RESUMEN
The tumor microenvironment presents many obstacles to effective chimeric antigen receptor (CAR) T cell therapy, including glucose competition from tumor and myeloid cells. Using mouse models of acute lymphoblastic leukemia (ALL), renal cell carcinoma (RCC), and glioblastoma (GBM), we show that enforced expression of the glucose transporter GLUT1 enhances anti-tumor efficacy and promotes favorable CAR-T cell phenotypes for two clinically relevant CAR designs, 19-28z and IL13Rα2-BBz. In the NALM6 ALL model, 19-28z-GLUT1 promotes T stem cell-like memory formation and prolongs survival. RNA sequencing of these CAR-T cells reveals that the overexpression of GLUT1, but not GLUT3, enriches for genes involved in glycolysis, mitochondrial respiration, and memory precursor phenotypes. Extending these data, 19-28z-GLUT1 CAR-T cells improve tumor control and response to rechallenge in an RCC patient-derived xenograft model. Furthermore, IL13Rα2-BBz CAR-T cells overexpressing GLUT1 prolong the survival of mice bearing orthotopic GBMs and exhibit decreased exhaustion markers. This novel engineering approach can offer a competitive advantage to CAR-T cells in harsh tumor environments where glucose is limiting.
Asunto(s)
Transportador de Glucosa de Tipo 1 , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Humanos , Ratones , Línea Celular Tumoral , Modelos Animales de Enfermedad , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Inmunoterapia Adoptiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
The fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.
Asunto(s)
Proteína de la Leucemia Mieloide-Linfoide , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Epigénesis Genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Genes Reguladores , CromatinaRESUMEN
Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia , Glucosa/metabolismo , Glucólisis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Vía de Pentosa Fosfato , Proteínas Serina-Treonina Quinasas/metabolismo , Estrés Fisiológico , Aminoácidos/deficiencia , Aminoácidos/metabolismo , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/deficiencia , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Biomarcadores de Tumor/metabolismo , Muerte Celular , Proteínas de Unión al ADN/metabolismo , Femenino , Fructosa-Bifosfatasa/metabolismo , Genotipo , Células HCT116 , Hexoquinasa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Células MCF-7 , Masculino , Ratones Noqueados , Fenotipo , Fosfofructoquinasa-1/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Understanding pore-scale morphology and distribution of remaining oil in pore space are of great importance to carry out in-depth tapping of oil potential. Taking two water-wet cores from a typical clastic reservoir in China as an example, X-ray CT imaging is conducted at different experimental stages of water flooding and polymer-surfactant (P-S) flooding by using a high-resolution X-ray microtomography. Based on X-ray micro-CT image processing, 3D visualization of rock microstructure and fluid distribution at the pore scale is achieved. The integral geometry newly developed is further introduced to characterize pore-scale morphology and distribution of remaining oil in pore space. The underlying mechanism of oil recovery by P-S flooding is further explored. The results show that the average diameter of oil droplets gradually decreases, and the topological connectivity becomes worse after water flooding and P-S flooding. Due to the synergistic effect of "1 + 1 > 2" between the strong sweep efficiency of surfactant and the enlarged swept volume of the polymer, oil droplets with a diameter larger than 124.58 µm can be gradually stripped out by the polymer-surfactant system, causing a more scattered distribution of oil droplets in pore spaces of the cores. The network-like oil clusters are still dominant when water flooding is continued to 98% of water cut, but the dominant pore-scale oil morphology has evolved from network-like to porous-type and isolated-type after P-S flooding, which can provide strong support for further oil recovery in the later stage of chemical flooding.
