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Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the “bad seed in bad soil” hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis.

Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. There is a need to explore new therapeutic pathways to reduce renal fibrogenesis. To study this, we used unilateral ureteral obstruction (UUO) in mice as an experimental model of renal fibrosis and microarray analysis to compare gene expression in fibrotic and normal kidneys. The cannabinoid receptor 1 (CB1) was among the most upregulated genes in mice, and the main endogenous CB1 ligand (2-arachidonoylglycerol) was significantly increased in the fibrotic kidney. Interestingly, CB1 expression was highly increased in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis. Both genetic and pharmacological knockout of CB1 induced a profound reduction in renal fibrosis during UUO. While CB2 is also involved in renal fibrogenesis, it did not potentiate the role of CB1. CB1 expression was significantly increased in myofibroblasts, the main effector cells in renal fibrogenesis, upon TGF-β1 stimulation. The decrease in renal fibrosis during CB1 blockade could be explained by a direct action on myofibroblasts. CB1 blockade reduced collagen expression in vitro. Rimonabant, a selective CB1 endocannabinoid receptor antagonist, modulated the macrophage infiltrate responsible for renal fibrosis in UUO through a decrease in monocyte chemoattractant protein-1 synthesis. Thus, CB1 has a major role in the activation of myofibroblasts and may be a new target for treating chronic kidney disease.

Despite the major progress obtained in prognosis with the use of tyrosine kinase inhibitors (TKI), the great majority of patients with CML remain on long-term therapy and progression occurs in patients with either primary or secondary resistance. The mechanisms of progression towards accelerated phase (AP) and blast crisis (BC) have been studied so far only in primary patient samples in BC. Currently, there is no in vitro model to study sequentially the molecular events leading from CP to BC as only some primary sequential samples are amenable to analysis. Using induced Pluripotent Stem Cell (iPSC) technology, it is now possible to reprogram the primary leukemic cells to pluripotency and generate a major source of stem cells. To determine the feasibility of studying progression of CML towards AP and BC, we have used a patient-specific iPSC line that we have generated from the primary leukemic cells of a patient who later showed a TKI-resistance requiring an allogeneic stem cell transplant. These iPSC expressed BCR-ABL, exhibited all pluripotency markers and after injection in NSG mice, generated teratoma with differentiation into three germ layers. In hematopoietic differentiation assays using day 19-embryoid bodies (EB), increased numbers of hematopoietic progenitors were found as compared to control iPSC (5-fold increase n= 3). We have then treated leukemic iPSC with the mutagenic agent N-ethyl-N-nitrosourea (ENU) during regular medium changes. After 61 days in ENU cultures, day-19 derived embryoid bodies generated hematopoietic cells (>90% CD45+, CD43+) which proliferated in liquid cultures with myeloid and some blast cell morphology. Cytogenetic analyses of iPSC revealed chromosomal abnormalities such as loss of Y and loss of der q9+, both alterations known to occur in CML during progression. They exhibited increased numbers of micronuclei (MN) as compared to leukemic iPSC without ENU (X 3 Fold increase) suggesting acquisition of a progressive chromosomal instability. CGH array analyses were performed using ENU-treated iPSC-derived hematopoietic cells in two different timepoints as compared to leukemic iPSC cultured without ENU. Genomic aberrations were analyzed by Agilent Cytogenomics software with Mosaicism workflow on HG19 genome. 249 gene loci alterations were detected after polymorphism filtration on European population. These analyses showed DNA losses and DNA gains in genes implicated in mesoderm development and hematopoietic lineage as well as genes implicated in DNA damage response. Several loci of transcription factors were found to be involved such as IKZF1 described in imatinib-refractory chronic myeloid leukemia (Bolton-Gillespie et al. 2013). The aberrations included SIRT1and BLM which is implicated in DNA repair. Several cancer genes were found to be involved, some known to be altered in leukemia (BLM, IKZF1, NCOA2, ALK, EP300, ERG, MKL1, PHF6 and TET1). Remarkably, transcriptome geodataset GSE4170 (Radich et al. 2006) allowed us to associate 125 of 249 of the aberrations that we detected in CML iPSC, with the CML progression genes already described during progression from chronic and AP to BC (p-value =9.43E-32, after ANOVA with 1000 permutations). 38 most predictive aberrations allowed perfect reclassification of BC and chronic phase samples by unsupervised classification. Among these candidates, eleven of them have been described in CML physiopathology and connected to TKI resistance and genomic instability. Majority of them ( 7/11) are connected to chronic phase (FAS, ACTB, TRIM21, ANPEP, MLK1, CSF2RA, and MAGEC2) whereas a minority of them (4/11) are connected to BC (ACP1, SH3YL1, FHL1, IL3RA). Interestingly, these experiments also allowed us to discover the connection of a new multidrug resistance molecule associated to BC and having the ability to modify interferon pathway connected to the TKI sensitivity. Thus, genomic instability pattern that we have generated using a single leukemic iPSC allowed duplication of genomic abnormalities described in CML progression and allowed identification of some novel genes. Overall, these results demonstrated that we have generated for the first time to our knowledge, an in vitro BC model, reproducing genomic events described in patients with BC. This "blast crisis in a dish" tool using patient-derived iPSC will be of major interest to study CML progression and eventually to test novel therapies. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Heterotopic ossification (HO) is a pathology characterized by the formation of ectopic bone in soft tissues generally in muscles surrounding joints such hip, knee, elbow or shoulder. The etiology of this pathology is not known but it is well established that the risk of HO is increased after traumatic brain or spinal cord injuries and also after fractures or burns. Patients with HOs suffer from pain and the range of motion from limbs with ectopic bone is highly reduced. Currently, the only effective treatment for HOs is surgery but recurrences are frequent underpinning that a better understanding of its pathophysiology is required to cure patients. Hematopoietic marrow-like tissue is frequently observed on histological section of HO. Therefore, we investigated whether HOs from patients with traumatic brain or spinal cord injury represent an ectopic model of hematopoietic stem cell (HSC) niche as defined by the presence of HSC-supportive stromal cells. HOs were obtained from patients after surgical resection in Garches Hospital (France) with their informed consent. HOs were cut in small pieces and hematopoietic as well as endothelial cells were extracted from the HO marrow by gentle washing. Mononuclear cells were then recovered by ficoll gradient and were either directly analyzed by flow cytometry or purified by immunomagnetic selection for in vitro and in vivo experiments. Mesenchymal stromal cells (MSCs) were obtained by plastic adherence from HO explants. We first confirmed the presence of hematopoietic cells from different lineages in HO marrow such as neutrophils, monocytes/macrophages, B and T lymphocytes as evidenced by membrane expression of CD45, CD15, CD11/CD14, CD19, CD3/CD4 and CD3/CD8 antigens. HSC presence within HO marrow was further demonstrated by: (i) in vitro clonogenic assays, (ii) presence of quiescent CD45+CD34+CD38- cells with a side-population phenotype and, (iii) in vivo human hematopoietic reconstitution assays in immunodeficient NSG mice. We also show that human HO marrow contained functional MSCs and endothelial progenitors (EPs) as demonstrated by phenotypic and functional analyses. MSC expressing the classical mesenchymal markers CD73, CD90, CD105 and capable to in vitro and in vivo differentiate into osteoblasts, adipocytes and chondrocytes could be isolated from human HOs. These MSCs were also able to support in vitro long term human hematopoiesis and in vivo murine hematopoiesis when seeded on hydroxyapatite scaffolds and implanted into nude mice. Endothelial progenitors with a characteristic CD31+CD144+CD34+CD45-CD90+ phenotype were detected in the mononuclear cell fraction from HO marrows. After sorting, these cells were able to form colonies on plastic and vascular networks when cultured in a Matrigel and to express high levels of VCAM-1 and ICAM-1 adhesion molecules after TNFα stimulation, confirming their EP functional capability. In conclusion, we show for the first time that human HOs contain a marrow tissue with quiescent HSC that can proliferate and differentiate within a suitable and functional mesenchymal and endothelial microenvironment, acknowledging that HO marrows are ectopic HSC niches. We have recently demonstrated the involvement of macrophages in the development of neurogenic HOs in spinalized mice1. Taking into account the role of immune cells and the neurogenic origin of human HOs, our results bring new evidences for the role of interactions between bone and neuro-immune systems in the initiation and development of adult hematopoiesis. Mechanisms underlying these processes are currently studied in our laboratory. 1 Genet F et al., J Pathol. 2015 Jun;236(2):229-40 Disclosures No relevant conflicts of interest to declare.

