RESUMO
A large fraction of the proteins that are being identified as key tumor dependencies represent poor pharmacological targets or lack clinically-relevant small-molecule inhibitors. Availability of fully generalizable approaches for the systematic and efficient prioritization of tumor-context specific protein activity inhibitors would thus have significant translational value. Unfortunately, inhibitor effects on protein activity cannot be directly measured in systematic and proteome-wide fashion by conventional biochemical assays. We introduce OncoLead, a novel network based approach for the systematic prioritization of candidate inhibitors for arbitrary targets of therapeutic interest. In vitro and in vivo validation confirmed that OncoLead analysis can recapitulate known inhibitors as well as prioritize novel, context-specific inhibitors of difficult targets, such as MYC and STAT3. We used OncoLead to generate the first unbiased drug/regulator interaction map, representing compounds modulating the activity of cancer-relevant transcription factors, with potential in precision medicine.
Assuntos
Antineoplásicos , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Linhagem Celular Tumoral , Humanos , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
The genome-wide identification of pairs of interacting proteins is an important step in the elucidation of cell regulatory mechanisms. Much of our present knowledge derives from high-throughput techniques such as the yeast two-hybrid assay and affinity purification, as well as from manual curation of experiments on individual systems. A variety of computational approaches based, for example, on sequence homology, gene co-expression and phylogenetic profiles, have also been developed for the genome-wide inference of protein-protein interactions (PPIs). Yet comparative studies suggest that the development of accurate and complete repertoires of PPIs is still in its early stages. Here we show that three-dimensional structural information can be used to predict PPIs with an accuracy and coverage that are superior to predictions based on non-structural evidence. Moreover, an algorithm, termed PrePPI, which combines structural information with other functional clues, is comparable in accuracy to high-throughput experiments, yielding over 30,000 high-confidence interactions for yeast and over 300,000 for human. Experimental tests of a number of predictions demonstrate the ability of the PrePPI algorithm to identify unexpected PPIs of considerable biological interest. The surprising effectiveness of three-dimensional structural information can be attributed to the use of homology models combined with the exploitation of both close and remote geometric relationships between proteins.
Assuntos
Algoritmos , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteínas/química , Proteínas/metabolismo , Proteômica/métodos , Animais , Teorema de Bayes , Encéfalo/metabolismo , Caderinas/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Modelos Moleculares , PPAR gama/metabolismo , Filogenia , Ligação Proteica , Conformação Proteica , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteoma/química , Proteoma/metabolismo , Curva ROC , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
We previously identified the natural products isopomiferin and pomiferin as powerful, indirect MYCN-ablating agents. In this work, we expand on their mechanism of action and find that casein kinase 2 (CK2), phosphoinositide 3-kinase (PI3K), checkpoint kinase 1 (CHK1) and serine/threonine protein kinase 38-like (STK38L), as well as STK38, work synchronously to create a field effect that maintains MYCN stability. By systematically inhibiting these kinases, we degraded MYCN and induced cell death. Additionally, we synthesized and tested several simpler and more cost-effective pomiferin analogues, which successfully emulated the compound's MYCN ablating activity. Our work identified and characterized key kinases that can be targeted to interfere with the stability of the MYCN protein in NBL cells, demonstrating the efficacy of an indirect approach to targeting "undruggable" cancer drivers.
RESUMO
The various functions of the skeleton are influenced by extracellular cues, hormones, and neurotransmitters. One type of neuronal regulation favors bone mass accrual by inhibiting sympathetic nervous system (SNS) activity. This observation raises questions about the transcriptional mechanisms regulating catecholamine synthesis. Using a combination of genetic and pharmacological studies, we found that the histone deacetylase sirtuin 1 (SIRT1) is a transcriptional modulator of the neuronal control of bone mass. Neuronal SIRT1 reduced bone mass by increasing SNS signaling. SIRT1 did so by increasing expression of monoamine oxidase A (MAO-A), a SIRT1 target that reduces brain serotonin levels by inducing its catabolism and by suppressing tryptophan hydroxylase 2 (Tph2) expression and serotonin synthesis in the brain stem. SIRT1 upregulated brain catecholamine synthesis indirectly through serotonin, but did not directly affect dopamine ß hydroxylase (Dbh) expression in the locus coeruleus. These results help us to understand skeletal changes associated with selective serotonin reuptake inhibitors (SSRIs) and may have implications for treating skeletal and metabolic diseases.
