RESUMO
The tumor suppressor protein p53 is mutated in close to 50% of human tumors and is dysregulated in many others, for instance by silencing or loss of p14ARF. Under steady-state conditions, the two E3 ligases MDM2/MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 in tumors with wild-type (WT) p53 has therefore been considered a therapeutic strategy. Moreover, studies indicate that p53 reactivation may synergize with radiation and increase tumor immunogenicity. In vivo studies of most MDM2 inhibitors have utilized immunodeficient xenograft mouse models, preventing detailed studies of action of these molecules on the immune response. The mouse melanoma cell line B16-F10 carries functional, WT p53 but does not express the MDM2 regulator p19ARF. In this study, we tested a p53-MDM2 protein-protein interaction inhibitor, the small molecule Navtemadlin, which is currently being tested in phase II clinical trials. Using mass spectrometry-based proteomics and imaging flow cytometry, we identified specific protein expression patterns following Navtemadlin treatment of B16-F10 melanoma cells compared with their p53 CRISPR-inactivated control cells. In vitro, Navtemadlin induced a significant, p53-dependent, growth arrest but little apoptosis in B16-F10 cells. When combined with radiotherapy, Navtemadlin showed synergistic effects and increased apoptosis. In vivo, Navtemadlin treatment significantly reduced the growth of B16-F10 melanoma cells implanted in C57Bl/6 mice. Our data highlight the utility of a syngeneic B16-F10 p53+/+ mouse melanoma model for assessing existing and novel p53-MDM2/MDM4 inhibitors and in identifying new combination therapies that can efficiently eliminate tumors in vivo. Significance: The MDM2 inhibitor Navtemadlin arrests mouse tumor growth and potentiates radiotherapy. Our results support a threshold model for apoptosis induction that requires a high, prolonged p53 signaling for cancer cells to become apoptotic.
Assuntos
Antineoplásicos , Melanoma Experimental , Humanos , Animais , Camundongos , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/metabolismo , Melanoma Experimental/tratamento farmacológico , Modelos Animais de Doenças , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
Scavenger receptors are pattern recognition receptors that recognize both foreign and self-ligands, and initiate different mechanisms of cellular activation, often as co-receptors. The function of scavenger receptor CD36 in the immune system has mostly been studied in macrophages but it is also highly expressed by innate type B cells where its function is less explored. Here we report that CD36 is involved in macro-autophagy/autophagy in B cells, and in its absence, the humoral immune response is impaired. We found that CD36-deficient B cells exhibit a significantly reduced plasma cell formation, proliferation, mitochondrial mobilization and oxidative phosphorylation. These changes were accompanied by impaired initiation of autophagy, and we found that CD36 regulated autophagy and colocalized with autophagosome membrane protein MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). When we investigated T-cell-dependent immune responses, we found that mice with CD36 deficiency, specifically in B cells, exhibited attenuated germinal center responses, class switching, and antibody production as well as autophagosome formation. These findings establish a critical role for CD36 in B cell responses and may also contribute to our understanding of CD36-mediated autophagy in other cells as well as in B cell lymphomas that have been shown to express the receptor.Abbreviations: AICDA/AID: activation-induced cytidine deaminase; ATG5: autophagy related 5; ATP: adenosine triphosphate; BCR: B-cell receptor; CPG: unmethylated cytosine-guanosine; CQ: chloroquine; DC: dendritic cells; FOB: follicular B cells; GC: germinal center; Ig: immunoglobulin; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MZB: marginal zone B cells; NP-CGG: 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin; OCR: oxygen consumption rate; oxLDL: oxidized low-density lipoprotein; PC: plasma cells; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; SRBC: sheep red blood cells; Tfh: follicular helper T cells; TLR: toll-like receptor.
