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The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, suggesting that MYB may be a therapeutic target in these diseases. However, realization of this potential requires a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis, and an approach for developing an effective therapeutic. We previously showed that the interactionof c-MybwiththecoactivatorCBP/p300 is essential for its transforming activity. Here, by using cells from Booreana mice which carry a mutant allele of c-Myb, we show that this interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type mice, Booreana cells transduced with AML1-ETO9a or MLL-AF9 retroviruses fail to generate leukemia upon transplantation into irradiated recipients. Finally, we have begun to explore themolecularmechanisms underlying these observations by gene expression profiling. This identified several genes previously implicated in myeloid leukemogenesis and HSC function as being regulated in a c-Myb-p300-dependent manner. These data highlight the importance of the c-Myb-p300 interaction inmyeloid leukemogenesis and suggest disruption of this interaction as a potential therapeutic strategy for acute myeloid leukemia. Refereed/Peer-reviewed

We have previously shown vascular E (endothelial)-selectin to play a key role in niche-mediated chemo-resistance in Acute Myeloid Leukaemia (AML). Now we report that the cell surface glycosylation of AML blasts -and thus their E-selectin-binding potential- alters during therapy and queried whether these variations influence treatment outcome. Using preclinical mouse models of 11q23-rearranged (MLL-AF9) monomyelocytic AML, we found that although AML blasts in untreated mice display a range of E-selectin-binding potentials, the blasts with highest E-selectin-binding potential dominate in bone marrow (BM) after 24hr cytarabine therapy. Indeed, the highest 10% of AML blasts for E-selectin-binding were >12-fold (p=0.0014) more likely to survive post-chemotherapy. These data raise the question whether E-selectin binding potential itself may prospectively identify AML blasts with heightened regenerative potential. An alternative explanation could be that high-binding AML blasts predominate after chemotherapy simply due to survival advantages mediated by vascular niche E-selectin interaction. To investigate this, BM AML blasts were FACs sorted from donor C57BL/6 mice based on E-selectin-binding potential (highest and lowest 10%) and transplanted into recipient mice (1,500 AML blasts/recipient, 8/gp). No significant differences in duration of disease-free survival was observed. Thus E-selectin-binding potential itself does not prognostically identify the most potent Leukemia Reconstituting Cells (LRC) that initiate relapse. To determine instead whether the AML blasts that bind E-selectin can dominate during stress because of the survival advantage of interacting with E-selectin at the niche, an identical parallel experiment was performed, the only difference being that donor mice had E-selectin blocked (GMI-1271 100mg/kg BiD) for the last 48h prior to BM harvest, FACs sort of AML blasts and recipient mouse transplant. This time we observed a significant (2-fold) extension in disease-free survival in the recipients of high-binding AMLs (from donors treated with GMI-1271) compared to all other groups (p=0.012, median disease-free survival 33 vs. 63 days). Together these data indicate that administration of E-selectin antagonists, even as a single agent, may potentially improve patient outcomes - in cases where heightened E-selectin binding potential is observed. Next we investigated the intracellular AML signaling pathways potentially dampened by E-selectin absence/blockade. Two common pathways used by malignant cells for survival/regeneration are the PI3K/AKT/mTOR/ NF-kB and RAS/MAPK/ERK pathways. We have already shown the absence (in Sele-/- mice), or therapeutic blockade of E-selectin (with GMI-1271) in mice significantly dampens PI3K/AKT/NF-kB signaling in BM AML blasts in vivo. However, AML blasts can utilize alternative pathways such as RAS/MAPK/ERK for survival signaling as well, especially in the 30% of AMLs with NRAS mutations. So we determined whether MAPK/ERK signaling in AML could be similarly altered by E-selectin absence/blockade. Indeed MAPK/ERK phosphorylation was significantly reduced (2-fold) in BM AML blasts from host mice treated 24hr with GMI-1271 and in Sele-/- hosts. Thus contact with vascular E-selectin induces a range of survival/regenerative signaling pathways within BM AML blasts that would be highly advantageous for the blast in times of stress. In summary we show, (1) vascular niche E-selectin blockade by GMI-1271 dampens malignant AML reconstitution/survival potential in vivo when administered as sole agent alone, (2) That E-selectin blockade mediates these effects via dampening a range of intracellular survival/regeneration signalling pathways in the malignant cell, and finally (3) these data suggest E-selectin blockade may synergise with other specific pathway inhibitors to improve treatment outcomes - but only for malignant cells that are appropriately glycosylated to interact with E-selectin. Disclosures Winkler: GlycoMimetics: Patents & Royalties. Levesque:GlycoMimetics: Equity Ownership. Magnani:GlycoMimetics Inc: Employment, Equity Ownership.

We have recently found that the vascular adhesion molecule E(endothelial)-selectin is a critical bone marrow niche component mediating acute myeloid leukemia (AML) chemo-resistance. Clinical trials involving the use of E-selectin mimetics to improve efficacy of conventional AML therapy are in progress. In this study we investigate the identity of the AML cell surface receptors mediating vascular E-selectin-induced chemo-resistance. E-selectin has two well-characterised receptors, CD162 also known as P-Selectin Glycoprotein Ligand-1 (PSGL-1), and CD44 isoform HCELL (Hematopoietic Cell E-selectin/L-selectin Ligand) with other cell surface glycoproteins (such as ESL-1) and glycolipids also binding E-selectin after Sialyl Lewisx/a glycosylation. To investigate which of these AML cell surface receptors are responsible for mediating vascular E-selectin survival signalling, we first investigated if each co-localized with E-selectin on AML cell surface by confocal imaging. Human CD34+ AML KG1a cells were labelled ± adhesion to fluorescently E-selectin-IgM. Confocal imaging revealed that although both canonical CD44 and CD162 receptors co-localized with E-selectin at the site of cell contact, only CD162 became strongly polarized at E-selectin binding site, while CD44 remained widely distributed across the cell surface. To dissect a functional role for each of these canonical receptors in human AML, CRISPR-Cas9 gene editing was used to selectively delete CD44 and/or CD162 from human AML KG1a cells. We found that although deletion of both CD44 and CD162 receptors reduced KG1a E-selectin-IgM binding potential (3-fold), deletion of either receptor alone did not. Next, we investigated whether deletion of either receptor reversed E-selectin-mediated chemo-resistance in an in vitro chemosensitivity assay. KG1a cells were seeded in wells pre-coated with a range of vascular adhesion molecules commonly expressed in the bone marrow niche, then monitored for cell survival after 48hr treatment ± cytarabine. In this in vitro assay, we found that adhesion to E-selectin significantly increased parental KG1a survival to chemotherapy (p=0.0035). No similar increase in survival was observed following adhesion to P-selectin, or with integrin ligands ICAM-1 and PE-CAM-1. When we repeated the assay using Crispr CD44 deleted KG1a AML we found significant E-selectin-mediated chemo-resistance was still observed (p=0.027) even in the absence of CD44. These results suggest CD44 is not the receptor mediating AML chemo-resistance. In contrast E-selectin-mediated chemo-resistance was abrogated in the CD162 Crispr deleted human KG1a AMLs. Together these data suggest CD162/PSGL-1 expressed on the surface of human AML KG1a appears to be the receptor mediating vascular E-selectin chemo-resistance. This would be a completely novel role described for CD162 which is conventionally known as a homing molecule. To confirm this new role for CD162 in mediating AML chemo-resistance can be replicated in pre-clinical models in vivo, we next generated (11q23-rearranged) AML from CD44-/- and/or CD162-/- gene-deleted mice by retroviral transduction of murine hematopoietic stem cells with MLL-AF9 which then were transplanted into wildtype mice. Cohorts of leukemic mice (n=8/gp) were administered induction therapy (cytarabine/doxorubicin) to monitor impact on disease-free survival. In contrast to AMLs from wildtype, we found absence of CD162 in murine AMLs lead to a pronounced chemo-sensitisation in vivo resulting in a significant (6-fold, p=0.0004) extension in overall disease-free survival duration, compared to either no chemotherapy gene-deleted AML controls, or to treated wildtype AML controls. These in vivo murine data confirm the identification of an exciting new role for CD162 as an important cell surface receptor mediating therapy resistance in AML. In conclusion, we describe a novel form of niche-mediated chemo-resistance and identify CD162 as a key AML cell surface receptor involved in both human and mouse AML therapy resistance. CD162/PSGL-1 expression has not previously been implicated in direct therapy resistance. Together these findings help extend our knowledge on the potential mechanisms by which therapeutic blockade of vascular E-selectin can significantly improves therapy outcomes. Disclosures Levesque: GlycoMimetics: Equity Ownership. Magnani:GlycoMimetics Inc: Employment, Equity Ownership. Winkler:GlycoMimetics: Patents & Royalties.

