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While the genomics era has allowed remarkable advances in understanding the mechanisms driving the biology and pathogenesis of numerous blood cancers, including acute lymphoblastic leukemia (ALL), metabolic studies are still lagging, especially regarding how the metabolism differs between healthy and diseased individuals. T-cell ALL (T-ALL) is an aggressive hematological neoplasm deriving from the malignant transformation of T-cell progenitors characterized by frequent NOTCH1 pathway activation. The aim of our study was to characterize tumor and plasma metabolomes during T-ALL development using a NOTCH1-induced murine T-ALL model (ΔE-NOTCH1). In tissue, we found a significant metabolic shift with leukemia development, as metabolites linked to glycolysis (lactic acid) and Tricarboxylic acid cycle replenishment (succinic and malic acids) were elevated in NOTCH1 tumors, while metabolites associated with lipid oxidation (e.g., carnitine) as well as purine and pyrimidine metabolism were elevated in normal thymic tissue. Glycine, serine, and threonine metabolism, glutathione metabolism, as well as valine, leucine, and isoleucine biosynthesis were enriched pathways in tumor tissue. Phenylalanine and tyrosine metabolism was highly enriched in plasma from leukemia-bearing mice compared to healthy mice. Further, we identified a metabolic signature consisting of glycine, alanine, proline, 3-hydroxybutyrate, and glutamic acid as potential biomarkers for leukemia progression in plasma. Hopefully, the metabolic differences detected in our leukemia model will apply to humans and contribute to the development of metabolism-oriented therapeutic approaches.
Assuntos
Biomarcadores Tumorais , Metabolômica , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Receptor Notch1 , Animais , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Camundongos , Receptor Notch1/metabolismo , Metabolômica/métodos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Metaboloma , Modelos Animais de DoençasRESUMO
Acute leukemias, classified as acute myeloid leukemia and acute lymphoblastic leukemia, represent the most prevalent hematologic tumors in adolescent and young adults. In recent years, new challenges have emerged in order to improve the clinical effectiveness of therapies already in use and reduce their side effects. In particular, in this scenario, metabolic reprogramming plays a key role in tumorigenesis and prognosis, and it contributes to the treatment outcome of acute leukemia. This review summarizes the latest findings regarding the most relevant metabolic pathways contributing to the continuous growth, redox homeostasis, and drug resistance of leukemia cells. We describe the main metabolic deregulations in acute leukemia and evidence vulnerabilities that could be exploited for targeted therapy.
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Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Resultado do Tratamento , Adulto JovemRESUMO
Numerous studies have shown that hedgehog inhibitors (iHHs) only partially block the growth of tumor cells, especially in vivo. Leukemia often expands in a nutrient-depleted environment (bone marrow and thymus). In order to identify putative signaling pathways implicated in the adaptive response to metabolically adverse conditions, we executed quantitative phospho-proteomics in T-cell acute lymphoblastic leukemia (T-ALL) cells subjected to nutrient-depleted conditions (serum starvation). We found important modulations of peptides phosphorylated by critical signaling pathways including casein kinase, mammalian target of rapamycin, and 5'AMP-activated kinase (AMPK). Surprisingly, in T-ALL cells, AMPK signaling was the most consistently downregulated pathway under serum-depleted conditions, and this coincided with increased GLI1 expression and sensitivity to iHHs, especially the GLI1/2 inhibitor GANT-61. Increased sensitivity to GANT-61 was also found following genetic inactivation of the catalytic subunit of AMPK (AMPKα1) or pharmacological inhibition of AMPK by Compound C. Additionally, patient-derived xenografts showing high GLI1 expression lacked activated AMPK, suggesting an important role for this signaling pathway in regulating GLI1 protein levels. Further, joint targeting of HH and AMPK signaling pathways in T-ALL cells by GANT-61 and Compound C significantly increased the therapeutic response. Our results suggest that metabolic adaptation that occurs under nutrient starvation in T-ALL cells increases responsiveness to HH pathway inhibitors through an AMPK-dependent mechanism and that joint therapeutic targeting of AMPK signaling and HH signaling could represent a valid therapeutic strategy in rapidly expanding tumors where nutrient availability becomes limiting.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Hedgehog/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Morte Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Jurkat , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
Loss-of-function mutations and deletions in Wilms tumor 1 (WT1) gene are present in approximately 10% of T-cell acute lymphoblastic leukemia. Clinically, WT1 mutations are enriched in relapsed series and are associated to inferior relapse-free survival in thymic T-cell acute lymphoblastic leukemia cases. Here we demonstrate that WT1 plays a critical role in the response to DNA damage in T-cell leukemia. WT1 loss conferred resistance to DNA damaging agents and attenuated the transcriptional activation of important apoptotic regulators downstream of TP53 in TP53-competent MOLT4 T-leukemia cells but not in TP53-mutant T-cell acute lymphoblastic leukemia cell lines. Notably, WT1 loss positively affected the expression of the X-linked inhibitor of apoptosis protein, XIAP, and genetic or chemical inhibition with embelin (a XIAP inhibitor) significantly restored sensitivity to γ-radiation in both T-cell acute lymphoblastic leukemia cell lines and patient-derived xenografts. These results reveal an important role for the WT1 tumor suppressor gene in the response to DNA damage, and support the view that anti-XIAP targeted therapies could have a role in the treatment of WT1-mutant T-cell leukemia.
