RESUMO
Considering the role played by the heat shock protein of 70 kDa (HSP70) in cancer, we characterized this protein and its major regulator, the heat shock factor 1 (HSF1), in chronic lymphocytic leukemia (CLL). We found both HSP70 and HSF1 overexpressed in CLL patients, correlated to poor prognosis and abnormally localized in the nucleus of leukemic B cells. The two proteins were strictly correlated each other and their levels decreased consensually in those patients responding to in vivo therapeutic regimens. HSP70 and HSF1 inhibition was proved to be effective in inducing a dose-dependent in vitro apoptosis of CLL B cells. Considering that HSF1 is finely regulated by kinases belonging to pathways triggered by rat sarcoma (RAS), we benefited from a previous proteomic study performed in CLL patients aiming to assess the activation/expression of key signaling proteins. We found that patients showing high levels of HSP70 also expressed high Akt-Ser473, thus activating HSF1. Inhibition of PI3K, which activates AKT, reduced the expression of HSF1 and HSP70. By contrast, HSP70-low patients displayed high activation of MEK1/2 and ERK1/2, known to negatively regulate HSF1. These data demonstrate that the HSP70 expression is regulated by the modulation of HSF1 activity through the activation of RAS-regulated pathways and suggest the HSP70/HSF1 interplay as an interesting target for antileukemic therapies. Finally, inhibition of PI3K, that activates AKT, reduced the expression of HSF1 and HSP70.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Transdução de Sinais , Adenina/análogos & derivados , Estudos de Casos e Controles , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Flavonoides/farmacologia , Flavonóis , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Terapia de Alvo Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Piperidinas , Prognóstico , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Pediatric T-acute lymphoblastic leukemia (T-ALL) patients often display resistance to glucocorticoid (GC) treatment. These patients, classified as prednisone poor responders (PPR), have poorer outcome than do the other pediatric T-ALL patients receiving a high-risk adapted therapy. Because glucocorticoids are administered to ALL patients during all the different phases of therapy, GC resistance represents an important challenge to improving the outcome for these patients. Mechanisms underlying resistance are not yet fully unraveled; thus our research focused on the identification of deregulated signaling pathways to point out new targeted approaches. We first identified, by reverse-phase protein arrays, the lymphocyte cell-specific protein-tyrosine kinase (LCK) as aberrantly activated in PPR patients. We showed that LCK inhibitors, such as dasatinib, bosutinib, nintedanib, and WH-4-023, are able to induce cell death in GC-resistant T-ALL cells, and remarkably, cotreatment with dexamethasone is able to reverse GC resistance, even at therapeutic drug concentrations. This was confirmed by specific LCK gene silencing and ex vivo combined treatment of cells from PPR patient-derived xenografts. Moreover, we observed that LCK hyperactivation in PPR patients upregulates the calcineurin/nuclear factor of activated T cells signaling triggering to interleukin-4 (IL-4) overexpression. GC-sensitive cells cultured with IL-4 display an increased resistance to dexamethasone, whereas the inhibition of IL-4 signaling could increase GC-induced apoptosis in resistant cells. Treatment with dexamethasone and dasatinib also impaired engraftment of leukemia cells in vivo. Our results suggest a quickly actionable approach to supporting conventional therapies and overcoming GC resistance in pediatric T-ALL patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glucocorticoides/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Criança , Dasatinibe/farmacologia , Dexametasona/farmacologia , Xenoenxertos , Humanos , Interleucina-4/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/enzimologia , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prednisona/farmacologiaRESUMO
