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Supplementary Figure 8 from E2F1 Expression Is Deregulated and Plays an Oncogenic Role in Sporadic Burkitt's Lymphoma

Current treatments of sporadic Burkitt's lymphoma (sBL) are associated with severe toxicities. A better understanding of sBL formation would facilitate development of less toxic therapies. The etiology of sBL remains, however, largely unknown, C-MYC up-regulation being the only lesion known to occur in all sBL cases. Several studies examining the role of C-MYC in the pathogenesis of BL have concluded that C-MYC translocation is not the only critical event and that additional unidentified factors are expected to be involved in the formation of this tumor. We herein report that a gene distinct from C-MYC, E2F1, is involved in the formation of all or most sBL tumors. We found that E2F1 is highly expressed in Burkitt's lymphoma cell lines and sBL lymphoma specimens. Our data indicate that its elevated expression is not merely the consequence of the presence of more cycling cells in this tumor relative to other cell lines or to other neoplasias. In fact, we show that reduction of its expression in sBL cells inhibits tumor formation and decreases their proliferation rate. We also provide data suggesting that E2F1 collaborates with C-MYC in sBL formation. E2F1 expression down-regulation did not affect, however, the proliferation of human primary diploid fibroblasts. Because E2F1 is not needed for cell proliferation of normal cells, our results reveal E2F1 as a promising therapeutic target for sBL. [Cancer Res 2009;69(9):4052–8]

Supplementary Figure 6 from E2F1 Expression Is Deregulated and Plays an Oncogenic Role in Sporadic Burkitt's Lymphoma

Supplementary Figure 7 from E2F1 Expression Is Deregulated and Plays an Oncogenic Role in Sporadic Burkitt's Lymphoma

Supplementary Figure 9 from E2F1 Expression Is Deregulated and Plays an Oncogenic Role in Sporadic Burkitt's Lymphoma

Supplementary Figures 1-5 from E2F1 Expression Is Deregulated and Plays an Oncogenic Role in Sporadic Burkitt's Lymphoma

This study concerns the impact of microphysics on the HARMONIE-AROME NWP model. In particular, the representation of cloud droplets in the single-moment bulk microphysics scheme is examined in relation to fog forecasting. We focus on the shape parameters of the cloud droplet size distribution and recent changes to the representation of the cloud droplet number concentration (CDNC). Two configurations of CDNC are considered: a profile that varies with height and a constant one. These aspects are examined together since few studies have considered their combined impact during fog situations. We present a set of six experiments performed for two non-idealised three-dimensional case studies over the Iberian Peninsula and the North Sea. One case displays both low clouds and fog, and the other shows a persistent fog field above sea. The experiments highlight the importance of the considered parameters that affect droplet sedimentation, which plays a key role in modelled fog. We show that none of the considered configurations can simultaneously represent all aspects of both cases. Hence, continued efforts are needed to introduce relationships between the governing parameters and the relevant atmospheric conditions.

The continuous relationship between blood pressure (BP) and cardiovascular events makes the distinction between elevated BP and hypertension based on arbitrary cut-off values for BP. Even mild BP elevations manifesting as high-normal BP have been associated with cardiovascular risk. We hypothesize that persistent elevated BP increases atherosclerotic plaque development. To evaluate this causal link, we developed a new mouse model of elevated BP based on adeno-associated virus (AAV) gene transfer. We constructed AAV vectors to support transfer of the hRenin and hAngiotensinogen genes. A single injection of AAV-Ren/Ang (1011 total viral particles) induced sustained systolic BP increase (130 ± 20 mmHg, vs. 110 ± 15 mmHg in controls, p = 0.05). In ApoE−/− mice, AAV-induced mild BP elevation caused larger atherosclerotic lesions evaluated by histology (10-fold increase vs. normotensive controls). In this preclinical model, atheroma plaques development was attenuated by BP control with a calcium channel blocker, indicating that a small increase in BP within a physiological range has a substantial impact on plaque development in a preclinical model of atherosclerosis. These data support that non-optimal BP represents a risk for atherosclerosis development. Earlier intervention in elevated BP may prevent or delay morbidity and mortality associated with atherosclerosis.

