RESUMO
Even though SYK and ZAP70 kinases share high sequence homology and serve analogous functions, their expression in B and T cells is strictly segregated throughout evolution. Here, we identified aberrant ZAP70 expression as a common feature in a broad range of B cell malignancies. We validated SYK as the kinase that sets the thresholds for negative selection of autoreactive and premalignant clones. When aberrantly expressed in B cells, ZAP70 competes with SYK at the BCR signalosome and redirects SYK from negative selection to tonic PI3K signaling, thereby promoting B cell survival. In genetic mouse models for B-ALL and B-CLL, conditional expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Inducible activation of Zap70 during B cell development compromised negative selection of autoreactive B cells, resulting in pervasive autoantibody production. Strict segregation of the two kinases is critical for normal B cell selection and represents a central safeguard against the development of autoimmune disease and B cell malignancies.
Assuntos
Autoimunidade , Neoplasias/enzimologia , Neoplasias/prevenção & controle , Quinase Syk/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Animais , Antígenos CD19/metabolismo , Linfócitos B , Cálcio/metabolismo , Diferenciação Celular , Transformação Celular Neoplásica , Ativação Enzimática , Humanos , Tolerância Imunológica , Linfoma de Células B/enzimologia , Linfoma de Células B/patologia , Camundongos , Modelos Genéticos , Fatores de Transcrição NFATC/metabolismo , Proteínas de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR) or hyperactivation above maximum (for example, self-reactive BCR) thresholds of signalling strength causes negative selection. In â¼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival. We tested the hypothesis that targeted hyperactivation--above a maximum threshold--will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR-ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1), we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.
Assuntos
Linfócitos B/metabolismo , Linfócitos B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transdução de Sinais , Motivos de Aminoácidos/genética , Animais , Antígenos CD/metabolismo , Linfócitos B/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteínas de Fusão bcr-abl/genética , Deleção de Genes , Humanos , Inositol Polifosfato 5-Fosfatases , Peptídeos e Proteínas de Sinalização Intracelular/agonistas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/deficiência , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk , Tirosina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The transcription factor Foxp1 is critical for early B cell development. Despite frequent deregulation of Foxp1 in B cell lymphoma, the physiological functions of Foxp1 in mature B cells remain unknown. Here, we used conditional gene targeting in the B cell lineage and report that Foxp1 disruption in developing and mature B cells results in reduced numbers and frequencies of follicular and B-1 B cells and in impaired antibody production upon T cell-independent immunization in vivo. Moreover, Foxp1-deficient B cells are impaired in survival even though they exhibit an increased capacity to proliferate. Transcriptional analysis identified defective expression of the prosurvival Bcl-2 family gene Bcl2l1 encoding Bcl-xl in Foxp1-deficient B cells, and we identified Foxp1 binding in the regulatory region of Bcl2l1 Transgenic overexpression of Bcl2 rescued the survival defect in Foxp1-deficient mature B cells in vivo and restored peripheral B cell numbers. Thus, our results identify Foxp1 as a physiological regulator of mature B cell survival mediated in part via the control of Bcl-xl expression and imply that this pathway might contribute to the pathogenic function of aberrant Foxp1 expression in lymphoma.