The presence of the chromosomal rearrangement t(12;21)(ETV6-RUNX1) in childhood B-acute lymphoblastic leukemia (B-ALL) is an independent predictor of favorable prognosis, however relapses still occur many years later after stopping therapy, and patients often display resistance to current treatments. Since spleen tyrosine kinase (SYK), a cytosolic nonreceptor tyrosine kinase interacting with immune receptors, has been previously associated with malignant transformation and cancer cell proliferation, we aimed to assess its role in ETV6-RUNX1 cell survival and prognosis. We evaluated the effects on cell survival of three SYK inhibitors and showed that all of them, in particular entospletinib, are able to induce cell death and enhance the efficacy of conventional chemotherapeutics. By using reverse phase protein arrays we next revealed that activated SYK is upregulated at diagnosis in pediatric ETV6-RUNX1 patients who will experience relapse, and, importantly, hyperactivation is maintained at a high level also at relapse occurrence. We thus treated primary cells from patients both at diagnosis and relapse with the combination entospletinib + chemotherapeutics and observed that SYK inhibition is able to sensitize resistant primary cells to conventional drugs. Entospletinib could thus represent a new therapeutic option supporting conventional chemotherapy for relapsed ETV6-RUNX1 patients, and these evidences encourage further studies on SYK for treatment of other relapsed resistant acute lymphoblastic leukemia (ALL) subgroups.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Recidiva Local de Neoplasia/tratamento farmacológico , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Quinase Syk/antagonistas & inibidores , Aminopiridinas , Proliferação de Células/efeitos dos fármacos , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Cicloexilaminas/farmacologia , Humanos , Indazóis/farmacologia , Morfolinas , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Proteínas de Fusão Oncogênica/genética , Oxazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Pirazinas/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Células Tumorais CultivadasAssuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Benzamidas , Humanos , Mesilato de Imatinib , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapiaAssuntos
Linfoma Anaplásico de Células Grandes , MicroRNAs , Quinase do Linfoma Anaplásico , Linhagem Celular Tumoral , Criança , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma Anaplásico de Células Grandes/genética , MicroRNAs/genética , Receptores Proteína Tirosina Quinases/genética , Fatores de Transcrição/genéticaRESUMO
We assessed signaling protein mapping in total T cells, to analyze the proportions of T regulatory (Treg) and TCD4+ effector (Teff) cell phenotypes, and the respective interleukin 6Rα (IL-6Rα) expression in the inflammatory microenvironment of synovial fluid (SF) of patients with sustained psoriatic arthritis (PsA). Our approach was to measure the IL-6 level in SF using a multiplex bead immunoassay. Reverse-phase protein array was used to assess Janus kinase (JAK) 1 and JAK2, extra-cellular regulated kinase (ERK) 1 and 2, protein kinase Cδ (PKCδ), signal transducer and activator and transcription (STAT) 1, STAT3, and STAT5 phosphoproteins in total T cell lysates from SF of patients with PsA. Frequencies of CD4+IL-17A-F+IL-23+ CD4+ Th cells producing IL-17A and IL-17F (Th17) and CD4+CD25high intracellular forkhead box transcription factor+ (FOXP3+) phenotypes, and the percentage of Treg- and Teff- cells were quantified in SF and matched peripheral blood (PB) of patients with PsA and PB of healthy controls (HC) by flow cytometry. Our results were the following: In PsA SF samples, a coordinate increase of JAK1, ERK1/2, STAT1, STAT3, and STAT5 phosphoproteins was found in total T cells in SF of PsA; where IL-6 levels were higher than in PB from HC. Expanded CD4+IL-17A-F+IL-23+ Th17, CD4+ CD25- Teff- and CD4+CD25(high) FoxP3+Treg subsets, showing similar levels of enhanced IL-6Rδ expression, were confined to PsA joints. In our studies, the transcriptional network profile identified by ex vivo signaling protein mapping in T lymphocytes in PsA joints revealed the complex interplay between IL-1, IL-6, and IL-23 signaling and differentiation of Th17 cells and CD4+Tregs in sustained joint inflammation in PsA.
