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1.
Cell ; 184(15): 4032-4047.e31, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34171309

RESUMO

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.


Assuntos
Neoplasias/genética , Neoplasias/imunologia , Splicing de RNA/genética , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Epitopos/imunologia , Etilenodiaminas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoterapia , Inflamação/patologia , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Isoformas de Proteínas/metabolismo , Pirróis/farmacologia , Splicing de RNA/efeitos dos fármacos , Sulfonamidas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
2.
Nat Immunol ; 23(10): 1424-1432, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36138187

RESUMO

B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies-including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab-targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to 'antigen escape'-the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR-Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL.


Assuntos
Linfoma de Burkitt , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Antígenos CD19/genética , Antígenos CD19/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Humanos , Imunoterapia Adotiva/efeitos adversos , Glicoproteínas de Membrana/metabolismo , Poliadenilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Transativadores/metabolismo
3.
Nat Rev Mol Cell Biol ; 22(9): 589-607, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34140671

RESUMO

The human genome contains over one million short tandem repeats. Expansion of a subset of these repeat tracts underlies over fifty human disorders, including common genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72), polyglutamine-associated ataxias and Huntington disease, myotonic dystrophy, and intellectual disability disorders such as Fragile X syndrome. In this Review, we discuss the four major mechanisms by which expansion of short tandem repeats causes disease: loss of function through transcription repression, RNA-mediated gain of function through gelation and sequestration of RNA-binding proteins, gain of function of canonically translated repeat-harbouring proteins, and repeat-associated non-AUG translation of toxic repeat peptides. Somatic repeat instability amplifies these mechanisms and influences both disease age of onset and tissue specificity of pathogenic features. We focus on the crosstalk between these disease mechanisms, and argue that they often synergize to drive pathogenesis. We also discuss the emerging native functions of repeat elements and how their dynamics might contribute to disease at a larger scale than currently appreciated. Lastly, we propose that lynchpins tying these disease mechanisms and native functions together offer promising therapeutic targets with potential shared applications across this class of human disorders.


Assuntos
Expansão das Repetições de DNA/genética , Doenças Neurodegenerativas/genética , Animais , Inativação Gênica , Instabilidade Genômica , Humanos , Mutação , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Especificidade de Órgãos , Biossíntese de Proteínas , Estruturas R-Loop , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo
4.
Cell ; 170(2): 284-297.e18, 2017 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-28689640

RESUMO

Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.


Assuntos
Prosencéfalo Basal/fisiopatologia , Depressão/patologia , Neurônios/patologia , Animais , Aprendizagem da Esquiva , Prosencéfalo Basal/patologia , Depressão/fisiopatologia , Transtorno Depressivo Maior/patologia , Transtorno Depressivo Maior/fisiopatologia , Feminino , Técnicas In Vitro , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Parvalbuminas/metabolismo
5.
Mol Cell ; 84(10): 1886-1903.e10, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38688280

RESUMO

Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.


Assuntos
Neoplasias Hematológicas , Proteínas Musculares , Mutação , Fosfoproteínas , Fatores de Processamento de RNA , Animais , Humanos , Camundongos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/metabolismo , Hematopoese/genética , Íntrons , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Splicing de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo
6.
Mol Cell ; 83(3): 324-329, 2023 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-36736306

RESUMO

Pathogenic repeat sequences underlie several human disorders, including amyotrophic lateral sclerosis, Huntington's disease, and myotonic dystrophy. Here, we speak to several researchers about how repeat sequences have been implicated in affecting all aspects of the Central Dogma of molecular biology through their effects on DNA, RNA, and protein.


Assuntos
Esclerose Lateral Amiotrófica , Doença de Huntington , Distrofia Miotônica , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteínas/genética , Doença de Huntington/genética , RNA/genética , Distrofia Miotônica/genética , Expansão das Repetições de Trinucleotídeos/genética
7.
Cell ; 163(7): 1716-29, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26686653

RESUMO

Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.


Assuntos
Colesterol/metabolismo , Imunidade Inata , Interferon gama/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Humanos , Interferon beta-1b , Proteínas de Membrana/metabolismo , Ácido Mevalônico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
8.
Cell ; 158(3): 534-48, 2014 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-25018104

RESUMO

Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic ß cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.


Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Endorribonucleases/química , Endorribonucleases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Retina/metabolismo , Ribonucleases/antagonistas & inibidores
11.
Nature ; 601(7893): 428-433, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34937946

RESUMO

Although deregulation of transfer RNA (tRNA) biogenesis promotes the translation of pro-tumorigenic mRNAs in cancers1,2, the mechanisms and consequences of tRNA deregulation in tumorigenesis are poorly understood. Here we use a CRISPR-Cas9 screen to focus on genes that have been implicated in tRNA biogenesis, and identify a mechanism by which altered valine tRNA biogenesis enhances mitochondrial bioenergetics in T cell acute lymphoblastic leukaemia (T-ALL). Expression of valine aminoacyl tRNA synthetase is transcriptionally upregulated by NOTCH1, a key oncogene in T-ALL, underlining a role for oncogenic transcriptional programs in coordinating tRNA supply and demand. Limiting valine bioavailability through restriction of dietary valine intake disrupted this balance in mice, resulting in decreased leukaemic burden and increased survival in vivo. Mechanistically, valine restriction reduced translation rates of mRNAs that encode subunits of mitochondrial complex I, leading to defective assembly of complex I and impaired oxidative phosphorylation. Finally, a genome-wide CRISPR-Cas9 loss-of-function screen in differential valine conditions identified several genes, including SLC7A5 and BCL2, whose genetic ablation or pharmacological inhibition synergized with valine restriction to reduce T-ALL growth. Our findings identify tRNA deregulation as a critical adaptation in the pathogenesis of T-ALL and provide a molecular basis for the use of dietary approaches to target tRNA biogenesis in blood malignancies.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Valina-tRNA Ligase , Valina , Animais , Disponibilidade Biológica , Sistemas CRISPR-Cas , Dieta , Complexo I de Transporte de Elétrons/genética , Transportador 1 de Aminoácidos Neutros Grandes , Camundongos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , RNA de Transferência/genética , Valina/metabolismo , Valina-tRNA Ligase/metabolismo
12.
Plant Cell ; 36(10): 4511-4534, 2024 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-39102899

RESUMO

Elevated temperatures impair pollen performance and reproductive success, resulting in lower crop yields. The tomato (Solanum lycopersicum) anthocyanin reduced (are) mutant harbors a mutation in FLAVANONE 3-HYDROXYLASE (F3H), resulting in impaired flavonol antioxidant biosynthesis. The are mutant has reduced pollen performance and seed set relative to the VF36 parental line, phenotypes that are accentuated at elevated temperatures. Transformation of are with the wild-type F3H gene, or chemical complementation with flavonols, prevented temperature-dependent reactive oxygen species (ROS) accumulation in pollen and restored the reduced viability, germination, and tube elongation of are to VF36 levels. Overexpression of F3H in VF36 prevented temperature-driven ROS increases and impaired pollen performance, revealing that flavonol biosynthesis promotes thermotolerance. Although stigmas of are had reduced flavonol and elevated ROS levels, the growth of are pollen tubes was similarly impaired in both are and VF36 pistils. RNA-seq was performed at optimal and stress temperatures in are, VF36, and the F3H overexpression line at multiple timepoints across pollen tube elongation. The number of differentially expressed genes increased over time under elevated temperatures in all genotypes, with the greatest number in are. These findings suggest potential agricultural interventions to combat the negative effects of heat-induced ROS in pollen that lead to reproductive failure.


Assuntos
Flavonóis , Regulação da Expressão Gênica de Plantas , Germinação , Homeostase , Tubo Polínico , Pólen , Espécies Reativas de Oxigênio , Solanum lycopersicum , Termotolerância , Solanum lycopersicum/genética , Solanum lycopersicum/fisiologia , Solanum lycopersicum/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Flavonóis/metabolismo , Termotolerância/genética , Pólen/genética , Pólen/fisiologia , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo
13.
Cell ; 150(4): 710-24, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22901804

