RESUMO
Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). More effective strategies to increase HbF are needed because the current standard of care, hydroxyurea, is not effective in a significant proportion of patients. Treatment of the millions of patients projected worldwide would best be accomplished with an orally administered drug therapy that increased HbF. LSD1 is a component of corepressor complexes that repress γ-globin gene expression and are a therapeutic target for HbF reactivation. We have shown that subcutaneous administration of RN-1, a pharmacological LSD1 inhibitor, increased γ-globin expression in SCD mice and baboons, which are widely acknowledged as the best animal model in which to test the activity of HbF-inducing drugs. The objective of this investigation was to test the effect of oral administration of a new LSD1 inhibitor, ORY-3001. Oral administration of ORY-3001 to SCD mice (nâ¯=â¯3 groups) increased γ-globin expression, Fetal Hemoglobin (HbF)-containing (F) cells, and F reticulocytes (retics). In normal baboons (nâ¯=â¯7 experiments) treated with ORY-3001, increased F retics, γ-globin chain synthesis, and γ-globin mRNA were observed. Experiments in anemic baboons (nâ¯=â¯2) showed that ORY-3001 increased F retics (PA8695, predoseâ¯=â¯24%, postdoseâ¯=â¯66.8%; PA8698: predoseâ¯=â¯13%, postdoseâ¯=â¯93.6%), γ-globin chain synthesis (PA8695: predoseâ¯=â¯0.07 γ/γ+ß, postdoseâ¯=â¯0.20 γ/γ+ß; PA8698: predoseâ¯=â¯0.02 γ/γ+ß, postdoseâ¯=â¯0.44 γ/γ+ß), and γ-globin mRNA (PA8695: predoseâ¯=â¯0.06 γ/γ+ß, postdoseâ¯=â¯0.18 γ/γ+ß; PA8698: predoseâ¯=â¯0.03 γ/γ+ß, postdoseâ¯=â¯0.33 γ/γ+ß). We conclude that oral administration of ORY-3001 increases F retics, γ-globin chain synthesis, and γ-globin mRNA in baboons and SCD mice, supporting further efforts toward the development of this drug for SCD therapy.
Assuntos
Anemia Falciforme/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Hemoglobina Fetal/biossíntese , Histona Desmetilases/antagonistas & inibidores , gama-Globinas/biossíntese , Administração Oral , Anemia/sangue , Anemia/tratamento farmacológico , Anemia Falciforme/sangue , Animais , Contagem de Células Sanguíneas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Feminino , Hemoglobina Fetal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Papio , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reticulócitos/metabolismo , gama-Globinas/genéticaRESUMO
The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Citosina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Células Eritroides/citologia , Regiões Promotoras Genéticas/efeitos dos fármacos , gama-Globinas/metabolismo , 5-Metilcitosina/metabolismo , Animais , Animais Recém-Nascidos , Antineoplásicos/farmacologia , Ácido Ascórbico/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Células da Medula Óssea , Células Cultivadas , Citosina/metabolismo , Citosina/farmacologia , Decitabina , Dioxigenases/metabolismo , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Histona Desmetilases/metabolismo , Humanos , Hidroxiureia/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Papio anubis , Tranilcipromina/farmacologiaRESUMO
Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates.