RESUMO
In chronic lymphocytic leukaemia (CLL) the efficacy of SARS-CoV-2 vaccination remains unclear as most studies have focused on humoral responses. Here we comprehensively examined humoral and cellular responses to vaccine in CLL patients. Seroconversion was observed in 55.2% of CLL with lower rate and antibody titres in treated patients. T-cell responses were detected in a significant fraction of patients. CD4+ and CD8+ frequencies were significantly increased independent of serology with higher levels of CD4+ cells in patients under a Bruton tyrosine kinase (BTK) or a B-cell lymphoma 2 (BCL-2) inhibitor. Vaccination skewed CD8+ cells towards a highly cytotoxic phenotype, more pronounced in seroconverted patients. A high proportion of patients showed spike-specific CD4+ and CD8+ cells producing interferon gamma (IFNγ) and tumour necrosis factor alpha (TNFα). Patients under a BTK inhibitor showed increased production of IFNγ and TNFα by CD4+ cells. Vaccination induced a Th1 polarization reverting the Th2 CLL T-cell profile in the majority of patients with lower IL-4 production in untreated and BTK-inhibitor-treated patients. Such robust T-cell responses may have contributed to remarkable protection against hospitalization and death in a cohort of 540 patients. Combining T-cell metrics with seroprevalence may yield a more accurate measure of population immunity in CLL, providing consequential insights for public health.
Assuntos
Antineoplásicos , COVID-19 , Leucemia Linfocítica Crônica de Células B , Vacinas , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Vacinas contra COVID-19/uso terapêutico , Fator de Necrose Tumoral alfa , SARS-CoV-2 , Estudos Soroepidemiológicos , COVID-19/prevenção & controle , Antineoplásicos/uso terapêutico , Interferon gamaRESUMO
Key features of chronic lymphocytic leukemia (CLL) are defects in the immune system and the ability of leukemic cells to evade immune defenses and induce immunosuppression, resulting in increased susceptibility to infections and disease progression. Several immune effectors are impaired in CLL, including T and natural killer (NK) cells. The role of T cells in defense against CLL and in CLL progression and immunotherapy has been extensively studied. Less is known about the role of NK cells in this leukemia, and data on NK cell alterations in CLL are contrasting. Besides studies showing that NK cells have intrinsic defects in CLL, there is a large body of evidence indicating that NK cell dysfunctions in CLL mainly depend on the escape mechanisms employed by leukemic cells. In keeping, it has been shown that NK cell functions, including antibody-dependent cellular cytotoxicity (ADCC), can be retained and/or restored after adequate stimulation. Therefore, due to their preserved ADCC function and the reversibility of CLL-related dysfunctions, NK cells are an attractive source for novel immunotherapeutic strategies in this disease, including chimeric antigen receptor (CAR) therapy. Recently, satisfying clinical responses have been obtained in CLL patients using cord blood-derived CAR-NK cells, opening new possibilities for further exploring NK cells in the immunotherapy of CLL. However, notwithstanding the promising results of this clinical trial, more evidence is needed to fully understand whether and in which CLL cases NK cell-based immunotherapy may represent a valid, alternative/additional therapeutic option for this leukemia. In this review, we provide an overview of the current knowledge about phenotypic and functional alterations of NK cells in CLL and the mechanisms by which CLL cells circumvent NK cell-mediated immunosurveillance. Additionally, we discuss the potential relevance of using NK cells in CLL immunotherapy.
