Biologia de cèl·lules mare, leucèmia del desenvolupament i immunoteràpia

  • Menéndez Lab 2021

Laboratori Institut Josep Carreras

Ala Sud, Sector 2, Planta 2

Facultat de Medicina Universitat de Barcelona

C. Casanova 143

08036 Barcelona


Pablo Menéndez, Group Leader is an ICREA Research Professor





Our lab studies human pluripotent stem cells (hESCs and iPSCs) and human multipotent stem cells (hematopoietic and mesenchymal) derived from different tissue sources in order to understand the intrinsic and extrinsic factors, and developmentally conserved pathways, driving blood endothelium and mesenchymal differentiation. We are also actively involved in modeling t(4;11) MLL-AF4+ Acute Lymphoblastic Leukemia (with special interest in deciphering the cell-of-origin and cooperating oncogenic events) as well as other hematological malignances using cord blood-derived HSPCs and hESC/hiPSC-derived blood derivatives. In parallel, we are also studying the contribution of MSCs to hematological malignances and sarcomas.

Call for Postdoctoral and Associate Investigators


The Stem cells, mesenchymal cancerand development Group is actively recruiting postdoctoral and Associate investigators. If you are interested, please send a full CV and a letter explaining your research experience and your research proposal for the group to Pablo Menéndez See more information on our Work With Us Page.


Our laboratory strives to understand the pathogenesis of acute leukemia and to develop novel targeted therapeutics. There are currently 3 main active research programs: 

1.-Modeling  in utero  leukemogenesis and decoding fetal hematopoiesis as a platform to understand the etiology, pathogenesis and cell-of-origin of pediatric leukemias. We harness state-of-the-art multilayer-omics approaches, clinically relevant primary samples and  in vivo PDX models. 

2.-Novel immunotherapies for acute leukemias of both myeloid and lymphoid lineage. We have developed proprietary blocking MoAbs and CAR T-cell therapies with high efficiency and specificity in murine PDX models. We are also combining novel immunocheckpoints inhibitors with front-line treatments, and exploring BiTE-secreting T-cell therapies as an innovate approach to boost the therapeutic potential of untransduced bystander T-cells decorated by the secreted BiTEs. 

3.-Contribution of the BM-MSC to leukemia initiation/progression with a special focus on BM-MSC-mediated chemo- and immune-resistance.  We strive to resolve the transcriptional heterogeneity and developmental origin of the BM-MSCs. We are especially interested in understanding how pro-inflammatory vs immunosuppressive stromas control immune resistance and immune escape to immunotherapies.


Prof. Mel Greaves and Tony Ford, Institute of Cancer Research, London. UK
Dr. Tanja Gruber, St. Jude´s Children Hospital., Memphis, USA
Dr. Geertruy te Kronnie, Pediatrics Department, University of Padova, Italy
Dr. Manuel Ramírez Orellana, Niño Jesús Hospital, Madrid, Spain
Dr. Mireia Camos, Hospital Sant Joan de Deu, Barcelona, Spain
Dr. Jose Luis Fuster, Hospital Virgen de la Arrixaca, Murcia, Spain
Prof. Majlinda Lako, Newcastle University, UK
Dr. Juan Bueren and Jose Carlos Segovia, CIEMAT, Madrid, Spain
Dr. Consuelo del Cañizo, Hospital Clínico de Salamanca, Spain
Dr. Mario F Fraga and Agustín Fernández, IUOPA, Oviedo, Spain
Dr. René Rodríguez, IUOPA, Oviedo, Spain
Prof. Plácido Navas and Carlos Santos Ocaña, UPO, Sevilla, Spain
Dr. Pedro J Real & Dr. Verónica Ramos, GENyO. Granada, Spain
Dr. Juan Carlos Rodríguez-Manzaneque, GENyO, Granada, Spain
Prof. Mario Delgado, IPBLN-CSIC, Granada, Spain
Prof. María Luisa Toribio, CBMSO-CSIC, Madrid, Spain.
Prof. Alberto Orfao, CIC, Salamanca. Spain
Prof. Rolf Marschalek. Goethe University, Frankfurt, Germany
Dr. Ronald W. Stam, Erasmus University, Rotterdam, The Netherlands
Dr. Anna Bigas, IMIM, Barcelona, Spain
Dr. Ignacio Varela, IBBITEC, Santander, Spain.
Dr. Eduardo Anguita, Hospital Clínico, Madrid, Spain
Dr. Jose Lopez barneo, Sevilla, IBiS, Spain
Dr. Antonio Postigo, IDIBAPS, Barcelona, Spain
Dr. Jose A Peréz-Simón, IBIS-HUVR, Seville, Spain
Dr. Gianni Cazanniga/Michela Bardini, University of Milan Bicacca, Ospedale San Gerardo, Monza, Italy
Dr. Pablo m García-Roves, IDIBAPS, Barcelona, Spain
Dr. Josep Nomdedeu and Dr.Jordi Sierra, Hospital Sant Pau, Barcelona, Spain
Dr. Susanna Vives y Francesc Solé, Hospital Germansd Trias i Pujol/IJC, Badalona, Spain


Selected publications

Muñoz-López A, Romero-Moya D, Prieto C, Ramos-Mejía V, Agraz-Doblas A, Varela I, Buschbeck M, Palau A, Carvajal-Vergara X, Giorgetti A, Ford A, Lako M, Granada I, Ruiz-Xivillé N, Rodríguez-Perales S, Torres-Ruíz R, Stam RW, Fuster JL, Fraga MF, Nakanishi M, Cazzaniga G, Bardini M, Cobo I, Bayon GF, Fernandez AF, Bueno C, Menendez P

Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency.

Stem Cell Reports 11 Oct 2016, 7 (4) 602-618. Epub 22 Set 2016
Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming "boosters" also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.
Més informació
Prieto C, Stam RW, Agraz-Doblas A, Ballerini P, Camos M, Castaño J, Marschalek R, Bursen A, Varela I, Bueno C, Menendez P

Activated KRAS cooperates with MLLAF4 to promote extramedullary engraftment and migration of cord blood CD34+ HSPC but is insufficient to initiate leukemia.

Cancer Res. 2 Feb 2016, . Epub 2 Feb 2016
The MLL-AF4 (MA4) fusion gene is the genetic hallmark of an aggressive infant pro-B-acute lymphoblastic leukemia (B-ALL). Our understanding of MA4-mediated transformation is very limited. Whole-genome sequencing studies revealed a silent mutational landscape, which contradicts the aggressive clinical outcome of this hematological malignancy. Only RAS mutations were recurrently detected in patients and found to be associated with poorer outcome. The absence of MA4-driven B-ALL models further questions whether MA4 acts as a single oncogenic driver or requires cooperating mutations to manifest a malignant phenotype. We explored whether KRAS activation cooperates with MA4 to initiate leukemia in cord blood (CB)-derived CD34+ hematopoietic stem/progenitor cells (HSPCs). Clonogenic and differentiation/proliferation assays demonstrated that KRAS activation does not cooperate with MA4 to immortalize CD34+ HSPCs. Intra-bone marrow transplantation into immunodeficient mice further showed that MA4 and KRASG12V alone or in combination enhanced hematopoietic repopulation without impairing myeloid-lymphoid differentiation, and that mutated KRAS did not cooperate with MA4 to initiate leukemia. However, KRAS activation enhanced extramedullary hematopoiesis of MA4-expressing cell lines and CD34+ HSPCs that was associated with leukocytosis and central nervous system infiltration, both hallmarks of infant t(4;11)+B-ALL. Transcriptional profiling of MA4-expressing patients supported a cell migration gene signature underlying the mutant KRAS-mediated phenotype. Collectively, our findings demonstrate that KRAS impacts the homeostasis of MA4-expressing HSPCs, suggesting that KRAS activation in MA4+ B-ALL is important for tumor maintenance rather than initiation.
Més informació
Bueno C, Sardina JL, Di Stefano B, Romero-Moya D, Muñoz-López A, Ariza L, Chillón MC, Balanzategui A, Castaño J, Herreros A, Fraga MF, Fernández A, Granada I, Quintana-Bustamante O, Segovia JC, Nishimura K, Ohtaka M, Nakanishi M, Graf T, Menendez P

Reprogramming human B-cells into induced pluripotent stem cells and its enhancement by C/EBPα.

Leukemia 26 Oct 2015, . Epub 26 Oct 2015
B-cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B-cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B-cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus (SeV). Co-expression of c/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B-cells. We also demonstrate that myeloid cells are much easier to reprogram than B- and T-lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B-cells since biased reprogramming was avoided by using freshly-isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B-cells offers an unprecedented opportunity for studying developmental B-lymphopoiesis and modeling B-cell malignances.Leukemia accepted article preview online, 26 October 2015. doi:10.1038/leu.2015.294.
Més informació
Sanjuan-Pla A, Bueno C, Prieto C, Acha P, Stam RW, Marschalek R, Menéndez P

Revisiting the biology of infant t(4;11)/MLL-AF4+ B-cell acute lymphoblastic leukemia.