Granulocyte colony-stimulating factor (G-CSF) and the CXCR4 chemokine receptor antagonist Plerixafor are used to mobilize hematopoietic stem and progenitor cells (HSPC). However, in the autologous setting, prior chemotherapy and radiotherapy impair HSPC mobilizing response to G-CSF and Plerixafor thereby precluding autologous HSC transplantation. We now demonstrate in mice that oncostatin M (OSM) production in the bone marrow (BM) is dramatically increased in response to G-CSF and that OSM counteracts the mobilizing effect of G-CSF and Plerixafor indirectly through the BM stroma. Indeed, by immunohistochemistry, a 4-day G-CSF treatment strongly increased OSM protein expression in cell clusters containing F4/80+ macrophages in the endosteal and central region of the mobilized BM. OSM concentration was 7-fold higher in BM fluids of G-CSF mobilized mice compared to control mice as measured by ELISA. To investigate OSM effects on HSPC mobilization, C57BL/6 mice (WT) and mice lacking the OSM receptor gene (OSMR-/-) were treated with 125 μg/kg human G-CSF bidaily for 2-6 days and/or Plerixafor (10 mg/kg) one hour before harvest. Absence of OSMR increased HSPC mobilization with a 4-fold increase in colony forming cells (CFC), phenotypic Lin-Kit+Sca1+ HSPC (LKS+ cells) and LKS+Flt3-CD48-CD150+ HSC into the blood at days 2 and 4 of G-CSF treatment and increased accumulation of HSPC and HSC in the spleen of OSMR-/- mice at day 6 of G-CSF treatment. In a competitive repopulation assay with serial dilutions of mobilized blood, deletion of OSMR gene enhanced ≈4-fold mobilization of functional reconstituting HSC in response to G-CSF as shown by Poisson's distribution analysis. HSPC mobilization in response to Plerixafor was also significantly enhanced with a doubling of CFC, HSPC and HSC into the blood of OSMR-/- mice compared to WT. OSMR gene deletion also significantly enhanced HSPC mobilization (3-fold) in response to G-CSF plus Plerixafor combination, the most effective mobilization regimen currently approved. To further demonstrate that endogenous OSM restrains HSPC mobilization in response to G-CSF, we produced a recombinant mouse OSM-trap, a dimeric protein chimera made of the extracellular domain of mouse OSMR and mouse gp130 fused with a mutant Fc fragment of mouse IgG2a. Co-administration of this OSM-trap together with G-CSF also increased HSPC and HSC mobilization into the blood in response to G-CSF compared to mice treated with G-CSF and control trap construct. This negative effect of endogenous OSM on HSPC mobilization was indirectly mediated by BM stromal cells. qRT-PCR on BM myeloid and stromal cells sorted from steady-state or mobilized mice showed that Osm mRNA is mostly expressed by CD11b+F4/80+CD169+VCAM1+ macrophages and CD11b+F4/80-Ly6G+ neutrophils. In sharp contrast, Osmr mRNA was undetectable in any sorted BM leukocyte population, but abundantly expressed by CD45-Lin-CD31+Sca1+CD105+ endothelial and CD45-Lin-CD31-CD51+PDGFRα+/-Sca-1+/- mesenchymal progenitor cells. OSMR protein was expressed by vascular beds in the BM as detected by IHC. Confirming the indirect effect of OSM, we found that recombinant OSM had no effect on HSPC chemotaxis in response to CXCL12, or HSPC adhesion to immobilized VCAM-1 or selectins in vitro. However, HSPC freshly isolated from the BM of OSMR-/- mice had enhanced chemotaxis in response to CXCL12 compared to HSPC isolated from WT mice. In conclusion, G-CSF increases OSM expression in the BM, and this appears to restrain HSPC mobilization in response to G-CSF via OSMR-expressing endothelial and mesenchymal cells that decreases HSPC responsiveness to CXCL12 gradient. Disclosures Winkler: GlycoMimetics: Patents & Royalties. Levesque:GlycoMimetics: Equity Ownership.

Hematopoietic stem cells (HSC) reside in specific peri-vascular niches in the bone marrow (BM). We have shown interactions with the inflammatory vascular adhesion molecule E-(endothelial)-selectin awakens HSC (Nat Med 2012). We now report that BM vascular cell-surface E-selectin expression is strongly upregulated during HSC mobilization regimens raising the question of a role for endothelial E-selectin in HSC transplant outcome. G-CSF was administered to cohorts of E gene-deleted or wildtype C57BL/6 mice together with E-selectin antagonist Uproleselan (Upro, GMI-1271). We found absence (Sele-/- mice) or therapeutic blockade (Upro) of E-selectin alone in steady-state hosts did not alter levels of circulating peripheral blood (PB) HSC. In contrast absence or therapeutic blockade of E-selectin strongly synergized with mobilizing regimens such as G-CSF or cyclophosphamide+G-CSF by boosting long-term engraftment and reconstitution potential of mobilized blood. The effect was pronounced boosting reconstitution potential over G-CSF-alone-mobilized blood 24-fold ( Two hypotheses may explain how therapeutic E-selectin-blockade promotes potency of mobilized HSC;Dampening of inflammatory activation at the BM niche induced by G-CSF that drive HSC exhaustion,Directly preventing adhesion-mediated HSC commitment during trans-endothelial transmigration from BM into blood. To investigate inflammatory mediator profiles in the BM, cohorts of mice were administered saline control or G-CSF ± Upro (125ug/kg G-CSF, 40mg/kg Upro BiD for 3 days) then femoral BM fluids flushed for cytokine profiling (LegendPlex). As anticipated, G-CSF administration significantly increased concentration of classic pro-inflammatory cytokines (TNF-α, IL-1β, IL-23, IFN-β) in BM associated with HSC activation and loss of quiescence in the BM. No similar boost in inflammatory mediators was observed in BM from mobilized mice co-administered Upro. Similarly pronounced metabolic changes could be observed in BM HSC following in vivo G-CSF administration (such as a significant doubling in HSC Mitochondrial Membrane potential [MOMP] suggesting increased energy demands with HSC activation) was similarly reversed in vivo by Upro co-administration. Together these results suggest blockade of E-selectin on activated vasculature significantly dampens BM HSC activation following G-CSF administration - potentially shielding HSC self-renewal potential. Next we investigated whether the transient interactions with endothelial E-selectin during trans-endothelial transmigration from BM into blood also directly affects HSC potency. BM HSPC harvested from G-CSF plus Upro-treated mice were loaded in transwells pre-coated with recombinant adhesion molecules (P-selectin, E-selectin and CD14-Fc as control) and HSC induced to transmigrate through coated pores towards CXCL12 gradient. After 3hr cell numbers were enumerated and exactly 100 transmigrated HSC from each well transplanted/recipient in a LT-CR transplant assay. Analysis of % CD45.2+ donor HSC reconstitution confirmed a pronounced 10-fold drop in reconstitution potential of HSC transmigrated through E-selectin compared to P-selectin or control coated transwells (p=0.0027) confirming that transient adhesive interactions with E-selectin, such as would occur on activated vasculature during HSC transmigration from BM into the blood, strongly impact subsequent HSC reconstitution upon transplantation. In summary, transient interactions between intravasating HSC with E-selectin on BM vasculature inadvertently compromises reconstitution potential of up to 96% of conventionally harvested HSC, indicating an unexpected disadvantage with current HSC harvesting procedures. These studies also point the way forward to a simple remedy, administration of E-selectin antagonist (Upro) together with G-CSF during HSC mobilization, to improve HSC transplant outcomes. Disclosures Winkler: GlycoMimetics: Patents & Royalties. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Marlton:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlycoMimetics: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Perkins:Novartis Oncology: Honoraria. Levesque:GlycoMimetics: Equity Ownership, Patents & Royalties.

Haematopoietic stem cells (HSCs) are regulated by their immediate microenvironment or niche. The most potent functional HSCs are enriched at the endosteum near the bone, which comprises ~10% of total bone marrow (BM). To identify novel niche factors that regulate HSCs, we performed a gene expression microarray seeking genes that were >2-fold overexpressed in the endosteal BM relative to the central BM. In this screen, we uncovered known essential HSC niche factors overexpressed in the endosteal BM such as Scf, Cxcl12, and Angpt1, which validated our approach. Among the genes overexpressed in the endosteal BM, prostaglandin I2 (PGI2) synthase (Ptgis) was one of the highest enriched genes in the endosteum (>10-fold by qRT-PCR, p In initial experiments where we cultured fluorescence activated cell sorted (FACS) BM lineage- Kit+ Sca-1- (LKS+) haematopoietic stem and progenitor cells (HSPCs) for 7 days in serum free conditions, we found iloprost treatment potently reduced proliferation and differentiation compared to vehicle controls, assessed by flow cytometry phenotyping (p We next sought to determine whether iloprost affects HSC function in vivo. In steady state, low dose 0.1mg/kg iloprost administration to mice for 15 days did not alter BM HSPC phenotypes by flow cytometry nor HSC reconstitution potential in competitive transplants compared to vehicle controls suggesting PGI2 does not alter HSC function in homeostasis. To further test the effect of PGI2 following stress, mice were sub-lethally (6.5 Gy) irradiated or administered pro-inflammatory granulocyte colony stimulating factor (G-CSF) for 3 days. We found low dose iloprost administration partially rescued BM HSC reconstitution potential 21 days following irradiation (p To understand the extrinsic mechanisms through which PGI2 regulates HSC in the BM following stress, we performed a HSC homing assay using naïve donors transplanted into 2-day G-CSF administered recipients. Analysis of HSPC homing to the BM at 4 hours post-transplantation revealed ~4-fold greater proportion of LKS+ HSPC homing to the BM of iloprost co-administered recipients compared to vehicle controls (p In summary, we have identified PGI2 as a novel HSC niche factor abundant in the endosteal BM. PGI2 analogues like Iloprost are well-tolerated and used clinically to treat vascular diseases such as pulmonary arterial hypertension and Raynaud's phenomenon. Our research suggests that PGI2 analogues can be rapidly repurposed in the clinic to improve HSC transplant outcomes and protect against BM failure following acute stressors such as accidental irradiation or inflammation. Disclosures Levesque: GlycoMimetics: Equity Ownership, Patents & Royalties.

In the bone marrow, hematopoietic stem cells (HSCs) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate the regulation of these endosteal niches, we studied the mobilization of HSCs into the bloodstream in response to granulocyte colony-stimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts, leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression, and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed, in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the: (1) loss of endosteal osteoblasts and (2) marked reduction of HSC-trophic cytokines at the endosteum, with (3) HSC mobilization into the blood, as observed during G-CSF administration. Together, these results establish that bone marrow macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSCs into the blood.

Accumulating evidence indicates that interaction of stromal cell-derived factor 1 (SDF-1/CXCL12 [CXC motif, ligand 12]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During granulocyte colony-stimulating factor (G-CSF)–induced HPC mobilization, CXCL12 protein expression in the bone marrow decreases. Herein, we show that in a series of transgenic mice carrying targeted mutations of their G-CSF receptor and displaying markedly different G-CSF–induced HPC mobilization responses, the decrease in bone marrow CXCL12 protein expression closely correlates with the degree of HPC mobilization. G-CSF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. Cell sorting experiments showed that osteoblasts and to a lesser degree endothelial cells are the major sources of CXCL12 production in the bone marrow. Interestingly, osteoblast activity, as measured by histomorphometry and osteocalcin expression, is strongly down-regulated during G-CSF treatment. However, the G-CSF receptor is not expressed on osteoblasts; accordingly, G-CSF had no direct effect on osteoblast function. Collectively, these data suggest a model in which G-CSF, through an indirect mechanism, potently inhibits osteoblast activity resulting in decreased CXCL12 expression in the bone marrow. The consequent attenuation of CXCR4 signaling ultimately leads to HPC mobilization.

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9–deficient mice, NE × CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE × CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12α protein expression in the bone marrow of Ne × CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization.

