RESUMO
The lysine-specific histone demethylase 1 A (LSD1) is involved in antitumor immunity; however, its role in shaping CD8 + T cell (CTL) differentiation and function remains largely unexplored. Here, we show that pharmacological inhibition of LSD1 (LSD1i) in CTL in the context of adoptive T cell therapy (ACT) elicits phenotypic and functional alterations, resulting in a robust antitumor immunity in preclinical models in female mice. In addition, the combination of anti-PDL1 treatment with LSD1i-based ACT eradicates the tumor and leads to long-lasting tumor-free survival in a melanoma model, complementing the limited efficacy of the immune or epigenetic therapy alone. Collectively, these results demonstrate that LSD1 modulation improves antitumoral responses generated by ACT and anti-PDL1 therapy, providing the foundation for their clinical evaluation.
Assuntos
Linfócitos T CD8-Positivos , Histona Desmetilases , Imunoterapia Adotiva , Camundongos Endogâmicos C57BL , Animais , Histona Desmetilases/metabolismo , Histona Desmetilases/antagonistas & inibidores , Imunoterapia Adotiva/métodos , Camundongos , Feminino , Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Linhagem Celular Tumoral , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Antígeno B7-H1/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Humanos , Melanoma/imunologia , Melanoma/terapiaRESUMO
Innate immune responses to coronavirus infections are highly cell specific. Tissue-resident macrophages, which are infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients but are inconsistently infected in vitro, exert critical but conflicting effects by secreting both antiviral type I interferons (IFNs) and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for the treatment of coronavirus disease 2019 (COVID-19), indiscriminately suppress both responses, possibly impairing viral clearance. Here, we established in vitro cell culture systems that enabled us to separately investigate the cell-intrinsic and cell-extrinsic proinflammatory and antiviral activities of mouse macrophages infected with the prototypical murine coronavirus MHV-A59. We showed that the nuclear factor κB-dependent inflammatory response to viral infection was selectively inhibited by loss of the lysine demethylase LSD1, which was previously implicated in innate immune responses to cancer, with negligible effects on the antiviral IFN response. LSD1 ablation also enhanced an IFN-independent antiviral response, blocking viral egress through the lysosomal pathway. The macrophage-intrinsic antiviral and anti-inflammatory activity of Lsd1 inhibition was confirmed in vitro and in a humanized mouse model of SARS-CoV-2 infection. These results suggest that LSD1 controls innate immune responses against coronaviruses at multiple levels and provide a mechanistic rationale for potentially repurposing LSD1 inhibitors for COVID-19 treatment.
Assuntos
COVID-19 , Lisina , Animais , Humanos , Camundongos , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Citocinas/metabolismo , SARS-CoV-2/metabolismoRESUMO
Neuroendocrine tumors (NETs) are highly vascularized malignancies in which angiogenesis may entail cell proliferation and survival. Among the emerging compounds with antivascular properties, cabozantinib (CAB) appeared promising. We analyzed the antitumor activity of CAB against NETs utilizing in vitro and in vivo models. For cell cultures, we used BON-1, NCI-H727 and NCI-H720 cell lines. Cell viability was assessed by manual count coupled with quantification of cell death, performed through fluorescence-activated cell sorting analysis as propidium iodide exclusion assay. In addition, we investigated the modulation of the antiapoptotic myeloid cell leukemia 1 protein under CAB exposure, as a putative adaptive pro-survival mechanism, and compared the responses with sunitinib. The activity of CAB was also tested in mouse and zebrafish xenograft tumor models. Cabozantinib showed a dose-dependent and time-dependent effect on cell viability and proliferation in human NET cultures, besides a halting of cell cycle progression for endoduplication, never reported for other tyrosine kinase inhibitors. In a transplantable zebrafish model, CAB drastically inhibited NET-induced angiogenesis and migration of implanted cells through the embryo body. CAB showed encouraging activity in NETs, both in vitro and in vivo models. On this basis, we envisage future research to further investigate along these promising lines.
