RESUMO
Curtailed protein translation ensures stemness and multipotency in embryonic and adult tissue-specific stem cells. In this issue of Cell, a study led by Zhao and colleagues uncovered increased susceptibility of hematopoietic stem cells (HSC) to iron-dependent programmed necrotic cell death (ferroptosis) as a consequence of low protein synthesis.
Assuntos
Células-Tronco Hematopoéticas , Biossíntese de Proteínas , Proliferação de Células , FerroptoseRESUMO
The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells1. This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division2-4. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)5, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.
Assuntos
Autofagia Mediada por Chaperonas/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Adulto , Idoso , Envelhecimento , Animais , Autorrenovação Celular , Células Cultivadas , Autofagia Mediada por Chaperonas/efeitos dos fármacos , Autofagia Mediada por Chaperonas/genética , Metabolismo Energético , Feminino , Glicólise , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Ácido Linoleico/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Rejuvenescimento , Adulto JovemRESUMO
Dysregulated apoptosis and proliferation are fundamental properties of cancer, and microRNAs (miRNA) are critical regulators of these processes. Loss of miR-15a/16-1 at chromosome 13q14 is the most common genomic aberration in chronic lymphocytic leukemia (CLL). Correspondingly, the deletion of either murine miR-15a/16-1 or miR-15b/16-2 locus in mice is linked to B cell lymphoproliferative malignancies. However, unexpectedly, when both miR-15/16 clusters are eliminated, most double knockout (DKO) mice develop acute myeloid leukemia (AML). Moreover, in patients with CLL, significantly reduced expression of miR-15a, miR-15b, and miR-16 associates with progression of myelodysplastic syndrome to AML, as well as blast crisis in chronic myeloid leukemia. Thus, the miR-15/16 clusters have a biological relevance for myeloid neoplasms. Here, we demonstrate that the myeloproliferative phenotype in DKO mice correlates with an increase of hematopoietic stem and progenitor cells (HSPC) early in life. Using single-cell transcriptomic analyses, we presented the molecular underpinning of increased myeloid output in the HSPC of DKO mice with gene signatures suggestive of dysregulated hematopoiesis, metabolic activities, and cell cycle stages. Functionally, we found that multipotent progenitors (MPP) of DKO mice have increased self-renewing capacities and give rise to significantly more progeny in the granulocytic compartment. Moreover, a unique transcriptomic signature of DKO MPP correlates with poor outcome in patients with AML. Together, these data point to a unique regulatory role for miR-15/16 during the early stages of hematopoiesis and to a potentially useful biomarker for the pathogenesis of myeloid neoplasms.
Assuntos
Leucemia Linfocítica Crônica de Células B , Leucemia Mieloide Aguda , MicroRNAs , Transtornos Mieloproliferativos , Humanos , Animais , Camundongos , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Divisão Celular , Transtornos Mieloproliferativos/genéticaRESUMO
How hematopoietic stem cells (HSCs) coordinate the regulation of opposing cellular mechanisms such as self-renewal and differentiation commitment remains unclear. Here we identified the transcription factor and chromatin remodeler Satb1 as a critical regulator of HSC fate. HSCs lacking Satb1 had defective self-renewal, were less quiescent and showed accelerated lineage commitment, which resulted in progressive depletion of functional HSCs. The enhanced commitment was caused by less symmetric self-renewal and more symmetric differentiation divisions of Satb1-deficient HSCs. Satb1 simultaneously repressed sets of genes encoding molecules involved in HSC activation and cellular polarity, including Numb and Myc, which encode two key factors for the specification of stem-cell fate. Thus, Satb1 is a regulator that promotes HSC quiescence and represses lineage commitment.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Polaridade Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pbio.3000301.].
RESUMO
Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular â¼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.
