RESUMO
Fusion proteins involving Nucleoporin 98 (NUP98) are recurrently found in acute myeloid leukemia (AML) and are associated with poor prognosis. Lack of mechanistic insight into NUP98-fusion-dependent oncogenic transformation has so far precluded the development of rational targeted therapies. We reasoned that different NUP98-fusion proteins deregulate a common set of transcriptional targets that might be exploitable for therapy. To decipher transcriptional programs controlled by diverse NUP98-fusion proteins, we developed mouse models for regulatable expression of NUP98/NSD1, NUP98/JARID1A, and NUP98/DDX10. By integrating chromatin occupancy profiles of NUP98-fusion proteins with transcriptome profiling upon acute fusion protein inactivation in vivo, we defined the core set of direct transcriptional targets of NUP98-fusion proteins. Among those, CDK6 was highly expressed in murine and human AML samples. Loss of CDK6 severely attenuated NUP98-fusion-driven leukemogenesis, and NUP98-fusion AML was sensitive to pharmacologic CDK6 inhibition in vitro and in vivo. These findings identify CDK6 as a conserved, critical direct target of NUP98-fusion proteins, proposing CDK4/CDK6 inhibitors as a new rational treatment option for AML patients with NUP98-fusions.
Assuntos
Quinase 6 Dependente de Ciclina/metabolismo , Sistemas de Liberação de Medicamentos , Leucemia Mieloide Aguda/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Animais , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/genética , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genéticaRESUMO
Single-cell isolation and transcriptomic analysis of a specific cell type or tissue offers the possibility of studying cell function and heterogeneity in time-dependent processes with remarkable resolution. The reduced tissue complexity and highly stereotyped development of Caenorhabditis elegans, combined with an extensive genetic toolbox and the ease of growing large tightly synchronized populations makes it an exceptional model organism for the application of such approaches. However, the difficulty to dissociate and isolate single cells from larval stages has been a major constraint to this kind of studies. Here, we describe an improved protocol for dissociation and preparation of single cell suspensions from developmentally synchronized populations of C. elegans L1 larvae. Our protocol has been empirically optimized to allow efficient FACS-based purification of large number of single cells from rare cell types, for subsequent extraction and sequencing of their mRNA.