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm characterized by the occurrence of the Ph1 chromosome in a primitive hematopoietic stem cell with a major amplification of myeloid compartment. Despite the major progress obtained by the use of tyrosine kinase inhibitors (TKI), resistances to these drugs occur with progression towards blast crisis (BC) during which there is an increase of BCR-ABL expression. To evaluate the potential targets of BCR-ABL in this context, a gene profiling analysis of the UT7-11 cell line overexpressing BCR-ABL fusion protein was performed using a whole transcriptome microarray. One of the highly upregulated genes was ETS1 with a fold change of +35.86. ETS1 proto-oncogene, the founder member of the ETS-domain family of transcription factors such as TEL and FLI1, is the human homolog of the avian erythroblastosis virus E26. They play a crucial role in stem cell biology, tumorigenesis and ETS1 has been shown to be involved in the regulation of granulocytic differentiation. Its role in CML pathophysiology has not been studied so far. We first showed by Western blots that ETS1 protein was highly increased in UT7-11 cells as compared to parental UT7 cells. Using a DOX-Inducible BCR-ABL system, we have shown that ETS1 expression was downregulated upon inhibition of BCR-ABL expression. ETS1 expression was a tyrosine kinase dependent event as its expression was reduced in the presence of TKI. To determine if ETS1 expression is upregulated in primary leukemic cells, we have analyzed leukemic cells of CML patients at diagnosis (n= 40) as compared to healthy controls (n= 30) showing an increase of ETS1 mRNA expression in primary CML cells in a highly significant manner (p < 0.0007). We then analyzed ETS1 targets using chip-sequencing in K562 cells, performed on HG19 human genome allowing to predict 3209 proximal promoters (-3000 upstream pb, + 50pb around Transcription Starting Sites) which represents a promoter enrichment of +33.68 (p-value=4.9E-324) as compared to the distribution of peaks in the whole genome. Chromosomes 19, 16, 17, 11, 12 and 1 were found over-represented with ETS1 bound events in Chip-sequencing. Integration of BCR-ABL transcriptome with ETS1 promoters identified by Chip-Sequencing led to the identification of 130 ETS1-targets activated by BCR-ABL, allowing reclassification of BCR-ABL transfected samples with transcriptome data by unsupervised classification. ETS1 transcriptional program regulated by BCR-ABL analysis performed on CD34+ from CML patients during progression of the disease (GSE47927) allowed discrimination of the different states of the progression (from the chronic phase to accelerated phase and BC p-value=1.63E-33). This analysis was also found significant by Gene Set Enrichment Analysis ETS1_BC specific geneset (NES=+1.52, p-value Disclosures No relevant conflicts of interest to declare.

International audience; Cholangiocytes are biliary epithelial cells, which, like hepatocytes, originate from hepatoblasts during embryonic development. In this study we investigated the potential of human embryonic stem cells (hESCs) to differentiate into cholangiocytes and we report a new approach, which drives differentiation of hESCs toward the cholangiocytic lineage using feeder-free and defined culture conditions. After differentiation into hepatic progenitors, hESCs were differentiated further into cholangiocytes using growth hormone, epidermal growth factor, interleukin-6, and then sodium taurocholate. These conditions also allowed us to generate cholangiocytes from HepaRG-derived hepatoblasts. hESC- and HepaRG-derived cholangiocyte-like cells expressed markers of cholangiocytes including cytokeratin 7 and osteopontin, and the transcription factors SOX9 and hepatocyte nuclear factor 6. The cells also displayed specific proteins important for cholangiocyte functions including cystic fibrosis transmembrane conductance regulator, secretin receptor, and nuclear receptors. They formed primary cilia and also responded to hormonal stimulation by increase of intracellular Ca2+. We demonstrated by integrative genomics that the expression of genes, which signed hESC- or HepaRG-cholangiocytes, separates hepatocytic lineage from cholangiocyte lineage. When grown in a 3D matrix, cholangiocytes developed epithelial/apicobasal polarity and formed functional cysts and biliary ducts. In addition, we showed that cholangiocyte-like cells could also be generated from human induced pluripotent stem cells, demonstrating the efficacy of our approach with stem/progenitor cells of diverse origins. Conclusion: We have developed a robust and efficient method for differentiating pluripotent stem cells into cholangiocyte-like cells, which display structural and functional similarities to bile duct cells in normal liver. These cells will be useful for the in vitro study of the molecular mechanisms of bile duct development and have important potential for therapeutic strategies, including bioengineered liver approaches. (Hepatology 2014;60:700–714)

Background & Aims The mechanisms by which fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) develop during chronic hepatitis C virus (HCV) infection are not fully understood. We previously observed that HCV core protein induced a TGF-β-dependent epithelial mesenchymal transition, a process contributing to the promotion of cell invasion and metastasis by impacting TGF-β1 signalling. Here we investigated HCV core capacity to drive increased expression of the active form of TGF-β1n transgenic mice and hepatoma cell lines. Methods We used an in vivo model of HCV core expressing transgenic mice. Results We observed that about 50% of genes deregulated by core protein expression were TGF-β1 target genes. Active TGF-β levels were increased in HCV core transgenic mouse livers. Overexpression of core protein in hepatoma cells increased active TGF-β levels in culture supernatants and induced Smad2/3 phosphorylation, thus reflecting activation of the TGF-β signaling pathway. Moreover, our data showed the implication of thrombospondin-1 in core-dependent TGF-β activation. Finally, hepatoma cells expressing HCV core could activate stellate cells in co-culture and this activation was TGF-β dependent. Conclusions Collectively, these data delineate a novel paradigm where HCV may be related to liver pathogenesis through its ability to induce a local, intrahepatic TGF-β activation. They argue for a dual impact of HCV core on liver fibrosis and liver carcinogenesis: HCV core could act both as autocrine and paracrine factor modulating TGF-β responses within hepatocytes and in stromal environment through TGF-β activation.

Despite the major success obtained with their use in chronic myeloid leukemia (CML), recent data obtained from treatment discontinuation trials suggest that tyrosine kinase inhibitors (TKI) alone are not sufficient to eradicate the most primitive CML stem cells in the majority of the patients. The mechanisms of this inefficiency might involve cell autonomous (activation of alternate signaling, reduced BCR-ABL expression) or non-cell autonomous (niche-related) pathways. Several strategies of targeting the primitive stem cell compartment, in association with TKI, are currently being studied. Inecalcitol (ICC) is a vitamin D3 analog exerting antiproliferative effects in several types of cancer cells. ICC was tested in CD34+ cells isolated from CML patients at diagnosis (n=18) in clonogenic assays as well as in the more primitive LTC-IC-derived progenitors. ICC alone inhibits the clonogenic growth in the majority of the CML patients at diagnosis (15/18 patients). The combination of ICC with either, Imatinib (IM), Dasatinib (DA) or Nilotinib (NIL) in clonogenic assays showed a synergistic effect for the inhibition of CFC growth (10-25% CFC survival) with no toxicity on normal progenitors. Synergistic effects of ICC and TKI was also demonstrated in LTC-IC-derived progenitors with IM, NIL and DA. To determine possible mechanism of action of ICC in CML stem cells, we have performed a gene profiling analysis of CD34+ cells obtained at diagnosis from 4 CML patients. CD34+ cells were cultured for 7 days in the presence of growth factors with or without ICC. In phenotypic analyses, CD34+ cells treated in the presence of ICC showed an increase of monocyte/macrophage differentiation features with increased expression of CD13/CD14 as well as CD11b expression. Day 0 and Day7 RNA with or without ICC treatment were then studied by transcriptome hybridation on human-v2 (8*60k) Agilent technologies microarrays. Data were treated with Feature Extraction 11.5.11, Genespring GX12, Mev 4.9, R software and GSEA 2.2.20. Gene set enrichment analysis performed at day 7 of culture between ICC condition and control untreated cells showed an increase of cell differentiation markers under ICC (Normalized enrichment score = +1.94, p-value < 0.001) One way ANNOVA with False Discovery Rate (FDR) correction allowed to discover 7176 modulated probes between the 3 experimental conditions (Day 0, Day7 without ICC, Day 7 with ICC). CYP24A1 was the gene with the greatest induction by the treatment (Fold Change = +150). CYP24A1 is responsible of the degradation of 1.25(OH)2D3 by the monocyte/macrophage cells. Unsupervised classification with 372 macrophage connected genes (ANOVA p Disclosures Dufour-Lamartinie: Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Turhan:Bristol Myers Squibb: Consultancy; Novartis: Research Funding.

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm defined by the presence of BCR-ABL fusion gene. This oncogenic event occurs in a hematopoietic stem cell (HSC) involved in CML initiation, maintenance, relapse and progression. Several evidences suggest that inflammatory pathways may participate to the pathophysiology of CML as well as disease progression to blast crisis. It has been shown that NFKB/REL pathway is constitutively activated both in BCR-ABL positive leukemic cell lines as well as in primary blast cells from CML-BC patients. More recent works identified IL6 as key cytokine acting on CML multipotent progenitors and their normal bystander counterpart to favor their differentiation toward the myeloid lineage. In addition, high levels of autocrine TNFα secretion by quiescent CML stem/progenitor cells activate NFKB pathway and promote their survival. Although all of these observations are linked to inflammatory processes, a focused analysis of inflammatory pathways in primary CML stem cells has not been performed so far. In the present study we undertook a text-mining strategy using pubmed e-querying to generate an exhaustive set of genes linked to inflammation. Then we integrated transcriptome analysis of highly purified CML stem cells to evaluate the contribution of these genes in CML development and progression. Methods : We queried 6 key words (Inflammation, macrophages, inflammatory response, chemokines, leukocytes and interleukins) that returned a total of 332000 hits in Pubmed. A raw gene set of 918 genes was found significantly associated (p Results: Among the 588 genes related to inflammation we found 70 genes differentially expressed between the four groups (normal, CP, AP and BC, p0.75 and p-value Conclusion : Using a bio-integrative approach we identified a specific inflammatory signature in CD34+CD38-CD90+ CML stem cells. This inflammatory network is highly altered in blast crisis suggesting its contribution to disease evolution. We identified REL overexpression as a good predictor for disease progression to blast crisis and found NFKB1and RELA (p=3.2x10-13) as the best REL target candidates. RELA/NFKB1 was previously shown to be constitutively activated in CML and Ph+ ALL and this analysis suggests that this may also take place in the most primitive subset of CML cells although REL may be the main partner of NFKB in CML stem cells. These results which are currently validated using functional assays, could lead to identification of novel therapeutic strategies. Disclosures Turhan: Bristol Myers Squibb: Consultancy; Novartis: Research Funding.

BACKGROUND The dynamic relationship between HSC, their niches and the trabecular bone is disrupted in MPN as primary myelofibrosis (PMF). PMF is associated to extramedullary hematopoiesis (EMH) usually with splenomegaly. Several tracers are used in nuclear medicine to assess EMH and bone marrow (BM) activity. Colloids labelled with Technetium 99m (99mTc) show the reticulo-endothelial system (RES) and Indium111 chloride (111In-Cl3) transferrin scintigraphy reflects the hematopoietic activity. Cell imaging using single photon emission tomography (SPECT) utilizes gamma emitting radiopharmaceuticals and 3D acquisition allowing for imaging planes reconstruction. It is often coupled to a computed tomography (CT) (SPECT/CT). Few data exist about hematopoiesis assessment in PMF using hybrid imaging (HIm). We have shown its interest in EMH diagnosis. In PMF, we looked at patterns of active/inactive hematopoiesis in order to study later the impact of targeted therapies. MATERIAL & METHODS We performed HIm with 99mTc nanocolloids and 111In-Cl3 (coupled to SPECT/CT) in 5 untreated PMF patients and in 1 with essential thrombocytemia (ET) who was developing a secondary MF (Table1). RESULTS AND DISCUSSION We observed a lower fixation of both tracers in the axial skeleton (AxS) and conversely a hyperfixation of 111In-Cl3 in the distal skeleton (DiS) and the spleen, this latter considered as pathognomonic of PMF or secondary MF (Figs1-2. Table1). The more advanced the process of marrow fibrosis and HSC externalization the more 111In-Cl3 splenic and DiS fixation. When associated to a low 111In-Cl3 fixation in the AxS it is suggestive of PMF. No other EMH localizations besides spleen were observed. Liver fixation interpretation was uneasy because both tracers show a significant fixation. Some questions remain unsolved as the mechanisms involved in the DiS recruitment in circumstances that this hematopoietic area was progressively inactive since birth and replaced with adipose tissue. Is DiS reactivation a consequence of HSCs that cannot seed in the physiological homing hematopoietic areas, which take part essentially in the AxS? Or is the fruit of HSC reactivation (and their microenvironment) already present in the DiS but simply dormant? In this latter case we do not know if HSCs carry the same molecular abnormalities of the malignant PMF clone. Interestingly in the post ET MF patient, there was no 111In-Cl3 hyperfixation at the DiS. Conversely we observed a significant splenic fixation with both tracers and a low fixation with 99mTc in the AxS (Figs1-2). This evocates a sequence of hematopoietic reactivation starting in the spleen, followed by DiS recruitment and associated to a progressive hematopoietic regression in the AxS. HiM using these 2 radionuclides and SPECT/CT is a non-invasive procedure allowing larger and more precise visualization of the active/inactive hematopoietic areas in PMF. Moreover, regarding deep regions or high-weight patients, attenuation correction can show more uptake focus, potentially hidden because of soft-tissue interposition between emitting source and detector. This can represent an alternative to BM biopsies that also have the inconvenient to be limited in their investigation to the crista iliac. They may reflect the AxS but do not give any information neither on DiS nor on liver and spleen hematopoiesis. CONCLUSIONS The use of HIm allowed a good assessment of the extent and intensity of PMF hematopoiesis. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Activating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F–dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F–binding regulate JAK2-mediated signals in MPNs.

International audience; Most patients with polycythemia vera and half with idiopathic myelofibrosis and essential thrombocythemia have an acquired V617F mutation in JAK2. Using sensitive polymerase chain reaction (PCR)-based methods, we genotyped 152 patients with idiopathic myelofibrosis to establish whether there were differences in presentation and outcome between those with and those without the mutation. Patients positive for V617F had higher neutrophil and white cell counts (P = .02) than did patients negative for V617F, but other diagnostic features were comparable between the 2 groups. Patients positive for V617F were less likely to require blood transfusion during follow-up (P = .03). Despite this, patients positive for V617F had poorer overall survival, even after correction for confounding factors (P = .01).

Introduction. Primary myelofibrosis (PMF) can be associated to increased bloodstream traffic of some adult progenitor cells. The extramedullary hematopoiesis developed in this setting might remind some features of the fetal hematopoiesis. Material and methods. Some evidence of this hypothesis was searched by flow-cytometry and molecular analysis of blood samples from 12 untreated patients with PMF, 6 pre-fibrotic (PF) and 6 fibrotic (F). The PF or F phase was defined according to the WHO and Thiele histopathology scores. Results. As expected we detected high numbers of CD34+ cells (258x103±402x103/ml; range 1.2-1218x103/ml) as well as small mean numbers of Lin-/CD45-CD34+AC133+CXCR4+ progenitor cells (130±275/ml; range 0-882/ml) that are related to VSELs (very-small embryonic-like stem cells), which have been described as putative pluripotent adult stem cells. When we looked at some embryonic molecular markers by RT-PCR, NANOG and OCT4 expression was detected in the MNC fraction of all the patients tested (hES and CPRE2 cell lines were the positive and negative controls). OCT4 expression was half the level of hES being stronger in F-PMF than in PF-PMF. In all the tested patients NANOG expression was similar to that of hES, whereas SOX2 and LIN28 were not expressed in most patients. Regarding other circulating progenitor cells we observed that PF-PMF had higher mean values of progenitor endothelial cells (PEC) (130±271/ml; range 0-683) than F-PMF (7±16/ml; range 0-37). Mesenchymal progenitor cells (MPC) had also higher mean values in PF-PMF (289±452/ml; range 0-1165) than in F-PMF (111±199/ml; range 0-458). Conversely, Lin-/CD45-CD34+AC133+CXCR4+ cell subset detection was higher in F-PMF (185±381/ml; range 0-882) than in PF-PMF (38±60/ml; range 0-140). Patients V617F-JAK2pos presented lower levels of PEC and MPC (16±19/ml and 43±61/ml respectively) than those V617F-JAK2neg (173±340/ml and 515±470/ml respectively). Similar numbers of Lin-/CD45-CD34+AC133+CXCR4+ cells were observed in both V617F-JAK2pos and V617F-JAK2neg patients (133±331/ml and 123±115/ml respectively). Patients with elevated IPSS (>3) showed higher levels of PEC, MPC and Lin-/CD45-CD34+AC133+CXCR4+ cells (respectively 180±335/ml, 334±560/ml and 256±422/ml) than those with low IPSS ( We also performed a bio-informatics analysis of microarray databases comparing the molecular libraries of the gene expression ommibus dataseries GSE3410 and GSE31869 (VSELs (Lin-CD45-CXCR4+CD34-) and CD34+ VSELs (Lin-CD45-CXCR4+CD34+) with the molecular profiling of circulating CD34+ cells from PMF patients and bone marrow CD34+ cells from healthy donors and we observed a tight clustering between PMF and VSELs cells samples (Figure 1). Conclusions. Variations in the blood traffic of some progenitor cell subsets in patients with PMF were observed, including Lin-/CD45-CD34+AC133+CXCR4+ cells. The fact this cell subset was detected in higher number in F-PMF and in patients with a high IPSS, as well as a higher expression of OCT4 in F-PMF supports in part our hypothesis that PMF evolution can be associated to the recruitment and circulation of some primitive progenitors (dormant in the adult life) as it can be observed during the fetal period. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Primary myelofibrosis (PMF) is myeloproliferative neoplasm characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in bone marrow and spleen. Mesenchymal stromal cells (MSCs) are reported to play a pivotal role in fibrosis and stromal changes are considered as a reactive counterpart of the cytokine production by clonal hematopoietic cells. The present study shows that MSCs from patients demonstrate functional abnormalities that are unexpectedly maintained ex-vivo, in culture. Material and Methods: we studied MSCs and bone marrow sections from PMF patients (n=12) as compared to healthy donors (HDs) (n=6). We tested their proliferation, immunophenotype, hematopoiesis supporting capacities, differentiation abilities, in-vivo osteogenic assays, and performed secretome and transcriptome analysis. Results: We found that PMF-MSCs exhibit similar proliferative capacity and long-term hematopoiesis supporting abilities as compare to healthy donors. They overproduce interleukin 6, VEGF, RANTES, PDGF, BMP-2 and surprisingly TGF-beta1. MSCs from fibrotic PMF patients express high levels of glycosaminoglycans. Adipocytes and chondrocytes differentiation abilities were not different as compared to HDs but PMF-MSCs exhibit an increased in vitro potential. Implementation on scaffold in nude mice confirmed, in vivo, this increased osteogenic potential. We then looked into gene expression and discovered that PMF-MSCs show an original transcriptome signature related to osteogenic lineage and TGF-beta1. Indeed, osteogenic genes such as Runx2, Dlx5, Twist1, Noggin, Sclerostin, GDF5 and Serpine1 are deregulated and suggest a potential osteoprogenitor priming of PMF-MSCs. These molecular results also advocated for a TGF-beta1 impregnation that prompted us to study its impact on PMF-MSCs osteogenic differentiation. First, we then showed that Smad2 is intrinsically over-activated in PMF-MSC and that stimulation by TGF-beta1 is associated with an increase phospho-Smad2 level and an enhancement of bone master gene regulator Runx2 expression. Then, we inhibited TGF-beta1 pathway by by SB-431542 and evidenced a specific behavior of osteogenic MSCs differentiation in patients, suggesting involvement of TGF-beta1 in osteogenic impairment. Conclusion: Altogether, our results identify a signature of PMF-MSCs and suggest that they participate in PMF osteogenic dysregulation independently from in vivo local stimulation by clonal hematopoietic cells Disclosures No relevant conflicts of interest to declare.