Mutations in desmosomal Plakophilin-2 (PKP2) are the most prevalent drivers of arrhythmogenic cardiomyopathy (ACM) and a common cause of sudden cardiac death in young athletes. However, partner proteins that elucidate PKP2 cellular mechanism to understand cardiac dysfunction in ACM are mostly unknown. Here we identify the actin-based motor proteins Myh9 and Myh10 as key PKP2 interactors, and demonstrate that the expression of the ACM-related PKP2 mutant R735X alters actin fiber organization and cell mechanical stiffness. We also show that SARS-CoV-2 Nsp1 protein acts similarly to this known pathogenic R735X mutant, altering the actomyosin component distribution on cardiac cells. Our data reveal that the viral Nsp1 hijacks PKP2 into the cytoplasm and mimics the effect of delocalized R735X mutant. These results demonstrate that cytoplasmic PKP2, wildtype or mutant, induces the collapse of the actomyosin network, since shRNA-PKP2 knockdown maintains the cell structure, validating a critical role of PKP2 localization in the regulation of actomyosin architecture. The fact that Nsp1 and PKP2 mutant R735X share similar phenotypes also suggests that direct SARS-CoV-2 heart infection could induce a transient ACM-like disease in COVID-19 patients, which may contribute to right ventricle dysfunction, observed in patients with poor survival prognosis., The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). This study was supported by MCIU grant BFU2016-75144-R. The study was also partially supported by the “Ayudas a la Investigación Cátedra Real Madrid-Universidad Europea” (2017/RM01). C.M-L. and S.S. hold MCIU predoctoral contracts BES-2017- 079715, and BES-2017-079707 respectively. RG acknowledges funding from the European Research Council under grant ERC-AG-340177 (3DNanoMech) and from the MCIU under grant MAT2016-76507-R.

Ponencia presentada en: VI Simposio Nacional de Predicción, celebrado en los servicios centrales de AEMET, en Madrid, del 17 al 19 de septiembre de 2018. La convección es uno de los fenómenos meteorológicos más difíciles de predecir. Los modelos numéricos no hidrostáticos que se integran a escalas convectivas o convection-permitting han supuesto una mejora significativa en la simulación de la convección comparados con los modelos de escala sinóptica, que necesitan parametrizar la convección profunda. Se estudia el comportamiento de los modelos HARMONIE-AROME y del IFS del Centro Europeo de Predicción a Medio Plazo (ECMWF) utilizando métodos objetivos como la verificación puntual y la verificación espacial, así como métodos subjetivos. Se ve que los modelos convection-permitting como HARMONIE-AROME reproducen mucho mejor el ciclo diurno de la precipitación que los modelos que parametrizan la convección profunda como el del Centro Europeo. En promedio, el ciclo 40 obtiene mejores resultados que el ciclo 38 tanto con la verificación puntual como con la verificación espacial. El ciclo 38 tiende a producir más actividad convectiva y también a producir más falsas alarmas. Otro resultado interesante es que la resolución efectiva de HARMONIE para la precipitación parece estar en torno a los 50 km

Hematopoietic stem and progenitor cells (HSPCs) and leukocytes circulate between the bone marrow (BM) and peripheral blood following circadian oscillations. Autonomic sympathetic noradrenergic signals have been shown to regulate HSPC and leukocyte trafficking, but the role of the cholinergic branch has remained unexplored. We have investigated the role of the cholinergic nervous system in the regulation of day/night traffic of HSPCs and leukocytes in mice. We show here that the autonomic cholinergic nervous system (including parasympathetic and sympathetic) dually regulates daily migration of HSPCs and leukocytes. At night, central parasympathetic cholinergic signals dampen sympathetic noradrenergic tone and decrease BM egress of HSPCs and leukocytes. However, during the daytime, derepressed sympathetic noradrenergic activity causes predominant BM egress of HSPCs and leukocytes via β3–adrenergic receptor. This egress is locally supported by light-triggered sympathetic cholinergic activity, which inhibits BM vascular cell adhesion and homing. In summary, central (parasympathetic) and local (sympathetic) cholinergic signals regulate day/night oscillations of circulating HSPCs and leukocytes. This study shows how both branches of the autonomic nervous system cooperate to orchestrate daily traffic of HSPCs and leukocytes., This work was supported by core support grants from the Wellcome Trust and the Medical Research Council (MRC) to the Cambridge Stem Cell Institute, the Spanish Ministry of Economy and Competitiveness (SAF-2011-30308), the Pro-CNIC Foundation, Severo Ochoa Center of Excellence award SEV-2015-0505 to CNIC, Ramón y Cajal Program grant RYC-2009-04703, Marie Curie Career Integration grant FP7-PEOPLE-2011-RG-294096, ConSEPOC-Comunidad de Madrid S2010/BMD-2542, Red TerCel (Instituto de Salud Carlos III (ISCIII)-Spanish Cell Therapy Network), National Health Institute Blood and Transplant (United Kingdom), Horizon2020 grant ERC-2014-CoG-64765, and a Cancer Research UK Programme Foundation Award to S.M.-F., who was also supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute. A.G.-G. received fellowships from the Ramón Areces and LaCaixa Foundations. C.K. was supported by Marie Curie Career Integration grant H2020-MSCA-IF-2015-708411.