Assuntos
Modelos Animais de Doenças , GTP Fosfo-Hidrolases/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mielomonocítica Crônica/patologia , Proteínas de Membrana/genética , Mutação , Proteínas Repressoras/genética , Microambiente Tumoral , Animais , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/imunologia , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Fenótipo , Transdução de SinaisRESUMO
Chaperone-mediated autophagy (CMA) contributes to the lysosomal degradation of a selective subset of proteins. Selectivity lies in the chaperone heat shock cognate 71 kDa protein (HSC70) recognizing a pentapeptide motif (KFERQ-like motif) in the protein sequence essential for subsequent targeting and degradation of CMA substrates in lysosomes. Interest in CMA is growing due to its recently identified regulatory roles in metabolism, differentiation, cell cycle, and its malfunctioning in aging and conditions such as cancer, neurodegeneration, or diabetes. Identification of the subset of the proteome amenable to CMA degradation could further expand our understanding of the pathophysiological relevance of this form of autophagy. To that effect, we have performed an in silico screen for KFERQ-like motifs across proteomes of several species. We have found that KFERQ-like motifs are more frequently located in solvent-exposed regions of proteins, and that the position of acidic and hydrophobic residues in the motif plays the most important role in motif construction. Cross-species comparison of proteomes revealed higher motif conservation in CMA-proficient species. The tools developed in this work have also allowed us to analyze the enrichment of motif-containing proteins in biological processes on an unprecedented scale and discover a previously unknown association between the type and combination of KFERQ-like motifs in proteins and their participation in specific biological processes. To facilitate further analysis by the scientific community, we have developed a free web-based resource (KFERQ finder) for direct identification of KFERQ-like motifs in any protein sequence. This resource will contribute to accelerating understanding of the physiological relevance of CMA.
Assuntos
Motivos de Aminoácidos , Autofagia Mediada por Chaperonas , Proteoma/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Drosophila melanogaster/genética , Evolução Molecular , Humanos , Camundongos , Células NIH 3T3 , Proteoma/química , Saccharomyces cerevisiae/genéticaRESUMO
Epigenetic regulators are recurrently mutated and aberrantly expressed in acute myeloid leukemia (AML). Targeted therapies designed to inhibit these chromatin-modifying enzymes, such as the histone demethylase lysine-specific demethylase 1 (LSD1) and the histone methyltransferase DOT1L, have been developed as novel treatment modalities for these often refractory diseases. A common feature of many of these targeted agents is their ability to induce myeloid differentiation, suggesting that multiple paths toward a myeloid gene expression program can be engaged to relieve the differentiation blockade that is uniformly seen in AML. We performed a comparative assessment of chromatin dynamics during the treatment of mixed lineage leukemia (MLL)-AF9-driven murine leukemias and MLL-rearranged patient-derived xenografts using 2 distinct but effective differentiation-inducing targeted epigenetic therapies, the LSD1 inhibitor GSK-LSD1 and the DOT1L inhibitor EPZ4777. Intriguingly, GSK-LSD1 treatment caused global gains in chromatin accessibility, whereas treatment with EPZ4777 caused global losses in accessibility. We captured PU.1 and C/EBPα motif signatures at LSD1 inhibitor-induced dynamic sites and chromatin immunoprecipitation coupled with high-throughput sequencing revealed co-occupancy of these myeloid transcription factors at these sites. Functionally, we confirmed that diminished expression of PU.1 or genetic deletion of C/EBPα in MLL-AF9 cells generates resistance of these leukemias to LSD1 inhibition. These findings reveal that pharmacologic inhibition of LSD1 represents a unique path to overcome the differentiation block in AML for therapeutic benefit.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Leucemia Aguda Bifenotípica/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Leucemia Aguda Bifenotípica/genética , Leucemia Aguda Bifenotípica/metabolismo , Leucemia Aguda Bifenotípica/patologia , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas/genética , Elementos de Resposta , Transativadores/genéticaRESUMO
Recent studies have demonstrated that myelodysplastic syndromes (MDSs) arise from a small population of disease-initiating hematopoietic stem cells (HSCs) that persist and expand through conventional therapies and are major contributors to disease progression and relapse. MDS stem and progenitor cells are characterized by key founder and driver mutations and are enriched for cytogenetic alterations. Quantitative alterations in hematopoietic stem and progenitor cell (HSPC) numbers are also seen in a stage-specific manner in human MDS samples as well as in murine models of the disease. Overexpression of several markers such as interleukin-1 (IL-1) receptor accessory protein (IL1RAP), CD99, T-cell immunoglobulin mucin-3, and CD123 have begun to differentiate MDS HSPCs from healthy counterparts. Overactivation of innate immune components such as Toll-like receptors, IL-1 receptor-associated kinase/tumor necrosis factor receptor-associated factor-6, IL8/CXCR2, and IL1RAP signaling pathways has been demonstrated in MDS HSPCs and is being targeted therapeutically in preclinical and early clinical studies. Other dysregulated pathways such as signal transducer and activator of transcription 3, tyrosine kinase with immunoglobulinlike and EGF-like domains 1/angiopoietin-1, p21-activated kinase, microRNA 21, and transforming growth factor ß are also being explored as therapeutic targets against MDS HSPCs. Taken together, these studies have demonstrated that MDS stem cells are functionally critical for the initiation, transformation, and relapse of disease and need to be targeted therapeutically for future curative strategies in MDSs.
