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1.
Haematologica ; 105(5): 1317-1328, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31467126

RESUMO

Despite substantial progress in treatment of T-cell acute lymphoblastic leukemia (T-ALL), mortality remains relatively high, mainly due to primary or acquired resistance to chemotherapy. Further improvements in survival demand better understanding of T-ALL biology and development of new therapeutic strategies. The Notch pathway has been involved in the pathogenesis of this disease and various therapeutic strategies are currently under development, including selective targeting of NOTCH receptors by inhibitory antibodies. We previously demonstrated that the NOTCH1-specific neutralizing antibody OMP52M51 prolongs survival in TALL patient-derived xenografts bearing NOTCH1/FBW7 mutations. However, acquired resistance to OMP52M51 eventually developed and we used patient-derived xenografts models to investigate this phenomenon. Multi-level molecular characterization of T-ALL cells resistant to NOTCH1 blockade and serial transplantation experiments uncovered heterogeneous types of resistance, not previously reported with other Notch inhibitors. In one model, resistance appeared after 156 days of treatment, it was stable and associated with loss of Notch inhibition, reduced mutational load and acquired NOTCH1 mutations potentially affecting the stability of the heterodimerization domain. Conversely, in another model resistance developed after only 43 days of treatment despite persistent down-regulation of Notch signaling and it was accompanied by modulation of lipid metabolism and reduced surface expression of NOTCH1. Our findings shed light on heterogeneous mechanisms adopted by the tumor to evade NOTCH1 blockade and support clinical implementation of antibody-based target therapy for Notch-addicted tumors.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animais , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch1/genética , Transdução de Sinais , Linfócitos T , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Br J Cancer ; 118(7): 985-994, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29515258

RESUMO

BACKGROUND: Despite chemotherapy intensification, a subgroup of high-risk paediatric T-cell acute lymphoblastic leukemia (T-ALL) patients still experience treatment failure. In this context, we hypothesised that therapy resistance in T-ALL might involve aldo-keto reductase 1C (AKR1C) enzymes as previously reported for solid tumors. METHODS: Expression of NRF2-AKR1C signaling components has been analysed in paediatric T-ALL samples endowed with different treatment outcomes as well as in patient-derived xenografts of T-ALL. The effects of AKR1C enzyme modulation has been investigated in T-ALL cell lines and primary cultures by combining AKR1C inhibition, overexpression, and gene silencing approaches. RESULTS: We show that T-ALL cells overexpress AKR1C1-3 enzymes in therapy-resistant patients. We report that AKR1C1-3 enzymes play a role in the response to vincristine (VCR) treatment, also ex vivo in patient-derived xenografts. Moreover, we demonstrate that the modulation of AKR1C1-3 levels is sufficient to sensitise T-ALL cells to VCR. Finally, we show that T-ALL chemotherapeutics induce overactivation of AKR1C enzymes independent of therapy resistance, thus establishing a potential resistance loop during T-ALL combination treatment. CONCLUSIONS: Here, we demonstrate that expression and activity of AKR1C enzymes correlate with response to chemotherapeutics in T-ALL, posing AKR1C1-3 as potential targets for combination treatments during T-ALL therapy.


Assuntos
Aldo-Ceto Redutases/fisiologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , 20-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 20-Hidroxiesteroide Desidrogenases/fisiologia , Idade de Início , Membro C3 da Família 1 de alfa-Ceto Redutase/antagonistas & inibidores , Membro C3 da Família 1 de alfa-Ceto Redutase/fisiologia , Aldo-Ceto Redutases/antagonistas & inibidores , Animais , Criança , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Hidroxiesteroide Desidrogenases/fisiologia , Isoenzimas/fisiologia , Acetato de Medroxiprogesterona/administração & dosagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Oxirredutases/antagonistas & inibidores , Oxirredutases/fisiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/epidemiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Células Tumorais Cultivadas , Vincristina/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
3.
BMC Genomics ; 17: 228, 2016 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-26969675

RESUMO

BACKGROUND: Inference of gene regulation from expression data may help to unravel regulatory mechanisms involved in complex diseases or in the action of specific drugs. A challenging task for many researchers working in the field of systems biology is to build up an experiment with a limited budget and produce a dataset suitable to reconstruct putative regulatory modules worth of biological validation. RESULTS: Here, we focus on small-scale gene expression screens and we introduce a novel experimental set-up and a customized method of analysis to make inference on regulatory modules starting from genetic perturbation data, e.g. knockdown and overexpression data. To illustrate the utility of our strategy, it was applied to produce and analyze a dataset of quantitative real-time RT-PCR data, in which interferon-α (IFN-α) transcriptional response in endothelial cells is investigated by RNA silencing of two candidate IFN-α modulators, STAT1 and IFIH1. A putative regulatory module was reconstructed by our method, revealing an intriguing feed-forward loop, in which STAT1 regulates IFIH1 and they both negatively regulate IFNAR1. STAT1 regulation on IFNAR1 was object of experimental validation at the protein level. CONCLUSIONS: Detailed description of the experimental set-up and of the analysis procedure is reported, with the intent to be of inspiration for other scientists who want to realize similar experiments to reconstruct gene regulatory modules starting from perturbations of possible regulators. Application of our approach to the study of IFN-α transcriptional response modulators in endothelial cells has led to many interesting novel findings and new biological hypotheses worth of validation.