Myeloproliferation, myelofibrosis, and neoangiogenesis are the 3 major intrinsic pathophysiologic features of myeloid metaplasia with myelofibrosis (MMM). The myeloproliferation is characterized by an increased number of circulating CD34+ progenitors with the prominent amplification of dystrophic megakaryocytic (MK) cells and myeloid metaplasia in the spleen and liver. The various biologic activities of interleukin 8 (IL-8) in hematopoietic progenitor proliferation and mobilization as well as in neoangiogenesis prompted us to analyze its potential role in MMM. We showed that the level of IL-8 chemokine is significantly increased in the serum of patients and that various hematopoietic cells, including platelets, participate in its production. In vitro inhibition of autocrine IL-8 expressed by CD34+ cells with either a neutralizing or an antisense anti–IL-8 treatment increases the proliferation of MMM CD34+-derived cells and stimulates their MK differentiation. Moreover, addition of neutralizing anti–IL-8 receptor (CXC chemokine receptor 1 [CXCR1] or 2 [CXCR2]) antibodies to MMM CD34+ cells cultured under MK liquid culture conditions increases the proliferation and differentiation of MMM CD41+ MK cells and restores their polyploidization. Our results suggest that IL-8 and its receptors participate in the altered MK growth that features MMM and open new therapeutic prospects for this still incurable disease.

Infection with Aspergillus fumigatus is a common respiratory disease in birds. Any avian species, captive or free, domesticated or wild, is susceptible to this fungal infection (11). With turkeys, aspergillosis leads to consequential economic losses related to low productivity, mortality, and carcass condemnations at slaughter inspection. Two forms of the disease are regularly reported for turkeys. The first form is an acute aspergillosis leading to severe outbreaks in very young birds. Dramatic mortality is then observed in chicks a few days old. Clinical signs usually include dyspnea, gasping, and inappetence. Lesions are first located in the air sacs and the lungs, but dissemination rapidly occurs and accounts for additional clinical signs, such as diarrhea and encephalitis (11, 12). The second form is a chronic aspergillosis occurring in adult turkeys. Birds usually survive, but the resulting low productivity is a source a considerable monetary loss (6, 11). Molecular typing studies should help to elucidate the epidemiology of the different forms of avian aspergillosis. In the present study, a 16-week surveillance program was set in a turkey confinement house where several outbreaks of aspergillosis had previously occurred. Our purpose was to collect and genotype A. fumigatus isolates to characterize environmental sources in the confinement house and to assess whether pathogenic genotypes could be identified in turkeys. We used the polymorphic microsatellite marker (PMM) analysis because this technique has a very high level of discriminatory power, produces reproducible results, and is easy to use (1–3, 7). Animals. A flock comprising 4,500 breeding turkeys (2,250 males and 2,250 females) was subjected to analysis. At the age of 1 day, the animals were placed in a 600-m 2 confinement

To obtain a rapid genotyping method of Candida albicans , three polymorphic microsatellite markers were investigated by multiplex PCR. The three loci, called CDC3 , EF3 , and HIS3 , were chosen because they are on different chromosomes so as to improve the chances of finding polymorphisms. One set of primers was designed for each locus, and one primer of each set was dye-labeled to read PCR signals by using an automatic sequencer. Amplifications were performed directly from the colonies harvested on the agar plate without a sophisticated DNA extraction step. At total of 27 reference strains and 73 clinical independent isolates were tested. The numbers of allelic associations were 10, 22, and 25 for the loci CDC3 , EF3 , and HIS3 , respectively. The combined discriminatory power of the three microsatellites markers was 0.97. The markers were stable after 25 subcultures, and the amplifications were specific for C. albicans . An initial study of 17 clinical isolate pairs, including blood culture and peripheral sites, showed a similar genotype for 15 of them, confirming that candidemia usually originates from the colonizing isolate. Therefore, microsatellite marker analysis with multiplex PCR and automated procedures has a high throughput and should be suitable for large epidemiologic studies of C. albicans .

Chronic myeloid leukemia (CML) is the paradigm of malignancy treated by targeted therapies by the use of tyrosine kinase inhibitors (TKI), essentially Imatinib, Dasatinib and Nilotinib. Despite their major efficiency, especially as first line therapies, resistance to these drugs develop partly due to genetic instability inherent to CML. BCR-ABL-kinase mutations remain the first cause of resistance, which appears to be due to clonal selection of cells bearing a given mutation under TKI therapies. Amongst these mutations, the “gatekeeper” T315I mutant is a major concern as it confers resistance to all three TKI clinically used and patients with this mutation have a poor prognosis. The inaccessibility of the TKI to the ABL kinase pocket might not be the only “mechanistic” cause of resistance and it has been suggested that T315I-mutated BCR-ABL (Skaggs BJ et al, 2006) could induce a specific phosphoproteome signature. To evaluate this possibility, we decided to determine if a specific gene expression profiling can be associated with T315I-mutated BCR-ABL, as compared to native BCR-ABL. The human hematopoietic cell line UT7 was transfected with retroviral vectors encoding for native BCR-ABL (UT7.11) or BCR-ABL with the T315I mutation (UT7.T315I). The cell lines were characterized by their cell growth, Western blotting and sequencing. UT7.11 cells were sensitive to Imatinib, Dasatinib and Nilotinib as well as to Ponatinib whereas UT7-T315I cells were resistant to all three TKI except for Ponatinib. Affymetrix microarrays were performed in triplicate on each of three groups (UT7, UT7.11, UT7.T315I). The datas were normalized using the dchip software. Bioinformatics analyzes were performed with R software (packages FactoMineR, limma, PAMR) Mev in TM4 software, enrichment analysis with the GSEA software (Broad institute). The principal component analysis (PCA) showed that the overall RNA expression of UT7.T315I was different from that of UT7.11 (native BCR-ABL) and parental UT7. On factorial map, UT7.11 was found more distant from parental UT7 than UT7.T315I. The contrast analysis of the linear model by the algorithm limma between the 3 groups, showed a strong differential signature of UT7.11 as compared to parental UT7 and UT7.T315I (respectively 4792 and 4813 genes). Only 800 genes were found to be differentially expressed between UT7.T315I and parental UT7. In hierarchical clustering, the total signature obtained in limma confirmed a closed profile between parental UT7 and UT7.T315I. Among the results of the limma model, we identified a 286 specific genes signature for UT7.T315I (both different from parental UT7 and UT7.11 and also not regulated between UT7.11 and UT7). This specific list of UT7.T315I was validated with the T315I group sample segregation by different multivariate methods: PCA, hierarchical clustering and non-negative matrix factorization. Among this T315I-specific gene list limma, 34 ZNF family genes were found (11.88%). Predicting class algorithm based on shunkren centroid (PAMR) separated the three group samples with low classification error and a global list of 368 genes: only 75 genes predicted UT7.T315I group and from this list 13 were in the ZNF gene family (13.33%). By the method of gene set enrichment analysis (GSEA), we explored the top 100 ranked genes as upregulated in UT7.T315I by comparing the two other sample groups. This gene set showed a high representation of ZNF family genes (25%). The design of a gene set with ZNF family genes selected showed a positive enrichment of ZNF (NES = +1.35, p-value Disclosures: Turhan: Bristol Myers Squibb, Novartis: Consultancy, Honoraria.

