AML associated oncofusion proteins PML-RARA, AML1-ETO and CBFB-MYH11 target RUNX/ETS-factor binding sites to modulate H3ac levels and drive leukemogenesis

Chromosomal translocations are one of the hallmarks of acute myeloid leukemia (AML), often leading to gene fusions and expression of an oncofusion protein. Over recent years it has become clear that most of the AML associated oncofusion proteins molecularly adopt distinct mechanisms for inducing leukemogenesis. Still these unique molecular properties of the chimeric proteins converge and give rise to a common pathogenic molecular mechanism. In the present study we compared genome-wide DNA binding and transcriptome data associated with AML1-ETO, CBFB-MYH11 and PML-RARA oncofusion protein expression to identify unique and common features. Our analyses revealed targeting of oncofusion binding sites to RUNX1 and ETS-factor occupied genomic regions. In addition, it revealed a highly comparable global histone acetylation pattern, similar expression of common target genes and related enrichment of several biological pathways critical for maintenance of AML, suggesting oncofusion proteins deregulate common gene programs despite their distinct binding signatures and mechanisms of action.Peer reviewe

Nanopatterned acellular valve conduits drive the commitment of blood-derived multipotent cells

Considerable progress has been made in recent years toward elucidating the correlation among nanoscale topography, mechanical properties, and biological behavior of cardiac valve substitutes. Porcine TriCol scaffolds are promising valve tissue engineering matrices with demonstrated self-repopulation potentiality. In order to define an in vitro model for investigating the influence of extracellular matrix signaling on the growth pattern of colonizing blood-derived cells, we cultured circulating multipotent cells (CMC) on acellular aortic (AVL) and pulmonary (PVL) valve conduits prepared with TriCol method and under no-flow condition. Isolated by our group from Vietnamese pigs before heart valve prosthetic implantation, porcine CMC revealed high proliferative abilities, three-lineage differentiative potential, and distinct hematopoietic/endothelial and mesenchymal properties. Their interaction with valve extracellular matrix nanostructures boosted differential messenger RNA expression pattern and morphologic features on AVL compared to PVL, while promoting on both matrices the commitment to valvular and endothelial cell-like phenotypes. Based on their origin from peripheral blood, porcine CMC are hypothesized in vivo to exert a pivotal role to homeostatically replenish valve cells and contribute to hetero- or allograft colonization. Furthermore, due to their high responsivity to extracellular matrix nanostructure signaling, porcine CMC could be useful for a preliminary evaluation of heart valve prosthetic functionality

The acute myeloid leukemia associated AML1-ETO fusion protein alters the transcriptome and cellular progression in a single-oncogene expressing in vitro induced pluripotent stem cell based granulocyte differentiation model

Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect normal hematopoiesis. The analysis of human AMLs has mostly been performed using end-point materials, such as cell lines and patient derived AMLs that also carry additional contributing mutations. The molecular effects of a single oncogenic hit, such as expression of the AML associated oncoprotein AML1-ETO on hematopoietic development and transformation into a (pre-) leukemic state still needs further investigation. Here we describe the development and characterization of an induced pluripotent stem cell (iPSC) system that allows in vitro differentiation towards different mature myeloid cell types such as monocytes and granulocytes. During in vitro differentiation we expressed the AML1-ETO fusion protein and examined the effects of the oncoprotein on differentiation and the underlying alterations in the gene program at 8 different time points. Our analysis revealed that AML1-ETO as a single oncogenic hit in a non-mutated background blocks granulocytic differentiation, deregulates the gene program via altering the acetylome of the differentiating granulocytic cells, and induces t(8;21) AML associated leukemic characteristics. Together, these results reveal that inducible oncogene expression during in vitro differentiation of iPS cells provides a valuable platform for analysis of aberrant regulation in disease