The cellular and molecular mechanisms responsible for hematopoietic progenitor cell (HPC) mobilization from bone marrow (BM) into peripheral blood after administration of cytokines such as granulocyte colony-stimulating factor (G-CSF) are still unknown. In this study we show that high concentrations of soluble calcium induce the detachment of BM CD34+ HPC adherent on fibronectin, a major component of BM extracellular matrix. Because G-CSF has been shown to induce osteoporosis in patients with congenital neutropenia and in G-CSF–overexpressing transgenic mice, we hypothesized that short-term G-CSF administration may be sufficient to induce bone resorption, resulting in the release of soluble calcium in the endosteum leading in turn to the inhibition of attachment to fibronectin and the egress of HPC from the BM. We show herein that in humans, serum osteocalcin concentration, a specific marker of bone formation, is strongly reduced after 3 days of G-CSF administration. Furthermore, in patients mobilized with G-CSF either alone or in association with stem cell factor or interleukin-3, the reduction of serum osteocalcin is significantly correlated with the number of HPC mobilized in peripheral blood. Urine levels of deoxypyridinoline (DPyr), a specific marker of bone resorption, gradually elevated during the time course of G-CSF administration until day 7 after cessation of G-CSF, showing a simultaneous stimulation of bone degradation during G-CSF–induced HPC mobilization. In an in vivo murine model, we found that the number of osteoclasts was dramatically increased paralleling the elevation of DPyr after G-CSF administration. When pamidronate, an inhibitor of osteoclast-mediated bone resorption, was administered together with G-CSF in mice, the G-CSF–induced increase of DPyr levels was completely abolished whereas the numbers of colony-forming cells mobilized in peripheral blood were not decreased, but unexpectedly increased relative to the numbers elicited by G-CSF alone. Collectively, our data therefore show that short-term administration of G-CSF induces bone degradation by a simultaneous inhibition of bone formation and an enhanced osteoclast-mediated bone resorption. This increased bone resorption is inhibited by pamidronate without reducing G-CSF–induced HPC mobilization, suggesting that the activation of bone resorption after G-CSF administration is not the direct cause of HPC mobilization as initially hypothesized, but a parallel event.© 1998 by The American Society of Hematology.

The interaction between p145(o-KIT) and p210(bcr-abl), transduced cell lines, and the selective outgrowth of normal progenitors during long-term culture of chronic myeloid leukemia (CML) cells on stroma deficient in stem-cell factor (SCF) suggests that the response of CML cells to SCF may be abnormal. We examined the proliferative effect of SCF(100 ng/mL), provided as the sole stimulus, on individual CD34(+) cells from five normal donors and five chronic-phase CML patients. Forty-eight percent of isolated single CML CD34(+) cells proliferated after 6 days of culture to a mean of 18 cells, whereas only 8% of normal CD34(+) cells proliferated (mean number of cells generated was 4). SCF, as a single agent, supported the survival and expansion of colony-forming unit-granulocyte-macrophage (CFU-GM) from CML CD34(+)CD38(+) cells and the more primitive CML CD34(+)CD38(-) cells. These CFU-GM colonies were all bcr-abl positive, showing the specificity of SCF stimulation for the leukemic cell population. Coculture of CML and normal CD34(+) cells showed exclusive growth of Phi cells, suggesting that growth in SCF alone is not dependent on secretion of cytokines by CML cells. SCF augmentation of beta(1)-integrin-mediated adhesion of CML CD34(+) cells to fibronectin was not increased when compared with the effect on normal CD34(+) cells, suggesting that the proliferative and adhesive responses resulting from SCF stimulation are uncoupled. The increased proliferation may contribute to the accumulation of leukemic progenitors, which is a feature of CML, (C) 1998 by The American Society of Hematology.

Adhesive interactions between CD34+ hematopoietic progenitor cells (HPC) and bone marrow stroma are crucial for normal hematopoiesis, yet their molecular bases are still poorly elucidated. We have investigated whether cell surface proteoglycan CD44 can mediate adhesion of human CD34+ HPC to immobilized hyaluronan (HA), an abundant glycosaminoglycan of the bone marrow extracellular matrix. Our data show that, although CD34+ cells strongly express CD44, only 13.3% ± 1.1% spontaneously adheres to HA. Short-term methylcellulose assay showed that HA-adherent CD34+ cells comprised granulo-monocytic and erythroid committed progenitors (19.6% ± 2.5% and 7.3% ± 1.0% of the input, respectively). More primitive progenitors, such as pre–colony-forming units, also adhered to HA. Moreover, we found that CD44-mediated adhesion of CD34+ cells to HA could be enhanced by phorbol 12-myristate 13-acetate (PMA), the function-activating anti-CD44 monoclonal antibody H90, and cytokines such as granulocyte-monocyte colony-stimulating factor, interleukin-3 (IL-3), and stem cell factor. Enhancement through PMA required several hours, was protein-synthesis–dependent, and was associated with an increase of CD44 cell surface expression, whereas stimulation of adhesion by H90 monoclonal antibody and cytokines was very rapid and without alteration of CD44 expression. H90-induced activation occurred at 4°C and lasted for at least 2 hours, whereas activation by cytokines required incubation at 37°C and was transient. These data, which show for the first time that CD34+ HPC can directly adhere to HA via CD44, point out that this adhesive interaction to HA is a process that may also be physiologically regulated by cytokines.

In this issue of Blood , Corselli et al[1][1] purify CD146+ pericytes from human adipose or fetal bone marrow and demonstrate that these cells are capable of supporting the self-renewal and proliferation of transplantable human cord blood hematopoietic stem cells (HSCs). ![Figure][2] Human

Macrophage colony-stimulating factor (M-CSF or CSF-1) plays a role in regulating innate immune responses promoting macrophage growth and differentiation. We hypothesized CSF-1 may also play a role in growth and progression of Acute Myeloid Leukaemia (AML). The aim of this study is to investigate the role of CSF-1 in survival and chemo-resistance of leukaemia stem and progenitor cells (LSPC). We further hypothesized that blocking CSF-1R signalling in LSPC may dampen leukaemia survival in vitro and delay leukaemia progression in vivo. AML was induced in mice by injecting murine Haematopoietic Stem and Progenitor Cells (HSPC) transduced with either MLL-AF9 or AML1- ETO fusion oncogenes for the development of either monomyelocytic or granulocytic leukaemia respectively. We found CSF-1R, the main receptor for CSF-1, was expressed on these acute myeloid leukaemia cells, thus it is possible that CSF-1 provide supportive microenvironment for leukemic growth. To identify whether CSF-1 in the bone marrow (BM) niche is essential for growth of malignant LSPC, we harvested normal or- leukaemic blasts from BM for in vitro studies using Long-Term Culture-Initiating-Cell (LT-CIC) assays. In these assays AML LSPC or normal HSPC cells were co-cultured with mesenchymal stromal cells (MSC) from either wildtype mice (MSC that produce CSF-1) or MSC from OP/OP mice (unable to produce functional CSF-1). We found normal (wild-type) HSPC were able to proliferate, survive and produce LT-CIC in the absence of niche-provided CSF-1, however AML blasts could not, unless rescued by addition of recombinant CSF-1 (100 ng/mL) in vitro. Together these data suggest CSF-1 signalling may be critical for AML LSPC but not normal HSPC. Next we investigated in mice whether therapeutic CSF-1 blockade could similarly dampen AML survival or progression in vivo. Cohorts of mice were injected with luciferase-expressing monomyelocytic (MLL-AF9) BM leukaemic blasts, then 7 days later administered the small molecule CSF-1 antagonist (GW2580, 160mg/kg daily for 10 days) or vehicle control. Leukaemia progression was tracked by biweekly bioluminescence and testbleeds for appearance of GFP+ leukaemia blasts in blood. We found therapeutic blockade of CSF-1 significantly reduced tumour burden in these mice by both bioluminescence and testbleed analysis. Mice were also monitored for duration of survival. As anticipated by the observed reduction in leukaemia burden, therapeutic CSF-1 blockade also significantly extended the duration of overall mouse survival (P Together these studies suggest therapeutic CSF-1 blockade may show promise as an adjunct therapy to help reduce tumour burden and improve success of AML leukaemia therapies. Disclosures Winkler: GlycoMimetics: Research Funding.

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.

Erythropoiesis is a highly controlled process partly regulated in erythroblastic islands by the central erythroblastic island macrophage (EI MΦ) which provides iron, growth factors and mediates enucleation of the maturing erythroblasts. As macrophages are key effectors of inflammation, we investigated the effect of bacterial LPS in vivo on erythropoiesis and EI MΦ defined as CD11b+ F4/80+ VCAM1+ CD169+Ly6G+ in mice. C57BL/6 mice were injected i.p. with 2.5 mg/kg/day LPS from E. coli for 2 days and the effect on medullar erythropoiesis examined at various time-points after this. LPS administration caused a marked whitening of the bone marrow (BM) with decreased numbers of basophilic (9-fold), polychromatic (3.7-fold), orthochromatic erythroblasts (2.2-fold) and reticulocytes (2.5-fold) 48hrs after LPS challenge. Those remained significantly reduced up to 6 days post-LPS. Likewise, EI MΦ were suppressed in the BM 24-48hrs after LPS challenge and remained significantly reduced 6 days post-LPS. This loss of medullar erythropoiesis was compensated by increased number of EI MΦ (13.6-fold), pro-erythroblasts (1.5-fold), polychromatic (1.9-fold), orthochromatic (3.2-fold) and reticulocytes (2.3-fold) in the spleen. As this phenotype resembled suppression of medullar erythropoiesis following G-CSF treatment, we examined whether the mechanism could be indirect via endogenous G-CSF release. LPS induced a transient 80-fold increase in G-CSF concentration in the blood from 123pg/mL (n=6) to 10ng/ml (n=6) 2 days post-LPS. LPS was administered to TLR4 KO and G-CSF receptor (GCSFR) KO mice. These LPS-mediated responses were abrogated in TLR4 KO mice demonstrating that erythropoiesis suppression in response to LPS is fully TLR4-dependant. However responses in GCSFR KO mice were more contrasted. EI MΦ numbers did not change in GCSFR KO mice in response to LPS demonstrating that suppression of EI MΦ in response to LPS is an indirect effect of endogenous G-CSF release. In contrast, medullar erythropoiesis was still suppressed in GCSFR KO mice with significantly reduced numbers of basophilic, polychromatic and orthochromatic erythroblasts demonstrating that medullar erythroblast suppression 1) persists despite the presence of EI MΦ, and 2) is not G-CSF-dependent. Unexpectedly, the BM from GCSFR KO mice treated with LPS was not whitened with high numbers of reticulocytes/erythrocytes. To further understand how BM erythrocytes could be increased whilst erythropoiesis is suppressed in LPS-treated GCSFR KO mice, we measured vascular leakage by injecting Evans Blue i.v. Blood plasma volume in the BM of LPS-treated GCSFR KO mice was 2.9-fold higher compared to LPS-treated wild-type mice and untreated WT and KO mice (6.0±2.0 µL vs 2.1±0.9 µL blood plasma/femur, p=0.005) suggesting that GCSFR-mediated signaling is necessary to maintain the integrity of the BM vasculature in response to LPS. In conclusion LPS-mediated medullar erythropoiesis suppression involves at least two different TLR4-dependent mechanisms in regards to their requirement for GCSFR: 1) GCSFR-dependent suppression of EI MΦ, 2) GCSFR-independent and EI MΦ-independent suppression of maturing erythroblasts. We are currently investigating whether the latter mechanism involves hepcidin. Finally we also discovered that GCSFR-mediated signaling is necessary to maintain the BM vasculature integrity following LPS challenge. Disclosures Winkler: GlycoMimetics: Research Funding. Levesque:GlycoMimetics: Equity Ownership.

