Modelado molecular de la interacción de fármacos antitumorales y nucleasas con el ADN

Premio Extraordinario de Doctorado de la UAH en 2013Los métodos computacionales están convirtiéndose en herramientas muy importantes en ciertas áreas de la investigación como la caracterización de sitios de unión de ligandos a proteínas, acoplamiento de pequeñas moléculas en sitios de unión al ADN y proteínas y simulaciones de dinámica molecular. Los resultados obtenidos aportan información que, a veces, está más allá de las posibilidades puramente experimentales y pueden usarse para guiar y mejorar un gran número de experimentos. El objetivo de esta tesis es estudiar mediante técnicas de modelado molecular la interacción entre el ADN y distintos ligandos incluyendo diversos fármacos antitumorales, distintas familias de nucleasas como XPF y la nucleasa de Vibrio vulnificus y la ARN polimerasa II. Las investigaciones se llevaron a cabo en colaboración con distintos grupos experimentales de la Universidad de Alcalá, del grupo del Profesor Egly en el Institut de génétique et de biologie moléculaire et cellulaire de Estrasburgo y de la empresa biotecnológica PharmaMar. Los trabajos realizados se dividen en los siguientes puntos: i)Descripción de la interacción del ADN con el nuevo fármaco antitumoral Zalypsis y análisis de su especificidad de secuencia por técnicas de modelado molecular. ii)Descripción de la interacción del ADN con el nuevo fármaco antitumoral PM01183 y análisis de su especificidad de secuencia por técnicas de modelado molecular. iii)Estudio de la interacción de XPF y la ARN polimerasa II con el ADN. Consecuencias de la incorporación de un aducto covalente con el ADN. iv)Estudio mediante simulaciones de dinámica molecular de la fusión inducida por alta temperatura de un segmento de ADN en ausencia y presencia de fármacos. v)Mecanismo de formación de entrecruzamientos intercatenarios en el ADN por Mitomicina C. Efecto de la modificación de las citosinas en su reactividad. vi)Estudio mediante simulaciones de dinámica molecular del mecanismo de acción de la nucleasa de Vibrio vulnificus e implicaciones en distintas familias de endonucleasas

Targeted gene therapy and cell reprogramming in Fanconi anemia

Altres ajuts: European Regional Development FEDER Funds, Italian Ministry of Health, Fondo de Investigaciones Sanitarias, Dirección General de Investigación de la Comunidad de Madrid S2010/BMD-2420, La Fundació Privada La Marató de TV3 121430/31/32, Marató de TV3 464/C/2012Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies

Detectable clonal mosaicism in blood as a biomarker of cancer risk in Fanconi anemia

Detectable clonal mosaicism for large chromosomal events has been associated with aging and an increased risk of hematological and some solid cancers. We hypothesized that genetic cancer predisposition disorders, such as Fanconi anemia (FA), could manifest a high rate of chromosomal mosaic events (CMEs) in peripheral blood, which could be used as early biomarkers of cancer risk. We studied the prevalence of CMEs by single-nucleotide polymorphism (SNP) array in 130 FA patients' blood DNA and their impact on cancer risk. We detected 51 CMEs (4.4-159 Mb in size) in 16 out of 130 patients (12.3%), of which 9 had multiple CMEs. The most frequent events were gains at 3q (n = 6) and 1q (n = 5), both previously associated with leukemia, as well as rearrangements with breakpoint clustering within the major histocompatibility complex locus (P = 7.3 x 10(-9)). Compared with 15 743 age-matched population controls, FA patients had a 126 to 140 times higher risk of detectable CMEs in blood (P < 2.2 x 10(-16)). Prevalent and incident hematologic and solid cancers were more common in CME carriers (odds ratio [OR] = 11.6, 95% confidence interval [CI] = 3.4-39.3, P = 2.8 x 10(-5)), leading to poorer prognosis. The age-adjusted hazard risk (HR) of having cancer was almost 5 times higher in FA individuals with CMEs than in those without CMEs. Regarding survival, the HR of dying was 4 times higher in FA individuals having CMEs (HR = 4.0, 95% CI = 2.0-7.9, P = 5.7 x 10(-5)). Therefore, our data suggest that molecular karyotyping with SNP arrays in easy-to-obtain blood samples could be used for better monitoring of bone marrow clonal events, cancer risk, and overall survival of FA patients

