RESUMO
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
Epigenetic alterations in the pattern of DNA and histone modifications play a crucial role in cancer development. Analysis of patient samples, however, is hampered by technical limitations in the study of chromatin structure from pathology archives that usually consist of heavily fixed, paraffin-embedded material. Here, we present a methodology [pathology tissue-ChIP (PAT-ChIP)] to extract and immunoprecipitate chromatin from paraffin-embedded patient samples up to several years old. In a pairwise comparison with canonical ChIP, PAT-ChIP showed a high reproducibility of results for several histone marks and an identical ability to detect dynamic changes in chromatin structure upon pharmacological treatment. Finally, we showed that PAT-ChIP can be coupled with high-throughput sequencing (PAT-ChIP-Seq) for the genome-wide analysis of distinct chromatin modifications. PAT-ChIP therefore represents a versatile procedure and diagnostic tool for the analysis of epigenetic alterations in cancer and potentially other diseases.
Assuntos
Imunoprecipitação da Cromatina/métodos , Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Animais , Humanos , Neoplasias/genética , Neoplasias/patologia , Processamento de Proteína Pós-Traducional , Fixação de Tecidos/métodosRESUMO
Lysine-specific demethylase 1 (LSD1 or KDM1A) is a FAD-dependent enzyme that acts as a transcription corepressor or coactivator by regulating the methylation status of histone H3 lysines K4 and K9, respectively. KDM1A represents an attractive target for cancer therapy. While, in the past, the main medicinal chemistry strategy toward KDM1A inhibition was based on the optimization of ligands that irreversibly bind the FAD cofactor within the enzyme catalytic site, we and others have also identified reversible inhibitors. Herein we reported the discovery of 5-imidazolylthieno[3,2-b]pyrroles, a new series of KDM1A inhibitors endowed with picomolar inhibitory potency, active in cells and efficacious after oral administration in murine leukemia models.
RESUMO
We report the stereoselective synthesis and biological activity of a novel series of tranylcypromine (TCPA) derivatives (14a-k, 15, 16), potent inhibitors of KDM1A. The new compounds strongly inhibit the clonogenic potential of acute leukemia cell lines. In particular three molecules (14d, 14e, and 14g) showing selectivity versus MAO A and remarkably inhibiting colony formation in THP-1 human leukemia cells, were assessed in mouse for their preliminary pharmacokinetic. 14d and 14e were further tested in vivo in a murine acute promyelocytic leukemia model, resulting 14d the most effective. Its two enantiomers were synthesized: the (1S,2R) enantiomer 15 showed higher activity than its (1R,2S) analogue 16, in both biochemical and cellular assays. Compound 15 exhibited in vivo efficacy after oral administration, determining a 62% increased survival in mouse leukemia model with evidence of KDM1A inhibition. The biological profile of compound 15 supports its further investigation as a cancer therapeutic.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Histona Desmetilases/antagonistas & inibidores , Leucemia Promielocítica Aguda/tratamento farmacológico , Tranilcipromina/química , Tranilcipromina/uso terapêutico , Administração Oral , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Histona Desmetilases/metabolismo , Humanos , Leucemia Promielocítica Aguda/metabolismo , Relação Estrutura-Atividade , Tranilcipromina/administração & dosagem , Tranilcipromina/farmacologiaRESUMO
Mcl-1 is a unique Bcl-2 family member that plays crucial roles in apoptosis. Apoptosis-unrelated functions of Mcl-1 are however emerging, further justifying its tight regulation. Here we unravel a novel mechanism of Mcl-1 regulation mediated by the haplo-insufficient tumour suppressor Beclin 1. Beclin 1 negatively modulates Mcl-1 stability in a reciprocal manner whereby depletion of one leads to the stabilization of the other. This co-regulation is independent of autophagy and of their physical interaction. Both Beclin 1 and Mcl-1 are deubiquitinated and thus stabilized by binding to a common deubiquitinase, USP9X. Beclin 1 and Mcl-1 negatively modulate the proteasomal degradation of each other through competitive displacement of USP9X. The analysis of patient-derived melanoma cells and tissue samples shows that the levels of Beclin 1 decrease, while Mcl-1 levels subsequently increase during melanoma progression in a significant inter-dependent manner. The identified inverse co-regulation of Beclin 1 and Mcl-1 represents a mechanism of functional counteraction in cancer.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Carcinogênese , Melanoma/metabolismo , Proteínas de Membrana/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Autofagia , Proteína Beclina-1 , Células HEK293 , Haploinsuficiência , Células HeLa , Humanos , Melanoma/secundário , Camundongos , Transplante de Neoplasias , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas , Ubiquitina Tiolesterase/metabolismoRESUMO
Heat-shock protein 90 (Hsp90) is a molecular chaperone involved in the stabilization of key oncogenic signaling proteins, and therefore, inhibition of Hsp90 represents a new strategy in cancer therapy. 2-Amino-7-[4-fluoro-2-(3-pyridyl)phenyl]-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime is a racemic Hsp90 inhibitor that targets the N-terminal adenosine triphosphatase site. We developed a method to resolve the enantiomers and evaluated their inhibitory activity on Hsp90 and the consequent antitumor effects. The (S) stereoisomer emerged as a potent Hsp90 inhibitor in biochemical and cellular assays. In addition, this enantiomer exhibited high oral bioavailability in mice and excellent antitumor activity in two different human cancer xenograft models.
