RESUMO
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
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
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
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
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
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
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
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
Overcoming drug resistance remains a key challenge to cure patients with acute and chronic B cell malignancies. Here, we describe a stromal cell-autonomous signaling pathway, which contributes to drug resistance of malignant B cells. We show that protein kinase C (PKC)-ß-dependent signals from bone marrow-derived stromal cells markedly decrease the efficacy of cytotoxic therapies. Conversely, small-molecule PKC-ß inhibitors antagonize prosurvival signals from stromal cells and sensitize tumor cells to targeted and nontargeted chemotherapy, resulting in enhanced cytotoxicity and prolonged survival in vivo. Mechanistically, stromal PKC-ß controls the expression of adhesion and matrix proteins, required for activation of phosphoinositide 3-kinases (PI3Ks) and the extracellular signal-regulated kinase (ERK)-mediated stabilization of B cell lymphoma-extra large (BCL-XL) in tumor cells. Central to the stroma-mediated drug resistance is the PKC-ß-dependent activation of transcription factor EB, regulating lysosome biogenesis and plasma membrane integrity. Stroma-directed therapies, enabled by direct inhibition of PKC-ß, enhance the effectiveness of many antileukemic therapies.
Assuntos
Proteína Quinase C beta/metabolismo , Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Tumorais CultivadasRESUMO
Hematological malignancies (HM) are a collection of malignant transformations originating from cells in the primary or secondary lymphoid organs. Leukemia, lymphoma, and multiple myeloma comprise the three major types of HM. Current treatment consists of bone marrow transplantation, radiotherapy, immunotherapy and chemotherapy. Although, many chemotherapeutic drugs are clinically available for the treatment of HM, the use of these agents is limited due to dose-related toxicity and lack of specificity to tumor tissue. Moreover, the poor pharmacokinetic profile of most of the chemotherapeutics requires high dosage and frequent administration to maintain therapeutic levels at the target site, both increasing adverse effects. This underlines an urgent need for a suitable drug delivery system to improve efficacy, safety, and pharmacokinetic properties of conventional therapeutics. Nanomedicines have proven to enhance these properties for anticancer therapeutics. The most extensively studied nanomedicine systems are lipid-based nanoparticles and polymeric nanoparticles. Typically, nanomedicines are small sub-micron sized particles in the size range of 20-200 nm. The biocompatible and biodegradable nature of nanomedicines makes them attractive vehicles to improve drug delivery. Their small size allows them to extravasate and accumulate at malignant sites passively by means of the enhanced permeability and retention (EPR) effect, resulting from rapid angiogenesis and inflammation. Moreover, the specificity to the target tissue can be further enhanced by surface modification of nanoparticles. This review describes currently available therapies as well as limitations and potential advantages of nanomedicine formulations for treatment of various types of HM. Additionally, recent investigational and approved nanomedicine formulations and their limited applications in HM are discussed.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Neoplasias Hematológicas/terapia , Animais , Antineoplásicos/uso terapêutico , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/radioterapia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Imunoterapia/métodos , Lipossomos/química , Micelas , Nanomedicina/métodos , Microambiente Tumoral/efeitos dos fármacosRESUMO
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
The survival and proliferation of CLL cells depends on microenvironmental contacts in lymphoid organs. CD38 is a cell surface receptor that plays an important role in survival and proliferation signaling in CLL. In this study we demonstrate SYK's direct involvement in the CD38 signaling pathway in primary CLL samples. CD38 stimulation of CLL cells revealed SYK activation. SYK downstream target AKT was subsequently induced and MCL-1 expression was increased. Concomitant inhibition of SYK by the SYK inhibitor R406 resulted in reduced activation of AKT and prevented upregulation of MCL-1. Moreover, short-term CD38 stimulation enhanced BCR-signaling, as indicated by increased ERK phosphorylation. CXCL12-dependent migration was increased after CD38 stimulation. Treating CLL cells with R406 inhibited CD38-mediated migration. In addition, we observed marked downregulation of CD38 expression for CLL cells treated with R406 compared to vehicle control. Finally, we observed a clear correlation between CD38 expression on CLL cells and SYK-inhibitor efficacy. In conclusion, our study provides deeper mechanistic insight into the effect of SYK inhibition in CLL.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Glicoproteínas de Membrana/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , ADP-Ribosil Ciclase 1/biossíntese , ADP-Ribosil Ciclase 1/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular , Quimiocina CXCL12/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/farmacologia , Pessoa de Meia-Idade , Oxazinas/farmacologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Células Tumorais CultivadasRESUMO
