RESUMO
Differentiation therapy is a less toxic but still a very effective treatment for a subset of acute myeloid leukaemia (AML) cases. With the goal to identify novel compounds that can effectively and safely induce the terminal differentiation of non-acute promyelocytic leukaemia (APL) AML cells, we performed a chemical screening and identified albendazole (ABZ), a widely used anti-helminthic drug, as a promising lead compound that can differentiate non-APL AML cells by stimulating the Krüppel-like factor 4-dihydropyrimidinase-like 2A (KLF4-DPYSL2A) differentiation axis to the monocytes. Our in vitro and in vivo findings demonstrate that ABZ is an attractive candidate drug as a novel differentiation chemotherapy for patients with non-APL AML.
Assuntos
Albendazol/farmacologia , Anti-Helmínticos/farmacologia , Antineoplásicos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fator 4 Semelhante a Kruppel/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas do Tecido Nervoso/metabolismo , Albendazol/uso terapêutico , Animais , Anti-Helmínticos/uso terapêutico , Antineoplásicos/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Monócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Traditionally, the central nervous system (CNS) has been viewed as an immune-privileged environment with no lymphatic vessels. This view was partially overturned by the discovery of lymphatic vessels in the dural membrane that surrounds the brain, in contact with the interior surface of the skull. We here examine the distribution and developmental timing of these lymphatic vessels. RESULTS: Using the Prox1-GFP BAC transgenic reporter and immunostaining with antibodies to lymphatic markers LYVE-1, Prox1, and Podoplanin, we have carried out whole-mount imaging of dural lymphatic vasculature at postnatal stages. We have found that between birth and postnatal day (P) 13, lymphatic vessels extend alongside dural blood vessels from the side of the skull toward the midline. Between P13 and P20, lymphatic vessels along the transverse sinuses reach the superior sagittal sinus (SSS) and extend along the SSS toward the olfactory bulb. CONCLUSIONS: Compared with the embryonic developmental timing of lymphatic vessels in other tissues, e.g. skin, dural lymphatic vessel development is dramatically delayed. This study provides useful anatomical data for continuing investigations of the fundamental mechanisms that underlie dural lymphatic vessel development. Developmental Dynamics 247:741-753, 2018. © 2018 Wiley Periodicals, Inc.
Assuntos
Vasos Linfáticos/embriologia , Animais , Encéfalo/irrigação sanguínea , Meninges/embriologia , Camundongos , Camundongos Transgênicos , Pele/embriologiaRESUMO
BACKGROUND: Overexpression of CXC chemokine receptor 4 (CXCR4+) is a poor prognostic factor in adult acute myeloid leukemia (AML); however, its prognostic significance in pediatric AML is unclear. PROCEDURE: This retrospective study examined the prognostic significance of CXCR4+ in pediatric AML patients enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 study. RESULTS: In the total cohort (n = 248), no significant differences were observed between CXCR4+ patients (n = 81) and CXCR4- patients (n = 167) in terms of 3-year overall survival (OS) (69.4% vs. 75.2%, P = 0.44). However, there was a significant difference in 3-year OS between CXCR4+ and CXCR4- patients in the low-risk (LR) group (n = 93; 79.2% vs. 98.3%, P = 0.007). CXCR4+ patients in the t(8;21) AML without KIT mutation group had a significantly worse 3-year OS than CXCR4- patients (n = 44; 76.1% vs. 100.0%, P = 0.01). Multivariate Cox regression analysis identified CXCR4+ as a poor prognostic factor for OS in LR AML patients (hazard ratio, 11.47; P = 0.01). Consistent with the data for survival analysis, CXCR4+ patients in the t(8;21) AML group had a higher incidence of splenomegaly than CXCR4- patients (25.9% vs. 5.9%, P = 0.03). CONCLUSIONS: These results suggest that CXCR4+ is a poor prognostic factor for LR patients, particularly t(8;21) patients without KIT mutation. The poor outcome was only applicable to OS, not relapse-free survival (RFS); thus, CXCR4+ may be associated with a poor prognosis after recurrence. Intensive therapy, including administration of CXCR4 antagonists, may be promising for pediatric AML patients with LR.
