RESUMEN
Defect in the SMN1 gene causes spinal muscular atrophy (SMA), which shows loss of motor neurons, muscle weakness and atrophy. While current treatment strategies, including small molecules or viral vectors, have shown promise in improving motor function and survival, achieving a definitive and long-term correction of SMA's endogenous mutations and phenotypes remains highly challenging. We have previously developed a CRISPR-Cas9 based homology-independent targeted integration (HITI) strategy, enabling unidirectional DNA knock-in in both dividing and non-dividing cells in vivo. In this study, we demonstrated its utility by correcting an SMA mutation in mice. When combined with Smn1 cDNA supplementation, it exhibited long-term therapeutic benefits in SMA mice. Our observations may provide new avenues for the long-term and efficient treatment of inherited diseases.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Terapia Genética , Atrofia Muscular Espinal , Proteína 1 para la Supervivencia de la Neurona Motora , Atrofia Muscular Espinal/terapia , Atrofia Muscular Espinal/genética , Animales , Edición Génica/métodos , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Ratones , Terapia Genética/métodos , Modelos Animales de Enfermedad , Humanos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Mutación , Masculino , FemeninoRESUMEN
Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.
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Complejo CD3 , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares , Proteínas de la Membrana , Carcinoma Pulmonar de Células Pequeñas , Linfocitos T , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Carcinoma Pulmonar de Células Pequeñas/inmunología , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/terapia , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Complejo CD3/inmunología , Animales , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/inmunología , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Línea Celular Tumoral , Activación de Linfocitos/inmunología , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute myeloid leukemia (AML) is a heterogenous malignancy characterized by distinct lineage subtypes and various genetic/epigenetic alterations. As with other neoplasms, AML cells have well-known aerobic glycolysis, but metabolic variations depending on cellular lineages also exist. Lysine-specific demethylase-1 (LSD1) has been reported to be crucial for human leukemogenesis, which is currently one of the emerging therapeutic targets. However, metabolic roles of LSD1 and lineage-dependent factors remain to be elucidated in AML cells. Here, we show that LSD1 directs a hematopoietic lineage-specific metabolic program in AML subtypes. Erythroid leukemia (EL) cells particularly showed activated glycolysis and high expression of LSD1 in both AML cell lines and clinical samples. Transcriptome, chromatin immunoprecipitation-sequencing, and metabolomic analyses revealed that LSD1 was essential not only for glycolysis but also for heme synthesis, the most characteristic metabolic pathway of erythroid origin. Notably, LSD1 stabilized the erythroid transcription factor GATA1, which directly enhanced the expression of glycolysis and heme synthesis genes. In contrast, LSD1 epigenetically downregulated the granulo-monocytic transcription factor C/EBPα. Thus, the use of LSD1 knockdown or chemical inhibitor dominated C/EBPα instead of GATA1 in EL cells, resulting in metabolic shifts and growth arrest. Furthermore, GATA1 suppressed the gene encoding C/EBPα that then acted as a repressor of GATA1 target genes. Collectively, we conclude that LSD1 shapes metabolic phenotypes in EL cells by balancing these lineage-specific transcription factors and that LSD1 inhibitors pharmacologically cause lineage-dependent metabolic remodeling.
Asunto(s)
Leucemia Eritroblástica Aguda , Proteína alfa Potenciadora de Unión a CCAAT , Factor de Transcripción GATA1/genética , Histona Demetilasas/genética , Humanos , Leucemia Eritroblástica Aguda/genética , Proteínas Proto-Oncogénicas , Factores de TranscripciónRESUMEN
SKY59 or RO7112689 is a humanized monoclonal antibody against complement protein C5 with pH-dependent C5-binding and neonatal Fc receptor-mediated recycling capabilities, which result in long-lasting neutralization of C5. We developed and validated a novel total drug assay for quantification of target-binding competent SKY59 in the presence of endogenous C5 in cynomolgus monkey plasma. The target-binding competent SKY59 was determined after complex formation by the addition of recombinant monkey C5 using goat anti-human IgG-heavy chain monkey-adsorbed polyclonal antibody as a capture antibody and rabbit anti-C5 monoclonal antibody (mAb) non-competing with SKY59 for detection. The total SKY59 assay was shown to be accurate and precise over the range of 0.05-3.2 µg/mL as well as be tolerant to more than 400 µg/mL of C5 (~ 3000-fold molar excess of target). We also developed and validated a total C5 assay, confirmed selectivity and parallelism, and verified the utility of recombinant monkey C5 for the total C5 assay as well as the total SKY59 assay. Furthermore, we used these validated methods to measure SKY59 and C5 concentrations in cynomolgus monkey plasma samples in a toxicology study. This total drug assay can be applied not only to other antibody therapeutics against shed/soluble targets when a non-competing reagent mAb is available but also for clinical studies when a reagent mAb specific for engineered Fc region on a therapeutic mAb is available.
