Vilas, A. (Amaia)
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- MicroRNA expression profiling in Imatinib-resistant Chronic Myeloid Leukemia patients without clinically significant ABL1-mutations(BioMed Central, 2009) Vilas, A. (Amaia); Cordeu, L. (Lucía); Jimenez-Velasco, A. (A.); Roman-Gomez, J. (José); San-Jose-Eneriz, E. (Edurne); Martin, V. (Vanesa); Garate, L. (Leire); Rodriguez-Otero, P. (Paula); Prosper-Cardoso, F. (Felipe); Calasanz-Abinzano, M.J. (Maria Jose); Aguirre-Ena, X. (Xabier)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.
- Midbrain microglia mediate a specific immunosuppressive response under inflammatory conditions(Springer Science and Business Media LLC, 2019) Vilas, A. (Amaia); Abellanas-Sánchez, M.A. (Miguel Ángel); San-Martín-Uriz, P. (Patxi); Zamarbide-González, M. (Marta); Aymerich-Soler, M.S. (María Soledad); Basurco, L. (Leyre); Hervas-Stubbs, S. (Sandra); Garcia-Granero, M. (Marta); Mengual, E. (Elisa); Clavero, P. (P.); Luquin, E. (Esther)Background: Inflammation is a critical process for the progression of neuronal death in neurodegenerative disorders. Microglia play a central role in neuroinflammation and may affect neuron vulnerability. Next generation sequencing has shown the molecular heterogeneity of microglial cells; however, the variability in their response to pathological inputs remains unknown. Methods: To determine the effect of an inflammatory stimulus on microglial cells, lipopolysaccharide (LPS) was administered peripherally to mice and the inflammatory status of the cortex, hippocampus, midbrain, and striatum was assessed. Microglial activation and interaction with the immune system were analyzed in single cell suspensions obtained from the different brain regions by fluorescence-activated cell sorting, next generation RNA sequencing, real-time PCR, and immunohistochemical techniques. Antigen-presenting properties of microglia were evaluated by the ability of isolated cells to induce a clonal expansion of CD4+ T cells purified from OT-II transgenic mice. Results: Under steady-state conditions, the midbrain presented a high immune-alert state characterized by the presence of two unique microglial subpopulations, one expressing the major histocompatibility complex class II (MHC-II) and acting as antigen-presenting cells and another expressing the toll-like receptor 4 (TLR4), and by the presence of a higher proportion of infiltrating CD4+ T cells. This state was not detected in the cortex, hippocampus, or striatum. Systemic LPS administration induced a general increase in classic pro-inflammatory cytokines, in coinhibitory programmed death ligand 1 (PD-L1), and in cytotoxic T lymphocyte antigen 4 (CTLA-4) receptors, as well as a decrease in infiltrating effector T cells in all brain regions. Interestingly, a specific immune-suppressive response was observed in the midbrain which was characterized by the downregulation of MHC-II microglial expression, the upregulation of the anti-inflammatory cytokines IL10 and TGFβ, and the increase in infiltrating regulatory T cells. Conclusions: These data show that the midbrain presents a high immune-alert state under steady-state conditions that elicits a specific immune-suppressive response when exposed to an inflammatory stimulus. This specific inflammatory tone and response may have an impact in neuronal viability
- Down-Regulation of hsa-miR-10a in Chronic Myeloid Leukemia CD34+ Cells Increases USF2-Mediated Cell Growth(American Association for Cancer Research, 2008) Vilas, A. (Amaia); Cordeu, L. (Lucía); Jimenez-Velasco, A. (A.); Aparicio, O. (Óscar); Roman-Gomez, J. (José); San-Jose-Eneriz, E. (Edurne); Heiniger, A. (A.); Garate, L. (Leire); Navarro, G. (Germán); Bandres, E. (Eva); Garcia-Foncillas, J. (Jesús); Fortes, P. (Puri); Prosper-Cardoso, F. (Felipe); Calasanz-Abinzano, M.J. (Maria Jose); Aguirre-Ena, X. (Xabier); Perez-Roger, I. (Ignacio); Saez, B. (Borja)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.