The vascular adhesion molecule E-selectin is a key component of the Bone Marrow (BM) Haematopoietic Stem Cell (HSC) niche prompting HSC to proliferate at the expense of self-renewal (Winkler, Nat Med 2012).Only 3 - 5% of BM endothelial cells express E-selectin in steady state however E-selectin is greatly upregulated (5 - 10 fold) in BM of mice with acute myeloid leukemia (AML) raising the question; how do AML stem cells (LSC) respond to E-selectin at the vascular niche & does E-selectin signalling in AML & HSC differ? Using models of murine AML generated by retroviral transduction of MLL-AF9 or AML1-ETO oncogenes, we found leukemic blasts rapidly upregulate E-selectin-binding upon oncogenic transformation. Furthermore E-selectin adhesion promoted LSC survival to cytarabine in vitro as well as in vivo. LSC survival to chemotherapy in wildtype compared to E-selectin knockout (E-/-) mice quantified by rigorous limiting-dilution transplantation assay of 1%, 0.1%, 0.01% femur BM demonstrated that E-selectin deletion increased sensitivity of LSC to high-dose cytarabine therapy ~11-fold (900mg/kg cytarabine n=6 donors &15 recipients/gp p=0.0037). Thus E-selectin is a critical vascular niche component mediating LSC chemo resistance. Importantly these findings could be replicated by administration of a potent small molecule glycomimetic E-selectin antagonist (GMI-1271) to wt mice. Furthermore treatment with GMI-1271 (40mg/kg bidaily) for 10 days in combination with standard mouse version of 7+3 induction chemotherapy (5 days cytarabine 100mg/kg; 3 days doxorubicin 1mg/kg) was able to significantly double mouse survival over chemotherapy alone (p=0.0054; no chemotherapy median survival 25 d, AraC/Dox alone 32 d, AraC/Dox plus GMI-1271 survival 41 d; n=8 mice/gp). To understand mechanisms of this chemo-sensitisation, mice with MLL-AF9 monomyelocytic (11q23 translocation) or AML1-ETO granulocytic t(8;21) -induced AML were administered GMI-1271 or vehicle control for 5 days before sorting BM AML blasts for RNA sequencing. Analysis of differentially expressed transcripts by CuffDiff / DSeq2 revealed 170 RNAs differed following in vivo E-selectin blockade. KEGG pathway analysis indicated a pathway potentially dampened in AML blasts following GMI-1271 administration was PI3K - NF-kB signalling - raising the hypothesis that adhesion to E-selectin activates pro-survival NF-kB signalling in AML cells leading to enhanced chemoresistance. Using two in vitro assays, we confirmed E-selectin to be unique among vascular adhesion molecules tested in being able to directly activate NF-kB. Activation of NF-kB was only observed upon E-selectin mediated adhesion & was not observed following adhesion to P-selectin, PECAM-1 or VCAM-1 using either myeloid NF-kB GFP reporter cell lines or by induction of p65 NF-kB (Ser 536). Importantly E-selectin mediated NF-kB activation was completely inhibited when E-selectin antagonist GMI-1271 added. Assays repeated in presence of a specific NF-kB activation antagonist (BMS-345541 10uM 24hrs) demonstrated that NF-kB blockade alone reversed E-selectin-mediated chemoresistance in vitro. Upstream blockade of E-selectin by GMI-1271 not only inhibits NF-kB activation but also mobilizes LSC out of the protective BM niche & prevents re-entry thereby breaking the chemo resistance observed with these cells. A Phase I/II Clinical trial to study efficacy of GMI-1271 in combination with chemotherapy in AML patients (NCT02306291) is currently in progress Disclosures Winkler: GlycoMimetics: Research Funding. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Levesque:GlycoMimetics: Equity Ownership.

Quiescent hematopoietic stem cells (HSCs) preferentially reside in poorly perfused niches that may be relatively hypoxic. Most of the cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIFs). To investigate the effects of hypoxia on HSCs, we blocked O2-dependent HIF-1α degradation in vivo in mice by injecting 2 structurally unrelated prolyl hydroxylase domain (PHD) enzyme inhibitors: dimethyloxalyl glycine and FG-4497. Injection of either of these 2 PHD inhibitors stabilized HIF-1α protein expression in the BM. In vivo stabilization of HIF-1a with these PHD inhibitors increased the proportion of phenotypic HSCs and immature hematopoietic progenitor cells in phase G0 of the cell cycle and decreased their proliferation as measured by 5-bromo-2'-deoxyuridine incorporation. This effect was independent of erythropoietin, the expression of which was increased in response to PHD inhibitors. Finally, pretreatment of mice with a HIF-1α stabilizer before severe, sublethal 9.0-Gy irradiation improved blood recovery and enhanced 89-fold HSC survival in the BM of irradiated mice as measured in long-term competitive repopulation assays. The results of the present study demonstrate that the levels of HIF-1α protein can be manipulated pharmacologically in vivo to increase HSC quiescence and recovery from irradiation.HIF-1α protein stabilization increases HSC quiescence in vivo. HIF-1α protein stabilization increases HSC resistance to irradiation and accelerates recovery.

Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment. Controversy exists on N-cadherin's role in support of HSCs. Specifically, it is unknown whether microenvironmental N-cadherin is required for normal marrow microarchitecture and for hematopoiesis. To determine whether osteoblastic N-cadherin is required for HSC regulation, we used a genetic murine model in which deletion of Cdh2, the gene encoding N-cadherin, has been targeted to cells of the osteoblastic lineage. Targeted deletion of N-cadherin resulted in an age-dependent bone phenotype, ultimately characterized by decreased mineralized bone, but no difference in steady-state HSC numbers or function at any time tested, and normal recovery from myeloablative injury. Intermittent parathyroid hormone (PTH) treatment is well established as anabolic to bone and to increase marrow HSCs through microenvironmental interactions. Lack of osteoblastic N-cadherin did not block the bone anabolic or the HSC effects of PTH treatment. This report demonstrates that osteoblastic N-cadherin is not required for regulation of steady-state hematopoiesis, HSC response to myeloablation, or for rapid expansion of HSCs through intermittent treatment with PTH.

Transplantation with 2-5 × 106 mobilized CD34+cells/kg body weight lowers transplantation costs and mortality. Mobilization is most commonly performed with recombinant human G-CSF with or without chemotherapy, but a proportion of patients/donors fail to mobilize sufficient cells. BM disease, prior treatment, and age are factors influencing mobilization, but genetics also contributes. Mobilization may fail because of the changes affecting the HSC/progenitor cell/BM niche integrity and chemotaxis. Poor mobilization affects patient outcome and increases resource use. Until recently increasing G-CSF dose and adding SCF have been used in poor mobilizers with limited success. However, plerixafor through its rapid direct blockage of the CXCR4/CXCL12 chemotaxis pathway and synergy with G-CSF and chemotherapy has become a new and important agent for mobilization. Its efficacy in upfront and failed mobilizers is well established. To maximize HSC harvest in poor mobilizers the clinician needs to optimize current mobilization protocols and to integrate novel agents such as plerixafor. These include when to mobilize in relation to chemotherapy, how to schedule and perform apheresis, how to identify poor mobilizers, and what are the criteria for preemptive and immediate salvage use of plerixafor.

The effect of human fibrinogen on the proliferation of purified SBA- CD34+ human bone marrow progenitors was investigated in clonal cultures. Fibrinogen alone or in combination with erythropoietin had no significant effect. However, in the presence of recombinant human interleukin-3 (IL-3), fibrinogen increased significantly in a dose- dependent manner the number of mixed and burst-forming unit-ethrocyte-- derived colonies, whereas the number of other colonies did not significantly change. In the presence of fibrinogen, low concentrations of IL-3 (0.17 U/mL) produced three times more mixed colonies than without fibrinogen, reaching the number of colonies obtained with optimal concentrations of IL-3 (1.67 U/mL). Fibrinogen fragment D had the same effect in the presence of IL-3 as intact fibrinogen, whereas fibrinogen fragment E and human collagen IV did not. This effect was not mediated by integrins, because peptides or monoclonal antibodies that block fibrinogen binding on integrins alpha IIb beta 3, alpha v beta 3 (RGD-peptides), alpha m beta 2 (OKM-1), and alpha x beta 2 (HC1/1) did not affect the observed mitogenic effect. The mitogenic effect of fibrinogen and its D fragment was not mediated by induction of IL-6 or granulocyte--colony-stimulating factor secretion, because it was not inhibited by blocking antisera against these two growth factors. Our results indicate that fibrinogen potentiates the effect of IL-3 on primitive hematopoietic progenitors and suggest that the mitogenic effect of fibrinogen could be mediated via a specific mitogenic receptor that does not belong to the integrin family.