Assuntos
Células-Tronco Hematopoéticas/patologia , Síndromes Mielodisplásicas/patologia , Animais , Biomarcadores Tumorais/análise , Progressão da Doença , Humanos , Imunidade Inata , Síndromes Mielodisplásicas/genética , Células-Tronco Neoplásicas/patologiaRESUMO
Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Novel therapeutic targets against preleukemic stem cells need to be identified for potentially curative strategies. We conducted parallel transcriptional analysis of highly fractionated stem and progenitor populations in MDS, AML, and control samples and found interleukin 8 (IL8) to be consistently overexpressed in patient samples. The receptor for IL8, CXCR2, was also significantly increased in MDS CD34(+) cells from a large clinical cohort and was predictive of increased transfusion dependence. High CXCR2 expression was also an adverse prognostic factor in The Cancer Genome Atlas AML cohort, further pointing to the critical role of the IL8-CXCR2 axis in AML/MDS. Functionally, CXCR2 inhibition by knockdown and pharmacologic approaches led to a significant reduction in proliferation in several leukemic cell lines and primary MDS/AML samples via induction of G0/G1 cell cycle arrest. Importantly, inhibition of CXCR2 selectively inhibited immature hematopoietic stem cells from MDS/AML samples without an effect on healthy controls. CXCR2 knockdown also impaired leukemic growth in vivo. Together, these studies demonstrate that the IL8 receptor CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell-enriched cell fractions in MDS and AML.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Interleucina-8/metabolismo , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicas/metabolismo , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Interleucina-8/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Camundongos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Receptores de Interleucina-8B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist that has been approved recently for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Prior studies have shown that EP stimulates megakaryopoiesis in BM cells from patients with acute myeloid leukemia and myelodysplastic syndrome, and the results also suggested that it may inhibit leukemia cell growth. In the present study, we studied the effects of EP on leukemia cell proliferation and the mechanism of its antiproliferative effects. We found that EP leads to a decreased cell division rate, a block in G(1) phase of cell cycle, and increased differentiation in human and murine leukemia cells. Because EP is species specific in that it can only bind TPO-R in human and primate cells, these findings further suggested that the antileukemic effect is independent of TPO-R. We found that treatment with EP leads to a reduction in free intracellular iron in leukemic cells in a dose-dependent manner. Experimental increase of intracellular iron abrogated the antiproliferative and differentiation-inducing effects of EP, demonstrating that its antileukemic effects are mediated through modulation of intracellular iron content. Finally, determination of EP's antileukemic activity in vivo demonstrated its ability to prolong survival in 2 mouse models of leukemia.
Assuntos
Benzoatos/farmacologia , Hidrazinas/farmacologia , Ferro/metabolismo , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Pirazóis/farmacologia , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia/patologia , Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptores de Trombopoetina/agonistas , Células U937RESUMO
Cellular and interpatient heterogeneity and the involvement of different stem and progenitor compartments in leukemogenesis are challenges for the identification of common pathways contributing to the initiation and maintenance of acute myeloid leukemia (AML). Here we used a strategy of parallel transcriptional analysis of phenotypic long-term hematopoietic stem cells (HSCs), short-term HSCs, and granulocyte-monocyte progenitors from individuals with high-risk (-7/7q-) AML and compared them with the corresponding cell populations from healthy controls. This analysis revealed dysregulated expression of 11 genes, including IL-1 receptor accessory protein (IL1RAP), in all leukemic stem and progenitor cell compartments. IL1RAP protein was found to be overexpressed on the surface of HSCs of AML patients, and marked cells with the -7/7q- anomaly. IL1RAP was also overexpressed on HSCs of patients with normal karyotype AML and high-risk myelodysplastic syndrome, suggesting a pervasive role in different disease subtypes. High IL1RAP expression was independently associated with poor overall survival in 3 independent cohorts of AML patients (P = 2.2 × 10(-7)). Knockdown of IL1RAP decreased clonogenicity and increased cell death of AML cells. Our study identified genes dysregulated in stem and progenitor cells in -7/7q- AML, and suggests that IL1RAP may be a promising therapeutic and prognostic target in AML and high-risk myelodysplastic syndrome.