Blood 13 Oct 2015, . Epub 13 Oct 2015
Infant B-cell acute lymphoblastic leukemia (B-ALL) accounts for 10% of childhood ALL. The genetic hallmark of most infant B-ALL are chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene. Despite improvement in the clinical management and survival (~85-90%) of childhood B-ALL, the outcome of infants with MLL-rearranged (MLL-r) B-ALL remains dismal, with overall survival <35%. Among MLL-r infant B-ALL, t(4;11)+ patients harboring the fusion MLL-AF4 (MA4), display a particularly poor prognosis and a pro-B/mixed phenotype. Studies in monozygotic twins and archived blood spots have provided compelling evidence of a single cell of prenatal origin as target for MA4 fusion, explaining the brief leukemia latency. Despite its aggressiveness and short latency, current progress about its etiology, pathogenesis and cellular origin is limited as evidenced by the lack of mouse/human models recapitulating the disease phenotype/latency. We propose this is because infant cancer is from an etiological and pathogenesis standpoint distinct to adult cancer and should be seen as a developmental disease. This is supported by whole-genome sequencing studies suggesting that opposite to the view of cancer as "multiple-and-sequential-hit" model, t(4;11) alone might be sufficient to spawn leukemia. The stable genome of these patients suggests that in infant developmental cancer one "big-hit" might be sufficient for overt disease, and supports a key contribution of epigenetics and a prenatal cell-of-origin during a critical developmental window of stem cell vulnerability in the leukemia pathogenesis. Here, we revisit the biology of t(4;11)+ infant B-ALL with emphasis on its origin, genetics and disease models.
Més informació
Bueno C, Ayllón V, Montes R, Navarro-Montero O, Ramos-Mejia V, Real PJ, Romero-Moya D, Araúzo-Bravo MJ, Menendez P

FLT3 activation cooperates with MLL-AF4 fusion protein to abrogate the hematopoietic specification of human ESCs.

Blood 9 Mai 2013, 121 (19) 3867-78, S1-3. Epub 11 Mar 2013
Mixed-lineage leukemia (MLL)-AF4 fusion arises prenatally in high-risk infant acute pro-B-lymphoblastic leukemia (pro-B-ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hematoendothelial specification but was insufficient for transformation, suggesting that additional oncogenic insults seem required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL expresses enormous levels of FLT3, occasionally because of activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. Here, we explored the developmental impact of FLT3 activation alone, or together with MLL-AF4, in the hematopoietic fate of hESCs. FLT3 activation does not affect specification of hemogenic precursors but significantly enhances the formation of CD45(+) blood cells, and CD45(+)CD34(+) blood progenitors with clonogenic potential. However, overexpression of FLT3 mutations or wild-type FLT3 (FLT3-WT) completely abrogates hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly affects hESC specification rather than proliferation or survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling of hESC-derived CD45(+) cells supports the FLT3-mediated inhibition of hematopoiesis in MLL-AF4-expressing hESCs, which is associated with large transcriptional changes and downregulation of genes involved in hematopoietic system development and function. Importantly, FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells, suggesting the need of alternative (epi)-genetic cooperating hits.
Més informació
M Martinez-Lage, R Torres-Ruiz, P Puig P Moreno-Gaona M Martin, F Moya, O Quintana-Bustamante, S Garcia-Silva, Angel Carcaboso, P Petazzi, C Bueno, J Mora, H Peinado, JC Segovia, Menendez P, S Rodríguez-Perales

In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells

Nat Commun . 2020 Oct 8;11(1):5060 , .
Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells.
Més informació
O. Molina, MA Abad,, Sole F, Menendez P

Aneuploidy in Cancer: Lessons from Acute Lymphoblastic Leukemia

Trends Cancer . 2020 Sep 17;S2405-8033(20)30240-5 17 Set 2020, .
Aneuploidy, the gain or loss of chromosomes in a cell, is a hallmark of cancer. Although our understanding of the contribution of aneuploidy to cancer initiation and progression is incomplete, significant progress has been made in uncovering the cellular consequences of aneuploidy and how aneuploid cancer cells self-adapt to promote tumorigenesis. Aneuploidy is physiologically associated with significant cellular stress but, paradoxically, it favors tumor progression. Although more common in solid tumors, different forms of aneuploidy represent the initiating oncogenic lesion in patients with B cell acute lymphoblastic leukemia (B-ALL), making B-ALL an excellent model for studying the role of aneuploidy in tumorigenesis. We review the molecular mechanisms underlying aneuploidy and discuss its contributions to B-ALL initiation and progression.
Més informació
SR Zanetti, PA Romecín, M Vinyoles, M Juan, JL Fuster, M Camós, S Querol, M Delgado,, Menéndez P

Bone marrow MSC from pediatric patients with B-ALL highly immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity

J Immunother Cancer . 2020 Aug;8(2):e001419 , .
Background: Although adoptive transfer of CD19-directed chimeric antigen receptor (CAR) T-cells (CD19-CAR T-cells) achieves high rates of complete response in patients with B-cell acute lymphoblastic leukemia (B-ALL), relapse is common. Bone marrow (BM) mesenchymal stem/stromal cells (BM-MSC) are key components of the hematopoietic niche and are implicated in B-ALL pathogenesis and therapy resistance. MSC exert an immunosuppressive effect on T-cells; however, their impact on CD19-CAR T-cell activity is understudied. Methods: We performed a detailed characterization of BM-MSC from pediatric patients with B-ALL (B-ALL BM-MSC), evaluated their immunomodulatory properties and their impact on CD19-CAR T-cell activity in vitro using microscopy, qRT-PCR, ELISA, flow cytometry analysis and in vivo using a preclinical model of severe colitis and a B-ALL xenograft model. Results: While B-ALL BM-MSC were less proliferative than those from age-matched healthy donors (HD), the morphology, immunophenotype, differentiation potential and chemoprotection was very similar. Likewise, both BM-MSC populations were equally immunosuppressive in vitro and anti-inflammatory in an in vivo model of severe colitis. Interestingly, BM-MSC failed to impair CD19-CAR T-cell cytotoxicity or cytokine production in vitro using B-ALL cell lines and primary B-ALL cells. Finally, the growth of NALM6 cells was controlled in vivo by CD19-CAR T-cells irrespective of the absence/presence of BM-MSC. Conclusions: Collectively, our data demonstrate that pediatric B-ALL and HD BM-MSC equally immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity.
Més informació
T Velasco-Hernandez, S. R Zanetti, H Roca-Ho, F. Gutiérrez-Agüera, P. Petazzi, D. Sanchez-Martínez, O. Molina, ML. Baroni, JL, Fuster, P. Ballerini, C. Bueno, P. Engel, Menendez P

Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope

J Immunother Cancer . 2020 Aug;8(2):e000896. , .
Background: There are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19- either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22+CD19- B-ALL relapses and CD19- preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied. Methods: Here, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays. Results: Conformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy. Conclusions: We report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22high and CD22low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22-CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL.
Més informació
C. Bueno, P Ballerini, I. Varela, Menendez P, R. Bashford-Rogers

Shared D-J rearrangements reveal cell of origin of TCF3-ZNF384 and PTPN11 mutations in monozygotic twins with concordant BCP-ALL

Blood . 2020 Aug 27;136(9):1108-1111 , .
No abstract available
Més informació
M Baroni, D Sanchez-Martínez, F Gutierrez, M Castella, SR Zanetti, T Velasco-Hernández, R de la Guardia, J Castaño, E Anguita, S Vives, JF Nomdedeu, H Lapillonne, AE Bras, VHJ van Velden, J Junca, I. Jeremias, A López, M Sorigue, C Bueno, Menéndez P

41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo

Journal for Immunotherapy of Cancer, 01 Jun 2020, 8(1) , .
Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative. Methods: We set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs. Results: Here, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs. Conclusions: This study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML.
Més informació
P Petazzi, R Torres, A Fidanza, H Roca-Ho, F Gutierrez-Agüera, R Díaz de la Guardia, B López-Millán, A Bigas, L Forrester, C Bueno, Menéndez P

Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells

Molecular therapy. Nucleic Acids, 27 Feb 2020, 20:196-204 , .
Human pluripotent stem cells (hPSCs) and mesenchymal stromal/stem cells (hMSCs) are clinically relevant sources for cellular therapies and for modeling human development and disease. Many stem cell-based applications rely on the ability to activate several endogenous genes simultaneously to modify cell fate. However, genetic intervention of these cells remains challenging. Several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains can modulate gene expression when directed to their regulatory regions by a specific single-guide RNA (sgRNA). In this study, we have compared the ability of the first-generation dCas9-VP64 activator and the second-generation systems, dCas9-SAM and dCas9-SunTag, to induce gene expression in hPSCs and hMSCs. Several stem cell lines were tested for single and multiplexed gene activation. When the activation of several genes was compared, all three systems induced specific and potent gene expression in both single and multiplexed settings, but the dCas9-SAM and dCas9-SunTag systems resulted in the highest and most consistent level of gene expression. Simultaneous targeting of the same gene with multiple sgRNAs did not result in additive levels of gene expression in hPSCs nor hMSCs. We demonstrate the robustness and specificity of second-generation dCas9 activators as tools to simultaneously activate several endogenous genes in clinically relevant human stem cells.
Més informació
L Beneforti, E Dander, S Bresolin, C Bueno,D Acunzo, M Betagna, A Ford, B Gentner, G Te Kronnie, P Vergani, Menéndez P, A Biondi, G D’Amico, C Palmi, G Cazzaniga

Pro-inflammatory cytokines favor the emergence of ETV6-RUNX1-positive pre-leukemic cells in a model of mesenchymal niche

Br J Haematol . 2020 Jul;190(2):262-273 , .
ETV6-RUNX1 (E/R) fusion gene, arising in utero from translocation t(12;21)(p13:q22), is the most frequent alteration in childhood acute lymphoblastic leukemia (ALL). However, E/R is insufficient to cause overt leukemia since it generates a clinically silent pre-leukemic clone which persists in the bone marrow but fails to out-compete normal progenitors. Conversely, pre-leukemic cells show increased susceptibility to transformation following additional genetic insults. Infections/inflammation are the most accredited triggers for mutations accumulation and leukemic transformation in E/R+ pre-leukemic cells. However, precisely how E/R and inflammation interact in promoting leukemia is still poorly understood. Here we demonstrate that IL6/TNFα/ILβ pro-inflammatory cytokines cooperate with BM-MSC in promoting the emergence of E/R+ Ba/F3 over their normal counterparts by differentially affecting their proliferation and survival. Moreover, IL6/TNFα/ILβ-stimulated BM-MSC strongly attract E/R+ Ba/F3 in a CXCR2-dependent manner. Interestingly, E/R-expressing human CD34+ IL7R+ progenitors, a putative population for leukemia initiation during development, were preserved in the presence of BM-MSC and IL6/TNFα/ILβ compared to their normal counterparts. Finally, the extent of DNA damage increases within the inflamed niche in both control and E/R-expressing Ba/F3, potentially leading to transformation in the apoptosis-resistant pre-leukemic clone. Overall, our data provide new mechanistic insights into childhood ALL pathogenesis.
Més informació
R Diaz de la Guardia, L Gonzalez-Slva, B Lopez-Millán, J Rodríguez-Sevilla, M L Baroni, C Bueno, E Anguita, S Vives, L Palomo, H Lapillonne, I Varela,, Menéndez P