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Suscetibilidade a Doenças , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucemia Linfocítica Crônica de Células B/etiologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Biomarcadores , Comunicação Celular , Gerenciamento Clínico , Humanos , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/terapia , Ligantes , Ligação Proteica , Receptores de Células Matadoras Naturais/genética , Receptores de Células Matadoras Naturais/metabolismo , Resultado do Tratamento , Evasão Tumoral/genética , Evasão Tumoral/imunologiaRESUMO
Dysregulated NOTCH1 signaling, by either gene mutations or microenvironment interactions, has been increasingly linked to chronic lymphocytic leukemia (CLL). Thus, inhibiting NOTCH1 activity represents a potential therapeutic opportunity for this disease. Using gene expression-based screening, we identified the calcium channel modulator bepridil as a new NOTCH1 pathway inhibitor. In primary CLL cells, bepridil induced selective apoptosis even in the presence of the protective stroma. Cytotoxic effects of bepridil were independent of NOTCH1 mutation and other prognostic markers. The antitumor efficacy of bepridil was associated with inhibition of NOTCH1 activity through a decrement in trans-membrane and activated NOTCH1 protein levels with unchanged NOTCH2 protein levels. In a CLL xenotransplant model, bepridil significantly reduced the percentage of leukemic cells infiltrating the spleen via enhanced apoptosis and decreased NOTCH1 activation. In conclusion, we report in vitro and in vivo anti-leukemic activity of bepridil associated with inhibition of the NOTCH1 pathway in CLL. These data provide a rationale for the clinical development of bepridil as anti-NOTCH1 targeted therapy for CLL patients.
Assuntos
Antineoplásicos/farmacologia , Bepridil/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Quimiotaxia/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Mutação , Prognóstico , Receptor Notch1/genética , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Group B Streptococcus (GBS) has evolved several strategies to avoid host defences. We have shown that interaction of macrophages with GBS causes macrophage calpain activation, cytoskeletal disruption and apoptosis, consequences of intracellular calcium increase induced by membrane permeability alterations provoked by GBS-ß-haemolysin. Open question remains about what effect calcium influx has on other calcium-sensing proteins such as gelsolin, involved in cytoskeleton modulation and apoptosis. Therefore we analysed the effect of GBS-III-COH31:macrophage interaction on gelsolin expression. Here we demonstrate that an early macrophage response to GBS-III-COH31 is a very strong gelsolin increase, which occurs in a time- and infection-ratio-dependent manner. This is not due to transcriptional events, translation events, protein turnover alterations, or protein-kinase activation, but to calcium influx, calpain activation and caspase-3 degradation. In fact, EGTA and PD150606 (calpain inhibitor) prevented gelsolin increase while BAF (caspase inhibitor) enhanced it. Since gelsolin increase is induced by highly ß-haemolytic GBS-III-NEM316 and GBS-V-10/84, but not by weakly ß-haemolytic GBS, or GBS-III-COH31 in conditions suppressing ß-haemolysin expression/activity and the presence of dipalmitoylphosphatidylcholine (ß-haemolysin inhibitor), GBS-ß-haemolysin is solely responsible for gelsolin increase causing, through membrane permeability defects, calcium influx and calpain activation. Early gelsolin increase could represent a macrophage response to antagonize apoptosis since gelsolin knockdown increases macrophage susceptibility to GBS-induced apoptosis. This response seems to be GBS specific because macrophage apoptosis by Staurosporine or Cycloeximide does not induce gelsolin.
