Publications by authors named "Leire Garate"

36 Publications

Design and Synthesis of Novel Epigenetic Inhibitors Targeting Histone Deacetylases, DNA Methyltransferase 1, and Lysine Methyltransferase G9a with Efficacy in Multiple Myeloma.

J Med Chem 2021 Mar 4. Epub 2021 Mar 4.

Small Molecule Discovery Platform, Molecular Therapeutics Program, Center for Applied Medical Research (CIMA), University of Navarra, Avenida Pio XII 55, E-31008 Pamplona, Spain.

Concomitant inhibition of key epigenetic pathways involved in silencing tumor suppressor genes has been recognized as a promising strategy for cancer therapy. Herein, we report a first-in-class series of quinoline-based analogues that simultaneously inhibit histone deacetylases (from a low nanomolar range) and DNA methyltransferase-1 (from a mid-nanomolar range, IC < 200 nM). Additionally, lysine methyltransferase G9a inhibitory activity is achieved (from a low nanomolar range) by introduction of a key lysine mimic group at the 7-position of the quinoline ring. The corresponding epigenetic functional cellular responses are observed: histone-3 acetylation, DNA hypomethylation, and decreased histone-3 methylation at lysine-9. These chemical probes, multitarget epigenetic inhibitors, were validated against the multiple myeloma cell line MM1.S, demonstrating promising activity of (CM-444) with GI of 32 nM, an adequate therapeutic window (>1 log unit), and a suitable pharmacokinetic profile. , achieved significant antitumor efficacy in a xenograft mouse model of human multiple myeloma.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02255DOI Listing
March 2021

Characterization of complete lncRNAs transcriptome reveals the functional and clinical impact of lncRNAs in multiple myeloma.

Leukemia 2021 Feb 17. Epub 2021 Feb 17.

Área de Oncología, Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, IDISNA, Pamplona, Spain.

Multiple myeloma (MM) is an incurable disease, whose clinical heterogeneity makes its management challenging, highlighting the need for biological features to guide improved therapies. Deregulation of specific long non-coding RNAs (lncRNAs) has been shown in MM, nevertheless, the complete lncRNA transcriptome has not yet been elucidated. In this work, we identified 40,511 novel lncRNAs in MM samples. lncRNAs accounted for 82% of the MM transcriptome and were more heterogeneously expressed than coding genes. A total of 10,351 overexpressed and 9,535 downregulated lncRNAs were identified in MM patients when compared with normal bone-marrow plasma cells. Transcriptional dynamics study of lncRNAs in the context of normal B-cell maturation revealed 989 lncRNAs with exclusive expression in MM, among which 89 showed de novo epigenomic activation. Knockdown studies on one of these lncRNAs, SMILO (specific myeloma intergenic long non-coding RNA), resulted in reduced proliferation and induction of apoptosis of MM cells, and activation of the interferon pathway. We also showed that the expression of lncRNAs, together with clinical and genetic risk alterations, stratified MM patients into several progression-free survival and overall survival groups. In summary, our global analysis of the lncRNAs transcriptome reveals the presence of specific lncRNAs associated with the biological and clinical behavior of the disease.
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http://dx.doi.org/10.1038/s41375-021-01147-yDOI Listing
February 2021

Chromatin activation as a unifying principle underlying pathogenic mechanisms in multiple myeloma.

Genome Res 2020 Sep 20;30(9):1217-1227. Epub 2020 Aug 20.

Centro de Investigación Biomédica en Red de Cáncer, CIBERONC, 28029 Madrid, Spain.

Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (), a major regulator of cellular redox status and, in addition, identified as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype.
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http://dx.doi.org/10.1101/gr.265520.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545147PMC
September 2020

Inhibition of a G9a/DNMT network triggers immune-mediated bladder cancer regression.

Nat Med 2019 07 3;25(7):1073-1081. Epub 2019 Jul 3.

Molecular Oncology Unit CIEMAT, Madrid, Spain.

Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances. Recent molecular characterization has defined new (epi)genetic drivers and potential targets for bladder cancer. The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients. Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (Pten; Trp53; Rb1; Rbl1) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade.
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http://dx.doi.org/10.1038/s41591-019-0499-yDOI Listing
July 2019

Long non-coding RNAs discriminate the stages and gene regulatory states of human humoral immune response.

Nat Commun 2019 02 18;10(1):821. Epub 2019 Feb 18.

Department of Medicine, Division of Hematology/Oncology, Weill Cornell Medicine, New York, NY, 10021, USA.

lncRNAs make up a majority of the human transcriptome and have key regulatory functions. Here we perform unbiased de novo annotation of transcripts expressed during the human humoral immune response to find 30% of the human genome transcribed during this process, yet 58% of these transcripts manifest striking differential expression, indicating an lncRNA phylogenetic relationship among cell types that is more robust than that of coding genes. We provide an atlas of lncRNAs in naive and GC B-cells that indicates their partition into ten functionally categories based on chromatin features, DNase hypersensitivity and transcription factor localization, defining lncRNAs classes such as enhancer-RNAs (eRNA), bivalent-lncRNAs, and CTCF-associated, among others. Specifically, eRNAs are transcribed in 8.6% of regular enhancers and 36.5% of super enhancers, and are associated with coding genes that participate in critical immune regulatory pathways, while plasma cells have uniquely high levels of circular-RNAs accounted for by and reflecting the combinatorial clonal state of the Immunoglobulin loci.
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http://dx.doi.org/10.1038/s41467-019-08679-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379396PMC
February 2019

Showcasing the role of seawater in bacteria recruitment and microbiome stability in sponges.

