Publications by authors named "Estibaliz Miranda"

12 Publications

  • Page 1 of 1

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

Epigenomic profiling of myelofibrosis reveals widespread DNA methylation changes in enhancer elements and as a potential tumor suppressor gene that is epigenetically regulated.

Haematologica 2019 08 17;104(8):1572-1579. Epub 2019 Jan 17.

Área de Hemato-Oncología, Centro de Investigación Médica Aplicada, IDISNA, Universidad de Navarra, Pamplona

In this study we interrogated the DNA methylome of myelofibrosis patients using high-density DNA methylation arrays. We detected 35,215 differentially methylated CpG, corresponding to 10,253 genes, between myelofibrosis patients and healthy controls. These changes were present both in primary and secondary myelofibrosis, which showed no differences between them. Remarkably, most differentially methylated CpG were located outside gene promoter regions and showed significant association with enhancer regions. This aberrant enhancer hypermethylation was negatively correlated with the expression of 27 genes in the myelofibrosis cohort. Of these, we focused on the gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of patients and myeloid cell-lines. reporter assay and 5'-azacitidine treatment confirmed the functional relevance of hyper-methylation of enhancer. Furthermore, rescue of expression had an impact on cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of as a tumor suppressor gene in myelofibrosis. Collectively, we describe the DNA methylation profile of myelofibrosis, identifying extensive changes in enhancer elements and revealing as a novel candidate tumor suppressor gene.
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http://dx.doi.org/10.3324/haematol.2018.204917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669145PMC
August 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

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

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

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