Assuntos
Envelhecimento , Serotonina , Sirtuína 1 , Animais , Camundongos , Envelhecimento/genética , Catecolaminas , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Serotonina/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
Remodeling of the microenvironment by tumor cells can activate pathways that favor cancer growth. Molecular delineation and targeting of such malignant-cell nonautonomous pathways may help overcome resistance to targeted therapies. Herein we leverage genetic mouse models, patient-derived xenografts, and patient samples to show that acute myeloid leukemia (AML) exploits peripheral serotonin signaling to remodel the endosteal niche to its advantage. AML progression requires the presence of serotonin receptor 1B (HTR1B) in osteoblasts and is driven by AML-secreted kynurenine, which acts as an oncometabolite and HTR1B ligand. AML cells utilize kynurenine to induce a proinflammatory state in osteoblasts that, through the acute-phase protein serum amyloid A (SAA), acts in a positive feedback loop on leukemia cells by increasing expression of IDO1-the rate-limiting enzyme for kynurenine synthesis-thereby enabling AML progression. This leukemia-osteoblast cross-talk, conferred by the kynurenine-HTR1B-SAA-IDO1 axis, could be exploited as a niche-focused therapeutic approach against AML, opening new avenues for cancer treatment. SIGNIFICANCE: AML remains recalcitrant to treatments due to the emergence of resistant clones. We show a leukemia-cell nonautonomous progression mechanism that involves activation of a kynurenine-HTR1B-SAA-IDO1 axis between AML cells and osteoblasts. Targeting the niche by interrupting this axis can be pharmacologically harnessed to hamper AML progression and overcome therapy resistance. This article is highlighted in the In This Issue feature, p. 873.
Assuntos
Cinurenina , Leucemia Mieloide Aguda , Animais , Humanos , Cinurenina/metabolismo , Cinurenina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Osteoblastos/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
Anti-CD3 mAb can modulate graft rejection and attenuate autoimmune diseases but their mechanism(s) of action remain unclear. CD8(+) T cells with regulatory function are induced in vitro by Teplizumab, a humanized anti-CD3 antibody and inhibit responses of autologous and allogeneic T cells. They inhibit CD4(+) T-cell proliferation by mechanisms involving TNF and CCL4, and by blocking target cell entry into G2/M phase of cell cycle but neither kill them, nor compete for IL-2. CD8(+) Treg can be isolated from peripheral blood following treatment of patients with Type 1 diabetes with Teplizumab, but not from untreated patients. The induction of CD8(+) Treg by anti-CD3 mAb requires TNF and signaling through the NF-κB cascade. The CD8(+) Treg express CD25, glucocorticoid-induced TNF receptor family, CTLA-4, Foxp3, and TNFR2, and the combined expression of TNFR2 and CD25 identifies a potent subpopulation of CD8(+) Treg. These studies have identified a novel mechanism of immune regulation by anti-CD3 mAb and markers that may be used to track inducible CD8(+) Treg in settings such as chronic inflammation or immune therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Muromonab-CD3/farmacologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Anticorpos Monoclonais Humanizados , Antígenos CD/imunologia , Antígeno CTLA-4 , Criança , Diabetes Mellitus Tipo 1/tratamento farmacológico , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Humanos , Interleucina-10/imunologia , NF-kappa B/imunologia , Receptores de Fator de Crescimento Neural/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Fator de Crescimento Transformador beta1/imunologia , Fator de Necrose Tumoral alfa/imunologia , Adulto JovemRESUMO
Assembly of a transcriptional and post-translational molecular interaction network in B cells, the human B-cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC). Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre-replication, replication, and mitosis. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Genes Reguladores/genética , Centro Germinativo/citologia , Centro Germinativo/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Algoritmos , Apoptose/genética , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Replicação do DNA/genética , Retroalimentação Fisiológica , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Mitose , Complexos Multiproteicos/metabolismo , Ligação Proteica , Transcrição GênicaRESUMO
Regulation of food intake is a recently identified endocrine function of bone that is mediated by Lipocalin-2 (LCN2). Osteoblast-secreted LCN2 suppresses appetite and decreases fat mass while improving glucose metabolism. We now show that serum LCN2 levels correlate with insulin levels and ß-cell function, indices of healthy glucose metabolism, in obese mice and obese, prediabetic women. However, LCN2 serum levels also correlate with body mass index and insulin resistance in the same individuals and are increased in obese mice. To dissect this apparent discrepancy, we modulated LCN2 levels in mice. Silencing Lcn2 expression worsens metabolic dysfunction in genetic and diet-induced obese mice. Conversely, increasing circulating LCN2 levels improves metabolic parameters and promotes ß-cell function in mouse models of ß-cell failure acting as a growth factor necessary for ß-cell adaptation to higher metabolic load. These results indicate that LCN2 up-regulation is a protective mechanism to counteract obesity-induced glucose intolerance by decreasing food intake and promoting adaptive ß-cell proliferation.