Sci Rep 2018 10 12;8(1):15201. Epub 2018 Oct 12.

Centre d'Estudis Avançats de Blanes, CEAB-CSIC, Accés Cala St. Francesc, Blanes, Girona, 17300, Spain.

We studied the core bacterial communities of 19 sponge species from Nha Trang Bay (Central Vietnam), with particular emphasis on the contribution of planktonic seawater bacteria to the sponge core microbiomes. To ensure consistent sponge-microbe associations and accurate identification of planktonic bacteria transmitted from seawater, we were very restrictive with the definition of the sponge core microbiomes (present in all the replicates), and with the identification of valid biological 16S rRNA gene sequences (100% sequence identity) that belonged to potentially different bacterial taxa. We found a high overlap (>50% relative abundance) between the sponge species core microbiome and the seawater bacterial core in ca. a half of the studied species, including representatives of both, HMA and LMA sponges. From our restrictive analysis, we point to horizontal transmission as a relevant way of symbiont acquisition in sponges. Some species-specific recognition mechanisms may act in sponges to enrich specific seawater bacteria in their tissues. These mechanisms would allow the maintenance of bacterial communities in a species across geographical ranges. Moreover, besides contrasting preferences in bacteria selection from seawater, divergent physiological traits may also account for the different microbiomes in species of HMA and LMA sponges.
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http://dx.doi.org/10.1038/s41598-018-33545-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185911PMC
October 2018

Discovery of Reversible DNA Methyltransferase and Lysine Methyltransferase G9a Inhibitors with Antitumoral in Vivo Efficacy.

J Med Chem 2018 Aug 19;61(15):6518-6545. Epub 2018 Jul 19.

Departmento de Hematología, Clinica Universidad de Navarra , University of Navarra , Avenida Pio XII 36 , E-31008 Pamplona , Spain.

Using knowledge- and structure-based approaches, we designed and synthesized reversible chemical probes that simultaneously inhibit the activity of two epigenetic targets, histone 3 lysine 9 methyltransferase (G9a) and DNA methyltransferases (DNMT), at nanomolar ranges. Enzymatic competition assays confirmed our design strategy: substrate competitive inhibitors. Next, an initial exploration around our hit 11 was pursued to identify an adequate tool compound for in vivo testing. In vitro treatment of different hematological neoplasia cell lines led to the identification of molecules with clear antiproliferative efficacies (GI values in the nanomolar range). On the basis of epigenetic functional cellular responses (levels of lysine 9 methylation and 5-methylcytosine), an acceptable therapeutic window (around 1 log unit) and a suitable pharmacokinetic profile, 12 was selected for in vivo proof-of-concept ( Nat. Commun. 2017 , 8 , 15424 ). Herein, 12 achieved a significant in vivo efficacy: 70% overall tumor growth inhibition of a human acute myeloid leukemia (AML) xenograft in a mouse model.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01926DOI Listing
August 2018

Detailed Exploration around 4-Aminoquinolines Chemical Space to Navigate the Lysine Methyltransferase G9a and DNA Methyltransferase Biological Spaces.

J Med Chem 2018 Aug 19;61(15):6546-6573. Epub 2018 Jul 19.

Departmento de Hematología, Clinica Universidad de Navarra , University of Navarra , Avenida Pio XII 36 , E-31008 Pamplona , Spain.

Epigenetic regulators that exhibit aberrant enzymatic activities or expression profiles are potential therapeutic targets for cancers. Specifically, enzymes responsible for methylation at histone-3 lysine-9 (like G9a) and aberrant DNA hypermethylation (DNMTs) have been implicated in a number of cancers. Recently, molecules bearing a 4-aminoquinoline scaffold were reported as dual inhibitors of these targets and showed a significant in vivo efficacy in animal models of hematological malignancies. Here, we report a detailed exploration around three growing vectors born by this chemotype. Exploring this chemical space led to the identification of features to navigate G9a and DNMT1 biological spaces: not only their corresponding exclusive areas, selective compounds, but also common spaces. Thus, we identified from selective G9a and first-in-class DNMT1 inhibitors, >1 log unit between their IC values, with IC < 25 nM (e.g., 43 and 26, respectively) to equipotent inhibitors with IC < 50 nM for both targets (e.g., 13). Their ADME/Tox profiling and antiproliferative efficacies, versus some cancer cell lines, are also reported.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01925DOI Listing
August 2018

Reversible dual inhibitor against G9a and DNMT1 improves human iPSC derivation enhancing MET and facilitating transcription factor engagement to the genome.

PLoS One 2017 27;12(12):e0190275. Epub 2017 Dec 27.

Cell Therapy Program, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain.