Hematopoietic stem cell (HSC) niches have been reported at the endosteum or adjacent to bone marrow (BM) vasculature. To investigate functional attributes of these niches, mice were perfused with Hoechst 33342 (Ho) in vivo before BM cell collection in presence of pump inhibitors and antibody stained. We report that the position of phenotypic HSCs, multipotent and myeloid progenitors relative to blood flow, follows a hierarchy reflecting differentiation stage, whereas mesenchymal stromal cells are perivascular. Furthermore, during granulocyte colony-stimulating factor–induced mobilization, HSCs migrated closer to blood flow, whereas stromal cells did not. Interestingly, phenotypic Lin−Sca1+KIT+CD41−CD48−CD150+ HSCs segregated into 2 groups (Honeg or Homed), based on degree of blood/Ho perfusion of their niche. HSCs capable of serial transplantation and long-term bromodeoxyuridine label retention were enriched in Honeg HSCs, whereas Homed HSCs cycled more frequently and only reconstituted a single host. This suggests that the most potent HSC niches are enriched in locally secreted factors and low oxygen tension due to negligible blood flow. Importantly, blood perfusion of niches correlates better with HSC function than absolute distance from vasculature. This technique enables prospective isolation of serially reconstituting HSCs distinct from other less potent HSCs of the same phenotype, based on the in vivo niche in which they reside.

T-cells are critical mediators of immune defense against pathogens and cancer. Adoptive T cell immunotherapy and T-cell engineering have promising clinical applications but T cell survival and exhaustion are current limitations. Central memory cells (TCM CD62L+ CCR7+) and their precursors, stem central memory T-cells (TSCM) possess the stem-like properties needed to reconstitute and prolong an effective immune response long-term. These cells have been shown to significantly improve therapeutic efficacy of adoptive T-cell therapy. The challenge remains to harvest good quality TCM-cells for these immunotherapy approaches. The bone marrow (BM) is the major reservoir of CD8+ TCM and their precursors. We have previously shown that E-selectin is expressed in the BM vasculature and drives activation and differentiation of hematopoietic stem cells during G-CSF induced mobilization to the blood. We find therapeutic blockade of E-selectin promotes HSC self-renewal and reconstitution in vivo. We now examine the impact of E-selectin blockade on CD8+ T cell mobilization from the bone marrow to the blood and hypothesize that E-selectin blockade may also dampen the activation/differentiation of this subset. First we administered a standard G-CSF regime (filgastim 250ug/kg/day for 3 days) to mice and then dosed some cohorts with GMI-1271 (40mg/kg BID) from 12 to 72 hours within this 3 day period. Administration of G-CSF alone results in a near complete disappearance of bone marrow resident CD8+ TCM cells, and their apparent migration (increase in numbers) to the blood, while CD8+ subsets in the lymph nodes and spleen were barely affected by G-CSF. Furthermore among T-cell subsets, CD8+ but not CD4+ TCM were specifically mobilized into the blood when GMI-1271 was co-administered for the last 12 to 24 hours of G-CSF. These findings are consistent with reports demonstrating the bone marrow to be a major reservoir for CD8+ but not CD4+ central memory T-cells. Administration of GMI-1271 caused a marked enhancement in mobilization into the blood of CD8+ TCM/SCM (CD62Lhi, CCR7+) cells over treatment with G-CSF alone (p In a previous report we have shown that therapeutic blockade of E-selectin promotes HSC self-renewal in vivo. Thus, it is possible that E-selectin blockade boosts mobilization of CD8+ TCM/SCM with stem-like properties into the blood by loosening factors retaining CD8+ TCM/SCM in the bone marrow and/or blocking the E-selectin-mediated activation and differentiation of this T-cell subset. In summary, our studies identify E-selectin blockade as a novel target to improve harvesting of CD8+ TCM/SCM cells with stem-like properties. Blockade of this target with GMI-1271 significantly improves the in vivo reconstitution potential and regenerative properties of CD8+ T-cells from donor blood allowing a valuable source of desired T-cells for use in adoptive immunotherapy and T-cell engineering. Disclosures Winkler: GlycoMimetics Inc: Research Funding. Barbier:GlycoMimetics Inc: Research Funding. Davies:GlycoMimetics Inc: Research Funding. Smith:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Although both P- and E-selectin are constitutively expressed on bone marrow endothelial cells, their role in the regulation of hematopoiesis has only recently been investigated. We have previously shown that P-selectin glycoprotein ligand-l (PSGL-1/CD162) is expressed by primitive human bone marrow CD34+ cells, mediates their adhesion to P-selectin, and, more importantly, inhibits their proliferation. We now demonstrate that adhesion to E-selectin inhibits the proliferation of human CD34+ cells isolated either from human umbilical cord blood, adult mobilized blood, or steady-state bone marrow. Furthermore, a subpopulation, which does not contain the most primitive hematopoietic progenitor cells, undergoes apoptosis following E-selectin–mediated adhesion. The same phenomenon was observed in cells isolated from mouse bone marrow. Using lineage-negative Sca-1+ c-KIT+ bone marrow cells from PSGL-1–/– and wild-type mice, we establish that PSGL-1 is not the ligand involved in E-selectin–mediated growth inhibition and apoptosis. Moreover, stable transfection of the human myeloid cell line K562 (which does not express PSGL-1) with α(1,3) fucosyltransferase VII alone was sufficient to recapitulate the E-selectin–mediated growth inhibition and apoptosis observed in hematopoietic progenitor cells. These data demonstrate that an E-selectin ligand(s) other than PSGL-1 transduces growth inhibitory and proapoptotic signals and requires posttranslational fucosylation to be functional.

We recently reported that vascular adhesion molecule E-selectin is a key component of the bone marrow vascular niche, ‘awakening’ otherwise dormant Haematopoietic Stem Cells (HSC) (Winkler et al., Nat Med 2012). Following cytotoxic chemotherapy or radiation injury, E-selectin expression in the bone marrow increases ~10 to 20-fold during the recovery phase, at a time when HSC must cycle to replenish the blood and immune systems. When E-selectin is absent (in gene deleted mice) or E-selectin is therapeutically blocked using the small molecule glycomimetic antagonist GMI-1271, a greater proportion of HSC return to quiescence following radiation or chemotherapy. We now report cell surface E-selectin to be also upregulated 5 to 10-fold on the BM vasculature in mice with acute myeloid leukaemia (AML). This raises the interesting question: how do AML leukaemia stem cells (LSC) respond to E-selectin at the vascular niche? Using models of murine AML generated by retroviral transduction of the MLL-AF9 fusion oncogene into HSC, we found leukemic blasts rapidly upregulate E-selectin binding potential upon oncogenic transformation. In fact targeted disruption of these E-selectin-mediated interactions by administration of GMI-1271 injection distrupts adhesion and localization of AML cells and was sufficient to continually mobilise leukaemic blasts into the blood for at least 24 hours after a single injection at 40 mg/kg, suggesting that E-selectin-mediated interactions play a role in retaining LSC within BM niches. We next queried whether E-selectin-mediated signalling may help promote LSC survival following therapy. To test this, cohorts of 20 wildtype or 20 E-selectin knock-out mice were transplanted with the same AML cells, then 4 weeks later, half were treated with high dose cytarabine (2 x 900mg/kg at 12hour interval) while the other half remained untreated. At 24 hours after the first cytarabine injection, BM cells were harvested to measure numbers of surviving functional LSC by limiting-dilution transplantation assays in irradiated wild-type syngenic recipients and the proportion of these recipients that developed leukemia was used to calculate the original number of surviving LSC by Poisson’s distribution. We found that although the absence of E-selectin had no effect on total LSC numbers per femur, the absence of E-selectin dramatically increased sensitivity of LSC to cytarabine treatment (20-fold). These results indeed suggest that E-selectin is a key vascular niche component mediating LSC chemoresistance. Our data are also consistent with previous xenograft models in immune-deficient mice showing that the few human CD34+ AML LSC that survived chemotherapy, were observed clustered around endosteal vascular endothelium in recipient mice (Ishikawa et al., Nat BioTechnol 2007; Ninomiya et al., Leukemia 2007) where E-selectin is expressed. In summary our data confirm that niche factors alone can strongly influence LSC sensitivity to chemotherapy, and suggest a chemoprotective role for the vascular adhesion molecule E-selectin which is upregulated in the bone marrow of leukaemic mice, Taken together, these data identify E-selectin as a novel therapeutic target for the treatment of AML leukemic stem cells in that in vivo inhibition by the small molecule glycomimetic E-selectin antagonist GMI-1271 may improve chemosensitivity. Disclosures Winkler: FibroGen Inc.: Research Funding. Magnani:GlycoMimetics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

We have previously identified a novel function for the adhesion molecule E-selectin – awakening otherwise dormant hematopoietic stem cells (HSC) and inducing lineage commitment (Winkler et al., Nat Med 2012). Now we show that therapeutic blockade of E-selectin in vivo specifically augments the mobilisation of HSC with highest self-renewal potential following G-CSF administration, and markedly improves subsequent engraftment and reconstitution in mice. From these data we hypothesise that vascular E-selectin acts as a gatekeeper influencing activation of transmigrating HSC. Firstly we found that administration of mobilizing doses of G-CSF increased the level of E-selectin expressed on the surface of bone marrow (BM) endothelial cells. To determine whether E-selectin influenced HSC mobilisation, we then compared G-CSF-mediated mobilisation in wildtype and E-selectin knock-out mice. We found that although absence of E-selectin did not significantly alter the number of phenotypic HSC or colony-forming cells mobilized into the blood following G-CSF, the absence of E-selectin in mobilised mice did increase the subsequent engraftment and reconstitution potential of mobilised blood analysed by competitive repopulation transplant assays. Next we investigated whether this beneficial effect could similarly be achieved by transient E-selectin blockade using therapeutic doses of GMI-1271, a small synthetic mimetic that specifically blocks the binding of E-selectin to its receptors. Wild-type mice were administered human G-CSF alone (125µg/kg subcutaneously twice daily for 3 days) ± GMI-1271 injections (20 mg/kg BID). The number of mobilized HSC were quantified by rigorous limiting-dilution transplantation of 0.3, 1, 5 or 20 µL mobilised blood in competition with 200,000 congenic BM cells into lethally-irradiated congenic recipients, to enable quantification of long-term reconstituting cells per mL of mobilised blood by Poisson’s distribution. We found the mobilized blood of donor mice injected with both GMI-1271 and G-CSF, showed faster engraftment and 25-fold increased reconstitution potential over blood from G-CSF alone injected controls (p These data are consistent with the role of E-selectin in the bone marrow (to awaken otherwise dormant HSC) that we have previously reported, and also point the way forward to a simple remedy (administration of E-selectin antagonist together with G-CSF) during HSC mobilisation to improve short- and long-term engraftment thus accelerate recovery in transplant recipients. In conclusion we propose the novel concept of E-selectin as a vascular ‘gate-keeper’ dampening the potential of migratory HSC. Disclosures Winkler: FibroGen Inc.: Research Funding. Magnani:GlycoMimetics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Mobilized progenitor cells currently represent the most commonly used source of hematopoietic progenitor cells (HPCs) to effect hematopoietic reconstitution following myeloablative chemotherapies. Despite their widespread use, the molecular mechanisms responsible for the enforced egress of HPCs from the bone marrow (BM) into the circulation in response to mobilizing agents such as cytokines remain to be determined. Results of this study indicate that expression of vascular cell adhesion molecule-1 (VCAM-1) is strongly reduced in vivo in the BM during HPC mobilization by granulocyte colony-stimulating factor (G-CSF) and stem cell factor. Two serine proteases, namely, neutrophil elastase and cathepsin G, were identified, which cleave VCAM-1 and are released by neutrophils accumulating in the BM during the course of immobilization induced by G-CSF. The proposal is made that an essential step contributing to the mobilization of HPCs is the proteolytic cleavage of VCAM-1 expressed by BM stromal cells, an event triggered by the degranulation of neutrophils accumulating in the BM in response to the administration of G-CSF.