Assuntos
Lipocalina-2/fisiologia , Obesidade/metabolismo , Estado Pré-Diabético/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Glucose/metabolismo , Humanos , Resistência à Insulina , Células Secretoras de Insulina/metabolismo , Lipocalina-2/sangue , Lipocalina-2/metabolismo , Camundongos , Camundongos Obesos/sangue , Camundongos Obesos/metabolismo , Camundongos Obesos/fisiologia , Pessoa de Meia-Idade , Obesidade/sangue , Estado Pré-Diabético/sangueRESUMO
In the mouse, the osteoblast-derived hormone Lipocalin-2 (LCN2) suppresses food intake and acts as a satiety signal. We show here that meal challenges increase serum LCN2 levels in persons with normal or overweight, but not in individuals with obesity. Postprandial LCN2 serum levels correlate inversely with hunger sensation in challenged subjects. We further show through brain PET scans of monkeys injected with radiolabeled recombinant human LCN2 (rh-LCN2) and autoradiography in baboon, macaque, and human brain sections, that LCN2 crosses the blood-brain barrier and localizes to the hypothalamus in primates. In addition, daily treatment of lean monkeys with rh-LCN2 decreases food intake by 21%, without overt side effects. These studies demonstrate the biology of LCN2 as a satiety factor and indicator and anorexigenic signal in primates. Failure to stimulate postprandial LCN2 in individuals with obesity may contribute to metabolic dysregulation, suggesting that LCN2 may be a novel target for obesity treatment.
Obesity has reached epidemic proportions worldwide and affects more than 40% of adults in the United States. People with obesity have a greater likelihood of developing type 2 diabetes, cardiovascular disease or chronic kidney disease. Changes in diet and exercise can be difficult to follow and result in minimal weight loss that is rarely sustained overtime. In fact, in people with obesity, weight loss can lower the metabolism leading to increased weight gain. New drugs may help some individuals achieve 5 to 10% weight loss but have side effects that prevent long-term use. Previous studies in mice show that a hormone called Lipocalin-2 (LCN2) suppresses appetite. It also reduces body weight and improves sugar metabolism in the animals. But whether this hormone has the same effects in humans or other primates is unclear. If it does, LCN2 might be a potential obesity treatment. Now, Petropoulou et al. show that LCN2 suppressed appetite in humans and monkeys. In human studies, LCN2 levels increased after a meal in individuals with normal weight or overweight, but not in individuals with obesity. Higher levels of LCN2 in a person's blood were also associated with a feeling of reduced hunger. Using brain scans, Petropoulou et al. showed that LCN2 crossed the blood-brain barrier in monkeys and bound to the hypothalamus, the brain center regulating appetite and energy balance. LCN2 also bound to human and monkey hypothalamus tissue in laboratory experiments. When injected into monkeys, the hormone suppressed food intake and lowered body weight without toxic effects in short-term studies. The experiments lay the initial groundwork for testing whether LCN2 might be a useful treatment for obesity. More studies in animals will help scientists understand how LCN2 works, which patients might benefit, how it would be given to patients and for how long. Clinical trials would also be needed to verify whether it is an effective and safe treatment for obesity.
