ABSTRACT
The fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.
Subject(s)
Myeloid-Lymphoid Leukemia Protein , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Epigenesis, Genetic , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Genes, Regulator , ChromatinABSTRACT
The fusion oncogene RUNX1/RUNX1T1 encodes an aberrant transcription factor, which plays a key role in the initiation and maintenance of acute myeloid leukemia. Here we show that the RUNX1/RUNX1T1 oncogene is a regulator of alternative RNA splicing in leukemic cells. The comprehensive analysis of RUNX1/RUNX1T1-associated splicing events identifies two principal mechanisms that underlie the differential production of RNA isoforms: (i) RUNX1/RUNX1T1-mediated regulation of alternative transcription start site selection, and (ii) direct or indirect control of the expression of genes encoding splicing factors. The first mechanism leads to the expression of RNA isoforms with alternative structure of the 5'-UTR regions. The second mechanism generates alternative transcripts with new junctions between internal cassettes and constitutive exons. We also show that RUNX1/RUNX1T1-mediated differential splicing affects several functional groups of genes and produces proteins with unique conserved domain structures. In summary, this study reveals alternative splicing as an important component of transcriptome re-organization in leukemia by an aberrant transcriptional regulator.
Subject(s)
Alternative Splicing , Core Binding Factor Alpha 2 Subunit/genetics , Gene Expression Regulation, Leukemic , Leukemia, Myeloid/genetics , Oncogene Proteins, Fusion/genetics , RUNX1 Translocation Partner 1 Protein/genetics , Acute Disease , Cell Line, Tumor , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Expression Profiling/methods , Humans , Leukemia, Myeloid/pathology , Models, Genetic , Oncogene Proteins, Fusion/metabolism , RNA Interference , RNA Isoforms/genetics , RNA Isoforms/metabolism , RUNX1 Translocation Partner 1 Protein/metabolism , Transcription Initiation SiteABSTRACT
Oncogenic transcription factors such as the leukemic fusion protein RUNX1/ETO, which drives t(8;21) acute myeloid leukemia (AML), constitute cancer-specific but highly challenging therapeutic targets. We used epigenomic profiling data for an RNAi screen to interrogate the transcriptional network maintaining t(8;21) AML. This strategy identified Cyclin D2 (CCND2) as a crucial transmitter of RUNX1/ETO-driven leukemic propagation. RUNX1/ETO cooperates with AP-1 to drive CCND2 expression. Knockdown or pharmacological inhibition of CCND2 by an approved drug significantly impairs leukemic expansion of patient-derived AML cells and engraftment in immunodeficient murine hosts. Our data demonstrate that RUNX1/ETO maintains leukemia by promoting cell cycle progression and identifies G1 CCND-CDK complexes as promising therapeutic targets for treatment of RUNX1/ETO-driven AML.
Subject(s)
Cell Cycle Checkpoints/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Cyclin D2/genetics , Animals , Cell Line, Tumor , Chromosomes, Human, Pair 21/genetics , Gene Expression Regulation, Leukemic/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Male , Mice , Oncogene Proteins, Fusion/genetics , Oncogenes/genetics , Translocation, Genetic/geneticsABSTRACT
Acute myeloid leukemia (AML) is a heterogeneous disease caused by mutations in transcriptional regulator genes, but how different mutant regulators shape the chromatin landscape is unclear. Here, we compared the transcriptional networks of two types of AML with chromosomal translocations of the RUNX1 locus that fuse the RUNX1 DNA-binding domain to different regulators, the t(8;21) expressing RUNX1-ETO and the t(3;21) expressing RUNX1-EVI1. Despite containing the same DNA-binding domain, the two fusion proteins display distinct binding patterns, show differences in gene expression and chromatin landscape, and are dependent on different transcription factors. RUNX1-EVI1 directs a stem cell-like transcriptional network reliant on GATA2, whereas that of RUNX1-ETO-expressing cells is more mature and depends on RUNX1. However, both types of AML are dependent on the continuous expression of the fusion proteins. Our data provide a molecular explanation for the differences in clinical prognosis for these types of AML.
Subject(s)
Chromatin/metabolism , Chromosomes, Human/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Leukemia, Myeloid, Acute/genetics , Translocation, Genetic/genetics , Base Sequence , Binding Sites , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Cell Differentiation/genetics , Cell Survival/genetics , GATA2 Transcription Factor/metabolism , Gene Knockdown Techniques , Humans , PhenotypeABSTRACT
This unit provides information how to use short interfering RNA (siRNA) for sequence-specific gene silencing in mammalian cells. Several methods for siRNA generation and optimization, as well as recommendations for cell transfection and transduction, are presented.