Ponencia presentada en: XXXV Jornadas Científicas de la AME y el XIX Encuentro Hispano Luso de Meteorología celebrado en León, del 5 al 7 de marzo de 2018. Los modelos numéricos de la atmósfera son muy útiles para la predicción del tiempo a corto y medio plazo. La comunidad científica dentro del campo de la meteorología destina gran cantidad de recursos humanos y técnicos al desarrollo de los modelos de predicción numérica con el objetivo de lograr perfeccionar el pronóstico. Estos modelos de predicción constituyen una representación físico-matemática de la atmósfera, complementados con el uso de las observaciones existentes en el momento de iniciar el pronóstico del estado futuro de la atmósfera. Hoy día, las salidas de los modelos de predicción numérica del tiempo son utilizadas diariamente por pare de los predictores en los servicios meteorológicos. Por ello, es necesario conocer las características físicas y dinámicas en las que se basan estos modelos, para así, poder interpretar correctamente sus resultados. Desde junio de 2017, HARMONIE-AROME es el nuevo modelo operativo de alta resolución de AEMET. Es un modelo No-Hidrostático que resuelve la convección profunda explícitamente y que se integra con una resolución horizontal de 2.5 km. Se presenta en estas jornadas de la AME las principales características del nuevo modelo.

Myeloproliferative neoplasms (MPNs) are diseases caused by mutations in the haematopoietic stem cell (HSC) compartment. Most MPN patients have a common acquired mutation of Janus kinase 2 (JAK2) gene in HSCs that renders this kinase constitutively active, leading to uncontrolled cell expansion. The bone marrow microenvironment might contribute to the clinical outcomes of this common event. We previously showed that bone marrow nestin(+) mesenchymal stem cells (MSCs) innervated by sympathetic nerve fibres regulate normal HSCs. Here we demonstrate that abrogation of this regulatory circuit is essential for MPN pathogenesis. Sympathetic nerve fibres, supporting Schwann cells and nestin(+) MSCs are consistently reduced in the bone marrow of MPN patients and mice expressing the human JAK2(V617F) mutation in HSCs. Unexpectedly, MSC reduction is not due to differentiation but is caused by bone marrow neural damage and Schwann cell death triggered by interleukin-1β produced by mutant HSCs. In turn, in vivo depletion of nestin(+) cells or their production of CXCL12 expanded mutant HSC number and accelerated MPN progression. In contrast, administration of neuroprotective or sympathomimetic drugs prevented mutant HSC expansion. Treatment with β3-adrenergic agonists that restored the sympathetic regulation of nestin(+) MSCs prevented the loss of these cells and blocked MPN progression by indirectly reducing the number of leukaemic stem cells. Our results demonstrate that mutant-HSC-driven niche damage critically contributes to disease manifestation in MPN and identify niche-forming MSCs and their neural regulation as promising therapeutic targets.

Summary Estrogens are potent regulators of mature hematopoietic cells; however, their effects on primitive and malignant hematopoietic cells remain unclear. Using genetic and pharmacological approaches, we observed differential expression and function of estrogen receptors (ERs) in hematopoietic stem cell (HSC) and progenitor subsets. ERα activation with the selective ER modulator (SERM) tamoxifen induced apoptosis in short-term HSCs and multipotent progenitors. In contrast, tamoxifen induced proliferation of quiescent long-term HSCs, altered the expression of self-renewal genes, and compromised hematopoietic reconstitution after myelotoxic stress, which was reversible. In mice, tamoxifen treatment blocked development of JAK2 V617F -induced myeloproliferative neoplasm in vivo, induced apoptosis of human JAK2 V617F+ HSPCs in a xenograft model, and sensitized MLL-AF9 + leukemias to chemotherapy. Apoptosis was selectively observed in mutant cells, and tamoxifen treatment only had a minor impact on steady-state hematopoiesis in disease-free animals. Together, these results uncover specific regulation of hematopoietic progenitors by estrogens and potential antileukemic properties of SERMs.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma, accounting for 30–40% of all newly diagnosed lymphomas. DLBCL is considered a heterogeneous disease, with some specific clinicopathological variants of DLBCLs being associated with the presence of the EBV.1 EBV is a lymphotropic virus that has been implicated in the development of several lymphoid malignancies, mainly Burkitt lymphoma (BL) and Hodgkin's lymphoma and with low prevalence in DLBCL.1 BCL6 is a key transcriptional repressor during normal B-cell differentiation that has been shown to repress NF-kB in some DLBCLs.2 In some B-cell lymphomas, BCL6 expression was inversely correlated with LMP1 expression, and some evidences suggest that LMP1 can cause downregulation of BCL6(ref. 3), but other possible mechanisms have not been studied. We have found a strong inverse correlation between BCL6 protein expression and EBV infection (P