Assuntos
Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Receptores CXCR4/biossíntese , Adolescente , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Japão , Masculino , Mutação , Prognóstico , Proteínas Proto-Oncogênicas c-kit/genética , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Estudos Retrospectivos , Esplenomegalia/patologia , Análise de SobrevidaRESUMO
Children with Down syndrome (DS) are susceptible to two blood disorders, transient abnormal myelopoiesis (TAM) and Down syndrome-associated acute megakaryocytic leukemia (DS-AMKL). Mutations in GATA binding protein 1 (GATA1) have been identified as the cause of these diseases, and the expression levels of the resulting protein, short-form GATA1 (GATA1s), are known to correlate with the severity of TAM. On the other hand, despite the presence of GATA1 mutations in almost all cases of DS-AMKL, the incidence of DS-AMKL in TAM patients is inversely correlated with the expression of GATA1s. This discovery has required the need to clarify the role of GATA1s in generating the cells of origin linked to the risk of both diseases. Focusing on this point, we examined the characteristics of GATA1 mutant trisomy-21 pluripotent stem cells transfected with a doxycycline (Dox)-inducible GATA1s expression cassette in a stepwise hematopoietic differentiation protocol. We found that higher GATA1s expression significantly reduced commitment into the megakaryocytic lineage at the early hematopoietic progenitor cell (HPC) stage, but once committed, the effect was reversed in progenitor cells and acted to maintain the progenitors. These differentiation stage-dependent reversal effects were in contrast to the results of myeloid lineage, where GATA1s simply sustained and increased the number of immature myeloid cells. These results suggest that although GATA1 mutant cells cause the increase in myeloid and megakaryocytic progenitors regardless of the intensity of GATA1s expression, the pathways vary with the expression level. This study provides experimental support for the paradoxical clinical features of GATA1 mutations in the two diseases.
Assuntos
Síndrome de Down/sangue , Fator de Transcrição GATA1/metabolismo , Hematopoese/genética , Células-Tronco Embrionárias Humanas/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Síndrome de Down/genética , Doxiciclina/farmacologia , Fator de Transcrição GATA1/genética , Humanos , Leucemia Megacarioblástica Aguda/sangue , Leucemia Megacarioblástica Aguda/genética , Reação Leucemoide/sangue , Reação Leucemoide/genética , Megacariócitos/metabolismo , Células Mieloides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção/métodos , Trissomia/genéticaRESUMO
The t(8;21) translocation is the most common cytogenetic abnormality in acute myeloid leukemia (AML). Although t(8;21) AML patients have a relatively favorable prognosis, relapse is a frequent occurrence, underscoring the need to develop novel therapeutic approaches. Here, we showed that t(8;21) AML is characterized by frequent mutation and overexpression of CCND2. Analysis of 19 AML cell lines showed that t(8;21) AML cells had lower IC50 values for the selective CDK4/6 inhibitors palbociclib and abemaciclib than non-t(8;21) AML cells. CDK4/6 inhibitors caused cell cycle arrest at G1 phase and impaired cell proliferation in t(8;21) AML cells. CDK4/6 inhibition decreased MAP-ERK and PI3K-AKT-mTOR signaling pathway activity, induced LC3B-I to LC3B-II conversion, and enhanced autophagosome formation, suggesting autophagy induction. Treatment of t(8;21) AML cells with the autophagy inhibitors chloroquine (CQ) or LY294002 in combination with the CDK4/6 inhibitor abemaciclib significantly increased the percentage of apoptotic (Annexin V positive) cells, whereas CQ or LY294002 single treatment had no significant effects. The effectiveness of co-inhibiting CDK4/6 and autophagy was confirmed in primary t(8;21) AML cells. The results suggest that the combination of CDK4/6 and autophagy inhibitors had a synergistic effect on inducing apoptosis, suggesting a novel therapeutic approach for the treatment of t(8;21) AML.
Assuntos
Apoptose/genética , Autofagia/genética , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Leucemia Mieloide Aguda/genética , Translocação Genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Ciclina D2/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Interferência de RNA , Transdução de Sinais/efeitos dos fármacosRESUMO
Down syndrome (DS) is caused by the trisomy of chromosome 21. Among the many disabilities found in individuals with DS is an increased risk of early-onset Alzheimer's disease (AD). Although higher oxidative stress and an upregulation of amyloid ß (Aß) peptides from an extra copy of the APP gene are attributed to the AD susceptibility, the relationship between the two factors is unclear. To address this issue, we established an in vitro cellular model using neurons differentiated from DS patient-derived induced pluripotent stem cells (iPSCs) and isogenic euploid iPSCs. Neurons differentiated from DS patient-derived iPSCs secreted more Aß compared to those differentiated from the euploid iPSCs. Treatment of the neurons with an antioxidant, N-acetylcysteine, significantly suppressed the Aß secretion. These findings suggest that oxidative stress has an important role in controlling the Aß level in neurons differentiated from DS patient-derived iPSCs and that N-acetylcysteine can be a potential therapeutic option to ameliorate the Aß secretion.