G-CSF mobilizes hematopoietic stem cells (HSCs) from the bone marrow (BM) into the blood by suppressing a subset of HSC niche supportive macrophages. As macrophages are the central component of erythropoietic islands in BM, spleen and liver, we examined the effect of G-CSF on erythropoiesis in C57BL/6 mice. Mobilizing doses of G-CSF caused a marked whitening of the BM, a 15-fold decrease in the number of phenotypic erythroblasts, a 1.5-fold decrease in polychromatic and orthochromatic erythroblasts, and a 4.5-fold reduction in reticulocytes in the BM. Conversely, more immature pro-erythroblasts increased 4.4-fold. As the cell surface antigen ER-HR3 identifies erythroid island macrophages in mouse liver and spleen, while VCAM-1 and CD169 on macrophages have been independently reported to be critical for erythropoiesis, we followed the expression on these antigens on BM macrophages during mobilization. G-CSF treatment caused a 35-fold reduction in the number of CD11b+ F4/80+ VCAM1+ ER-HR3+ CD169+ Ly6G+ macrophages that paralleled the loss of erythroblasts. As a result, splenic erythropoiesis was up-regulated to compensate for the loss of medullary erythropoiesis with a 4-5 fold increase in pro-erythroblasts, all erythroblast subsets and reticulocytes. In another set of experiments, we quantified medullar erythropoiesis and macrophages during recovery after a 4 day G-CSF treatment. Erythroblasts and supportive macrophages significantly recovered as early as 24 hours after cessation of G-CSF but it took 7 days to normalize to pre-mobilization values. This suggests that mobilizing doses of G-CSF transiently block erythroblast differentiation specifically in the BM (but not the spleen) by affecting central macrophages in erythroid islands. To confirm that CD11b+ F4/80+ VCAM1+ ER-HR3+ CD169+ Ly6G+ macrophages are critical to the maturation of pro-erythroblasts into erythroblasts, we next performed a broad macrophage depletion by injecting clodronate-loaded liposomes, or a selective depletion of CD169+ macrophages in mice knocked-in with diphtheria toxin receptor into the Siglec1 (CD169) gene. Both clodronate liposome treatment in wild-type mice, and diphtheria toxin treatment in Siglec1DTR/+ mice caused a concomitant depletion of CD11b+ F4/80+ VCAM1+ ER-HR3+ CD169+ macrophages, loss of erythroblasts and accumulation of pro-erythroblasts. Unlike G-CSF, these two treatments also blocked splenic erythropoiesis. In conclusion, we propose that 1) CD11b+ F4/80+ VCAM1+ ER-HR3+ CD169+ Ly6G+ macrophages include nursing macrophages at the centre of erythroid islands and are essential for the maturation of pro-erythroblasts to erythroblasts and 2) mobilizing doses of G-CSF transiently stop medullary erythropoiesis by depleting CD11b+ F4/80+ VCAM1+ ER-HR3+ CD169+ Ly6G+ macrophages in erythropoietic islands in the BM, but not in the spleen. Disclosures: No relevant conflicts of interest to declare.

Hypoxia and hypoxia-inducible factors (HIFs) are implicated in the regulation of normal and malignant hematopoiesis. HIF-1α stabilization makes leukemia stem cells and normal HSC dormant and is necessary to maintain their self-renewal potential. In sharp contrast, HIF-2α, which shares 60% homology with HIF-1a, promotes proliferation of renal clear carcinoma and embryonic stem cells by enhancing expression of oct-4, sox2 and activating c-myc. In this study, we investigated the role of hypoxia and HIF-2α in leukemia. In normal mouse and human bone marrow (BM), HIF-2α mRNA expression was observed predominantly in non-hematopoietic stromal cells, while hematopoietic cells displayed low to undetectable levels. In contrast, HIF-2α mRNA and protein were detected in the BM of moribund NOD/SCID mice engrafted with 3 different human ALL, and in cultured human ALL and AML cell lines, suggesting that HIF-2α is abnormally expressed in leukemic cells. To investigate the potential roles of HIF-2α in leukemic cells, we cloned human HIF-2α cDNA into the MXIE retroviral vector. In a 1st model the GM-CSF-dependent mouse pre-leukemic cell line FDCP1, which does not express HIF-2α, was retrovirally transduced with HIF-2α. HIF-2α provided a significant proliferative advantage to FDCP1 cells in hypoxic or normoxic cultures and reduced GM-CSF dependency. We next transplanted retrovirally transduced FDCP1 cells into non-irradiated syngeneic DBA/2 mice. All recipients of FDCP1 transduced with HIF-2α-MXIE vector succumbed to leukemia by week 28 post-transplantation. In sharp contrast, mice receiving FDCP1 transduced with empty MXIE vector, displayed a leukemia penetrance of only 15% by week 45 (Fig. 1a; p=0.0001 log rank, hazard ratio = 12.28).Fig. 1Percent survival of recipients of (a) FDCP1 cells retrovirally transduced with HIF-2α-MXIE vector or MXIE control empty vector, (b) vavBcl2 HSC transduced with HIF-2α-MXIE vector or MXIE empty vector, and (c) HL60 cells transduced with HIF-2α knocked-down or scrambled control lentiviral vectors.Fig. 1. Percent survival of recipients of (a) FDCP1 cells retrovirally transduced with HIF-2α-MXIE vector or MXIE control empty vector, (b) vavBcl2 HSC transduced with HIF-2α-MXIE vector or MXIE empty vector, and (c) HL60 cells transduced with HIF-2α knocked-down or scrambled control lentiviral vectors. In a 2nd model, HSC from vavBcl2 transgenic mice were transduced with human HIF-2α-containing or empty MXIE retroviral vectors and subsequently transplanted into lethally irradiated wild-type recipients. Transduction of vavBcl2 HSC with HIF-2α resulted in the outgrowth of HIF-2α-expressing B cells which was not observed in recipients of vavBcl2 HSC transduced with empty vector. Consequently recipients of HIF-2α transduced vavBcl2 HSC succumbed more rapidly to spontaneous lymphoma compared to controls (Fig. 1b; p=0.036 log rank, hazard ratio = 2.971, MXIE median survival = 56 weeks, HIF2α median survival = 41 weeks). Finally, HIF-2α was knocked-down in human leukemia cell lines U937 and HL60 using a shRNA lentiviral vector. HIF-2α knock-down resulted in a 2-fold decrease in proliferation in vitro. We next transplanted HL60-HIF-2a shRNA and HL60-scrambled shRNA cells into NOD/SCID/ IL2Rγ-/- (NSG) mice for each group. Notably, all recipients of HL60-HIF-2a shRNA cells succumbed to leukemia significantly later than recipients of HL60-scrambled shRNA cells (Fig. 1c; p= Disclosures: No relevant conflicts of interest to declare.

Abstract 2402 The MYB oncogene is widely expressed in acute leukaemias and is important for the continued proliferation of leukemia cells, raising the possibility that MYB may be a therapeutic target. However realization of this potential requires (i) a significant therapeutic window for MYB inhibition, given its essential role in normal haemopoiesis; and (ii) an approach for developing an effective therapeutic. We previously showed that the interaction of Myb with the coactivator CBP/p300 is essential for Myb's intrinsic transforming activity (1). Here we use haemopoietic cells from the Booreana mouse strain, which carries a mutation in Myb that prevents interaction with CBP/p300 (see Fig. 1 and ref. 2), to examine the requirement for this interaction in myeloid transformation and leukaemogenesis. Using this strain and a strain (plt6) carrying a “complementary” mutation in p300 (3), we show that the Myb-p300 interaction is essential for in vitro transformation by the myeloid leukaemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type (WT) mice, Booreana cells fail to induce leukaemia upon transplantation into irradiated recipients following transduction with an AML1-ETO9a retrovirus (Fig. 2). These data, as well as reinforcing the notion that MYB is an essential cofactor for myeloid leukaemogenesis, highlight disruption of the Myb-p300 interaction as a potential therapeutic strategy for AML. Furthermore, our results suggest that such a strategy would have a useable therapeutic index since Booreana mice, unlike Myb null mice, are viable. We have started to explore the use of small peptides to provide proof-of-principle for this approach. We have also begun to explore the molecular basis of the requirement for MYB, and the Myb-p300 interaction, in AML using gene expression profiling. This has highlighted several Myb target genes – identified by our previous ChIP-Seq analysis (4) - that have been previously implicated in myeloid leukaemogenesis, as being differentially expressed between WT and Booreana cells transduced with AML1-ETO9a. One striking example is the apparent Myb-p300 dependent activation of Ets2 and Gfi1, accompanied by repression of Gata2 and Gfi1b; this pattern has previously been reported in MLL-AF9 and MOZ-TIF-induced murine AML (5). Disclosures: No relevant conflicts of interest to declare.