Assuntos
Tumores Neuroendócrinos , Peixe-Zebra , Humanos , Animais , Camundongos , Transdução de Sinais , Tumores Neuroendócrinos/patologia , Linhagem Celular TumoralRESUMO
Combined inhibition of oxidative phosphorylation (OXPHOS) and glycolysis has been shown to activate a PP2A-dependent signaling pathway, leading to tumor cell death. Here, we analyze highly selective mitochondrial complex I or III inhibitors in vitro and in vivo to elucidate the molecular mechanisms leading to cell death following OXPHOS inhibition. We show that IACS-010759 treatment (complex I inhibitor) induces a reactive oxygen species (ROS)-dependent dissociation of CIP2A from PP2A, leading to its destabilization and degradation through chaperone-mediated autophagy. Mitochondrial complex III inhibition has analogous effects. We establish that activation of the PP2A holoenzyme containing B56δ regulatory subunit selectively mediates tumor cell death, while the arrest in proliferation that is observed upon IACS-010759 treatment does not depend on the PP2A-B56δ complex. These studies provide a molecular characterization of the events subsequent to the alteration of critical bioenergetic pathways and help to refine clinical studies aimed to exploit metabolic vulnerabilities of tumor cells.
Assuntos
Autofagia Mediada por Chaperonas , Complexo I de Transporte de Elétrons , Neoplasias , Humanos , Autoantígenos/metabolismo , Linhagem Celular Tumoral , Metabolismo Energético , Neoplasias/patologia , Fosforilação Oxidativa , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , Transdução de Sinais , Complexo I de Transporte de Elétrons/antagonistas & inibidoresRESUMO
According to a hierarchical model, targeting leukemia-initiating cells (LICs) was speculated to achieve complete remission (CR) or cure. Nonetheless, increasing evidence emphasized the plasticity of differentiated blasts undergoing interconversion into LICs. We exploited murine models of acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia driven by the promyelocytic leukemia/retinoic acid receptor (PML-RARα) oncofusion protein, which recruits histone deacetylase (HDAC)-containing complexes. We studied APLs with different LIC frequencies and investigated the effect of two HDAC inhibitors: valproic acid (VPA), with relative selectivity towards class I HDAC enzymes and vorinostat/suberoylanilide hydroxamic acid (SAHA) (pan-HDAC inhibitor) in combination with all-trans retinoic acid (ATRA), on the bulk APL cells and APL LICs. Indeed, we found that while VPA differentiates the bulk APL cells, SAHA selectively targets LICs. ATRA + VPA + SAHA combination efficiently induced CR in an APL model with lower LIC frequency. Substituting ATRA with synthetic retinoids as etretinate which promotes APL differentiation without downregulating PML/RARα compromised the therapeutic benefit of ATRA + VPA + SAHA regimen. Altogether, our study emphasizes the therapeutic power of co-targeting the plasticity and heterogeneity of cancer -herein demonstrated by tackling LICs and bulk leukemic blasts - to achieve and maintain CR.
Assuntos
Antineoplásicos , Leucemia Promielocítica Aguda , Animais , Antineoplásicos/uso terapêutico , Diferenciação Celular , Erradicação de Doenças , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Camundongos , Proteínas de Fusão Oncogênica/metabolismo , Tretinoína/farmacologia , Tretinoína/uso terapêutico , Ácido Valproico/farmacologiaRESUMO
The histone demethylase LSD1 is over-expressed in hematological tumors and has emerged as a promising target for anticancer treatment, so that several LSD1 inhibitors are under development and testing, in preclinical and clinical settings. However, the complete understanding of their complex mechanism of action is still unreached. Here, we unraveled a novel mode of action of the LSD1 inhibitors MC2580 and DDP-38003, showing that they can induce differentiation of AML cells through the downregulation of the chromatin protein GSE1. Analysis of the phenotypic effects of GSE1 depletion in NB4 cells showed a strong decrease of cell viability in vitro and of tumor growth in vivo. Mechanistically, we found that a set of genes associated with immune response and cytokine-signaling pathways are upregulated by LSD1 inhibitors through GSE1-protein reduction and that LSD1 and GSE1 colocalize at promoters of a subset of these genes at the basal state, enforcing their transcriptional silencing. Moreover, we show that LSD1 inhibitors lead to the reduced binding of GSE1 to these promoters, activating transcriptional programs that trigger myeloid differentiation. Our study offers new insights into GSE1 as a novel therapeutic target for AML.