The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272) improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0190275PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744984PMC
February 2018

In-silico gene essentiality analysis of polyamine biosynthesis reveals APRT as a potential target in cancer.

Sci Rep 2017 10 30;7(1):14358. Epub 2017 Oct 30.

Bioinformatics Group, CEIT and TECNUN, University of Navarra, San Sebastian, 20018, Spain.

Constraint-based modeling for genome-scale metabolic networks has emerged in the last years as a promising approach to elucidate drug targets in cancer. Beyond the canonical biosynthetic routes to produce biomass, it is of key importance to focus on metabolic routes that sustain the proliferative capacity through the regulation of other biological means in order to improve in-silico gene essentiality analyses. Polyamines are polycations with central roles in cancer cell proliferation, through the regulation of transcription and translation among other things, but are typically neglected in in silico cancer metabolic models. In this study, we analysed essential genes for the biosynthesis of polyamines. Our analysis corroborates the importance of previously known regulators of the pathway, such as Adenosylmethionine Decarboxylase 1 (AMD1) and uncovers novel enzymes predicted to be relevant for polyamine homeostasis. We focused on Adenine Phosphoribosyltransferase (APRT) and demonstrated the detrimental consequence of APRT gene silencing on different leukaemia cell lines. Our results highlight the importance of revisiting the metabolic models used for in-silico gene essentiality analyses in order to maximize the potential for drug target identification in cancer.
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http://dx.doi.org/10.1038/s41598-017-14067-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662602PMC
October 2017

An in-silico approach to predict and exploit synthetic lethality in cancer metabolism.

Nat Commun 2017 09 6;8(1):459. Epub 2017 Sep 6.

CEIT and Tecnun, University of Navarra, Manuel de Lardizábal 13, 20018, San Sebastián, Spain.

Synthetic lethality is a promising concept in cancer research, potentially opening new possibilities for the development of more effective and selective treatments. Here, we present a computational method to predict and exploit synthetic lethality in cancer metabolism. Our approach relies on the concept of genetic minimal cut sets and gene expression data, demonstrating a superior performance to previous approaches predicting metabolic vulnerabilities in cancer. Our genetic minimal cut set computational framework is applied to evaluate the lethality of ribonucleotide reductase catalytic subunit M1 (RRM1) inhibition in multiple myeloma. We present a computational and experimental study of the effect of RRM1 inhibition in four multiple myeloma cell lines. In addition, using publicly available genome-scale loss-of-function screens, a possible mechanism by which the inhibition of RRM1 is effective in cancer is established. Overall, our approach shows promising results and lays the foundation to build a novel family of algorithms to target metabolism in cancer.Exploiting synthetic lethality is a promising approach for cancer therapy. Here, the authors present an approach to identifying such interactions by finding genetic minimal cut sets (gMCSs) that block cancer proliferation, and apply it to study the lethality of RRM1 inhibition in multiple myeloma.
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http://dx.doi.org/10.1038/s41467-017-00555-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587678PMC
September 2017

Contrasting biological features in morphologically cryptic Mediterranean sponges.

PeerJ 2017 29;5:e3490. Epub 2017 Jun 29.

Department of Marine Ecology, Centre d'Estudis Avançats de Blanes (CEAB-CSIC), Blanes, Girona, Spain.

Sponges are key organisms in the marine benthos where they play essential roles in ecological processes such as creating new niches, competition for resources, and organic matter recycling. Despite the increasing number of taxonomical studies, many sponge species remain hidden, whether unnoticed or cryptic. The occurrence of cryptic species may confound ecological studies by underestimating biodiversity. In this study, we monitored photographically growth, fusions, fissions, and survival of two morphologically cryptic species Uriz, Garate & Agell, 2017 and (Bowerbank, 1874). Additionally, we characterized the main environmental factors of the corresponding species habitats, trying to ascertain whether some abiotic factors were correlated with the distribution of these species. Sponge monitoring was performed monthly. Seawater samples were collected the same monitoring days in the vicinity of the target sponges. Results showed contrasting growth and survival patterns for each species: totally disappeared after larval release while 64% of individuals of survived the entire two years we monitored. The species also differed in the number of fissions and fusions. These events were evenly distributed throughout the year in the population but concentrated in cold months in . No measured environmental factor correlated with growth rates, while temperature and dissolved organic nitrogen were negatively correlated with growth rates. The strong differences in depth distribution, survival, growth, fusions, and fissions found between these two cryptic species, highlights the importance of untangling cryptic species before ecological studies are performed in particular when these species share geographical distribution.
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http://dx.doi.org/10.7717/peerj.3490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493970PMC
June 2017

Redescription and establishment of a holotype and three paratypes for the species sp. nov.

PeerJ 2017 8;5:e3426. Epub 2017 Jun 8.

Department of Marine Ecology, Centre for Advanced Studies of Blanes (CEAB-CSIC), Blanes, Girona, Spain.

Background: In a recent paper, we described a new sponge species named Uriz, Garate & Agell, 2017. However, we failed to designate a holotype and a type locality, as required by the International Commission on Zoological Nomenclature (ICZN). Although the validity of the previous conclusions remains unchanged, the species name cannot be considered available according to ICZN regulations until a holotype is designated.