- Activation-induced deaminase is critical for the establishment of DNA methylation patterns prior to the germinal center reaction(2021) Álvarez-Prado, A.F. (Ángel F.); Durandy, A. (Anne); Vilas, A. (Amaia); Català-Moll, F. (Francesc); Colobran, R. (Roger); Hammarstrom, L. (Lennart); Ballestar, E. (E.); Abolhassani, H. (Hassan); Martínez-Gallo, M. (Mónica); Ferreté-Bonastre, A.G. (Anna G.); Li, T. (Tianlu); Lutsik, P. (Pavlo); Martín-Nalda, A. (Andrea); Klemann, C. (Christian); Ciudad, L. (Laura); Rivière, J.G. (Jacques); Grimbacher, B. (Bodo); Plass, C. (Christoph); Prosper-Cardoso, F. (Felipe); Kracker, S. (Sven); Weichenhan, D. (Dieter); Rodríguez-Ubreva, J. (Javier); Dieli-Crimi, R. (Romina); Speckmann, C. (Carsten); Soler-Palacín, P. (Pere)Activation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naive and memory B cells both display widespread DNA methylation alterations, of which similar to 25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naive B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns.
- Epigenetic Silencing of the Tumor Suppressor MicroRNA Hsa-miR-124a Regulates CDK6 Expression and Confers a Poor Prognosis in Acute Lymphoblastic Leukemia(American Association for Cancer Research, 2009) Vilas, A. (Amaia); Cordeu, L. (Lucía); Jimenez-Velasco, A. (A.); Roman-Gomez, J. (José); San-Jose-Eneriz, E. (Edurne); Martin, V. (Vanesa); Heiniger, A. (A.); Garate, L. (Leire); Rifon, J. J. (Jose J.); Bandres, E. (Eva); Siebert, R. (Reiner); Rodriguez-Otero, P. (Paula); Fortes, P. (Puri); Prosper-Cardoso, F. (Felipe); Torres, A. (Antonio); Calasanz-Abinzano, M.J. (Maria Jose); Aguirre-Ena, X. (Xabier); Abizanda-Sarasa, G. (Gloria); Martin-Subero, J.I. (Jose Ignacio)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.T o explore the epigenetic alterations of miRNAs in ALL, we analyzed the methylation and chromatin status of the miR-124a loci in ALL.E xpression 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.Cyc lin-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 / ;c / mouse model.The clinical implications of these findings were analyzed in a group of 353 patients diagnosed with ALL.M ethylation 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.
- Epigenetic regulation of the non-canonical Wnt pathway in acute myeloid leukemia(Wiley-Blackwell, 2010) Vilas, A. (Amaia); Cervera, J. (Jose); Roman-Gomez, J. (José); San-Jose-Eneriz, E. (Edurne); Martin, V. (Vanesa); Valencia, A. (Ana); Rodriguez-Otero, P. (Paula); Sanz, M.A. (Miguel A.); Prosper-Cardoso, F. (Felipe); Torres, A. (Antonio); Aguirre-Ena, X. (Xabier); Herrera, C. (Concepción)Wnt5a is a member of the Wnt family of proteins that signals through the non-canonical Wnt ⁄ Ca2+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.
