RESUMO
Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.
Assuntos
Di-Hidropiridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Pirazóis/farmacologia , Regulação Alostérica , Sítio Alostérico , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ilhas de CpG , Cristalografia por Raios X , Citosina/química , Citosina/metabolismo , Metilação de DNA/efeitos dos fármacos , Di-Hidropiridinas/química , Di-Hidropiridinas/farmacocinética , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Granulócitos/efeitos dos fármacos , Granulócitos/enzimologia , Granulócitos/patologia , Humanos , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Cinética , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Modelos Moleculares , Mutação , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Cultura Primária de Células , Ligação Proteica , Pirazóis/química , Pirazóis/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Flow cytometry-based analysis of T-cell receptor (TCR) repertoires is an essential tool for the detection of clonal T-cell expansions in physiologic and pathologic conditions. Individual T-cell subsets can be investigated based on their surface properties. The aims of our study were to provide reference values for various disease settings and delineate the contribution of individual TCR repertoires to the human T-cell differentiation pathway. We analyzed blood of 66 healthy subjects aged 0 (cord blood) to 72 years. Lymphocyte subpopulations and TCR repertoires were simultaneously explored using antibodies specific to CD3, CD4, CD8, CD45RA, CCR7, CD27, CD57 and a set of 25 antibodies detecting human TCR-Vß chains. Statistical analysis included Wilcoxon, paired t and ANOVA tests. Initially, TCR expansion values were calculated based on the analysis of TCR-Vß distribution on CD4+ and CD8+ T cells. We then established gating strategies and an algorithm for data analysis allowing for discrimination of T-cell subsets and TCR distribution. Dominant TCR expansions were present within effector as opposed to central/effector memory or naive cells, e.g., median TCR-Vß expansion rate was highest on CD45RA+/CCR7- effector CD4+/8+ cells (eight and 11-fold, respectively). Remarkably, TCR expansions were missing (0) or very low (0.5) on CD4+ and CD8+ central memory population, respectively. No significant gender-related variability of TCR repertoires was identified, and significant impact of chronic cytomegalovirus infection was shown. Our results serve as reference for future studies elucidating clonal TCR dominance of T-cell subsets.