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ABSTRACT: Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which myc-associated zinc finger protein (MAZ) and nuclear transcription factor Y subunit alpha (NFY-A) are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.
Assuntos
Quinase 6 Dependente de Ciclina , Células-Tronco Hematopoéticas , Quinase 6 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Animais , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Humanos , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/citologia , Proliferação de Células , Diferenciação Celular , Camundongos Endogâmicos C57BL , Transplante de Células-Tronco Hematopoéticas , Autorrenovação Celular/efeitos dos fármacosRESUMO
ABSTRACT: Patients with T- and natural killer (NK)-cell neoplasms frequently have somatic STAT5B gain-of-function mutations. The most frequent STAT5B mutation is STAT5BN642H, which is known to drive murine T-cell leukemia, although its role in NK-cell malignancies is unclear. Introduction of the STAT5BN642H mutation into human NK-cell lines enhances their potential to induce leukemia in mice. We have generated a mouse model that enables tissue-specific expression of STAT5BN642H and have selectively expressed the mutated STAT5B in hematopoietic cells (N642Hvav/+) or exclusively in NK cells (N642HNK/NK). All N642Hvav/+ mice rapidly develop an aggressive T/NKT-cell leukemia, whereas N642HNK/NK mice display an indolent NK-large granular lymphocytic leukemia (NK-LGLL) that progresses to an aggressive leukemia with age. Samples from patients with NK-cell leukemia have a distinctive transcriptional signature driven by mutant STAT5B, which overlaps with that of murine leukemic N642HNK/NK NK cells. To our knowledge, we have generated the first reliable STAT5BN642H-driven preclinical mouse model that displays an indolent NK-LGLL progressing to aggressive NK-cell leukemia. This novel in vivo tool will enable us to explore the transition from an indolent to an aggressive disease and will thus permit the study of prevention and treatment options for NK-cell malignancies.
Assuntos
Células Matadoras Naturais , Leucemia Linfocítica Granular Grande , Fator de Transcrição STAT5 , Animais , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Camundongos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Humanos , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/patologia , Modelos Animais de Doenças , Linhagem da Célula/genética , Mutação , Camundongos TransgênicosRESUMO
Gain-of-function mutations in the signal transducer and activator of transcription 3 (STAT3) gene are recurrently identified in patients with large granular lymphocytic leukemia (LGLL) and in some cases of natural killer (NK)/T-cell and adult T-cell leukemia/lymphoma. To understand the consequences and molecular mechanisms contributing to disease development and oncogenic transformation, we developed murine hematopoietic stem and progenitor cell models that express mutated STAT3Y640F. These cells show accelerated proliferation and enhanced self-renewal potential. We integrated gene expression analyses and chromatin occupancy profiling of STAT3Y640F-transformed cells with data from patients with T-LGLL. This approach uncovered a conserved set of direct transcriptional targets of STAT3Y640F. Among these, strawberry notch homolog 2 (SBNO2) represents an essential transcriptional target, which was identified by a comparative genome-wide CRISPR/Cas9-based loss-of-function screen. The STAT3-SBNO2 axis is also present in NK-cell leukemia, T-cell non-Hodgkin lymphoma, and NPM-ALK-rearranged T-cell anaplastic large cell lymphoma (T-ALCL), which are driven by STAT3-hyperactivation/mutation. In patients with NPM-ALK+ T-ALCL, high SBNO2 expression correlates with shorter relapse-free and overall survival. Our findings identify SBNO2 as a potential therapeutic intervention site for STAT3-driven hematopoietic malignancies.
