Lara-Astiaso, D. (David)
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- Genome-wide analysis of the H3K27me3 epigenome and transcriptome in brassica rapa(Oxford University Press (OUP), 2019) Poza-Viejo, L. (Laura); San-Martín-Uriz, P. (Patxi); Lara-Astiaso, D. (David); Wilkinson, M.D. (Mark D.); Crevillén, P. (Pedro); Payá-Milans, M. (Miriam)Background Genome-wide maps of histone modifications have been obtained for several plant species. However, most studies focus on model systems and do not enforce FAIR data management principles. Here we study the H3K27me3 epigenome and associated transcriptome of Brassica rapa, an important vegetable cultivated worldwide. Findings We performed H3K27me3 chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis by 3′-end RNA sequencing from B. rapa leaves and inflorescences. To analyze these data we developed a Reproducible Epigenomic Analysis pipeline using Galaxy and Jupyter, packaged into Docker images to facilitate transparency and reuse. We found that H3K27me3 covers roughly one-third of all B. rapa protein-coding genes and its presence correlates with low transcript levels. The comparative analysis between leaves and inflorescences suggested that the expression of various floral regulatory genes during development depends on H3K27me3. To demonstrate the importance of H3K27me3 for B. rapa development, we characterized a mutant line deficient in the H3K27 methyltransferase activity. We found that braA.clf mutant plants presented pleiotropic alterations, e.g., curly leaves due to increased expression and reduced H3K27me3 levels at AGAMOUS-like loci. Conclusions We characterized the epigenetic mark H3K27me3 at genome-wide levels and provide genetic evidence for its relevance in B. rapa development. Our work reveals the epigenomic landscape of H3K27me3 in B. rapa and provides novel genomics datasets and bioinformatics analytical resources. We anticipate that this work will lead the way to further epigenomic studies in the complex genome of Brassica crops.
- Chromatin activation as a unifying principle underlying pathogenic mechanisms in multiple myeloma(2020) García-Torre, B. (Beatriz); Soler-Vila, P. (Paula); Taha, R.Y. (Ruba Y.); Beekman, R. (Renée); Rodriguez-Madoz, J.R. (Juan Roberto); El-Omri, H. (Halima); Charalampopoulou, S. (Stella); San-Jose-Eneriz, E. (Edurne); Martens, J.H.A. (Joost H. A.); Flicek, P. (Paul); Agirre, X. (Xabier); Stunnenberg, H.G. (Hendrik G.); Garate, L. (Leire); Kulis, M. (Marta); Mitsiades, C.S. (Constantine S.); Licht, J.D. (Jonathan D.); Carrasco-León, A. (Arantxa); Chapaprieta, V. (Vicente); Lara-Astiaso, D. (David); Ezponda, T. (Teresa); Ordóñez-Ciriza, R. (Raquel); Verdaguer-Dot, N. (Núria); Vilas-Zornoza, A. (Amaia); Campo, E. (Elías); Dupéré-Richer, D. (Daphné); Miranda, E. (Estibaliz); Duran-Ferrer, M. (Martí); Meydan, C. (Cem); Paiva, B. (Bruno); Vilarrasa-Blasi, R. (Roser); Gut, I. (Ivo); Melnick, A. (Ari); Prosper-Cardoso, F. (Felipe); Calasanz-Abinzano, M.J. (Maria Jose); Martínez-Turrilas, R. (Rebeca); Clot, G. (Guillem); San-Miguel, J.F. (Jesús F.); Martin-Subero, J.I. (Jose Ignacio); Russiñol, N. (Nuria)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 (TXN), a major regulator of cellular redox status and, in addition, identified PRDM5 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.