Asunto(s)
Anticuerpos Monoclonales Humanizados/sangre , Bioensayo/métodos , Complemento C5/antagonistas & inhibidores , Monitoreo de Drogas/métodos , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacocinética , Complemento C5/análisis , Complemento C5/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Antígenos de Histocompatibilidad Clase I/metabolismo , Inyecciones Intravenosas , Inyecciones Subcutáneas , Límite de Detección , Macaca fascicularis , Masculino , Modelos Animales , Receptores Fc/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.
Asunto(s)
Adenosina Trifosfato , Neoplasias , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Neoplasias , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológico , Microambiente Tumoral , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
Central nervous system tumors are classified based on an integrated diagnosis combining histology and molecular characteristics, including IDH1/2 and H3-K27M mutations, as well as 1p/19q codeletion. Here, we aimed to develop and assess the feasibility of a glioma-tailored 48-gene next-generation sequencing (NGS) panel for integrated glioma diagnosis. We designed a glioma-tailored 48-gene NGS panel for detecting 1p/19q codeletion and mutations in IDH1/2, TP53, PTEN, PDGFRA, NF1, RB1, CDKN2A/B, CDK4, and the TERT promoter (TERTp). We analyzed 106 glioma patients (grade II: 19 cases, grade III: 23 cases, grade IV: 64 cases) using this system. The 1p/19q codeletion was detected precisely in oligodendroglial tumors using our NGS panel. In a cohort of 64 grade â £ gliomas, we identified 56 IDH-wildtype glioblastomas. Within these IDH-wildtype glioblastomas, 33 samples (58.9%) showed a mutation in TERTp. Notably, PDGFRA mutations and their amplification were more commonly seen in TERTp-wildtype glioblastomas (43%) than in TERTp-mutant glioblastomas (6%) (P = .001). Hierarchical molecular classification of IDH-wildtype glioblastomas revealed 3 distinct groups of IDH-wildtype glioblastomas. One major cluster was characterized by mutations in PDGFRA, amplification of CDK4 and PDGFRA, homozygous deletion of CDKN2A/B, and absence of TERTp mutations. This cluster was significantly associated with older age (P = .021), higher Ki-67 score (P = .007), poor prognosis (P = .012), and a periventricular tumor location. We report the development of a glioma-tailored NGS panel for detecting 1p/19q codeletion and driver gene mutations on a single platform. Our panel identified distinct subtypes of IDH- and TERTp-wildtype glioblastomas with frequent PDGFRA alterations.