Assuntos
Lipocalina-2/metabolismo , Macaca/metabolismo , Obesidade/metabolismo , Papio/metabolismo , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Ingestão de Alimentos , Humanos , Lipocalina-2/genética , Obesidade/diagnóstico por imagem , Obesidade/genética , Obesidade/fisiopatologia , Tomografia por Emissão de Pósitrons , Transporte ProteicoRESUMO
Modified anti-CD3 mAbs are emerging as a possible means of inducing immunologic tolerance in settings including transplantation and autoimmunity such as in type 1 diabetes. In a trial of a modified anti-CD3 mAb [hOKT3gamma1(Ala-Ala)] in patients with type 1 diabetes, we identified clinical responders by an increase in the number of peripheral blood CD8+ cells following treatment with the mAb. Here we show that the anti-CD3 mAb caused activation of CD8+ T cells that was similar in vitro and in vivo and induced regulatory CD8+CD25+ T cells. These cells inhibited the responses of CD4+ cells to the mAb itself and to antigen. The regulatory CD8+CD25+ cells were CTLA4 and Foxp3 and required contact for inhibition. Foxp3 was also induced on CD8+ T cells in patients during mAb treatment, which suggests a potential mechanism of the anti-CD3 mAb immune modulatory effects involving induction of a subset of regulatory CD8+ T cells.
Assuntos
Anticorpos Monoclonais/farmacologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Imunossupressores/farmacologia , Muromonab-CD3/farmacologia , Receptores de Interleucina-2/imunologia , Anticorpos Monoclonais/imunologia , Antígenos CD , Antígenos de Diferenciação/imunologia , Antígeno CTLA-4 , Células Cultivadas , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/imunologia , Relação Dose-Resposta Imunológica , Fatores de Transcrição Forkhead/imunologia , Humanos , Imunossupressores/imunologia , Ativação Linfocitária/efeitos dos fármacos , Muromonab-CD3/imunologia , Transplante , Tolerância ao Transplante/efeitos dos fármacos , Tolerância ao Transplante/imunologiaRESUMO
The cancer-testis antigen NY-ESO-1 is one of the most promising candidates for generic vaccination of cancer patients. Here we analyzed the CD8(+) T cell response to a NY-ESO-1 peptide vaccine composed of the two previously defined peptides 157-165 and 157-167, administered with GM-CSF as a systemic adjuvant. The NY-ESO-1 peptide vaccine elicited a CD8(+) T cell response directed against multiple distinct epitopes in the 157-167 region, as revealed by using A2/peptide multimers incorporating overlapping A2 binding peptides in this region. However, only a minor fraction of the elicited CD8(+) T cells, namely those recognizing the peptide 157-165 with sufficiently high functional avidity, recognized the naturally processed target on NY-ESO-1(+) tumor cells. In contrast, the majority of peptide 157-165-specific CD8(+) T cells exhibited lower functional avidity and no tumor reactivity. In addition, vaccine-elicited CD8(+) T cells specific for other overlapping epitopes in the 157-167 region failed to significantly recognize NY-ESO-1-expressing tumor targets. Thus, because of the complexity of the CD8(+) T cell repertoire that can be elicited by vaccination with synthetic peptides, a precise definition of the targeted epitope, and hence, of the corresponding peptide to be used as immunogen, is required to ensure a precise tumor targeting.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Epitopos/metabolismo , Proteínas de Membrana , Neoplasias/imunologia , Proteínas/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/metabolismo , Células Cultivadas , Epitopos/imunologia , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Leiomiossarcoma/imunologia , Leiomiossarcoma/terapia , Masculino , Neoplasias/terapia , Ligação Proteica , Sarcoma Sinovial/imunologia , Sarcoma Sinovial/terapia , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
We have previously reported that premenopausal women with idiopathic osteoporosis (IOP) have profound microarchitectural deficiencies and heterogeneous bone remodeling. Those with the lowest bone formation rate have higher baseline serum insulin-like growth factor-1 (IGF-1) levels and less robust response to teriparatide. Because IGF-1 stimulates bone formation and is critical for teriparatide action on osteoblasts, these findings suggest a state of IGF-1 resistance in some IOP women. To further investigate the hypothesis that osteoblast and IGF-1-related mechanisms mediate differential responsiveness to teriparatide in IOP, we studied circulating osteoblast progenitor (COP) cells and their IGF-1 receptor (IGF-1R) expression. In premenopausal women with IOP, peripheral blood mononuclear cells (PBMCs) were obtained at baseline (n = 25) and over 24 months of teriparatide treatment (n = 11). Flow cytometry was used to identify and quantify COPs (non-hematopoetic lineage cells expressing osteocalcin and RUNX2) and to quantify IGF-1R expression levels. At baseline, both the percent of PBMCs that were COPs (%COP) and COP cell-surface IGF-1R expression correlated directly with several