Assuntos
Neoplasias Hematológicas , Fator de Transcrição STAT3 , Animais , Humanos , Camundongos , Quinase do Linfoma Anaplásico/metabolismo , Linhagem Celular Tumoral , Neoplasias Hematológicas/genética , Linfoma Anaplásico de Células Grandes/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Cyclin-dependent kinase 6 (CDK6) represents a novel therapeutic target for the treatment of certain subtypes of acute myeloid leukaemia (AML). CDK4/6 kinase inhibitors have been widely studied in many cancer types and their effects may be limited by primary and secondary resistance mechanisms. CDK4/6 degraders, which eliminate kinase-dependent and kinase-independent effects, have been suggested as an alternative therapeutic option. We show that the efficacy of the CDK6-specific protein degrader BSJ-03-123 varies among AML subtypes and depends on the low expression of the INK4 proteins p16INK4A and p18INK4C. INK4 protein levels are significantly elevated in KMT2A-MLLT3+ cells compared to RUNX1-RUNX1T1+ cells, contributing to the different CDK6 degradation efficacy. We demonstrate that CDK6 complexes containing p16INK4A or p18INK4C are protected from BSJ-mediated degradation and that INK4 levels define the proliferative response to CDK6 degradation. These findings define INK4 proteins as predictive markers for CDK6 degradation-targeted therapies in AML.
RESUMO
The transcription factors signal transducer and activator of transcription 5A (STAT5A) and STAT5B are critical in hematopoiesis and leukemia. They are widely believed to have redundant functions, but we describe a unique role for STAT5B in driving the self-renewal of hematopoietic and leukemic stem cells (HSCs/LSCs). We find STAT5B to be specifically activated in HSCs and LSCs, where it induces many genes associated with quiescence and self-renewal, including the surface marker CD9. Levels of CD9 represent a prognostic marker for patients with STAT5-driven leukemia, and our findings suggest that anti-CD9 antibodies may be useful in their treatment to target and eliminate LSCs. We show that it is vital to consider STAT5A and STAT5B as distinct entities in normal and malignant hematopoiesis.
Assuntos
Células-Tronco Hematopoéticas/patologia , Leucemia/patologia , Células-Tronco Neoplásicas/patologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Tetraspanina 29/metabolismo , Animais , Autorrenovação Celular , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Células Tumorais CultivadasRESUMO
Mutations in the gene for calreticulin (CALR) were identified in the myeloproliferative neoplasms (MPNs) essential thrombocythaemia (ET) and primary myelofibrosis (MF) in 2013; in combination with previously described mutations in JAK2 and MPL, driver mutations have now been described for the majority of MPN patients. In subsequent years, researchers have begun to unravel the mechanisms by which mutant CALR drives transformation and to understand their clinical implications. Mutant CALR activates the thrombopoietin receptor (MPL), causing constitutive activation of Janus kinase 2 (JAK2) signaling and cytokine independent growth in vitro. Mouse models show increased numbers of hematopoietic stem cells (HSCs) and overproduction of megakaryocytic lineage cells with associated thrombocytosis. In the clinic, detection of CALR mutations has been embedded in World Health Organization and other international diagnostic guidelines. Distinct clinical and laboratory associations of CALR mutations have been identified together with their prognostic significance, with CALR mutant patients showing increased overall survival. The discovery and subsequent study of CALR mutations have illuminated novel aspects of megakaryopoiesis and raised the possibility of new therapeutic approaches.
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Current next-generation sequencing (NGS) technologies allow unprecedented insights into the mutational profiles of tumors. Recent studies in myeloproliferative neoplasms have further demonstrated that, not only the mutational profile, but also the order in which these mutations are acquired is relevant for our understanding of the disease. Our ability to assign mutation order from NGS data alone is, however, limited. Here, we present a strategy of highly multiplexed genotyping of burst forming unit-erythroid colonies based on NGS results to assess subclonal tumor structure. This allowed for the generation of complex clonal hierarchies and determination of order of mutation acquisition far more accurately than was possible from NGS data alone.
Assuntos
Análise Mutacional de DNA/métodos , DNA de Neoplasias/genética , Células Precursoras Eritroides/química , Técnicas de Genotipagem/métodos , Neoplasias Hematológicas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Policitemia Vera/genética , Análise de Sequência de DNA/métodos , Alelos , Células Clonais , Análise por Conglomerados , Granulócitos/química , Neoplasias Hematológicas/patologia , Humanos , Janus Quinase 2/genética , Mutação de Sentido Incorreto , Policitemia Vera/patologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF.