Assuntos
Dano ao DNA/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteínas WT1/deficiência , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Raios gama , Xenoenxertos , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/fisiologia , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/radioterapia , Proteína Supressora de Tumor p53/fisiologia , Proteínas WT1/fisiologiaRESUMO
Liquid biopsy has recently emerged as an important tool in clinical practice particularly for lung cancer patients. We retrospectively evaluated cell-free DNA analyses performed at our Institution by next generation sequencing methodology detecting the major classes of genetic alterations. Starting from the graphical representation of chromosomal alterations provided by the analysis software, we developed a support vector machine classifier to automatically classify chromosomal profiles as stable (SCP) or unstable (UCP). High concordance was found between our binary classification and tumor fraction evaluation performed using shallow whole genome sequencing. Among clinical features, UCP patients were more likely to have ≥ 3 metastatic sites and liver metastases. Longitudinal assessment of chromosomal profiles in 33 patients with lung cancer receiving immune checkpoint inhibitors (ICIs) showed that only patients that experienced early death or hyperprogressive disease retained or acquired an UCP within 3 weeks from the beginning of ICIs. UCP was not observed following ICIs among patients that experienced progressive disease or clinical benefit. In conclusion, our binary classification, applied to whole copy number alteration profiles, could be useful for clinical risk stratification during systemic treatment for non-small cell lung cancer patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Variações do Número de Cópias de DNA , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Feminino , Biópsia Líquida/métodos , Idoso , Pessoa de Meia-Idade , Estudos Retrospectivos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inibidores de Checkpoint Imunológico/uso terapêutico , Idoso de 80 Anos ou mais , Máquina de Vetores de SuporteRESUMO
The molecular mechanisms involved in disease progression and relapse in T-cell acute lymphoblastic leukemia (T-ALL) are poorly understood. We used single nucleotide polymorphism array analysis to analyze paired diagnostic and relapsed T-ALL samples to identify recurrent genetic alterations in T-ALL. This analysis showed that diagnosis and relapsed cases have common genetic alterations, but also that relapsed samples frequently lose chromosomal markers present at diagnosis, suggesting that relapsed T-ALL emerges from an ancestral clone different from the major leukemic population at diagnosis. In addition, we identified deletions and associated mutations in the WT1 tumor suppressor gene in 2 of 9 samples. Subsequent analysis showed WT1 mutations in 28 of 211 (13.2%) of pediatric and 10 of 85 (11.7%) of adult T-ALL cases. WT1 mutations present in T-ALL are predominantly heterozygous frameshift mutations resulting in truncation of the C-terminal zinc finger domains of this transcription factor. WT1 mutations are most prominently found in T-ALL cases with aberrant rearrangements of the oncogenic TLX1, TLX3, and HOXA transcription factor oncogenes. Survival analysis demonstrated that WT1 mutations do not confer adverse prognosis in pediatric and adult T-ALL. Overall, these results identify the presence of WT1 mutations as a recurrent genetic alteration in T-ALL.