The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease

Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal.We thank members of the Cambridge BioResource Scientific Advisory Board and Management Committee for their support of our study and the National Institute for Health Research Cambridge Biomedical Research Centre for funding. K.D. is funded as a HSST trainee by NHS Health Education England. M.F. is funded from the BLUEPRINT Grant Code HEALTH-F5-2011-282510 and the BHF Cambridge Centre of Excellence [RE/13/6/30180]. J.R.S. is funded by a MRC CASE Industrial studentship, co-funded by Pfizer. J.D. is a British Heart Foundation Professor, European Research Council Senior Investigator, and National Institute for Health Research (NIHR) Senior Investigator. S.M., S.T, M.H, K.M. and L.D. are supported by the NIHR BioResource-Rare Diseases, which is funded by NIHR. Research in the Ouwehand laboratory is supported by program grants from the NIHR to W.H.O., the European Commission (HEALTH-F2-2012-279233), the British Heart Foundation (BHF) to W.J.A. and D.R. under numbers RP-PG-0310-1002 and RG/09/12/28096 and Bristol Myers-Squibb; the laboratory also receives funding from NHSBT. W.H.O is a NIHR Senior Investigator. The INTERVAL academic coordinating centre receives core support from the UK Medical Research Council (G0800270), the BHF (SP/09/002), the NIHR and Cambridge Biomedical Research Centre, as well as grants from the European Research Council (268834), the European Commission Framework Programme 7 (HEALTH-F2-2012-279233), Merck and Pfizer. DJR and DA were supported by the NIHR Programme ‘Erythropoiesis in Health and Disease’ (Ref. NIHR-RP-PG-0310-1004). N.S. is supported by the Wellcome Trust (Grant Codes WT098051 and WT091310), the EU FP7 (EPIGENESYS Grant Code 257082 and BLUEPRINT Grant Code HEALTH-F5-2011-282510). The INTERVAL study is funded by NHSBT and has been supported by the NIHR-BTRU in Donor Health and Genomics at the University of Cambridge in partnership with NHSBT. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health of England or NHSBT. D.G. is supported by a “la Caixa”-Severo Ochoa pre-doctoral fellowship