histomorphometric indices of bone formation in tetracycline-labeled transiliac biopsies. In treated subjects, both %COP and IGF-1R expression increased promptly after teriparatide, returning toward baseline by 18 months. Although neither baseline %COP nor increase in %COP after 3 months predicted the bone mineral density (BMD) response to teriparatide, the percent increase in IGF-1R expression on COPs at 3 months correlated directly with the BMD response to teriparatide. Additionally, lower IGF-1R expression after teriparatide was associated with higher body fat, suggesting links between teriparatide resistance, body composition, and the GH/IGF-1 axis. In conclusion, these assays may be useful to characterize bone remodeling noninvasively and may serve to predict early response to teriparatide and possibly other bone formation-stimulating medications. These new tools may also have utility in the mechanistic investigation of teriparatide resistance in premenopausal IOP and perhaps in other populations. © 2017 American Society for Bone and Mineral Research.
Assuntos
Osteoblastos/metabolismo , Osteogênese , Osteoporose/tratamento farmacológico , Osteoporose/fisiopatologia , Pré-Menopausa/fisiologia , Receptor IGF Tipo 1/metabolismo , Células-Tronco/metabolismo , Teriparatida/uso terapêutico , Tecido Adiposo/efeitos dos fármacos , Adolescente , Adulto , Biópsia , Composição Corporal/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Estudos de Coortes , Feminino , Humanos , Pessoa de Meia-Idade , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Pré-Menopausa/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Teriparatida/farmacologia , Adulto JovemRESUMO
Despite advances in understanding autoimmune diabetes in animal models, there has been little progress in altering the natural course of the human disease, which involves progression to insulin deficiency. Studies with immunosuppressive agents have shown short-term effectiveness, but they have not induced tolerance, and continuous treatment is needed. We studied the effects of hOKT3gamma1(Ala-Ala), a humanized Fc mutated anti-CD3 monoclonal antibody, on the progression of type 1 diabetes in patients with recent-onset disease in a randomized controlled trial. In general, the drug was well tolerated. A single course of treatment, within the first 6 weeks after diagnosis, preserved C-peptide responses to a mixed meal for 1 year after diagnosis (97 +/- 9.6% of response at study entry in drug-treated patients vs. 53 +/- 7.6% in control subjects, P < 0.01), with significant improvement in C-peptide responses to a mixed meal even 2 years after treatment (P < 0.02). The improved C-peptide responses were accompanied by reduced HbA(1c) and insulin requirements. Clinical responses to drug treatment were predicted by an increase in the relative number of CD8(+) T-cells in the peripheral blood after the lymphocyte count recovered 2 weeks after the last dose of drug. We conclude that treatment with the anti-CD3 monoclonal antibody hOKT3gamma1(Ala-Ala) results in improved C-peptide responses and clinical parameters in type 1 diabetes for at least 2 years in the absence of continued immunosuppressive medications.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Muromonab-CD3/uso terapêutico , Adolescente , Adulto , Anticorpos Monoclonais Humanizados , Peptídeo C/sangue , Criança , Diabetes Mellitus Tipo 1/sangue , Feminino , Humanos , Insulina/uso terapêutico , Masculino , Fatores de TempoRESUMO
Follicular lymphoma, the most common indolent subtype of non-Hodgkin lymphoma, is associated with a relatively long overall survival rate ranging from 6 to 10 years from the time of diagnosis. However, in 20% to 60% of follicular lymphoma patients, transformation to aggressive diffuse large B-cell lymphoma (DLBCL) reduces median survival to only 1.2 years. The specific functional and genetic determinants of follicular lymphoma transformation remain elusive, and genomic alterations underlying disease advancement have only been identified for a subset of cases. Therefore, to identify candidate drivers of follicular lymphoma transformation, we performed systematic analysis of a B-cell-specific regulatory model exhibiting follicular lymphoma transformation signatures using the Master Regulator Inference algorithm (MARINa). This analysis revealed FOXM1, TFDP1, ATF5, HMGA1, and NFYB to be candidate master regulators (MR) contributing to disease progression. Accordingly, validation was achieved through synthetic lethality assays in which RNAi-mediated silencing of MRs individually or in combination reduced the viability of (14;18)-positive DLBCL (t-DLBCL) cells. Furthermore, specific combinations of small-molecule compounds targeting synergistic MR pairs induced loss of viability in t-DLBCL cells. Collectively, our findings indicate that MR analysis is a valuable method for identifying bona fide contributors to follicular lymphoma transformation and may therefore guide the selection of compounds to be used in combinatorial treatment strategies.