Results: The present work fulfills the requirements of the ICZN by designating a holotype, three paratypes and the type locality for the new species and has been registered in ZooBank.
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http://dx.doi.org/10.7717/peerj.3426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466809PMC
June 2017

Molecular phylogenies confirm the presence of two cryptic species in the Mediterranean and reveal the polyphyly of the genera and (Demospongiae: Poecilosclerida).

PeerJ 2017 7;5:e2958. Epub 2017 Mar 7.

Department of Marine Ecology, Centre for Advanced Studies of Blanes (CEAB-CSIC) , Blanes, Girona , Spain.

Background: Sponges are particularly prone to hiding cryptic species as their paradigmatic plasticity often favors species phenotypic convergence as a result of adaptation to similar habitat conditions. is a sponge genus (Family Hymedesmiidae, Order Poecilosclerida) with four formally described species, from which only has been recorded in the Atlanto-Mediterranean basin, on shallow to 80 m deep bottoms. Contrasting biological features between shallow and deep individuals of suggested larger genetic differences than those expected between sponge populations. To assess whether shallow and deep populations indeed belong to different species, we performed a phylogenetic study of across the Mediterranean. We also included other and species from the Red Sea, with the additional aim of clarifying the relationships of the genus .

Methods: was sampled across the Mediterranean, and Adriatic Seas. and were collected from the Red Sea and Pacific. From two to three specimens per species and locality were extracted, amplified for Cytochrome C Oxidase I (COI) (M1-M6 partition), 18S rRNA, and 28S (D3-D5 partition) and sequenced. Sequences were aligned using Clustal W v.1.81. Phylogenetic trees were constructed under neighbor joining (NJ), Bayesian inference (BI), and maximum likelihood (ML) criteria as implemented in Geneious software 9.01. Moreover, spicules of the target species were observed through a Scanning Electron microscope.

Results: The several phylogenetic reconstructions retrieved both and polyphyletic. Strong differences in COI sequences indicated that from the Red Sea might belong in a different genus, closer to than to the Atlanto-Mediterranean spp. Molecular and external morphological differences between and the Atlanto-Mediterranean also suggest that fit in a separate genus. On the other hand, the Atlanto-Mediterranean Crellidae appeared in 18S and 28S phylogenies as a sister group of the Atlanto-Mediterranean . Moreover, what was known up to now as is formed by two cryptic species with contrasting bathymetric distributions. Some small but consistent morphological differences allow species distinction.

Conclusions: A new family (Hemimycalidae) including the genus and the two purported new genera receiving and might be proposed according to our phylogenetic results. However, the inclusion of additional Operational Taxonomic Unit (OTUs) appears convenient before taking definite taxonomical decisions. A new cryptic species ( sp. nov.) is described. Morphologically undifferentiated species with contrasting biological traits, as those here reported, confirm that unidentified cryptic species may confound ecological studies.
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http://dx.doi.org/10.7717/peerj.2958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5344016PMC
March 2017

Endosymbiotic calcifying bacteria across sponge species and oceans.

Sci Rep 2017 03 6;7:43674. Epub 2017 Mar 6.

Centre d'Estudis Avançats de Blanes. Access Cala St Francesc, 14. 17300. Blanes (Girona) Spain.

From an evolutionary point of view, sponges are ideal targets to study marine symbioses as they are the most ancient living metazoans and harbour highly diverse microbial communities. A recently discovered association between the sponge Hemimycale columella and an intracellular bacterium that generates large amounts of calcite spherules has prompted speculation on the possible role of intracellular bacteria in the evolution of the skeleton in early animals. To gain insight into this purportedly ancestral symbiosis, we investigated the presence of symbiotic bacteria in Mediterranean and Caribbean sponges. We found four new calcibacteria OTUs belonging to the SAR116 in two orders (Poecilosclerida and Clionaida) and three families of Demospongiae, two additional OTUs in cnidarians and one more in seawater (at 98.5% similarity). Using a calcibacteria targeted probe and CARD-FISH, we also found calcibacteria in Spirophorida and Suberitida and proved that the calcifying bacteria accumulated at the sponge periphery, forming a skeletal cortex, analogous to that of siliceous microscleres in other demosponges. Bacteria-mediated skeletonization is spread in a range of phylogenetically distant species and thus the purported implication of bacteria in skeleton formation and evolution of early animals gains relevance.
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http://dx.doi.org/10.1038/srep43674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5337934PMC
March 2017

TET2 mutations are associated with specific 5-methylcytosine and 5-hydroxymethylcytosine profiles in patients with chronic myelomonocytic leukemia.

PLoS One 2012 6;7(2):e31605. Epub 2012 Feb 6.

Laboratory of Myeloproliferative Syndromes, Oncology Area, University of Navarra, Pamplona, Spain.

Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has been recently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031605PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273467PMC
June 2012

Down-regulated expression of hsa-miR-181c in Fanconi anemia patients: implications in TNFα regulation and proliferation of hematopoietic progenitor cells.

Blood 2012 Mar 6;119(13):3042-9. Epub 2012 Feb 6.

Hematopoiesis and Gene Therapy Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain.