Current treatments of sporadic Burkitt's lymphoma (sBL) are associated with severe toxicities. A better understanding of sBL formation would facilitate development of less toxic therapies. The etiology of sBL remains, however, largely unknown, C-MYC up-regulation being the only lesion known to occur in all sBL cases. Several studies examining the role of C-MYC in the pathogenesis of BL have concluded that C-MYC translocation is not the only critical event and that additional unidentified factors are expected to be involved in the formation of this tumor. We herein report that a gene distinct from C-MYC, E2F1, is involved in the formation of all or most sBL tumors. We found that E2F1 is highly expressed in Burkitt's lymphoma cell lines and sBL lymphoma specimens. Our data indicate that its elevated expression is not merely the consequence of the presence of more cycling cells in this tumor relative to other cell lines or to other neoplasias. In fact, we show that reduction of its expression in sBL cells inhibits tumor formation and decreases their proliferation rate. We also provide data suggesting that E2F1 collaborates with C-MYC in sBL formation. E2F1 expression down-regulation did not affect, however, the proliferation of human primary diploid fibroblasts. Because E2F1 is not needed for cell proliferation of normal cells, our results reveal E2F1 as a promising therapeutic target for sBL. [Cancer Res 2009;69(9):4052–8]

Aberrant inhibition of B-cell receptor (BCR)-induced programmed cell death pathways is frequently associated with the development of human auto-reactive B-cell lymphomas. Here, we integrated loss-of-function, genomic, and bioinformatics approaches for the identification of oncogenic mechanisms linked to the inhibition of BCR-induced clonal deletion pathways in human B-cell lymphomas. Lentiviral (HIV)-based RNA interference screen identified MCL1 as a key survival molecule linked to BCR signaling. Loss of MCL1 by RNA interference rendered human B-cell lymphomas sensitive to BCR-induced programmed cell death. Conversely, MCL1 overexpression blocked programmed cell death on BCR stimulation. To get insight into the mechanisms of MCL1-induced survival and transformation, we screened 41 000 human genes in a genome-wide gene expression profile analysis of MCL1-overexpressing B-cell lymphomas. Bioinformatic gene network reconstruction illustrated reprogramming of relevant oncoproteins within β-catenin–T-cell factor signaling pathways induced by enforced MCL1 expression. Overall, our findings not only illustrate MCL1 as an aberrantly expressed reprogramming oncoprotein in follicular lymphomas but also highlight MCL1 as key therapeutic target.