Assuntos
Genes do Tumor de Wilms , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Adulto , Criança , Aberrações Cromossômicas , Células Clonais/química , Metilação de DNA , Análise Mutacional de DNA , DNA de Neoplasias/genética , Progressão da Doença , Genes Homeobox , Humanos , Estimativa de Kaplan-Meier , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Oncogenes , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Prognóstico , Recidiva , Proteínas WT1/química , Proteínas WT1/genética , Dedos de Zinco/genéticaRESUMO
Genetic screening of somatic mutations in circulating free DNA (cfDNA) opens up new opportunities for personalized medicine. In this study, we aim to illustrate the implementation of NGS-based liquid biopsy in clinical practice for the detection of somatic alterations in selected genes. Our work is particularly relevant for the diagnosis and treatment of NSCLC. Beginning in 2020, we implemented the use of Roche's Avenio ctDNA expanded panel in our diagnostic routine. In this study, we retrospectively review NGS-based clinical genetic tests performed in our laboratory, focusing on key analytical parameters. Avenio ctDNA kits demonstrated 100% sensitivity in detecting single nucleotide variants (SNVs) at >0.5% variant allele frequency (VAF), and high consistency in reproducibility. Since 2020, we performed cfDNA genotyping test in 86 NSCLC patients, and we successfully sequenced 96.5% (83/86) of samples. We observed consistency in sequencing performance based upon sequencing depth and on-target rate. At least one gene variant was identified in 52 samples (63%), and one or more actionable variants were detected in 21 out of 83 (25%) of analysed patients. We demonstrated the feasibility of implementing an NGS-based liquid biopsy assay for routine genetic characterization of metastatic NSCLC patients.
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T-cell acute lymphoblastic leukemia (T-ALL) is a highly malignant pediatric leukemia, where few therapeutic options are available for patients which relapse. We find that therapeutic targeting of GLI transcription factors by GANT-61 is particularly effective against NOTCH1 unmutated T-ALL cells. Investigation of the functional role of GLI1 disclosed that it contributes to T-ALL cell proliferation, survival, and dissemination through the modulation of AKT and CXCR4 signaling pathways. Decreased CXCR4 signaling following GLI1 inactivation was found to be prevalently due to post-transcriptional mechanisms including altered serine 339 CXCR4 phosphorylation and cortactin levels. We also identify a novel cross-talk between GLI transcription factors and FOXC1. Indeed, GLI factors can activate the expression of FOXC1 which is able to stabilize GLI1/2 protein levels through attenuation of their ubiquitination. Further, we find that prolonged GLI1 deficiency has a double-edged role in T-ALL progression favoring disease dissemination through the activation of a putative AKT/FOXC1/GLI2 axis. These findings have clinical significance as T-ALL patients with extensive central nervous system dissemination show low GLI1 transcript levels. Further, T-ALL patients having a GLI2-based Hedgehog activation signature are associated with poor survival. Together, these findings support a rationale for targeting the FOXC1/AKT axis to prevent GLI-dependent oncogenic Hedgehog signaling.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Apoptose , Biópsia , Pontos de Checagem do Ciclo Celular , Biologia Computacional/métodos , Modelos Animais de Doenças , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Proteínas Hedgehog/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Prognóstico , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores CXCR4/metabolismo , Fatores de TranscriçãoRESUMO
Several studies strongly suggest that DC differentiated in vitro in the presence of type I IFN acquire more potent immune stimulatory properties, compared with DC differentiated in vitro with IL-4. However, little is known about the molecular mechanisms underlying this phenomenon. To address this question, we compared the Ag-processing machinery (APM) profile in human DC grown in the presence of IFN-alpha ((IFN)DC) or IL-4 ((IL-4)DC). Using a panel of APM component-specific mAb in Western blot experiments, we found that (IFN)DC preferentially express inducible proteasome subunits (LMP2, LMP7, and MECL1) both at immature and mature stages. In contrast, immature (IL-4)DC co-express both constitutive (beta1, beta2, and beta5) and inducible subunits, as shown by Western blotting analysis. In addition, immature (IFN)DC express higher levels of TAP1, TAP2, calnexin, calreticulin, tapasin, and HLA class I molecules than (IL-4)DC. The different proteasome profiles of (IFN)DC and (IL-4)DC were associated with a greater ability of (IFN)DC to present an immunodominant epitope that requires LMP7 expression for its processing. In general, these data show the impact of cytokines on APM component expression and hence the Ag-processing ability of DC.
