Egress Of Hematopoietic Stem Cells Research Articles

Interferon-alpha2 treatment of patients with polycythemia vera and related neoplasms favorably impacts deregulation of oxidative stress genes and antioxidative defense mechanisms.

Chronic inflammation is considered a major driving force for clonal expansion and evolution in the Philadelphia-negative myeloproliferative neoplasms, which include essential thrombocythemia, polycythemia vera and primary myelofibrosis (MPNs). One of the key mutation drivers is the JAK2V617F mutation, which has been shown to induce the generation of reactive oxygen species (ROS). Using whole blood gene expression profiling, deregulation of several oxidative stress and anti-oxidative defense genes has been identified in MPNs, including significant downregulation of TP53, the NFE2L2 or NRF2 genes. These genes have a major role for maintaining genomic stability, regulation of the oxidative stress response and in modulating migration or retention of hematopoietic stem cells. Therefore, their deregulation might give rise to increasing genomic instability, increased chronic inflammation and disease progression with egress of hematopoietic stem cells from the bone marrow to seed in the spleen, liver and elsewhere. Interferon-alpha2 (rIFNα) is increasingly being recognized as the drug of choice for the treatment of patients with MPNs. Herein, we report the first gene expression profiling study on the impact of rIFNα upon oxidative stress and antioxidative defense genes in patients with MPNs (n = 33), showing that rIFNα downregulates several upregulated oxidative stress genes and upregulates downregulated antioxidative defense genes. Treatment with rIFNα induced upregulation of 19 genes in ET and 29 genes in PV including CXCR4 and TP53. In conclusion, this rIFNα- mediated dampening of genotoxic damage to hematopoietic cells may ultimately diminish the risk of additional mutations and accordingly clonal evolution and disease progression towards myelofibrotic and leukemic transformation.

GPR182 is an endothelium-specific atypical chemokine receptor that maintains hematopoietic stem cell homeostasis

Significance G protein–coupled receptors (GPCRs) are important regulators of cellular and biological functions and are primary targets of therapeutic drugs. About 100 mammalian GPCRs are still considered orphan receptors because they lack a known endogenous ligand. We report the deorphanization of GPR182, which is expressed in endothelial cells of the microvasculature. We show that GPR182 is an atypical chemokine receptor, which binds CXCL10, 12, and 13. However, binding does not induce downstream signaling. Consistent with a scavenging function of GPR182, mice lacking GPR182 have increased plasma levels of chemokines. In line with the crucial role of CXCL12 in hematopoietic stem cell homeostasis, we found that loss of GPR182 results in increased egress of hematopoietic stem cells from the bone marrow.

Mobilized peripheral blood: an updated perspective.

Enforced egress of hematopoietic stem cells (HSCs) out of the bone marrow (BM) into the peripheral circulation, termed mobilization, has come a long way since its discovery over four decades ago. Mobilization research continues to be driven by the need to optimize the regimen currently available in the clinic with regard to pharmacokinetic and pharmacodynamic profile, costs, and donor convenience. In this review, we describe the most recent findings in the field and how we anticipate them to affect the development of mobilization strategies in the future. Furthermore, the significance of mobilization beyond HSC collection, i.e. for chemosensitization, conditioning, and gene therapy as well as a means to study the interactions between HSCs and their BM microenvironment, is reviewed. Open questions, controversies, and the potential impact of recent technical progress on mobilization research are also highlighted.

1022 - SYSTEMATIC GENETIC DISSECTION OF THE HEMATOPOIETIC STEM CELL NICHE IN THE FETAL LIVER

The liver is the main hematopoietic organ and the site of hematopoietic stem cell (HSC) expansion and maturation in mammalian fetuses. However, little is known about the nature of the HSC niche in the developing liver in vivo. Here, we genetically dissected the cellular components of the developing liver niche by determining the cellular sources of a key HSC niche factor, stem cell factor (SCF). We found that Scf was primarily expressed by endothelial cells and hepatic stellate cells. Conditional deletion of Scf from hematopoietic cells, hepatocytes, Ng2+ cells or endothelial cells did not affect HSC number or function. Deletion of Scf from hepatic stellate cells depleted HSCs. Nearly all HSCs were lost when Scf was deleted from both endothelial cells and hepatic stellate cells in the developing liver. Hepatic stellate cells significantly downregulate SCF and other candidate HSC-supporting factors around birth, coinciding with HSC egress from the liver. Strikingly, the transcriptome of fetal stellate cells resembled that of adult bone marrow mesenchymal stromal cells, but not adult stellate cells, suggesting that the HSC-supporting activity of stellate cells is specific to fetal development. Thus, hepatic stellate and endothelial cells create perivascular HSC niche in developing livers in vivo by producing SCF.