Bone Marrow Clonogenic Myeloid Progenitors from NPM1-Mutated AML Patients Do Not Harbor the NPM1 Mutation: Implication for the Cell-Of-Origin of NPM1+ AML

Genes (Basel) . 2020 Jan 9;11(1):73 , .
The cell-of-origin of NPM1- and FLT3-mutated acute myeloid leukemia (AML) is still a matter of debate. Here, we combined in vitro clonogenic assays with targeted sequencing to gain further insights into the cell-of-origin of NPM1 and FLT3-ITD-mutated AML in diagnostic bone marrow (BM) from nine NPM1+/FLT3-ITD (+/-) AMLs. We reasoned that individually plucked colony forming units (CFUs) are clonal and reflect the progeny of a single stem/progenitor cell. NPM1 and FLT3-ITD mutations seen in the diagnostic blasts were found in only 2/95 and 1/57 individually plucked CFUs, suggesting that BM clonogenic myeloid progenitors in NPM1-mutated and NPM1/FLT3-ITD-mutated AML patients do not harbor such molecular lesions. This supports previous studies on NPM1 mutations as secondary mutations in AML, likely acquired in an expanded pool of committed myeloid progenitors, perhaps CD34-, in line with the CD34-/low phenotype of NPM1-mutated AMLs. This study has important implications on the cell-of-origin of NPM1+ AML, and reinforces that therapeutic targeting of either NPM1 or FLT3-ITD mutations might only have a transient clinical benefit in debulking the leukemia, but is unlikely to be curative since will not target the AML-initiating/preleukemic cells. The absence of NPM1 and FLT3-ITD mutations in normal clonogenic myeloid progenitors is in line with their absence in clonal hematopoiesis of indeterminate potential.
Més informació
I Mirones, L Sisinni, M García-Morín, J Anguita, M Ramírez, MA Díaz, M González, L Moreno, L Alonso, S, Rives, M M Alonso, A Patiño-García, P Palomo, J Verdú-Amorós, I Martínez, G Lizeaga, P guerra-García, J L Fuster, J M Moraleda, A Sánchez-Salinas, M Blanquer, J García-castro, ML Toribio, H M van Santen, Menéndez P, A Pérez-Martínez

Immunotherapy with CAR-T cells in paediatric haematology-oncology

An Pediatr (Barc) . 2020 Jul;93(1):59.e1-59.e10. , .
Despite being a rare disease, cancer is the first cause of mortality due to disease during the paediatric age in the developed countries. The current, great increase in new treatments, such as immunotherapy, constitutes a new clinical and regulatory paradigm. Cellular immunotherapy is one of these types of immunotherapy. In particular, the advanced therapy drugs with chimeric antigen receptors in the T-lymphocytes (CAR-T), and particularly the CAR-T19 cells, has opened up a new scenario in the approach to haematology tumours like acute lymphoblastic leukaemia and the B-Cell lymphomas. The approval of tisagenlecleucel and axicabtagene ciloleucel by the regulatory authorities has led to the setting up of the National Plan for Advanced Therapies-CAR-T drugs in Spain. There is evidence of, not only the advantage of identifying the centres most suitable for their administration, but also the need for these to undergo a profound change in order that their healthcare activity is extended, in some cases, to the ability for the in-house manufacture of these types of therapies. The hospitals specialised in paediatric haematology-oncology thus have the challenge of progressing towards a healthcare model that integrates cellular immunotherapy, having the appropriate capacity to manage all aspects relative to their use, manufacture, and administration of these new treatments.
Més informació
Godfrey L, Crump NT, O'Byrne S, Lau IJ, Rice S, Harman JR, Jackson T, Elliott N, Buck G, Connor C, Thorne R, Knapp DJHF, Heidenreich O, Vyas P, Menendez P, Inglott S, Ancliff P, Geng H, Roberts I, Roy A, Milne TA

H3K79me2/3 controls enhancer-promoter interactions and activation of the pan-cancer stem cell marker PROM1/CD133 in MLL-AF4 leukemia cells.

Leukemia 2 Abr 2020, . Epub 2 Abr 2020
MLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer-promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.
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Molina O, Vinyoles M, Granada I, Roca-Ho H, Gutierrez-Agüera F, Valledor L, López-López CM, Rodríguez-González P, Trincado JL, Tirados-Menéndez S, Pal D, Ballerini P, Den Boer ML, Plensa I, Perez-Iribarne MDM, Rodriguez-Perales S, Calasanz MJ, Ramírez M, Rodríguez R, Camos M, Calvo M, Bueno C, Menendez P

Impaired Condensin Complex and Aurora B kinase underlie mitotic and chromosomal defects in hyperdiploid B-cell ALL.

Blood 22 Abr 2020, . Epub 22 Abr 2020
B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, and high-hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD is an initiating oncogenic event affiliated to childhood B-ALL, the mitotic and chromosomal defects associated to HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated to chromosome alignment defects at the metaphase plate leading to robust chromosome segregation defects and non-modal karyotypes. Mechanistically, biochemical, functional and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness and mis-localization of the chromosome passenger complex proteins Aurora B Kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and impaired spindle assembly checkpoint (SAC) thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated to defective condensin complex, AURKB and SAC.
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Vidal-Crespo A, Matas-Céspedes A, Rodriguez V, Rossi C, Valero JG, Serrat N, Sanjuan-Pla A, Menéndez P, Roué G, López-Guillermo A, Giné E, Campo E, Colomer D, Bezombes C, van Bueren JL, Chiu C, Doshi P, Pérez-Galán P

Daratumumab displays in vitro and in vivo anti-tumor activity in models of B-cell non-Hodgkin lymphoma and improves responses to standard chemo-immunotherapy regimens.

Haematologica Abr 2020, 105 (4) 1032-1041. Epub 11 Jul 2019
CD38 is expressed in several types of non-Hodgkin lymphoma (NHL) and constitutes a promising target for antibody-based therapy. Daratumumab (Darzalex) is a first-in-class anti-CD38 antibody approved for the treatment of relapsed/refractory (R/R) multiple myeloma (MM). It has also demonstrated clinical activity in Waldenström macroglobulinaemia and amyloidosis. Here, we have evaluated the activity and mechanism of action of daratumumab in preclinical
Més informació
O'Byrne S, Elliott N, Rice S, Buck G, Fordham N, Garnett C, Godfrey L, Crump NT, Wright G, Inglott S, Hua P, Psaila B, Povinelli B, Knapp DJHF, Agraz-Doblas A, Bueno C, Varela I, Bennett P, Koohy H, Watt SM, Karadimitris A, Mead AJ, Ancliff P, Vyas P, Menendez P, Milne TA, Roberts I, Roy A

Discovery of a CD10-negative B-progenitor in human fetal life identifies unique ontogeny-related developmental programs.

Blood 26 Set 2019, 134 (13) 1059-1071. Epub 5 Ago 2019
Human lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks postconception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD19
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J Castaño, S Aranda, C Bueno, FJ Calero-Nieto, E Mejia-Ramirez, E Blanco, X Wang, C Prieto, L Zabaleta, M Rovira, B Gӧttgens, L Di Croce, Menéndez P, A Raya, A Giorgetti

GATA2 Promotes Hematopoietic Development and Represses Cardiac Differentiation of Human Mesoderm

Stem Cell Reports . 2019 Sep 10;13(3):515-529 , .
In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.
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A. Agraz-Doblas, C.Bueno, R.Bashford-Rogers, A. Roy, P.Scheneider, P. Ballerini, G. Cazzaniga, M.Bardini, I.Robers, R. Pieters, I. Varela, RW.Stam, Menéndez P

Unraveling the cellular origin and clinical prognostic markers of infant B-cell acute lymphoblastic leukemia using genome-wide analysis

Haematologica . 2019 Jun;104(6):1176-1188 , .
B-cell acute lymphoblastic leukemia is the commonest childhood cancer. In infants, B-cell acute lymphoblastic leukemia remains fatal, especially in patients with t(4;11), present in ~80% of cases. The pathogenesis of t(4;11)/KMT2A-AFF1+ (MLL-AF4+) infant B-cell acute lymphoblastic leukemia remains difficult to model, and the pathogenic contribution in cancer of the reciprocal fusions resulting from derivative translocated-chromosomes remains obscure. Here, "multi-layered" genome-wide analyses and validation were performed on a total of 124 de novo cases of infant B-cell acute lymphoblastic leukemia uniformly diagnosed and treated according to the Interfant 99/06 protocol. These patients showed the most silent mutational landscape reported so far for any sequenced pediatric cancer. Recurrent mutations were exclusively found in K-RAS and N-RAS, were subclonal and were frequently lost at relapse, despite a larger number of non-recurrent/non-silent mutations. Unlike non-MLL-rearranged B-cell acute lymphoblastic leukemias, B-cell receptor repertoire analysis revealed minor, non-expanded B-cell clones in t(4;11)+ infant B-cell acute lymphoblastic leukemia, and RNA-sequencing showed transcriptomic similarities between t(4;11)+ infant B-cell acute lymphoblastic leukemias and the most immature human fetal liver hematopoietic stem and progenitor cells, confirming a "pre-VDJ" fetal cellular origin for both t(4;11) and RAS mut The reciprocal fusion AF4-MLL was expressed in only 45% (19/43) of the t(4;11)+ patients, and HOXA cluster genes are exclusively expressed in AF4-MLL-expressing patients. Importantly, AF4-MLL/HOXA-expressing patients had a significantly better 4-year event-free survival (62.4% vs 11.7%, P=0.001), and overall survival (73.7 vs 25.2%, P=0.016). AF4-MLL expression retained its prognostic significance when analyzed in a Cox model adjusting for risk stratification according to the Interfant-06 protocol based on age at diagnosis, white blood cell count and response to prednisone. This study has clinical implications for disease outcome and diagnostic risk-stratification of t(4;11)+ infant B-cell acute lymphoblastic leukemia.
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C. Bueno, T. Velasco, F Gutierrez, S. Zanetti, D. Sánchez, M. Baroni, O. Molina, A. Closa, A. Agraz, E. Eyras, I. Varela,, Menéndez P