Assuntos
Linfócitos B/classificação , Linfócitos B/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Proteínas Repressoras/metabolismo , Animais , Anticorpos/metabolismo , Antígenos CD19/metabolismo , Fatores de Transcrição Forkhead/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteínas Repressoras/genética , Linfócitos T/fisiologia , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Pre-B cells within the bone marrow represent the normal counterpart for most acute lymphoblastic leukemia (ALL). During normal early B-cell development, survival and proliferation signals are dominated by cytokines, particularly interleukin-7 (IL-7) for murine developing B cells. With expression of a functional pre-B-cell receptor (BCR), cytokine signaling is attenuated and the tonic/autonomous pre-BCR signaling pathway provides proliferation as well as differentiation signals. In this review, we first describe checkpoint mechanisms during normal B-cell development and then discuss how genetic lesions in these pathways function as oncogenic mimicries and allow transformed pre-B cells to bypass checkpoint control. We focus on cytokine receptor signaling that is mimicked by activating lesions in receptor subunits or downstream mediators as well as aberrant activation of non-B lymphoid cytokine receptors. Furthermore, we describe the molecular switch from cytokine receptor to pre-BCR signaling, how this pathway is of particular importance for certain ALL subtypes, and how pre-BCR signaling is engaged by genetic lesions, such as BCR-ABL1. We discuss the transcriptional control mechanisms downstream of both cytokine- and pre-BCR signaling and how normal checkpoint control mechanisms are circumvented in pre-B ALL. Finally, we highlight new therapeutic concepts for targeted inhibition of oncogenic cytokine or pre-BCR signaling pathways.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Receptores de Células Precursoras de Linfócitos B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Células Precursoras de Linfócitos B/fisiologia , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Proteínas de Fusão bcr-abl/genética , Humanos , Camundongos , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacosRESUMO
B-cell antigen receptor (BCR) expression is an important feature of chronic lymphocytic leukaemia (CLL), one of the most prevalent B-cell neoplasias in Western countries. The presence of stereotyped and quasi-identical BCRs in different CLL patients suggests that recognition of specific antigens might drive CLL pathogenesis. Here we show that, in contrast to other B-cell neoplasias, CLL-derived BCRs induce antigen-independent cell-autonomous signalling, which is dependent on the heavy-chain complementarity-determining region (HCDR3) and an internal epitope of the BCR. Indeed, transferring the HCDR3 of a CLL-derived BCR provides autonomous signalling capacity to a non-autonomously active BCR, whereas mutations in the internal epitope abolish this capacity. Because BCR expression was required for the binding of secreted CLL-derived BCRs to target cells, and mutations in the internal epitope reduced this binding, our results indicate a new model for CLL pathogenesis, with cell-autonomous antigen-independent signalling as a crucial pathogenic mechanism.
Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Autoantígenos/imunologia , Autoantígenos/metabolismo , Sinalização do Cálcio , Regiões Determinantes de Complementaridade/imunologia , Regiões Determinantes de Complementaridade/metabolismo , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Receptores de Antígenos de Linfócitos B/imunologiaRESUMO
PURPOSE OF REVIEW: Normal B cells that have failed to productively rearrange immunoglobulin V region genes encoding a functional B-cell receptor (BCR) are destined to die. Likewise, the majority of B-cell malignancies remain dependent on functional BCR signaling, whereas in some subtypes BCR expression is missing and, apparently, counterselected. Here, we summarize the recent experimental evidence for the importance of BCR signaling and clinical concepts to target the BCR pathway in B-cell leukemia and lymphoma. RECENT FINDINGS: Although the dependency on pre-BCR signaling in pre-B acute lymphoblastic leukemia (ALL) seems to be limited to few ALL subtypes (e.g. TCF3-PBX1), most mature B-cell lymphomas rely on BCR signaling provided by different stimuli, for example tonic B-cell signaling, chronic (auto)-antigen exposure, and self-binding properties of the BCR. The finding that in chronic lymphocytic leukemia, BCRs bind to an epitope on the BCR itself unravels a novel concept for chronic lymphocytic leukemia pathogenesis. SUMMARY: Targeting of the B-cell receptor tyrosine kinases spleen tyrosine kinase, Bruton's tyrosine kinase, and phosphatidylinositol 3-kinase achieve promising clinical responses in various mature B-cell malignancies and might also be useful in defined subsets of ALL. However, further understanding of the BCR signal integration in the different disease groups is required to accurately predict which groups of patients will benefit from BCR pathway inhibition.
Assuntos
Leucemia de Células B/metabolismo , Linfoma de Células B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Humanos , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/genética , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/genética , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacosRESUMO
Richter's transformation (RT) is a progression of chronic lymphocytic leukemia (CLL) to aggressive lymphoma. MGA (Max gene associated), a functional MYC suppressor, is mutated at 3% in CLL and 36% in RT. However, genetic models and molecular mechanisms of MGA deletion that drive CLL to RT remain elusive. We established an RT mouse model by knockout of Mga in the Sf3b1/Mdr CLL model using CRISPR-Cas9 to determine the role of Mga in RT. Murine RT cells exhibited mitochondrial aberrations with elevated oxidative phosphorylation (OXPHOS). Through RNA sequencing and functional characterization, we identified Nme1 (nucleoside diphosphate kinase) as an Mga target, which drives RT by modulating OXPHOS. Given that NME1 is also a known MYC target without targetable compounds, we found that concurrent inhibition of MYC and electron transport chain complex II substantially prolongs the survival of RT mice in vivo. Our results suggest that the Mga-Nme1 axis drives murine CLL-to-RT transition via modulating OXPHOS, highlighting a potential therapeutic avenue for RT.