RESUMO

The muscleblind-like (Mbnl) family of RNA-binding proteins plays important roles in muscle and eye development and in myotonic dystrophy (DM), in which expanded CUG or CCUG repeats functionally deplete Mbnl proteins. We identified transcriptome-wide functional and biophysical targets of Mbnl proteins in brain, heart, muscle, and myoblasts by using RNA-seq and CLIP-seq approaches. This analysis identified several hundred splicing events whose regulation depended on Mbnl function in a pattern indicating functional interchangeability between Mbnl1 and Mbnl2. A nucleotide resolution RNA map associated repression or activation of exon splicing with Mbnl binding near either 3' splice site or near the downstream 5' splice site, respectively. Transcriptomic analysis of subcellular compartments uncovered a global role for Mbnls in regulating localization of mRNAs in both mouse and Drosophila cells, and Mbnl-dependent translation and protein secretion were observed for a subset of mRNAs with Mbnl-dependent localization. These findings hold several new implications for DM pathogenesis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Distrofia Miotônica/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcriptoma , Regiões 3' não Traduzidas , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Éxons , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Mioblastos/metabolismo , Distrofia Miotônica/genética , Proteínas Nucleares , Especificidade de Órgãos , Sítios de Splice de RNA , Proteínas de Ligação a RNA/genética
14.
Nature ; 595(7866): 283-288, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34010947

RESUMO

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-6. Although pathological innate immune activation is well-documented in severe disease1, the effect of autoantibodies on disease progression is less well-defined. Here we use a high-throughput autoantibody discovery technique known as rapid extracellular antigen profiling7 to screen a cohort of 194 individuals infected with SARS-CoV-2, comprising 172 patients with COVID-19 and 22 healthcare workers with mild disease or asymptomatic infection, for autoantibodies against 2,770 extracellular and secreted proteins (members of the exoproteome). We found that patients with COVID-19 exhibit marked increases in autoantibody reactivities as compared to uninfected individuals, and show a high prevalence of autoantibodies against immunomodulatory proteins (including cytokines, chemokines, complement components and cell-surface proteins). We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signalling and by altering peripheral immune cell composition, and found that mouse surrogates of these autoantibodies increase disease severity in a mouse model of SARS-CoV-2 infection. Our analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics. Our findings suggest a pathological role for exoproteome-directed autoantibodies in COVID-19, with diverse effects on immune functionality and associations with clinical outcomes.


Assuntos
Autoanticorpos/análise , Autoanticorpos/imunologia , COVID-19/imunologia , COVID-19/metabolismo , Proteoma/imunologia , Proteoma/metabolismo , Animais , Antígenos de Superfície/imunologia , COVID-19/patologia , COVID-19/fisiopatologia , Estudos de Casos e Controles , Proteínas do Sistema Complemento/imunologia , Citocinas/imunologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Especificidade de Órgãos/imunologia
15.
Proc Natl Acad Sci U S A ; 121(19): e2321438121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38687782

RESUMO

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.


Assuntos
Sistemas CRISPR-Cas , Núcleo Celular , Edição de Genes , Terapia Genética , Músculo Esquelético , Distrofia Muscular de Duchenne , Edição de Genes/métodos , Animais , Camundongos , Músculo Esquelético/metabolismo , Núcleo Celular/metabolismo , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Humanos , Sinais de Localização Nuclear/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Modelos Animais de Doenças , Mioblastos/metabolismo
16.
Genes Dev ; 33(23-24): 1635-1640, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31624084

RESUMO

Short tandem repeats (STRs) are prone to expansion mutations that cause multiple hereditary neurological and neuromuscular diseases. To study pathomechanisms using mouse models that recapitulate the tissue specificity and developmental timing of an STR expansion gene, we used rolling circle amplification and CRISPR/Cas9-mediated genome editing to generate Dmpk CTG expansion (CTGexp) knockin models of myotonic dystrophy type 1 (DM1). We demonstrate that skeletal muscle myoblasts and brain choroid plexus epithelial cells are particularly susceptible to Dmpk CTGexp mutations and RNA missplicing. Our results implicate dysregulation of muscle regeneration and cerebrospinal fluid homeostasis as early pathogenic events in DM1.