Introduction Primary myelofibrosis (PMF) is accompanied by an increase in the bloodstream circulation of some adult progenitor cells. Extramedullary hematopoiesis observed in this setting might remind some features related to foetal hematopoiesis. Material and methods We looked for evidence in favour of this hypothesis in blood samples of a small cohort of untreated patients with PMF (4 pre-fibrotic (PF) and 4 fibrotic (F), defined according to the WHO and Thiele's histopathology score (Blood, 2011)). Patient baseline characteristics are shown below. We performed a) flow-cytometric analysis for cell subsets related to VSEL, PEC, MPC, HPC; b) RT-PCR for embryonic transcriptional factors NANOG, OCT4, SOX2, LIN28 from MNC fraction (positive control hES, negative control CPRE2 c) in-vitro development of embryonic stem like cells (ESlC) under specific culture conditions. In addition we looked for SRSF2 mutations in order to better characterize PMF stages. Results As expected we detected high numbers of circulating CD34+ cells (HPC) (mean 233083±307148/ml (range 4600-783000), with similar numbers in PF- (231125±289553/ml) and F-PMF (235040 ±369156/ml). We were able to detect small numbers of the following cell subsets related to VSEL (size 2-4m) (Fig 1) Lin-/CD45-/CD34+ (mean 124±239/ml), Lin-/CD45-/CD133+ (mean 1178±971/ml), Lin-/CD45-/CXCR4+ (mean 1572±1622/ml). Lin-/CD45-CD34+AC133+CXCR4+ cells were detected in 6 of 8 patients (mean 186±375/ml) with F-PMF patients showing higher numbers (279±416/ml) than PF-PMF (63±71/ml). NANOG and OCT4 expression was detected by RT-PCR in all the patients tested. Mean OCT4 expression was about 50% the level of hES, but F-PMF showed higher levels. NANOG expression was similar to that of hES, whereas Sox2 and Lin28 were not expressed in most patients. We failed to observe the in-vitro development of ESlC in the 2 tested patients. PEC (Lin-/CD45-CD34+AC133+KDR+) were detected in all the PF-PMF (185±332/ml) and in 1 of 4 F-PMF (mean 9±18/ml). MPC (Lin-/CD45-CD90+CD105+) were detected in higher numbers in PF-PMF (mean 413±528/ml) than in F-PMF (mean 157±216/ml). We were not able to detect mutations in the hot spot of SRSF2 (codons 93,94,95). Conclusions Small numbers of cell subsets displaying morphologic and immunophenotypic features of VSELs were detected in PMF patients. However, we are not able to define these as fully specific VSELs according to previous works that defined them (Kucia, Leukemia 2006). Interestingly Lin-/CD45-CD34+AC133+CXCR4+ cells were observed in higher numbers in F-PMF, supporting in part our hypothesis that PMF evolution can be associated to the recruitment and circulation of some primitive progenitors (dormant in the adult life) as it can be observed during the foetal period. This recruitment also involves HPC. Moreover although all patients expressed OCT4 and NANOG, OCT4 expression was higher in F-PMF. As expected PEC circulate in higher numbers in PF-PMF compared to F-PMF. Interestingly both F-PMF and PF-PMF were associated to the circulation of significant numbers of MPC but higher numbers observed in PF-MFP might be interpreted either as a necessary recruitment to establish extra-medullary stroma or due to the exit from bone marrow of highly proliferative MPC. Whether all these different circulating progenitor cells, are clonally or not clonally related to the PMF pathogenesis or to unspecific mobilisation secondary to bone marrow microenvironment injury cannot be determined from these preliminary results. Disclosures: No relevant conflicts of interest to declare.

Results: Approximately 2 million, 76bp reads were randomly generated per sample, providing 5% coverage of the human genome. CNAs were identified and showed no significant aberrations in Macroregenerative nodules (MRN) (n =8). Dysplastic nodules (DN) (n =5) showed development of spikes of amplifications and deletions, which became increasingly complex and involved larger areas of the chromosome as HCC developed (n =22). This progression was demonstrated on chromosomes 1, 5, 7, 14, 22 for amplifications and 1, 4, 6, 14, 16, 22 for deletions (figure 1). Conclusion: NGS can provide CNA data from FFPE material with reasonable costs (£50/sample). Genetic profiles of pre-malignant nodules revealed no changes within MRNs. However lesions start to occur with the onset of dysplasia and become increasingly complex. Initial amplifications and deletions expanded in size with progression. More work is needed to expand cohorts and investigate heterogeneity within individual lesions.

Abstract 3448 Introduction SDF-1/CXCL12 chemokine exhibits a well-known effect on retention, migration and homing of hematopoietic stem/progenitor cells (HSC/HP). We have previously demonstrated that it is also a key regulator of hematopoiesis homeostasis, acting, at low concentrations, as a survival and cell cycle promoting factor for human CD34+ HP. It has long been considered that CXCR4 was responsible for SDF-1 induced biological effects until the recent discovery of its second receptor, CXCR7. In the present study, we explored the respective role of CXCR4 and CXCR7 in the cell cycling and survival promoting effect of SDF-1/CXCL12 on human CD34+HP. Material and Methods We used CD34+ HP purified from the peripheral blood (PB) of healthy un-mobilized donors since they are mainly in G0. This allows to study the role of CXCR4 and CXCR7 receptors in 0.5ng/ml SDF-1/CXCL12 induced G0-G1 transition in synchronized quiescent cells. Gene expression was detected by RT-QPCR. Protein expression was detected and quantified using confocal microscopy, flow cytometry, immunoblotting and immunoprecipitation. Cell cycling experiments were performed using a Ki67 antibody and CXCR7 binding assay was performed using SDF-1/CXCL12AF647. Neutralization experiments were performed using a specific CXCR4 antibody or CXCR7 chemical inhibitors, a kind gift from ChemoCentryx, Inc (CCX771 and CCX733) and their respective controls (IgG and CCX704). Results Flow cytometry and confocal analysis showed that CXCR7 and CXCR4 are differentially distributed in PB CD34+ cells. In contrast to CXCR4 that is present at both the plasma membrane and intracellular level, CXCR7 expression is mainly restricted to the intracellular compartment. Confocal analysis suggested the presence of CXCR4/CXCR7 heterodimers on these cells the presence of which were confirmed by immunoprecipitation in a HP cell line. Despite its very low expression at the surface of CD34+ cells, we found that CXCR7 is capable of binding to exogenous SDF-1/CXCL12. Indeed, pretreatment with CXCR7 antibody or a chemical inhibitor reduces the mean fluorescence of bound fluorescent SDF-1/CXCL12AF647, a fully functional and specific chemokine with similar effects compared to unlabeled SDF-1/CXCL12. Neutralizing either CXCR4 or CXCR7 in PB CD34+ cells strongly reduced Akt activation induced by SDF-1/CXCL12 (0.5 ng/ml) as well as the percentage of cells in cycle (G1 and S + G2/M), colony formation and cell survival. This demonstrates that both receptors cooperate in SDF-1/CXCL12 induced functional effects. We further analyzed the respective role of CXCR4 and CXCR7 in SDF-1/CXCL12 signalization. In contrast to CXCR4, CXCR7 is reported not to activate G protein signaling pathways in response to SDF-1/CXCL12. However, it can transduce cell signaling through the β-arrestin pathway. In the present study, we showed that CXCR7 and β-arrestin 2 colocalize near the plasma membrane in freshly purified PB CD34+ cells, suggesting that CXCR7 is constitutively activated. After SDF-1/CXCL12 treatment, the majority of β-arrestin 2 was translocated to the nucleus and only a partial colocalization persisted in the cytoplasm. Using neutralizing antibodies and specific inhibitors, we showed that β-arrestin 2 nuclear translocation was dependent on both CXCR7 and CXCR4 receptors. Reducing β-arrestin 2 expression using siRNA decreased SDF-1/CXCL12 induced Akt activation in PB CD34+cells indicating the involvement of β-arrestin 2 in this process. Conclusion Altogether, our results demonstrate for the first time the role of CXCR7 together with CXCR4 in SDF-1/CXCL12-induced CD34+ cell cycling/proliferation. They also suggest the involvement of β-arrestin 2 as signalling hubs, downstream of both receptors. Disclosures: No relevant conflicts of interest to declare.

