triagem em Larga Escala , Homeostase , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Precursores de Proteínas/metabolismo , Proteína C Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Proteínas Associadas a Surfactantes Pulmonares/metabolismo
13.
Cell Chem Biol ; 27(12): 1472-1482.e6, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-32905759

RESUMO

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that compromise its chloride channel activity. Here, we present a therapeutic strategy to ameliorate RNA splicing deficiency of CFTR with a small molecule. The 3,849 + 10 kb C>T is the most common splicing mutation in CF, creating a pseudo exon with premature stop codon. We reveal that the 3,849 + 10 kb C>T-induced CFTR pseudo exon is regulated by phosphorylation of serine/arginine-rich splicing factors, and their functional inhibition by a CDC-like kinase inhibitor restores normal splicing of CFTR. Subsequent screening of our focused chemical library identified CaNDY as a rectifier of the aberrant splicing. CaNDY treatment restored normal splicing of CFTR with the 3,849 + 10 kb C>T in CF patient cells and functional CFTR protein expression in the CF model cells. Our findings open the door for mechanism-based personalized medicine for pseudo-exon-type genetic diseases.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Éxons/genética , Medicina de Precisão , Humanos , Fosforilação/efeitos dos fármacos
14.
Br J Cancer ; 122(2): 245-257, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31819188

RESUMO

BACKGROUND: The fatty acid (FA) composition of phosphatidylinositols (PIs) is tightly regulated in mammalian tissue since its disruption impairs normal cellular functions. We previously found its significant alteration in breast cancer by using matrix-assisted laser desorption and ionisation imaging mass spectrometry (MALDI-IMS). METHODS: We visualised the histological distribution of PIs containing different FAs in 65 primary breast cancer tissues using MALDI-IMS and investigated its association with clinicopathological features and gene expression profiles. RESULTS: Normal ductal cells (n = 7) predominantly accumulated a PI containing polyunsaturated FA (PI-PUFA), PI(18:0/20:4). PI(18:0/20:4) was replaced by PIs containing monounsaturated FA (PIs-MUFA) in all non-invasive cancer cells (n = 12). While 54% of invasive cancer cells (n = 27) also accumulated PIs-MUFA, 46% of invasive cancer cells (n = 23) accumulated the PIs-PUFA, PI(18:0/20:3) and PI(18:0/20:4). The accumulation of PI(18:0/20:3) was associated with higher incidence of lymph node metastasis and activation of the PD-1-related immune checkpoint pathway. Fatty acid-binding protein 7 was identified as a putative molecule controlling PI composition. CONCLUSIONS: MALDI-IMS identified PI composition associated with invasion and nodal metastasis of breast cancer. The accumulation of PI(18:0/20:3) could affect the PD-1-related immune checkpoint pathway, although its precise mechanism should be further validated.


Assuntos
Neoplasias da Mama/genética , Ácidos Graxos/metabolismo , Fosfatidilinositóis/metabolismo , Transcriptoma/genética , Idoso , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Ácidos Graxos/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Espectrometria de Massas , Pessoa de Meia-Idade , Fosfatidilinositóis/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
15.
Trials ; 20(1): 489, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399147

RESUMO

BACKGROUND: Human papilloma viruses (HPVs) infect squamous epithelial cells and form verrucous lesions, or warts. Besides the psychosocial problems caused by the disfiguring warts, a subset of HPVs can be the primary etiologic agent for malignancies such as cervical cancer. However, there is no curative antiviral therapy for HPV infection. We recently found that the viral RNA transcription of DNA viruses requires cyclin dependent kinase 9 (CDK9) in the host cells, and that FIT039, a specific inhibitor of CDK9, suppressed the proliferation of DNA viruses such as herpes simplex virus 1 (HSV-1), HSV-2, human adenovirus, human cytomegalovirus, hepatitis virus B, and HPVs. Here, we describe a protocol to evaluate the safety and antiviral effect of FIT039 on common warts in human skin. METHODS AND DESIGN: A multi-institutional, single-blind, placebo-controlled randomized phase I/II clinical trial was designed to evaluate the safety and efficacy of FIT039 on common warts on the extremities. A total of 44 adults with a primary diagnosis of verruca vulgaris on the extremities without serious comorbidities will be randomized into either the intervention group with an FIT039-releasing transdermal patch or a control group for a duration of 14 days. Outcomes will be assessed at baseline and postintervention. Participants will be further assessed at 2 months follow-up. The primary endpoint for efficacy is the resolution of the warts. The safety endpoint is the incidence of adverse events and adverse drug reactions. The secondary endpoints are changes in the dimensions of the wart, the cross-sectional area of the wart, and the number of clots within the area of the wart. DISCUSSION: This study is the first to assess the efficacy of FIT039 and will contribute to the development of antiviral agents that can cure refractory common warts in immunocompromised patients. TRIAL REGISTRATION: UMIN Clinical Trials, UMIN000029695 . Registered on 1 November 2017.


Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Piridinas/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Verrugas/tratamento farmacológico , Adulto , Humanos , Piridinas/efeitos adversos , Projetos de Pesquisa , Método Simples-Cego
16.
Clin Drug Investig ; 39(1): 55-61, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30284700

RESUMO

BACKGROUND: Cutaneous warts are caused by human papilloma virus (HPV) infection. FIT039, a specific inhibitor of CDK9, suppresses the proliferation of DNA viruses in vitro. PURPOSE: We evaluated the safety, plasma concentrations, and efficacy of FIT039 delivered by single application of an adhesive skin patch on normal back skin and cutaneous warts. PATIENTS AND METHODS: In this placebo-controlled, dose-escalation, open-label, two-cohort phase I/II clinical trial, after a single administration of a 1% FIT039 patch, 3% FIT039 patch, or placebo on back skin, patients with cutaneous warts were treated with cryotherapy followed by a 1% FIT039 patch for 24 h in the first cohort. In the second cohort, cutaneous warts were treated with cryotherapy followed by a 3% FIT039 patch for 24 h. Adverse events and adverse drug reactions, the concentrations of FIT039, and surface area of cutaneous warts were evaluated. RESULTS: Neither irritant reactions nor symptoms related to FIT039 occurred when the FIT039 patches were applied to patients' backs or on warts in ten patients. The concentrations of FIT039 were under 0.1 ng/ml at every time point. The median wart surface area at 1 week after application of the 1% FIT039 patch was similar to baseline, while that of the 3% FIT039 patch was smaller than baseline. CONCLUSION: The FIT039 patch showed no topical or systemic adverse reactions when applied on normal skin or cutaneous warts. The safety and good adherence of the FIT039 patch are encouraging and support further studies to evaluate the efficacy of FIT039 in patients with cutaneous warts.


Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Infecções por Papillomavirus/tratamento farmacológico , Piridinas/uso terapêutico , Verrugas/tratamento farmacológico , Adesivos , Administração Cutânea , Adulto , Idoso , Crioterapia/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adesivo Transdérmico , Adulto Jovem
17.
J Clin Invest ; 129(2): 583-597, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30422821

RESUMO

X-linked dominant incontinentia pigmenti (IP) and X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) are caused by loss-of-function and hypomorphic IKBKG (also known as NEMO) mutations, respectively. We describe a European mother with mild IP and a Japanese mother without IP, whose 3 boys with EDA-ID died from ID. We identify the same private variant in an intron of IKBKG, IVS4+866 C>T, which was inherited from and occurred de novo in the European mother and Japanese mother, respectively. This mutation creates a new splicing donor site, giving rise to a 44-nucleotide pseudoexon (PE) generating a frameshift. Its leakiness accounts for NF-κB activation being impaired but not abolished in the boys' cells. However, aberrant splicing rates differ between cell types, with WT NEMO mRNA and protein levels ranging from barely detectable in leukocytes to residual amounts in induced pluripotent stem cell-derived (iPSC-derived) macrophages, and higher levels in fibroblasts and iPSC-derived neuronal precursor cells. Finally, SRSF6 binds to the PE, facilitating its inclusion. Moreover, SRSF6 knockdown or CLK inhibition restores WT NEMO expression and function in mutant cells. A recurrent deep intronic splicing mutation in IKBKG underlies a purely quantitative NEMO defect in males that is most severe in leukocytes and can be rescued by the inhibition of SRSF6 or CLK.


