RESUMO
The endothelial cell adhesion molecule E-selectin is a key component of the bone marrow hematopoietic stem cell (HSC) vascular niche regulating balance between HSC self-renewal and commitment. We now report in contrast, E-selectin directly triggers signaling pathways that promote malignant cell survival and regeneration. Using acute myeloid leukemia (AML) mouse models, we show AML blasts release inflammatory mediators that upregulate endothelial niche E-selectin expression. Alterations in cell-surface glycosylation associated with oncogenesis enhances AML blast binding to E-selectin and enable promotion of pro-survival signaling through AKT/NF-κB pathways. In vivo AML blasts with highest E-selectin binding potential are 12-fold more likely to survive chemotherapy and main contributors to disease relapse. Absence (in Sele-/- hosts) or therapeutic blockade of E-selectin using small molecule mimetic GMI-1271/Uproleselan effectively inhibits this niche-mediated pro-survival signaling, dampens AML blast regeneration, and strongly synergizes with chemotherapy, doubling the duration of mouse survival over chemotherapy alone, whilst protecting endogenous HSC.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Selectina E/antagonistas & inibidores , Selectina E/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Medula Óssea , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Glicolipídeos/uso terapêutico , Glicosilação , Células-Tronco Hematopoéticas/citologia , Humanos , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Myeloproliferative neoplasms (MPNs) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells and an increased risk of late transformation to acute myeloid leukemia or primary myelofibrosis. Approximately 15% of MPN cases do not carry mutations in JAK2, CALR, or MPL and are thus often referred to as triple-negative cases. These are caused by a diverse set of rare mutations in cytokine receptors, JAK-STAT signaling pathway components, or epigenetic modifiers. In addition, some cases diagnosed as MPN are reactive rather than clonal disorders, so a negative result from a genetic screen can be informative. To obtain a comprehensive rapid molecular diagnosis for most MPNs, we developed an assay to detect genetic mutations (single nucleotide variants and/or small insertions/deletions) in 86 genes using targeted exon resequencing (AmpliSeq) and a bench-top semiconductor machine (Ion Torrent Personal Genome Machine). Our assay reliably detects well characterized mutations in JAK2, CALR, and MPL, but also rarer mutations in ASXL1, TET2, SH2B3, and other genes. Some of these mutations are novel. We find multiple mutations in advanced cases, suggesting co-operation between Janus kinase-STAT pathway mutations and epigenetic mutations in disease progression. This assay can be used to follow molecular progression, clonal heterogeneity, and drug resistance in MPNs.
Assuntos
Epigênese Genética , Éxons , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genética , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Biomarcadores , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Janus Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Mutação , Transtornos Mieloproliferativos/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição STAT/metabolismo , Sensibilidade e Especificidade , Transdução de SinaisRESUMO
KLF1 (formerly known as EKLF) regulates the development of erythroid cells from bi-potent progenitor cells via the transcriptional activation of a diverse set of genes. Mice lacking Klf1 die in utero prior to E15 from severe anemia due to the inadequate expression of genes controlling hemoglobin production, cell membrane and cytoskeletal integrity, and the cell cycle. We have recently described the full repertoire of KLF1 binding sites in vivo by performing KLF1 ChIP-seq in primary erythroid tissue (E14.5 fetal liver). Here we describe the KLF1-dependent erythroid transcriptome by comparing mRNA-seq from Klf1(+/+) and Klf1(-/-) erythroid tissue. This has revealed novel target genes not previously obtainable by traditional microarray technology, and provided novel insights into the function of KLF1 as a transcriptional activator. We define a cis-regulatory module bound by KLF1, GATA1, TAL1, and EP300 that coordinates a core set of erythroid genes. We also describe a novel set of erythroid-specific promoters that drive high-level expression of otherwise ubiquitously expressed genes in erythroid cells. Our study has identified two novel lncRNAs that are dynamically expressed during erythroid differentiation, and discovered a role for KLF1 in directing apoptotic gene expression to drive the terminal stages of erythroid maturation.
Assuntos
Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Kruppel-Like/genética , RNA Mensageiro/genética , Transcriptoma , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Diferenciação Celular , Mapeamento Cromossômico , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Células Eritroides/citologia , Células Eritroides/metabolismo , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Perfilação da Expressão Gênica , Marcação In Situ das Extremidades Cortadas , Fatores de Transcrição Kruppel-Like/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Commitment towards megakaryocyte versus erythroid blood cell lineages occurs in the megakaryocyte-erythroid progenitor, where mutually exclusive expression of either EKLF (Klf1) or Fli1 defines alternative outcomes. Here we show there is a marked increase in the number of circulating platelets in mice lacking the erythroid transcription factor EKLF. In addition, committed erythroid cells retain key signatures of megakaryocytes both on the cell surface and at the mRNA level. We also show that the effect of EKLF on megakaryocyte-erythroid progenitor lineage decision and commitment is cell autonomous in bone marrow reconstitution assays where stem cells lacking EKLF favor the megakaryocyte differentiation pathway. We conclude the megakaryocyte program is aberrantly activated in EKLF null erythroid cells.
Assuntos
Linhagem da Célula/genética , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Fatores de Transcrição Kruppel-Like/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos TransgênicosRESUMO
Differentiation of erythroid cells requires precise control over the cell cycle to regulate the balance between cell proliferation and differentiation. The zinc finger transcription factor, erythroid Krüppel-like factor (EKLF/KLF1), is essential for proper erythroid cell differentiation and regulates many erythroid genes. Here we show that loss of EKLF leads to aberrant entry into S-phase of the cell cycle during both primitive and definitive erythropoiesis. This cell cycle defect was associated with a significant reduction in the expression levels of E2f2 and E2f4, key factors necessary for the induction of S-phase gene expression and erythropoiesis. We found and validated novel intronic enhancers in both the E2f2 and E2f4 genes, which contain conserved CACC, GATA, and E-BOX elements. The E2f2 enhancer was occupied by EKLF in vivo. Furthermore, we were able to partially restore cell cycle dynamics in EKLF(-/-) fetal liver upon additional genetic depletion of Rb, establishing a genetic causal link between reduced E2f2 and the EKLF cell cycle defect. Finally, we propose direct regulation of the E2f2 enhancer is a generic mechanism by which many KLFs regulate proliferation and differentiation.