Fanconi anemia (FA) is an inherited genetic disorder associated with BM failure and cancer predisposition. In the present study, we sought to elucidate the role of microRNAs (miRNAs) in the hematopoietic defects observed in FA patients. Initial studies showed that 3 miRNAs, hsa-miR-133a, hsa-miR-135b, and hsa-miR-181c, were significantly down-regulated in lymphoblastoid cell lines and fresh peripheral blood cells from FA patients. In vitro studies with cells expressing the luciferase reporter fused to the TNFα 3'-untranslated region confirmed in silico predictions suggesting an interaction between hsa-miR-181c and TNFα mRNA. These observations were consistent with the down-regulated expression of TNFα mediated by hsa-miR-181c in cells from healthy donors and cells from FA patients. Because of the relevance of TNFα in the hematopoietic defects of FA patients, in the present study, we transfected BM cells from FA patients with hsa-miR-181c to evaluate the impact of this miRNA on their clonogenic potential. hsa-miR-181c markedly increased the number and size of the myeloid and erythroid colonies generated by BM cells from FA patients. Our results offer new clues toward understanding the biologic basis of BM failure in FA patients and open new possibilities for the treatment of the hematologic dysfunction in FA patients based on miRNA regulation.
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http://dx.doi.org/10.1182/blood-2011-01-331017DOI Listing
March 2012

Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia.

PLoS One 2011 Feb 28;6(2):e17012. Epub 2011 Feb 28.

Hematology Service and Area of Cell Therapy, Clínica Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain.

Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2'-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017012PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046174PMC
February 2011

MicroRNA expression profiling in Imatinib-resistant Chronic Myeloid Leukemia patients without clinically significant ABL1-mutations.

Mol Cancer 2009 Sep 1;8:69. Epub 2009 Sep 1.

Foundation for Applied Medical Research, Division of Cancer, Area of Cell Therapy and Hematology Service, Clínica Universitaria, Universidad de Navarra, Spain.

The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML.
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http://dx.doi.org/10.1186/1476-4598-8-69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743636PMC
September 2009

Epigenetic silencing of the tumor suppressor microRNA Hsa-miR-124a regulates CDK6 expression and confers a poor prognosis in acute lymphoblastic leukemia.

Cancer Res 2009 May 12;69(10):4443-53. Epub 2009 May 12.

Hematology Department and Area of Cell Therapy, Clinica Universitaria and Division of Gene Therapy and Hepatology, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain.

Whereas transcriptional silencing of genes due to epigenetic mechanisms is one of the most important alterations in acute lymphoblastic leukemia (ALL), some recent studies indicate that DNA methylation contributes to down-regulation of miRNAs during tumorigenesis. To explore the epigenetic alterations of miRNAs in ALL, we analyzed the methylation and chromatin status of the miR-124a loci in ALL. Expression of miR-124a was down-regulated in ALL by hypermethylation of the promoter and histone modifications including decreased levels of 3mk4H3 and AcH3 and increased levels of 2mK9H3, 3mK9H3, and 3mK27H3. Epigenetic down-regulation of miR-124a induced an up-regulation of its target, CDK6, and phosphorylation of retinoblastoma (Rb) and contributed to the abnormal proliferation of ALL cells both in vitro and in vivo. Cyclin-dependent kinase 6 (CDK6) inhibition by sodium butyrate or PD-0332991 decreased ALL cell growth in vitro, whereas overexpression of pre-miR124a led to decreased tumorigenicity in a xenogeneic in vivo Rag2(-/-)gammac(-/-) mouse model. The clinical implications of these findings were analyzed in a group of 353 patients diagnosed with ALL. Methylation of hsa-miR-124a was observed in 59% of the patients, which correlated with down-regulation of miR-124a (P < 0.001). Furthermore, hypermethylation of hsa-miR-124a was associated with higher relapse rate (P = 0.001) and mortality rate (P < 0.001), being an independent prognostic factor for disease-free survival (P < 0.001) and overall survival (P = 0.005) in the multivariate analysis. These results provide the grounds for new therapeutic strategies in ALL either targeting the epigenetic regulation of microRNAs and/or directly targeting the CDK6-Rb pathway.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-4025DOI Listing
May 2009

Epigenetic down-regulation of BIM expression is associated with reduced optimal responses to imatinib treatment in chronic myeloid leukaemia.

Eur J Cancer 2009 Jul 4;45(10):1877-89. Epub 2009 May 4.

Hematology Department and Area of Cell Therapy, Clinica Universitaria, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain.

Background: Expression of the pro-apoptotic BCL-2-interacting mediator (BIM) has recently been implicated in imatinib-induced apoptosis of BCR-ABL1(+) cells. However, the mechanisms involved in the regulation of BIM in CML and its role in the clinical setting have not been established.

Design And Methods: We analysed the mRNA expression of BIM in 100 newly diagnosed patients with CML in chronic phase by Q-RT-PCR and the protein levels by Western blot analysis. Methylation status was analysed by bisulphite genomic sequencing and MSP. CML cell lines were treated with imatinib and 5-aza-2'-deoxycytidine, and were transfected with two different siRNAs against BIM and cell proliferation and apoptosis were analysed.