Modulation of oxidative stress as well as metabolic adjustments are key aspects in cancer stem cell-derived diseases, such as acute myelogenous leukemia (AML). Metabolic reprogramming in leukemia seems to be influenced by the crosstalk of malignant cells with their surrounding microenvironment. Reactive oxygen species (ROS), mainly generated by the mitochondria, are relatively high in malignant cells, without reaching a cytotoxic level that would compromise their survival. In addition, chemotherapy efficacy lies on increased ROS-mediated cellular damage. Cumulative evidence shows that AML often exhibits poor response to chemotherapy partly due to the support of leukemic stem cells (LSCs) by the bone marrow (BM) microenvironment, which includes BM mesenchymal stem cells (BMSCs). However, how exactly this happens, i.e. the specific mechanism(s) and cell populations implicated, remains incompletely understood. Hematopoietic stem cell (HSC)-niche forming BMSCs can be identified by the expression of the intermediate filament protein nestin and can be isolated and propagated as non-adherent 'mesenspheres', which can self-renew and support HSCs ex vivo. Here we have studied the contribution of nestin+ BMSCs to MLL-AF9-driven AML development and resistance. Immunohistochemistry for nestin on BM samples from AML patients (N=35) showed that the number of nestin+ niches per square millimeter was 2.4-fold higher as compared to control samples (N=12). To study the role of BMSCs in AML, we used a doxycycline-inducible iMLL-AF9 mouse strain that closely mimics the human disease (Stavropoulou V et al., Cancer Cell 2016;30:43-58). Development of AML in iMLL-AF9 mice caused a significant reduction of stromal cells in the BM but it did not reduce the number of BM nestin+ cells. To study the possible contribution of nestin+ cells to leukemia development, we partially depleted nestin+ cells by inducing diphtheria toxin expression in these cells using Nestin-creERT2 ; iDTA mice previously transplanted in a competitive fashion with pre-leukemic and normal BM cells. Nestin+ cell depletion selectively diminished leukemia burden in BM, spleen and blood, and reduced the number of primitive leukemic cells, without affecting normal residual hematopoiesis. Combined nestin+ cell depletion and standard chemotherapy further exaggerated the elimination of BM leukemic cells, suggesting that nestin+ cells contribute to chemotherapy resistance. To investigate the crosstalk of BMSCs and leukemic blasts, we developed a novel in vitro co-culture system using BM mesenspheres and iMLL-AF9+ leukemic blasts. Co-cultures with nestin+ mesenspheres promoted leukemic blast survival under metabolic stress (FBS deprivation) and chemotherapy (cytarabine, Ara-C). Increased survival correlated with a strong protection against excessive ROS levels and lipid peroxidation. Particularly, reduced glutathione (GSH), a key antioxidant element, increased in leukemic blasts co-cultured with nestin+ mesenspheres. Mitochondrial transfer from BMSCs has also recently been proposed as a metabolic-related mechanism contributing to chemoresistance in AML (Moschoi R et al. Blood 2016;128:253-64). Using the Seahorse Extracellular Flux Analyzer, we actually revealed that leukemic blast bioenergetics increased in co-culture with mesenspheres. Surprisingly, we found that chemotherapy-induced transfer of mitochondria from BMSCs to AML cells correlated with decreased ROS levels in the leukemic blasts, pointing out mitochondria transfer as an important ROS detoxifying mechanism to allow chemoresistance. We demonstrated that cell-to-cell contact could be required for Ara-C-mediated protective effects of mesenspheres on leukemic blasts and their mitochondria transfer. Accordingly, either the inhibition of GSH generation or mitochondrial transfer (by avoiding cell contact) may reduce BMSCs survival support after chemotherapy. Altogether, these results suggest that simultaneous co-opting of energy sources and anti-oxidant mechanisms of BMSCs contribute to chemoresistance in AML. Disclosures No relevant conflicts of interest to declare.

Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin− MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin+ cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP+ Pdgfrα− cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation. DOI: http://dx.doi.org/10.7554/eLife.03696.001, eLife digest During the earliest phases of development, the embryo is formed by groups of stem cells that can develop into all the different types of tissue in the body—from bones to brain tissue. Later in life, small stockpiles of adult stem cells are found in various tissues and provide a reservoir of new cells available for replacing old or damaged cells. The most important source of blood stem cells is the bone marrow, which produces and stores cells that are capable of developing into blood and immune system cells. These processes are assisted by different bone marrow cells called stromal cells, which create a specialized local environment or ‘niche’. But are the stromal stem cells that form the skeleton the same ones that form this niche during development? Or do the various types of stromal stem cells develop from distinct groups of cells in the embryo? Furthermore, it is unclear which cells guide blood stem cells towards the forming bones. Other types of cells, including some of the cells of the nervous system, can communicate with the stem cells in the adult marrow and influence their behavior. This led scientists to wonder whether the stem cells in the bone marrow niche and the cells that communicate with them developed from the same type of embryonic stem cell. Isern et al. tracked down the developmental origins of different types of bone marrow stromal stem cells by examining the bone marrow from the long bones (for example, the bones in the leg) of unborn and infant mice. It turns out that not all stromal stem cells in the developing bone marrow are alike. In fact, one pool of stromal stem cells forms the skeleton and loses stem cell activity in the process. In contrast, a different population of stromal stem cells develops from the same group of embryonic cells that gives rise to the cells of the nervous system. The stromal stem cells in this second group function as a niche to recruit and store the incoming blood stem cells and retain their stem cell activity throughout life. The findings of Isern et al. help to explain why the nervous system is able to communicate with stem cells in the adult marrow, and provide a model for understanding how stem cell niches in organs that contain nerve tissue are established. DOI: http://dx.doi.org/10.7554/eLife.03696.002