Assuntos
Calreticulina/genética , Autorrenovação Celular/genética , Células-Tronco Hematopoéticas/fisiologia , Mielofibrose Primária/genética , Trombocitose/genética , Animais , Células Cultivadas , Homozigoto , Leucocitose/genética , Leucocitose/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto , Esplenomegalia/genética , Esplenomegalia/patologia , Trombocitemia Essencial/genética , Trombocitemia Essencial/patologiaAssuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Transtornos Mieloproliferativos/genética , Análise Mutacional de DNA , Família , Feminino , Humanos , Masculino , Transtornos Mieloproliferativos/diagnóstico , Linhagem , Ubiquitina-Proteína LigasesRESUMO
Genotoxic chemotherapy is the most common cancer treatment strategy. However, its untargeted generic DNA-damaging nature and associated systemic cytotoxicity greatly limit its therapeutic applications. Here, we used a haploid genetic screen in human cells to discover an absolute dependency of the clinically evaluated anticancer compound YM155 on solute carrier family member 35 F2 (SLC35F2), an uncharacterized member of the solute carrier protein family that is highly expressed in a variety of human cancers. YM155 generated DNA damage through intercalation, which was contingent on the expression of SLC35F2 and its drug-importing activity. SLC35F2 expression and YM155 sensitivity correlated across a panel of cancer cell lines, and targeted genome editing verified SLC35F2 as the main determinant of YM155-mediated DNA damage toxicity in vitro and in vivo. These findings suggest a new route to targeted DNA damage by exploiting tumor and patient-specific import of YM155.
Assuntos
Dano ao DNA/efeitos dos fármacos , Imidazóis/farmacologia , Substâncias Intercalantes/farmacologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Naftoquinonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular , Clonagem Molecular , Ensaio Cometa , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Haploidia , Humanos , Imidazóis/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos SCID , Naftoquinonas/metabolismo , RNA Neoplásico/química , RNA Neoplásico/genéticaRESUMO
Patients with essential thrombocythemia may carry JAK2 (V617F), an MPL substitution, or a calreticulin gene (CALR) mutation. We studied biologic and clinical features of essential thrombocythemia according to JAK2 or CALR mutation status and in relation to those of polycythemia vera. The mutant allele burden was lower in JAK2-mutated than in CALR-mutated essential thrombocythemia. Patients with JAK2 (V617F) were older, had a higher hemoglobin level and white blood cell count, and lower platelet count and serum erythropoietin than those with CALR mutation. Hematologic parameters of patients with JAK2-mutated essential thrombocythemia or polycythemia vera were related to the mutant allele burden. While no polycythemic transformation was observed in CALR-mutated patients, the cumulative risk was 29% at 15 years in those with JAK2-mutated essential thrombocythemia. There was no significant difference in myelofibrotic transformation between the 2 subtypes of essential thrombocythemia. Patients with JAK2-mutated essential thrombocythemia and those with polycythemia vera had a similar risk of thrombosis, which was twice that of patients with the CALR mutation. These observations are consistent with the notion that JAK2-mutated essential thrombocythemia and polycythemia vera represent different phenotypes of a single myeloproliferative neoplasm, whereas CALR-mutated essential thrombocythemia is a distinct disease entity.