CD133-directed CAR T-cells for MLL leukemia: on-target, off-tumor myeloablative toxicity

Leukemia . 2019 Aug;33(8):2090-2125 , .
No abstract available
Més informació
Urdinguio, R.G., Lopez, V., Bayón, G.F., Sierra, M., García-Toraño,E., Fernandez, R., García, M., Carella, A., Cueto, P., Prieto, P., Dmitrieva, M., Santamarina, P., Mangas, C., Diaconou, E., Ferrero, C., Tejedor, J.R., Bravo, C., Bueno, C., Sanjuán, A., Rodríguez, R.M., Suarez, B., López-Larrea, C., Bernal, T., Colado, E., Balbín, M., García-Suarez, O., Chiara, M.D., Sáenz-de-Santa-María, I., Rodríguez, F., Pando-Sandoval, A., Rodrigo, L., Santos, L., Salas, A., Vallejo, J., Carrera, A.C., Rico, D., Hernández-López, I., Vayá, A., Ricart, J.M., Seto, A., Vaquero A, Sima, N., Pisano, D., Graña, O., Thomas, T., Voss, A.K., Villar-Garea, A., Deutzmann, R., Fernandez, A.F., Fraga, M.F, Menéndez P

Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment

Nucleic Acids Res. 47(10):5016-5037 (2019). , .
Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.
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D Sánchez-Martínez, ML Baroni, F Gutierrez-Agüera, H Roca-Ho, O Blanch-Lombarte, T Velasco, C Bueno, JL Fuster, J Garcia-Prado, J Cools, M Camos, F Pflumio, ML Toribio, Menéndez P

Fratricide-resistant CD1a-specific CAR T cells for the treatment of cortical T-cell acute lymphoblastic leukemia

Blood . 2019 May 23;133(21):2291-2304 , .
Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient-derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.
Més informació
M. Martinez-Lage, P. Puig-Serra, Menéndez P, R. Torres-Ruiz, S. Rodriguez-Perales.

CRISPR/Cas9 for Cancer Therapy: Hopes and Challenges

Biomedicines . 2018 Nov 12;6(4):105 , .
Cancer is the second leading cause of death globally and remains a major economic and social burden. Although our understanding of cancer at the molecular level continues to improve, more effort is needed to develop new therapeutic tools and approaches exploiting these advances. Because of its high efficiency and accuracy, the CRISPR-Cas9 genome editing technique has recently emerged as a potentially powerful tool in the arsenal of cancer therapy. Among its many applications, CRISPR-Cas9 has shown an unprecedented clinical potential to discover novel targets for cancer therapy and to dissect chemical-genetic interactions, providing insight into how tumours respond to drug treatment. Moreover, CRISPR-Cas9 can be employed to rapidly engineer immune cells and oncolytic viruses for cancer immunotherapeutic applications. Perhaps more importantly, the ability of CRISPR-Cas9 to accurately edit genes, not only in cell culture models and model organisms but also in humans, allows its use in therapeutic explorations. In this review, we discuss important considerations for the use of CRISPR/Cas9 in therapeutic settings and major challenges that will need to be addressed prior to its clinical translation for a complex and polygenic disease such as cancer.
Més informació
R. Diaz de la Guardia, B Lopez-Millan, H Roca-Ho, C Bueno, F Gutiérrez-Agüera, JL Fuster, E Anguita, S Vives, J Nomdedeu, R Sackstein, J Lavoie, E Gónzalez-Rey, M Delgado, M Rosu-Myles,, Menéndez P

Bone marrow mesenchymal stem/stromal cells from risk-stratified acute myeloid leukemia patients are anti-inflammatory in in vivo preclinical models of hematopoietic reconstitution and severe colitis

Haematologica . 2019 Feb;104(2):e54-e58 , .
No abstract available
Més informació
Lopez-Millán, H. Roca-Ho, R. Diaz Guardia, D. Sánchez-Martínez, F. Gutiérrez-Agüera, JL. Fuster, P. Ballerini, C. Nombela-Arrieta, C. Bueno, Menéndez P

NG2 antigen is a therapeutic target for MLL-rearranged B-cell acute lymphoblastic leukemia

Leukemia . 2019 Jul;33(7):1557-1569. , .
B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer, with cure rates of ∼80%. MLL-rearranged (MLLr) B-ALL (MLLr-B-ALL) has, however, an unfavorable prognosis with common therapy refractoriness and early relapse, and therefore new therapeutic targets are needed for relapsed/refractory MLLr-B-ALL. MLLr leukemias are characterized by the specific expression of chondroitin sulfate proteoglycan-4, also known as neuron-glial antigen-2 (NG2). NG2 was recently shown involved in leukemia invasiveness and central nervous system infiltration in MLLr-B-ALL, and correlated with lower event-free survival (EFS). We here hypothesized that blocking NG2 may synergize with established induction therapy for B-ALL based on vincristine, glucocorticoids, and L-asparaginase (VxL). Using robust patient-derived xenograft (PDX) models, we found that NG2 is crucial for MLLr-B-ALL engraftment upon intravenous (i.v.) transplantation. In vivo blockade of NG2 using either chondroitinase-ABC or an anti-NG2-specific monoclonal antibody (MoAb) resulted in a significant mobilization of MLLr-B-ALL blasts from bone marrow (BM) to peripheral blood (PB) as demonstrated by cytometric and 3D confocal imaging analysis. When combined with either NG2 antagonist, VxL treatment achieved higher rates of complete remission, and consequently higher EFS and delayed time to relapse. Mechanistically, anti-NG2 MoAb induces neither antibody-dependent cell-mediated not complement-dependent cytotoxicity. NG2 blockade rather overrides BM stroma-mediated chemoprotection through PB mobilization of MLLr-B-ALL blasts, thus becoming more accessible to chemotherapy. We provide a proof of concept for NG2 as a therapeutic target for MLLr-B-ALL.
Més informació
C Bueno, F Calero-Nieto, X Wang, R Valdés-Mas, H Roca-Ho, F Gutiérrez-Agüero, V Ayllon, PJ Real, D Arambilet, L Espinosa, A Bigas, B Gottgens, R Marschalek, Menéndez P

Enhanced hemato-endothelial specification during human embryonic differentiation through developmental cooperation between AF4-MLL and MLL-AF4 fusions

Haematologica . 2019 Jun;104(6):1189-1201 , .
The t(4;11)(q21;q23) translocation is associated with high-risk infant pro-B-cell acute lymphoblastic leukemia and arises prenatally during embryonic/fetal hematopoiesis. The developmental/pathogenic contribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) fusions remains unclear; MA4 is always expressed in patients with t(4;11)+ B-cell acute lymphoblastic leukemia, but the reciprocal fusion A4M is expressed in only half of the patients. Because prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, we took advantage of well-established human embryonic stem cell-based hematopoietic differentiation models to study whether the A4M fusion cooperates with MA4 during early human hematopoietic development. Co-expression of A4M and MA4 strongly promoted the emergence of hemato-endothelial precursors, both endothelial- and hemogenic-primed. Double fusion-expressing hemato-endothelial precursors specified into significantly higher numbers of both hematopoietic and endothelial-committed cells, irrespective of the differentiation protocol used and without hijacking survival/proliferation. Functional analysis of differentially expressed genes and differentially enriched H3K79me3 genomic regions by RNA-sequencing and H3K79me3 chromatin immunoprecipitation-sequencing, respectively, confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing hemato-endothelial precursors. Importantly, chromatin immunoprecipitation-sequencing analysis revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in double fusion-expressing differentiating hematopoietic cells. Overall, these results establish a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development.
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Recasens-Zorzo C, Cardesa-Salzmann T, Petazzi P, Ros-Blanco L, Esteve-Arenys A, Clot G, Guerrero-Hernández M, Rodríguez V, Soldini D, Valera A, Moros A, Climent F, González-Barca E, Mercadal S, Arenillas L, Calvo X, Mate JL, Gutiérrez-García G, Casanova I, Mangues R, Sanjuan-Pla A, Bueno C, Menéndez P, Martínez A, Colomer D, Tejedor RE, Teixidó J, Campo E, López-Guillermo A, Borrell JI, Colomo L, Pérez-Galán P, Roué G

Pharmacological modulation of CXCR4 cooperates with BET bromodomain inhibition in diffuse large B-cell lymphoma.

Haematologica Abr 2019, 104 (4) 778-788. Epub 28 Jun 2018
Constitutive activation of the chemokine receptor CXCR4 has been associated with tumor progression, invasion, and chemotherapy resistance in different cancer subtypes. Although the CXCR4 pathway has recently been suggested as an adverse prognostic marker in diffuse large B-cell lymphoma, its biological relevance in this disease remains underexplored. In a homogeneous set of 52 biopsies from patients, an antibody-based cytokine array showed that tissue levels of CXCL12 correlated with high microvessel density and bone marrow involvement at diagnosis, supporting a role for the CXCL12-CXCR4 axis in disease progression. We then identified the tetra-amine IQS-01.01RS as a potent inverse agonist of the receptor, preventing CXCL12-mediated chemotaxis and triggering apoptosis in a panel of 18 cell lines and primary cultures, with superior mobilizing properties in vivo than those of the standard agent. IQS-01.01RS activity was associated with downregulation of p-AKT, p-ERK1/2 and destabilization of MYC, allowing a synergistic interaction with the bromodomain and extra-terminal domain inhibitor, CPI203. In a xenotransplant model of diffuse large B-cell lymphoma, the combination of IQS-01.01RS and CPI203 decreased tumor burden through MYC and p-AKT downregulation, and enhanced the induction of apoptosis. Thus, our results point out an emerging role of CXCL12-CXCR4 in the pathogenesis of diffuse large B-cell lymphoma and support the simultaneous targeting of CXCR4 and bromodomain proteins as a promising, rationale-based strategy for the treatment of this disease.
Més informació
Petazzi P, Menéndez P

A NEWral approach for HSC production in vitro?