Assuntos
Leucemia Linfocítica Crônica de Células B , Mitocôndrias , Fosforilação Oxidativa , Animais , Mitocôndrias/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos , Deleção de Genes , Humanos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Modelos Animais de DoençasRESUMO
B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), remain incurable, with MM particularly prone to relapse. Our study introduces a novel mouse model with active RANK signaling and the TCL1 oncogene, displaying both CLL and MM phenotypes. In younger mice, TCL1 and RANK expression expands CLL-like B1-lymphocytes, while MM originates from B2-cells, becoming predominant in later stages and leading to severe disease progression and mortality. The induced MM mimics human disease, exhibiting features like clonal plasma cell expansion, paraproteinemia, anemia, and kidney and bone failure, as well as critical immunosurveillance strategies that promote a tumor-supportive microenvironment. This research elucidates the differential impacts of RANK activation in B1- and B2-cells and underscores the distinct roles of single versus combined oncogenes in B-cell malignancies. We also demonstrate that human MM cells express RANK and that inhibiting RANK signaling can reduce MM progression in a xenotransplantation model. Our study provides a rationale for further investigating the effects of RANK signaling in B-cell transformation and the shaping of a tumor-promoting microenvironment.
Assuntos
Linfócitos B , Transformação Celular Neoplásica , Mieloma Múltiplo , Proteínas Proto-Oncogênicas , Transdução de Sinais , Animais , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Humanos , Linfócitos B/metabolismo , Linfócitos B/imunologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Linhagem da Célula , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Modelos Animais de Doenças , Microambiente TumoralRESUMO
Patients with chronic lymphocytic leukemia (CLL) treated with B-cell pathway inhibitors and anti-CD20 antibodies exhibit low humoral response rates following SARS-CoV-2 vaccination. To investigate this observation, a prospective single-institution study was conducted comparing peripheral blood mononuclear cell transcriptional response with antibody and T-cell response rates following heterologous BNT162b2/ChAdOx1 vaccination of 15 patients with CLL/small lymphocytic lymphoma (SLL). Two-dose antibody response rate was 40%, increasing to 53% after booster. Patients on Bruton tyrosine kinase inhibitor (BTKi) and venetoclax ± anti-CD20 antibody within 12 months of vaccination responded inferiorly compared with those under BTKi alone. The 2-dose-T-cell response rate was 80%, which increased to 93% after the booster dose. Key transcriptional findings were that interferon-mediated signaling activation including activation of the JAK-STAT pathway generally occurred within days of vaccination, but was independent from the magnitude of the antibody response. Increasing counts of IGHV genes were associated with B-cell reconstitution and improved humoral response rate in the vaccinated patients. T-cell responses in patients with CLL appeared independent of treatment status, whereas higher humoral response rate was associated with BTKi treatment and B-cell reconstitution. Boosting was particularly effective when intrinsic immune status was improved by CLL treatment. Limitations included studying a relatively small cohort, with different treatments and vaccination schedules.
Assuntos
COVID-19 , Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Vacinas contra COVID-19 , Vacina BNT162 , Janus Quinases , Leucócitos Mononucleares , Estudos Prospectivos , COVID-19/prevenção & controle , SARS-CoV-2 , Fatores de Transcrição STAT , Transdução de Sinais , Anticorpos , ImunidadeRESUMO
Despite available targeted treatments for the disease, drug-resistant chronic lymphocytic leukemia (CLL) poses a clinical challenge. The objective of this study is to examine whether the dual-specific phosphatases DUSP1 and DUSP6 are required to negatively regulate mitogen-activated protein kinases (MAPKs) and thus counterbalance excessive MAPK activity. We show that high expression of DUSP6 in CLL correlates with poor clinical prognosis. Importantly, genetic deletion of the inhibitory phosphatase DUSP1 or DUSP6 and blocking DUSP1/6 function using a small-molecule inhibitor reduces CLL cell survival in vitro and in vivo. Using global phospho-proteome approaches, we observe acute activation of MAPK signaling by DUSP1/6 inhibition. This promotes accumulation of mitochondrial reactive oxygen species and, thereby, DNA damage and apoptotic cell death in CLL cells. Finally, we observe that DUSP1/6 inhibition is particularly effective against treatment-resistant CLL and therefore suggest transient DUSP1/6 inhibition as a promising treatment concept to eliminate drug-resistant CLL cells.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Retroalimentação , Proteínas Quinases Ativadas por MitógenoRESUMO
Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.
