Lineage-specific function of Engrailed-2 in the progression of chronic myelogenous leukemia to T-cell blast crisis

In hematopoietic malignancies, oncogenic alterations interfere with cellular differentiation and lead to tumoral development. Identification of the proteins regulating differentiation is essential to understand how they are altered in malignancies. Chronic myelogenous leukemia (CML) is a biphasic disease initiated by an alteration taking place in hematopoietic stem cells. CML progresses to a blast crisis (BC) due to a secondary differentiation block in any of the hematopoietic lineages. However, the molecular mechanisms of CML evolution to T-cell BC remain unclear. Here, we have profiled the changes in DNA methylation patterns in human samples from BC-CML, in order to identify genes whose expression is epigenetically silenced during progression to T-cell lineage-specific BC. We have found that the CpG-island of the ENGRAILED-2 (EN2) gene becomes methylated in this progression. Afterwards, we demonstrate that En2 is expressed during T-cell development in mice and humans. Finally, we further show that genetic deletion of En2 in a CML transgenic mouse model induces a T-cell lineage BC that recapitulates human disease. These results identify En2 as a new regulator of T-cell differentiation whose disruption induces a malignant T-cell fate in CML progression, and validate the strategy used to identify new developmental regulators of hematopoiesis.Research at C.C.’s lab was partially supported by FEDER, Fondo de Investigaciones Sanitarias, CSIC P.I.E., Junta de Castilla y León, and from an institutional grant from the Fundación Ramón Areces. Research in ISG group is partially supported by FEDER and by MICINN (SAF2012-32810), by MEC OncoBIO Consolider-Ingenio 2010 (Ref. CSD2007-0017), by NIH grant (R01 CA109335-04A1), the ARIMMORA project (FP7-ENV-2011, European Union Seventh Framework Program), by Junta de Castilla y León (BIO/SA06/13) and by “Proyecto en red de investigación en células madre tumorales” supported by Obra Social Kutxa y Consejería de Sanidad de la Junta de Castilla y Leon. C.V.D.’s research is supported by Junta de Castilla y León (proyecto de investigación en biomedicina SAN/39/2010). J.A.M.C.’s research is supported by the Instituto de Salud Carlos III (ISCIII), grants FIS-PI12/00202 and RTICC-RD12/0036/0063. All Spanish funding is co-sponsored by the European Union FEDER program. I.S.G. is an API lab of the EuroSyStem project and a partner of DECIDE European network. F.A.-J. and E.C.S. were supported by Spanish Ministry of Science and Innovation fellowships. E.C.-S. was a “Residencia de Estudiantes” Fellow. A.T.N. was the recipient of a “Beca de Postgrado de la Fundación Ramón Areces/UAM.”Peer Reviewe

Generation of glucocorticoid-producing cells derived from human pluripotent stem cells

Adrenal insufficiency is a life-threatening condition resulting from the inability to produce adrenal hormones in a dose- and time-dependent manner. Establishing a cell-based therapy would provide a physiologically responsive approach for the treatment of this condition. We report the generation of large numbers of human-induced steroidogenic cells (hiSCs) from human pluripotent stem cells (hPSCs). Directed differentiation of hPSCs into hiSCs recapitulates the initial stages of human adrenal development. Following expression of steroidogenic factor 1, activation of protein kinase A signaling drives a steroidogenic gene expression profile most comparable to human fetal adrenal cells, and leads to dynamic secretion of steroid hormones, in vitro. Moreover, expression of the adrenocorticotrophic hormone (ACTH) receptor/co-receptor (MC2R/MRAP) results in dose-dependent ACTH responsiveness. This protocol recapitulates adrenal insufficiency resulting from loss-of-function mutations in AAAS, which cause the enigmatic triple A syndrome. Our differentiation protocol generates sufficient numbers of hiSCs for cell-based therapy and offers a platform to study disorders causing adrenal insufficiency

Generation of glucocorticoid-producing cells derived from human pluripotent stem cells.