Assuntos
Diferenciação Celular/imunologia , Monócitos/enzimologia , Complexos Multienzimáticos/biossíntese , Complexo de Endopeptidases do Proteassoma/biossíntese , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Epitopos/imunologia , Humanos , Fatores Imunológicos/farmacologia , Interferon-alfa/farmacologia , Interleucina-4/farmacologia , Proteínas Sensoras de Cálcio Intracelular/efeitos dos fármacos , Proteínas Sensoras de Cálcio Intracelular/imunologia , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Membrana Transportadoras/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Monócitos/imunologia , Complexos Multienzimáticos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacosRESUMO
Heterodimerization domain (HD) mutations in NOTCH1 induce ligand-independent activation of the receptor and contribute to the pathogenesis of one-third of human T-cell lymphoblastic leukemias (T-ALLs). Here we report a novel class of activating mutations in NOTCH1 leading to aberrant activation of NOTCH1 signaling in T-cell lymphoblasts. These so-called juxtamembrane expansion (JME) alleles consist of internal duplication insertions in the vicinity of exon 28 of the NOTCH1 gene encoding the extracellular juxtamembrane region of the receptor. Notably, structure-function analysis of leukemia-derived and synthetic JME mutants demonstrated that the aberrant activation of NOTCH1 signaling is dependent on the number of residues introduced in the extracellular juxtamembrane region of the receptor and not on the specific amino acid sequence of these insertions. JME NOTCH1 mutants are effectively blocked by gamma-secretase inhibitors and require an intact metalloprotease cleavage site for activation. Overall, these results show a novel mechanism of NOTCH1 activation in T-ALL and provide further insight on the mechanisms that control the activation of NOTCH1 signaling.
Assuntos
Leucemia-Linfoma de Células T do Adulto/genética , Mutação , Receptor Notch1/genética , Sequências de Repetição em Tandem , Secretases da Proteína Precursora do Amiloide/fisiologia , Sequência de Bases , Linhagem Celular , Membrana Celular , Análise Mutacional de DNA , Espaço Extracelular , Humanos , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/etiologia , Receptor Notch1/metabolismo , TransfecçãoRESUMO
Notwithstanding intensified therapy, a considerable fraction of T-cell acute lymphoblastic leukemia (T-ALL) patients face a dismal prognosis due to primary resistance to treatment and relapse, raising the need for more efficient and targeted therapies. Hedgehog (HH) signaling is a major developmental pathway frequently deregulated in cancer, for which a role in T-ALL is emerging. Mounting evidence suggests that ligand-independent activation of HH pathway occurs in cancer including T-ALL, emphasizing the necessity of dissecting the complex interplay between HH and other signaling pathways regulating activation. In this work, we present a therapeutically relevant crosstalk between HH signaling and the glucocorticoid receptor (NR3C1) pathway acting at the level of GLI1 transcription factor. GLI inhibitor GANT61 and dexamethasone were shown to exert a synergistic anti-leukemic effect in vitro in T-ALL cell lines and patient-derived xenografts. Mechanistically, dexamethasone-activated NR3C1 impaired GLI1 function by dynamically modulating the recruitment of PCAF acetyltransferase and HDAC1 deacetylase. Increased GLI1 acetylation was associated with compromised transcriptional activity and reduced protein stability. In summary, our study identifies a novel crosstalk between GLI1 and NR3C1 signaling pathway which could be exploited in HH-dependent malignancies to increase therapeutic efficacy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Hedgehog/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acetilação , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Sinergismo Farmacológico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/antagonistas & inibidores , Histona Desacetilase 1/metabolismo , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Estabilidade Proteica/efeitos dos fármacos , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Receptores de Glucocorticoides/agonistas , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Proteína GLI1 em Dedos de Zinco/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive disease arising from T-cell precursors. NOTCH1 plays an important role both in T-cell development and leukemia progression, and more than 60% of human T-ALLs harbor mutations in components of the NOTCH1 signaling pathway, leading to deregulated cell growth and contributing to cell transformation. Besides multiple NOTCH1 target genes, microRNAs have also been shown to regulate T-ALL initiation and progression. Using an established mouse model of T-ALL induced by NOTCH1 activation, we identified several microRNAs downstream of NOTCH1 activation. In particular, we found that NOTCH1 inhibition can induce miR-22-3p in NOTCH1-dependent tumors and that this regulation is also conserved in human samples. Importantly, miR-22-3p overexpression in T-ALL cells can inhibit colony formation in vitro and leukemia progression in vivo. In addition, miR-22-3p was found to be downregulated in T-ALL specimens, both T-ALL cell lines and primary samples, relative to immature T-cells. Our results suggest that miR-22-3p is a functionally relevant microRNA in T-ALL whose modulation can be exploited for therapeutic purposes to inhibit T-ALL progression.