Assuntos
Processamento Alternativo/genética , Repetições de Microssatélites/genética , Músculo Esquelético/fisiopatologia , Distrofia Miotônica/genética , Distrofia Miotônica/fisiopatologia , Splicing de RNA/genética , Regiões 3' não Traduzidas/genética , Animais , Plexo Corióideo/fisiopatologia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/citologia , Mutação , Miotonina Proteína Quinase/genética , Miotonina Proteína Quinase/metabolismo , Proteínas de Ligação a RNA/genética
17.
Nature ; 588(7837): 315-320, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32846427

RESUMO

There is increasing evidence that coronavirus disease 2019 (COVID-19) produces more severe symptoms and higher mortality among men than among women1-5. However, whether immune responses against severe acute respiratory syndrome coronavirus (SARS-CoV-2) differ between sexes, and whether such differences correlate with the sex difference in the disease course of COVID-19, is currently unknown. Here we examined sex differences in viral loads, SARS-CoV-2-specific antibody titres, plasma cytokines and blood-cell phenotyping in patients with moderate COVID-19 who had not received immunomodulatory medications. Male patients had higher plasma levels of innate immune cytokines such as IL-8 and IL-18 along with more robust induction of non-classical monocytes. By contrast, female patients had more robust T cell activation than male patients during SARS-CoV-2 infection. Notably, we found that a poor T cell response negatively correlated with patients' age and was associated with worse disease outcome in male patients, but not in female patients. By contrast, higher levels of innate immune cytokines were associated with worse disease progression in female patients, but not in male patients. These findings provide a possible explanation for the observed sex biases in COVID-19, and provide an important basis for the development of a sex-based approach to the treatment and care of male and female patients with COVID-19.


Assuntos
COVID-19/imunologia , Citocinas/imunologia , Imunidade Inata/imunologia , SARS-CoV-2/imunologia , Caracteres Sexuais , Linfócitos T/imunologia , COVID-19/sangue , COVID-19/virologia , Quimiocinas/sangue , Quimiocinas/imunologia , Estudos de Coortes , Citocinas/sangue , Progressão da Doença , Feminino , Humanos , Ativação Linfocitária , Masculino , Monócitos/imunologia , Fenótipo , Prognóstico , RNA Viral/análise , SARS-CoV-2/patogenicidade , Carga Viral
18.
J Cell Sci ; 136(19)2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37795818

RESUMO

Emergent cell behaviors that drive tissue morphogenesis are the integrated product of instructions from gene regulatory networks, mechanics and signals from the local tissue microenvironment. How these discrete inputs intersect to coordinate diverse morphogenic events is a critical area of interest. Organ-on-chip technology has revolutionized the ability to construct and manipulate miniaturized human tissues with organotypic three-dimensional architectures in vitro. Applications of organ-on-chip platforms have increasingly transitioned from proof-of-concept tissue engineering to discovery biology, furthering our understanding of molecular and mechanical mechanisms that operate across biological scales to orchestrate tissue morphogenesis. Here, we provide the biological framework to harness organ-on-chip systems to study tissue morphogenesis, and we highlight recent examples where organ-on-chips and associated microphysiological systems have enabled new mechanistic insight in diverse morphogenic settings. We further highlight the use of organ-on-chip platforms as emerging test beds for cell and developmental biology.


Assuntos
Sistemas Microfisiológicos , Engenharia Tecidual , Humanos , Engenharia Tecidual/métodos , Morfogênese
19.
N Engl J Med ; 386(8): 735-743, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35196427

RESUMO

BACKGROUND: Covalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood. METHODS: We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors. RESULTS: Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors. CONCLUSIONS: Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.).


Assuntos
Tirosina Quinase da Agamaglobulinemia , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B , Mutação , Fosfolipase C gama , Inibidores de Proteínas Quinases , Humanos , Pessoa de Meia-Idade , Adenina/análogos & derivados , Adenina/farmacologia , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/ultraestrutura , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Fosfolipase C gama/genética , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Antígenos de Linfócitos B/metabolismo , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos
20.
Mol Cell ; 68(3): 479-490.e5, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29056323

RESUMO

Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endonucleases/metabolismo , Terapia Genética/métodos , Repetições de Microssatélites , Distrofia Miotônica/terapia , Transcrição Gênica , Processamento Alternativo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Antígeno CD24/genética , Antígeno CD24/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Regulação para Baixo , Ativação Enzimática , Feminino , Vetores Genéticos , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos Transgênicos , Mioblastos/metabolismo , Mioblastos/patologia , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , RNA Guia de Cinetoplastídeos/biossíntese , RNA Guia de Cinetoplastídeos/genética , Transdução Genética , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo
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