- In vivo screening characterizes chromatin factor functions during normal and malignant hematopoiesis(2023) Calvo, I.A. (Isabel A.); Torrea, N. (Natalia); Navarro-Alonso, M. (Mar); Fortelny, N. (Nikolaus); López, A. (Andrés); Taylor-King, J.P. (Jake P.); Lopez, C.K. (Cecile K.); Valle, C. (Cynthia) del; Gross, T. (Torsten); Beinortas, T. (Tumas); Lara-Astiaso, D. (David); Aguado-Álvaro, L.P. (Laura Pilar); Narayan, N. (Nisha); Alignani, D. (Diego); Zazpe, J. (Jon); Giotopoulos, G. (George); Marchese, F.P. (Francesco P.); Goñi-Salaverri, A. (Ainhoa); Prosper-Cardoso, F. (Felipe); Mendieta-Esteban, J. (Julen); Huntly, B.J.P. (Brian J. P.); Saez, B. (Borja)Bulk ex vivo and single-cell in vivo CRISPR knockout screens are used to characterize 680 chromatin factors during mouse hematopoiesis, highlighting lineage-specific and normal and leukemia-specific functions. Cellular differentiation requires extensive alterations in chromatin structure and function, which is elicited by the coordinated action of chromatin and transcription factors. By contrast with transcription factors, the roles of chromatin factors in differentiation have not been systematically characterized. Here, we combine bulk ex vivo and single-cell in vivo CRISPR screens to characterize the role of chromatin factor families in hematopoiesis. We uncover marked lineage specificities for 142 chromatin factors, revealing functional diversity among related chromatin factors (i.e. barrier-to-autointegration factor subcomplexes) as well as shared roles for unrelated repressive complexes that restrain excessive myeloid differentiation. Using epigenetic profiling, we identify functional interactions between lineage-determining transcription factors and several chromatin factors that explain their lineage dependencies. Studying chromatin factor functions in leukemia, we show that leukemia cells engage homeostatic chromatin factor functions to block differentiation, generating specific chromatin factor-transcription factor interactions that might be therapeutically targeted. Together, our work elucidates the lineage-determining properties of chromatin factors across normal and malignant hematopoiesis.
- CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I(2018) Vilas, A. (Amaia); González-Aseguinolaza, G. (Gloria); Zabaleta-Lasarte, N. (Nerea); Betancor, I. (Isabel); Rodriguez-Madoz, J.R. (Juan Roberto); Vales, A. (África); Rodriguez, S. (Saray); Lara-Astiaso, D. (David); Martínez-Turrillas, R. (Rebeca); Castro-Labrador, L. (Laura); Olagüe, M. (María); Torella, L. (Laura); Salido, E. (Eduardo); Barberia, M. (Miren); Prosper-Cardoso, F. (Felipe); Martin-Higueras, C. (Cristina); Zapata-Linares, N.M. (Natalia María)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.
- The phospholipid transporter PITPNC1 links KRAS to MYC to prevent autophagy in lung and pancreatic cancer(2023) Halberg, N. (Nils); Vicent, S. (Silvestre); Vallejo-Blanco, A. (Adrián); Cueto-Ureña, C. (Cristina); Entrialgo-Cadierno, R. (Rodrigo); Vera, L. (Laura); Ambrogio, C. (Chiara); Vietti-Michelina, S. (Sandra); Cortés-Dominguez, I. (Iván); Scaparone, P. (Pietro); Lara-Astiaso, D. (David); Erice, O. (Oihane); Macaya, I. (Irati); Darbo, E. (Elodie); Feliu, I. (Iker); Morales-Urteaga, X. (Xabier); Guruceaga, E. (Elizabeth); Moreno, H. (Haritz); Goñi-Salaverri, A. (Ainhoa); Lecanda, F. (Fernando); Welch, C. (Connor); Lopez, I. (Inés)BackgroundThe discovery of functionally relevant KRAS effectors in lung and pancreatic ductal adenocarcinoma (LUAD and PDAC) may yield novel molecular targets or mechanisms amenable to inhibition strategies. Phospholipids availability has been appreciated as a mechanism to modulate KRAS oncogenic potential. Thus, phospholipid transporters may play a functional role in KRAS-driven oncogenesis. Here, we identified and systematically studied the phospholipid transporter PITPNC1 and its controlled network in LUAD and PDAC.MethodsGenetic modulation of KRAS expression as well as pharmacological inhibition of canonical effectors was completed. PITPNC1 genetic depletion was performed in in vitro and in vivo LUAD and PDAC models. PITPNC1-deficient cells were RNA sequenced, and Gene Ontology and enrichment analyses were applied to the output data. Protein-based biochemical and subcellular localization assays were run to investigate PITPNC1-regulated pathways. A drug repurposing approach was used to predict surrogate PITPNC1 inhibitors that were tested in combination with KRASG12C inhibitors in 2D, 3D, and in vivo models.ResultsPITPNC1 was increased in human LUAD and PDAC, and associated with poor patients' survival. PITPNC1 was regulated by KRAS through MEK1/2 and JNK1/2. Functional experiments showed PITPNC1 requirement for cell proliferation, cell cycle progression and tumour growth. Furthermore, PITPNC1 overexpression enhanced lung colonization and liver metastasis. PITPNC1 regulated a transcriptional signature which highly overlapped with that of KRAS, and controlled mTOR localization via enhanced MYC protein stability to prevent autophagy. JAK2 inhibitors were predicted as putative PITPNC1 inhibitors with antiproliferative effect and their combination with KRASG12C inhibitors elicited a substantial anti-tumour effect in LUAD and PDAC.ConclusionsOur data highlight the functional and clinical relevance of PITPNC1 in LUAD and PDAC. Moreover, PITPNC1 constitutes a new mechanism linking KRAS to MYC, and controls a druggable transcriptional network for combinatorial treatments.