Assuntos
Histona DesmetilasesRESUMO
Synthetic lethality is an innovative framework for discovering novel anticancer drug candidates. One example is the use of PARP inhibitors (PARPi) in oncology patients with BRCA mutations. Here, we exploit a new paradigm based on the possibility of triggering synthetic lethality using only small organic molecules (dubbed "fully small-molecule-induced synthetic lethality"). We exploited this paradigm to target pancreatic cancer, one of the major unmet needs in oncology. We discovered a dihydroquinolone pyrazoline-based molecule (35d) that disrupts the RAD51-BRCA2 protein-protein interaction, thus mimicking the effect of BRCA2 mutation. 35d inhibits the homologous recombination in a human pancreatic adenocarcinoma cell line. In addition, it synergizes with olaparib (a PARPi) to trigger synthetic lethality. This strategy aims to widen the use of PARPi in BRCA-competent and olaparib-resistant cancers, making fully small-molecule-induced synthetic lethality an innovative approach toward unmet oncological needs.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Proteína BRCA2/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rad51 Recombinase/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Antineoplásicos/química , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Descoberta de Drogas , Sinergismo Farmacológico , Recombinação Homóloga/efeitos dos fármacos , Humanos , Modelos Moleculares , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Ftalazinas/química , Piperazinas/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Mutações Sintéticas Letais/efeitos dos fármacosRESUMO
Lysine specific demethylase-1 (LSD1) has been shown to be critical in acute myeloid leukemia (AML) pathogenesis and this has led to the development of LSD1 inhibitors (LSD1i) which are currently tested in clinical trials. Nonetheless, preclinical studies reported that AML cells frequently exhibit intrinsic resistance to LSD1 inhibition, and the molecular basis for this phenomenon is largely unknown. We explored the potential involvement of mammalian target of rapamycin (mTOR) in mediating the resistance of leukemic cells to LSD1i. Strikingly, unlike sensitive leukemias, mTOR complex 1 (mTORC1) signaling was robustly triggered in resistant leukemias following LSD1 inhibition. Transcriptomic, chromatin immunoprecipitation and functional studies revealed that insulin receptor substrate 1(IRS1)/extracellular-signal regulated kinases (ERK1/2) signaling critically controls LSD1i induced mTORC1 activation. Notably, inhibiting mTOR unlocked the resistance of AML cell lines and primary patient-derived blasts to LSD1i both in vitro and in vivo In conclusion, mTOR activation might act as a novel pro-survival mechanism of intrinsic as well as acquired resistance to LSD1i, and combination regimens co-targeting LSD1/mTOR could represent a rational approach in AML therapy.
Assuntos
Leucemia Mieloide Aguda , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Transdução de Sinais , SirolimoRESUMO
Olaparib is a PARP inhibitor (PARPi). For patients bearing BRCA1 or BRCA2 mutations, olaparib is approved to treat ovarian cancer and in clinical trials to treat breast and pancreatic cancers. In BRCA2-defective patients, PARPi inhibits DNA single-strand break repair, while BRCA2 mutations hamper double-strand break repair. Recently, we identified a series of triazole derivatives that mimic BRCA2 mutations by disrupting the Rad51-BRCA2 interaction and thus double-strand break repair. Here, we have computationally designed, synthesized, and tested over 40 novel derivatives. Additionally, we designed and conducted novel biological assays to characterize how they disrupt the Rad51-BRCA2 interaction and inhibit double-strand break repair. These compounds synergized with olaparib to target pancreatic cancer cells with functional BRCA2. This supports the idea that small organic molecules can mimic genetic mutations to improve the profile of anticancer drugs for precision medicine. Moreover, this paradigm could be exploited in other genetic pathways to discover innovative anticancer targets and drug candidates.
Assuntos
Antineoplásicos/química , Proteína BRCA2/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Rad51 Recombinase/metabolismo , Triazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteína BRCA2/genética , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Mimetismo Molecular , Mutação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Triazóis/síntese químicaRESUMO
In BRCA2-defective cells, poly(adenosine diphosphate [ADP]-ribose) polymerase inhibitors can trigger synthetic lethality, as two independent DNA-repairing mechanisms are simultaneously impaired. Here, we have pharmacologically induced synthetic lethality, which was triggered by combining two different small organic molecules. When administered with a BRCA2-Rad51 disruptor in nonmutant cells, Olaparib showed anticancer activity comparable to that shown when administered alone in BRCA2-defective cells. This strategy could represent an innovative approach to anticancer drug discovery and could be extended to other synthetic lethality pathways.