Asunto(s)
Petróleo , Surfactantes Pulmonares , Tensoactivos , Polímeros , Microtomografía por Rayos X/métodos , AguaRESUMEN
T-cell Acute Lymphoblastic Leukemia (T-ALL) is frequently characterized by glucocorticoid (GC) resistance, which is associated with inferior outcomes, thus highlighting the need for novel therapeutic approaches for GC resistant T-ALL. The pTCR/TCR signaling pathways play a critical role in cell fate decisions during physiological thymocyte development, with an interplay between TCR and glucocorticoid receptor (GR) signaling determining the T-lymphocyte selection process. We performed an shRNA screen in vitro and in vivo in T-ALL cell lines and patient derived xenograft (PDX) samples to identify vulnerabilities in the pTCR/TCR pathway and identified a critical role for the kinase LCK in cell proliferation. LCK knockdown or inhibition with dasatinib (DAS) caused cell cycle arrest. Combination of DAS with dexamethasone (DEX) resulted in significant drug synergy leading to cell death. The efficacy of this drug combination was underscored in a randomized phase II-like murine trial, recapitulating an early phase human clinical trial. T-ALL expansion in immunocompromised mice was significantly impaired using this drug combination, relative to mice receiving control vehicle or single drug treatment, highlighting the immediate clinical relevance of this drug combination for high risk T-ALL patients. Our results thus provide a strategy to improve the efficacy of current chemotherapy platforms and circumvent GC resistance.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animales , Apoptosis , Línea Celular Tumoral , Dasatinib/farmacología , Dexametasona/farmacología , Resistencia a Antineoplásicos , Glucocorticoides/farmacología , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Linfocitos TRESUMEN
Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a life-threatening disease without an effective drug at present. Fibroblast growth factor 21 (FGF21) was reported to be protective against inflammation in metabolic disease in recent studies. However, the role of FGF21 in ALI has been rarely investigated. In this study, it was found that the expression of FGF21 was markedly increased in lung tissue under lipopolysaccharide (LPS) stimulation in vivo, whereas it was decreased in lung epithelial cells under LPS stimulation in vitro. Therefore, our research aimed to elucidate the potential role of FGF21 in LPS-induced ALI and to detect possible underlying mechanisms. The results revealed that the deficiency of FGF21 aggravated pathological damage, inflammatory infiltration, and pulmonary function in LPS-induced ALI, while exogenous administration of FGF21 improved these manifestations. Moreover, through RNA sequencing and enrichment analysis, it was unveiled that FGF21 might play a protective role in LPS-induced ALI via JAK2/STAT3 signaling pathway. The therapeutic effect of FGF21 was weakened after additional usage of JAK2 activator in vivo. Further investigation revealed that FGF21 significantly inhibited STAT3 phosphorylation and impaired the nuclear translocation of STAT3 in vitro. In addition, the aggravation of inflammation caused by silencing FGF21 can be alleviated by JAK2 inhibitor in vitro. Collectively, these findings unveil a potent protective effect of FGF21 against LPS-induced ALI by inhibiting the JAK2/STAT3 pathway, implying that FGF21 might be a novel and effective therapy for ALI.
Asunto(s)
Lesión Pulmonar Aguda , Factores de Crecimiento de Fibroblastos , Síndrome de Dificultad Respiratoria , Humanos , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/patología , Janus Quinasa 2/metabolismo , Lipopolisacáridos/farmacología , Pulmón/metabolismo , Transducción de Señal , Factor de Transcripción STAT3/metabolismoRESUMEN
Chimeric antigen receptor T cells have dramatically improved the treatment of hematologic malignancies. T cell antigen receptor (TCR)-based cell therapies are yet to achieve comparable outcomes. Importantly, chimeric antigen receptors not only target selected antigens but also reprogram T cell functions through the co-stimulatory pathways that they engage upon antigen recognition. We show here that a fusion receptor comprising the CD80 ectodomain and the 4-1BB cytoplasmic domain, termed 80BB, acts as both a ligand and a receptor to engage the CD28 and 4-1BB pathways, thereby increasing the antitumor potency of human leukocyte antigen-independent TCR (HIT) receptor- or TCR-engineered T cells and tumor-infiltrating lymphocytes. Furthermore, 80BB serves as a switch receptor that provides agonistic 4-1BB co-stimulation upon its ligation by the inhibitory CTLA4 molecule. By combining multiple co-stimulatory features in a single antigen-agnostic synthetic receptor, 80BB is a promising tool to sustain CD3-dependent T cell responses in a wide range of targeted immunotherapies.