Abstract 1762 Introduction The primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by myeloproliferation, splenomegaly with hematopoietic metaplasia and dysmegakaryopoieisis. We have previously described an increase in Flt3 ligand (FL) in plasma and spleen of patients with PMF and its role in the dysmegakaryopoiesis (Desterke and al, Cancer Res, 2011). Account the importance of FL in development of splenic myeloid dendritic cells (mDC), we studied the differentiation of mDC in patients and its potential impact on dysmegakaryopoiesis. Patients and Methods The mDCs were obtained from cell culture of blood and spleen mononuclear cells in presence of fetal calf serum (FCS) and bacterial lipolysaccharides (LPS). The megakaryocytes were obtained by culturing CD34+ cells from blood or spleen in specific medium serum free in presence of IL-3, IL-6, IL-11 and Tpo. Gene expression was quantified by microarray and RT-QPCR, protein analysis by immunofluorescence and flow cytometry, and migration experiments were performed in Boyden chamber. Results Transcriptome of circulating CD34+ cells from PMF patients showed an increase in expression of genes encoding for integrin CD11c and also TLR4 and a decrease in the expression of gene encoding TLR9: suggesting the presence of progenitor mDCs in the blood. These results have been confirmed 1/ in cytometry by an increase in the number of CD34+ CD11c+ HLA-Dr+ cells in the blood; 2/ in cell culture by the presence of adherent cell colonies positives for TLR4+ CD11c+ HLA-Dr+ in the blood. The in vitro differentiation of mDC cells and the proportion of mature mDCs HLA-Dr+ CD11c+ cells are decreased in blood of PMF patients. Myeloid nature of circulating DCs was confirmed by the absence expression of the plasmacytoid membrane marker CD123 and by an increased of TLR4 and myeloid PU-1 (myeloid transcription factor) expression in opposite to a decreased of plasmacytoid markers: IL-23, HMGB1 and TLR9. Moreover in PMF patients, circulating adherent mDCs overexpressed CXCL12 chemokine and also FL which have abnormal chemottractant ability with respect to PMF megakaryocytic precursors still expressing flt3 receptor. Finally, in PMF patients, coculture of MK with mDC promotes their survival, differentiation and maturation (MK ploidy and transcriptional program). Primary results confirmed the presence of these mDCs precursors (CD34+ CD11c+ HLA-Dr+) in the spleen of PMF patients which harbored also an extramedullary megakaryopoiesis. These mDCs precursors are absent of the spleen from healthy subjects. Conclusion Our results show an increased presence of mDC progenitor population CD34+ HLA-Dr+ CD11c+ in the blood and the spleen of PMF patients. They also suggest that these cells are involved in migration, survival and differentiation/maturation of megakaryocytes, particulary in the spleen of patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 3834 Introduction: CD9, a four transmembrane glycoprotein belonging to the tetraspanin family, is suggested to regulate cell motility and adhesion and to play a role in megakaryopoiesis. It has been reported to be a molecular marker of primary myelofibrosis (PMF) being characterized by myeloproliferation, dysmegakaryopoiesis, alterated bone marrow/spleen stroma and extramedullary haematopoiesis. CD9 mRNA has been shown to be overexpressed in CD34+ PMF HPs and its membrane expression level was correlated with platelet counts. Our recent data evidencing an alteration of CD9 expression in PMF megakaryocytes (MK) have encouraged us to investigate whether CD9 participates in the dysmegakaryopoiesis and whether it is involved in the dialogue between MK and stromal cells in PMF patients. Patients and Methods: CD34+ cells were MACS selected from the peripheral blood of PMF patients (n=67) and of unmobilized healthy donors (n=61). Functional studies were performed on MK precursor-derived from CD34+ cells cultured in MK medium with ou without monoclonal antibodies (Syb mAb) or siRNAs targeting CD9. CXCL12-induced MK migration was performed in Boyden chambers. Bone marrow mesenchymal stromal cells (MSC) from healthy donors and PMF patients were cultured in DMEM+10%FCS. Results: Our results showed that CD9 membrane expression was altered on CD34+ cells and on MK precursor-derived from PMF CD34+ cells. Binding of CD9 with Syb mAb restored the in vitro megakaryocyte differentiation process that was altered in patients as shown by an increase in: i) megakaryocytic colony formation in semisolid medium, ii) CD41 and CD62p MK differentiation marker and GATA-1 expression, iii) MK cytoplasmic maturation, iv) apoptotic MK number (reduced AKT phosphorylation and Bcl-XL expression and increased percentage of Annexin+ cells). Activation of CD9 was also associated with regulation of MAPK and AKT-GSK3β pathways whose balance is involved in MK differentiation. Treatment of PMF MK precursors by Syb modulated activation of the MAPK pathway as shown by an increased of p38, JNK and GSK3β phosphorylation and of AP-1 mRNA expression. Taking into account the structure of the tetraspanin molecular network, binding with Syb mAb might also impact the effects associated to the multimolecular complex in which CD9 is involved. This prompted us to study the effects of a molecular silencing of CD9 on the PMF MK differentiation. We showed that, in contrast to the Syb mAb, addition of CD9 siRNA to PMF megakaryocytes reduced their transcriptional program including c-Myb, a transcription factor that is involved in CD9 regulation during megakaryopoiesis. Given the role of CD9 in cell migration, we further investigated whether it could be involved in the megakaryocytic precursor migration observed in patients. We showed that silencing CD9 reduced the CXCL12-dependent megakaryocytic precursor migration as well as the CXCR4 and CXCL12 transcription and that this migration involved actin polymerization. c-Myb siRNA restored CXCR4 and CXCL12 expression and reduced actin polymerization suggesting that CD9 was involved, via c-Myb, in the CXCL12-dependent megakaryocytic precursor migration. Effect of CD9 on cell migration is often interpreted as related to modulation of integrins participating in the integrin/tetraspanin network and of their interaction with mesenchymal stromal cells (MSC). We showed that several genes involving the CD9 partner interactome were over-expressed in MSC from PMF bone marrow as compared to MSC from healthy donors. Preliminary results showing that PMF MK precursors display different behaviour in terms of cell survival and adhesion when co-cultured on bone marrow MSC from PMF patients as compared to healthy donors suggest that interactions between MKs and bone marrow MSC is involved in PMF dysmegakaryopoiesis. Addition of Syb reverses these alterations suggesting the participation of CD9 in the abnormal dialogue between MK and MSC. Conclusion: Our results show a deregulation of CD9 expression in megakaryocytes from PMF patients. They also suggest that CD9 i) participates in PMF dysmegakaryopoieis in terms of MK differentiation and survival and ii) is involved in the increased MK precursor migration through alterations of the CXCL12/CXCR4 axis. Our data further support the role of bone marrow stroma in PMF dysmegakaryopoeisis through CD9 interactions. Disclosures: No relevant conflicts of interest to declare.