Assuntos
Antineoplásicos , Apoptose , Processamento de Proteína Pós-Traducional , Proteômica , Antígenos CD20/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteômica/métodos , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , HumanosRESUMO
The microenvironment provides essential growth and survival signals to chronic lymphocytic leukemia (CLL) cells and contributes to their resistance to cytotoxic agents. Pharmacologic inhibition of spleen tyrosine kinase (SYK), a key mediator of B-cell receptor (BCR) signaling, induces apoptosis in primary CLL cells and prevents stroma contact-mediated cell survival. This report demonstrates a role of SYK in molecularly defined pathways that mediate the CLL-microenvironmental crosstalk independent from the BCR. Chemokine and integrin stimulation induced SYK phosphorylation, SYK-dependent Akt phosphorylation, and F-actin formation in primary CLL cells. Inhibition of SYK by 2 pharmacologic inhibitors and siRNA-knockdown abrogated downstream SYK signaling and morphologic changes induced by these stimuli. CLL cell migration toward CXCL12, the major homing attractor, and CLL cell adhesion to VCAM-1, a major integrin ligand expressed on stromal cells, were markedly reduced by SYK inhibition. In combination with fludarabine, the SYK inhibitor R406 abrogated stroma-mediated drug resistance by preventing up-regulation of the antiapoptotic factor Mcl-1 in CLL cells. SYK blockade in CLL is a promising therapeutic principle not only for its inhibition of the BCR signaling pathway, but also by inhibiting protective stroma signals in a manner entirely independent of BCR signaling.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Oxazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Actinas/metabolismo , Idoso , Animais , Apoptose/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Quimiocinas/metabolismo , Quimiotaxia , Técnicas de Cocultura , Feminino , Fibronectinas/metabolismo , Humanos , Integrina alfa4/metabolismo , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides , Prognóstico , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Quinase Syk , Células Tumorais Cultivadas , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
OBJECTIVE: Spleen tyrosine kinase (SYK) has come into focus as a potential therapeutic target in chronic inflammatory diseases, such as rheumatoid arthritis and asthma, as well as in B-cell lymphomas. SYK has also been involved in the signaling of immunoreceptors, cytokine receptors, and integrins. We therefore hypothesized that inhibition of SYK attenuates the inflammatory process underlying atherosclerosis and reduces plaque development. METHODS AND RESULTS: Low-density lipoprotein receptor-deficient mice consuming a high-cholesterol diet supplemented with 2 doses of the orally available SYK inhibitor fostamatinib for 16 weeks showed a dose-dependent reduction in atherosclerotic lesion size by up to 59±6% compared with the respective controls. Lesions of fostamatinib-treated animals contained fewer macrophages but more smooth muscle cells and collagen-characteristics associated with more stable plaques in humans. Mechanistically, fostamatinib attenuated adhesion and migration of inflammatory cells and limited macrophage survival. Furthermore, fostamatinib normalized high-cholesterol diet -induced monocytosis and inflammatory gene expression. CONCLUSIONS: We present the novel finding that the SYK inhibitor fostamatinib attenuates atherogenesis in mice. Our data identify SYK inhibition as a potentially fruitful antiinflammatory therapeutic strategy in atherosclerosis.