Subject(s)
Gene Silencing , RNA Interference , Animals , Cell Line , Humans , Mammals , RNA, Small Interfering/genetics , RNA, Small Interfering/isolation & purification , RNA, Small Interfering/metabolism , Transduction, Genetic/methods , Transfection/methodsABSTRACT
Oncogenic transcription factors such as RUNX1/ETO, which is generated by the chromosomal translocation t(8;21), subvert normal blood cell development by impairing differentiation and driving malignant self-renewal. Here, we use digital footprinting and chromatin immunoprecipitation sequencing (ChIP-seq) to identify the core RUNX1/ETO-responsive transcriptional network of t(8;21) cells. We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion. Perturbation of this equilibrium in t(8;21) cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation. Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21) is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.
Subject(s)
Leukemia, Myeloid, Acute/pathology , Translocation, Genetic , Adaptor Proteins, Signal Transducing/metabolism , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Cell Line, Tumor , Chromatin Immunoprecipitation , Chromosome Mapping , Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 8 , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Regulatory Networks , Humans , LIM Domain Proteins/metabolism , Leukemia, Myeloid, Acute/metabolism , Protein Binding , Proto-Oncogene Proteins/metabolism , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering , Sequence Analysis, RNA , Trans-Activators/metabolismABSTRACT
INTRODUCTION: Crossed cerebellar diaschisis is a functional deficit in an area that is remote from that of a supratentorial brain lesion, although the two are anatomically and functionally connected. Deactivation of the contralateral cerebellar hemisphere occurs and is believed to be caused by a transneuronal metabolic depression of the cortico-ponto-cerebellar pathway. A reduction in the blood flow in the brain takes place and this gives rise to a diminished oxygenation of the cerebellar hemisphere. This finding can be evaluated by different functional imaging methods. CASE REPORT: A 32-year-old female, without any relevant events in her personal history, presented an extensive supra-tentorial ischaemic lesion on the right-hand side. An MR angiography scan was performed, in which the homolateral middle cerebral artery and internal carotid artery were not identified. With no specific causation and suspecting a probable case of vasculitis (two days after the first ischaemic episode, the patient suffered another one in the left-hand occipital area, although this time it was smaller and clinically asymptomatic), a positron emission tomography/computerised tomography (PET/CT) scan was performed. The results revealed hypoenhancement of the radiotracer in the contralateral cerebellar hemisphere, which was interpreted as hypometabolism. CONCLUSIONS: Determined by a possible prognostic value in the presence of crossed cerebellar diaschisis in supratentorial vascular episodes, with respect to the final clinical outcome of the patients, it is interesting to take this possibility into account when evaluating patients with an acute cerebrovascular accident, either by means of PET/CT, MR imaging (perfusion) or CT (perfusion).
Subject(s)
Brain Ischemia/etiology , Carotid Stenosis/complications , Cerebellum/physiopathology , Magnetic Resonance Angiography , Multimodal Imaging , Positron-Emission Tomography , Tomography, X-Ray Computed , Vasculitis, Central Nervous System/complications , Adult , Brain Ischemia/diagnostic imaging , Brain Ischemia/pathology , Brain Ischemia/physiopathology , Carotid Artery, Internal/diagnostic imaging , Carotid Artery, Internal/pathology , Carotid Stenosis/diagnostic imaging , Carotid Stenosis/pathology , Carotid Stenosis/physiopathology , Cerebellum/diagnostic imaging , Cerebellum/metabolism , Cerebellum/pathology , Cerebrovascular Circulation , Dominance, Cerebral , Dysarthria/etiology , Female , Fluorine Radioisotopes , Fluorodeoxyglucose F18 , Humans , Infarction, Middle Cerebral Artery/diagnostic imaging , Infarction, Middle Cerebral Artery/etiology , Infarction, Middle Cerebral Artery/pathology , Infarction, Middle Cerebral Artery/physiopathology , Neural Pathways/physiopathology , Paresis/etiology , RadiopharmaceuticalsABSTRACT