Stem cells in molecular and regenerative medicine

Stem Cells are rare cells with the crucial ability to self-renew and to generate mature cells of any tissue through differentiation. Adult stem cells hold great promise for regenerative medicine, tissue repair, and gene therapy. Adult bone marrow cells (BMCs) include two populations of bone marrow stem cells (BMCs): hematopoietic stem cells (HSCs), which give rise to all mature lineages of blood, and mesenchymal stem cells (MSCs), which can differentiate into osteoblasts, chondrocytes, adipocytes, myocytes, tenocytes, and haematopoiesis supporting stromal cells. Under normal condition these stem cells are tightly regulated by both intrinsic and extrinsic signals and malfunctioning in this balance can result in cancer. In this thesis we focused on two different aspects of stem cells: the leukemia stem/initiating cells in acute myeloid leukemia (AML) and the usage of stem cells in regenerative medicine. In the first part we focused on the molecular mechanism of AML-ETO, a results from the t(8:21) translocation which has been associated with leukemic transformation. Acute myeloid leukaemia (AML) is defined as a heterogeneous group of clonal disorders caused by malignant transformation of a bone marrow-derived self-renewing stem or progenitor cell, which demonstrates an enhanced proliferation as well as aberrant differentiation resulting in haematopoietic insufficiency (i.e. granulocytopenia, thrombocytopenia or anaemia). These leukaemias are suggested to result from the acquisition of chromosomal rearrangements and multiple gene mutations in either a hematopoietic multipotent cell or a more differentiated, lineage-restricted progenitor cell that is transformed in a so-called leukaemic stem or initiating cell, which keeps the ability to self-renewal. AML is generally regarded as a stem cell disease and is commonly altered both at the epigenetic as well as the genetic level. AML is the most common acute leukemia affecting adults, and its incidence increases with age. Therapies based on the current knowledge target the bulk leukemic population and spare the leukemic stem cells. It is therefore critical to determine and characterize the exact molecular mechanism involved in leukemic transformation for the development of novel therapeutic targets. AML patients harboring the t(8:21) translocation has intermediate prognosis and the identification of genome wide events in this subset of AML is clinically relevant and would lead to the understanding of molecular mechanism of disease progression. To this end we analyzed the DNA binding pattern of AML1-ETO in AML cell lines and in primary AML blasts. We demonstrate that AML1-ETO preferentially binds regions that contain RUNX1/AML1 and ETS core consensus sequences and that the AML1-ETO binding sites invariably consist of HEB and partially CBFβ, RUNX1/AML1 as well as of ETS factors such as ERG and FLI1. Subsequent analysis in t(8;21) and t(15;17) (another AML associated translocation) cells revealed cell type specific ETS factor binding and preferential AML1-ETO binding to the cell type specific ETS factor binding sites. In addition, we uncovered that binding of the ETS factor ERG correlates with the ‘active’ histone acetylation mark. Together our results suggest that ETS factors demarcate hematopoietic regulatory sites that provide a target for (aberrant) epigenetic regulation by oncofusion proteins. In the second part we attempted to evaluate the possibility to obtain in vitro an implantable tissue-engineered esophagus composed of acellular esophageal matrix and Mesenchymal stem cells (MSCs). Mesenchymal Stem Cells (MSCs) are multipotent precursors to many mesodermal cell lineages in vertebrate animals and are most often obtained from bone marrow. Certain attributes of MSCs, including migration toward sites of inflammation, ease of transduction, and lack of immunogenicity, suggest these cells may be potentially useful for regenerative medicine. Putative therapeutic uses include regeneration of damaged tissue, acting as a vessel for delivering a therapeutic transgene, support of other cell types for tissue repair, and modulating the immune reaction to co-transplanted cells or tissues. The use of MSCs in tissue engineering approaches avoids the moral and technical issues associated with the use of those from embryonic source and MSCs have already demonstrated their efficacy in preliminary tissue engineering application. Artificial materials and autologous tissues used for esophageal reconstruction often induce complications like stenosis and leakage at long-term follow-up. In the present study we attempted to evaluate the adhesion of MSCs on acellular esophageal matrix for esophagus tissue engineering. MSCs were isolated from rabbit bone marrow, characterized, expanded in vitro, and seeded onto rabbit acellular esophageal matrix. Acellular matrices obtained by detergent-enzymatic method did not present any major histocompatibility complex marker. Moreover, they supported cell adhesion, and in as much as just after 24 h from seeding, the scaffold appeared completely covered by MSCs in static as well as in bioreactor. Collectively, these results suggest that patches composed of homologous esophageal acellular matrix and autologous MSCs may represent a promising tissue engineering approach for the repair of esophageal injuriesLe cellule staminali sono una popolazione cellulare con la particolare capacità di moltiplicarsi indefinitamente autorinnovandosi e di differenziarsi in cellule mature di qualsiasi altro tessuto attraverso il processo di differenziazione. In particolare l'utilizzo delle cellule staminali adulte costituisce una promettente applicazione nel campo della medicina rigenerativa, la riparazione dei tessuti e la terapia genica. Le cellule staminali adulte da midollo osseo (BMCs) comprendono due popolazioni cellulari: le cellule staminali ematopoietiche (HSCs), dalle quali originano tutte le cellule mature del sangue, e le cellule staminali mesenchimali (MSCs) che possono differenziare in osteoblasti, condrociti, adipociti, miociti, tenociti e cellule stromali di supporto per l'ematopoiesi. In condizioni normali l'autorinnovamento della popolazione staminale è strettamente regolato sia da segnali estrinseci che intrinseci ed un'alterazione di questo equilibrio può portare all'instaurarsi di un cancro. In questa tesi abbiamo analizzato due differenti aspetti delle cellule staminali: le cellule staminali che danno origine a leucemia nella leucemia mieloide acuta (AML) e l'utilizzo delle cellule staminali nella medicina rigenerativa. Nella prima parte del lavoro abbiamo approfondito il meccanismo molecolare dell' AML-ETO, risultato della traslocazione genica t(8:21) che viene associata alla trasformazione leucemica. La leucemia mieloide acuta (AML) è definita come un gruppo eterogeneo di disordini clonali causati dalla trasformazione maligna di cellule staminali o progenitori staminali di derivazione midollare, che mostrano un aumento della capacità proliferativa così come un differenziamento aberrante che porta ad una insufficienza ematopoietica (per esempio: granulocitopenia, trombocitopenia o anemia). Questi tipi di leucemia sembrano essere il risultato dell'acquisizione di riarrangiamenti cromosomici e mutazioni geniche multiple da parte delle cellule ematopoietiche multipotenti o di progenitori cellulari più differenziati e indirizzati verso una linea cellulare specifica, che risultano così trasformati in cellule staminali leucemiche o cellule inizianti la leucemia, che mantengono la capacità di autorinnovamento. L' AML è solitamente considerata una malattia delle cellule staminali e comunemente presenta alterazioni sia a livello genetico che epigenetico. L' AML è la forma più comune di leucemia acuta che colpisce soprattutto la popolazione adulta e la sua incidenza aumenta con l'età. Gli attuali approcci terapeutici hanno come target le cellule staminali leucemiche e la popolazione leucemica per intero. E' quindi di cruciale importanza riuscire a determinare e caratterizzare l'esatto meccanismo molecolare coinvolto nella trasformazione leucemica per lo sviluppo di nuovi bersagli terapeutici. I pazienti affetti da AML che manifestano la traslocazione t(8:21) hanno una prognosi intermedia e l'identificazione di ampi eventi genici in questo subset delle AML è clinicamente rilevante in quanto potrebbe portare alla comprensione dei meccanismi molecolari della progressione della malattia. A questo scopo sono stati analizzati i pattern di legame al DNA di AML1-ETO nelle cellule di linea AML e nei blasti di AML. Abbiamo dimostrato che AML1-ETO lega preferenzialmente le regioni che contengono le sequenze di consenso RUNX1/AML1 e ETS e che i siti di legame di AML1-ETO si sovrappongono invariabilmente a quelli di HEB e parzialmente a quelli di CBFβ, RUNX1/AML1 così come accade per i fattori ETS, quali ERG e FLI1. Le successive analisi sulle cellule t(8;21) e t(15;17) (un'altra traslocazione associata con l' AML) hanno evidenziato il legame di fattori ETS specifici per questi tipi cellulari e il legame preferenziale di AML1-ETO ai siti di legame per i fattori ETS specifici per il tipo cellulare. Inoltre è stato anche scoperto che il legame di un fattore ETS, ERG, correla con un segnale di acetilazione istonica "attiva". Presi insieme questi risultati suggeriscono che i fattori ETS demarcano i siti regolatori ematopoietici che forniscono un target per la regolazione epigenetica (aberrante) da parte delle proteine di oncofusione. Nella seconda parte di questa tesi è stata testata la possibilità di ottenere in vitro un esofago ingegnerizzato composto da matrice acellulare esofagea e cellule staminali mesenchimali (MSCs) che potesse essere impiantato in vivo. Le cellule staminali mesenchimali (MSCs) nei vertebrati sono precursori multipotenti di molte linee cellulari di origine mesodermica e vengono ottenute per la maggior parte dal midollo osseo. Alcune caratteristiche delle MSCs, inclusa la capacità di migrare verso i siti di infiammazione, la facilità di trasduzione e la perdita di immunogenicità, suggeriscono che queste cellule possano essere potenzialmente utilizzabili nella medicina rigenerativa. I probabili usi terapeutici includono la possibilità di rigenerare un tessuto danneggiato, agendo come veicolo per il trasporto di transgeni terapeutici, di supportare altri tipi cellulari per il riparo tessutale, e di modulare la reazione immunitaria dell'ospite nei confronti delle cellule o dei tessuti co-trapiantati. L'uso delle MSCs permette di evitare i problemi di natura etica e morale associati all'utilizzo delle cellule staminali di origine embrionale; inoltre le MSCs hanno già dimostrato la loro efficacia in studi preliminari che prevedevano la loro applicazione in ingegneria tessutale. I materiali artificiali e i tessuti autologhi utilizzati per la ricostruzione dell'esofago spesso comportano complicazioni come stenosi e rottura dell'impianto nei follow-up a lungo termine. Nel presente studio è stata valutata l'adesione delle MSCs ad una matrice acellulare di esofago per la costruzione di un tessuto esofageo ingegnerizzato. Le MSCs sono state isolate da midollo osseo di coniglio, caratterizzate, espanse in vitro e seminate su una matrice esofagea di coniglio. Le matrici acellulari ottenute attraverso un metodo detergente-enzimatico non presentavano marker per il complesso maggiore di istocompatibilità. Inoltre supportavano l'adesione cellulare e in non più di 24 ore dalla semina lo scaffold appariva completamente coperto dalle MSCs sia in condizione statica che in bioreattore. Complessivamente questi risultati suggeriscono che i tessuti ingegnerizzati composti da matrice acellulare omologa e MSCs autologhe possono rappresentare un promettente approccio per il riparo di danni all'esofag

AML associated oncofusion proteins PML-RARA, AML1-ETO and CBFB-MYH11 target RUNX/ETS-factor binding sites to modulate H3ac levels and drive leukemogenesis

Chromosomal translocations are one of the hallmarks of acute myeloid leukemia (AML), often leading to gene fusions and expression of an oncofusion protein. Over recent years it has become clear that most of the AML associated oncofusion proteins molecularly adopt distinct mechanisms for inducing leukemogenesis. Still these unique molecular properties of the chimeric proteins converge and give rise to a common pathogenic molecular mechanism. In the present study we compared genome-wide DNA binding and transcriptome data associated with AML1-ETO, CBFB-MYH11 and PML-RARA oncofusion protein expression to identify unique and common features. Our analyses revealed targeting of oncofusion binding sites to RUNX1 and ETSfactor occupied genomic regions. In addition, it revealed a highly comparable global histone acetylation pattern, similar expression of common target genes and related enrichment of several biological pathways critical for maintenance of AML, suggesting oncofusion proteins deregulate common gene programs despite their distinct binding signatures and mechanisms of action