Results: We demonstrated that down-regulation of BIM expression was present in 36% of the patients and was significantly associated with a lack of optimal response to imatinib as indicated by the decrease in cytogenetic and molecular responses at 6, 12 and 18 months in comparison with patients with normal BIM expression (p<0.05). Expression of BIM was mediated by promoter hypermethylation as demonstrated by restoration of BIM expression after treatment of CML cells with 5-aza-2'-deoxycytidine. Using CML cell lines with low and normal expression of BIM we further demonstrated that the expression of BIM is required for imatinib-induced CML apoptosis.

Conclusion: Our data indicate that down-regulation of BIM is epigenetically controlled by methylation in a percentage of CML patients and has an unfavourable prognostic impact, and that the combination of imatinib with a de-methylating agent may result in improved responses in patients with decreased expression of BIM.
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http://dx.doi.org/10.1016/j.ejca.2009.04.005DOI Listing
July 2009

Epigenetic regulation of microRNAs in acute lymphoblastic leukemia.

J Clin Oncol 2009 Mar 21;27(8):1316-22. Epub 2009 Jan 21.

Hematology Department. Reina Sofia Hospital. Avda. Menendez Pidal s/n. 14004 Cordoba. Spain.

Purpose: To identify microRNAs (miRNAs) epigenetically regulated in acute lymphoblastic leukemia (ALL).

Methods: We first examined ALL-derived cell lines for the presence of abnormal levels of two different histone modifications (trimethylation of H3 lysine 4 [K4H3me3] and dimethylation of H3 lysine 9 [K9H3me2]) in the 5'UTR regions around CpG islands of 78 miRNAs by chromatin immunoprecipitation (ChIP)-on-ChIP analysis. Methylation status (methylation-specific polymerase chain reaction [PCR]) and expression (quantitative PCR) of miRNAs showing a pattern of histone modifications linked to a closed chromatin structure were analyzed in a panel of six ALL cell lines and in 353 ALL patients.

Results: CpG islands around 13 miRNAs disclosed high levels of K9H3me2 and/or low levels of K4H3me3, a pattern of histone modifications underlying a closed chromatin structure associated with repressive gene expression. Complete consistency in the correlation between both histone marks, the presence of DNA methylation around these miRNAs, and their expression patterns was confirmed in the six ALL cell lines. Treatment with 5-Aza-2'-deoxycytidine upregulated the expression levels of these genes, suggesting that epigenetic mechanisms deregulate the expression of these miRNAs. A total of 65% of the ALL samples had at least one miRNA methylated (methylated group). Estimated disease-free survival (DFS) and overall survival (OS) at 14 years were 78% and 71% for nonmethylated patients and 24% and 28% for methylated patients (P = .00001 for both). Multivariate analysis demonstrated that methylation profile was an independent prognostic factor for predicting DFS (P = .0001) and OS (P = .0001).

Conclusion: Aberrant miRNA methylation is a common phenomenon in ALL that affects the clinical outcome of these patients.
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http://dx.doi.org/10.1200/JCO.2008.19.3441DOI Listing
March 2009

Down-regulation of hsa-miR-10a in chronic myeloid leukemia CD34+ cells increases USF2-mediated cell growth.

Mol Cancer Res 2008 Dec;6(12):1830-40

Foundation for Applied Medical Research, Division of Cancer, Clínica Universitaria, University of Navarre, Pamplona, Spain.

MicroRNAs (miRNA) are small noncoding, single-stranded RNAs that inhibit gene expression at a posttranscriptional level, whose abnormal expression has been described in different tumors. The aim of our study was to identify miRNAs potentially implicated in chronic myeloid leukemia (CML). We detected an abnormal miRNA expression profile in mononuclear and CD34(+) cells from patients with CML compared with healthy controls. Of 157 miRNAs tested, hsa-miR-10a, hsa-miR-150, and hsa-miR-151 were down-regulated, whereas hsa-miR-96 was up-regulated in CML cells. Down-regulation of hsa-miR-10a was not dependent on BCR-ABL1 activity and contributed to the increased cell growth of CML cells. We identified the upstream stimulatory factor 2 (USF2) as a potential target of hsa-miR-10a and showed that overexpression of USF2 also increases cell growth. The clinical relevance of these findings was shown in a group of 85 newly diagnosed patients with CML in which expression of hsa-miR-10a was down-regulated in 71% of the patients, whereas expression of USF2 was up-regulated in 60% of the CML patients, with overexpression of USF2 being significantly associated with decreased expression of hsa-miR-10a (P = 0.004). Our results indicate that down-regulation of hsa-miR-10a may increase USF2 and contribute to the increase in cell proliferation of CML implicating a miRNA in the abnormal behavior of CML.
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http://dx.doi.org/10.1158/1541-7786.MCR-08-0167DOI Listing
December 2008

Methylation status of Wnt signaling pathway genes affects the clinical outcome of Philadelphia-positive acute lymphoblastic leukemia.

Cancer Sci 2008 Sep 28;99(9):1865-8. Epub 2008 Jun 28.

Hematology Department, Reina Sofia Hospital, 14004-Cordoba, Spain.