Assuntos
Calreticulina/genética , Janus Quinase 2/genética , Mutação , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Transformação Celular Neoplásica/genética , Códon , Éxons , Feminino , Granulócitos , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/genética , Policitemia Vera/genética , Mielofibrose Primária/genética , Prognóstico , Receptores de Trombopoetina/genética , Trombocitemia Essencial/mortalidade , Trombose/genética , Adulto JovemRESUMO
BACKGROUND: Approximately 50 to 60% of patients with essential thrombocythemia or primary myelofibrosis carry a mutation in the Janus kinase 2 gene (JAK2), and an additional 5 to 10% have activating mutations in the thrombopoietin receptor gene (MPL). So far, no specific molecular marker has been identified in the remaining 30 to 45% of patients. METHODS: We performed whole-exome sequencing to identify somatically acquired mutations in six patients who had primary myelofibrosis without mutations in JAK2 or MPL. Resequencing of CALR, encoding calreticulin, was then performed in cohorts of patients with myeloid neoplasms. RESULTS: Somatic insertions or deletions in exon 9 of CALR were detected in all patients who underwent whole-exome sequencing. Resequencing in 1107 samples from patients with myeloproliferative neoplasms showed that CALR mutations were absent in polycythemia vera. In essential thrombocythemia and primary myelofibrosis, CALR mutations and JAK2 and MPL mutations were mutually exclusive. Among patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 or MPL, CALR mutations were detected in 67% of those with essential thrombocythemia and 88% of those with primary myelofibrosis. A total of 36 types of insertions or deletions were identified that all cause a frameshift to the same alternative reading frame and generate a novel C-terminal peptide in the mutant calreticulin. Overexpression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with mutated CALR had a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. CONCLUSIONS: Most patients with essential thrombocythemia or primary myelofibrosis that was not associated with a JAK2 or MPL alteration carried a somatic mutation in CALR. The clinical course in these patients was more indolent than that in patients with the JAK2 V617F mutation. (Funded by the MPN Research Foundation and Associazione Italiana per la Ricerca sul Cancro.).
Assuntos
Calreticulina/genética , Mutação , Mielofibrose Primária/genética , Trombocitemia Essencial/genética , Doenças da Medula Óssea/genética , Éxons , Humanos , Janus Quinase 2/genética , Leucemia Mieloide/genética , Reação em Cadeia da Polimerase , Mielofibrose Primária/mortalidade , Modelos de Riscos Proporcionais , Receptores de Trombopoetina/genética , Análise de Sequência de DNA , Trombocitemia Essencial/complicações , Trombocitemia Essencial/mortalidade , Trombose/etiologiaRESUMO
Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 11/genética , Proteínas de Ligação a DNA/genética , Proteínas com Domínio LIM/genética , Proteínas Proto-Oncogênicas/genética , Exoma/genética , Histona-Lisina N-Metiltransferase , Humanos , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Proteína de Leucina Linfoide-Mieloide/genética , Policitemia Vera/genética , Reação em Cadeia da Polimerase , Mielofibrose Primária/genética , Proteínas Proto-Oncogênicas c-cbl/genética , Trombocitose/genéticaRESUMO
The study aimed to identify genetic lesions associated with secondary acute myeloid leukemia (sAML) in comparison with AML arising de novo (dnAML) and assess their impact on patients' overall survival (OS). High-resolution genotyping and loss of heterozygosity mapping was performed on DNA samples from 86 sAML and 117 dnAML patients, using Affymetrix Genome-Wide Human SNP 6.0 arrays. Genes TP53, RUNX1, CBL, IDH1/2, NRAS, NPM1, and FLT3 were analyzed for mutations in all patients. We identified 36 recurrent cytogenetic aberrations (more than five events). Mutations in TP53, 9pUPD, and del7q (targeting CUX1 locus) were significantly associated with sAML, while NPM1 and FLT3 mutations associated with dnAML. Patients with sAML carrying TP53 mutations demonstrated lower 1-year OS rate than those with wild-type TP53 (14.3% ± 9.4% vs. 35.4% ± 7.2%; P = 0.002), while complex karyotype, del7q (CUX1) and del7p (IKZF1) showed no significant effect on OS. Multivariate analysis confirmed that mutant TP53 was the only independent adverse prognostic factor for OS in sAML (hazard ratio 2.67; 95% CI: 1.33-5.37; P = 0.006). Patients with dnAML and complex karyotype carried sAML-associated defects (TP53 defects in 54.5%, deletions targeting FOXP1 and ETV6 loci in 45.4% of the cases). We identified several co-occurring lesions associated with either sAML or dnAML diagnosis. Our data suggest that distinct genetic lesions drive leukemogenesis in sAML. High karyotype complexity of sAML patients does not influence OS. Somatic mutations in TP53 are the only independent adverse prognostic factor in sAML. Patients with dnAML and complex karyotype show genetic features associated with sAML and myeloproliferative neoplasms.