Assuntos
Displasia Ectodérmica , Mutação da Fase de Leitura , Quinase I-kappa B , Incontinência Pigmentar , Íntrons , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Displasia Ectodérmica/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Quinase I-kappa B/deficiência , Quinase I-kappa B/metabolismo , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Síndromes de Imunodeficiência/patologia , Incontinência Pigmentar/genética , Incontinência Pigmentar/metabolismo , Incontinência Pigmentar/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino
18.
Front Genet ; 9: 368, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30233648

RESUMO

Williams-Beuren syndrome (WBS) is a multisystemic neurodevelopmental disorder caused by a hemizygous deletion on chromosome 7q11.23. Though at present there is a limited number of reports on WBS patients with tumors, most cases are related to blood cancer in children with WBS. We describe a case of Burkitt lymphoma in a 21-year-old man with WBS. In addition to providing a summary of published reports describing tumors observed in patients with WBS, we present a hypothesis about a possible mechanism of oncogenesis. In particular, we identified some significantly dysregulated cancer-related genes using blood samples from this patient at the age of 19 years (who have not yet developed Burkitt lymphoma). Our findings may provide a new perspective on the relation between WBS and Burkitt lymphoma.

19.
Clin Cancer Res ; 24(18): 4518-4528, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29712686

RESUMO

Purpose: Cervical cancer is one of the leading causes of cancer-related deaths among women worldwide. The purpose of this study is to assess the therapeutic effect of the newly developed cyclin-dependent kinase 9 (CDK9) inhibitor FIT-039 on cervical neoplasia induced by human papillomavirus (HPV) infection.Experimental Design: We examined FIT-039 for its effect on HPV gene expression in HPV+ cervical cancer cells. Primary keratinocytes monolayer and organotypic raft culture models were used to evaluate HPV viral replication and cervical intraepithelial neoplasia (CIN) phenotypes. Preclinical pharmacokinetics and toxicity tests for FIT-039 were also conducted. Finally, the anti-HPV effect of FIT-039 was further examined in vivo, using HPV+ cervical cancer xenografts.Results: FIT-039 inhibits HPV replication and expression of E6 and E7 viral oncogenes, restoring tumor suppressors p53 and pRb in HPV+ cervical cancer cells. The therapeutic effect of FIT-039 was demonstrated in CIN model of an organotypic raft culture, where FIT-039 suppressed HPV18-induced dysplasia/hyperproliferation with reduction in viral load. FIT-039 also repressed growth of HPV16+, but not HPV- cervical cancer xenografts without any significant adverse effects. Safety and pharmacokinetics of FIT-039 were confirmed for systemic and topical routes.Conclusions: The CDK9 inhibitor FIT-039 showed potent anti-HPV activity without significant toxicity in preclinical studies. Thus, FIT-039 is expected to be a novel therapeutic for CIN to prevent cervical cancer. Clin Cancer Res; 24(18); 4518-28. ©2018 AACR.


Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Infecções por Papillomavirus/tratamento farmacológico , Piridinas/farmacologia , Displasia do Colo do Útero/tratamento farmacológico , Animais , Proliferação de Células/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/genética , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/virologia , Camundongos , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Cultura Primária de Células , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genética , Replicação Viral/efeitos dos fármacos , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/patologia , Displasia do Colo do Útero/virologia
20.
Cell Rep ; 23(5): 1326-1341, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29719248

RESUMO

Genes specifically expressed in neurons contain members with extended long introns. Longer genes present a problem with respect to fulfilment of gene length transcription, and evidence suggests that dysregulation of long genes is a mechanism underlying neurodegenerative and psychiatric disorders. Here, we report the discovery that RNA-binding protein Sfpq is a critical factor for maintaining transcriptional elongation of long genes. We demonstrate that Sfpq co-transcriptionally binds to long introns and is required for sustaining long-gene transcription by RNA polymerase II through mediating the interaction of cyclin-dependent kinase 9 with the elongation complex. Phenotypically, Sfpq disruption caused neuronal apoptosis in developing mouse brains. Expression analysis of Sfpq-regulated genes revealed specific downregulation of developmentally essential neuronal genes longer than 100 kb in Sfpq-disrupted brains; those genes are enriched in associations with neurodegenerative and psychiatric diseases. The identified molecular machinery yields directions for targeted investigations of the association between long-gene transcriptopathy and neuronal diseases.


Assuntos
Encéfalo , Transtornos Mentais , Doenças Neurodegenerativas , Neurônios/metabolismo , Fator de Processamento Associado a PTB/deficiência , Elongação da Transcrição Genética , Animais , Apoptose , Encéfalo/metabolismo , Encéfalo/patologia , Quinase 9 Dependente de Ciclina/metabolismo , Íntrons , Transtornos Mentais/genética , Transtornos Mentais/metabolismo , Transtornos Mentais/patologia , Camundongos , Camundongos Knockout , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Fator de Processamento Associado a PTB/metabolismo , RNA Polimerase II/metabolismo
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