Asunto(s)
Glioblastoma/genética , Isocitrato Deshidrogenasa/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Telomerasa/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Glioblastoma/clasificación , Glioblastoma/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genéticaAsunto(s)
Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Glucuronidasa/uso terapéutico , Osteoartritis de la Rodilla/tratamiento farmacológico , Receptor Tipo II de Factor de Crecimiento Transformador beta/uso terapéutico , Animales , Cartílago Articular/patología , Técnicas de Cultivo de Célula , Condrocitos/patología , Humanos , Proteínas Klotho , Osteoartritis de la Rodilla/inducido químicamente , Papaína , Ratas , Proteínas Recombinantes/uso terapéuticoRESUMEN
Tolerance to severe tumor microenvironments, including hypoxia and nutrient starvation, is a common feature of aggressive cancer cells and can be targeted. However, metabolic alterations that support cancer cells upon nutrient starvation are not well understood. Here, by comprehensive metabolome analyses, we show that glutamine deprivation leads to phosphoethanolamine (PEtn) accumulation in cancer cells via the downregulation of PEtn cytidylyltransferase (PCYT2), a rate-limiting enzyme of phosphatidylethanolamine biosynthesis. PEtn accumulation correlated with tumor growth under nutrient starvation. PCYT2 suppression was partially mediated by downregulation of the transcription factor ELF3. Furthermore, PCYT2 overexpression reduced PEtn levels and tumor growth. In addition, PEtn accumulation and PCYT2 downregulation in human breast tumors correlated with poor prognosis. Thus, we show that glutamine deprivation leads to tumor progression by regulating PE biosynthesis via the ELF3-PCYT2 axis. Furthermore, manipulating glutamine-responsive genes could be a therapeutic approach to limit cancer progression.
Asunto(s)
Regulación hacia Abajo/genética , Etanolaminas/metabolismo , Glutamina/metabolismo , ARN Nucleotidiltransferasas/genética , Inanición/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Progresión de la Enfermedad , Células HeLa , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células MCF-7 , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-ets/genética , Transcripción Genética/genéticaRESUMEN
Transcriptional and epigenetic regulation is fundamentally involved in initiating and maintaining progression of cellular differentiation. The 2 types of thermogenic adipocytes, brown and beige, are thought to be of different origins but share functionally similar phenotypes. Here, we report that lysine-specific demethylase 2 (LSD2) regulates the expression of genes associated with lineage identity during the differentiation of brown and beige adipogenic progenitors in mice. In HB2 mouse brown preadipocytes, short hairpin RNA-mediated knockdown (KD) of LSD2 impaired formation of lipid droplet-containing adipocytes and down-regulated brown adipogenesis-associated genes. Transcriptomic analysis revealed that myogenesis-associated genes were up-regulated in LSD2-KD cells under adipogenic induction. In addition, loss of LSD2 during later phases of differentiation had no obvious influence on adipogenic traits, suggesting that LSD2 functions during earlier phases of brown adipocyte differentiation. Using adipogenic cells from the brown adipose tissues of LSD2-knockout (KO) mice, we found reduced expression of brown adipogenesis genes, whereas myogenesis genes were not affected. In contrast, when LSD2-KO cells from inguinal white adipose tissues were subjected to beige induction, these cells showed a dramatic rise in myogenic gene expression. Collectively, these results suggest that LSD2 regulates distinct sets of genes during brown and beige adipocyte formation.-Takase, R., Hino, S., Nagaoka, K., Anan, K., Kohrogi, K., Araki, H., Hino, Y., Sakamoto, A., Nicholson, T. B., Chen, T., Nakao, M. Lysine-specific demethylase-2 is distinctively involved in brown and beige adipogenic differentiation.
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Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Histona Demetilasas/metabolismo , ARN Interferente Pequeño/metabolismo , Adipogénesis/genética , Adipogénesis/fisiología , Animales , Western Blotting , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Inmunoprecipitación de Cromatina , Femenino , Histona Demetilasas/genética , Lentivirus/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , PPAR gamma/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The antibody drug market is rapidly expanding, and various antibody engineering technologies are being developed to create antibodies that can provide better benefit to patients. Although bispecific antibody drugs have been researched for more than 30â¯years, currently only a limited number of bispecific antibodies have achieved regulatory approval. Of the few successful examples of industrially manufacturing a bispecific antibody, the "common light chain format" is an elegant technology that simplifies the purification of a whole IgG-type bispecific antibody. Using this IgG format, the bispecific function can be introduced while maintaining the natural molecular shape of the antibody. In this article, we will first introduce the outline, prospects, and limitations of the common light chain format. Then, we will describe the identification and optimization process for ERY974, an anti-glypican-3â¯×â¯anti-CD3ε T cell-redirecting bispecific antibody with a common light chain. This format includes one of Chugai's proprietary technologies, termed ART-Ig technology, which consists of a method to identify a common light chain, isoelectric point (pI) engineering to purify the desired bispecific IgG antibody from byproducts, and Fc heterodimerization by an electrostatic steering effect. Furthermore, we describe some tips for de-risking the antibody when engineering a T cell redirecting antibody.