Assuntos
Redes Reguladoras de Genes , Interferon-alfa/genética , Interferência de RNA , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Helicase IFIH1 Induzida por Interferon , Modelos Genéticos , Receptor de Interferon alfa e beta/genética , Fator de Transcrição STAT1/genética
4.
Carcinogenesis ; 36(1): 115-21, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25355291

RESUMO

Activation of the NOTCH pathway occurs commonly in T acute lymphoblastic leukemia (T-ALL) mainly due to mutations in NOTCH1 or alterations in FBW7 and is involved in the regulation of cell proliferation and survival. Since mutations hit different domains of the receptor, they are predicted to heterogeneously perturb ligand-induced NOTCH1 activity. Moreover, T-ALL cells also co-express NOTCH3 receptors which could be triggered by different ligands. In this study, we aimed to investigate the role of DLL4 in the regulation of NOTCH signaling in T-ALL cells in the context of different types of NOTCH1 mutation or wild-type NOTCH receptor, as well as the effects of DLL4 neutralization on T-ALL engraftment in mice. We found that NOTCH signaling can be stimulated in T-ALL cells in vitro by either human or murine DLL4 with heterogeneous effects, according to NOTCH1/FBW7 mutation status, and that these effects can be blocked by antibodies neutralizing DLL4, NOTCH1 or NOTCH2/3. In vivo, DLL4 is expressed in the spleen and the bone marrow (BM) of NOD/SCID mice bearing T-ALL xenografts as well as the BM of T-ALL patients. Importantly, DLL4 blockade impaired growth of T-ALL cells in NOD/SCID mice and increased leukemia cell apoptosis. These results show that DLL4 is an important component of the tumor microenvironment which contributes to the early steps of T-ALL cell growth.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptores Notch/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Western Blotting , Proteínas de Ligação ao Cálcio , Proliferação de Células , Citometria de Fluxo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Notch/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Exp Hematol Oncol ; 12(1): 76, 2023 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-37667380

RESUMO

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic tumor, characterized by several genetic alterations, that constitutes 15% of pediatric and 25% of adult ALL. While with current therapeutic protocols children and adults' overall survival (OS) rates reach 85-90% and 40-50%, respectively, the outcome for both pediatric and adult T-ALL patients that relapse or are refractory to induction therapy, remains extremely poor, achieving around 25% OS for both patient groups. About 60% of T-ALL patients show increased NOTCH1 activity, due to activating NOTCH1 mutations or alterations in its ubiquitin ligase FBXW7. NOTCH signaling has been shown to contribute to chemotherapy resistance in some tumor models. Hence, targeting the NOTCH1 signaling pathway may be an effective option to overcome relapsed and refractory T-ALL.Here, we focused on the therapeutic activity of the NOTCH1-specific monoclonal antibody OMP-52M51 in combination either with drugs used during the induction, consolidation, or maintenance phase in mice xenografts established from pediatric and adult relapsed NOTCH1 mutated T-ALL samples. Interestingly, from RNAseq data we observed that anti-NOTCH1 treatment in vivo affects the purine metabolic pathway. In agreement, both in vitro and in vivo, the greatest effect on leukemia growth reduction was achieved by anti-NOTCH1 therapy in combination with antimetabolite drugs. This result was further corroborated by the longer life span of mice treated with the anti-NOTCH1 in combination with antimetabolites, indicating a novel Notch-targeted therapeutic approach that could ameliorate pediatric and adult T-ALL patients outcome with relapse disease for whom so far, no other therapeutic options are available.

6.
Front Oncol ; 8: 195, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29915721

RESUMO

The liver kinase B1 (LKB1) gene is a tumor suppressor with an established role in the control of cell metabolism and oxidative stress. However, whether dis-regulated oxidative stress promotes growth of LKB1-deficient tumors remains substantially unknown. Through in vitro studies, we observed that loss of LKB1 perturbed expression of several genes involved in reactive oxygen species (ROS) homeostasis. In particular, this analysis evidenced strongly up-modulated NADPH oxidase 1 (NOX1) transcript levels in tumor cells lacking LKB1. NOX1 accounted in part for enhanced cytotoxic effects of H2O2-induced oxidative stress in A549 LKB1-deficient tumor cells. Notably, genetic and pharmacologic inhibition of NOX1 activity reduced angiogenesis and growth of A549 tumors in mice. These results suggest that NOX1 inhibitors could counteract ROS production and the angiogenic switch in LKB1-deficient tumors.

7.
Oncogene ; 37(28): 3839-3851, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29643474

RESUMO

Several studies have revealed that endosomal sorting controls the steady-state levels of Notch at the cell surface in normal cells and prevents its inappropriate activation in the absence of ligands. However, whether this highly dynamic physiologic process can be exploited to counteract dysregulated Notch signaling in cancer cells remains unknown. T-ALL is a malignancy characterized by aberrant Notch signaling, sustained by activating mutations in Notch1 as well as overexpression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Here we show that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pTα, CR2, and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of dynein with ciliobrevin D. Pharmacologic studies with HDAC1, 6, and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL cells. Finally, HDAC6 silencing impaired leukemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. These results connect HDAC6 activity to regulation of total and surface Notch3 levels and suggest HDAC6 as a potential novel therapeutic target to lower Notch signaling in T-ALL and other Notch3-addicted tumors.


Assuntos
Desacetilase 6 de Histona/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Transporte Proteico/fisiologia , Receptor Notch3/metabolismo , Linfócitos T/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/patologia , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/patologia
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