Assuntos
Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Progressão da Doença , Humanos , Linfoma Folicular/genética , Terapia de Alvo Molecular/métodosRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0109569.].
RESUMO
PURPOSE: In this study, we have compared patterns of gene expression and functional activity of human dendritic cells (DCs) cultured under defined conditions in IFN-alpha-2b and recombinant human granulocyte macrophage colony-stimulating factor (DCA) with cells grown in granulocyte macrophage colony-stimulating factor and IL-4 (DC4) as an initial step in evaluating the clinical utility of DCA in cancer immunotherapy. EXPERIMENTAL DESIGN AND RESULTS: Comparison of mRNA transcript profiles between DCA and DC4 revealed different expression patterns for cytokines, chemokines, chemokine receptors, costimulatory molecules, and adhesion proteins. Many genes involved in antigen (Ag) processing were equally expressed in both populations; however, expression of transcripts involved in Ag presentation was increased in DCA. DCA also showed up-regulation of Toll-like receptor 2 and 3, as well as several tumor necrosis factor family ligands. Consistent with expression profiling, functional assays demonstrated that DCAs were more potent stimulators of naive T-cell responses than DC4 in an interleukin 15 and interleukin 1beta-dependent manner. DCA-mediated tumor cell-directed cytotoxicity induced apoptosis in different human tumor cell lines and internalized apoptotic bodies to a greater extent than DC4. Lastly, in vitro priming experiments, using apoptotic cells or peptide as sources of Ag, showed that DCA drove the expansion of tumor peptide Ag-specific autologous CD8+ T cells to a greater extent than DC4. CONCLUSIONS: The unique phenotype conferred by culturing DCs in IFN-alpha-2b may be useful in adoptive transfer regimens where the destruction of tumor cells in situ, initiation of T-cell responses toward tumor tissue with unknown Ags, and/or enhancement of pre-existing Ag-specific memory responses are desired outcomes.
Assuntos
Células Dendríticas/fisiologia , Perfilação da Expressão Gênica , Imunoterapia Adotiva/métodos , Interferon-alfa/farmacologia , Neoplasias/terapia , Apoptose , Proteínas Reguladoras de Apoptose , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imunofenotipagem , Interferon alfa-2 , Interleucina-4/farmacologia , Ativação Linfocitária , Glicoproteínas de Membrana/fisiologia , Proteínas Recombinantes , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Achieving immunologic tolerance to autoimmune diabetes is the goal of therapies for treatment and prevention of the disease. However, whether this can be achieved with an antigen-specific approach is still unproven in humans. Other approaches, including treatment with anti-CD3 monoclonal antibody, have focused on regulation of an active immune response. Preclinical studies with anti-CD3 mAb showed the ability to reverse diabetes and induce tolerance to autoimmunity, even at the time of presentation with hyperglycemia. These studies also suggested that mAb treatment induced an active regulatory process. Based on these and other preclinical data, we have carried out a Phase I/II trial of the humanized FcR non-binding anti-CD3 mAb hOKT3gamma1(Ala-Ala) in patients with new-onset type 1 diabetes. mAb treatment prevented the loss of insulin production over the first two years of the disease with reduced hemoglobin A1c levels and insulin usage. Studies have suggested that the mechanism of drug action involves induction of regulatory cells. CD4(+)IL-10(+) T cells can be found in patients after treatment; in addition, the CD8(+) T cells are induced by the mAb, and these cells may regulate antigen- specific responses. These initial studies have shown clinical efficacy of treatment with anti-CD3 mAb and suggest a novel mechanism that may account for the lasting effects of treatment.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Complexo CD3/metabolismo , Diabetes Mellitus Tipo 1/terapia , Animais , Anticorpos Monoclonais/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ensaios Clínicos como Assunto , Diabetes Mellitus Tipo 1/imunologia , Hemoglobinas Glicadas/análise , Humanos , Tolerância Imunológica , Insulina/sangue , Insulina/metabolismo , Interleucina-10/biossíntese , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptores Fc/metabolismo , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