- Signature-driven repurposing of Midostaurin for combination with MEK1/2 and KRASG12C inhibitors in lung cancer(2023) Gil-Bazo, I. (Ignacio); Drosten, M. (Mathias); Barbacid, M. (Mariano); Kovalski, J. (Joanna); Pineda-Lucena, A. (Antonio); Román, M. (Marta); Jantus-Lewintre, E. (Eloisa); Calabuig-Fariñas, S. (Silvia); Vicent, S. (Silvestre); Ludwig, I.A. (Iziar Amaia); Entrialgo-Cadierno, R. (Rodrigo); Palomino-Echeverría, S. (Sara); Salmon, M. (Marina); Fernandez-Irigoyen, J. (Joaquín); Ruggero, D. (Davide); Lara-Astiaso, D. (David); Santos, A. (Alba); Ponz-Sarvise, M. (Mariano); Macaya, I. (Irati); Feliu, I. (Iker); Rodríguez-Remírez, M. (M.); Guruceaga, E. (Elizabeth); Paz-Ares, L. (Luis); Ferrer, I. (Irene); Narayanan, S. (Shruthi); Ferrero, M. (Macarena); Lecanda, F. (Fernando); Welch, C. (Connor); Lopez, I. (Inés); Lonfgren, S.M. (Shane M.); Santamaria, E. (Enrique); Khatri, P. (Purvesh)Drug combinations are key to circumvent resistance mechanisms compromising response to single anti-cancer targeted therapies. The implementation of combinatorial approaches involving MEK1/2 or KRASG12C inhibitors in the context of KRAS-mutated lung cancers focuses fundamentally on targeting KRAS proximal activators or effectors. However, the antitumor effect is highly determined by compensatory mechanisms arising in defined cell types or tumor subgroups. A potential strategy to find drug combinations targeting a larger fraction of KRAS-mutated lung cancers may capitalize on the common, distal gene expression output elicited by oncogenic KRAS. By integrating a signature-driven drug repurposing approach with a pairwise pharmacological screen, here we show synergistic drug combinations consisting of multi-tyrosine kinase PKC inhibitors together with MEK1/2 or KRASG12C inhibitors. Such combinations elicit a cytotoxic response in both in vitro and in vivo models, which in part involves inhibition of the PKC inhibitor target AURKB. Proteome profiling links dysregulation of MYC expression to the effect of both PKC inhibitor-based drug combinations. Furthermore, MYC overexpression appears as a resistance mechanism to MEK1/2 and KRASG12C inhibitors. Our study provides a rational framework for selecting drugs entering combinatorial strategies and unveils MEK1/2- and KRASG12C-based therapies for lung cancer.
- Correction: The phospholipid transporter PITPNC1 links KRAS to MYC to prevent autophagy in lung and pancreatic cancer(2023) Halberg, N. (Nils); Vicent, S. (Silvestre); Vallejo-Blanco, A. (Adrián); Cueto-Ureña, C. (Cristina); Entrialgo-Cadierno, R. (Rodrigo); Vera, L. (Laura); Ambrogio, C. (Chiara); Vietti-Michelina, S. (Sandra); Cortés-Dominguez, I. (Iván); Scaparone, P. (Pietro); Lara-Astiaso, D. (David); Erice, O. (Oihane); Macaya, I. (Irati); Darbo, E. (Elodie); Feliu, I. (Iker); Morales-Urteaga, X. (Xabier); Guruceaga, E. (Elizabeth); Moreno, H. (Haritz); Goñi-Salaverri, A. (Ainhoa); Lecanda, F. (Fernando); Welch, C. (Connor); Lopez, I. (Inés)
- Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD)(2020) Gomez-Cabrero, D. (David); Paramo, J.A. (José Antonio); Saenz-Pipaón, G. (Goren); Ravassa, S. (Susana); Orbe, J. (Josune); Rodriguez, J.A. (José Antonio); Lara-Astiaso, D. (David); Planell, N. (Núria); Dupéré-Richer, D. (Daphné); Maillo, A. (Alberto); Alameda, D. (Daniel); Martínez-Aguilar, E. (Esther); Roncal, C. (Carmen); Prosper-Cardoso, F. (Felipe); San Martín, P. (Patxi)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 plateletfree 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 plateletfree plasma. Transcriptomic analysis of EVs identified 15 differentially expressed genes. Among them, serum calprotectin was elevated in PAD patients (p < 0.05) and associated with increased amputation risk before and after covariate adjustment (mean follow-up 3.6 years, p < 0.01). The combination of calprotectin with hs-CRP in the multivariate analysis further improved risk stratification (p < 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.