Adrenal insufficiency is a life-threatening condition resulting from the inability to produce adrenal hormones in a dose- and time-dependent manner. Establishing a cell-based therapy would provide a physiologically responsive approach for the treatment of this condition. We report the generation of large numbers of human-induced steroidogenic cells (hiSCs) from human pluripotent stem cells (hPSCs). Directed differentiation of hPSCs into hiSCs recapitulates the initial stages of human adrenal development. Following expression of steroidogenic factor 1, activation of protein kinase A signaling drives a steroidogenic gene expression profile most comparable to human fetal adrenal cells, and leads to dynamic secretion of steroid hormones, in vitro. Moreover, expression of the adrenocorticotrophic hormone (ACTH) receptor/co-receptor (MC2R/MRAP) results in dose-dependent ACTH responsiveness. This protocol recapitulates adrenal insufficiency resulting from loss-of-function mutations in AAAS, which cause the enigmatic triple A syndrome. Our differentiation protocol generates sufficient numbers of hiSCs for cell-based therapy and offers a platform to study disorders causing adrenal insufficiency

FUS-DDIT3 Prevents the Development of Adipocytic Precursors in Liposarcoma by Repressing PPARγ and C/EBPα and Activating eIF4E

FUS-DDIT3 is a chimeric protein generated by the most common chromosomal translocation t(12;16)(q13;p11) linked to liposarcomas, which are characterized by the accumulation of early adipocytic precursors. Current studies indicate that FUS-DDIT3- liposarcoma develops from uncommitted progenitors. However, the precise mechanism whereby FUS-DDIT3 contributes to the differentiation arrest remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Here we have characterized the adipocyte regulatory protein network in liposarcomas of FUS-DITT3 transgenic mice and showed that PPARgamma2 and C/EBPalpha expression was altered. Consistent with in vivo data, FUS-DDIT3 MEFs and human liposarcoma cell lines showed a similar downregulation of both PPARgamma2 and C/EBPalpha expression. Complementation studies with PPARgamma but not C/EBPalpha rescued the differentiation block in committed adipocytic precursors expressing FUS-DDIT3. Our results further show that FUS-DDIT3 interferes with the control of initiation of translation by upregulation of the eukaryotic translation initiation factors eIF2 and eIF4E both in FUS-DDIT3 mice and human liposarcomas cell lines, explaining the shift towards the truncated p30 isoform of C/EBPalpha in liposarcomas. Suppression of the FUS-DDIT3 transgene did rescue this adipocyte differentiation block. Moreover, eIF4E was also strongly upregulated in normal adipose tissue of FUS-DDIT3 transgenic mice, suggesting that overexpression of eIF4E may be a primary event in the initiation of liposarcomas. Reporter assays showed FUS-DDIT3 is involved in the upregulation of eIF4E in liposarcomas and that both domains of the fusion protein are required for affecting eIF4E expression. CONCLUSIONS/SIGNIFICANCE: Taken together, this study provides evidence of the molecular mechanisms involve in the disruption of normal adipocyte differentiation program in liposarcoma harbouring the chimeric gene FUS-DDIT3.Research in ISG group is supported partially by FEDER and by MEC (SAF2006-03726), Junta de Castilla y León (CSI03A05), FIS (PI050087, PI050116), Fundación de Investigación MMA, Federación de Cajas de Ahorro Castilla y León (I Convocatoria de Ayudas para Proyectos de Investigación Biosanitaria con Células Madre), CDTEAM project (CENIT-Ingenio 2010) and MEC Consolider-Ingenio 2010 (Ref. CSD2007-0017).Research in ISG group is supported partially by FEDER and by MEC (SAF2006-03726 and PETRI N° 95-0913.OP), Junta de Castilla y León (CSI03A05), FIS (PI050087, PI050116), Fundación de Investigación MMA, Federación de Cajas de Ahorro Castilla y León (I Convocatoria de Ayudas para Proyectos de Investigación Biosanitaria con Células Madre), CDTEAM project (CENIT-Ingenio 2010) and MEC Consolider-Ingenio 2010 (Ref. CSD2007-0017). MSM is supported by the Ramon y Cajal Scientific Spanish Program, Fondo Investigacion Sanitaria (FIS PI04-1271), Junta de Castilla y León (SA085A06) and Fundación Manuel Solorzano, University of Salamanca.Peer reviewe