Blood . 2020 Dec 17;136(25):2845-2847 , .
No abstract available
Més informació
K Otterbasch, A Sanjuan-Pla, R Torres-Ruíz, C Bueno, T Velasco, Menéndez P

The "Never-Ending" Mouse Models for MLL-Rearranged Acute Leukemia Are Still Teaching Us

Hemasphere . 2018 Jun 19;2(4):e57 , .
No abstract available
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E. Garcia-Alegría, S Menegatti, MZH Fadlullah, Menéndez P, G Lacaud, V. Kouskoff

Early Human Hemogenic Endothelium Generates Primitive and Definitive Hematopoiesis In Vitro

Stem Cell Reports . 2018 Nov 13;11(5):1061-1074 , .
The differentiation of human embryonic stem cells (hESCs) to hematopoietic lineages initiates with the specification of hemogenic endothelium, a transient specialized endothelial precursor of all blood cells. This in vitro system provides an invaluable model to dissect the emergence of hematopoiesis in humans. However, the study of hematopoiesis specification is hampered by a lack of consensus in the timing of hemogenic endothelium analysis and the full hematopoietic potential of this population. Here, our data reveal a sharp decline in the hemogenic potential of endothelium populations isolated over the course of hESC differentiation. Furthermore, by tracking the dynamic expression of CD31 and CD235a at the onset of hematopoiesis, we identified three populations of hematopoietic progenitors, representing primitive and definitive subsets that all emerge from the earliest specified hemogenic endothelium. Our data establish that hemogenic endothelium populations endowed with primitive and definitive hematopoietic potential are specified simultaneously from the mesoderm in differentiating hESCs.
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Castella M, Boronat A, Martín-Ibáñez R, Rodríguez V, Suñé G, Caballero M, Marzal B, Pérez-Amill L, Martín-Antonio B, Castaño J, Bueno C, Balagué O, González-Navarro EA, Serra-Pages C, Engel P, Vilella R, Benitez-Ribas D, Ortiz-Maldonado V, Cid J, Tabera J, Canals JM, Lozano M, Baumann T, Vilarrodona A, Trias E, Campo E, Menendez P, Urbano-Ispizua Á, Yagüe J, Pérez-Galán P, Rives S, Delgado J, Juan M

Development of a Novel Anti-CD19 Chimeric Antigen Receptor: A Paradigm for an Affordable CAR T Cell Production at Academic Institutions.

Mol Ther Methods Clin Dev 15 Mar 2019, 12 134-144. Epub 6 Des 2018
Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells
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Fernandez AF, Bayón GF, Sierra MI, Urdinguio RG, Toraño EG, García M, Carella A, Lopez V, Santamarina P, Pérez RF, Belmonte T, Ramon Tejedor J, Cobo I, Menendez P, Mangas C, Ferrero C, Rodrigo L, Astudillo A, Ortea I, Cueto Díaz S, Rodríguez-Gonzalez P, Ignacio García Alonso J, Mollejo M, Meléndez B, Dominguez G, Bonilla F, Fraga MF

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma.

Hum. Mol. Genet. 5 Jun 2018, . Epub 5 Jun 2018
Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5mC in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4,627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island (CGI) shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.
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Lopez-Millan B, Diaz de la Guardia R, Roca-Ho H, Anguita E, Islam ABMMK, Romero-Moya D, Prieto C, Gutierrez-Agüera F, Bejarano-Garcia JA, Perez-Simon JA, Costales P, Rovira M, Marín P, Menendez S, Iglesias M, Fuster JL, Urbano-Ispizua A, Anjos-Afonso F, Bueno C, Menendez P

IMiDs mobilize acute myeloid leukemia blasts to peripheral blood through downregulation of CXCR4 but fail to potentiate AraC/Idarubicin activity in preclinical models of non del5q/5q- AML.

Oncoimmunology 2018, 7 (9) e1477460. Epub 26 Jul 2018
Treatment for acute myeloid leukemia (AML) remains suboptimal and many patients remain refractory or relapse upon standard chemotherapy based on nucleoside analogs plus anthracyclines. The crosstalk between AML cells and the BM stroma is a major mechanism underlying therapy resistance in AML. Lenalidomide and pomalidomide, a new generation immunomodulatory drugs (IMiDs), possess pleiotropic anti-leukemic properties including potent immune-modulating effects and are commonly used in hematological malignances associated with intrinsic dysfunctional BM such as myelodysplastic syndromes and multiple myeloma. Whether IMiDs may improve the efficacy of current standard treatment in AML remains understudied. Here, we have exploited
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Meyer C, Burmeister T, Gröger D, Tsaur G, Fechina L, Renneville A, Sutton R, Venn NC, Emerenciano M, Pombo-de-Oliveira MS, Barbieri Blunck C, Almeida Lopes B, Zuna J, Trka J, Ballerini P, Lapillonne H, De Braekeleer M, Cazzaniga G, Corral Abascal L, van der Velden VHJ, Delabesse E, Park TS, Oh SH, Silva MLM, Lund-Aho T, Juvonen V, Moore AS, Heidenreich O, Vormoor J, Zerkalenkova E, Olshanskaya Y, Bueno C, Menendez P, Teigler-Schlegel A, Zur Stadt U, Lentes J, Göhring G, Kustanovich A, Aleinikova O, Schäfer BW, Kubetzko S, Madsen HO, Gruhn B, Duarte X, Gameiro P, Lippert E, Bidet A, Cayuela JM, Clappier E, Alonso CN, Zwaan CM, van den Heuvel-Eibrink MM, Izraeli S, Trakhtenbrot L, Archer P, Hancock J, Möricke A, Alten J, Schrappe M, Stanulla M, Strehl S, Attarbaschi A, Dworzak M, Haas OA, Panzer-Grümayer R, Sedék L, Szczepański T, Caye A, Suarez L, Cavé H, Marschalek R

The MLL recombinome of acute leukemias in 2017.

Leukemia Feb 2018, 32 (2) 273-284. Epub 13 Jul 2017
Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.
Més informació
de la Guardia RD, Correa JG, López-Millán B, Juan M, Bueno C, Cervantes F, Menéndez P

Detection of inflammatory monocytes but not mesenchymal stem/stromal cells in peripheral blood of patients with myelofibrosis.

Br. J. Haematol. 21 Feb 2017, . Epub 21 Feb 2017Més informació
Prieto C, López-Millán B, Roca-Ho H, Stam RW, Romero-Moya D, Rodríguez-Baena FJ, Sanjuan-Pla A, Ayllón V, Ramírez M, Bardini M, De Lorenzo P, Valsecchi MG, Stanulla M, Iglesias M, Ballerini P, Carcaboso ÁM, Mora J, Locatelli F, Bertaina A, Padilla L, Carlos Rodríguez-Manzaneque J, Bueno C, Menéndez P

NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL.

Leukemia Mar 2018, 32 (3) 633-644. Epub 25 Set 2017
Mixed-lineage leukemia (MLL)-rearranged (MLLr) infant B-cell acute lymphoblastic leukemia (iMLLr-B-ALL) has a dismal prognosis and is associated with a pro-B/mixed phenotype, therapy refractoriness and frequent central nervous system (CNS) disease/relapse. Neuron-glial antigen 2 (NG2) is specifically expressed in MLLr leukemias and is used in leukemia immunophenotyping because of its predictive value for MLLr acute leukemias. NG2 is involved in melanoma metastasis and brain development; however, its role in MLL-mediated leukemogenesis remains elusive. Here we evaluated whether NG2 distinguishes leukemia-initiating/propagating cells (L-ICs) and/or CNS-infiltrating cells (CNS-ICs) in iMLLr-B-ALL. Clinical data from the Interfant cohort of iMLLr-B-ALL demonstrated that high NG2 expression associates with lower event-free survival, higher number of circulating blasts and more frequent CNS disease/relapse. Serial xenotransplantation of primary MLL-AF4
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García-Peydró M, Fuentes P, Mosquera M, García-León MJ, Alcain J, Rodríguez A, de Miguel PG, Menéndez P, Weijer K, Spits H, Scadden DT, Cuesta-Mateos C, Muñoz-Calleja C, Sánchez-Madrid F, Toribio ML

The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model.

J. Clin. Invest. 21 Mai 2018, . Epub 21 Mai 2018
NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T-ALL), but crucial NOTCH1 downstream signals and target genes contributing to T-ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia-initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T-ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T-ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T-ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T-ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T-ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T-ALL pathogenesis. The observed therapeutic benefit of anti-CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T-ALL relapse.
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Tejedor JR, Bueno C, Cobo I, Bayón GF, Prieto C, Mangas C, Pérez RF, Santamarina P, Urdinguio RG, Menéndez P, Fraga MF, Fernández AF

Epigenome-wide analysis reveals specific DNA hypermethylation of T cells during human hematopoietic differentiation.

Epigenomics 5 Abr 2018, . Epub 5 Abr 2018
Epigenetic regulation plays an important role in cellular development and differentiation. A detailed map of the DNA methylation dynamics that occur during cell differentiation would contribute to decipher the molecular networks governing cell fate commitment.
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Body S, Esteve-Arenys A, Miloudi H, Recasens-Zorzo C, Tchakarska G, Moros A, Bustany S, Vidal-Crespo A, Rodriguez V, Lavigne R, Com E, Casanova I, Mangues R, Weigert O, Sanjuan-Pla A, Menéndez P, Marcq B, Picquenot JM, Pérez-Galán P, Jardin F, Roué G, Sola B

Cytoplasmic cyclin D1 controls the migration and invasiveness of mantle lymphoma cells.