Assuntos
Proteína BRCA2/antagonistas & inibidores , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rad51 Recombinase/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Modelos Moleculares , Mutação , Ftalazinas/química , Piperazinas/química , Conformação Proteica , Rad51 Recombinase/metabolismoRESUMO
Purpose: Histone deacetylase inhibitors (HDACi) are promising anticancer drugs. Although some HDACi have entered the clinic, the mechanism(s) underlying their tumor selectivity are poorly understood.Experimental Design and Results: Using gene expression analysis, we define a core set of six genes commonly regulated in acute myeloid leukemia (AML) blasts and cell lines. MYC, the most prominently modulated, is preferentially altered in leukemia. Upon HDACi treatment, c-Myc is acetylated at lysine 323 and its expression decreases, leading to TRAIL activation and apoptosis. c-Myc binds to the TRAIL promoter on the proximal GC box through SP1 or MIZ1, impairing TRAIL activation. HDACi exposure triggers TRAIL expression, altering c-Myc-TRAIL binding. These events do not occur in normal cells. Excitingly, this inverse correlation between TRAIL and c-Myc is supported by HDACi treatment ex vivo of AML blasts and primary human breast cancer cells. The predictive value of c-Myc to HDACi responsiveness is confirmed in vivo in AML patients undergoing HDACi-based clinical trials.Conclusions: Collectively, our findings identify a key role for c-Myc in TRAIL deregulation and as a biomarker of the anticancer action of HDACi in AML. The potential improved patient stratification could pave the way toward personalized therapies. Clin Cancer Res; 23(10); 2542-55. ©2016 AACR.
Assuntos
Histona Desacetilase 1/genética , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Acetilação , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/antagonistas & inibidores , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Fatores de Transcrição Kruppel-Like/genética , Neoplasias/patologia , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição Sp1/genéticaRESUMO
Most patients who initially respond to treatment with the multi-tyrosine kinase inhibitor sunitinib eventually relapse. Therefore, developing a deeper understanding of the contribution of sunitinib's numerous targets to the clinical response or to resistance is crucial. Here, we have shown that cancer cells respond to clinically relevant doses of sunitinib by enhancing the stability of the antiapoptotic protein MCL-1 and inducing mTORC1 signaling, thus evoking little cytotoxicity. Inhibition of MCL-1 or mTORC1 signaling sensitized cells to clinically relevant doses of sunitinib in vitro and was synergistic with sunitinib in impairing tumor growth in vivo, indicating that these responses are triggered as prosurvival mechanisms that enable cells to tolerate the cytotoxic effects of sunitinib. Furthermore, higher doses of sunitinib were cytotoxic, triggered a decline in MCL-1 levels, and inhibited mTORC1 signaling. Mechanistically, we determined that sunitinib modulates MCL-1 stability by affecting its proteasomal degradation. Dual modulation of MCL-1 stability at different dose ranges of sunitinib was due to differential effects on ERK and GSK3ß activity, and the latter also accounted for dual modulation of mTORC1 activity. Finally, comparison of patient samples prior to and following sunitinib treatment suggested that increases in MCL-1 levels and mTORC1 activity correlate with resistance to sunitinib in patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/tratamento farmacológico , Pirróis/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Estabilidade Enzimática , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Sunitinibe , Serina-Treonina Quinases TOR/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: The identification of genes maintaining cancer growth is critical to our understanding of tumorigenesis. We report the first in vivo genetic screen of patient-derived tumors, using metastatic melanomas and targeting 236 chromatin genes by expression of specific shRNA libraries. Our screens revealed unprecedented numerosity of genes indispensable for tumor growth (â¼50% of tested genes) and unexpected functional heterogeneity among patients (<15% in common). Notably, these genes were not activated by somatic mutations in the same patients and are therefore distinguished from mutated cancer driver genes. We analyzed underlying molecular mechanisms of one of the identified genes, the Histone-lysine N-methyltransferase KMT2D, and showed that it promotes tumorigenesis by dysregulating a subset of transcriptional enhancers and target genes involved in cell migration. The assembly of enhancer genomic patterns by activated KMT2D was highly patient-specific, regardless of the identity of transcriptional targets, suggesting that KMT2D might be activated by distinct upstream signaling pathways. SIGNIFICANCE: Drug targeting of biologically relevant cancer-associated mutations is considered a critical strategy to control cancer growth. Our functional in vivo genetic screens of patient-derived tumors showed unprecedented numerosity and interpatient heterogeneity of genes that are essential for tumor growth, but not mutated, suggesting that multiple, patient-specific signaling pathways are activated in tumors. Cancer Discov; 6(6); 650-63. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561.
