Publications by authors named "Amaia Vilas-Zornoza"

24 Publications

  • Page 1 of 1

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

Single-Cell RNA Sequencing Analysis Reveals a Crucial Role for CTHRC1 (Collagen Triple Helix Repeat Containing 1) Cardiac Fibroblasts After Myocardial Infarction.

Circulation 2020 Nov 25;142(19):1831-1847. Epub 2020 Sep 25.

Program of Regenerative Medicine (A.R.-V., S.C.H., P.G.-O., E.I., G.A., G.M., B.P., F.P.), Program of Hemato-Oncology, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain.

Background: Cardiac fibroblasts (CFs) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Because of the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is lacking. The purpose of this study was to define CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate CF function.

Methods: Collagen1α1-GFP (green fluorescent protein)-positive CFs were characterized after myocardial infarction (MI) by single-cell and bulk RNA sequencing, assay for transposase-accessible chromatin sequencing, and functional assays. Swine and patient samples were studied using bulk RNA sequencing.

Results: We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear profibrotic signature, express high levels of Cthrc1 (collagen triple helix repeat containing 1), and localize into the scar. Noncanonical transforming growth factor-β signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Absence of CTHRC1 results in pronounced lethality attributable to ventricular rupture. A population of CFs with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy.

Conclusions: We report CF heterogeneity and their dynamics during the course of MI and redefine the CFs that respond to cardiac injury and participate in myocardial remodeling. Our study identifies as a novel regulator of the healing scar process and a target for future translational studies.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.044557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730974PMC
November 2020

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

Deep MRD profiling defines outcome and unveils different modes of treatment resistance in standard- and high-risk myeloma.

Blood 2021 Jan;137(1):49-60

Clinica Universidad de Navarra, Centro de Investigación Medica Aplicada (CIMA), Instituto de Investigación Sanitaria de Navarra (IDISNA), Centro de Investigación Biomédica en Red-Cáncer (CIBER-ONC) CB16/12/00369, Pamplona, Spain.

Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of ∼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species-mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.
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http://dx.doi.org/10.1182/blood.2020006731DOI Listing
January 2021

Immunogenomic identification and characterization of granulocytic myeloid-derived suppressor cells in multiple myeloma.

Blood 2020 07;136(2):199-209

Clinica Universidad de Navarra, Centro de Investigacion Medica Aplicada (CIMA), Instituto de Investigacion Sanitaria de Navarra (IDISNA), CIBER-ONC numbers CB16/12/00369, CB16/12/00489, Pamplona, Spain.

Granulocytic myeloid-derived suppressor cells (G-MDSCs) promote tumor growth and immunosuppression in multiple myeloma (MM). However, their phenotype is not well established for accurate monitoring or clinical translation. We aimed to provide the phenotypic profile of G-MDSCs based on their prognostic significance in MM, immunosuppressive potential, and molecular program. The preestablished phenotype of G-MDSCs was evaluated in bone marrow samples from controls and MM patients using multidimensional flow cytometry; surprisingly, we found that CD11b+CD14-CD15+CD33+HLADR- cells overlapped with common eosinophils and neutrophils, which were not expanded in MM patients. Therefore, we relied on automated clustering to unbiasedly identify all granulocytic subsets in the tumor microenvironment: basophils, eosinophils, and immature, intermediate, and mature neutrophils. In a series of 267 newly diagnosed MM patients (GEM2012MENOS65 trial), only the frequency of mature neutrophils at diagnosis was significantly associated with patient outcome, and a high mature neutrophil/T-cell ratio resulted in inferior progression-free survival (P < .001). Upon fluorescence-activated cell sorting of each neutrophil subset, T-cell proliferation decreased in the presence of mature neutrophils (0.5-fold; P = .016), and the cytotoxic potential of T cells engaged by a BCMA×CD3-bispecific antibody increased notably with the depletion of mature neutrophils (fourfold; P = .0007). Most interestingly, RNA sequencing of the 3 subsets revealed that G-MDSC-related genes were specifically upregulated in mature neutrophils from MM patients vs controls because of differential chromatin accessibility. Taken together, our results establish a correlation between the clinical significance, immunosuppressive potential, and transcriptional network of well-defined neutrophil subsets, providing for the first time a set of optimal markers (CD11b/CD13/CD16) for accurate monitoring of G-MDSCs in MM.
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http://dx.doi.org/10.1182/blood.2019004537DOI Listing
July 2020

Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD).

J Extracell Vesicles 2020 19;9(1):1729646. Epub 2020 Feb 19.