Identificación y caracterización funcional de nuevos factores de transcripción implicados en la progresión leucémica y la hematopoyesis

[ES] Esta tesis tiene como objetivos: - Identificación de aquellos genes cuyas islas CpG se metilan en muestras de DNA procedentes de pacientes con crisis blásticas, en comparación con muestras procedenntes de fases crónicas. Analizar el estado de metilación de los genes identificados en muestras humanas de diferentes orígenes. Este análisis, realizado a partir de muestras de leucemias "in vivo" permitirá establecer la existencia de un modelo molecular de progresion hacia la crisis blástica en el que la hipermetilación de promotores de genes esenciales para la diferenciación celular juega un papel fundamental en el desarrollo de la patología leucémica. Al mismo tiempo posibilitará la identificación de nuevos genes implicados en el desarrollo hematopoyético (y, en condiciones patológicas, en desarrollo tumoral) o asignar un nuevo papel en hematopoyesis a genes previamente conocidos por su función en otros sistemas. - Una vez probado que existe metilación diferencia de genes en la progresión leucémica, para el/los genes de mayor interés potencial se realizará la validación funcional del papel que dichos genes desempeñan en la hematopoyesis mediante el estudio inicial del patrón de expresión de los genes identificados en la hematopoyesis normal y el análisis de ratones knockout como modelos de pérdida de función y transgénicos como modelos de ganancia de función.[EN] This thesis aims to: - Identification of those genes whose CpG islands are methylated in DNA samples from patients with blast crisis compared with chronic phase procedenntes samples. To analyze the methylation status of the genes identified in human samples of different origins. This analysis, performed on samples of leukemia "in vivo" will establish the existence of a molecular model of progression to blast crisis in which the promoter hypermethylation of genes essential for cell differentiation plays a fundamental role in the development of the leukemic disease. At the same time enable the identification of new genes involved in hematopoietic development (and, in pathological conditions in tumor development) or assign a new role in hematopoiesis genes previously known for its role in other systems. - Once there is evidence that gene methylation difference in the progression of leukemia, for the / gene of interest will be made to validate potential functional role that these genes play in hematopoiesis by the initial study of the expression pattern of genes identified in normal hematopoiesis and analysis of knockout mice as models of transgenic loss of function and gain as role models

Physiological cellular reprogramming and cancer

The traditional approaches to cancer research and therapy have been primarily focused in the aspect of aberrant, uncontrolled, proliferation. Although this is clearly a very important issue, however, the emphasis on this characteristic has led to a relative neglect of an essential aspect of cancer biology: the alteration of normal differentiation processes. The oncogenic alterations that arise in an otherwise healthy cell lead to a whole reprogramming of the normal cellular fate and open a new pathologic developmental program. In this way cancer, reprogramming and cellular plasticity are tightly intertwined, since only some cells posses the necessary plasticity so as to allow the tumoral reprogramming to take place, and only some oncogenes have, in the right cellular context, the required tumoral reprogramming capacity. Research in the field of induced pluripotency is shedding a new light on the molecular mechanisms of tumor initiation and differentiation. In this review we discuss the latest findings in the area of cellular reprogramming and their implications from the point of view of tumor biology. © 2010 Elsevier Ltd.Research in the group is supported partially by FEDER (Fondo de Investigaciones Sanitarias PI080164), Proyectos Intramurales Especiales (CSIC), Fundación Mutua Madrileña and Junta de Castilla y León (SA060A09 and Proyecto Biomedicina 2009–2010). F.A.J. is the recipient of an FPU fellowship from Ministerio de Ciencia e Innovación.Peer Reviewe