Assuntos
Antineoplásicos/química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Oximas/química , Quinazolinonas/química , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sítios de Ligação , Linhagem Celular Tumoral , Feminino , Células HCT116 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Camundongos Nus , Microssomos/metabolismo , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Oximas/farmacologia , Oximas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Estereoisomerismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of spiro[chromane-2,4'-piperidine] derivatives based on a previously published lead benzyl spirocycle 1 and bearing various N-aryl and N-alkylaryl substituents on the piperidine ring were prepared as novel histone deacetylase (HDAC) inhibitors. The compounds were evaluated for their abilities to inhibit nuclear HDACs, their in vitro antiproliferative activities, and in vitro ADME profiles. Based on these activities, 4-fluorobenzyl and 2-phenylethyl spirocycles were selected for further characterization. In vivo pharmacokinetic (PK) studies showed that both compounds exhibit an overall lower clearance rate, an increased half-life, and higher AUCs after intravenous and oral administration than spiropiperidine 1 under the conditions used. The improved PK behavior of these two compounds also correlated with superior in vivo antitumor activity in an HCT-116 xenograft model.
Assuntos
Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Piperidinas/química , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Proteínas Sanguíneas/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450 , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/farmacocinética , Injeções Intravenosas , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Estrutura Molecular , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
New spiro[chromane-2,4'-piperidine] and spiro[benzofuran-2,4'-piperidine] hydroxamic acid derivatives as HDAC inhibitors have been identified by combining privileged structures with a hydroxamic acid moiety as zinc binding group. The compounds were evaluated for their ability to inhibit nuclear extract HDACs and for their in vitro antiproliferative activity on different tumor cell lines. This work resulted in the discovery of spirocycle 30d that shows good oral bioavailability and tumor growth inhibition in an HCT-116 murine xenograft model.
Assuntos
Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores de Histona Desacetilases/síntese química , Humanos , Ácidos Hidroxâmicos/síntese química , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Two major mechanisms have been causally implicated in the establishment of cellular senescence: the activation of the DNA damage response (DDR) pathway and the formation of senescence-associated heterochromatic foci (SAHF). Here we show that in human fibroblasts resistant to premature p16(INK4a) induction, SAHF are preferentially formed following oncogene activation but are not detected during replicative cellular senescence or on exposure to a variety of senescence-inducing stimuli. Oncogene-induced SAHF formation depends on DNA replication and ATR (ataxia telangiectasia and Rad3-related). Inactivation of ATM (ataxia telangiectasia mutated) or p53 allows the proliferation of oncogene-expressing cells that retain increased heterochromatin induction. In human cancers, levels of heterochromatin markers are higher than in normal tissues, and are independent of the proliferative index or stage of the tumours. Pharmacological and genetic perturbation of heterochromatin in oncogene-expressing cells increase DDR signalling and lead to apoptosis. In vivo, a histone deacetylase inhibitor (HDACi) causes heterochromatin relaxation, increased DDR, apoptosis and tumour regression. These results indicate that heterochromatin induced by oncogenic stress restrains DDR and suggest that the use of chromatin-modifying drugs in cancer therapies may benefit from the study of chromatin and DDR status of tumours.