RESUMEN
Cancer treatment has been transformed by checkpoint blockade therapies, with the highest anti-tumor activity of anti-programmed death 1 (PD-1) antibody therapy seen in Hodgkin lymphoma. Disappointingly, response rates have been low in the non-Hodgkin lymphomas, with no activity seen in relapsed/refractory chronic lymphocytic leukemia (CLL) with PD-1 blockade. Thus, identifying more powerful combination therapy is required for these patients. Here, we preclinically demonstrate enhanced anti-CLL activity following combinational therapy with anti-PD-1 or anti-PD-1 ligand (PD-L1) and avadomide, a cereblon E3 ligase modulator (CELMoD). Avadomide induced type I and II interferon (IFN) signaling in patient T cells, triggering a feedforward cascade of reinvigorated T-cell responses. Immune modeling assays demonstrated that avadomide stimulated T-cell activation, chemokine expression, motility and lytic synapses with CLL cells, as well as IFN-inducible feedback inhibition through upregulation of PD-L1. Patient-derived xenograft tumors treated with avadomide were converted to CD8+ T cell-inflamed tumor microenvironments that responded to anti-PD-L1/PD-1-based combination therapy. Notably, clinical analyses showed increased PD-L1 expression on T cells, as well as intratumoral expression of chemokine signaling genes in B-cell malignancy patients receiving avadomide-based therapy. These data illustrate the importance of overcoming a low inflammatory T-cell state to successfully sensitize CLL to checkpoint blockade-based combination therapy.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico/farmacología , Leucemia Linfocítica Crónica de Células B/inmunología , Activación de Linfocitos/efectos de los fármacos , Piperidonas/farmacología , Quinazolinonas/farmacología , Linfocitos T/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Humanos , Inmunoterapia/métodos , Interferones/inmunología , Ratones , Transducción de Señal/efectos de los fármacos , Linfocitos T/inmunología , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Reduced bioavailability of nitric oxide due to impaired endothelial nitric oxide synthase (eNOS) activity is a leading cause of endothelial dysfunction in diabetes. Enhancing eNOS activity in diabetes is a potential therapeutic target. This study investigated basal cerebral blood flow and cerebrovascular reactivity in wild-type mice, diabetic mice (Ins2(Akita+/-)), nondiabetic eNOS-overexpressing mice (TgeNOS), and the cross of two transgenic mice (TgeNOS-Ins2(Akita+/-)) at six months of age. The cross was aimed at improving eNOS expression in diabetic mice. The major findings were: (i) Body weights of Ins2(Akita+/-) and TgeNOS-Ins2(Akita+/-) were significantly different from wild-type and TgeNOS mice. Blood pressure of TgeNOS mice was lower than wild-type. (ii) Basal cerebral blood flow of the TgeNOS group was significantly higher than cerebral blood flow of the other three groups. (iii) The cerebrovascular reactivity in the Ins2(Akita+/-) mice was significantly lower compared with wild-type, whereas that in the TgeNOS-Ins2(Akita+/-) was significantly higher compared with the Ins2(Akita+/-) and TgeNOS groups. Overexpression of eNOS rescued cerebrovascular dysfunction in diabetic animals, resulting in improved cerebrovascular reactivity. These results underscore the possible role of eNOS in vascular dysfunction in the brain of diabetic mice and support the notion that enhancing eNOS activity in diabetes is a potential therapeutic target.