Sphingosine-1-Phosphate Receptor 3 (S1PR3) Promotes Myeloid Commitment of Human Hematopoietic and Leukemic Stem Cells

Understanding the mechanisms underlying the abnormal differentiation of human acute myeloid leukemia (AML) may reveal novel therapies to eradicate leukemic stem cells (LSC), which are often resistant to standard treatments and contribute to relapse. Cellular metabolism is recognized as a hallmark of cancer and is known to be distinct between hematopoietic stem cells (HSC) and downstream progenitors. In particular, sphingosine-1-phosphate (S1P) is a bioactive lipid produced from sphingolipid metabolism that regulates proliferation and survival and is implicated in HSC egress, lymphocyte trafficking and mouse lymphoid lineage determination primarily through binding to S1PR1, one of five S1P G-protein coupled receptors. However, sphingolipids are understudied in human LSC and HSC. We recently found that sphingolipid perturbation governs HSC self-renewal and influences lineage outcome. Here, we show that S1PR3 governs myeloid commitment of LSC in a subset of human AML and is thus an attractive therapeutic target.Lipidomic analysis of LSC+ and LSC‒ fractions derived from AML patient samples and validated by xenotransplantation assays showed distinct sphingolipid alterations when compared to each other and to normal human cord blood (CB) CD34+CD38‒ stem cells and CD34+CD38+ progenitor populations. Interestingly, LSC+ fractions have increased S1P levels, prompting us to wonder if S1P signaling contributes to LSC maintenance. To gain a better understanding of the role of S1P in stemness, we examined S1P signaling in normal CB. Analysis of gene expression of S1PR1-5 and S1P transporters (SPNS2 and MFSD2B) within a comprehensive transcriptional roadmap of human hematopoiesis comprising thirteen normal populations from HSC to mature blood lineages demonstrated distinct expression patterns in specific blood lineages. S1PR1 and MFSD2B were most highly expressed in lymphoid and erythroid lineages, respectively, consistent with murine data. Notably, S1PR3 expression is specific to mature monocytes and granulocytes in human CB. S1PR3 protein was absent from the surface of long-term (LT) and short-term (ST)-HSC as determined by flow cytometry. Remarkably, lentiviral overexpression (OE) of S1PR3 was sufficient to promote myelopoiesis at the expense of erythropoiesis from LT- and ST-HSC in vitro in liquid culture, single cell assays and colony forming assays. To ascertain the mechanisms promoting myeloid commitment, we performed gene expression profiling by RNA-seq of LT- and ST-HSC following S1PR3 OE. This yielded a subset of shared genes similarly upregulated following S1PR3 OE relative to controls, including the known myeloid differentiation and AML-associated factors Early Growth Response 1 and 2 (EGR1/2) and Tribbles 1 (TRIB1). Thus, promiscuous expression of S1PR3 promotes a myeloid fate program in human HSC.S1PR3 protein expression was higher in AML patient samples relative to human CB and bone marrow cells by flow cytometry. Bioinformatic analysis of three independent AML cohorts revealed that AML patient samples with high S1PR3 gene expression also had high EGR2 and TRIB1 expression. Limiting dilution xenotransplantation assays of LSC-containing fractions sorted based on surface expression of S1PR3 demonstrated lower LSC frequency in S1PR3high versus S1PR3low/- LSC fractions. Moreover, S1PR3 OE in LSC+ fractions virtually abolished leukemic engraftment in xenotranplantation assays. These results suggest that higher S1PR3 levels in LSC are associated with a more mature myeloid state and that further increasing S1PR3 levels disrupts LSC maintenance. Treatment of mice bearing primary AML xenografts with FTY720, a S1P mimetic that targets S1P receptors including S1PR3, decreased leukemia burden in a subset of patient samples tested, including those obtained from relapsed and treatment-refractory patients. 70% of AML samples tested showed decreased LSC frequency in serial repopulation assays following FTY720 treatment. Importantly, treatment with FTY720 did not alter normal hematopoietic xenografts, demonstrating the existence of a therapeutic window. Collectively, our results provide the first direct evidence that sphingolipids govern myeloid commitment in human HSC and LSC, and demonstrate the potential of S1PR3 as a novel therapeutic target in AML for eradication of LSC while sparing HSC. DisclosuresNo relevant conflicts of interest to declare.