Abstract 216 Up to 5% allogeneic healthy donors and up to 40–60% of chemotherapy-treated patients in autologous setting, fail to reach minimal threshold of 2×106 blood CD34+cells/kg in response to G-CSF, precluding transplantation. Plerixafor, a small inhibitor of the chemokine receptor CXCR4, used for 4 days in combination with G-CSF enables this minimal threshold to be reached in up to 60% patients who previously failed to mobilise in response to G-CSF alone. However, the remaining 40% of patients who failed to mobilise in response to G-CSF alone, still fail to mobilize adequately with G-CSF + Plerixafor. In an attempt to further boost HSC mobilization in response to combinations of G-CSF and Plerixafor, we have investigated the role of the hypoxia-sensing pathway in HSC mobilization. HIF-1α (Hypoxia-inducible factor-1α) controls HSC proliferation and self-renewal in poorly perfused hypoxic bone marrow (BM) niches where very quiescent HSC with highest self-renewal potential reside. When O2 concentration is above 2% in the cell microenvironment, HIF-1α protein is rapidly hydroxylated on Pro residues by prolyl hydroxylases PHD1-3. This recruits the E3 ubiquitin ligase VHL, which targets HIF-1α to rapid proteasomal degradation. When O2concentration is below 2% (hypoxia), PHDs are inactive; HIF-1α protein is stabilized, associates with its β subunit ARNT, translocates to the nucleus and activates of transcription and hypoxia-responsive genes. In this study, we have investigated the effect of pharmacological stabilization of HIF-1α protein on HSC mobilization in mice using the HIF-PHD inhibitor FG-4497. We report that FG-4497 treatment stabilizes HIF-1α protein in mouse BM. We find that FG-4497 synergizes with G-CSF and Plerixafor to enhance HSC mobilization. C57/Bl6 mice were in 4 treatment groups: (G) 250μg/kg/day G-CSF alone for 2 days; (GF) G-CSF for 2 days + 20mg/kg/day FG-4497 for 3 days; (GP) G-CSF for 2 days together with16mg/kg Plerixafor 1 hour prior harvest; (GPF) G-CSF together with Plerixafor and FG-4497 with same dosing as above. Mobilization of colony-forming cells (CFC), phenotypic Lin-CD41-Sca1+Kit+CD48-CD150+ HSC, and functional HSC in long-term competitive transplantation assays were measured. Mice in the GF group (G-CSF + FG-4497) mobilized CFC to the blood 4-fold and phenotypic HSC 3-fold more than mice mobilized with G-CSF alone (p To determine which cell types drive HSC mobilization in a HIF-1α-dependant manner, we crossed HIF1αflox/floxmice with osxCre (HIF-1α gene deletion in osteoprogenitors), LysMCre (deletion in myeloid cells), or with SclCreER mice (tamoxifen-induced deletion in HSC). While studies in LysMCre and SclCreER mice are ongoing, we find that deletion of HIF-1α gene in osteoprogenitors (osxCre mice) decreased 2.5-fold the number of CFU/mL blood following 2 and 3 days treatment with G-CSF. This suggests that HIF-1α in osteoprogenitors and their osteoblastic progenies is necessary for optimal mobilization in response to G-CSF. In conclusion, our data highlight the importance of HIF-1α in HSC mobilization and provide a novel therapeutic strategy for increasing HSC mobilization above levels obtained with combinations of G-CSF and Plerixafor. Thus PHD inhibitors could be useful agents in patients who still fail to mobilize in response to G-CSF and plerixafor. Disclosures: Walkinshaw: Fibrogen Inc.: Employment, Equity Ownership.

Abstract 1889 We have previously shown that G-CSF administration impairs HSC niches in the mobilized bone marrow (BM). G-CSF causes rapid suppression (within 2 days) of endosteal osteoblasts and bone formation with concomitant down-regulation of Kit ligand, CXCL12 and angiopoietin-1. This effect is mediated by the depletion of specific populations of BM macrophages1. Considering the very rapid impairment of HSC niches in response to G-CSF, we hypothesized that 1) the most primitive HSC should also mobilize at this very early stage within the first 48 hours of G-CSF treatment, and 2) that down-regulation of HSC niche function should also alter the number or function of HSC remaining in the mobilized BM. To test this, 125μg/kg rhuG-CSF was injected twice daily to C57BL/6 mice; blood and BM harvested at days 2 and 5 of G-CSF treatment to be transplanted into congenic recipients in a long-term competitive repopulation assay (LT-CR). Transplantation of 25μL blood showed a gradual increase in the number of LT-CR cells mobilized in response to G-CSF as measured by donor chimerism in myeloid and B lineages at 16 weeks post-transplant. Expectedly repopulating units (RU) per mL blood progressively increased from 0.2 ± 0.0 (n=6) in steady-state to 2.9 ± 1.6 (n=9) and 82.6 ± 40.4 (n=9) at days 2 and 5 of G-CSF treatment respectively. At 16 weeks post-transplant, BM from primary recipients were transplanted into secondary recipients. Surprisingly, secondary recipients of blood samples collected after 2 and 5 days of G-CSF treatment had equivalent levels of donor chimerism (37.2% ± 6.6% for 2 days G-CSF and 47.1% ± 7.8% for 5 days G-CSF, n = 8 per group). Therefore, although the absolute number of RU mobilized at day 2 of G-CSF was 28-fold lower than at day 5 of G-CSF administration, more primitive serially reconstituting HSC were mobilized at equivalent levels at days 2 and 5 of G-CSF treatment. This supports our hypothesis that most potent serially reconstituting HSC are mobilized as early as day 2 of G-CSF treatment consistent with the disappearance of osteoblasts1. To test the potential of HSC remaining in the BM, BM cells from G-CSF mobilized mice were transplanted in competition with BM cells from congenic mice in steady-state. Donor chimerism at 16 weeks post-transplant showed that competitive repopulation of BM cells was severely impaired at day 5 of G-CSF treatment with the number of RU per 200,000 BM cells decreasing from 4.1 ± 1.4 in steady-state and 5.2 ± 1.6 at day 2 of G-CSF treatment, to only 0.14 ± 0.05 at day 5 of G-CSF treatment. To test whether this 29-fold decrease in competitive repopulation was due to increased HSC proliferation, we measured BrdU incorporation for the last 2.5 days prior to BM harvest as well as cell cycle analysis with Ki67 and Hoechst33342. The proportion of quiescent Lin- Sca1+ Kit+ CD48- phenotypic HSC in G0 phase decreased from 62.8 ±4.0% in steady-state to 43.5±8.2% at day 2 of G-CSF, but surged back to 80.5±1.9% and 75.1±3.5% at days 3.5 and 5 of G-CSF treatment. The proportion of HSC in G1 and S/G2/M phases followed the opposite pattern, up at day 2, down at days 3.5 and 5. This was confirmed by BrdU incorporation for 2.5 days with the number of BrdU+ cells among Lin- Sca1+ KIT+ CD48- cells rising from 35.1±4.0% in steady-state, to 51.2±4.5% at day 2 of G-CSF and going down to 18.1±1.9% at day 3.5 and 23.3±5.5% at day 5 of G-CSF. Therefore, G-CSF recruits phenotypic HSC into cell cycle within the first 2 days of administration, but HSC return to quiescence despite continuing G-CSF. Therefore decreased repopulation potential at day5 of G-CSF is not due to increased cycling. Finally, we noted that the number of Lin-Sca1+KIT+CD48-CD150+ HSC and Lin-Sca1+KIT+CD48-CD150- multipotent progenitors were reduced 2.4- and 2.8-fold respectively (p Disclosures: No relevant conflicts of interest to declare.

Abstract 2334 The endosteal region of the bone marrow (BM) is hypoxic in steady-state and most quiescent hematopoietic stem cells (HSC) reside in hypoxic, poorly perfused niches. Mobilizing doses of G-CSF renders most of the BM space hypoxic. Most cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIF). At O2 concentration above 2%, HIF-α is rapidly hydroxylated on Pro residues by the prolyl hydroxylases PHD1, PHD2, and PHD3. HIF-α prolyl hydroxylation recruits the E3 ubiquitin ligase VHL, which targets HIF-α to the proteasome. In hypoxia (O2 < 2%), HIF-α proteins are stable, associate with the β subunit ARNT, and translocate to the nucleus to activate transcription. It has emerged that HIF-1α regulates HSC proliferation and is critical to maintain long-term HSC self-renewal in vivo. In this study, we investigated the effect of pharmacological stabilization of HIF-1α protein on HSC cycling and mobilization in mice using two different HIF prolyl hydroxylase (PHD) inhibitors, dimethyloxalyl glycine (DMOG) and FG-4497. We first assessed whether DMOG and FG-4497 stabilized HIF-1α protein in BM leukocytes in vivo by western-blot. Following a single injection of 400mg/kg DMOG, HIF-1α protein was stabilized for up to 6 hrs in BM leukocytes. With 20mg/kg FG-4497, HIF-1α protein persisted over 12 hours. HIF-1α protein was below detection in the BM from saline injected animals. C57BL/6 mice were injected daily with 400mg/kg DMOG to measure effect on HSC cycling and BrdU incorporation by flow cytometry. Using Hoecht33342 and FITC-conjugated anti-Ki67 antibody, a 18 day DMOG treatment increased the proportion of Lin-negative Kit+ Sca1+ CD48- (L-K+S+48-) HSC in phase G0 from 61±11% to 84±6% (p Since HIF-PHD inhibitors slow HSC cycling in vivo, we tested whether they could protect HSC from sublethal irradiation. Mice were treated with 400mg/kg DMOG or saline for 22 days and then irradiated with 9.0Gy. Both cohorts were leukopenic betweens days 7 and 14 post-irradiation but DMOG treated mice had significantly higher blood leukocytes at days 22 and 30, and higher platelet numbers day 22 and all subsequent time-points suggesting that DMOG enhances HSC survival with higher blood recovery. Finally mice were injected with DMOG for 4 days and pegylated rhuG-CSF 3 and 1.5 days before harvest. DMOG doubled mobilization in response to G-CSF with 1,620±530 CFC / mL blood in response G-CSF alone, and 3,250±830 CFC / mL in the G-CSF + DMOG group (p In conclusion, these data highlight the importance of the hypoxia pathway and HIF in the regulation of HPSC cycling and trafficking in vivo. Furthermore HIF-PHD inhibitors may provide therapeutic opportunities to protect HSC from deleterious effects of irradiation as well as increasing mobilization efficiency for transplantation. Disclosures: Walkinshaw: Fibrogen Inc.: Employment, Equity Ownership.