Asunto(s)
Circulación Cerebrovascular/genética , Trastornos Cerebrovasculares/tratamiento farmacológico , Diabetes Mellitus Tipo 1/complicaciones , Óxido Nítrico Sintasa de Tipo III/genética , Animales , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Trastornos Cerebrovasculares/genética , Diabetes Mellitus Experimental , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Expresión Génica , Ratones , Ratones Transgénicos , Terapia Molecular Dirigida/métodosRESUMEN
The balance of nitric oxide (NO) versus superoxide generation has a major role in the initiation and progression of endothelial dysfunction. Under conditions of high glucose, endothelial nitric oxide synthase (eNOS) functions as a chief source of superoxide rather than NO. In order to improve NO bioavailability within the vessel wall in type-1 diabetes, we investigated treatment strategies that improve eNOS phosphorylation and NO-dependent vasorelaxation. We evaluated methods to increase the eNOS activity by (1) feeding Ins2(Akita) spontaneously diabetic (type-1) mice with l-arginine in the presence of sepiapterin, a precursor of tetrahydrobiopterin; (2) preventing eNOS/NO deregulation by the inclusion of inhibitor kappa B kinase beta (IKKß) inhibitor, salsalate, in the diet regimen in combination with l-arginine and sepiapterin; and (3) independently increasing eNOS expression to improve eNOS activity and associated NO production through generating Ins2(Akita) diabetic mice that overexpress human eNOS predominantly in vascular endothelial cells. Our results clearly demonstrated that diet supplementation with l-arginine, sepiapterin along with salsalate improved phosphorylation of eNOS and enhanced vasorelaxation of thoracic/abdominal aorta in type-1 diabetic mice. More interestingly, despite the overexpression of eNOS, the in-house generated transgenic eNOS-GFP (TgeNOS-GFP)-Ins2(Akita) cross mice showed an unanticipated effect of reduced eNOS phosphorylation and enhanced superoxide production. Our results demonstrate that enhancement of endogenous eNOS activity by nutritional modulation is more beneficial than increasing the endogenous expression of eNOS by gene therapy modalities.
Asunto(s)
Diabetes Mellitus Tipo 1/prevención & control , Suplementos Dietéticos , Endotelio Vascular/metabolismo , Hipoglucemiantes/uso terapéutico , Óxido Nítrico Sintasa de Tipo III/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Procesamiento Proteico-Postraduccional , Animales , Aorta/citología , Aorta/metabolismo , Aorta/fisiopatología , Arginina/metabolismo , Arginina/uso terapéutico , Bovinos , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatología , Endotelio Vascular/citología , Endotelio Vascular/fisiopatología , Femenino , Heterocigoto , Humanos , Hipoglucemiantes/metabolismo , Insulina/genética , Insulina/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo III/química , Óxido Nítrico Sintasa de Tipo III/genética , Fosforilación , Inhibidores de Proteínas Quinasas/metabolismo , Pterinas/metabolismo , Pterinas/uso terapéutico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Salicilatos/metabolismo , Salicilatos/uso terapéutico , DesteteRESUMEN
B-precursor acute lymphoblastic leukemia (B-ALL) lymphoblast (blast) internalization of anti-cytokine receptor-like factor 2 (CRLF2) antibody-armored biodegradable nanoparticles (AbBNPs) are investigated. First, AbBNPsaere synthesized by adsorbing anti-CRLF2 antibodies to poly(D,L-lactide- co -glycolide) (PLGA) nanoparticles of various sizes and antibody surface density (Ab/BNP) ratios. Second, AbBNPs are incubated with CRLF2-overexpressing (CRLF2+) or control blasts. Third, internalization of AbBNPs by blasts is evaluated by multicolor flow cytometry as a function of receptor expression, AbBNP size, and Ab/BNP ratio. Results from these experiments are con-firmed by electron microscopy, fluorescence microscopy, and Western blotting. The optimal size and Ab/BNP for internalization of AbBNPs by CRLF2+ blasts is 50 nm with 10 Ab/BNP and 100 nm with 25 Ab/BNP. These studies show that internalization of AbBNPs in childhood B-ALL blasts is AbBNP size-and Ab/BNP ratio-dependent. All AbBNP combinations are non-cytotoxic. It is also shown that CD47 is very slightly up-regulated by blasts exposed to AbBNPs. CD47 is "the marker of self" overexpressed by blasts to escape phagocytosis, or "cellular devouring", by beneficial macrophages. The results indicate that precise engineering of AbBNPs by size and Ab/BNP ratio may improve the internalization and selectivity of future biodegradable nanoparticles for the treatment of leukemia patients, including drug-resistant minority children and Down's syndrome patients with CRLF2+B-ALL.