Assuntos
Transformação Celular Neoplásica/genética , Estudos de Associação Genética , Testes Genéticos , Neoplasias/diagnóstico , Neoplasias/genética , Fenótipo , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Elementos Facilitadores Genéticos , Epigênese Genética , Epigenômica/métodos , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Reprodutibilidade dos TestesRESUMO
The RET (REarranged during Transfection) receptor tyrosine kinase is targeted by oncogenic rearrangements in thyroid and lung adenocarcinoma. Recently, a RET (exon 12) rearrangement with FGFR1OP [fibroblast growth factor receptor 1 (FGFR1) oncogene partner] (exon 12) was identified in one chronic myelomonocytic leukemia (CMML) patient. We report the molecular cloning and functional characterization of a novel FGFR1OP (exon 11)-RET (exon 11) gene fusion event (named FGFR1OP-RET), mediated by a reciprocal translocation t(6; 10)(q27; q11), in a patient affected by primary myelofibrosis (PMF) with secondary acute myeloid leukemia (AML). The FGFR1OP-RET fusion protein displayed constitutive tyrosine kinase and transforming activity in NIH3T3 fibroblasts, and induced IL3-independent growth and activation of PI3K/STAT signaling in hematopoietic Ba/F3 cells. FGFR1OP-RET supported cytokine-independent growth, protection from stress and enhanced self-renewal of primary murine hematopoietic progenitor and stem cells in vitro. In vivo, FGFR1OP-RET caused a spectrum of disease phenotypes, with >50% of mice showing a fatal myeloproliferative disorder (MPD). Other phenotypes were leukemia transplantable in secondary recipients, dramatic expansion of the mast cell lineage, and reduction of repopulating activity upon lethal irradiation. In conclusion, FGFR1OP-RET chimeric oncogenes are endowed with leukemogenic potential and associated to myeloid neoplasms (CMML and PMF/AML).
Assuntos
Cromossomos Humanos Par 10 , Cromossomos Humanos Par 6 , Neoplasias Hematológicas , Proteínas de Fusão Oncogênica , Proteínas Proto-Oncogênicas c-ret , Proteínas Proto-Oncogênicas , Translocação Genética , Animais , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 10/metabolismo , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 6/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Camundongos , Células NIH 3T3 , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismoRESUMO
Aberrant recruitment of histone deacetylases (HDACs) by the oncogenic fusion protein PML-RAR is involved in the pathogenesis of acute promyelocytic leukemia (APL). PML-RAR, however, is not sufficient to induce disease in mice but requires additional oncogenic lesions during the preleukemic phase. Here, we show that knock-down of Hdac1 and Hdac2 dramatically accelerates leukemogenesis in transgenic preleukemic mice. These events are not restricted to APL because lymphomagenesis driven by deletion of p53 or, to a lesser extent, by c-myc overexpression, was also accelerated by Hdac1 knock-down. In the preleukemic phase of APL, Hdac1 counteracts the activity of PML-RAR in (1) blocking differentiation; (2) impairing genomic stability; and (3) increasing self-renewal in hematopoietic progenitors, as all of these events are affected by the reduction in Hdac1 levels. This led to an expansion of a subpopulation of PML-RAR-expressing cells that is the major source of leukemic stem cells in the full leukemic stage. Remarkably, short-term treatment of preleukemic mice with an HDAC inhibitor accelerated leukemogenesis. In contrast, knock-down of Hdac1 in APL mice led to enhanced survival duration of the leukemic animals. Thus, Hdac1 has a dual role in tumorigenesis: oncosuppressive in the early stages, and oncogenic in established tumor cells.