Assuntos
Gelsolina/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Streptococcus agalactiae/imunologia , Relação Dose-Resposta Imunológica , Proteínas Hemolisinas/metabolismo , Interações Hospedeiro-Patógeno , Macrófagos/metabolismoRESUMO
Notch1 signaling is involved in regulatory T (Treg)-cell differentiation. We previously demonstrated that, when cocultured with CD3(+) cells, mesenchymal stem cells (MSCs) induced a T-cell population with a regulatory phenotype. Here, we investigated the molecular mechanism underlying MSC induction of human Treg cells. We show that the Notch1 pathway is activated in CD4(+) T cells cocultured with MSCs. Inhibition of Notch1 signaling through GSI-I or the Notch1 neutralizing antibody reduced expression of HES1 (the Notch1 downstream target) and the percentage of MSC-induced CD4(+) CD25(high) FOXP3(+) cells in vitro. Moreover, we demonstrate that FOXP3 is a downstream target of Notch signaling in human cells. No crosstalk between Notch1 and TGF-ß signaling pathways was observed in our experimental system. Together, these findings indicate that activation of the Notch1 pathway is a novel mechanism in the human Treg-cell induction mediated by MSCs.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Mesenquimais/imunologia , Receptor Notch1/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Anticorpos Bloqueadores/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Antígenos CD4/metabolismo , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Contagem de Linfócitos , Células-Tronco Mesenquimais/efeitos dos fármacos , Oligopeptídeos/farmacologia , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/imunologia , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Fatores de Transcrição HES-1RESUMO
γ-Secretase inhibitors (GSIs) have been proposed for combined therapies of malignancies with a dysregulated Notch signaling. GSI I (Z-Leu-Leu-Nle-CHO) induces apoptosis of some tumor cells by inhibiting proteasome and Notch activity. Alterations in these two cell survival regulators contribute to apoptosis resistance of chronic lymphocytic leukemia (CLL) cells. Here, we investigated the mechanisms whereby GSI I increases apoptosis of primary CLL cells. Time-course studies indicate that initial apoptotic events are inhibition of proteasome activity, concomitant with an increased endoplasmic reticulum (ER) stress apoptotic signaling, and a consistent Noxa protein up-regulation. These events precede, and some of them contribute to, mitochondrial alterations, which occur notwithstanding Mcl-1 accumulation induced by GSI I. In CLL cells, GSI I inhibits Notch1 and Notch2 activation only in the late apoptotic phases, suggesting that this event does not initiate CLL cell apoptosis. However, Notch inhibition may contribute to amplify GSI I-induced CLL cell apoptosis, given that Notch activation sustains the survival of these cells, as demonstrated by the evidence that both Notch1 and Notch2 down-regulation by small-interfering RNA accelerates spontaneous CLL cell apoptosis. Overall, our results show that GSI I triggers CLL cell apoptosis by inhibiting proteasome activity and enhancing ER stress, and amplifies it by blocking Notch activation. These findings suggest the potential relevance of simultaneously targeting these three important apoptosis regulators as a novel therapeutic strategy for CLL.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/metabolismo , MAP Quinase Quinase 4/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
Group B Streptococcus (GBS) causes severe infection in the central nervous system. In this study, brain mitochondrial function was investigated by simulating infection of isolated mitochondria with GBS, which resulted in loss of mitochondrial activity. The ß-hemolysin expressing strains GBS-III-NEM316 and GBS-III-COH31, but not the gGBS-III-COH31 that does not express ß-hemolysin, caused dissipation of preformed mitochondrial membrane potential (Δψm). This indicates that ß-hemolysin is responsible for decreasing of the reducing power of mitochondria. GBS-III-COH31 interacted with mitochondria causing increase of oxygen consumption, due to uncoupling of respiration, blocking of ATP synthesis, and cytochrome c release outside mitochondria. Moreover, the mitochondrial systems contributing to the control of cellular Ca(2+) uptake were lost. In spite of these alterations, mitochondrial phospholipid content and composition did not change significantly, as evaluated by MALDI-TOF mass spectrometry. However, exogenous cardiolipin (CL) and dipalmitoylphosphatidylcholine (DPPC) attenuated the uncoupling effect of GBS-III-COH31, although with different mechanisms. CL was effective only when fused to the inner mitochondrial membrane, probably reducing the extent of GBS-induced proton leakage. DPPC, which is not able to fuse with mitochondrial membranes, exerted its effect outside mitochondria, likely by shielding mitochondria against GBS ß-hemolysin attack.