Assuntos
Autofagia , Linfócitos B/fisiologia , Antígenos CD36/fisiologia , Imunidade Humoral , Proteínas Associadas aos Microtúbulos/fisiologia , Animais , Autofagossomos/metabolismo , Autofagossomos/fisiologia , Autofagia/fisiologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos CD36/metabolismo , Diferenciação Celular , Proliferação de Células , Humanos , Switching de Imunoglobulina , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Plasmócitos/fisiologia , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich Syndrome protein (WASp). XLN patients have reduced numbers of cytotoxic cells in peripheral blood; however, their capacity to kill tumor cells remains to be determined. Here, we examined NK and T cells from 2 patients with XLN harboring the activating WASpL270P mutation. XLN patient NK and T cells had increased granzyme B content and elevated degranulation and IFN-γ production when compared with healthy control cells. Murine WASpL272P NK and T cells formed stable synapses with YAC-1 tumor cells and anti-CD3/CD28-coated beads, respectively. WASpL272P mouse T cells had normal degranulation and cytokine response whereas WASpL272P NK cells showed an enhanced response. Imaging experiments revealed that while WASpL272P CD8+ T cells had increased accumulation of actin upon TCR activation, WASpL272P NK cells had normal actin accumulation at lytic synapses triggered through NKp46 signaling but had impaired response to lymphocyte function associated antigen-1 engagement. When compared with WT mice, WASpL272P mice showed reduced growth of B16 melanoma and increased capacity to reject MHC class I-deficient cells. Together, our data suggest that cytotoxic cells with constitutively active WASp have an increased capacity to respond to and kill tumor cells.
Assuntos
Degranulação Celular , Granzimas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/imunologia , Animais , Estudos de Casos e Controles , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T Citotóxicos/imunologia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologiaRESUMO
The glycosphingolipid α-galactosylceramide (α-GalCer) is a well-described immune activator with strong anti-tumor properties in animal models. It is presented on CD1d and acts by stimulating the invariant, type I, natural killer T (iNKT) lymphocytes to rapidly secrete TH1 and TH2 associated cytokines. This in turn promotes activation of a diversity of immune cells including natural killer (NK) cells with anti-tumor functions. Prior to tumor development, iNKT cells can also perform tumor surveillance and naturally protect from emergence of cancer. In contrast, we have recently demonstrated that iNKT cells naturally promote polyps in the spontaneous murine adenomatous polyposis coli (Apc) ApcMin/+ model for colon cancer, associated with suppressed TH1 immunity and enhanced immunoregulation. Here we investigated whether iNKT cell directed immunotherapy could subvert the polyp promoting function of iNKT cells and reduce polyp growth in this model. We treated ApcMin/+ mice with α-GalCer, or synthetic derivatives of this ligand (C-glycoside and C20:2) that have enhanced immunoregulatory properties. Treatment with iNKT cell ligands led to increased iNKT cell division, but reduced iNKT cell frequencies, lower NK1.1 expression and elevation of PD-1. ApcMin/+ mice that had been treated either long-term (5-15 weeks of age), or short-term (12-15 weeks of age) with α-GalCer demonstrated a significant decrease in polyp burden. Surprisingly, long-term treatment with the TH1 biasing ligand C-glycoside did not have significant effects on polyps, while long-term treatment with the TH2 biasing ligand C20:2 enhanced polyp growth. In stark contrast, short-term treatment with C20:2 led to reduction in polyp numbers and size. Reduced polyp burden after long-term treatment was associated with increased expression of genes indicating a pro-inflammatory polyp microenvironment. Polyp-reducing short-term treatment led to CD8 T cell activation specifically in polyps, and decreased tumor infiltrating and splenic macrophages, and a switch toward a pro-inflammatory phenotype. Thus, iNKT cell directed therapy could subvert the natural polyp enhancing function of iNKT cells, overcome immunosuppression, and reduce polyps. However, different iNKT cell activating ligands had opposite effects, and the timing of treatment had a major influence on outcomes.