Assuntos
Aberrações Cromossômicas , Leucemia Mieloide Aguda/genética , Segunda Neoplasia Primária/genética , Aberrações Cromossômicas/estatística & dados numéricos , DNA/genética , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Estimativa de Kaplan-Meier , Cariotipagem , Leucemia Mieloide Aguda/mortalidade , Perda de Heterozigosidade , Análise Multivariada , Segunda Neoplasia Primária/mortalidade , Nucleofosmina , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , PrognósticoRESUMO
Chronic myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) have an inherent tendency to progress to acute myeloid leukemia (AML). Using high-resolution SNP microarrays, we studied a total of 517 MPN and MDS patients in different disease stages, including 77 AML cases with previous history of MPN (N = 46) or MDS (N = 31). Frequent chromosomal deletions of variable sizes were detected, allowing the mapping of putative tumor suppressor genes involved in the leukemic transformation process. We detected frequent deletions on the short arm of chromosome 6 (del6p). The common deleted region on 6p mapped to a 1.1-Mb region and contained only the JARID2 gene--member of the polycomb repressive complex 2 (PRC2). When we compared the frequency of del6p between chronic and leukemic phase, we observed a strong association of del6p with leukemic transformation (P = 0.0033). Subsequently, analysis of deletion profiles of other PRC2 members revealed frequent losses of genes such as EZH2, AEBP2, and SUZ12; however, the deletions targeting these genes were large. We also identified two patients with homozygous losses of JARID2 and AEBP2. We observed frequent codeletion of AEBP2 and ETV6, and similarly, SUZ12 and NF1. Using next generation exome sequencing of 40 patients, we identified only one somatic mutation in the PRC2 complex member SUZ12. As the frequency of point mutations in PRC2 members was found to be low, deletions were the main type of lesions targeting PRC2 complex members. Our study suggests an essential role of the PRC2 complex in the leukemic transformation of chronic myeloid disorders.
Assuntos
Transformação Celular Neoplásica/genética , Deleção Cromossômica , Cromossomos Humanos Par 6/ultraestrutura , Genes Supressores de Tumor , Síndromes Mielodisplásicas/genética , Transtornos Mieloproliferativos/genética , Proteínas de Neoplasias/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Doença Aguda , Idoso , Proteínas de Transporte/genética , Aberrações Cromossômicas , Mapeamento Cromossômico , Cromossomos Humanos Par 6/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Genótipo , Humanos , Leucemia Mieloide/genética , Masculino , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Análise de Sequência de DNA , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
Imprinted macro non-protein-coding (nc) RNAs are cis-repressor transcripts that silence multiple genes in at least three imprinted gene clusters in the mouse genome. Similar macro or long ncRNAs are abundant in the mammalian genome. Here we present the full coding and non-coding transcriptome of two mouse tissues: differentiated ES cells and fetal head using an optimized RNA-Seq strategy. The data produced is highly reproducible in different sequencing locations and is able to detect the full length of imprinted macro ncRNAs such as Airn and Kcnq1ot1, whose length ranges between 80-118 kb. Transcripts show a more uniform read coverage when RNA is fragmented with RNA hydrolysis compared with cDNA fragmentation by shearing. Irrespective of the fragmentation method, all coding and non-coding transcripts longer than 8 kb show a gradual loss of sequencing tags towards the 3' end. Comparisons to published RNA-Seq datasets show that the strategy presented here is more efficient in detecting known functional imprinted macro ncRNAs and also indicate that standardization of RNA preparation protocols would increase the comparability of the transcriptome between different RNA-Seq datasets.