Assuntos
Proteínas de Bactérias/biossíntese , Encéfalo/microbiologia , Cardiolipinas/farmacologia , Proteínas Hemolisinas/biossíntese , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/microbiologia , Fosfatidilcolinas/farmacologia , Streptococcus agalactiae/metabolismo , Animais , Apoptose , Respiração Celular/fisiologia , Espectrometria de Massas , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/microbiologia , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Infecções Estreptocócicas/tratamento farmacológicoRESUMO
Introduction: Constitutive activation of NOTCH1-wild-type (NT1-WT) signaling is associated with poor outcomes in chronic lymphocytic leukemia (CLL), and NOTCH1 mutation (c.7541_7542delCT), which potentiates NOTCH1 signaling, worsens the prognosis. However, the specific mechanisms of NOTCH1 deregulation are still poorly understood. Accumulative evidence mentioned endoplasmic reticulum (ER) stress/unfolded protein response (UPR) as a key targetable pathway in CLL. In this study, we investigated the impact of NOTCH1 deregulation on CLL cell response to ER stress induction, with the aim of identifying new therapeutic opportunities for CLL. Methods: We performed a bioinformatics analysis of NOTCH1-mutated (NT1-M) and NT1-WT CLL to identify differentially expressed genes (DEGs) using the rank product test. Quantitative real-time polymerase chain reaction (qPCR), Western blotting, cytosolic Ca2+, and annexin V/propidium iodide (PI) assay were used to detect curcumin ER stress induction effects. A median-effect equation was used for drug combination tests. The experimental mouse model Eµ-TCL1 was used to evaluate the impact of ER stress exacerbation by curcumin treatment on the progression of leukemic cells and NOTCH1 signaling. Results and discussion: Bioinformatics analysis revealed gene enrichment of the components of the ER stress/UPR pathway in NT1-M compared to those in NT1-WT CLL. Ectopic expression of NOTCH1 mutation upregulated the levels of ER stress response markers in the PGA1 CLL cell line. Primary NT1-M CLL was more sensitive to curcumin as documented by a significant perturbation in Ca2+ homeostasis and higher expression of ER stress/UPR markers compared to NT1-WT cells. It was also accompanied by a significantly higher apoptotic response mediated by C/EBP homologous protein (CHOP) expression, caspase 4 cleavage, and downregulation of NOTCH1 signaling in NT1-M CLL cells. Curcumin potentiated the apoptotic effect of venetoclax in NT1-M CLL cells. In Eµ-TCL1 leukemic mice, the administration of curcumin activated ER stress in splenic B cells ex vivo and significantly reduced the percentage of CD19+/CD5+ cells infiltrating the spleen, liver, and bone marrow (BM). These cellular effects were associated with reduced NOTCH1 activity in leukemic cells and resulted in prolonged survival of curcumin-treated mice. Overall, our results indicate that ER stress induction in NT1-M CLL might represent a new therapeutic opportunity for these high-risk CLL patients and improve the therapeutic effect of drugs currently used in CLL.
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BACKGROUND: Chronic lymphocytic leukemia (CLL) is an incurable disorder associated with alterations in several pathways essential for survival and proliferation. Despite the advances made in CLL therapy with the new target agents, in some cases, relapses and resistance could occur, making the discovery of new alternatives to manage CLL refractoriness necessary. To provide new therapeutic strategies for CLL, we investigated the anti-leukemic activity of silver nanoparticles (AgNPs), whose impact on CLL cells has been poorly explored. METHODS: We studied the action mechanisms of AgNPs in vitro through flow cytometry and molecular analyses. To improve the bioavailability of AgNPs, we generated AgNPs coated with the anti-CD20 antibody Rituximab (AgNPs@Rituximab) and carried out imaging-based approaches and in vivo experiments to evaluate specificity, drug uptake, and efficacy. RESULTS: AgNPs reduced the viability of primary CLL cells and the HG-3 cell line by inducing an intrinsic apoptotic pathway characterized by Bax/Bcl-2 imbalance, caspase activation, and PARP degradation. Early apoptotic events triggered by AgNPs included enhanced Ca2+ influx and ROS overproduction. AgNPs synergistically potentiated the cytotoxicity of Venetoclax, Ibrutinib, and Bepridil. In vitro, the AgNPs@Rituximab conjugates were rapidly internalized within CLL cells and strongly prolonged the survival of CLL xenograft models compared to each unconjugated single agent. CONCLUSIONS: AgNPs showed strong anti-leukemic activity in CLL, with the potential for clinical translation in combination with agents used in CLL. The increased specificity of AgNPs@Rituximab toward CLL cells could be relevant for overcoming in vivo AgNPs' non-specific distribution and increasing their efficacy.