Assuntos
Artrite Psoriásica/enzimologia , Articulações/enzimologia , Proteínas Quinases/análise , Fatores de Transcrição STAT/análise , Transdução de Sinais , Líquido Sinovial/enzimologia , Linfócitos T Reguladores/enzimologia , Artrite Psoriásica/imunologia , Estudos de Casos e Controles , Citometria de Fluxo , Humanos , Imunofenotipagem/métodos , Interleucina-6/análise , Articulações/imunologia , Fenótipo , Fosforilação , Análise Serial de Proteínas , Mapas de Interação de Proteínas , Líquido Sinovial/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
The cAMP response element-binding protein (CREB) is a nuclear transcription factor that is critical for normal and neoplastic hematopoiesis. Previous studies have demonstrated that CREB is a proto-oncogene whose overexpression promotes cellular proliferation in hematopoietic cells. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative disease with splenomegaly and aberrant myelopoiesis. However, CREB overexpressing mice do not spontaneously develop acute myeloid leukemia. In this study, we used retroviral insertional mutagenesis to identify genes that accelerate leukemia in CREB transgenic mice. Our mutagenesis screen identified several integration sites, including oncogenes Gfi1, Myb, and Ras. The Sox4 transcription factor was identified by our screen as a gene that cooperates with CREB in myeloid leukemogenesis. We show that the transduction of CREB transgenic mouse bone marrow cells with a Sox4 retrovirus increases survival and self-renewal of cells in vitro. Furthermore, leukemic blasts from the majority of acute myeloid leukemia patients have higher CREB, phosphorylated CREB, and Sox 4 protein expression. Sox4 transduction of mouse bone marrow cells results in increased expression of CREB target genes. We also demonstrate that CREB is a direct target of Sox4 by chromatin immunoprecipitation assays. These results indicate that Sox4 and CREB cooperate and contribute to increased proliferation of hematopoietic progenitor cells.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Células Mieloides/metabolismo , Fatores de Transcrição SOXC/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proliferação de Células , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Feminino , Células HL-60 , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/citologia , Fosforilação/fisiologia , Gravidez , Proto-Oncogene Mas , Retroviridae/genéticaRESUMO
According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro. Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2(+) neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Diferenciação Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Humanos , Naftóis/farmacologia , Neurônios/citologia , Organofosfatos/farmacologia , Sinapses/efeitos dos fármacosAssuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1 , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismoRESUMO
Bone marrow mesenchymal cells (MSCs) can protect leukemic cells from chemotherapy, thus increasing their survival rate. We studied the potential molecular mechanisms underlying this effect in acute lymphoblastic leukemia (ALL) cells. Coculture of ALL cells with MSCs induced on the lymphoblast plasma membrane the expression of a signaling complex formed by hERG1 (human ether-à-go-go-related gene 1) channels, the ß(1)-integrin subunit, and the chemokine receptor CXC chemokine receptor-4. The assembly of such a protein complex activated both the extracellular signal-related kinase 1/2 (ERK1/2) and the phosphoinositide 3-kinase (PI3K)/Akt prosurvival signaling pathways. At the same time, ALL cells became markedly resistant to chemotherapy-induced apoptosis. hERG1 channel function appeared to be important for both the initiation of prosurvival signals and the development of drug resistance, because specific channel blockers decreased the protective effect of MSCs. NOD/SCID mice engrafted with ALL cells and treated with channel blockers showed reduced leukemic infiltration and had higher survival rates. Moreover, hERG1 blockade enhanced the therapeutic effect produced by corticosteroids. Our findings provide a rationale for clinical testing of hERG1 blockers in the context of antileukemic therapy for patients with ALL.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Animais , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Técnicas de Cocultura , Doxorrubicina/farmacologia , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Humanos , Integrina beta1/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Complexos Multiproteicos/metabolismo , Piperidinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prednisona/farmacologia , Piridinas/farmacologia , Interferência de RNA , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To find candidate biomarkers of psoriatic arthritis (PsA). A panel of synovial fluid (SF) and synovial tissue (ST) biomarkers was analyzed in patients with resistant peripheral PsA, in relation to clinical and imaging outcomes of synovitis response following serial intraarticular (IA) etanercept injections (12.5 mg). METHODS: Fourteen PsA patients with resistant knee joint synovitis were treated with 4 IA etanercept injections in a single knee joint, once every 2 weeks. Primary outcome (Thompson's knee index: THOMP) and secondary outcomes were assessed at baseline and end of study: C-reactive protein, Knee Joint Articular Index (KJAI), Health Assessment Questionnaire disability index, maximal synovial thickness (MST) by gray-scale ultrasonography, contrast-enhanced magnetic resonance imaging (C+MRI), ST-cluster differentiation (CD)45+ mononuclear cell, ST-CD31+ vessels, and ST-CD105+ angiogenic endothelial cells, along with levels of SF interleukin 1ß (IL-1ß), IL-1 receptor antagonist (Ra), and IL-6. RESULTS: At the end of the study, clinical and imaging outcomes, ST and SF biological markers were significantly reduced compared to baseline. There was a significant association between IL-6 and either THOMP or KJAI; between either ST-CD31+ or ST-CD105+ or ST-CD45+; between ST and SF biomarkers expression (CD45+ and IL-1ß) and between ST-CD45+ and both KJAI and MRI-MST. Comparing pre- versus post-IA etanercept injection changes (Δ), Δ IL-1ß was significantly correlated with both Δ IL-6 and with Δ IL-1Ra and Δ IL-6 with Δ IL-1Ra. CONCLUSION: The association to disease activity and the changes following IA treatment indicate that ST-CD45+ and ST-CD31+, along with SF-IL-6 and SF-IL-1ß, may represent candidate biomarkers of the knee synovitis response to IA tumor necrosis factor-α blockade.