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Impressão Genômica , Cabeça/fisiologia , RNA não Traduzido/genética , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feto , Perfilação da Expressão Gênica , Genoma , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These disorders may undergo phenotypic shifts, and may specifically evolve into secondary myelofibrosis (MF) or acute myeloid leukemia (AML). We studied genomic changes associated with these transformations in 29 patients who had serial samples collected in different phases of disease. Genomic DNA from granulocytes, i.e., the myeloproliferative genome, was processed and hybridized to genome-wide human SNP 6.0 arrays. Most patients in chronic phase had chromosomal regions with uniparental disomy (UPD) and/or copy number changes. Disease progression to secondary MF or AML was associated with the acquisition of additional chromosomal aberrations in granulocytes (P = 0.002). A close relationship was observed between aberrations of chromosome 9p (UPD and/or gain) and progression from PV to post-PV MF (P = 0.002). The acquisition of one or more aberrations involving chromosome 5, 7, or 17p was specifically associated with progression to AML (OR 5.9, 95% CI 1.2-27.7, P = 0.006), and significantly affected overall survival (HR 18, 95% CI 1.9-164, P = 0.01). These observations indicate that disease progression from chronic-phase MPN to secondary MF or AML is associated with specific chromosomal aberrations that can be detected by means of high-resolution SNP array analysis of granulocyte DNA.
Assuntos
Transformação Celular Neoplásica/genética , Aberrações Cromossômicas , Transtornos Mieloproliferativos/genética , Polimorfismo de Nucleotídeo Único , Crise Blástica/etiologia , Crise Blástica/genética , Crise Blástica/metabolismo , DNA/química , DNA/metabolismo , Progressão da Doença , Feminino , Estudo de Associação Genômica Ampla , Granulócitos/metabolismo , Humanos , Itália , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/fisiopatologia , Masculino , Mutação , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Transtornos Mieloproliferativos/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos , Policitemia Vera/etiologia , Policitemia Vera/genética , Policitemia Vera/metabolismo , Mielofibrose Primária/etiologia , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , Análise de Sobrevida , Trombocitemia Essencial/etiologia , Trombocitemia Essencial/genética , Trombocitemia Essencial/metabolismoRESUMO
Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are clonal myeloid disorders with increased production of terminally differentiated cells. The disease course is generally chronic, but some patients show disease progression (secondary myelofibrosis or accelerated phase) and/or leukemic transformation. We investigated chromosomal aberrations in 408 MPN samples using high-resolution single-nucleotide polymorphism microarrays to identify disease-associated somatic lesions. Of 408 samples, 37.5% had a wild-type karyotype and 62.5% harbored at least 1 chromosomal aberration. We identified 25 recurrent aberrations that were found in 3 or more samples. An increased number of chromosomal lesions was significantly associated with patient age, as well as with disease progression and leukemic transformation, but no association was observed with MPN subtypes, Janus kinase 2 (JAK2) mutational status, or disease duration. Aberrations of chromosomes 1q and 9p were positively associated with disease progression to secondary myelofibrosis or accelerated phase. Changes of chromosomes 1q, 7q, 5q, 6p, 7p, 19q, 22q, and 3q were positively associated with post-MPN acute myeloid leukemia. We mapped commonly affected regions to single target genes on chromosomes 3p (forkhead box P1 [FOXP1]), 4q (tet oncogene family member 2 [TET2]), 7p (IKAROS family zinc finger 1 [IKZF1]), 7q (cut-like homeobox 1 [CUX1]), 12p (ets variant 6 [ETV6]), and 21q (runt-related transcription factor 1 [RUNX1]). Our data provide insight into the genetic complexity of MPNs and implicate new genes involved in disease progression.