Sci Rep 24 Oct 2017, 7 (1) 13946. Epub 24 Oct 2017
Mantle cell lymphoma (MCL) is a hematologic neoplasm characterised by the t(11;14)(q13;q32) translocation leading to aberrant cyclin D1 expression. The cell functions of cyclin D1 depend on its partners and/or subcellular distribution, resulting in different oncogenic properties. We observed the accumulation of cyclin D1 in the cytoplasm of a subset of MCL cell lines and primary cells. In primary cells, this cytoplasmic distribution was correlated with a more frequent blastoid phenotype. We performed immunoprecipitation assays and mass spectrometry on enriched cytosolic fractions from two cell lines. The cyclin D1 interactome was found to include several factors involved in adhesion, migration and invasion. We found that the accumulation of cyclin D1 in the cytoplasm was associated with higher levels of migration and invasiveness. We also showed that MCL cells with high cytoplasmic levels of cyclin D1 engrafted more rapidly into the bone marrow, spleen, and brain in immunodeficient mice. Both migration and invasion processes, both in vivo and in vitro, were counteracted by the exportin 1 inhibitor KPT-330, which retains cyclin D1 in the nucleus. Our data reveal a role of cytoplasmic cyclin D1 in the control of MCL cell migration and invasion, and as a true operator of MCL pathogenesis.
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Martin-Lopez M, Maeso-Alonso L, Fuertes-Alvarez S, Balboa D, Rodríguez-Cortez V, Weltner J, Diez-Prieto I, Davis A, Wu Y, Otonkoski T, Flores ER, Menéndez P, Marques MM, Marin MC

p73 is required for appropriate BMP-induced mesenchymal-to-epithelial transition during somatic cell reprogramming.

Cell Death Dis 7 Set 2017, 8 (9) e3034. Epub 7 Set 2017
The generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming holds great potential for modeling human diseases. However, the reprogramming process remains very inefficient and a better understanding of its basic biology is required. The mesenchymal-to-epithelial transition (MET) has been recognized as a crucial step for the successful reprogramming of fibroblasts into iPSCs. It has been reported that the p53 tumor suppressor gene acts as a barrier of this process, while its homolog p63 acts as an enabling factor. In this regard, the information concerning the role of the third homolog, p73, during cell reprogramming is limited. Here, we derive total Trp73 knockout mouse embryonic fibroblasts, with or without Trp53, and examine their reprogramming capacity. We show that p73 is required for effective reprogramming by the Yamanaka factors, even in the absence of p53. Lack of p73 affects the early stages of reprogramming, impairing the MET and resulting in altered maturation and stabilization phases. Accordingly, the obtained p73-deficient iPSCs have a defective epithelial phenotype and alterations in the expression of pluripotency markers. We demonstrate that p73 deficiency impairs the MET, at least in part, by hindering BMP pathway activation. We report that p73 is a positive modulator of the BMP circuit, enhancing its activation by DNp73 repression of the Smad6 promoter. Collectively, these findings provide mechanistic insight into the MET process, proposing p73 as an enhancer of MET during cellular reprogramming.
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Sierra MI, Rubio L, Bayón GF, Cobo I, Menendez P, Morales P, Mangas C, Urdinguio RG, Lopez V, Valdes A, Vales G, Marcos R, Torrecillas R, Fernández AF, Fraga MF

DNA methylation changes in human lung epithelia cells exposed to multi-walled carbon nanotubes.

Nanotoxicology Set 2017, 11 (7) 857-870. Epub 13 Set 2017
Humans are increasingly exposed to nanoparticles and, although many of their physiological effects have been described, the molecular mechanisms underlying them are still largely unknown. The present study aimed to determine the possible role of certain epigenetic mechanisms in the cellular response of human lung epithelial cells that are triggered by long-term exposure to titanium dioxide nanoparticles (TiO
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Domingo-Reines J, López-Ornelas A, Montes R, Romero T, Rodriguez-Llamas JL, Lara-Rodarte R, González-Pozas F, Ayllon V, Menendez P, Velasco I, Ramos-Mejia V

Hoxa9 and EGFP reporter expression in human Embryonic Stem Cells (hESC) as useful tools for studying human development.

Stem Cell Res Des 2017, 25 286-290. Epub 5 Ago 2017
HoxA9 is an evolutionarily conserved homeobox gene implicated in embryo development. To study the roles of Hoxa9 during human development we generated a transgenic H9 (hESC) line that overexpresses HoxA9 and the Enhanced Green Fluorescent Protein (EGFP), and a control H9 with a stable expression of the EGFP. The resulting H9-HoxA9-EGFP and H9-EGFP cell lines allow an efficient visualization of hESCs by fluorescent microscopy, quantification by flow cytometry and cell differentiation tracking. Both transgenic cell lines maintained the pluripotent phenotype, the ability to differentiate into all three germ layers and a normal karyotype.
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Varela I, Menendez P, Sanjuan-Pla A

Intratumoral heterogeneity and clonal evolution in blood malignancies and solid tumors.

Oncotarget 12 Set 2017, 8 (39) 66742-66746. Epub 16 Ago 2017
This meeting held at the University of Barcelona in March 2017, brought together scientists and clinicians worldwide to discuss current and future clinico-biological implications of intratumoral heterogeneity (ITH) and subclonal evolution in cancer diagnosis, patient stratification, and treatment resistance in diagnosis, treatment and follow-up. There was consensus that both longitudinal and tumor multi-region studies in matched samples are needed to better understand the dynamics of ITH. The contribution of the epigenome and microenvironment to ITH and subclone evolution remains understudied. It was recommended to combine computational, pathology and imaging tools to study the role of the microenvironment in subclone selection/evolution.
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Navarro-Montero O, Ayllon V, Lamolda M, López-Onieva L, Montes R, Bueno C, Ng E, Guerrero-Carreno X, Romero T, Romero-Moya D, Stanley E, Elefanty A, Ramos-Mejia V, Menendez P, Real PJ

RUNX1c Regulates Hematopoietic Differentiation of Human Pluripotent Stem Cells Possibly in Cooperation with Proinflammatory Signaling.

Stem Cells Nov 2017, 35 (11) 2253-2266. Epub 23 Set 2017
Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essential for hematopoietic stem cell (HSC) emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here, we have used human pluripotent stem cells (hPSCs) as model to study the role of RUNX1 in human embryonic hematopoiesis. Although the three RUNX1 isoforms a, b, and c were induced in CD45+ hematopoietic cells, RUNX1c was the only isoform induced in hematoendothelial progenitors (HEPs)/hemogenic endothelium. Constitutive expression of RUNX1c in human embryonic stem cells enhanced the appearance of HEPs, including hemogenic (CD43+) HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c dramatically reduced the generation of hematopoietic cells from HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced proinflammatory molecular signature, supporting previous studies demonstrating proinflammatory signaling as a regulator of HSC emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hPSCs, possibly in cooperation with proinflammatory signaling. Stem Cells 2017;35:2253-2266.
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Torres-Ruiz R, Rodriguez-Perales S, Bueno C, Menendez P

Modeling mixed-lineage-rearranged leukemia initiation in CD34

Haematologica Set 2017, 102 (9) 1467-1468. Més informació
Bueno C, Menendez P

Human acute leukemia induced pluripotent stem cells: a unique model for investigating disease development and pathogenesis.

Stem Cell Investig 2017, 4 55. Epub 13 Jun 2017Més informació
Gentilella A, Morón-Duran FD, Fuentes P, Zweig-Rocha G, Riaño-Canalias F, Pelletier J, Ruiz M, Turón G, Castaño J, Tauler A, Bueno C, Menéndez P, Kozma SC, Thomas G

Autogenous Control of 5′TOP mRNA Stability by 40S Ribosomes.

Mol. Cell 6 Jul 2017, 67 (1) 55-70.e4. Epub 29 Jun 2017
Ribosomal protein (RP) expression in higher eukaryotes is regulated translationally through the 5′TOP sequence. This mechanism evolved to more rapidly produce RPs on demand in different tissues. Here we show that 40S ribosomes, in a complex with the mRNA binding protein LARP1, selectively stabilize 5′TOP mRNAs, with disruption of this complex leading to induction of the impaired ribosome biogenesis checkpoint (IRBC) and p53 stabilization. The importance of this mechanism is underscored in 5q− syndrome, a macrocytic anemia caused by a large monoallelic deletion, which we found to also encompass the LARP1 gene. Critically, depletion of LARP1 alone in human adult CD34+ bone marrow precursor cells leads to a reduction in 5′TOP mRNAs and the induction of p53. These studies identify a 40S ribosome function independent of those in translation that, with LARP1, mediates the autogenous control of 5′TOP mRNA stability, whose disruption is implicated in the pathophysiology of 5q− syndrome.
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Prieto C, Marschalek R, Kühn A, Bursen A, Bueno C, Menéndez P

The AF4-MLL fusion transiently augments multilineage hematopoietic engraftment but is not sufficient to initiate leukemia in cord blood CD34

Oncotarget 10 Oct 2017, 8 (47) 81936-81941. Epub 26 Jul 2017
The translocation t(4;11)(q21;q23) is the hallmark genetic abnormality associated with infant pro-B acute lymphoblastic leukemia (B-ALL) and has the highest frequency of rearrangement in Mixed-lineage leukemia (MLL) leukemias. Unlike other MLL translocations, MLL-AF4-induced proB-ALL is exceptionally difficult to model in mice/humans. Previous work has investigated the relevance of the reciprocal translocation fusion protein AF4-MLL for t(4;11) leukemia, finding that AF4-MLL is capable of inducing proB-ALL without requirement for MLL-AF4 when expressed in murine hematopoietic stem/progenitor cells (HSPCs). Therefore, AF4-MLL might represent a key genetic lesion contributing to t(4;11)-driven leukemogenesis. Here, we aimed to establish a humanized mouse model by using AF4-MLL to analyze its transformation potential in human cord blood-derived CD34
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Pelkonen O, Terron A, Hernandez AF, Menendez P, Bennekou SH

Chemical exposure and infant leukaemia: development of an adverse outcome pathway (AOP) for aetiology and risk assessment research.