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A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8-mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.
Assuntos
Apoptose/fisiologia , Retículo Endoplasmático/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Regulação para Baixo , Retículo Endoplasmático/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Rearranjo Gênico do Linfócito B , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Humanos , Técnicas In Vitro , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/terapia , Proteínas de Membrana/metabolismo , Modelos Biológicos , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico , Tapsigargina/farmacologia , Tunicamicina/farmacologiaRESUMO
Group B Streptococcus (GBS) has evolved several strategies to avoid host defences where macrophages are one of main targets. Since pathogens frequently target the cytoskeleton to evade immune defences, we investigated if GBS manipulates macrophage cytoskeleton. GBS-III-COH31 in a time- and infection ratio-dependent manner induces great macrophage cytoskeleton alterations, causing degradation of several structural and regulatory cytoskeletal proteins. GBS ß-haemolysin is involved in cytoskeleton alterations causing plasma membrane permeability defects which allow calcium influx and calpain activation. In fact, cytoskeleton alterations are not induced by GBS-III-COH31 in conditions that suppress ß-haemolysin expression/activity and in presence of dipalmitoylphosphatidylcholine (ß-haemolysin inhibitor). Calpains, particularly m-calpain, are responsible for GBS-III-COH31-induced cytoskeleton disruption. In fact, the calpain inhibitor PD150606, m-calpain small-interfering-RNA and EGTA which inhibit calpain activation prevented cytoskeleton degradation whereas µ-calpain and other protease inhibitors did not. Finally, calpain inhibition strongly increased the number of viable intracellular GBS-III-COH31, showing that cytoskeleton alterations reduced macrophage phagocytosis. Marked macrophage cytoskeleton alterations are also induced by GBS-III-NEM316 and GBS-V-10/84 through ß-haemolysin-mediated plasma membrane permeability defects which allow calpain activation. This study suggests a new GBS strategy to evade macrophage antimicrobial responses based on cytoskeleton disruption by an unusual mechanism mediated by calcium influx and calpain activation.
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Actinas/antagonistas & inibidores , Actinas/metabolismo , Calpaína/metabolismo , Citoesqueleto/metabolismo , Macrófagos Peritoneais/microbiologia , Microtúbulos/metabolismo , Streptococcus agalactiae/patogenicidade , Animais , Proteínas de Bactérias/toxicidade , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Hemolisinas/toxicidade , CamundongosRESUMO
NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3ß downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in CLL cells. Indeed, GSK3ß silencing by small interfering RNA increases N1-ICD levels, whereas expression of an active GSK3ß mutant reduces them. Additionally, the GSK3ß inhibitor SB216763 enhances N1-ICD stability at a concentration at which it also increases CLL cell viability. We also show that N1-ICD is physically associated with GSK3ß in CLL cells. SB216763 reduces GSK3ß/N1-ICD interactions and the levels of ubiquitinated N1-ICD, indicating a reduction in N1-ICD proteasomal degradation when GSK3ß is less active. We then modulated the activity of two upstream regulators of GSK3ß and examined the impact on N1-ICD levels and CLL cell viability. Specifically, we inhibited AKT that is a negative regulator of GSK3ß and is constitutively active in CLL cells. Furthermore, we activated the protein phosphatase 2 A (PP2A) that is a positive regulator of GSK3ß, and has an impaired activity in CLL. Results show that either AKT inhibition or PP2A activation reduce N1-ICD expression and CLL cell viability in vitro, through mechanisms mediated by GSK3ß activity. Notably, for PP2A activation, we used the highly specific activator DT-061, that also reduces leukemic burden in peripheral blood, spleen and bone marrow in the Eµ-TCL1 adoptive transfer model of CLL, with a concomitant decrease in N1-ICD expression. Overall, we identify in GSK3ß a key component of the network regulating N1-ICD stability in CLL, and in AKT and PP2A new druggable targets for disrupting NOTCH1 signaling with therapeutic potential.