Myeloproliferative neoplasms (MPNs) are hematological malignancies originated by hematopoietic stem cells (HSCs) carrying mutations, most frequently in JAK2, CALR or MPL genes. These diseases, which cannot be cured in most cases, are characterized by overproduction of myeloid and erythroid-megakaryocytic lineages. However, it remains unclear how some mutations, that are frequently common in disparate MPNs, can cause different clinical outcomes. Moreover, current treatments are mostly symptomatic and are only capable of slowing down the disease, but generally they cannot eradicate it. This suggests that other factors critically contribute to MPN progression. Our research group has demonstrated specific alterations of the microenvironment of HSCs in MPN. We have shown that mutated HSCs can only expand and trigger disease after damaging bone marrow (BM) nerve terminals and mesenchymal stem cells (BMSCs), overcoming the control that this environment can exert over mutated HSC proliferation and migration. Therefore, preserving the HSC niche might be a complementary strategy to treat these diseases. This approach is being currently tested in a multicenter phase-II clinical trial (clinicaltrials.gov identifier NCT02311569). MPNs can be considered a pre-leukemic state since MPN patients have a higher risk of developing acute myeloid leukemia (AML), which is highly aggressive and exhibits poor response to chemotherapy. Work from other groups and our preliminary data suggest that acute leukemic cells can transform the BM microenvironment to promote leukemogenesis. We have studied the contribution of nestin+ BMSCs to the development of acute myeloid leukemia (AML) induced by the MLL-AF9 oncogene. For this purpose we have used a doxycycline-inducible rtTA;MLL-AF9 mouse strain, which closely mimics the human disease. Development of AML in rtTA;MLL-AF9 mice caused a significant reduction of stromal cells in the BM but, contrasting our previous findings in MPN, it did not seem to affect the number of BM nestin+ cells. To study the possible contribution of nestin+ cells to leukemia development, we partially depleted nestin+ cells by inducing diphtheria toxin expression in these cells using Nestin-creERT2;iDTR mice previously transplanted in a competitive fashion with preleukemic and normal BM cells. We have shown previously that nestin+ cell depletion using this strategy can accelerate MPN. In contrast, nestin+ cell depletion had the opposite effect in AML, since it selectively diminished leukemia burden in BM, spleen and blood, and reduced the number of primitive leukemic cells, without affecting normal residual hematopoiesis. Combined nestin+ cell depletion and standard chemotherapy further exaggerated the elimination of BM leukemic cells. Among primitive leukemic cells, MLL-AF9+ lin- ckitlow sca1- cells were most significantly expanded in leukemia and were most significantly decreased upon nestin+ cell ablation, an effect that seemed to be further enhanced by simultaneous chemotherapy. This was not associated with detectable changes in the cell cycle profile of leukemic progenitors. Moreover, non-proliferative (ki67-) lin- cells were not found enriched in the vicinity of nestin+ cells, and Cxcl12 deletion in nestin+ cells, which increased thrombocytosis in our previous MPN studies, did not affect AML progression. In order to identify changes in leukemic progenitors following Nes+ cell depletion, we compared the gene expression profiles of lin- ckitlow sca1- cells isolated from control and Nes+ cell-depleted, MLL-AF9 expressing mice. Next-generation sequencing assays identified downregulation of gene sets related to HSC self-renewal, protein synthesis and degradation, and mitochondrial respiration in leukemic progenitors obtained from Nes+ cell-depleted bone marrow. The data suggests that AML-AF9+ leukemic progenitors impose changes in Nes+ cells that promote leukemogenesis. Using a novel co-culture system, we are currently testing possible mediators of the pro-survival effect of BMSCs over leukemic progenitors. Citation Format: Abel Sánchez-Aguilera, Vaia Stavropoulou, Daniel Martín-Pérez, Alexandar Tzankov, Jürg Schwaller, Simón Méndez-Ferrer. Leukemia development and the microenvironment. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA23.

The Polycomb group (PcG) of proteins is a major mechanism of epigenetic regulation that has been broadly linked to cancer. This system can repress gene expression by chromatin modification and is essential for establishing cell identity. PcG proteins are important for stem cell function and differentiation and have a profound impact during hematopoiesis. In recent years, several published studies have deepened our knowledge of the biology of the PcG in health and disease. In this article, we review the current understanding of the mechanisms of PcG-mediated repression and their relation to DNA methylation, and we discuss the role of the PcG system in hematopoiesis and hematologic malignancies. We suggest that alteration of different PcG members is a frequent event in leukemia and lymphomas that confers the stem cell properties on tumor cells. Thus, drugs targeting Polycomb complexes could be useful for treating patients with these diseases.