La diasquisis cerebelosa cruzada es una deficiencia funcional en un área remota a la de una lesión cerebral supratentorial, pero anatómica y funcionalmente conectadas. Se produce una desactivación del hemisferio cerebeloso contralateral, y se considera que la causa una depresión metabólica transneuronal de la vía corticopontocerebelosa. Existe una reducción del flujo sanguíneo cerebral y, por consiguiente, de la oxigenación del hemisferio cerebeloso. Este hallazgo puede evaluarse a través de diferentes métodos de imágenes funcionales. Caso clínico. Mujer de 32 años, sin antecedentes personales relevantes, que presentó una extensa lesión isquémica supratentorial derecha. Se estudió mediante angiorresonancia, en la que no se identificaron la arteria cerebral media y la arteria carótida interna homolaterales. Sin etiología específica, pensando en una probable vasculitis (dos días después del inicial, la paciente sufrió otro episodio isquémico en el área occipital izquierda, de menor tamaño y clínicamente asintomático), se realizó una tomografía por emisión de positrones/tomografía computarizada (PET/TC) y se observó hipocaptación del radiotrazador del hemisferio cerebeloso contralateral, interpretado como hipometabolismo. Conclusión. En función de un posible valor pronóstico en la presencia de diasquisis cerebelosa cruzada en episodios vasculares supratentoriales, con respecto al resultado clínico final de los pacientes, es interesante tener presente esta posibilidad en la evaluación de pacientes con accidente cerebrovascular agudo, ya sea mediante PET/TC, resonancia magnética (perfusión) o TC (perfusión) (AU)
Introduction. Crossed cerebellar diaschisis is a functional deficit in an area that is remote from that of a supratentorial brain lesion, although the two are anatomically and functionally connected. Deactivation of the contralateral cerebellar hemisphere occurs and is believed to be caused by a transneuronal metabolic depression of the cortico-ponto-cerebellar pathway. A reduction in the blood flow in the brain takes place and this gives rise to a diminished oxygenation of the cerebellar hemisphere. This finding can be evaluated by different functional imaging methods. Case report. A 32-year-old female, without any relevant events in her personal history, presented an extensive supratentorial ischaemic lesion on the right-hand side. An MR angiography scan was performed, in which the homolateral middle cerebral artery and internal carotid artery were not identified. With no specific causation and suspecting a probable case of vasculitis (two days after the first ischaemic episode, the patient suffered another one in the left-hand occipital area, although this time it was smaller and clinically asymptomatic), a positron emission tomography/computerised tomography (PET/CT) scan was performed. The results revealed hypoenhancement of the radiotracer in the contralateral cerebellar hemisphere, which was interpreted as hypometabolism. Conclusions. Determined by a possible prognostic value in the presence of crossed cerebellar diaschisis in supratentorial vascular episodes, with respect to the final clinical outcome of the patients, it is interesting to take this possibility into account when evaluating patients with an acute cerebrovascular accident, either by means of PET/CT, MR imaging (perfusion) or CT (perfusion) (AU)
Subject(s)
Humans , Female , Adult , Cerebellar Diseases/diagnosis , Brain Diseases, Metabolic, Inborn/diagnosis , Positron-Emission Tomography/methods , Magnetic Resonance Spectroscopy/methods , Aphasia/etiologyABSTRACT
We describe the natural history of lupus nephritis (LN) in a historical cohort of 190 white patients with the diagnosis of biopsy-proven LN followed in a single reference center.We evaluated 670 patients with systemic lupus erythematosus (SLE) consecutively followed in our department from 1970 until 2006. All patients fulfilled the 1997 revised criteria for the classification of SLE. White patients (Spanish-born) with biopsy-proven LN were selected as the study population.The cohort included 190 patients (170 female patients and 20 male) with a mean age at LN diagnosis of 31 years. Renal biopsy revealed type I LN in 8 (4%) patients, type II in 33 (17%), type III in 46 (24%), type IV in 72 (38%), type V in 28 (15%), and type VI in 3 (2%) patients. Induction remission was achieved in 85% of patients with types I and II, 78% with type III, 70% with type IV, and 32% of patients with type V. After a mean follow-up of 2391 patient-years, 62 (33%) patients developed chronic renal failure and 18 (9%) evolved to end-stage renal disease. Adjusted multivariate Cox regression analysis identified male sex (hazard ratio [HR], 4.33) and elevated creatinine at LN diagnosis (HR, 5.18) as independent variables for renal failure. Survival was 92% at 10 years of follow-up, 80% after 20 years, and 72% after 30 years.Our results suggest that biopsy-proven LN in white patients has an excellent prognosis. Ethnicity should be considered a key factor when evaluating the prognosis and therapeutic response to different agents in patients with LN.