Laboratory of Atherothrombosis, Program of Cardiovascular Diseases, Cima Universidad de Navarra, Pamplona, Spain.

Peripheral arterial disease (PAD) is associated with a high risk of cardiovascular events and death and is postulated to be a critical socioeconomic cost in the future. Extracellular vesicles (EVs) have emerged as potential candidates for new biomarker discovery related to their protein and nucleic acid cargo. In search of new prognostic and therapeutic targets in PAD, we determined the prothrombotic activity, the cellular origin and the transcriptomic profile of circulating EVs. This prospective study included control and PAD patients. Coagulation time (Procoag-PPL kit), EVs cellular origin and phosphatidylserine exposure were determined by flow cytometry in platelet-free plasma (n = 45 PAD). Transcriptomic profiles of medium/large EVs were generated using the MARS-Seq RNA-Seq protocol (n = 12/group). The serum concentration of the differentially expressed gene S100A9, in serum calprotectin (S100A8/A9), was validated by ELISA in control (n = 100) and PAD patients (n = 317). S100A9 was also determined in EVs and tissues of human atherosclerotic plaques (n = 3). Circulating EVs of PAD patients were mainly of platelet origin, predominantly Annexin V positive and were associated with the procoagulant activity of platelet-free plasma. Transcriptomic analysis of EVs identified 15 differentially expressed genes. Among them, serum calprotectin was elevated in PAD patients ( < 0.05) and associated with increased amputation risk before and after covariate adjustment (mean follow-up 3.6 years,  < 0.01). The combination of calprotectin with hs-CRP in the multivariate analysis further improved risk stratification ( < 0.01). Furthermore, S100A9 was also expressed in femoral plaque derived EVs and tissues. In summary, we found that PAD patients release EVs, mainly of platelet origin, highly positive for AnnexinV and rich in transcripts related to platelet biology and immune responses. Amputation risk prediction improved with calprotectin and was significantly higher when combined with hs-CRP. Our results suggest that EVs can be a promising component of liquid biopsy to identify the molecular signature of PAD patients.
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http://dx.doi.org/10.1080/20013078.2020.1729646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048174PMC
February 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

CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I.

Nat Commun 2018 12 21;9(1):5454. Epub 2018 Dec 21.

Regenerative Medicine Program, Center for Applied Medical Research (CIMA), University of Navarra, IdiSNA, Pamplona, 31008, Spain.

CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1 mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.
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http://dx.doi.org/10.1038/s41467-018-07827-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303323PMC
December 2018

Inhibitor of Differentiation-1 Sustains Mutant -Driven Progression, Maintenance, and Metastasis of Lung Adenocarcinoma via Regulation of a FOSL1 Network.

Cancer Res 2019 02 18;79(3):625-638. Epub 2018 Dec 18.

Department of Oncology, Clínica Universidad de Navarra, Pamplona, Spain.

Because of the refractory nature of mutant lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of was a marker of poor survival in patients harboring mutant, but not wild-type . Abrogation of Id1 induced G-M arrest and apoptosis in mutant LUAD cells. , loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the oncogene through JNK, and loss of Id1 resulted in downregulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring as a critical gene in mutant LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD. SIGNIFICANCE: These findings highlight the prognostic significance of the transcriptional regulator Id1 in -mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1479DOI Listing
February 2019

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

Deregulation of in acute lymphoblastic leukemia is implicated in abnormal proliferation of leukemic cells.

Oncotarget 2018 Feb 5;9(16):12842-12852. Epub 2018 Feb 5.

Laboratory of Myeloproliferative Syndromes, Oncology Area, Foundation for Applied Medical Research, IDISNA, CIBERONC, University of Navarra, Pamplona, Spain.

Long Non-Coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. Several lncRNAs are involved in cell proliferation and are deregulated in several human tumors. Few lncRNAs have been described to play a role in Acute Lymphoblastic Leukemia (ALL). In this study, we carried out a genome wide lncRNA expression profiling in ALL samples and peripheral blood samples obtained from healthy donors. We detected 43 lncRNAs that were aberrantly expressed in ALL. Interestingly, among them, showed a significant downregulation in T and B-ALL. Re-expression of in ALL cells induced inhibition of leukemic cell growth that was associated with apoptosis induction and cell cycle arrest in G/M phase. induced the transcription of which reduced the viability of ALL cells. Intriguingly, we observed that treatment with anti-tumoral epigenetic drugs like LBH-589 (Panobinostat) and Curcumin induced the expression of and in ALL. These results indicate that the downregulation of plays a relevant role in the pathogenesis of ALL, and re-expression may be one of the mechanisms exerted by epigenetic drugs to reduce cell proliferation in ALL.
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http://dx.doi.org/10.18632/oncotarget.24401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849178PMC
February 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

Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies.