Arch. Toxicol. Ago 2017, 91 (8) 2763-2780. Epub 23 Mai 2017
Infant leukaemia (<1 year old) is a rare disease of an in utero origin at an early phase of foetal development. Rearrangements of the mixed-lineage leukaemia (MLL) gene producing abnormal fusion proteins are the most frequent genetic/molecular findings in infant B cell-acute lymphoblastic leukaemia. In small epidemiological studies, mother/foetus exposures to some chemicals including pesticides have been associated with infant leukaemia; however, the strength of evidence and power of these studies are weak at best. Experimental in vitro or in vivo models do not sufficiently recapitulate the human disease and regulatory toxicology studies are unlikely to capture this kind of hazard. Here, we develop an adverse outcome pathway (AOP) based substantially on an analogous disease-secondary acute leukaemia caused by the topoisomerase II (topo II) poison etoposide-and on cellular and animal models. The hallmark of the AOP is the formation of MLL gene rearrangements via topo II poisoning, leading to fusion genes and ultimately acute leukaemia by global (epi)genetic dysregulation. The AOP condenses molecular, pathological, regulatory and clinical knowledge in a pragmatic, transparent and weight of evidence-based framework. This facilitates the interpretation and integration of epidemiological studies in the process of risk assessment by defining the biologically plausible causative mechanism(s). The AOP identified important gaps in the knowledge relevant to aetiology and risk assessment, including the specific embryonic target cell during the short and spatially restricted period of susceptibility, and the role of (epi)genetic features modifying the initiation and progression of the disease. Furthermore, the suggested AOP informs on a potential Integrated Approach to Testing and Assessment to address the risk caused by environmental chemicals in the future.
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Romero-Moya D, Santos-Ocaña C, Castaño J, Garrabou G, Rodríguez-Gómez JA, Ruiz-Bonilla V, Bueno C, González-Rodriguez P, Giorgetti A, Perdiguero E, Prieto C, Moren-Nuñez C, Fernández-Ayala DJ, Cascajo MV, Velasco I, Canals JM, Montero R, Yubero D, Jou C, López-Barneo J, Cardellach F, Muñoz-Cánoves P, Artuch R, Navas P, Menéndez P

Genetic rescue of Mitochondrial and Skeletal Muscle Impairment in an IPSCs Model of Coenzyme Q10 Deficiency.

Stem Cells 4 Mai 2017, . Epub 4 Mai 2017
Coenzyme Q10 (CoQ10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC), and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a 4-year old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G>C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ10 ], CoQ10 biosynthesis, MRC activity affecting complexes I/II+III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4(ed) -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4(ed) -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. This article is protected by copyright. All rights reserved.
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Torres-Ruiz R, Martinez-Lage M, Martin MC, Garcia A, Bueno C, Castaño J, Ramirez JC, Menendez P, Cigudosa JC, Rodriguez-Perales S

Efficient Recreation of t(11;22) EWSR1-FLI1(+) in Human Stem Cells Using CRISPR/Cas9.

Stem Cell Reports 9 Mai 2017, 8 (5) 1408-1420.
Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma.
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Castaño J, Bueno C, Jiménez-Delgado S, Roca-Ho H, Fraga MF, Fernandez AF, Nakanishi M, Torres-Ruiz R, Rodríguez-Perales S, Menéndez P

Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the "safe harbor" AAVS1 locus.

Stem Cell Res Mai 2017, 21 137-140. Epub 22 Abr 2017
We report the generation-characterization of a fetal liver (FL) B-cell progenitor (BCP)-derived human induced pluripotent stem cell (hiPSC) line CRISPR/Cas9-edited to carry/express a single copy of doxycycline-inducible Cas9 gene in the "safe locus" AAVS1 (iCas9-FL-BCP-hiPSC). Gene-edited iPSCs remained pluripotent after CRISPR/Cas9 genome-edition. Correct genomic integration of a unique copy of Cas9 was confirmed by PCR and Southern blot. Cas9 was robustly and specifically expressed on doxycycline exposure. T7-endonuclease assay demonstrated that iCas9 induces robust gene-edition when gRNAs against hematopoietic transcription factors were tested. This iCas9-FL-BCP-hiPSC will facilitate gene-editing approaches for studies on developmental biology, drug screening and disease modeling.
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Ockleford C, Adriaanse P, Berny P, Brock T, Duquesne S, Grilli S, Hernandez-Jerez AF, Bennekou SH, Klein M, Kuhl T, Laskowski R, Machera K, Pelkonen O, Pieper S, Smith R, Stemmer M, Sundh I, Teodorovic I, Tiktak A, Topping CJ, Wolterink G, Angeli K, Fritsche E, Hernandez-Jerez AF, Leist M, Mantovani A, Menendez P, Pelkonen O, Price A, Viviani B, Chiusolo A, Ruffo F, Terron A, Bennekou SH

Investigation into experimental toxicological properties of plant protection products having a potential link to Parkinson's disease and childhood leukaemia

EFSA J . 2017 Mar 16;15(3):e04691 , .
In 2013, EFSA published a literature review on epidemiological studies linking exposure to pesticides and human health outcome. As a follow up, the EFSA Panel on Plant Protection Products and their residues (PPR Panel) was requested to investigate the plausible involvement of pesticide exposure as a risk factor for Parkinson's disease (PD) and childhood leukaemia (CHL). A systematic literature review on PD and CHL and mode of actions for pesticides was published by EFSA in 2016 and used as background documentation. The Panel used the Adverse Outcome Pathway (AOP) conceptual framework to define the biological plausibility in relation to epidemiological studies by means of identification of specific symptoms of the diseases as AO. The AOP combines multiple information and provides knowledge of biological pathways, highlights species differences and similarities, identifies research needs and supports regulatory decisions. In this context, the AOP approach could help in organising the available experimental knowledge to assess biological plausibility by describing the link between a molecular initiating event (MIE) and the AO through a series of biologically plausible and essential key events (KEs). As the AOP is chemically agnostic, tool chemical compounds were selected to empirically support the response and temporal concordance of the key event relationships (KERs). Three qualitative and one putative AOP were developed by the Panel using the results obtained. The Panel supports the use of the AOP framework to scientifically and transparently explore the biological plausibility of the association between pesticide exposure and human health outcomes, identify data gaps, define a tailored testing strategy and suggests an AOP's informed Integrated Approach for Testing and Assessment (IATA).
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Diaz de la Guardia R, Lopez-Millan B, Lavoie JR, Bueno C, Castaño J, Gómez-Casares M, Vives S, Palomo L, Juan M, Delgado J, Blanco ML, Nomdedeu J, Chaparro A, Fuster JL, Anguita E, Rosu-Myles M, Menéndez P

Detailed Characterization of Mesenchymal Stem/Stromal Cells from a Large Cohort of AML Patients Demonstrates a Definitive Link to Treatment Outcomes.

Stem Cell Reports 6 Jun 2017, 8 (6) 1573-1586. Epub 18 Mai 2017
Bone marrow mesenchymal stem/stromal cells (BM-MSCs) are key components of the hematopoietic niche thought to have a direct role in leukemia pathogenesis. BM-MSCs from patients with acute myeloid leukemia (AML) have been poorly characterized due to disease heterogeneity. We report a functional, genetic, and immunological characterization of BM-MSC cultures from 46 AML patients, stratified by molecular/cytogenetics into low-risk (LR), intermediate-risk (IR), and high-risk (HR) subgroups. Stable MSC cultures were successfully established and characterized from 40 of 46 AML patients irrespective of the risk subgroup. AML-derived BM-MSCs never harbored tumor-specific cytogenetic/molecular alterations present in blasts, but displayed higher clonogenic potential than healthy donor (HD)-derived BM-MSCs. Although HD- and AML-derived BM-MSCs equally provided chemoprotection to AML cells in vitro, AML-derived BM-MSCs were more immunosuppressive/anti-inflammatory, enhanced suppression of lymphocyte proliferation, and diminished secretion of pro-inflammatory cytokines. Multivariate analysis revealed that the level of interleukin-10 produced by AML-derived BM-MSCs as an independent prognostic factor negatively affected overall survival. Collectively our data show that AML-derived BM-MSCs are not tumor related, but display functional differences contributing to therapy resistance and disease evolution.
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Macia A, Widmann TJ, Heras SR, Ayllon V, Sanchez L, Benkaddour-Boumzaouad M, Munoz-Lopez M, Rubio A, Amador-Cubero S, Blanco-Jimenez E, Garcia-Castro J, Menendez P, Ng P, Muotri AR, Goodier JL, Garcia-Perez JL

Engineered LINE-1 retrotransposition in non-dividing human neurons.

Genome Res. 13 Des 2016, . Epub 13 Des 2016
Half of the human genome is made of Transposable Elements (TEs) whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embyogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to Neuronal Precursor Cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature non-dividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.
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Lopez-Millan B, Diaz de la Guardia R, Roca-Ho H, García-Herrero CM, Lavoie JR, Rosu-Myles M, Gonzalez-Rey E, O'Valle F, Criado G, Delgado M, Menendez P

Therapeutic effect of the immunomodulatory drug lenalidomide, but not pomalidomide, in experimental models of rheumatoid arthritis and inflammatory bowel disease.