"Bioinformática con Ñ v1.0": a collaborative project of young Spanish scientists to write a complete book about Bioinformatics

Here we present a project aiming to provide specialized educational bibliography on Bioinformatics for Spanish speakers. The idea of writing a book in Spanish language covering the most important topics in the field of Bioinformatics was born in the XIth Spanish Symposium on Bioinformatics in Barcelona two years ago. Different scientists have been involved in the project, from senior scientists to PhD students from different countries. The book intends to be the beginning of an open project, where all the chapters are susceptible of being updated and new topics can be incorporated in future versions. Current book version can be accessed online at http://goo.gl/UYG0o7.Peer Reviewe

Bcr/Abl Interferes with the Fanconi Anemia/BRCA Pathway: Implications in the Chromosomal Instability of Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34+ cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34+ cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

CIBERER: Spanish national network for research on rare diseases: A highly productive collaborative initiative

13 páginas,1 figura, 3 tablas, 1 apéndice. Se extraen los autores pertenecientes a The CIBERER network que trabajan en Centros del CSIC del Appendix ACIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research.This study has been funded by Instituto de Salud Carlos III (ISCIII) and Spanish Ministry of Science and InnovationPeer reviewe

Rationale for the opposite stereochemistry of the major monoadducts and interstrand crosslinks formed by mitomycin C and its decarbamoylated analogue at CpG steps in DNA and the effect of cytosine modification on reactivity

Mitomycin C (MMC) is a potent antitumour agent that forms a covalent bond with the 2-amino group of selected guanines in the minor groove of double-stranded DNA following intracellular reduction of its quinone ring and opening of its aziridine moiety. At some 5′-CG-3′ (CpG) steps the resulting monofunctional adduct can evolve towards a more deleterious bifunctional lesion, which is known as an interstrand crosslink (ICL). MMC reactivity is enhanced when the cytosine bases are methylated (5 MC) and decreased when they are replaced with 5-F-cytosine (5FC) whereas the stereochemical preference of alkylation changes upon decarbamoylation. We have studied three duplex oligonucleotides of general formula d(CGATAAXGCTAACG) in which X stands for C, 5MC or 5FC. Using a combination of molecular dynamics simulations in aqueous solution, quantum mechanics and continuum electrostatics, we have been able to (i) obtain a large series of snapshots that facilitate an understanding in atomic detail of the distinct stereochemistry of monoadduct and ICL formation by MMC and its decarbamoylated analogue, (ii) provide an explanation for the altered reactivity of MMC towards DNA molecules containing 5MC or 5FC, and (iii) show the distinct accommodation in the DNA minor groove of the different covalent modifications, particularly the most cytotoxic C1α and C1β ICLs.Peer reviewe

Elevated levels of IL-1β in Fanconi Anemia group A patients due to a constitutively active PI3K-AKT pathway are capable of promoting tumor cell proliferation

International audienceFanconi Anemia is a hereditary disease characterized by congenital malformations, progressive bone marrow failure and an extraordinary elevated predisposition to develop cancer. In the present article we describe an anomalous high level of the proinflammatory cytokine IL-1β present in the serum of Fanconi Anemia patients. The elevated levels of IL-1β were completely reverted by transduction of a wild type copy of the FancA cDNA into FA-A lymphocytes. Although the transcription factor NF-κB is a well established regulator of IL-1β expression, our experiments did not show any proof of elevated NF-κB activity in FA-A cells. However, we found that the overexpression of IL-1β in FA-A cells is related to a constitutively activated PI3K-AKT pathway in these cells. We provide evidence that the effect of AKT on IL-1β activation is mediated by the inhibition of GSK3β. Finally, our data indicate that the levels of IL-1β produced by FA-A lymphoblasts are enough to promote an activation of the cell cycle in primary glioblastoma progenitor cells. Together, these data demonstrate that the constitutive activation of the PI3K-AKT pathway in FA cells upregulates the expression of IL-1β through a NF-κB-independent mechanism and that this overproduction activates the proliferation of tumor cells