Assuntos
Aterosclerose/tratamento farmacológico , Inflamação/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Oxazinas/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/uso terapêutico , Receptores de LDL/deficiência , Administração Oral , Aminopiridinas , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Colesterol na Dieta/administração & dosagem , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Morfolinas , Pirimidinas , Quinase SykRESUMO
In the present work, we aim at developing an in vitro release assay to predict circulation times of hydrophobic drugs loaded into polymeric micelles (PM), upon intravenous (i.v.) administration. PM based on poly (ethylene glycol)-b-poly (N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) block copolymer were loaded with a panel of hydrophobic anti-cancer drugs and characterized for size, loading efficiency and release profile in different release media. Circulation times in mice of two selected drugs loaded in PM were evaluated and compared to the in vitro release profile. Release of drugs from PM was evaluated over 7 days in PBS containing Triton X-100 and in PBS containing albumin at physiological concentration (40 g/L). The results were utilized to identify crucial molecular features of the studied hydrophobic drugs leading to better micellar retention. For the best and the worst retained drugs in the in vitro assays (ABT-737 and BCI, respectively), the circulation of free and entrapped drugs into PM was examined after i.v. administration in mice. We found in vivo drug retention at 24 h post-injection similar to the retention found in the in vitro assays. This demonstrates that in vitro release assay in buffers supplemented with albumin, and to a lesser degree Triton X-100, can be employed to predict the in vivo circulation kinetics of drugs loaded in PM. Utilizing media containing acceptor molecules for hydrophobic compounds, provide a first screen to understand the stability of drug-loaded PM in the circulation and, therefore, can contribute to the reduction of animals used for circulation kinetics studies.
Assuntos
Portadores de Fármacos , Micelas , Albuminas , Animais , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Camundongos , Octoxinol , Polietilenoglicóis/química , Polímeros/químicaRESUMO
Patients with chronic lymphocytic leukemia (CLL) treated with B-cell pathway inhibitors and anti-CD20 antibodies exhibit low humoral response rate (RR) following SARS-CoV-2 vaccination. To investigate the relationship between the initial transcriptional response to vaccination with ensuing B and T cell immune responses, we performed a comprehensive immune transcriptome analysis flanked by antibody and T cell assays in peripheral blood prospectively collected from 15 CLL/SLL patients vaccinated with heterologous BNT162b2/ChAdOx1 with follow up at a single institution. The two-dose antibody RR was 40% increasing to 53% after booster. Patients on BTKi, venetoclax ± anti-CD20 antibody within 12 months of vaccination responded less well than those under BTKi alone. The two-dose T cell RR was 80% increasing to 93% after booster. Transcriptome studies revealed that seven patients showed interferon-mediated signaling activation within 2 days and one at 7 days after vaccination. Increasing counts of COVID-19 specific IGHV genes correlated with B-cell reconstitution and improved humoral RR. T cell responses in CLL patients appeared after vaccination regardless of treatment status. A higher humoral RR was associated with BTKi treatment and B-cell reconstitution. Boosting was particularly effective when intrinsic immune status was improved by CLL-treatment.
RESUMO
The idiotypes of B cell lymphomas represent tumor-specific antigens. T cell responses induced by idiotype vaccination in vivo are directed predominantly against CDR peptides, whereas in vitro T cells also recognize framework-derived epitopes. To investigate the mechanisms regulating the specificity of idiotype-specific T cells, BALB/c or B10.D2 mice were immunized with mature dendritic cells loaded with H-2K(d)-restricted peptides from influenza hemagglutinin, or from shared (J region) or unique (CDR3) structures of the A20 lymphoma idiotype. Antigen-specific T cells were induced in vivo by the CDR3 and influenza epitopes, but not by the J peptide. Gene expression profiling of splenic regulatory T cells revealed vaccination-induced Treg activation and proliferation. Treg activity involved J epitope-dependent IL-10 secretion and functional suppression of peptide-specific effector T cells. Vaccination-induced in vivo proliferation of transgenic hemagglutinin-specific T cells was suppressed by co-immunization with the J peptide and was restored in CD25-depleted animals. In conclusion, Treg induced by a shared idiotype epitope can systemically suppress T cell responses against idiotype-derived and immunodominant foreign epitopes in vivo. The results imply that tumor vaccines should avoid epitopes expressed by normal cells in the draining lymph node to achieve optimal anti-tumor efficacy.