- Preclinical activity of LBH589 alone or in combination with chemotherapy in a xenogeneic mouse model of human acute lymphoblastic leukemia.(Nature Publishing Group, 2012) Vilas, A. (Amaia); Cigudosa, J.C. (Juan Cruz); Roman-Gomez, J. (José); San-Jose-Eneriz, E. (Edurne); Blanco-Prieto, M.J. (María José); Garate, L. (Leire); Martinez-Climent, J.A. (José Ángel); Rifon, J. J. (Jose J.); Ribera, J.M. (José María); Miranda, E. (Estibaliz); Martino-Rodriguez, A. (Alba) de; Garcia-de-Jalon, J.A. (José A.); Prosper-Cardoso, F. (Felipe); Rio, P. (Paula); Segura, V. (Víctor); Calasanz-Abinzano, M.J. (Maria Jose); Aguirre-Ena, X. (Xabier); Abizanda-Sarasa, G. (Gloria); Martin-Subero, J.I. (Jose Ignacio); Moreno, C. (Cristina)Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here, we analyzed the therapeutic effect of LBH589, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced dose-dependent antiproliferative and apoptotic effects, which were associated with increased H3 and H4 histone acetylation. Intravenous administration of LBH589 in immunodeficient BALB/c-RAG2(-/-)γc(-/-) mice in which human-derived T and B-ALL cell lines were injected induced a significant reduction in tumor growth. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2(-/-)γc(-/-) mice was established, allowing continuous passages of transplanted cells to several mouse generations. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving vincristine and dexamethasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with vincristine and dexamethasone. Our results show the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL.
- Preneoplastic somatic mutations including MYD88(L265P) in lymphoplasmacytic lymphoma(2022) Reinhardt, H.C. (Hans Christian); Vilas, A. (Amaia); Sanchez-Garcia, I. (Isidro); Perez, C. (Cristina); Paiva, A. (Artur); López-de-Arancibia, A. (Aitziber); Sacco, A. (Antonio); Santos, S. (S.); Celay, J. (Jon); Sarvide, S. (Sarai); García-Sanz, R. (Ramón); Goicoechea, I. (Ibai); García-Barchino, M.J. (María José); Martinez-Climent, J.A. (José Ángel); Gárate-Luzuriaga, S. (Sonia); Carrasco, Y.R. (Yolanda R.); Panizo, C. (Carlos); Larrayoz, M. (Marta); Vitoria, H. (Helena); Puig, N. (Noemí); Botta, C. (Cirino); Rodríguez-Díaz, S. (Saray); Garcés-Latre, J.J. (Juan José); Motta, M. (Marina); Geraldes, C. (Catarina); Lamo-de-Espinosa-Vázquez-de-Sola, J.M. (José María); Alignani, D. (Diego); Duarte, S. (Sara); Paiva, B. (Bruno); Larrayoz, M.J. (María J.); Prosper-Cardoso, F. (Felipe); Calasanz-Abinzano, M.J. (Maria Jose); Roccaro, A.M. (Aldo M.); Fuerte, G. (Gema); Tucci, A. (Alessandra); San-Miguel, J.F. (Jesús F.); Gentile, M. (Massimo); Jiménez, C. (Cristina)Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88(L265P) in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88(L265P) is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas.
- Epigenetic Regulation of MicroRNAs in Acute Lymphoblastic Leukemia(American Society of Clinical Oncology, 2009) Vilas, A. (Amaia); Cordeu, L. (Lucía); Jimenez-Velasco, A. (A.); Arqueros, V. (V.); Roman-Gomez, J. (José); San-Jose-Eneriz, E. (Edurne); Martin, V. (Vanesa); Heiniger, A. (A.); Garate, L. (Leire); Bandres, E. (Eva); Castillejo, J.A. (J.A.); Siebert, R. (Reiner); Rodriguez-Otero, P. (Paula); Prosper-Cardoso, F. (Felipe); Torres, A. (Antonio); Calasanz-Abinzano, M.J. (Maria Jose); Aguirre-Ena, X. (Xabier); Martin-Subero, J.I. (Jose Ignacio)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.
- Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia(Public Library of Science, 2011) Vilas, A. (Amaia); Cigudosa, J.C. (Juan Cruz); Roman-Gomez, J. (José); Alvarez, S. (Sara); San-Jose-Eneriz, E. (Edurne); Garate, L. (Leire); Rifon, J. J. (Jose J.); Martin-Palanco, V. (Vanesa); Miranda, E. (Estibaliz); Rodriguez-Otero, P. (Paula); Prosper-Cardoso, F. (Felipe); Torres, A. (Antonio); Calasanz-Abinzano, M.J. (Maria Jose); Aguirre-Ena, X. (Xabier); Martin-Subero, J.I. (Jose Ignacio)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-29-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.