Asunto(s)
Anticuerpos Biespecíficos , Inmunoglobulina G , Cadenas Ligeras de Inmunoglobulina , Ingeniería de Proteínas/métodos , Animales , Complejo CD3/inmunología , Glipicanos/inmunología , Humanos , RatonesRESUMEN
Large cutaneous ulcers are, in severe cases, life threatening1,2. As the global population ages, non-healing ulcers are becoming increasingly common1,2. Treatment currently requires the transplantation of pre-existing epithelial components, such as skin grafts, or therapy using cultured cells2. Here we develop alternative supplies of epidermal coverage for the treatment of these kinds of wounds. We generated expandable epithelial tissues using in vivo reprogramming of wound-resident mesenchymal cells. Transduction of four transcription factors that specify the skin-cell lineage enabled efficient and rapid de novo epithelialization from the surface of cutaneous ulcers in mice. Our findings may provide a new therapeutic avenue for treating skin wounds and could be extended to other disease situations in which tissue homeostasis and repair are impaired.
Asunto(s)
Reprogramación Celular , Células Epiteliales/citología , Úlcera Cutánea/patología , Piel/citología , Heridas y Lesiones/patología , Animales , Linaje de la Célula , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Medicina Regenerativa , Piel/patología , Úlcera Cutánea/terapia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Cicatrización de Heridas , Heridas y Lesiones/terapiaRESUMEN
The metabolic properties of cells are formed under the influence of environmental factors such as nutrients and hormones. Although such a metabolic program is likely initiated through epigenetic mechanisms, the direct links between metabolic cues and activities of chromatin modifiers remain largely unknown. In this study, we show that lysine-specific demethylase-1 (LSD1) controls the metabolic program in myogenic differentiation, under the action of catabolic hormone, glucocorticoids. By using transcriptomic and epigenomic approaches, we revealed that LSD1 bound to oxidative metabolism and slow-twitch myosin genes, and repressed their expression. Consistent with this, loss of LSD1 activity during differentiation enhanced the oxidative capacity of myotubes. By testing the effects of various hormones, we found that LSD1 levels were decreased by treatment with the glucocorticoid dexamethasone (Dex) in cultured myoblasts and in skeletal muscle from mice. Mechanistically, glucocorticoid signaling induced expression of a ubiquitin E3 ligase, JADE-2, which was responsible for proteasomal degradation of LSD1. Consequently, in differentiating myoblasts, chemical inhibition of LSD1, in combination with Dex treatment, synergistically de-repressed oxidative metabolism genes, concomitant with increased histone H3 lysine 4 methylation at these loci. These findings demonstrated that LSD1 serves as an epigenetic regulator linking glucocorticoid action to metabolic programming during myogenic differentiation.
Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Histona Demetilasas/metabolismo , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Histona Demetilasas/antagonistas & inhibidores , Histonas/metabolismo , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Oxidación-Reducción , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Scratching is an important factor exacerbating skin lesions through the so-called itch-scratch cycle in atopic dermatitis (AD). In mice, interleukin (IL)-31 and its receptor IL-31 receptor A (IL-31RA) are known to play a critical role in pruritus and the pathogenesis of AD; however, study of their precise roles in primates is hindered by the low sequence homologies between primates and mice and the lack of direct evidence of itch sensation by IL-31 in primates. We showed that administration of cynomolgus IL-31 induces transient scratching behaviour in cynomolgus monkeys and by that were able to establish a monkey model of scratching. We then showed that a single subcutaneous injection of 1 mg/kg nemolizumab, a humanized anti-human IL-31RA monoclonal antibody that also neutralizes cynomolgus IL-31 signalling and shows a good pharmacokinetic profile in cynomolgus monkeys, suppressed the IL-31-induced scratching for about 2 months. These results suggest that the IL-31 axis and IL-31RA axis play as critical a role in the induction of scratching in primates as in mice and that the blockade of IL-31 signalling by an anti-human IL-31RA antibody is a promising therapeutic approach for treatment of AD. Nemolizumab is currently under investigation in clinical trials.