Assuntos
Progressão da Doença , MicroRNAs/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/metabolismo , Regulação para Cima/genéticaRESUMO
Multi-agent combination chemotherapy can be curative in acute lymphoblastic leukemia (ALL). Still, patients with primary refractory disease or with relapsed leukemia have a very poor prognosis. Here we integrate an in-depth dissection of the mutational landscape across diagnostic and relapsed pediatric and adult ALL samples with genome-wide CRISPR screen analysis of gene-drug interactions across seven ALL chemotherapy drugs. By combining these analyses, we uncover diagnostic and relapse-specific mutational mechanisms as well as genetic drivers of chemoresistance. Functionally, our data identifies common and drug-specific pathways modulating chemotherapy response and underscores the effect of drug combinations in restricting the selection of resistance-driving genetic lesions. In addition, by identifying actionable targets for the reversal of chemotherapy resistance, these analyses open novel therapeutic opportunities for the treatment of relapse and refractory disease.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto , Criança , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Prognóstico , RecidivaRESUMO
The chemokine receptor CXCR4 plays a central role in organ-specific homing and tumor spreading and is induced by hypoxia. B lymphocytes are exposed to low oxygen tensions during their development, but the influence of hypoxia on their physiology is poorly understood. Here, we show that hypoxia is associated with up-regulation of CXCR4 expression in human normal and malignant B cells, through both transcriptional and posttranslational mechanisms. However, a dichotomic functional response to CXCR4 triggering was observed: both peripheral B cells and lymphomas arising from mature B cells displayed increased responses to CXCR4 triggering under hypoxia, whereas germinal center (GC) B cells as well as GC-derived lymphomas showed CXCR4 receptor desensitization. This phenomenon was associated with differential modulation of key signal-transducing molecules, including mitogen-activated protein kinase phosphatase-1 and regulator of G protein signaling molecule-1. The unresponsiveness of GC-derived lymphomatous B cells to CXCR4 triggering under hypoxia may have implications for the development and pathogenesis of GC-derived lymphoid tumors.
Assuntos
Linfócitos B/metabolismo , Linfócitos B/fisiologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Receptores CXCR4/biossíntese , Animais , Linfócitos B/patologia , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Fosfatase 1 de Especificidade Dupla/biossíntese , Fosfatase 1 de Especificidade Dupla/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linfoma de Células B/genética , Camundongos , Camundongos SCID , Proteínas RGS/biossíntese , Proteínas RGS/genética , RNA Interferente Pequeno/genética , Receptores CXCR4/genética , Transcrição Gênica , Regulação para CimaRESUMO
The NOTCH signaling pathway is a conserved signaling cascade that regulates many aspects of development and homeostasis in multiple organ systems. Aberrant activity of this signaling pathway is linked to the initiation and progression of several hematological malignancies, exemplified by T-cell acute lymphoblastic leukemia (T-ALL). Interestingly, frequent non-mutational activation of NOTCH1 signaling has recently been demonstrated in B-cell chronic lymphocytic leukemia (B-CLL), significantly extending the pathogenic significance of this pathway in B-CLL. Leukemia patients often present with high-blood cell counts, diffuse disease with infiltration of the bone marrow, secondary lymphoid organs, and diffusion to the central nervous system (CNS). Chemokines are chemotactic cytokines that regulate migration of cells between tissues and the positioning and interactions of cells within tissue. Homeostatic chemokines and their receptors have been implicated in regulating organ-specific infiltration, but may also directly and indirectly modulate tumor growth. Recently, oncogenic NOTCH1 has been shown to regulate infiltration of leukemic cells into the CNS hijacking the CC-chemokine ligand 19/CC-chemokine receptor 7 chemokine axis. In addition, a crucial role for the homing receptor axis CXC-chemokine ligand 12/CXC-chemokine receptor 4 has been demonstrated in leukemia maintenance and progression. Moreover, the CCL25/CCR9 axis has been implicated in the homing of leukemic cells into the gut, particularly in the presence of phosphatase and tensin homolog tumor suppressor loss. In this review, we summarize the latest developments regarding the role of NOTCH signaling in regulating the chemotactic microenvironmental cues involved in the generation and progression of T-ALL and compare these findings to B-CLL.