Hematopoietic stem cells (HSC) reside in specialized niches in the bone marrow (BM), that regulate their survival, proliferation and differentiation. Two types of HSC niches have been reported: endosteal niches in close contact with osteoblasts, and endothelial niches near vascular sinuses. Whether these niches have distinct functions in controlling HSC fate remains unknown. One difference between these two niches is the constitutive expression of E-selectin and P-selectin by BM endothelial cells. E- and P-selectin are two cell adhesion molecules that modulate hematopoietic progenitor cell (HPC) survival, proliferation and differentiation in vitro. We now show that deletion of E-selectin, but not P-selectin, delays HSC turn-over in the BM in vivo. Mice lacking either E-selectin (E−/ −), P-selectin (P−/ −) or both (PE−/ −) were given bromo-deoxyuridine (BrdU) in their drinking water for up to 14 days. Lineage-negative c-KIT+ Sca-1+ CD34− (LKS34) cells were sorted from the BM and stained for BrdU incorporation into genomic DNA. Although it took only 3.6 days for 50% of LKS34 cells from wild-type (WT) and P−/ − mice to incorporate BrdU, 9 days were required for 50% BrdU incorporation in LKS34 cells from E−/ − and PE−/ − double KO mice. Thus, HSC cycling time is 2.5 times slower in the absence of E-selectin. To confirm these findings, LKS cells were stained with rhodamine123, a vital dye that is retained by metabolically active cells but effluxed from quiescent HSC. A higher proportion of LKS cells from E−/ − mice were rhodamine dull (34±2%) than WT LKS (23±1%; p=0.037) confirming that a greater proportion of HSC from E−/ − mice are quiescent. To further support these findings, we determined the effect of E-selectin deletion on HSC recovery following cytotoxic stress with a single dose of 5-fluorouracil (5FU 150mg/kg). As KIT is strongly down-regulated in the BM of 5FU-treated mice, we examined frequency and BrdU incorporation in Lin− Sca1+ CD41− CD48− CD150+ long-term reconstituting HSC. We found HSC recovery to be enhanced in E−/ − mice with a 5-fold increase in HSC numbers per femur compared to WT mice at day 7 post-5FU. Despite the more rapid recovery of E−/ − HSC, BrdU incorporation remained significantly lower in E−/ − HSC on days 3 and 7 post-5FU suggesting the decreased HSC turn-over in the absence of E-selectin protects them from the cytotoxic effect of 5FU. To determine whether this effect was mediated by the two described E-selectin receptors PSGL-1 and/or CD44, BrdU incorporation experiments were repeated with mice lacking both the PSGL-1 and CD44 genes. LKS cell turnover in these mice was identical to that of WT suggesting that the effect is mediated by a distinct unknown receptor(s) on HSC. The fact that a novel E-selectin receptor on HSC/HPC is involved was confirmed both using flow cytometry with selectin-IgM chimeras as well as cell adhesion assays using plastic-adsorbed selectin-IgG chimeras. In both assays, 90-95% of LKS cells from CD44−/ − PSGL-1−/ − double KO mice bound E-selectin whereas adhesion to P-selectin was completely lost. Taken together our findings suggest that E-selectin, whose constitutive expression is restricted to BM endothelial cells, plays an important role in the regulation HSC turnover in vivo, endothelial niches, where E-selectin is expressed, support more rapid HSC turn-over within the BM, and this effect is mediated by unknown E-selectin receptors distinct from PSGL-1 or CD44.

Although the molecular mechanisms responsible for hematopoietic stem cell (HSC) mobilization in response to G-CSF or chemotherapy have not been fully elucidated, various murine models demonstrate that both the expansion of the neutrophil pool within the bone marrow (BM) and the down-regulation of the chemokine CXCL12/SDF-1 play a critical role. The main function of neutrophils is to destroy pathogens and necrotic tissues during inflammation. Inflamed and damaged tissues are often very hypoxic due to local disruption of blood supply. Although most cells are stressed in hypoxic conditions, neutrophils and macrophages are attracted and activated by hypoxia, a property dependent on the stabilization of hypoxia-inducible transcription factor-1α (HIF-1α) which is absolutely required for neutrophil and macrophage function. Mice with a conditional deletion of the HIF-1α gene specifically in myeloid cells cannot mount an inflammatory response with neutrophils and macrophage displaying impaired motility, invasiveness and bacterial killing. We therefore hypothesized that hypoxia may play a major role in HSC mobilization. Considering that granulocyte/monocyte progenitors consume 30 times more O2 than their fully differentiated progeny and that mobilized BM is enriched in those progenitors, we calculated that mobilized BM should consume twice more O2 than steady-state BM (3.40μmole O2/femur/hour vs 1.64μmole O2/femur/hour in the mouse). According to the Krogh’s model that predicts O2 distribution in solid tissues, this increased O2 consumption should lead to O2 exhaustion resulting in hypoxia within mobilized BM. We demonstrated this in vivo by injecting the hypoxia-specific probe pimonidazole hydrochloride into mobilized and non-mobilized BALB/c mice 3 hours prior to sacrifice. Pimonidazole covalently binds to tissue proteins when O2 tension is below 10mmHg (O2 tension is 95mmHg in arterial blood, 40mmHg in venous and capillary blood and 150mmHg in the atmosphere at sea level). Staining of femoral BM with a monoclonal antibody (mAb) specific for pimonidazole showed that the hypoxic area (≤10mmHg or

Mobilization of hematopoietic stem cells (HSC) involves the disruption of 1) the adhesive interaction between VCAM-1 and α4-integrins and 2) the chemotactic interaction between CXCL12 and CXCR4, interactions which are both required for the retention of HSC within the bone marrow (BM). Experiments in mice deficient in neutrophil proteases have shown that while the disruption of the VCAM-1/α4 integrin interaction is entirely due to the proteolytic cleavage of VCAM-1 by proteases released from neutrophils accumulating in mobilized BM, the down-regulation of CXCL12 involves protease-independent mechanisms. We have recently shown that osteoblasts are the main source of CXCL12 in the BM and that both the number of osteoblasts lining the endosteum and bone formation are dramatically reduced during G-CSF administration as reflected by bone pain often experienced by mobilized donors. Consequently, the decreased level of CXCL12 levels in mobilized BM could be due to inhibition of osteoblasts, an essential component of the hematopoietic niche. Quantitative real-time RT-PCR were performed on mouse BM cells to follow osteocalcin mRNA levels. Osteocalcin mRNA dropped 2–3 logs during mobilization induced by either G-CSF or cyclophosphamide showing that the inhibition of osteoblast function is not restricted to G-CSF-induced mobilization. Morphometric analyses of tibia sections showed a quasi disappearance of osteoblasts and osteoid as early as day 2 of G-CSF injection. In humans, we observed a significant reduction of osteocalcin protein concentration in the plasma during mobilization induced by either G-CSF alone, G-CSF+ KIT ligand or IL3+GM-CSF, showing that in both humans and mice this effect is not restricted to G-CSF. In cultures of purified human osteoblasts, neither G-CSF, KIT ligand, IL3 nor GM-CSF inhibited osteocalcin production demonstrating that inhibition of osteoblast function is not a direct effect of these cytokines. In parallel experiments, addition of differentiated BM CD34− leukocytes to osteoblast cultures, resulted in a dose-dependant inhibition of osteocalcin production showing that the effect is mediated by mature leukocytes. Since IL6 and soluble IL6 receptor (sIL6R) are important mediators of bone formation, we tested these two cytokines on purified osteoblasts and found that the combination of IL6+sIL6R was a potent inhibitor of osteocalcin production while these cytokines had no effect when used alone. Furthermore, we find cocultures of osteoblasts and BM leukocytes results in a 30-fold increase in IL6 production compared to monocultures of osteoblasts or BM leukocytes. Finally, in humans, plasma concentration of sIL6R is significantly increased during HSC mobilization and this increase is significantly correlated with the number of circulating CFU-GM. Taken together, these data indicate that the inhibition of osteoblast function during HSC mobilization is an indirect effect involving mature BM leukocytes, IL6 and sIL6R.

There is accumulating evidence that interaction of stromal cell derived factor-1 (SDF-1/CXCL12) with its cognate receptor, CXCR4, generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During G-CSF induced HPC mobilization, SDF-1 protein expression in the bone marrow decreases, thereby attenuating CXCR4 signaling. We recently reported that G-CSF treatment induced a decrease in bone marrow SDF-1 mRNA that closely mirrored the fall in SDF-1 protein, suggesting that G-CSF targets one or more SDF-1 producing cell population in the bone marrow. However, the identity of cell populations in the bone marrow that express SDF-1 is controversial. In the present study, we address this issue by sorting cells into mature hematopoietic, hematopoietic progenitor, endothelial, and osteoblast cell populations. Real time RT-PCR analyses showed that osteoblasts and to a lesser degree endothelial cells are the major sources of SDF-1 production in the bone marrow. Surprisingly, on a per cell basis, SDF-1 expression per osteoblast was only modestly (less than two-fold) reduced in mice treated with G-CSF. These data raised the possibility that, rather than affecting SDF-1 expression per osteoblast, G-CSF regulated the number of osteoblasts in the bone marrow. To explore this possibility, osteoblast number in the bone marrow was measured by histomorphometry. Indeed, after 5 days of G-CSF treatment, a significant reduction in the number of endosteal osteoblasts was observed [number of osteoblasts per mm bone perimeter ± SEM: 74.8 ± 13.5 (untreated) versus 33.3 ± 3.8 (G-CSF)]. Moreover, expression of osteocalcin (a specific marker of mature osteoblasts) in the bone marrow was sharply reduced during G-CSF treatment: a 47 ± 12 fold reduction in osteocalcin mRNA (relative to b-actin mRNA) was observed in the bone marrow of G-CSF-treated mice compared with untreated mice. Finally, calcein double-labeling experiments showed that the mineral apposition rate was significantly reduced in G-CSF-treated mice. However, RT-PCR analyses showed that the G-CSF receptor is not expressed on osteoblasts. Accordingly, G-CSF had no direct effect on osteoblast activity in vitro. Collectively, these data show that G-CSF potently suppresses osteoblast number/activity in the bone marrow through an indirect mechanism. Since osteoblasts are thought to play a key role in establishing and maintaining the stem cell niche in the bone marrow, these data raise the possibility that G-CSF, by regulating osteoblast function (including SDF-1 expression), may have profound effects on the stem cell niche that ultimately contribute to HPC mobilization.

By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM- CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.