Polycomb proteins are known to be of great importance in human cancer pathogenesis. SUZ12 is a component of the Polycomb PRC2 complex that, along with EZH2, is involved in embryonic stem cell differentiation. EZH2 plays an essential role in many cancer types, but an equivalent involvement of SUZ12 has not been as thoroughly demonstrated. Here we show that SUZ12 is anomalously expressed in human primary tumors, especially in mantle cell lymphoma (MCL), pulmonary carcinomas and melanoma, and is associated with gene locus amplification in some cases. Using MCL as a model, functional and genomic studies demonstrate that SUZ12 loss compromises cell viability, increases apoptosis, and targets genes involved in central oncogenic pathways associated with MCL pathogenesis. Our results support the hypothesis that the abnormal expression of SUZ12 accounts for some of the unexplained features of MCL, such as abnormal DNA repair and increased resistance to apoptosis.

Myeloproliferative neoplasms (MPNs) are originated by mutations in a hematopoietic stem cell (HSC), frequently in the Janus kinase 2 (JAK2) gene. Different outcomes of this common event and limited efficacy of JAK2 inhibitors suggest the contribution of other factors. Additional HSC mutations and HSC-niche interaction might influence MPN progression, characterized by sequential expansion of HSCs, blood cells and megakaryocytes. Ensuing bone marrow (BM) fibrosis and osteosclerosis, which are contributed by osteoblastic lineage cells in a BCR/ABL CML model (Schepers et al Cell Stem Cell 2013), impede normal hematopoiesis. We have previously shown that BM nestin+ mesenchymal stem cells (MSCs) innervated by sympathetic nerve fibers regulate HSCs (Méndez-Ferrer et al Nature 2008 & 2010). Here we demonstrate that damage to this regulatory network is required for MPN manifestation. Nestin+ MSCs and NESTIN mRNA expression were rapidly reduced in the BM of MPN patients and mice expressing the human JAK2-V617F mutation. This reduction was not due to nestin+ MSC differentiation into fibroblasts or osteoblasts, as shown by 25-week lineage-tracing studies using Nes-CreERT2;RCE-loxP mice, but instead caused by early MSC apoptosis. In turn, nestin+ cell reduction stimulated MPN progression; selective nestin+ cell depletion using Nes-CreERT2;iDTA mice increased peripheral white and red blood cells, megakaryocytic invasion of spleen germinal centers and BM osteosclerosis. Our recent results indicate that the neural crest contributes during development to BM MSCs with specialized HSC niche function and that postnatal murine BM Nestin-GFP+ cells do not only contain MSCs but also Schwann cell precursor-like cells (Isern et al, ISSCR Annual Meeting 2013). BM Nestin-GFP+ cells from MPN mice showed reduced expression of HSC maintenance and mesenchymal genes, and increased expression of genes related to axon guidance and Schwann cell differentiation. Principal component analyses of independent biological samples further showed that control BM nestin+ cells clustered together with MSCs, whereas MPN BM nestin+ cells resembled Schwann cell precursors. These data suggested alterations to the neural component of the BM HSC niche in MPN. Indeed, BM sympathetic nerve fibers and Schwann cells, closely associated but different from Nestin-GFP+ cells, were rapidly reduced in the BM of diseased animals. Symptomatic MPN mice were treated with selective β3-adrenergic agonists to compensate for the loss of sympathetic stimulation of nestin+ MSCs. Treatment with BRL37344 or the recently FDA approved drug Mirabegron prevented MPN-associated neutrophilia and thrombocytosis, while it did not affect peripheral blood counts of wild-type mice. While vehicle-injected animals showed severe BM fibrosis, long-term BRL37344 treatment led to virtual absence of focal reticulin deposits or excessive fibroblasts. To further confirm the contribution of BM neural damage to MPN pathogenesis, diseased mice were treated with a neuroprotective agent. Sympathetic nerve-ensheathing Schwann cells were strongly reduced in the BM of vehicle-injected animals but preserved in 4-methylcatechol-treated mice. Like in BRL37344-treated animals, this was associated with prevention of very early MPN events, including neutrophilia and BM overproduction of the pro-inflammatory cytokine interleukin-1β. Since MPN is originated by a mutant HSC, we reasoned that sympathetic neuropathy might contribute to MPN pathogenesis through early disruption of the HSC niche. The chemokine Cxcl12 regulates HSC migration and proliferation. At early MPN stage, HSC expansion and mobilization correlated with decreased BM Cxcl12 expression and protein levels. Concomitantly, BM nestin+ MSC number and their Cxcl12 expression were significantly reduced. BRL37344 treatment completely restored the number of BM nestin+ cells and improved Cxcl12 BM levels. Treatment with 4-methylcatechol or BRL37344 prevented the early expansion of mutant hematopoietic progenitors, whereas long-term BRL37344 treatment efficiently reduced mutant hematopoietic progenitor numbers in BM and peripheral blood. These results demonstrate that damage of the niche, induced by the mutated HSCs, critically contributes to JAK2-V617F+ MPN pathogenesis. They also unravel HSC niche-forming MSCs and their neural regulation as promising novel therapeutic targets in MPN. Disclosures: Arranz: Centro Nacional de Investigaciones Cardiovasculares (CNIC): National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013, National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013 Patents & Royalties. Off Label Use: Beta-3-adrenergic agonists (e.g. FDA-approved Mirabegron) and neuroprotective drugs for the treatment of myeloprolifeative diseases. Isern:Centro Nacional de Investigaciones Cardiovasculares (CNIC): National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013, National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013 Patents & Royalties. Méndez-Ferrer:Centro Nacional de Investigaciones Cardiovasculares (CNIC): National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013, National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013 Patents & Royalties.