The age of the target cell affects B-cell leukaemia malignancy

This is an open‐access article distributed under the terms of the Creative Commons Attribution License.-- et al.The incidence, malignancy and treatment resistance of many types of human B-cell leukaemias (B-ALL) are directly related to patient age. A major obstacle to elucidate the contribution of age to the development and evolution of leukaemias is the lack of appropriate mouse models where precise control of the timing of oncogene expression is possible. Here we present proof-of-principle experiments showing how a conditional transgenic mouse model of BCR-ABLp190-driven B-ALL offers the opportunity to test the hypothesis that the age of the leukemic cells-of-origin of B-ALL influences B-ALL malignancy. B-ALLs generated from 12- and 20-month-old progenitors gave rise to a more invasive B-ALL than the one developed from 4-month old precursors. This was evidenced by survival analysis revealing the increased malignancy of B-ALLs generated from 20 or 12-month-old transformed progenitors compared with the 4-month equivalents (median survival of 88 days versus 50.5 and 33 days, respectively). Our study shows that the age of target cells at the time of transformation affects B-ALL malignancy.Research in ISG group was partially supported by FEDER and by MICINN (SAF2009-08803 to ISG), by Junta de Castilla y León (Proyecto Biomedicina 2009- 2010 to ISG), by MEC OncoBIO Consolider-Ingenio 2010 (Ref. CSD2007-0017), by NIH grant (R01 CA109335-04A1), by Sandra Ibarra Foundation, and by Group of Excellence Grant (GR15) from Junta de Castilla y Leon. CVD research is supported by Junta de Castilla y León (proyecto de investigación en biomedicina SAN/39/2010). ISG is an API lab of the EuroSyStem project. Research at C.C.´s lab was partially supported by FEDER, Fondo de Investigaciones Sanitarias (PI080164), CSIC P.I.E., Junta de Castilla y León (SA060A09 and proyecto Biomedicina 2009-2010) and from an institutional grant from the Fundación Ramón Areces. F.A-J. was supported by an FPU fellowship from the Spanish Ministerio de Ciencia e Innovacion.Peer Reviewe

Stem-cell driven cancer: "Hands-off" regulation of cancer development

This is an open access article.A cancer dogma states that inactivation of oncogene(s) can cause cancer remission, implying that oncogenes are the Achilles' heel of cancers. This current "hands on" model of cancer has kept oncogenes firmly in focus as therapeutic targets and is in agreement with the fact that in human cancers all cancerous cells, with independence of the cellular heterogeneity existing within the tumor, carry the same oncogenic genetic lesions. This rule has now been broken in a study of the effect of the BCR-ABL oncogene in cancer development in a mouse model in which oncogene expression is restricted to the stem cell compartment. BCR-ABL is linked to chronic myeloid leukemia (CML) disease in humans, and this study shows that by limiting the oncogene expression to Sca1+ cells CML arises, indicating that maintenance of oncogene expression is not critical for the generation of differentiated tumor cells and showing a "hands off" role for BCR-ABL in regulating cancer formation. Here we provide an update on the use of this system for modeling human cancer and its potential application for therapeutic targeting of cancer stem cells (CSCs) and the hands-off function of oncogenes. ©2009 Landes Bioscience.Research in our group is supported partially by FEDER and by MEC (SAF2006-03726), Junta de Castilla y León (CSI13A08), FIS (PI050087), CDTEAM project (CENIT-Ingenio 2010) and MEC OncoBIO Consolider-Ingenio 2010 (Ref. CSD2007-0017) and by Group of Excellence Grant (GR15) from Junta de Castilla y Leon. C.C. is a Spanish “Ramón y Cajal” investigator from the Spanish Ministerio de Educación y Ciencia. Research at C.C.’s lab is partially supported by Fondo de Investigaciones Sanitarias (PI04/0261; PI080164), Junta de Castilla y León (SA087A06) and Fundación de Investigación Médica MM.Peer Reviewe