Nat Commun 2017 05 26;8:15424. Epub 2017 May 26.

Area de Hemato-Oncología, Centro de Investigación Médica Aplicada, IDISNA, Ciberonc, Universidad de Navarra, Avenida Pío XII, 55 31008 Pamplona, Spain.

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.
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http://dx.doi.org/10.1038/ncomms15424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458547PMC
May 2017

Homeobox NKX2-3 promotes marginal-zone lymphomagenesis by activating B-cell receptor signalling and shaping lymphocyte dynamics.

Nat Commun 2016 06 14;7:11889. Epub 2016 Jun 14.

Division of Hemato-Oncology, Center for Applied Medical Research CIMA, University of Navarra, IDISNA, Pamplona 31008, Spain.

NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas.
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http://dx.doi.org/10.1038/ncomms11889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911677PMC
June 2016

Deregulation of FGFR1 and CDK6 oncogenic pathways in acute lymphoblastic leukaemia harbouring epigenetic modifications of the MIR9 family.

Br J Haematol 2011 Oct 2;155(1):73-83. Epub 2011 Aug 2.

Division of Cancer, Foundation for Applied Medical Research, Universidad de Navarra, Avenida Pio XII 36, Pamplona, Spain.

The role of epigenetic mechanisms in the regulation of microRNAs (miRNAs) with a tumour-suppressor function in human neoplasms has recently been established. Several miRNAs have been found to be inappropriately regulated by DNA methylation in patients with acute lymphoblastic leukaemia (ALL). We analysed the methylation status of the three members of the MIR9 family (MIR9-1, MIR9-2 and MIR9-3) in a uniformly treated cohort of 200 newly diagnosed ALLs. MIR9 was methylated in 54% of the patients and was associated with downregulation of MIR9 (P < 0·01). Hypermethylation of MIR9 was an independent prognostic factor for disease-free survival, overall survival and event-free survival in a multivariate analysis (P < 0·01). Epigenetic downregulation of MIR9 induced upregulation of its targets, FGFR1 and CDK6, while treatment of ALL cells with FGFR1 (PD-173074) and CDK6 (PD-0332991) inhibitors induced a decrease in cell proliferation and an increase in apoptosis of ALL cells. Our results indicate that the MIR9 family is involved in the pathogenesis and clinical behaviour of ALL and provide the basis for new therapeutic strategies in the treatment of ALL, targeting the epigenetic regulation of miRNAs and/or the FGFR1 or CDK6-RB pathway directly.
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http://dx.doi.org/10.1111/j.1365-2141.2011.08812.xDOI Listing
October 2011

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

Epigenetic regulation of the non-canonical Wnt pathway in acute myeloid leukemia.

Cancer Sci 2010 Feb 29;101(2):425-32. Epub 2009 Oct 29.

Hematology Department, Cellular Therapy Area, Reina Sofia Hospital, Maimonides Institute for Biomedical Research, 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. Deregulation of this pathway has been found in animal models suggesting that it acts as tumour suppressor in acute myeloid leukemia (AML). Although DNA methylation is the main mechanism of regulation of the canonical Wnt pathway in AML, the role of WNT5A abnormalities has never been evaluated in this clinical setting. The methylation status of WNT5A promoter-exon 1 was analyzed by methylation-specific PCR and sequencing in eleven AML-derived cell lines and 252 AML patients. We observed WNT5A hypermethylation in seven cell lines and in 43% (107/252) of AML patients. WNT5A methylation was associated with decreased WNT5A expression (P < 0.001) that was restored after exposure to 5-Aza-2'-deoxycytidine. Moreover, WNT5A hypermethylation correlated with upregulation of CYCLIN D1 expression (P < 0.001). Relapse (15%vs 37%, P < 0.001) and mortality (61%vs 79%, P = 0.004) rates were lower for patients in the non-methylated group. Disease-free survival and overall survival at 6 and 7 years, respectively, were 60% and 27% for unmethylated patients and 20% and 0% for hypermethylated patients (P = 0.0001 and P = 0.04, respectively). Interestingly, significant differences were also observed when the analysis was carried out according to cytogenetic risk groups. We demonstrate that WNT5A, a putative tumor suppressor gene in AML, 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 patients with AML.
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http://dx.doi.org/10.1111/j.1349-7006.2009.01413.xDOI Listing
February 2010

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 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

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