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Sistema Nervoso Central/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Receptor Notch1/metabolismo , Animais , Carcinogênese , Movimento Celular , Quimiocinas/metabolismo , Quimiotaxia , Humanos , Transdução de Sinais , Microambiente TumoralRESUMO
MYC-translocated T-lineage acute lymphoblastic leukemia (T-ALL) is a rare subgroup of T-ALL associated with CDKN2A/B deletions, PTEN inactivation, and absence of NOTCH1 or FBXW7 mutations. This subtype of T-ALL has been associated with induction failure and aggressive disease. Identification of drug targets and mechanistic insights for this disease are still limited. Here, we established a human NOTCH1-independent MYC-translocated T-ALL cell line that maintains the genetic and phenotypic characteristics of the parental leukemic clone at diagnosis. The University of Padua T-cell acute lymphoblastic leukemia 13 (UP-ALL13) cell line has all the main features of the above described MYC-translocated T-ALL. Interestingly, UP-ALL13 was found to harbor a heterozygous R882H DNMT3A mutation typically found in myeloid leukemia. Chromatin immunoprecipitation coupled with high-throughput sequencing for histone H3 lysine 27 (H3K27) acetylation revealed numerous putative super-enhancers near key transcription factors, including MYC, MYB, and LEF1. Marked cytotoxicity was found following bromodomain-containing protein 4 (BRD4) inhibition with AZD5153, suggesting a strict dependency of this particular subtype of T-ALL on the activity of super-enhancers. Altogether, this cell line may be a useful model system for dissecting the signaling pathways implicated in NOTCH1-independent T-ALL and for the screening of targeted anti-leukemia agents specific for this T-ALL subgroup.
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Local gene therapy could be a therapeutic option for ovarian carcinoma, a life-threatening malignancy, because of disease containment within the peritoneal cavity in most patients. Lentiviral vectors, which are potentially capable of stable transgene expression, may be useful to vehicle therapeutic molecules requiring long-term production in these tumors. To investigate this concept, we used lentiviral vectors to deliver the enhanced green fluorescent protein (EGFP) gene to ovarian cancer cells. Their efficiency of gene transfer was compared with that of a retroviral vector carrying the same envelope. In vitro, both vectors infected ovarian cancer cells with comparable efficiency under standard culture conditions; however, the lentiviral vector was much more efficient in transducing growth-arrested cells when compared with the retroviral vector. Gene transfer was fully neutralized by an anti-VSV-G antibody, and in vitro stability was similar. In vivo, the lentiviral vector delivered the transgene 10-fold more efficiently to ovarian cancer cells growing i.p. in SCID mice, as evaluated by real-time PCR analysis of the tumors. Confocal microscopy analysis of tumor sections showed a dramatic difference at the level of transgene expression, because abundant EGFP(+) cells were detected only in mice receiving the lentiviral vector. Quantitative analysis by flow cytometry confirmed this and indicated 0.05 and 5.6% EGFP(+) tumor cells after administration of the retroviral and lentiviral vector, respectively. Injection of ex vivo transduced tumor cells, sorted for EGFP expression, indicated that the lentiviral vector was considerably more resistant to in vivo silencing in comparison with the retroviral vector. Finally, multiple administrations of a murine IFN-alpha(1)-lentiviral vector to ovarian carcinoma-bearing mice significantly prolonged the animals' survival, indicating the therapeutic efficacy of this approach. These findings indicate that lentiviral vectors deserve attention in the design of future gene therapy approaches to ovarian cancer aimed at achieving long-term expression of therapeutic genes.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Lentivirus/genética , Neoplasias Ovarianas/terapia , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Vetores Genéticos/farmacocinética , Proteínas de Fluorescência Verde , Humanos , Interferon-alfa/biossíntese , Interferon-alfa/genética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/virologia , Distribuição Tecidual , Transcrição Gênica , Transgenes , Células Tumorais CultivadasRESUMO
The TS/A mouse mammary adenocarcinoma is a poorly immunogenic tumor widely used in preclinical models of cancer immunotherapy. CTLs have often been indicated as important in TS/A tumor destruction, but their generation in this model has been rarely studied, nor have their precise target(s) been identified. We hypothesized that the gp70 Env product of an endogenous murine leukemia virus could be a target antigen for TS/A-specific CTLs and investigated this possibility in four different TS/A cell lines engineered with the genes that encode IFN-alpha, IFN-gamma, interleukin-4, and B7.1, respectively. All tumor cell lines expressed gp70, albeit at different levels, as demonstrated by reverse transcription-PCR analysis. Transfected tumor cells exhibited a delayed growth in vivo, and partial tumor regression. Spleen cells from mice that displayed tumor regression had high percentages of CD8(+) T cells that were specifically stained with L(d) tetramers loaded with gp70(423-431), the antigenic epitope of gp70 protein. Mixed leukocyte-peptide and mixed leukocyte-tumor cultures, set up by stimulating splenocytes with the immunogenic peptide and with transfected TS/A tumor cells, respectively, resulted in similar large increases in tetramer-reactive CD8(+) T cells and showed high lytic activity specific for gp70(423-431). Finally, in a Cold Target Inhibition assay, lytic activity of a mixed leukocyte-tumor culture was inhibited in an overlapping fashion by both the TS/A line used for restimulation and 293L(d) cells loaded with gp70(423-431) peptide, but not by 293L(d) cells pulsed with an irrelevant H-2 L(d) epitope, thus demonstrating that all or most of the cytotoxic activity was directed exclusively against this antigenic epitope.