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Interleucinas/farmacología , Prurito/inducido químicamente , Receptores de Interleucina/metabolismo , Células A549 , Animales , Células CHO , Línea Celular , Cricetulus , ADN Complementario/metabolismo , Humanos , Cinética , Macaca fascicularis , Masculino , Ratones , Prurito/metabolismo , Transducción de Señal , Piel/inmunología , Piel/patología , Enfermedades de la Piel/inmunología , Enfermedades de la Piel/patologíaRESUMEN
Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell-redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid "on-target off-tumor" toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G-structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein-4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Glipicanos/inmunología , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Biespecíficos/farmacocinética , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Complejo CD3/metabolismo , Citocinas/metabolismo , Humanos , Inmunocompetencia/efectos de los fármacos , Inyecciones Intravenosas , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Macaca fascicularis , Ratones Transgénicos , Esteroides/farmacología , Esteroides/uso terapéutico , Linfocitos T/efectos de los fármacosRESUMEN
Dysregulation of the complement system is linked to the pathogenesis of a variety of hematological disorders. Eculizumab, an anti-complement C5 monoclonal antibody, is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). However, because of high levels of C5 in plasma, eculizumab has to be administered biweekly by intravenous infusion. By applying recycling technology through pH-dependent binding to C5, we generated a novel humanized antibody against C5, SKY59, which has long-lasting neutralization of C5. In cynomolgus monkeys, SKY59 suppressed C5 function and complement activity for a significantly longer duration compared to a conventional antibody. Furthermore, epitope mapping by X-ray crystal structure analysis showed that a histidine cluster located on C5 is crucial for the pH-dependent interaction with SKY59. This indicates that the recycling effect of SKY59 is driven by a novel mechanism of interaction with its antigen and is distinct from other known pH-dependent antibodies. Finally, SKY59 showed neutralizing effect on C5 variant p.Arg885His, while eculizumab does not inhibit complement activity in patients carrying this mutation. Collectively, these results suggest that SKY59 is a promising new anti-C5 agent for patients with PNH and other complement-mediated disorders.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Complemento C5/antagonistas & inhibidores , Complemento C5/inmunología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/química , Complemento C5/química , Cristalografía por Rayos X , Hemoglobinuria Paroxística/tratamiento farmacológico , Humanos , Macaca fascicularis , Unión Proteica , Conformación ProteicaRESUMEN
Cellular senescence is an irreversible growth arrest that contributes to development, tumor suppression, and age-related conditions. Senescent cells show active metabolism compared with proliferating cells, but the underlying mechanisms remain unclear. Here we show that the SETD8/PR-Set7 methyltransferase, which catalyzes mono-methylation of histone H4 at lysine 20 (H4K20me1), suppresses nucleolar and mitochondrial activities to prevent cellular senescence. SETD8 protein was selectively downregulated in both oncogene-induced and replicative senescence. Inhibition of SETD8 alone was sufficient to trigger senescence. Under these states, the expression of genes encoding ribosomal proteins (RPs) and ribosomal RNAs as well as the cyclin-dependent kinase (CDK) inhibitor p16INK4A was increased, with a corresponding reduction of H4K20me1 at each locus. As a result, the loss of SETD8 concurrently stimulated nucleolar function and retinoblastoma protein-mediated mitochondrial metabolism. In conclusion, our data demonstrate that SETD8 acts as a barrier to prevent cellular senescence through chromatin-mediated regulation of senescence-associated metabolic remodeling.