Abstract 2897 Poster Board II-873 Introduction: We have shown in a deficient mouse model that the adaptor protein Lnk had an important role as negative regulator of cytokine signaling during hematopoiesis (Velazquez et al., J. Exp Med 2002). Lnk-/- animals display abnormal megakaryopoiesis sharing many features with that found in MPN patients. This phenotype is due to loss of Lnk inhibition of thrombopoietin (TPO)-mediated JAK2 activation (Tong et al., J Exp Med 2004). Recent studies have shown that Lnk, when expressed in hematopoietic cell lines, could bind and regulate two mutant proteins found in MPNs, JAK2V617F and MPLW515L. However, the role of Lnk in MPN pathogenesis is still unclear. In the present study, we studied in detail both Lnk expression and function in MPNs. Patients and methods: The study included a total of 82 MPN patients (pts), including 41 essential thrombocytemia (ET), 29 polycythemia vera (PV) and 12 primary myelofibrosis (PMF). Lnk expression was assessed by quantitative RT-PCR. Biochemical and cellular analyses of Lnk and JAK2 interaction were carried out using co-immunoprecipitation, GST pull-down and proliferation assays on primary hematopoietic cells and cell lines expressing either wild-type (WT) or mutant forms of both Lnk and JAK2. Results: Lnk mRNA was clearly overexpressed in platelets and CD34+ cells of most MPN pts compared to controls (P=0.005 and P=0.03, respectively). Moreover, this increased Lnk expression strongly correlated with JAK2V617F allele burden (P=0.02). In contrast, Lnk mRNA levels were reduced in the 18 pts treated with interferon-α compared to the 34 pts treated with hydroxyurea (P=0.04). TPO specifically upregulated Lnk expression at both mRNA and protein levels in both primary and UT7/Mpl megakaryocytic (MK) cells. Analysis of TPO-stimulated platelets from ET patients revealed the existence of a new interaction site between Lnk and JAK2 located in the N-terminal region of Lnk, in addition to the previously known interaction mediated by Lnk SH2 domain. This interaction resulted in Lnk phosphorylation. In JAK2V617F expressing platelets or cell lines, we observed both increased phosphorylation of Lnk, and stronger binding of JAK2 to the N-terminal region of Lnk compared to WT-JAK2 cells. Overexpression of Lnk in JAK2V617F cells showed a dose dependent growth inhibition, as seen in JAK2 WT cells. In addition, overexpression of various mutant forms of Lnk showed that this inhibition required a fully functional SH2 domain. Finally, expression of either WT or mutant forms of Lnk also demonstrated the crucial role of Lnk SH2 domain in growth inhibition of myeloid and MK progenitors in Lnk-/- hematopoietic cells. Conclusion: This first study of a large cohort of 82 patients allowed us to investigate the role of Lnk in MPN: (1) Lnk mRNA was found to be significantly overexpressed in MPN derived platelets and CD34+ cells, and correlated with JAK2V617F allele burden. (2) Lnk expression is upregulated by TPO, an effect likely mediated by JAK2 activation. (3) The Lnk SH2 domain plays a major role in the down-regulation of both normal and MPN-derived hematopoiesis. (4) We describe here a novel interaction site between the N-terminal region of Lnk and JAK2. Stronger interaction of the JAK2V617F mutant form with this N-terminal binding site may account for the dysregulated hematopoiesis observed in MPN patients despite Lnk overexpression. Disclosures: No relevant conflicts of interest to declare.

Abstract 963 Introduction: Flt3, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis. Primary myelofibrosis (PMF) is a Ph-negative (Ph−) myeloproliferative neoplasm (MPN) characterized by a myeloproliferation with increased hematopoietic progenitors (HPs) and a prominent proliferation of “dystrophic” megakaryocytes (MK). The JAK2V617F mutation is present in about 50% of PMF patients. Previous results from our group have revealed a MAPK pathway gene deregulation associated with an Flt3 transcript modulation in PMF patients. Since activation of Flt3 receptor is known to activate MAPK pathway, which plays a role in megakaryopoiesis, we studied the functional impact of MAPK and Flt3 abnormalities on PMF dysmegakaryopoiesis. Patients and Methods: The study included a group of 106 PMF patients. Transcriptome and QRT-PCR studies were performed on MACS selected CD34+ HPs, megakaryocytes (MK)-derived from CD34+ cell cultures and peripheral blood mononuclear cells (PBMNC) from PMF patients and healthy donors. Cell phenotype associated with Flt3 expression as well as phosphorylation levels of Flt3 and MAPK effectors were analyzed by flow cytometry. Functional studies (FL-induced stimulation and migration) were performed on MK-precursors at day 6 of CD34+ culture. Effect of i) MAPK inhibitors: PD98059, targeting ERK1/2; SB202190, SB203580, PD169316, targeting p38 and, SP600129, targeting JNK, ii) Flt3 inhibitors and iii) Flt3 monoclonal antibody was tested on PMF MK cell cultures. MAPK-induced transcripts were quantified by QRT-PCR in MK-precursors during an 18-hour FL-stimulation kinetic. FL mRNA level was evaluated by using QRT-PCR in bone marrow stromal cells and FL protein was quantified in plasma by using ELISA. Results: Comparative transcriptome analysis of CD34+ HPs and MK cells from PMF patients (with or without JAK2 mutation) and healthy donors showed that the MAPK pathway gene deregulation was independent of the presence of the JAK2V617F mutation. This alteration was associated with a modulation of mRNA Flt3 level in both types of cells. PMF patients also had a higher proportion of circulating Flt3+CD34+CD41+ cells as compared to healthy donors. This population demonstrated an increased phosphorylation of Flt3 on tyr591 and of MAPK (p38, p42/p44, JNK). MAPK effector phosphorylation was also increased in PMF CD34+ cells and MK-derived from CD34+ cell cultures, independently of JAK2 mutational status. In contrast to healthy donors, Flt3 membrane expression was maintained at all stages of in vitro megakaryocyte differentiation in PMF patients. The FL level was increased in the plasma of patients and was mainly expressed by bone marrow stromal cells. In contrast to healthy donors, in MK-derived from PMF CD34+ cell cultures, activation of Flt3/FL axis by addition of exogenous FL induced a MAPK hyper-phosphorylation, especially of p38 and p42/p44 as well as an up-regulation of downstream p38 transcripts (ATF-2, NFATC4, p53, AP-1, IL-8). Addition of chemical inhibitors targeting either MAPK or Flt3 and of an antibody directed against Flt3 reduced the phosphorylation of p38 and of its pathway effectors (MKK3/MKK6, MSK1, ATF2, HSP27 and MAPKAPK2) and normalized the PMF altered megakaryopoiesis. Lastly, in contrast to healthy donors, MK-derived from PMF CD34+ cells showed a FL-induced migration that was reversed by addition of p38αβ inhibitors. Conclusion: Our results demonstrated an increase in the FL circulating level in PMF patients that was mainly secreted by stromal cells. This was associated with an aberrant expression of Flt3 in CD34+ and MK cells and an alteration of the MAPK pathway activation in patients, independent of their JAK2 mutational status. The persistence of Flt3-mediated MAPK activation that participates in the PMF dysmegakaryopoiesis, suggests that drugs targeting “FL/Flt3-MAPK” axis could be promising agents for rescuing the altered megakaryopoiesis observed in patients. Our demonstration that FL, a cytokine mainly produced by stromal cells, participates in the altered megakaryopoiesis in PMF patients strengthens the hypothesis highlighting the crucial role of stroma cells in the hematopoietic deregulation that characterizes the disease. Disclosures: No relevant conflicts of interest to declare.

Control of cell cycling is a key process in stem cell fate. A balance between cell quiescence and proliferation within the stem cell niche in interaction with microenvironment is critical for sustaining long-term hematopoiesis and for protection against stress. Among growth factors regulating haematopoietic stem/progenitor cell (HSC/HP) cell cycling, TGF-β is known to be a major negative regulator by blocking cell cycle progression during the G1 phase. Besides its effect on HSC/PH and lymphocyte trafficking, the SDF-1/CXCL12 chemokine is another key regulator which helps maintaining hematopoiesis homeostasis. We have previous demonstrated that SDF-1 acts as a survival and cell cycle promoting factor for CD34+ HSC/HP by triggering the G0-G1 transition and then regulating early cell cycle phases suggesting that it may act as an antagonist of TGF-β on cell cycle progression (Lataillade et al., Blood 2000 and 2002). The general aim of this study was to investigate the mechanisms of this antagonist effect on cell cycle. This work was performed on CD34+ cells purified from the peripheral blood of healthy un-mobilized donors. Since they are mainly in G0, they allow dissecting the early cell cycle phases including the G0-G1 transition. We demonstrated that SDF-1 and TGF-β exert an opposite effect on the expression of cell cycle key regulators such as cyclins and CDKI. We showed that cross-talk between SDF-1 and TGF-β signaling pathways involving PI3K/Akt phosphorylation participates in the control of CD34+ cell cycling. We demonstrated a pivotal role of FoxO3 and mTOR in the TGF-β/SDF-1 control of the quiescence/cycling of hematopoietic progenitors. A model integrating a pivotal role for the activation of two Akt substrates, FoxO3 and mTORC1, in the control of CD34+ cells quiescence/cycling by TGF-β and SDF-1 is proposed. Altogether, our results shed new light on the intracellular signaling mechanisms of SDF-1 and of its implication, together with TGF-β, in cell cycle promotion in primary CD34+ cells. Regarding their opposite effect on cell cycle and survival, we suggest that SDF-1 and TGF-β can be considered as natural mutual competitors, both acting as organizers of the stem cell niche where a balance between HSC hibernation and activation is an essential part of the stemness control.