Assuntos
Proteína Acessória do Receptor de Interleucina-1/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , Células-Tronco Neoplásicas/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/fisiologia , Estudos de Coortes , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HL-60 , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Proteína Acessória do Receptor de Interleucina-1/fisiologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Modelos Biológicos , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/mortalidade , Células-Tronco Neoplásicas/patologia , Prognóstico , Análise de Sobrevida , Células Tumorais Cultivadas , Regulação para Cima/genéticaRESUMO
Even though hematopoietic stem cell (HSC) dysfunction is presumed in myelodysplastic syndrome (MDS), the exact nature of quantitative and qualitative alterations is unknown. We conducted a study of phenotypic and molecular alterations in highly fractionated stem and progenitor populations in a variety of MDS subtypes. We observed an expansion of the phenotypically primitive long-term HSCs (lineage(-)/CD34(+)/CD38(-)/CD90(+)) in MDS, which was most pronounced in higher-risk cases. These MDS HSCs demonstrated dysplastic clonogenic activity. Examination of progenitors revealed that lower-risk MDS is characterized by expansion of phenotypic common myeloid progenitors, whereas higher-risk cases revealed expansion of granulocyte-monocyte progenitors. Genome-wide analysis of sorted MDS HSCs revealed widespread methylomic and transcriptomic alterations. STAT3 was an aberrantly hypomethylated and overexpressed target that was validated in an independent cohort and found to be functionally relevant in MDS HSCs. FISH analysis demonstrated that a very high percentage of MDS HSC (92% ± 4%) carry cytogenetic abnormalities. Longitudinal analysis in a patient treated with 5-azacytidine revealed that karyotypically abnormal HSCs persist even during complete morphologic remission and that expansion of clonotypic HSCs precedes clinical relapse. This study demonstrates that stem and progenitor cells in MDS are characterized by stage-specific expansions and contain epigenetic and genetic alterations.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 7/genética , Células-Tronco Hematopoéticas , Síndromes Mielodisplásicas/genética , Fator de Transcrição STAT3/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Azacitidina/administração & dosagem , Estudos de Casos e Controles , Linhagem da Célula , Metilação de DNA , Epigênese Genética , Citometria de Fluxo , Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Cariotipagem , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/patologia , Cultura Primária de Células , Recidiva , Fator de Transcrição STAT3/metabolismoRESUMO
Regulatory elements located within an â¼28-kb region 3' of the Igh gene cluster (3' regulatory region) are required for class switch recombination and for high levels of IgH expression in plasma cells. We previously defined novel DNase I hypersensitive sites (hs) 5, 6, 7 immediately downstream of this region. The hs 5-7 region (hs5-7) contains a high density of binding sites for CCCTC-binding factor (CTCF), a zinc finger protein associated with mammalian insulator activity, and is an anchor for interactions with CTCF sites flanking the D(H) region. To test the function of hs5-7, we generated mice with an 8-kb deletion encompassing all three hs elements. B cells from hs5-7 knockout (KO) (hs5-7KO) mice showed a modest increase in expression of the nearest downstream gene. In addition, Igh alleles in hs5-7KO mice were in a less contracted configuration compared with wild-type Igh alleles and showed a 2-fold increase in the usage of proximal V(H)7183 gene families. Hs5-7KO mice were essentially indistinguishable from wild-type mice in B cell development, allelic regulation, class switch recombination, and chromosomal looping. We conclude that hs5-7, a high-density CTCF-binding region at the 3' end of the Igh locus, impacts usage of V(H) regions as far as 500 kb away.
Assuntos
Linfócitos B/imunologia , Genes de Cadeia Pesada de Imunoglobulina/genética , Mutação em Linhagem Germinativa , Sequências Reguladoras de Ácido Nucleico/imunologia , Animais , Fator de Ligação a CCCTC , Citometria de Fluxo , Genes de Cadeia Pesada de Imunoglobulina/imunologia , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Proteínas Repressoras/genética , Proteínas Repressoras/imunologiaRESUMO
PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages.
Assuntos
Células Precursoras Eritroides/citologia , Redes Reguladoras de Genes , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Transcrição Gênica , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Imunoprecipitação da Cromatina , Células Precursoras Eritroides/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismoRESUMO
Hematopoietic stem cells (HSCs) are routinely mobilized from the bone marrow (BM) to the blood circulation for clinical transplantation. However, the precise mechanisms by which individual stem cells exit the marrow are not understood. This study identified cell-extrinsic and molecular determinants of a mobilizable pool of blood-forming stem cells. We found that a subset of HSCs displays macrophage-associated markers on their cell surface. Although fully functional, these HSCs are selectively niche-retained as opposed to stem cells lacking macrophage markers, which exit the BM upon forced mobilization. Macrophage markers on HSCs could be acquired through direct transfer by trogocytosis, regulated by receptor tyrosine-protein kinase C-Kit (CD117), from BM-resident macrophages in mouse and human settings. Our study provides proof of concept that adult stem cells utilize trogocytosis to rapidly establish and activate function-modulating molecular mechanisms.