CREB coactivators CRTC2 and CRTC3 modulate bone marrow hematopoiesis

Populations of circulating immune cells are maintained in equilibrium through signals that enhance the retention or egress of hematopoietic stem cells (HSCs) from bone marrow (BM). Prostaglandin E2 (PGE2) stimulates HSC renewal and engraftment through, for example, induction of the cAMP pathway. Triggering of PGE2 receptors increases HSC survival in part via the PKA-mediated induction of the cAMP response element-binding protein (CREB) signaling pathway. PKA stimulates cellular gene expression by phosphorylating CREB at Ser133 and by promoting the dephosphorylation of the cAMP- responsive transcriptional coactivators (CRTCs). We show here that disruption of both CRTC2 and CRTC3 causes embryonic lethality, and that a single allele of either CRTC2 or CRTC3 is sufficient for viability. CRTC2 knockout mice that express one CRTC3 allele (CRTC2/3m mice) develop neutrophilia and splenomegaly in adulthood due to the up-regulation of granulocyte-colony stimulating factor (G-CSF); these effects are reversed following administration of neutralizing anti-G-CSF antiserum. Adoptive transfer of CRTC2/3m BM conferred the splenomegaly/neutrophilia phenotype in WT recipients. Targeted disruption of both CRTC2 and CRTC3 in stromal cells with a mesenchymal Prx1-Cre transgene also promoted this phenotype. Depletion of CRTC2/3 was found to decrease the expression of Suppressor of Cytokine Signaling 3 (SOCS3), leading to increases in STAT3 phosphorylation and to the induction of CEBPβ, a key regulator of the G-CSF gene. As small molecule inhibition of JAK activity disrupted CEBPβ induction and reduced G-CSF expression in CRTC2/3m stromal cells, our results demonstrate how cross-coupling between the CREB/CRTC and JAK/STAT pathways contributes to BM homeostasis.

Sphingosine-1-Phosphate Prevents Egress of Hematopoietic Stem Cells From Liver to Reduce Fibrosis

Background & AimsThere is growing interest in the use of bone marrow cells to treat liver fibrosis, however, little is known about their antifibrotic efficacy or the identity of their effector cell(s). Sphingosine-1-phosphate (S1P) mediates egress of immune cells from the lymphoid organs into the lymphatic vessels; we investigated its role in the response of hematopoietic stem cells (HSCs) to liver fibrosis in mice.MethodsPurified (c-kit+/sca1+/lin-) HSCs were infused repeatedly into mice undergoing fibrotic liver injury. Chronic liver injury was induced in BoyJ mice by injection of carbon tetrachloride (CCl4) or placement on a methionine-choline–deficient diet. Some mice were irradiated and given transplants of bone marrow cells from C57BL6 mice, with or without the S1P antagonist FTY720; we then studied HSC mobilization and localization. Migration of HSC lines was quantified in Transwell assays. Levels of S1P in liver, bone marrow, and lymph fluid were measured using an enzyme-linked immunosorbent assay. Liver tissues were collected and analyzed by immunohistochemical quantitative polymerase chain reaction and sphingosine kinase activity assays. We performed quantitative polymerase chain reaction analyses of the expression of sphingosine kinase 1 and 2, sphingosine-1-phosphate lyase 1, and sphingosine-1-phosphate phosphatase 1 in normal human liver and cirrhotic liver from patients with alcohol-related liver disease (n = 6).ResultsInfusions of HSCs into mice with liver injury reduced liver scarring based on picrosirius red staining (49.7% reduction in mice given HSCs vs control mice; P < .001), and hepatic hydroxyproline content (328 mg/g in mice given HSCs vs 428 mg/g in control mice; P < .01). HSC infusion also reduced hepatic expression of α-smooth muscle actin (0.19 ± 0.007-fold compared with controls; P < .0001) and collagen type I α 1 chain (0.29 ± 0.17-fold compared with controls; P < .0001). These antifibrotic effects were maintained with infusion of lymphoid progenitors that lack myeloid potential and were associated with increased numbers of recipient neutrophils and macrophages in liver. In studies of HSC cell lines, we found HSCs to recruit monocytes, and this process to require C-C motif chemokine receptor 2. In fibrotic liver tissue from mice and patients, hepatic S1P levels increased owing to increased hepatic sphingosine kinase-1 expression, which contributed to a reduced liver:lymph S1P gradient and limited HSC egress from the liver. Mice given the S1P antagonist (FTY720) with HSCs had increased hepatic retention of HSCs (1697 ± 247 cells in mice given FTY720 vs 982 ± 110 cells in controls; P < .05), and further reductions in fibrosis.ConclusionsIn studies of mice with chronic liver injury, we showed the antifibrotic effects of repeated infusions of purified HSCs. We found that HSCs promote recruitment of endogenous macrophages and neutrophils. Strategies to reduce SIP signaling and increase retention of HSCs in the liver could increase their antifibrotic activities and be developed for treatment of patients with liver fibrosis.