Assuntos
Adenocarcinoma/imunologia , Epitopos Imunodominantes/imunologia , Neoplasias Mamárias Experimentais/imunologia , Proteínas Oncogênicas de Retroviridae/imunologia , Retroviridae/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Antígenos de Neoplasias/imunologia , Antígeno B7-1/biossíntese , Feminino , Interferon-alfa/biossíntese , Interferon gama/biossíntese , Interleucina-4/biossíntese , Ativação Linfocitária/imunologia , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Oncogênicas de Retroviridae/biossíntese , Proteínas Oncogênicas de Retroviridae/genética , Transfecção , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genéticaRESUMO
Calcineurin (Cn) is a calcium activated protein phosphatase involved in many aspects of normal T cell physiology, however the role of Cn and/or its downstream targets in leukemogenesis are still ill-defined. In order to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-cell acute lymphoblastic leukemia (T-ALL) we used tandem affinity chromatography, followed by mass spectrometry to purify novel Cn-interacting partners. We found the Cn-interacting proteins to be part of numerous cellular signaling pathways including eIF2 signaling and mTOR signaling. Coherently, modulation of Cn activity in T-ALL cells determined alterations in the phosphorylation status of key molecules implicated in protein translation such as eIF-2α and ribosomal protein S6. Joint targeting of PI3K-mTOR, eIF-2α and 14-3-3 signaling pathways with Cn unveiled novel synergistic pro-apoptotic drug combinations. Further analysis disclosed that the synergistic interaction between PI3K-mTOR and Cn inhibitors was prevalently due to AKT inhibition. Finally, we showed that the synergistic pro-apoptotic response determined by jointly targeting AKT and Cn pathways was linked to down-modulation of key anti-apoptotic proteins including Mcl-1, Claspin and XIAP. In conclusion, we identify AKT inhibition as a novel promising drug combination to potentiate the pro-apoptotic effects of Cn inhibitors.
Assuntos
Calcineurina/metabolismo , Fatores de Transcrição NFATC/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Transdução de Sinais/fisiologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Calcineurina/farmacologia , Linhagem Celular Tumoral , Ciclosporina/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ovarian cancer represents a suitable disease for gene therapy because of the containment of neoplastic cells in the peritoneal cavity even at advanced tumor stages. The aim of this study was to investigate whether intraperitoneal administration of a lentiviral vector encoding murine interferon-alpha (LV-IFN) could have therapeutic activity in a transplantable ovarian cancer model. Multiple injections of low amounts of LV-IFN into severe combined immunodeficiency (SCID) mice bearing IGROV-1 or OC316 ovarian cancer cells elicited remarkable antitumor activity, leading to prolongation of survival in the majority of animals. A definitive cure was obtained in animals bearing PD-OVA#1 tumors, generated by injecting tumor cells isolated from the ascitic fluid of a patient into SCID mice. Interferon-alpha levels were detected in the peritoneal fluids but not in the serum of treated mice, indicating that production of the cytokine is mainly local, by both tumor and normal cells of the host. Antitumor effects were associated with a remarkable decrease in the formation of hemorrhagic ascites, an increase in ischemic tumor necrosis, and a reduction in microvessel density. In conclusion, our findings show that intracavitary IFN-alpha gene therapy, using a lentiviral vector, provides strong antitumor effects in murine models of ovarian cancer and reinforces the evidence that angiogenesis inhibition is a promising strategy for the treatment of localized tumors.