The use of cultured tumor cells rather than original tumor tissue for the preparation of therapeutic cancer vaccines represents an obvious solution to the problem of availability of adequate quantities of autologous tumor. In this study we investigated possible changes in gene expression accompanying the transition of renal cell carcinoma cells from the original tissue to cell populations in culture. In our study we employed cDNA microarray technology to compare the gene expression pattern of ex vivo cultured renal carcinoma cells to that of the original solid tumor tissue from which the cells were derived. Using this approach we detected changes in the expression of many genes mostly related to the cell lines' physiological properties. Some of the products of those genes showing differential expression between tumor-derived cell line and original tumor are known human autoantigens or tumor-associated antigens. Furthermore, analysis of overexpressed genes revealed the presence of several transcripts with restricted normal tissue distribution, representing self-antigens with potential to elicit autoimmunity. Our results suggest that adapting tumor tissue to culture can result in changes in the level of transcripts specific for known antigens and that more information regarding the composition of tumor cells and their byproducts used in vaccine trials is needed before the efficacy and safety of such procedures can truly be determined.
Assuntos
Técnicas de Cultura de Células , Perfilação da Expressão Gênica , Imunoterapia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Adaptação Fisiológica/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Regulação para Baixo , Humanos , Neoplasias Renais/imunologia , Neoplasias Renais/terapia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Neoplásico/análise , RNA Neoplásico/genética , Transcrição Gênica/genética , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Systematic, high-throughput dissection of causal post-translational regulatory dependencies, on a genome wide basis, is still one of the great challenges of biology. Due to its complexity, however, only a handful of computational algorithms have been developed for this task. Here we present CINDy (Conditional Inference of Network Dynamics), a novel algorithm for the genome-wide, context specific inference of regulatory dependencies between signaling protein and transcription factor activity, from gene expression data. The algorithm uses a novel adaptive partitioning methodology to accurately estimate the full Condition Mutual Information (CMI) between a transcription factor and its targets, given the expression of a signaling protein. We show that CMI analysis is optimally suited to dissecting post-translational dependencies. Indeed, when tested against a gold standard dataset of experimentally validated protein-protein interactions in signal transduction networks, CINDy significantly outperforms previous methods, both in terms of sensitivity and precision.
Assuntos
Modelos Biológicos , Proteínas/genética , Algoritmos , Teorema de Bayes , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Biologia de SistemasRESUMO
A key role of signal transduction pathways is to control transcriptional programs in the nucleus as a function of signals received by the cell via complex post-translational modification cascades. This determines cell-context specific responses to environmental stimuli. Given the difficulty of quantitating protein concentration and post-translational modifications, signaling pathway studies are still for the most part conducted one interaction at the time. Thus, genome-wide, cell-context specific dissection of signaling pathways is still an open challenge in molecular systems biology. In this manuscript we extend the MINDy algorithm for the identification of posttranslational modulators of transcription factor activity, to produce a first genome-wide map of the interface between signaling and transcriptional regulatory programs in human B cells. We show that the serine-threonine kinase STK38 emerges as the most pleiotropic signaling protein in this cellular context and we biochemically validate this finding by shRNA-mediated silencing of this kinase, followed by gene expression profile analysis. We also extensively validate the inferred interactions using protein-protein interaction databases and the kinase-substrate interaction prediction algorithm NetworKIN.