Assuntos
Glicogênio Sintase Quinase 3 beta , Leucemia Linfocítica Crônica de Células B , Receptor Notch1 , Sobrevivência Celular/genética , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteína Fosfatase 2/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismoRESUMO
Notch signaling is involved in tumorigenesis, but its role in B-chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor-kappa B (NF-kappaB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the gamma-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-kappaB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.
Assuntos
Apoptose , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Receptores Notch/metabolismo , Transdução de Sinais , Linfócitos B/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Sobrevivência Celular , Regulação para Baixo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteína Jagged-2 , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Proteínas Serrate-Jagged , Células Tumorais CultivadasRESUMO
NOTCH1 mutations and deregulated signal have been commonly found in chronic lymphocytic leukemia (CLL) patients. Whereas the impact of NOTCH1 mutations on clinical course of CLL has been widely studied, the prognostic role of NOTCH1 activation in CLL remains to be defined. Here, we analyzed the activation of NOTCH1/NOTCH2 (ICN1/ICN2) and the expression of JAGGED1 (JAG1) in 163 CLL patients and evaluated their impact on TTFT (Time To First Treatment) and OS (Overall Survival). NOTCH1 activation (ICN1+) was found in 120/163 (73.6%) patients. Among them, 63 (52.5%) were NOTCH1 mutated (ICN1+/mutated) and 57 (47.5%) were NOTCH1 wild type (ICN1+/WT). ICN1+ patients had a significant reduction of TTFT compared to ICN1-negative (ICN1-). In the absence of NOTCH1 mutations, we found that the ICN1+/WT group had a significantly reduced TTFT compared to ICN1- patients. The analysis of IGHV mutational status showed that the distribution of the mutated/unmutated IGHV pattern was similar in ICN1+/WT and ICN1- patients. Additionally, TTFT was significantly reduced in ICN1+/ICN2+ and ICN1+/JAG1+ patients compared to ICN1-/ICN2- and ICN1-/JAG1- groups. Our data revealed for the first time that NOTCH1 activation is a negative prognosticator in CLL and is not correlated to NOTCH1 and IGHV mutational status. Activation of NOTCH2 and JAGGED1 expression might also influence clinical outcomes in this group, indicating the need for further dedicated studies. The evaluation of different NOTCH network components might represent a new approach to refine CLL risk stratification.
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BACKGROUND AIMS: The impact of chronic lymphatic leukemia (CLL) tumor burden on the autologous immune system has already been demonstrated. This study attempted to elucidate the molecular mechanisms underlying T-cell immunologic deficiencies in CLL. METHODS: Freshly isolated CD3(+) T cells from patients with a diagnosis of CLL and healthy donors were analyzed by gene expression profiling. Activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity against mutated and unmutated autologous B cells and DAUDI, K562 and P815 cell lines. To investigate T-cell mediated cytotoxicity in vivo, we co-transplanted OKT3-activated T lymphocytes and autologous B-cell CLL (B-CLL) cells into NOD/SCID mice. RESULTS: Gene expression profiles of peripheral blood T cells from B-CLL patients showed 25 down-regulated, and 31 up-regulated, genes that were mainly involved in cell differentiation, proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T-cell activation. After culture, the T-cell count remained unchanged, CD8 cells expanded more than CD4 and a cytotoxicity index >30% was present in 5/20 patients. Cytotoxicity against B autologous leukemic cells did not correlate with B-cell mutational status. Only activated T cells exerting cytotoxicity against autologous leukemic B cells prevented CLL in a human-mouse chimera. CONCLUSIONS: This study indicates that patients with CLL are affected by a partial immunologic defect that might be somewhat susceptible to repair. This study identifies the molecular pathways underlying T-cell deficiencies in CLL and shows that cytotoxic T-cell functions against autologous B-CLL can be rebuilt at least in part in vitro and in vivo.