Hematopoietic stem cells (HSC) are characterized by their potential to reconstitute in vivo haematopoiesis in NOD/SCID mice and to give rise to differentiated cells of all haematopoietic lineages. They are usually defined by a CD34+CD38−CD90+ antigenic profile; however taking into account the versatility of antigen membrane expression (Lataillade et al., J. Leukoc. Biol. 2005), more reliable methods based on their selective functional/metabolic activities such as ABCG2 activity and Aldehyde Deshydrogenase expression have been developed. Actually, HSC can be purified on basis of Hoechst 33342 die exclusion property due to the membrane ABCG2/BRCP1 transporter expression (SP cells). SP cells could be isolated from a wide variety of human and mammalian tissues and in many cases have been shown to contain multipotent stem cells; SP cells are heterogeneous since the lowest Hoechst fluorescent profile exhibited the highest primitive hierarchical level. Hematopoietic progenitors also expressed ALDH enzymatic activity and the use of BODIPY-AminoAcetAldehyde (BAAA), a fluorescent substrate of the ALDH enzyme, allows analyzing its expression by using Fluorescence Activated Cell Sorting (FACS). It has been reported that there is a close association between high ALDH expression (ALDHbr) and high HSC activity. Based on the hypothesis that the more primitive HSC should express both ALDH high expression level and SP profile, we developed a new multiparamatric flow cytometric assay combining an ALDH/SP co-labelling to identify different haematopoietic progenitor subpopulations. Our strategy allows identifying six lineage negative (Lin−) haematopoietic sub-populations from human bone marrow cells according to the co-expression of ALDH levels and SP phenotype: SP+ALDHBr, SP−ALDHBr, SP+ALDHMid, SP−ALDHMid, SP+ALDHLo and SP-ALDHLo. We showed that these different sub-populations exhibited specific antigenic patterns and demonstrated variable differentiation properties in long term cultures and clonogenic assays. Whatever the ALDH expression level, SP+ and SP−cells expressed the CD34 antigen, however, the SP phenotype identify a higher proportion of CD34+CD38− cells. Furthermore, in contrast to the other subpopulations, Lin-SP+ALDHBr cells were highly enriched with CD34+CD38−CD90+ progenitor cells and demonstrated a higher amplification and high clonogenic efficiency in liquid LTC-IC cultures than SP-ALDHBr cells. This subpopulation allowed obtaining all myeloid haematopoietic lineage differentiated cells after 30 days culture in liquid culture, including osteoclastic cells. Interestingly, in contrast to ALDHBr cells, ALDHLo sub-populations were highly enriched with megakaryocytic CD34+CD41+ progenitors that gave rise to CD41+CD42a+ cells. SP+ALDHMid and SP-ALDHMid have intermediate amplification, clonogenic and differentiation abilities as compared with the other subpopulations. In conclusion, our results show that the combined SP/ALDH technology allows discriminating a hierarchy within the human hematopoietic stem/progenitor compartment, the most primitive one being SP+ALDHBr. The stemness of these subpopulations is currently explored by testing their capability to reconstitute long term haematopoiesis in NOD/SCID mice.

Myeloid Metaplasia with Myelofibrosis (MMM) is a myeloproliferative disorder (MPD) associating ineffective and extramedullary hematopoiesis with progressive splenomegaly, bone marrow fibrosis and neoangiogenesis. The myeloproliferation is characterized by an increased number of circulating CD34+ cells with the prominent amplification of dystrophic megakaryocytes (Mk). We compared the transcriptome of CD34+ and Mk cell from the peripheral blood (PB) of MMM patients and from the PB and bone-marrow (BM) of unmobilized healthy donors. Application of multivariate analyses (principal component analysis and classification by partitioning around medoids algorithm) on Gene Ontology annotation of differential genes allowed a global functionally approach of the main cellular dysregulated pathways in MMM. Each sample cRNA probe was individually and differentially hybridized to the cRNA reference probe on a Human Oligo-microarray 22K (Agilent) and data were normalized by application of the local LOWESS algorithm. Comparison of the CD34+ cell transcriptome between MMM and healthy donors revealed a global down regulation of 2/3 of the expressed genes in contrast to 1/3 of genes that are up-regulated after data filtration by significance analysis microarray (SAM) algorithm (threshold p

Myeloid Metaplasia with Myelofibrosis (MMM) is a Philadelphia negative chronic myeloproliferative disorder associated with myeloid metaplasia in the spleen and liver, bone marrow fibrosis and neoangiogenesis. The myeloproliferation is characterized by an increased number of circulating CD34+ hematopoietic progenitors (up to 200 folds) with a prominent amplification of dystrophic megakaryocytes (Mk). Alteration of tyrosine kinase signals is suspected to play a key role in the myeloproliferation and in the increased sensitivity of hematopoietic progenitors to growth factors. Glivec is a small-molecule tyrosine kinase inhibitor of the ABL fusion gene, platelet derived growth factor receptor beta (PDGFRB) and SCF receptor (c-kit). In this study, we investigated the effect of Glivec/STI571 on the megakaryopoiesis and the kinase expression of 11 MMM patients enrolled in a European EUMNET clinical trial. We showed that 50 percents of the treated patients exhibited a tendency to a decrease in the CD34+ cell number from 261/μL to 56/μL with a normalization of the CD34+ cell count (1–10/μL) in 20 percents of these patients (2/10). We also observed a reduction of a CD34+CD41+ Mk subpopulation co-expressing the CD9 tetraspanin that is suggested to be a marker of MMM. Normalization of the CD9 gene expression level was further confirmed by quantitative RT-PCR in MMM mononuclear cells. In contrast, 55 percents of MMM patients (5/9 patients) showed an increase in their platelet count from 191 x 109/L to 282 x 109/L during treatment with Glivec. Interestingly, an increase in the platelet count was also observed in three of the five thrombopenic patients who exhibited an increase greater than 50 x 109/L (p= 0.02) being associated with an augmentation in the number of clonogenic megakaryocytic progenitors (CFU-Mk). The effect of Glivec treatment on megakaryopoiesis was further illustrated by a morphometric analysis on bone marrow histological sections showing a normalization of the size and shape of the dystrophic MMM Mk. We also analyzed by quantitative RT-PCR the effect of Glivec treatment on receptor kinase gene expression in circulating mononuclear cells from MMM patients and demonstrated a normalization of the receptor beta of the PDGF, a cytokine involved in Mk differentiation. Actually, the PDGFB expression that was down-regulated in MMM PBMC before Glivec as compared to normal PBMC (−1.14 +/− 0.76 and − 0.14 +/− 0.42, respectively; p= 0.02) was significantly increased during therapy, reaching a normal level in 60% of tested patients. In conclusion, our results show that Glivec treatment reduced the CD34+ cell number and stimulated the megakaryopoiesis of MMM thrombopenic patients, probably by restoring Mk progenitor differentiation.

MMM is a myeloproliferative disorder characterized by extramedullary hematopoiesis and reactive myelofibrosis. Recently, HMGA2 dysregulation has been demonstrated in 2 MMM patients showing 12q15 rearrangement and confirmed in 25 consecutive MMM patients without cytogenetic abnormalities (Andrieux, 2004). HMGA2 proteins belong to the high mobility group A (HMGA) family of architectural transcription factors regulating the expression of several genes. As MMM is a clonal disorder of CD34+ hematopoietic progenitors, we analyzed HMGA2 expression in peripheral blood sub-populations of 5 MMM patients and 7 healthy donors to determine in which sub-population HMGA2 was dysregulated. RNA was extracted from peripheral blood mononuclear cells (PBMC) and CD15+ granulocytic cells (PBCD15+) separated through Ficoll centrifugation or from immunomagnetically selected circulating CD34+ cells (PBCD34+). Real-time quantitative PCR (RQ-PCR) using Taqman technology was performed on cDNA. As different isoforms were described in malignancies, we used two primer sets : the first one allowing the amplification of all HMGA2 isoforms (exon 1 to 3) (HMGA2 1–3), the second one allowing the amplification of the full length HMGA2 isoform (exon 1 to 5)(HMGA2 1–5). In healthy donors and in MMM, PBMC HMGA2 expression levels were heterogeneous, depending of the cellular sub-population purity. HMGA2 1–3 or HMGA2 1–5 were both expressed in MMM and normal PBCD34+ cells, but with a higher expression level for HMGA2 1–3 as compared to HMGA2 1–5. Furthermore, both HMGA2 1–3 and HMGA2 1–5 expression levels were significantly increased in PBCD34+ MMM patients (p98%) nor in PB neutrophils from other myeloproliferative disorders (Polycythemia Vera and Essential Thrombocythemia). To determine if HMGA2 level was modified during hematopoietic differentiation, we quantified HMGA2 1–3 and 1–5 isoform expression on purified healthy donor PB CD34+ and MMM CD34+ before and after culture with specific lineage growth factors (12 day-culture). Primary results showed that both HMGA2 isoform expression levels were higher during granulocytic differentiation. Our results demonstrate that HMGA2 1–5 isoform is discriminately overexpressed in MMM PBCD15+. The persistence of this HMGA2 full length expression in MMM myeloid lineage could be considered as a marker of the disease.