Abstract 314 Introduction: Epstein Barr Virus (EBV) is able to transform B-cells by disrupting the normal B-cell differentiation programme, leading to the development of different types of B-cell lymphoma. BCL6 is a key transcriptional repressor during normal B-cell differentiation that is required for germinal centre reaction and it has been shown to repress NF-kB in Diffuse Large B-Cell Lymphoma (DLBCL). In some B-cell lymphomas the expression of BCL6 and infection with EBV are mutual exclusive occurrences, although the causal mechanism of this phenomenon and its biological significance remain elusive. Patients and methods: A microRNA expression profile was investigated using Agilent's Human miRNA microarray kit. microRNA targets were predicted with the miRanda algorithm (http://www.microrna.org/). Immunohistochemical and in situ hybridization analyses were performed on 149 DLBCL samples to investigate the expression of BCL6 and EBV status, respectively. Luciferase assays were carried out by cloning the BCL6 3'-UTR into the pGL3-Control vector. Results: 22 viral microRNAs were found to be upregulated specifically in EBV-positive cases of DLBCL. By applying the miRanda algorithm, 10 of these 22 microRNAs were predicted potentially to target BCL6. To explore this possibility, immunohistochemical analysis and in situ hybridization were carried out on 149 cases of DLBCL. The results showed an almost perfect inverse correlation between BCL6 protein expression and EBV infection, even in the absence of LMP1 protein expression (p Conclusions: DLBCL cases infected by EBV express a restricted set of viral microRNAs. Several of these microRNAs can potentially target the BCL6 gene, highlighting the importance of BCL6 downregulation in the context of EBV infection. Functional studies demonstrate that at least three of these microRNAs are able to downregulate BCL6 expression. The reason why EBV downregulates BCL6 is still unknown, but we propose that it might be required for DLBCL cells to survive in the context of EBV-induced NF-κB pathway activation. Disclosures: No relevant conflicts of interest to declare.

Strategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45(-) CD31(-) CD71(-) CD146(+) CD105(+) nestin(+) cells but could also be simply grown from fetal and adult BM CD45(-)-enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34(+) cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs.

Summary Like normal hematopoietic stem cells, leukemic stem cells depend on their bone marrow (BM) microenvironment for survival, but the underlying mechanisms remain largely unknown. We have studied the contribution of nestin+ BM mesenchymal stem cells (BMSCs) to MLL-AF9-driven acute myeloid leukemia (AML) development and chemoresistance in vivo. Unlike bulk stroma, nestin+ BMSC numbers are not reduced in AML, but their function changes to support AML cells, at the expense of non-mutated hematopoietic stem cells (HSCs). Nestin+ cell depletion delays leukemogenesis in primary AML mice and selectively decreases AML, but not normal, cells in chimeric mice. Nestin+ BMSCs support survival and chemotherapy relapse of AML through increased oxidative phosphorylation, tricarboxylic acid (TCA) cycle activity, and glutathione (GSH)-mediated antioxidant defense. Therefore, AML cells co-opt energy sources and antioxidant defense mechanisms from BMSCs to survive chemotherapy., Graphical Abstract, Highlights • Nestin+ BMSCs support leukemogenesis and chemoresistance • BMSCs support metabolic requirements of LSCs • BMSCs provide LSCs with essential antioxidant defense from chemotherapy • GSH and GSH peroxidases underlie BMSC-derived antioxidant AML protection, Forte et al. reveal that nestin+ bone marrow stromal cells directly contribute to leukemogenesis and chemotherapy resistance in an in vivo model of acute myeloid leukemia. Nestin+ BMSCs support leukemic stem cells through a dual mechanism of increased bioenergetic capacity through OXPHOS and TCA and glutathione-dependent antioxidant defense.