Assuntos
Citotoxicidade Imunológica/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/genética , Regulação para Baixo/genética , Regulação para Baixo/fisiologia , Perfilação da Expressão Gênica , Rearranjo Gênico do Linfócito B/genética , Rearranjo Gênico do Linfócito B/imunologia , Humanos , Fatores Imunológicos/farmacologia , Interleucina-2/farmacologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/fisiologia , Camundongos , Camundongos SCID , Muromonab-CD3/farmacologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Regulação para Cima/genética , Regulação para Cima/fisiologiaRESUMO
PURPOSE: Ibrutinib, a Bruton tyrosine kinase inhibitor (BTKi), has improved the outcomes of chronic lymphocytic leukemia (CLL), but primary resistance or relapse are issues of increasing significance. While the predominant mechanism of action of BTKi is the B-cell receptor (BCR) blockade, many off-target effects are unknown. We investigated potential interactions between BCR pathway and NOTCH1 activity in ibrutinib-treated CLL to identify new mechanisms of therapy resistance and markers to monitor disease response. EXPERIMENTAL DESIGN: NOTCH activations was evaluated either in vitro and ex vivo in CLL samples after ibrutinib treatment by Western blotting. Confocal proximity ligation assay (PLA) experiments and analyses of down-targets of NOTCH1 by qRT-PCR were used to investigate the cross-talk between BTK and NOTCH1. RESULTS: In vitro ibrutinib treatment of CLL significantly reduced activated NOTCH1/2 and induced dephosphorylation of eIF4E, a NOTCH target in CLL. BCR stimulation increased the expression of activated NOTCH1 that accumulated in the nucleus leading to HES1, DTX1, and c-MYC transcription. Results of in situ PLA experiments revealed the presence of NOTCH1-ICD/BTK complexes, whose number was reduced after ibrutinib treatment. In ibrutinib-treated CLL patients, leukemic cells showed NOTCH1 activity downregulation that deepened over time. The NOTCH1 signaling was restored at relapse and remained activated in ibrutinib-resistant CLL cells. CONCLUSIONS: We demonstrated a strong clinical activity of ibrutinib in a real-life context. The ibrutinib clinical efficacy was associated with NOTCH1 activity downregulation that deepened over time. Our data point to NOTCH1 as a new molecular partner in BCR signaling with potential to further improve CLL-targeted treatments.
Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Adenina/análogos & derivados , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Piperidinas , Inibidores de Proteínas Quinases/farmacologia , Receptor Notch1/genética , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
As previously reported, chronic lymphocytic leukemia (CLL) cells show constitutive Notch1/2 activation and express the Notchligand Jagged1. Despite increasing knowledge of the impact of Notch alterations on CLL biology and pathogenesis, the role of Jagged1 expressed in CLL cells remains undefined. In other cell types, it has been shown that after Notch engagement, Jagged1 not only activates Notch in signal-receiving cell, but also undergoes proteolytic activation in signal-sending cell, triggering a signaling with biological effects. We investigated whether Jagged1 expressed in CLL cells undergoes proteolytic processing and/or is able to induce Notch activation through autocrine/paracrine loops, focusing on the effect that CLL prosurvival factor IL-4 could exert on the Notch-Jagged1 system in these cells. We found that Jagged1 was constitutively processed in CLL cells and generated an intracellular fragment that translocated into the nucleus, and an extracellular fragment released into the culture supernatant. IL-4 enhanced expression of Jagged1 and its intracellular fragments, as well as Notch1/2 activation. The IL-4-induced increase in Notch1/2 activation was independent of the concomitant upregulated Jagged1 levels. Indeed, blocking Notch-Jagged1 interactions among CLL cells with Jagged1 neutralizing antibodies did not affect the expression of the Notch target Hes1. Notably, anti-Jagged1 antibodies partially prevented the IL-4-induced increase in Jagged1 processing and cell viability, suggesting that Jagged1 processing is one of the events contributing to IL-4-induced CLL cell survival. Consistent with this, Jagged1 silencing by small interfering RNA partially counteracted the capacity of IL-4 to promote CLL cell survival. Investigating the pathways whereby IL-4 promoted Notch1/2 activation in CLL cells independent of Jagged1, we found that PI3Kδ/AKT and PKCδ were involved in upregulating Notch1 and Notch2 proteins, respectively. Overall, this study provides new insights into the Notch-ligand system in CLL cells and suggests that targeting this system may be exploited as a novel/additional therapy approach for CLL.
Assuntos
Interleucina-4/genética , Proteína Jagged-1/genética , Leucemia Linfocítica Crônica de Células B/genética , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Feminino , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Proteína Quinase C-delta/genética , RNA Interferente Pequeno/genética , Receptor Notch1/genética , Receptor Notch2/genética , Transdução de SinaisRESUMO
Chronic lymphocytic leukemia (CLL) is an incurable B-cell neoplasm characterized by highly variable clinical outcomes. In recent years, genomic and molecular studies revealed a remarkable heterogeneity in CLL, which mirrored the clinical diversity of this disease. These studies profoundly enhanced our understanding of leukemia cell biology and led to the identification of new biomarkers with potential prognostic and therapeutic significance. Accumulating evidence indicates a key role of deregulated NOTCH1 signaling and NOTCH1 mutations in CLL. This review highlights recent discoveries that improve our understanding of the pathophysiological NOTCH1 signaling in CLL and the clinical impact of NOTCH1 mutations in retrospective and prospective trials. In addition, we discuss the rationale for a therapeutic strategy aiming at inhibiting NOTCH1 signaling in CLL, along with an overview on the currently available NOTCH1-directed approaches.
RESUMO
In allogeneic hematopoietic stem cell transplantation, which is the major curative therapy for hematological malignancies, T cells play a key role in the development of graft-versus-host disease (GvHD). NOTCH pathway is a conserved signal transduction system that regulates T cell development and differentiation. The present review analyses the role of the NOTCH signaling as a new regulator of acute GvHD. NOTCH signaling could also represent a new therapeutic target for GvHD.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Receptores Notch/metabolismo , Linfócitos T/imunologia , Animais , Diferenciação Celular , Antígenos HLA/imunologia , Humanos , Receptores Notch/genética , Transdução de Sinais , Transplante HomólogoRESUMO
To investigate chronic lymphocytic leukemia (CLL)-initiating cells, we assessed NOTCH1 mutation/expression in hematopoietic stem cells (HSCs). In NOTCH1-mutated CLL, we detected subclonal mutations in 57% CD34+/CD38- HSCs. NOTCH1 mutation was present in 66% CD34+/CD38+ progenitor cells displaying an increased mutational burden compared to HSCs. Flow cytometric analysis revealed significantly higher NOTCH1 activation in CD34+/CD38- and CD34+/CD38+ cells from CLL patients, regardless NOTCH1 mutation compared to healthy donors. Activated NOTCH1 resulted in overexpression of the NOTCH1 target c-MYC. We conclude that activated NOTCH1 is an early event in CLL that may contribute to aberrant HSCs in this disease.