Assuntos
Pólipos Intestinais/imunologia , Células T Matadoras Naturais/imunologia , Animais , Antígenos CD1d/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Feminino , Galactosilceramidas/imunologia , Terapia de Imunossupressão/métodos , Imunoterapia/métodos , Inflamação/imunologia , Ligantes , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Células Th1/imunologia , Células Th2/imunologia , Microambiente Tumoral/imunologiaRESUMO
The original PDF version of this Article listed the authors as "Marcus J.G.W. Ladds," where it should have read "Marcus J. G. W. Ladds, Ingeborg M. M. van Leeuwen, Catherine J. Drummond et al.#".Also in the PDF version, it was incorrectly stated that "Correspondence and requests for materials should be addressed to S. Lín.", instead of the correct "Correspondence and requests for materials should be addressed to S. Laín."This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication.
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The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Indazóis/farmacologia , Neoplasias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteólise/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
A hallmark of immunity to worm infections and many allergies is a strong type 2 immune response. This is characterized by the production of cytokines interleukin-5 (IL-5) and IL-13 by adaptive T helper 2 (TH2) cells and/or type 2 innate lymphoid cells. Peroxisome proliferator activated receptor-γ (PPAR-γ) is typically regarded as an anti-inflammatory factor. We report that TH2 cells express high levels of PPAR-γ in response to the allergen house dust mite and after infection with the parasite Heligmosomoides polygyrus Mice lacking PPAR-γ in T cells failed to effectively differentiate into IL-5- and IL-13-secreting cells and, hence, did not develop TH2 cell-associated pathologies, including goblet cell metaplasia and eosinophilia, in response to allergen challenge. Furthermore, these mice could not mount protective immune responses to nematode infection. In addition, mice lacking PPAR-γ in T cells had greatly reduced frequencies of TH2 cells in visceral adipose tissue. Mechanistically, PPAR-γ appeared to promote the expression of the IL-33 receptor on the surface of TH2 cells. These results pinpoint PPAR-γ as a factor that drives type 2 responses in allergy, worm infection, and visceral adipose tissue.
RESUMO
The innate responsiveness of the immune system is important not only for quick responses to pathogens but also for the initiation and shaping of the subsequent adaptive immune response. Activation via the cytokine IL-18, a product of inflammasomes, gives rise to a rapid response that includes the production of self-reactive antibodies. As increased concentrations of this cytokine are found in inflammatory diseases, we investigated the origin of the B cell response and its regulation. We identified an accumulation of B cell-helper neutrophils in the spleen that interacted with innate-type invariant natural killer T cells (iNKT cells) to regulate B cell responses. We found that neutrophil-dependent expression of the death-receptor ligand FasL by iNKT cells was needed to restrict autoantibody production. Neutrophils can thus license iNKT cells to regulate potentially harmful autoreactive B cell responses during inflammasome-driven inflammation.
Assuntos
Linfócitos B/imunologia , Proteína Ligante Fas/metabolismo , Inflamação/imunologia , Células T Matadoras Naturais/imunologia , Neutrófilos/imunologia , Imunidade Adaptativa , Animais , Autoanticorpos/biossíntese , Células Cultivadas , Proteína Ligante Fas/genética , Imunidade Inata , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Inflammation serves as the first line of defense in response to tissue injury, guiding the immune system to ensure preservation of the host. The inflammatory response can be divided into a quick initial phase mediated mainly by innate immune cells including neutrophils and macrophages, followed by a late phase that is dominated by lymphocytes. Early in the new millennium, a key component of the inflammatory reaction was discovered with the identification of a number of cytosolic sensor proteins (Nod-like receptors) that assembled into a common structure, the 'inflammasome'. This structure includes an enzyme, caspase-1, which upon activation cleaves pro-forms of cytokines leading to subsequent release of active IL-1 and IL-18. This review focuses on the role of IL-18 in inflammatory responses with emphasis on autoimmune diseases.