The clinical significance of aberrant promoter methylation of the canonical Wnt pathway antagonist genes (sFRP1, sFRP2, sFRP4, sFRP5, Wif1, Dkk3, and Hdpr1) and also putative tumor-suppressor gene Wnt5a, belonging to the non-canonical Wnt signaling pathway, was investigated in a large series of 75 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia by methylation-specific polymerase chain reaction. At least one methylated gene was observed in cells from 66% (49/75) of patients (methylated group). Disease-free survival and overall survival at 9 years were 51 and 40%, respectively, for the unmethylated group and 3 and 2%, respectively, for the methylated group (both P < 0.0001). Multivariate analysis demonstrated that the Wnt methylation profile was an independent prognostic factor predicting disease-free survival (P = 0.007) and overall survival (P = 0.039). Abnormal DNA methylation of promoter-associated CpG islands in the Wnt signaling pathway is very common in Philadelphia chromosome-positive acute lymphoblastic leukemia and potentially defines subgroups with distinct clinical characteristics.
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http://dx.doi.org/10.1111/j.1349-7006.2008.00884.xDOI Listing
September 2008

BCR-ABL1-induced expression of HSPA8 promotes cell survival in chronic myeloid leukaemia.

Br J Haematol 2008 Aug 5;142(4):571-82. Epub 2008 Jun 5.

Foundation for Applied Medical Research, Division of Cancer, Area of Cell Therapy and Haematology Service, Clínica Universitaria, Universidad de Navarra, Spain.

In order to determine new signal transduction pathways implicated in chronic myeloid leukaemia (CML), we performed a gene expression profile comparison between CD34+ cells from CML patients and healthy donors. Functional studies were performed using the Mo7e and Mo7e-p210 cell lines. Expression of CCND1 (Cyclin D1), as well as the chaperone HSPA8, which is important for regulation of CCND1, were significantly upregulated in CD34+ CML cells. Upregulation of HSPA8 was dependent, at least in part, on STAT5 (signal transducer and activator of transcrition 5)-dependent transcriptional activation, as demonstrated by chromatin immunoprecipitation. The presence of HSPA8 in the nuclear protein fraction as well as its binding to CCND1 suggests that it may contribute to stabilization of the CCND1/CDK4 complex, which, in turn, may participate in proliferation of CML cells. Treatment of CML cells with the specific HSPA8 inhibitor 15-deoxyspergualin induced inhibition of CML cell viability but did not induce apoptosis. In conclusion, our studies suggest that STAT5-mediated activation of HSPA8 induces nuclear translocation and activation of the CCND1/CDK4 complex leading to increased proliferation of CML cells, deciphering a new pathway implicated in CML and supporting a potential role of chaperone inhibitors in the treatment of CML.
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http://dx.doi.org/10.1111/j.1365-2141.2008.07221.xDOI Listing
August 2008

WNT5A, a putative tumour suppressor of lymphoid malignancies, is inactivated by aberrant methylation in acute lymphoblastic leukaemia.

Eur J Cancer 2007 Dec;43(18):2736-46

Hematology Department, Reina Sofia Hospital, Avda. Menendez Pidal s/n, 14004 Cordoba, Spain.

Wnt5a is a member of the Wnt family of proteins that signals through the non-canonical Wnt/Ca(2+) pathway to suppress cyclin D1 expression and negatively regulate B cell proliferation suggesting that it acts as an tumour suppressor for lymphoid leukemogenesis. Although canonical Wnt pathway is a 'hot spot' for methylation in acute lymphoblastic leukaemia (ALL), the role of Wnt5a abnormalities has never been evaluated in this clinical setting. The methylation status of the WNT5A promoter was analysed by methylation-specific PCR (MSP) and sequencing in six ALL-derived cell lines (TOM-1, NALM-20, MY, LOUCY, JURKAT and TANOUE) and in 307 ALL patients. WNT5A and CYCLIN D1 expressions were assessed by quantitative RT-PCR. We observed WNT5A hypermethylation in all cell lines and in cells from 43% (132/307) of ALL patients. WNT5A methylation was associated with decreased WNT5A mRNA expression (P<0.001) and this expression was restored after exposure to the demethylating agent 5-Aza-2'-deoxycytidine. Moreover, WNT5A hypermethylation correlated with upregulation of CYCLIN D1 expression (P=0.002). Disease-free survival (DFS) and overall survival (OS) at 13 and 14 years, respectively, were 59% and 53% for unmethylated patients and 28% and 31% for hypermethylated patients (P=0.0003 and P=0.003). Multivariate analysis demonstrated that WNT5A methylation was an independent prognostic factor predicting DFS (P=0.003) and OS (P=0.04). We have demonstrated that WNT5A, a putative tumour suppressor gene in ALL, is silenced by methylation in this disease and that this epigenetic event is associated with upregulation of CYCLIN D1 expression and confers poor prognosis in this group of patients.
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http://dx.doi.org/10.1016/j.ejca.2007.10.004DOI Listing
December 2007

Resistance to Imatinib Mesylate-induced apoptosis in acute lymphoblastic leukemia is associated with PTEN down-regulation due to promoter hypermethylation.