Asunto(s)
Senescencia Celular/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Metiltransferasas/metabolismo , Línea Celular , Nucléolo Celular/metabolismo , Cromatina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Replicación del ADN/fisiología , Regulación hacia Abajo/fisiología , Regulación de la Expresión Génica/fisiología , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Mitocondrias/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismoRESUMEN
Lysine-specific demethylase-1 (LSD1) removes the methyl groups from mono- and di-methylated lysine 4 of histone H3. Previous studies have linked LSD1 to malignancy in several human tumors, and LSD1 is considered to epigenetically regulate the energy metabolism genes in adipocytes and hepatocellular carcinoma. This study investigates the function of LSD1 in the invasive activity and the metabolism of esophageal cancer cells. We investigated whether LSD1 immunohistochemical expression levels are related to clinical and pathological features, including the maximum standard uptake value in fluorodeoxyglucose positron emission tomography assay. The influence of LSD1 on cell proliferation, invasion and glucose uptake was evaluated in vitro by using specific small interfering RNA for LSD1, and an LSD1 inhibitor. We also evaluated two major energy pathways (glycolytic pathway and mitochondrial respiration) by measuring the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) with an extracellular flux analyzer. High LSD1 immunohistochemical expression was significantly associated with high tumor stage, lymphovascular invasion, poor prognosis, and high maximum standard uptake value in esophageal cancer patients. In the in vitro analysis, LSD1 knockdown significantly suppressed the invasive activity and glucose uptake of cancerous cells, reduced their ECAR and increased their OCR and OCR/ECAR. LSD1 may contribute to malignant behavior by regulating the invasive activity and metabolism, activating the glycolytic pathway and inhibiting the mitochondrial respiration of esophageal cancer cells. The results support LSD1 as a potential therapeutic target.
Asunto(s)
Neoplasias Esofágicas/enzimología , Neoplasias Esofágicas/patología , Histona Demetilasas/metabolismo , Invasividad Neoplásica/patología , Anciano , Western Blotting , Neoplasias Esofágicas/diagnóstico por imagen , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/diagnóstico por imagen , Tomografía de Emisión de Positrones , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Metabolism is closely linked with cellular state and biological processes, but the mechanisms controlling metabolic properties in different contexts remain unclear. Cellular senescence is an irreversible growth arrest induced by various stresses, which exhibits active secretory and metabolic phenotypes. Here, we show that retinoblastoma protein (RB) plays a critical role in promoting the metabolic flow by activating both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) in cells that have undergone oncogene-induced senescence (OIS). A combination of real-time metabolic monitoring, and metabolome and gene expression analyses showed that OIS-induced fibroblasts developed an accelerated metabolic flow. The loss of RB downregulated a series of glycolytic genes and simultaneously reduced metabolites produced from the glycolytic pathway, indicating that RB upregulates glycolytic genes in OIS cells. Importantly, both mitochondrial OXPHOS and glycolytic activities were abolished in RB-depleted or downstream glycolytic enzyme-depleted OIS cells, suggesting that RB-mediated glycolytic activation induces a metabolic flux into the OXPHOS pathway. Collectively, our findings reveal that RB essentially functions in metabolic remodeling and the maintenance of the active energy production in OIS cells.
Asunto(s)
Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes ras , Glucólisis/genética , Fosforilación Oxidativa , Proteína de Retinoblastoma/genética , Línea Celular Tumoral , Senescencia Celular/genética , Ciclo del Ácido Cítrico/genética , Células Epiteliales/patología , Perfilación de la Expresión Génica , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Proteína de Retinoblastoma/deficiencia , Transducción de SeñalRESUMEN
The hallmark of most cancer cells is the metabolic shift from mitochondrial to glycolytic metabolism for adapting to the surrounding environment. Although epigenetic modification is intimately linked to cancer, the molecular mechanism, by which epigenetic factors regulate cancer metabolism, is poorly understood. Here, we show that lysine-specific demethylase-1 (LSD1, KDM1A) has an essential role in maintaining the metabolic shift in human hepatocellular carcinoma cells. Inhibition of LSD1 reduced glucose uptake and glycolytic activity, with a concurrent activation of mitochondrial respiration. These metabolic changes coexisted with the inactivation of the hypoxia-inducible factor HIF1α, resulting in a decreased expression of GLUT1 and glycolytic enzymes. In contrast, during LSD1 inhibition, a set of mitochondrial metabolism genes was activated with the concomitant increase of methylated histone H3 at lysine 4 in the promoter regions. Consistently, both LSD1 and GLUT1 were significantly overexpressed in carcinoma tissues. These findings demonstrate the epigenetic plasticity of cancer cell metabolism, which involves an LSD1-mediated mechanism.