RESUMO
The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regard to oncogenic alterations that do not only serve as prognostic markers but also as therapeutic targets in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Because of sequence-dependent protein conformational changes FLT3-ITD tends to autophosphorylate and displays a constitutive intracellular localization. Here, we analyzed the effect of tyrosine kinase inhibitors (TKIs) on the localization of the FLT3 receptor and its mutants. TKI treatment increased the surface expression through upregulation of FLT3 and glycosylation of FLT3-ITD and FLT3-D835Y mutants. In T cell-mediated cytotoxicity (TCMC) assays, using a bispecific FLT3 × CD3 antibody construct, the combination with TKI treatment increased TCMC in the FLT3-ITD-positive AML cell lines MOLM-13 and MV4-11, patient-derived xenograft cells and primary patient samples. Our findings provide the basis for rational combination of TKI and FLT3-directed immunotherapy with potential benefit for FLT3-ITD-positive AML patients.
Assuntos
Leucemia Mieloide Aguda/terapia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Sequências de Repetição em Tandem/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoterapia/métodos , Leucemia Mieloide Aguda/metabolismo , Mutação/efeitos dos fármacos , Mutação/genética , Prognóstico , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, P<0.0001). This phenomenon was cytokine-driven as the sole addition of interferon (IFN)-γ and tumor necrosis factor-α also induced expression. Through blockade of the PD-1/PD-L1 interaction, AMG 330-mediated lysis (n=9, P=0.03), T-cell proliferation (n=9, P=0.01) and IFN-γ secretion (n=8, P=0.008) were significantly enhanced. The combinatorial approach was most beneficial in settings of protracted AML cell lysis. Taken together, we have characterized a critical resistance mechanism employed by primary AML cells under AMG 330-mediated proinflammatory conditions. Our results support the evaluation of checkpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/imunologia , Evasão Tumoral/efeitos dos fármacos , Animais , Antígeno B7-H1/análise , Antígeno B7-H1/fisiologia , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Camundongos , Receptor de Morte Celular Programada 1/análise , Receptor de Morte Celular Programada 1/fisiologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/análiseRESUMO
The PhoP/PhoQ two-component regulatory system controls transcription of several key virulence genes essential for Salmonella survival in the host cell phagosome. Here, we determine that the PhoP/PhoQ system also regulates virulence in the aetiological agent of bacillary dysentery, Shigella flexneri, even though this pathogen escapes from the phagosome into the cytoplasm of the host cell. A phoP mutant of Shigella established infections and induced an acute inflammatory response in two different animal models. However, infections with phoP mutant bacteria were resolved more rapidly than infections with wild-type Shigella. Moreover, the Shigella phoP mutant was more sensitive than the wild-type strain to killing by polymorphonuclear leucocytes (PMNs), cationic polypeptides extracted from PMNs and other animal-derived antimicrobial peptides. The phoP mutant, however, invaded epithelial cells, spread intercellularly, induced apoptosis in macrophages and tolerated extreme acid pH as efficiently as the wild-type strain. PhoP appears to regulate Shigella susceptibility to PMNs and antimicrobial molecules that are important for the late stages of infection with this enteric bacterium.
Assuntos
Proteínas de Bactérias/metabolismo , Disenteria Bacilar/microbiologia , Regulação Bacteriana da Expressão Gênica , Inflamação/imunologia , Shigella flexneri/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Ceratoconjuntivite/imunologia , Ceratoconjuntivite/microbiologia , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos , Neutrófilos/imunologia , Transdução de Sinais , Virulência/genéticaRESUMO
A temperature-sensitive, protein synthesis-defective mutant of Escherichia coli exhibiting an altered ribosomal protein L22 has been investigated. The temperature-sensitive mutation was mapped to the rplV gene for protein L22. The genes from the wild type and mutant strains were amplified by the polymerase chain reaction and the products were sequenced. A cytosine to thymine transition at position 22 of the coding sequence was found in the mutant DNA, predicting an arginine to cysteine alteration in the protein. A single cysteine residue was found in the isolated mutant protein. This amino acid change accounts for the altered mobility of the mutant protein in two-dimensional gels and during reversed-phase HPLC. The temperature-sensitive phenotype was fully complemented by a plasmid carrying the wild type L22 gene. Ribosomes from the complemented cells showed only wild type protein L22 by two dimensional gel analysis and were as heat-resistant as control ribosomes in a translation assay. The point mutation in the L22 gene is uniquely responsible for the temperature-sensitivity of this strain.