Asunto(s)
Antígenos CD28 , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Humanos , Receptores Quiméricos de Antígenos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Antígenos CD28/inmunología , Animales , Ratones , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Antígeno B7-1/inmunología , Linfocitos T/inmunología , Antígeno CTLA-4/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Inmunoterapia Adoptiva/métodos , Activación de Linfocitos/inmunología , Tratamiento Basado en Trasplante de Células y Tejidos/métodosRESUMEN
Suboptimal functional persistence limits the efficacy of adoptive T-cell therapies. CD28-based chimeric antigen receptors (CAR) impart potent effector function to T cells but with a limited lifespan. We show here that the genetic disruption of SUV39H1, which encodes a histone-3, lysine-9 methyl-transferase, enhances the early expansion, long-term persistence, and overall antitumor efficacy of human CAR T cells in leukemia and prostate cancer models. Persisting SUV39H1-edited CAR T cells demonstrate improved expansion and tumor rejection upon multiple rechallenges. Transcriptional and genome accessibility profiling of repeatedly challenged CAR T cells shows improved expression and accessibility of memory transcription factors in SUV39H1-edited CAR T cells. SUV39H1 editing also reduces expression of inhibitory receptors and limits exhaustion in CAR T cells that have undergone multiple rechallenges. Our findings thus demonstrate the potential of epigenetic programming of CAR T cells to balance their function and persistence for improved adoptive cell therapies. SIGNIFICANCE: T cells engineered with CD28-based CARs possess robust effector function and antigen sensitivity but are hampered by limited persistence, which may result in tumor relapse. We report an epigenetic strategy involving disruption of the SUV39H1-mediated histone-silencing program that promotes the functional persistence of CD28-based CAR T cells. See related article by López-Cobo et al., p. 120. This article is featured in Selected Articles from This Issue, p. 5.
Asunto(s)
Leucemia , Receptores Quiméricos de Antígenos , Masculino , Humanos , Linfocitos T , Receptores de Antígenos de Linfocitos T , Histonas/metabolismo , Antígenos CD28/genética , Antígenos CD28/metabolismo , Inmunoterapia Adoptiva , Leucemia/metabolismo , Metilación , Ensayos Antitumor por Modelo de Xenoinjerto , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T-cell-derived induced pluripotent stem cells (TiPS) are a promising source for the generation of 'off-the-shelf' CAR T cells, but the in vitro differentiation of TiPS often yields T cells with suboptimal features. Here we show that the premature expression of the T-cell receptor (TCR) or a constitutively expressed CAR in TiPS promotes the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive differentiation. Delaying CAR expression and calibrating its signalling strength in TiPS enabled the generation of human TCR- CD8αß+ CAR T cells that perform similarly to CD8αß+ CAR T cells from peripheral blood, achieving effective tumour control on systemic administration in a mouse model of leukaemia and without causing graft-versus-host disease. Driving T-cell maturation in TiPS in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αß+ T cells for a broad range of immunotherapies.
Asunto(s)
Células Madre Pluripotentes Inducidas , Receptores Quiméricos de Antígenos , Ratones , Animales , Humanos , Linfocitos T , Células Madre Pluripotentes Inducidas/metabolismo , Receptores de Antígenos de Linfocitos T , Antígenos CD8/metabolismo , Receptores Quiméricos de Antígenos/metabolismoRESUMEN
The AMP-activated protein kinase (AMPK) is a master regulator of metabolic homeostasis by sensing cellular energy status. AMPK is mainly activated via phosphorylation by LKB1 when cellular AMP/ADP levels are increased. However, how AMP/ADP brings about AMPK phosphorylation remains unclear. Here, we show that it is AMP, but not ADP, that drives AXIN to directly tether LKB1 to phosphorylate AMPK. The complex formation of AXIN-AMPK-LKB1 is greatly enhanced in glucose-starved or AICAR-treated cells and in cell-free systems supplemented with exogenous AMP. Depletion of AXIN abrogated starvation-induced AMPK-LKB1 colocalization. Importantly, adenovirus-based knockdown of AXIN in the mouse liver impaired AMPK activation and caused exacerbated fatty liver after starvation, underscoring an essential role of AXIN in AMPK activation. These findings demonstrate an initiating role of AMP and demonstrate that AXIN directly transmits AMP binding of AMPK to its activation by LKB1, uncovering the mechanistic route for AMP to elicit AMPK activation by LKB1.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Monofosfato/farmacología , Proteína Axina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/deficiencia , Proteínas Quinasas Activadas por AMP/genética , Acetil-CoA Carboxilasa/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Proteína Axina/antagonistas & inhibidores , Proteína Axina/genética , Línea Celular , Sistema Libre de Células , Activación Enzimática , Células HEK293 , Humanos , Metabolismo de los Lípidos/fisiología , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Fosforilación/efectos de los fármacos , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Peroxisome proliferator-activated receptor gamma (PPARγ) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPARγ agonist, thereby uncovering a novel signaling route for this steroid drug. Similar to rosiglitazone, RU-486 modulates the expression of key PPARγ target genes and promotes adipocyte differentiation, but with a lower adipogenic activity. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPARγ ligand-binding pocket with distinctive properties and epitopes, providing the molecular mechanisms for the discrimination of RU-486 from thiazolidinediones (TZDs) drugs. Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPARγ ligands in the treatment of insulin resistance.