Exp. Mol. Med. 3 Feb 2017, 49 (2) e290. Epub 3 Feb 2017
Thalidomide is an immunomodulatory drug (IMiD) with proven therapeutic action in several autoimmune/inflammatory diseases; however, its inherent high toxicity has led to the development of more powerful and safer thalidomide analogs, including lenalidomide and pomalidomide. These are new generation IMiDs that exhibit direct antitumor activity as well as anti-inflammatory/immunomodulatory properties, and are FDA-approved for the treatment of several hematological malignances. Here we investigated the potential therapeutic effects of lenalidomide and pomalidomide in several experimental murine models of autoimmune/inflammatory diseases: 2,4,6-trinitrobenzene sulfonic acid- and dextran sulfate sodium-induced inflammatory bowel disease and type II collagen-induced arthritis. Lenalidomide displayed a strong therapeutic effect in all these models of autoimmune/inflammatory diseases, while the effect of pomalidomide was less pronounced. In vitro experiments confirmed the immunosuppressive effect of both IMiDs on the proliferative response of stimulated human lymphocytes and on the balance of secreted cytokines toward an anti-inflammatory profile. We conclude that lenalidomide may offer a therapeutic opportunity against autoimmune/inflammatory diseases.
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Giorgetti A, Castaño J, Bueno C, Diaz de la Guardia R, Delgado M, Bigas A, Espinosa L, Menendez P

Proinflammatory signals are insufficient to drive definitive hematopoietic specification of human HSCs in vitro.

Exp. Hematol. 28 Set 2016, . Epub 28 Set 2016
Recent studies in zebrafish and mice have revealed that proinflammatory signaling is a positive regulator of definitive hematopoietic development. Whether proinflammatory signaling also regulates human hematopoietic specification remains unknown. Here, we explored the impact of the proinflammatory cytokines tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), and interleukin-1β (IL1β) on in vitro hematopoietic differentiation using human pluripotent stem cells. Gene expression analysis and enzyme-linked immunosorbent assay revealed the absence of a proinflammatory signature during hematopoietic development of human pluripotent stem cells. Functionally, the emergence of hemogenic endothelial progenitors (CD31(+)CD34(+)CD45(-) or CD34(+)CD43(-)CD73(-)) and hematopoietic cells (CD43(+)CD45(+)) was not affected by treatment with increasing doses of TNFα, IFNγ, and IL1β irrespective of the developmental window or the differentiation protocol used (embryoid body or OP9 co-culture based). Similarly, knockdown of endogenous NF-kB signaling had no impact on hematopoietic differentiation of human pluripotent stem cells. This study serves as a demonstration that TNFα, IFNγ, and IL1β signals do not improve hematopoietic differentiation of human pluripotent stem cells using current protocols and suggests that proinflammatory signaling is insufficient to drive definitive hematopoietic specification of human hematopoietic stem cells in vitro.
Més informació
Matas-Céspedes A, Vidal-Crespo A, Rodriguez V, Villamor N, Delgado J, Giné E, Roca-Ho H, Menéndez P, Campo E, López-Guillermo A, Colomer D, Roué G, Wiestner A, Parren PW, Doshi P, Lammerts-van Bueren JJ, Perez-Galan P

The human CD38 monoclonal antibody daratumumab shows anti-tumor activity and hampers leukemia-microenvironment interactions in chronic lymphocytic leukemia.

Clin. Cancer Res. 16 Set 2016, . Epub 16 Set 2016
To establish a proof-of-concept for the efficacy of the anti-CD38 antibody daratumumab in the poor prognosis CD38+ CLL subtype.
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Current projects

Genomic, cellular and developmental reconstruction of infant MLL-AF4+ Acute Lymphoblastic Leukemia (INFANTLEUKEMIA)

Responsable:Pablo Menéndez
Data d'inici:01/01/2016
Data de finalització:30/06/2021

LEukaemia GENe Discovery by data sharing, mining and collaboration

Responsable:Pablo Menéndez
Data d'inici:26/10/2017
Data de finalització:25/10/2021

Redirecting CAR T-Cells to bone marrow: IMPROVED

Responsable:Pablo Menéndez
Data d'inici:01/10/2019
Data de finalització:30/09/2021

Recreació funcional de LA-B t (4; 11) en cèl·lules mare hematopoètiques en diferents estadis del desenvolupament

Responsable:Raúl Torres
Data d'inici:01/12/2017
Data de finalització:30/11/2021

ID-VITRORED: Obtención de Hematíes in vitro a partir de iPSCs de donantes con fenotipos eritrocitarios seleccionados y optimizados mediante edición Genómica, como alternativa a los paneles de hematíes actuales

Responsable:Pablo Menéndez
Data d'inici:01/09/2018
Data de finalització:30/06/2022

Childhood Leukemia: Overcoming distance between South America and Europe Regions (CLOSER)

Responsable:Pablo Menéndez
Data d'inici:01/01/2019
Data de finalització:31/12/2023

Recreación del nicho embrionario para la producción de células madre hematopoyéticas y sus derivados en gastruloides humanos.

Responsable:Clara Bueno
Data d'inici:01/12/2021
Data de finalització:30/11/2024

Novel and innovative therapeutic strategies for patients with childhood B acute lymphoblastic leukemia harboring MLL rearrangements

Responsable:Clara Bueno
Data d'inici:26/07/2022
Data de finalització:25/07/2024

TIM3, una nueva y prometedora diana inmunoterapéutica en leucemia linfoblástica aguda B de novo y en recaída (TIMBALL)

Responsable:Pablo Menéndez
Data d'inici:01/06/2020
Data de finalització:31/05/2023

Previous projects

Célula DIANA y eventos oncogénicos secundarios en la leucemia linfoblástica aguda del lactante con reordenamiento MLL-AF4

Responsable:Pablo Menéndez
Data d'inici:01/10/2014
Data de finalització:31/12/2016

Embryonic or fetal origin of the target cell for MLL-AF4 transformation in infant acute lymphoblastic leukemia

Responsable:Pablo Menéndez
Data d'inici:01/01/2015
Data de finalització:31/12/2016

Reconstrucció genòmica i cel·lular de la lecuemia linfblástica aguda de l'lactant amb reordenament MLLAF4.

Responsable:Clara Bueno
Data d'inici:01/09/2015
Data de finalització:31/08/2019

Project ERANET

Responsable:Pablo Menéndez
Data d'inici:01/01/2013
Data de finalització:31/03/2016

Project AECC

Responsable:Pablo Menéndez
Codi:AECC Cancer infantil 0023
Data d'inici:01/09/2013
Data de finalització:30/09/2015

Pro-inflammatory bone marrow stroma in Acute Myeloid Leukemia: implication in the onset, evolution and drug resistance (Co-leader, Michael Rosu-Myles, Ottawa)

Responsable:Pablo Menéndez
Data d'inici:01/10/2014
Data de finalització:31/12/2019

Fundación Sandra Ibarra para cáncer infantil 2013

Responsable:Pablo Menéndez
Data d'inici:01/01/2014
Data de finalització:31/12/2014

Grupo SGR330 (Support Research Group)

Responsable:Pablo Menéndez
Data d'inici:01/11/2014
Data de finalització:31/12/2016

Reconstrucción genómica y celular de la leucemia linfoblástica aguda del lactante con reordenamiento MLLAF4

Responsable:Clara Bueno
Data d'inici:01/11/2014
Data de finalització:31/08/2019

Career Integration Grant (CIG) European Commission

Responsable:Alejandra Sanjuan
Data d'inici:01/01/2014
Data de finalització:31/12/2017

Inmunoterapia adoptiva con células T CAR-NG2 para la leucemia aguda con reordenamiento MLL

Responsable:Pablo Menéndez
Data d'inici:01/01/2017
Data de finalització:31/12/2020

Inmunoterapia adoptiva con células T CAR-NG2 para la leucemia aguda con reordenamiento MLL.

Responsable:Clara Bueno
Data d'inici:01/01/2017
Data de finalització:31/12/2017

Innovative Therapeutic Strategies for Mixed Lineage Leukemia-rearranged B-cell Acute Lymphoblastic Leukemia (InTheMLLrBALL)

Responsable:Samanta Romina Zanetti
Data d'inici:01/04/2018
Data de finalització:31/03/2020

Escalado del suministro y estudios de eficacia del indolocarbazol EC-70124 en modelos animales predictivos. (INDOLKIN)

Responsable:Pablo Menéndez
Data d'inici:01/01/2016
Data de finalització:31/12/2019

Grupo SGR330 (Support Research Group 2018)

Responsable:Pablo Menéndez
Codi:2017 SGR 221
Data d'inici:01/01/2017
Data de finalització:31/12/2019

Inmunoterapia celular adoptiva con CAR CD5 "síngular o dual" para tratamiento de LLA-T pediátrica e infecciones fúngicas post-transplante alogénico de progenitores hematopoiéticos

Responsable:Clara Bueno
Data d'inici:01/01/2018
Data de finalització:31/12/2020

Regeneració hematopoetica a partir de célul·les mare pluripotents.

Responsable:Pablo Menéndez
Data d'inici:01/04/2017
Data de finalització:31/12/2019

Acute Myeloid Leukemia Leukemic Initiating Cells: Contribution of hypoxia/HIF pathway to chemoresistance and relapse (HifLICs)

Responsable:Talia Velasco
Data d'inici:01/01/2019
Data de finalització:31/12/2019

Therapeutic immunotherapy targeting NG2 and CD22 antigens for MLL-rearranged and MLL-germline B-cell Acute Lymphoblastic Leukemia (IT4B-ALL)

Responsable:Pablo Menéndez
Data d'inici:01/01/2019
Data de finalització:30/06/2020

Simulación genómica y celular de la leucemia linfoblástica aguda del lactante con reordenamiento MLLAF4.

Responsable:Clara Bueno
Data d'inici:01/01/2015
Data de finalització:31/12/2017

Towards a clinical translation of the CD1a-directed CAR for relapse/refractory cortical T-cell Acute Lymphoblastic Leukemia and Langerhans Cell Histioscytosis: feasibility, efficacy and safety

Responsable:Pablo Menéndez
Data d'inici:01/06/2019
Data de finalització:31/05/2021