Subject(s)
Lupus Nephritis/epidemiology , Lupus Nephritis/pathology , White People/statistics & numerical data , Adult , Anti-Inflammatory Agents/adverse effects , Bacterial Infections/epidemiology , Biopsy , Comorbidity , Female , Glomerular Filtration Rate , Humans , Immunosuppressive Agents/adverse effects , Kidney/pathology , Kidney Failure, Chronic/drug therapy , Kidney Failure, Chronic/epidemiology , Kidney Failure, Chronic/pathology , Life Expectancy , Lupus Nephritis/drug therapy , Male , Treatment Outcome , Virus Diseases/epidemiologyABSTRACT
OBJECTIVE: To analyze the prevalence of neutropenia in a large cohort of patients with primary Sjögren's syndrome (SS) and its association with clinical and immunological disease expression and adverse outcomes. METHODS: The study cohort included 300 patients diagnosed with primary SS in our department between 1984 and 2002. The outcomes measured after the first laboratory evidence of neutropenia (<2.5 x 10(9)/L) were first hospital admission caused by infection, development of systemic manifestations, neoplasia, and death. RESULTS: Ninety-nine (33%) patients had neutropenia during the follow-up, which was related to neoplasia or drugs in 9 (3%) patients and was considered idiopathic in the remaining 90 (30%). Patients with neutropenia had a lower mean age at diagnosis of SS (51.9 versus 59.4 years, P < 0.001) and a higher prevalence of anti-Ro/La antibodies (53% versus 22%, P < 0.001), rheumatoid factor (49% versus 32%, P = 0.009), and low C4 levels (17% versus 8%, P = 0.044) than those without neutropenia. Patients with neutropenia had a higher incidence of hospital admission caused by infection (24% versus 9%, P = 0.002), especially those with neutropenia <1 x 10(9)/L (50% versus 9%, P = 0.002), and a higher rate of admission (log rank = 0.0023) in comparison with those without neutropenia. Agranulocytosis was found in 7 (2%) patients, predominantly related to neoplasia (5 cases). One (1%) of the 90 patients with SS-related neutropenia developed large granular lymphocyte T-cell leukemia. CONCLUSION: Neutropenia should be considered a relevant hematologic finding of primary SS, due both to its elevated prevalence and to its clinical significance (close association with anti-Ro/La antibodies, coexistence with other cytopenias, and development of severe infections).
Subject(s)
Neutropenia/immunology , Neutropenia/mortality , Sjogren's Syndrome/immunology , Sjogren's Syndrome/mortality , Adolescent , Adult , Age Distribution , Aged , Aged, 80 and over , Cohort Studies , Female , Hospitalization/statistics & numerical data , Humans , Infections/immunology , Infections/mortality , Leukemia, T-Cell/immunology , Leukemia, T-Cell/mortality , Lymphoma/immunology , Lymphoma/mortality , Male , Middle Aged , Prevalence , Young AdultABSTRACT
Tumor necrosis factor (TNF)-targeted therapies are increasingly used for a rapidly expanding number of rheumatic and autoimmune diseases. With this use and longer follow-up periods of treatment, there are a growing number of reports of the development of autoimmune processes related to anti-TNF agents. We have analyzed the clinical characteristics, outcomes, and patterns of association with the different anti-TNF agents used in all reports of autoimmune diseases developing after TNF-targeted therapy found through a MEDLINE search of articles published between January 1990 and December 2006. We identified 233 cases of autoimmune diseases (vasculitis in 113, lupus in 92, interstitial lung diseases in 24, and other diseases in 4) secondary to TNF-targeted therapies in 226 patients. The anti-TNF agents were administered for rheumatoid arthritis (RA) in 187 (83%) patients, Crohn disease in 17, ankylosing spondylitis in 7, psoriatic arthritis in 6, juvenile RA in 5, and other diseases in 3. The anti-TNF agents administered were infliximab in 105 patients, etanercept in 96, adalimumab in 21, and other anti-TNF agents in 3. We found 92 reported cases of lupus following anti-TNF therapy (infliximab in 40 cases, etanercept in 37, and adalimumab in 15). Nearly half the cases fulfilled 4 or more classification criteria for systemic lupus erythematosus (SLE), which fell to one-third after discarding preexisting lupus-like features. One hundred thirteen patients developed vasculitis after receiving anti-TNF agents (etanercept in 59 cases, infliximab in 47, adalimumab in 5, and other agents in 2). Leukocytoclastic vasculitis was the most frequent type of vasculitis, and purpura was the most frequent cutaneous lesion. A significant finding was that one-quarter of patients with vasculitis related to anti-TNF agents had extracutaneous involvement. Twenty-four cases of interstitial lung disease associated with the use of anti-TNF agents were reported. In these patients, 2 specific characteristics should be highlighted: the poor prognosis in spite of cessation of anti-TNF therapy, and the possible adjuvant role of concomitant methotrexate. In conclusion, the use of anti-TNF agents has been associated with an increasing number of cases of autoimmune diseases, principally cutaneous vasculitis, lupus-like syndrome, SLE, and interstitial lung disease.
