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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.

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.

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.