Assuntos
Acetilcisteína/farmacologia , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/farmacologia , Síndrome de Down/genética , Regulação para Baixo/efeitos dos fármacos , Doença de Alzheimer/etiologia , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Síndrome de Down/complicações , Síndrome de Down/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacosRESUMO
Although the biological importance of Krüppel-like factor 4 (KLF4) transcription factor in the terminal differentiation of hematopoietic cells to the monocytes has been well established, the underlying mechanisms remain elusive. To clarify the molecular basis of KLF4-mediated monocytic differentiation, we performed detailed genetic studies in acute myeloid leukemia (AML) cells. Here, we report that dihydropyrimidinase like 2 (DPYSL2), also known as CRMP2, is a novel key differentiation mediator downstream of KLF4 in AML cells. Interestingly, we discovered that KLF4-mediated monocytic differentiation is selectively dependent on one specific isoform, DPYSL2A, but not on other DPYSL family genes. Terminal differentiation to the monocytes and proliferation arrest in AML cells induced by genetic or pharmacological upregulation of KLF4 were significantly reversed by short hairpin RNA (shRNA)-mediated selective depletion of DPYSL2A. Chromatin immunoprecipitation assay revealed that KLF4 associates with the proximal gene promoter of DPYSL2A and directly transactivates its expression. Together with the unique expression patterns of KLF4 and DPYSL2 limited to the differentiated monocytes in the hematopoietic system both in human and mouse, the identified KLF4-DPYSL2 axis in leukemia cells may serve as a potential therapeutic target for the development of novel differentiation therapies for patients with AML.
Assuntos
Diferenciação Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Leucemia Mieloide Aguda/patologia , Monócitos/citologia , Proteínas do Tecido Nervoso/fisiologia , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica , Humanos , Fator 4 Semelhante a Kruppel , Leucemia Mieloide Aguda/genética , Regiões Promotoras GenéticasRESUMO
The functional effect of a gene edit by designer nucleases depends on the DNA repair outcome at the targeted locus. While non-homologous end joining (NHEJ) repair results in various mutations, microhomology-mediated end joining (MMEJ) creates precise deletions based on the alignment of flanking microhomologies (µHs). Recently, the sequence context surrounding nuclease-induced double strand breaks (DSBs) has been shown to predict repair outcomes, for which µH plays an important role. Here, we survey naturally occurring human deletion variants and identify that 11 million or 57% are flanked by µHs, covering 88% of protein-coding genes. These biologically relevant mutations are candidates for precise creation in a template-free manner by MMEJ repair. Using CRISPR-Cas9 in human induced pluripotent stem cells (hiPSCs), we efficiently create pathogenic deletion mutations for demonstrable disease models with both gain- and loss-of-function phenotypes. We anticipate this dataset and gene editing strategy to enable functional genetic studies and drug screening.
Assuntos
Reparo do DNA por Junção de Extremidades/genética , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Deleção de Sequência/genética , Sequência de Bases , Sistemas CRISPR-Cas , Mutação com Ganho de Função , Humanos , Mutação com Perda de FunçãoRESUMO
Obtaining highly purified differentiated cells via directed differentiation from human pluripotent stem cells (hPSCs) is an essential step for their clinical application. Among the various conditions that should be optimized, the precise role and contribution of the extracellular matrix (ECM) during differentiation are relatively unclear. Here, using a short fragment of laminin 411 (LM411-E8), an ECM predominantly expressed in the vascular endothelial basement membrane, we demonstrate that the directed switching of defined ECMs robustly yields highly-purified (>95%) endothelial progenitor cells (PSC-EPCs) without cell sorting from hPSCs in an integrin-laminin axis-dependent manner. Single-cell RNA-seq analysis revealed that LM411-E8 resolved intercellular transcriptional heterogeneity and escorted the progenitor cells to the appropriate differentiation pathway. The PSC-EPCs gave rise to functional endothelial cells both in vivo and in vitro. We therefore propose that sequential switching of defined matrices is an important concept for guiding cells towards desired fate.