Assuntos
Vacinas Anticâncer , Idiótipos de Imunoglobulinas/imunologia , Linfoma de Células B/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Perfilação da Expressão Gênica , Antígenos H-2/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Imunização , Idiótipos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Vigilância Imunológica , Ativação Linfocitária , Linfoma de Células B/terapia , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Clinical evidence suggests alterations in receptor activator of NF-κB (RANK) signaling are key contributors to B cell autoimmunity and malignancy, but the pathophysiological consequences of aberrant B cell-intrinsic RANK signaling remain unknown. We generated mice that express a human lymphoma-derived, hyperactive RANKK240E variant in B lymphocytes in vivo. Forced RANK signaling disrupted B cell tolerance and induced a fully penetrant systemic lupus erythematosus-like disease in addition to the development of chronic lymphocytic leukemia (CLL). Importantly, RANKK240E transgenic CLL cells as well as CLL cells of independent murine and of human origin depend on microenvironmental RANK ligand (RANKL) for tumor cell survival. Consequently, inhibition of the RANKL-RANK axis with anti-RANKL antibodies killed murine and human CLL cells in vitro and in vivo. These results establish pathological B cell-intrinsic RANK signaling as a potential driver of autoimmunity and B cell malignancy, and they suggest the exploitation of clinically available anti-RANKL compounds for CLL treatment.
Assuntos
Autoimunidade/imunologia , Linfócitos B/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Animais , Linfócitos B/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Feminino , Células HEK293 , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Transdução de Sinais/imunologiaRESUMO
Current therapeutic approaches for chronic lymphocytic leukemia (CLL) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the phosphatidylinositol-3-phosphate/AKT (PI3K/AKT)-signaling pathway may be leveraged to trigger CLL cell death. Though counterintuitive, our data show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SH2-containing-inositol-5'-phosphatase-1 (SHIP1) induces acute cell death in CLL cells. Our mechanistic studies reveal that increased AKT activity upon inhibition of SHIP1 leads to increased mitochondrial respiration and causes excessive accumulation of reactive oxygen species (ROS), resulting in cell death in CLL with immunogenic features. Our results demonstrate that CLL cells critically depend on mechanisms to fine-tune PI3K/AKT activity, allowing sustained proliferation and survival but avoid ROS-induced cell death and suggest transient SHIP1-inhibition as an unexpectedly promising concept for CLL therapy.
Assuntos
Morte Celular/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Progressão da Doença , Humanos , Imuno-Histoquímica , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno , RNA-Seq , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Transplante Homólogo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The non-signalling chemokine receptors, including receptors DARC, D6 and CCX-CKR, have recently been shown to be involved in chemokine clearance and activity regulation. The human chemokine receptor CRAM (also known as HCR or CCRL2) is the most recently identified member of this atypical group. CRAM is expressed on B cells in a maturation-stage dependent manner and absent on T cells. We have recently shown that it competitively binds CCL19. CCL19 and its signalling receptor CCR7 are critical components involved in cell recruitment to secondary lymphoid organs and in maturation. B cell Chronic Lymphocytic Leukemia (B-CLL) is a low-grade lymphoma characterized by proliferative centres (or pseudofollicles). Proliferative centres develop due to abnormal cellular localisation and they are involved in the development of malignant cells. CCR7 is highly expressed on B cells from CLL patients and mediates migration towards its ligands CCL19 and CCL21, while CRAM expression and potential interferences with CCR7 are yet to be characterized. RESULTS: In this study, we show that B cells from patients with B-CLL present highly variable degrees of CRAM expression in contrast to more consistently high levels of CCR7. We investigated the hypothesis that, similar to the atypical receptor DARC, CRAM can modulate chemokine availability and/or efficacy, resulting in the regulation of cellular activation. We found that a high level of CRAM expression was detrimental to efficient chemotaxis with CCL19. MAP-kinase phosphorylation and intracellular calcium release induced by CCL19 were also altered by CRAM expression. In addition, we demonstrate that CRAM-induced regulation of CCL19 signalling is maintained over time. CONCLUSIONS: We postulate that CRAM is a factor involved in the fine tuning/control of CCR7/CCL19 mediated responses. This regulation could be critical to the pivotal role of CCL19 induced formation of proliferation centres supporting the T/B cells encounter as well as disease progression in B-CLL.