Assuntos
Transformação Celular Neoplásica/genética , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Leucemia Promielocítica Aguda/etiologia , Leucemia Promielocítica Aguda/prevenção & controle , Proteína Supressora de Tumor p53/fisiologia , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Feminino , Citometria de Fluxo , Instabilidade Genômica , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Leucemia Promielocítica Aguda/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas , Ácido Valproico/farmacologiaRESUMO
Although epigenetic drugs have been approved for use in selected malignancies, there is significant need for a better understanding of their mechanism of action. Here, we study the action of a clinically approved DNA-methyltransferase inhibitor - decitabine (DAC) - in acute myeloid leukemia (AML) cells. At low doses, DAC treatment induced apoptosis of NB4 Acute Promyelocytic Leukemia (APL) cells, which was associated with the activation of the extrinsic apoptotic pathway. Expression studies of the members of the Death Receptor family demonstrated that DAC induces the expression of TNF-related apoptosis-inducing ligand (TRAIL). Upregulation of TRAIL, upon DAC treatment, was associated with specific epigenetic modifications induced by DAC in the proximity of the TRAIL promoter, as demonstrated by DNA demethylation, increased DNaseI sensitivity and histone acetylation of a non-CpG island, CpG-rich region located 2kb upstream to the transcription start site. Luciferase assay experiments showed that this region behave as a DNA methylation sensitive transcriptional regulatory element. The CpG regulatory element was also found methylated in samples derived from APL patients. These findings have been confirmed in the non-APL, AML Kasumi cell line, suggesting that this regulatory mechanism may be extended to other AMLs. Our study suggests that DNA methylation is a regulatory mechanism relevant for silencing of the TRAIL apoptotic pathway in leukemic cells, and further elucidates the mechanism by which epigenetic drugs mediate their anti-leukemic effects.
Assuntos
Apoptose/efeitos dos fármacos , Azacitidina/análogos & derivados , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide Aguda/fisiopatologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Azacitidina/farmacologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Decitabina , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
The urokinase plasminogen activator receptor (uPAR) fragments D1 and D2D3 are often found in biological fluids from normal individuals and patients of cancer and other diseases. The D2D3 fragment may possess chemotactic activity depending on its N-terminal sequence. We have developed a sensitive and specific immunoassay for the chemotactic form of D2D3 and show that its level can be measured with high specificity and sensitivity in human serum and urine. Synthetic peptides (residues 84-92) derived from the linker region between domains 1 and 2 of uPAR were used as immunogens to generate mouse monoclonal antibodies. Recombinant soluble uPAR (D1D2D3(1-277)) was used to immunize rabbits to obtain polyclonal antibodies. A sandwich-type immunofluorimetric assay was developed with these antibodies. The assay specifically measures D2D3 containing the 84-88 residues, has a detection limit of 0.25 ng/ml and shows no cross-reactivity with D2D3(93-274). The assay is linear at 0-30 ng/ml, with an intra-assay CV of 10% (n=20), inter-assay CV of 15% (n=9) and a recovery of D2D3(84-274) added to urine samples of between 94% and 105%. A statistically significant difference level of D2D3(84-274) was found in two groups of tumor patients versus healthy volunteers (pAssuntos
Fluorimunoensaio/métodos
, Receptores de Superfície Celular/análise
, Receptores de Superfície Celular/imunologia
, Sequência de Aminoácidos
, Animais
, Anticorpos Monoclonais/biossíntese
, Western Blotting
, Células CHO
, Células COS
, Linhagem Celular
, Fatores Quimiotáticos/análise
, Fatores Quimiotáticos/genética
, Fatores Quimiotáticos/imunologia
, Chlorocebus aethiops
, Cricetinae
, Ensaio de Imunoadsorção Enzimática
, Epitopos/análise
, Epitopos/genética
, Citometria de Fluxo
, Fluorimunoensaio/estatística & dados numéricos
, Humanos
, Imunoprecipitação
, Camundongos
, Fragmentos de Peptídeos/análise
, Fragmentos de Peptídeos/genética
, Fragmentos de Peptídeos/imunologia
, Coelhos
, Receptores de Superfície Celular/genética
, Receptores de Ativador de Plasminogênio Tipo Uroquinase
, Proteínas Recombinantes/genética
, Proteínas Recombinantes/imunologia
, Sensibilidade e Especificidade