Assuntos
Artrite Psoriásica/diagnóstico , Biomarcadores/metabolismo , Articulação do Joelho/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Adulto , Antígenos CD/metabolismo , Artrite Psoriásica/tratamento farmacológico , Artrite Psoriásica/metabolismo , Biópsia , Proteína C-Reativa/metabolismo , Avaliação da Deficiência , Esquema de Medicação , Endoglina , Etanercepte , Feminino , Humanos , Imunoglobulina G/administração & dosagem , Imuno-Histoquímica , Imunossupressores/administração & dosagem , Injeções Intra-Articulares , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/patologia , Antígenos Comuns de Leucócito/metabolismo , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Necrose Tumoral/administração & dosagem , Inquéritos e Questionários , Membrana Sinovial/diagnóstico por imagem , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Fatores de Tempo , Resultado do Tratamento , UltrassonografiaRESUMO
Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy, characterized by a heterogeneous genetic landscape and complex clonal evolution, with poor outcomes. Mutation at the internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic alterations in AML, associated with high relapse rates and poor survival due to the constitutive activation of the FLT3 receptor tyrosine kinase and its downstream effectors, such as PI3K signaling. Thus, aberrantly activated FLT3-kinase is regarded as an attractive target for therapy for this AML subtype, and a number of small molecule inhibitors of this kinase have been identified, some of which are approved for clinical practice. Nevertheless, acquired resistance to these molecules is often observed, leading to severe clinical outcomes. Therapeutic strategies to tackle resistance include combining FLT3 inhibitors with other antileukemic agents. Here, we report on the preclinical activity of the combination of the FLT3 inhibitor quizartinib with the dual PI3K/mTOR inhibitor PF-04691502 in FLT3-ITD cells. Briefly, we show that the association of these two molecules displays synergistic cytotoxicity in vitro in FLT3-ITD AML cells, triggering 90% cell death at nanomolar concentrations after 48 h.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mutação , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Serina-Treonina Quinases TOR/genética , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/uso terapêuticoRESUMO
Lymphoblastic lymphoma (LBL) is the second most common type of non-Hodgkin lymphoma in childhood, mainly of T cell origin (T-LBL). Although current treatment protocols allow a complete remission in 85% of cases, the second-line treatment overall survival for patients with progressive or relapsed disease is around 14%, making this the major issue to be confronted. Thus, we performed a Reverse Phase Protein Array study in a cohort of 22 T-LBL patients to find reliable disease risk marker(s) and new therapeutic targets to improve pediatric T-LBL patients' outcome. Interestingly, we pinpointed JAK2 Y1007-1008 as a potential prognosis marker as well as a therapeutic target in poor prognosis patients. Hence, the hyperactivation of the JAK1/2-STAT6 pathway characterizes these latter patients. Moreover, we functionally demonstrated that STAT6 hyperactivation contributes to therapy resistance by binding the glucocorticoid receptor, thus inhibiting its transcriptional activity. This was further confirmed by specific STAT6 gene silencing followed by dexamethasone treatment. Finally, JAK1/2-STAT6 pathway inhibition by ruxolitinib, an FDA approved drug, in cell line models and in one T-LBL primary sample led to cell proliferation reduction and increased apoptosis. Globally, our results identify a new potential prognostic marker and suggest a novel therapeutic approach to overcome therapy resistance in pediatric T-LBL patients.
RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The Activation-Induced Cell Death (AICD) is a stimulation-dependent form of apoptosis used by the organism to shutdown T-cell response once the source of inflammation has been eliminated, while allowing the generation of immune memory. AICD is thought to progress through the activation of the extrinsic Fas/FasL pathway of cell death, leading to cytochrome-C release through caspase-8 and Bid activation. We recently described that, early upon AICD induction, mitochondria undergo structural alterations, which are required to promote cytochrome-C release and execute cell death. Here, we found that such alterations do not depend on the Fas/FasL pathway, which is instead only lately activated to amplify the cell death cascade. Instead, such alterations are primarily dependent on the MAPK proteins JNK1 and ERK1/2, which, in turn, regulate the activity of the pro-fission protein Drp1 and the pro-apoptotic factor Bim. The latter regulates cristae disassembly and cooperate with Drp1 to mediate the Mitochondrial Outer Membrane Permeabilization (MOMP), leading to cytochrome-C release. Interestingly, we found that Bim is also downregulated in T-cell Acute Lymphoblastic Leukemia (T-ALL) cells, this alteration favouring their escape from AICD-mediated control.
Assuntos
Dinaminas/metabolismo , Mitocôndrias/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras , Linfócitos T/imunologia , Animais , Morte Celular , Linhagem Celular Tumoral , Feminino , Humanos , Ativação Linfocitária , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Linfócitos T/citologiaRESUMO
The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although its oncogenic mechanism has not been well characterized. Phosphorylated level of RSK (T573) was increased in acute myeloid leukemia (AML) patients and associated with poor survival. To examine the role of RSK in AML, we analyzed apoptosis and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment increased the G2/M population and induced apoptosis in AML cell lines and patient AML cells. Characterization of mitotic phases showed that the metaphase/anaphase transition was significantly inhibited by BI-D1870. BI-D1870 treatment impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to CDC20, preventing mitotic exit. Moreover, the inactivation of spindle assembly checkpoint or MAD2 knockdown released cells from BI-D1870-induced metaphase arrest. Therefore, we investigated whether BI-D1870 potentiates the anti-leukemic activity of vincristine by targeting mitotic exit. Combination treatment of BI-D1870 and vincristine synergistically increased mitotic arrest and apoptosis in acute leukemia cells. These data show that BI-D1870 induces apoptosis of AML cells alone and in combination with vincristine through blocking mitotic exit, providing a novel approach to overcoming vincristine resistance in AML cells.
RESUMO
Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL. SIGNIFICANCE: Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery.This article is highlighted in the In This Issue feature, p. 1241.
Assuntos
Processamento Alternativo/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Fosfoproteínas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Processamento Alternativo/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sinergismo Farmacológico , Éxons/genética , Humanos , Células Jurkat , Masculino , Camundongos , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Estudo de Prova de Conceito , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. EXPERIMENTAL DESIGN: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. RESULTS: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. CONCLUSIONS: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.
Assuntos
Histona Desmetilases com o Domínio Jumonji/genética , Leucemia de Células T/genética , Receptor Notch1/genética , Peptidase 7 Específica de Ubiquitina/genética , Animais , Carcinogênese/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Terapia Genética , Humanos , Células Jurkat , Leucemia de Células T/patologia , Leucemia de Células T/terapia , Camundongos , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dysregulation of the cyclin D1-CDK4/CDK6 complex is frequently observed in almost all human cancer and contributes to aberrant cell proliferation and consequent tumorigenesis. Although many reports described the importance of CDK4/CDK6 in different set of human tumors, only few studies have been performed on leukemia. By gene expression analysis performed in a cohort of childhood patients affected by B-acute lymphoblastic leukemia (B-ALL) we found that both CDK4 and CDK6 are highly expressed. Moreover, reverse phase protein array (RPPA) analysis showed that cyclin D1 levels are higher in patients undergoing relapse. Starting from these considerations, we evaluated the effect of dual inhibition of CDK4/CDK6 in B-ALL and if this inhibition could enhance cytotoxic killing of leukemia cells after combination treatment with dexamethasone. We treated B-ALL cell lines with ribociclib, a highly specific CDK4/6 inhibitor. As expected, treatment with ribociclib induced growth inhibition of B-ALL cell lines, accompanied by strong cell cycle arrest in G1 phase, along with a dose-dependent decrease in phosphorylated retinoblastoma protein. Ribociclib exposure strongly synergizes with dexamethasone in SEM and RCH-ACV, two dexamethasone-resistant cell lines, along with a strong decrease in proliferation and a significant increase in apoptotic cell death. These results were also confirmed on primary cultures derived from bone marrow of pediatric patients affected by B-ALL. Immunoblot analysis showed a significant increase in glucocorticoid receptor (GR) along with some of its target genes, after combined treatment with ribociclib and dexamethasone. Altogether our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanism of glucocorticoid resistance.