Leuk Res 2008 May 17;32(5):709-16. Epub 2007 Oct 17.

Foundation for Applied Medical Research, Division of Cancer, Area of Cell Therapy and Hematology Service, Clínica Universitaria, Universidad de Navarra, Pamplona, Spain.

The aim of our study was to determine the potential mechanism(s) implicated in Imatinib resistance in patients with Ph+ ALL. Resistance of Ph+ ALL cells to Imatinib-induced apoptosis was associated with lack of inhibition of Akt phosphorylation. Addition of the PI3K inhibitor LY294002 to Imatinib significantly increased apoptosis of Ph+ ALL cells. Interestingly, expression of PTEN was reduced in Ph+ ALL cells which was due to PTEN promoter hypermethylation. Treatment of Ph+ ALL cells with 5-Aza-2'-deoxycytidine was associated with an increased expression of PTEN and an increase in cell apoptosis. These results suggest that Imatinib resistance in patients with ALL may be dependent at least in part to PTEN down-regulation due to the abnormal promoter hypermethylation and support the potential role of de-methylating agents for the treatment of patients with Ph+ ALL.
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http://dx.doi.org/10.1016/j.leukres.2007.09.005DOI Listing
May 2008

Multipotent Adult Progenitor Cells (MAPC) contribute to hepatocarcinoma neovasculature.

Biochem Biophys Res Commun 2007 Dec 2;364(1):92-9. Epub 2007 Oct 2.

Department of Medicine and Division of Hepatology and Gene Therapy, Clínica Universitaria/School of Medicine and Centre for Applied Medical Research (CIMA), University of Navarra, Spain.

The use of stem cells as a vehicle of therapeutic genes is an attractive approach for the development of new antitumoral strategies based on gene therapy. The aim of our study was to assess the potential of bone marrow-derived Multipotent Adult Progenitor Cells (rMAPCs) to differentiate in vitro and in vivo into endothelial cells and to be recruited to areas of tumor vasculogenesis. In vitro, rMAPCs obtained from Buffalo rats differentiated into cells expressing endothelial markers and demonstrated functional endothelial capacity. Intravenous injection of undifferentiated rMAPC transduced with a lentivirus expressing GFP in an orthotopic rat model of hepatocellular carcinoma, resulted in tumor recruitment of the injected cells and in vivo differentiation into endothelial cells in the tumor area with contribution to vasculogenesis. In summary, our results suggest that rMAPCs can be efficiently recruited by vascularized tumors and differentiate to endothelium and thus may represent a useful vehicle for delivery of therapeutic genes to sites of active tumor neovascularization.
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http://dx.doi.org/10.1016/j.bbrc.2007.09.106DOI Listing
December 2007

Repetitive DNA hypomethylation in the advanced phase of chronic myeloid leukemia.

Leuk Res 2008 Mar 4;32(3):487-90. Epub 2007 Sep 4.

Hematology Department, Reina Sofia Hospital, Avda. Menendez Pidal s/n, 14004 Cordoba, Spain.

Repetitive elements are heavily methylated in normal tissues, but hypomethylated in malignant tissues, driving the global genomic hypomethylation found in cancer. This hypomethylation results in chromosomal instability, a well-characterized feature of the advanced phases of chronic myeloid leukemia (CML). We investigated methylation changes of DNA repetitive elements (LINE1, Alu, Satellite-alpha and Satellite-2) during the progression of CML from chronic phase (CP) to blast crisis (BC). CP-CML samples were significantly more hypomethylated for all repetitive sequences compared with normal samples. Furthermore, a more profound level of hypomethylation was observed among BC samples compared with CP samples. Our data suggest that repetitive DNA hypomethylation are closely associated with CML progression.
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http://dx.doi.org/10.1016/j.leukres.2007.07.021DOI Listing
March 2008

Epigenetic regulation of PRAME gene in chronic myeloid leukemia.

Leuk Res 2007 Nov 26;31(11):1521-8. Epub 2007 Mar 26.

Hematology Department, Reina Sofia Hospital, Avda, Menendez Pidal s/n, 14004, Cordoba, Spain.

Tumor associated antigens (TAA) provide attractive targets for cancer-specific immunotherapy. PRAME is a TAA gene up-regulated in advanced phases of chronic myeloid leukemia (CML). To date, molecular mechanisms for the expression of PRAME have never been studied. We found that some Ph'-positive cell lines did not express PRAME. The expression of PRAME was restored in these cell lines by treatment with 5'-aza-2'-deoxycytidine, suggesting that the expression of PRAME is mainly suppressed by hypermethylation. Bisulfite sequencing analysis of the CpG sites of the PRAME exon 2 in these cancer cell lines revealed a close relationship between the methylation status of the PRAME gene and its expression. A methylation-specific PCR analysis demonstrated that hypomethylation of PRAME was significantly more frequent in CML blast crisis (70%) than in chronic phase (36%) (P=0.01) and was correlated with high expression levels of PRAME transcripts (P<0.0001). These results suggest that hypomethylation of PRAME up-regulates its expression in CML and might play a significant role in the progression of the disease.
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http://dx.doi.org/10.1016/j.leukres.2007.02.016DOI Listing
November 2007