Phosphatase and tensin homolog (PTEN) is a negative regulator of the phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) signaling pathway and a potent tumor suppressor in many types of cancer. To test a tumor suppressive role for PTEN in pre-B acute lymphoblastic leukemia (ALL), we induced Cre-mediated deletion of Pten in mouse models of pre-B ALL. In contrast to its role as a tumor suppressor in other cancers, loss of one or both alleles of Pten caused rapid cell death of pre-B ALL cells and was sufficient to clear transplant recipient mice of leukemia. Small-molecule inhibition of PTEN in human pre-B ALL cells resulted in hyperactivation of AKT, activation of the p53 tumor suppressor cell cycle checkpoint and cell death. Loss of PTEN function in pre-B ALL cells was functionally equivalent to acute activation of autoreactive pre-B cell receptor signaling, which engaged a deletional checkpoint for the removal of autoreactive B cells. We propose that targeted inhibition of PTEN and hyperactivation of AKT triggers a checkpoint for the elimination of autoreactive B cells and represents a new strategy to overcome drug resistance in human ALL.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , PTEN Fosfo-Hidrolase/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Proto-Oncogênicas c-akt/genética , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Células Precursoras de Linfócitos B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Studying mechanisms of malignant transformation of human pre-B cells, we found that acute activation of oncogenes induced immediate cell death in the vast majority of cells. Few surviving pre-B cell clones had acquired permissiveness to oncogenic signaling by strong activation of negative feedback regulation of Erk signaling. Studying negative feedback regulation of Erk in genetic experiments at three different levels, we found that Spry2, Dusp6, and Etv5 were essential for oncogenic transformation in mouse models for pre-B acute lymphoblastic leukemia (ALL). Interestingly, a small molecule inhibitor of DUSP6 selectively induced cell death in patient-derived pre-B ALL cells and overcame conventional mechanisms of drug-resistance.
Assuntos
Transformação Celular Neoplásica/genética , Sistema de Sinalização das MAP Quinases , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Animais , Antineoplásicos/farmacologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Fator C1 de Célula Hospedeira , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Prognóstico , Proteínas Serina-Treonina Quinases , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Despite recent advances in the cure rate of acute lymphoblastic leukaemia (ALL), the prognosis for patients with relapsed ALL remains poor. Here we identify FOXM1 as a candidate responsible for an aggressive clinical course. We show that FOXM1 levels peak at the pre-B-cell receptor checkpoint but are dispensable for normal B-cell development. Compared with normal B-cell populations, FOXM1 levels are 2- to 60-fold higher in ALL cells and are predictive of poor outcome in ALL patients. FOXM1 is negatively regulated by FOXO3A, supports cell survival, drug resistance, colony formation and proliferation in vitro, and promotes leukemogenesis in vivo. Two complementary approaches of pharmacological FOXM1 inhibition-(i) FOXM1 transcriptional inactivation using the thiazole antibiotic thiostrepton and (ii) an FOXM1 inhibiting ARF-derived peptide-recapitulate the findings of genetic FOXM1 deletion. Taken together, our data identify FOXM1 as a novel therapeutic target, and demonstrate feasibility of FOXM1 inhibition in ALL.
Assuntos
Antineoplásicos/farmacologia , Fatores de Transcrição Forkhead/antagonistas & inibidores , Regulação Leucêmica da Expressão Gênica , Peptídeos/síntese química , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Tioestreptona/farmacologia , Adulto , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Criança , Ensaios Clínicos como Assunto , Inibidor p16 de Quinase Dependente de Ciclina/química , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Forkhead Box M1 , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Camundongos , Peptídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
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
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.