Cholinergic Signals from the CNS Regulate G-CSF-Mediated HSC Mobilization from Bone Marrow via a Glucocorticoid Signaling Relay

Hematopoietic stem cells (HSCs) are mobilized from niches in the bone marrow (BM) to the blood circulation by the cytokine granulocyte colony-stimulating factor (G-CSF) through complex mechanisms. Among these, signals from the sympathetic nervous system regulate HSC egress via its niche, but how the brain communicates with the BM remains largely unknown. Here we show that muscarinic receptor type-1 (Chrm1) signaling in the hypothalamus promotes G-CSF-elicited HSC mobilization via hormonal priming of the hypothalamic-pituitary-adrenal (HPA) axis. Blockade of Chrm1 in the CNS, but not the periphery, reduces HSC mobilization. Mobilization is impaired in Chrm1-∕- mice and rescued by parabiosis with wild-type mice, suggesting a relay by a blood-borne factor. We have identified the glucocorticoid (GC) hormones as critical for optimal mobilization. Physiological levels of corticosterone promote HSC migration via the GC receptor Nr3c1-dependent signaling and upregulation of actin-organizing molecules. These results uncover long-range regulation of HSC migration emerging from the brain.

Abstract A04: Differential dependence on sphingolipid metabolism in the normal and leukemic human hematopoietic hierarchy

Abstract Metabolic alterations are a cancer hallmark that are typically evaluated in bulk tissues. However most normal tissues and cancers are hierarchically organized and the metabolic requirements of both normal and cancer stem cells are poorly understood, particularly beyond energy metabolism. By analyzing a comprehensive transcriptional roadmap of human hematopoiesis, and comparing this to a leukemia stem cell (LSC) signature developed from 84 human acute myeloid leukemia (AML) samples, we found that: (1) several metabolic pathways, specifically in bioactive lipids, distinguish normal hematopoietic stem cells (HSC) from progenitors; and (2) while LSC are similar to HSC, specific metabolic pathways are more comparable to those of normal progenitors. We defined a lipid stem signature of 24 lipid genes, including sphingolipid genes, whose expression is higher in HSC than progenitors. Interestingly, sphingosine-1-phosphate (S1P) is known to play a role in HSC egress, and ceramide vs S1P levels serve as a rheostat to regulate cell growth and survival. To determine if sphingolipids play a functional role in the primitive hematopoietic compartment, we altered sphingolipid signaling by plating sorted populations of HSC or granulocyte-myeloid progenitors (GMP) in methylcellulose containing myriocin, which inhibits the first step of de novo sphingolipid synthesis, or FTY720, a S1P mimetic. Myriocin decreased GMP colony output but did not affect CFC derived from HSC. By contrast FTY720 affected HSC-derived CFC but not those from GMP, suggesting differential sensitivity to sphingolipid pathway inhibition between stem and progenitor cells. In vitro treatment of lineage depleted cord blood (Lin- CB) with myriocin for 8 days limited only myeloid differentiation compared to control treated cells. In contrast, FTY720 treatment reduced levels of immunophenotypic stem cells, erythroid and myeloid cells, as would be expected with inhibition of S1P proliferative signaling. However, in vitro FTY720-treated Lin- CB cells exhibit 16-week engraftment capacity comparable to that of controlled-treated cells, suggesting that any effects on HSC function are reversible after drug withdrawal. Furthermore, in vivo treatment with FTY720 in mice with established CB grafts did not decrease engraftment. Our lipid stem signature is enriched in LSC gene expression profiles from our 84 AML cohort by GSEA analysis, suggesting that differences in lipid metabolism may also exist in LSC vs. non-LSC. To determine if sphingolipids play a functional role in AML biology, we transplanted mice with peripheral blood cells from 13 AML patients, including those with therapy resistant and relapsed disease, and treated engrafted mice with myriocin or FTY720. We observed heterogeneous responses in our cohort, with reduction of leukemic burden in 3 and 5 samples following treatment with FTY720 and myriocin, respectively. Remarkably, serial transplantation of FTY720 responders into untreated secondary mice at limiting dilution demonstrated decreased LSC frequency in FTY720-treated primary mice compared to vehicle-treated controls, whereas myriocin responders showed no alteration of LSC frequency. These results suggest that FTY720 but not myriocin treatment affects LSC number and/or function. To stratify responders from nonresponders, we performed transcriptional analysis of untreated patient samples and compared these data to transcriptional signatures generated from the normal and AML hierarchy. Bioinformatic analysis demonstrated that FTY720 responders had an enriched LSC signature compared to nonresponders. In contrast, myriocin responders exhibited a strong GMP signature compared to nonresponders. Thus, normal human hematopoietic stem and progenitor cells display a variable dependence on sphingolipid biology that is also distinct between LSC and HSC, pointing to targeting of bioactive sphingolipids as a novel therapeutic strategy in AML to eradicate LSC while sparing HSC. Citation Format: Stephanie Z. Xie, Elisa Laurenti, Robin Ferrari, John E. Dick. Differential dependence on sphingolipid metabolism in the normal and leukemic human hematopoietic hierarchy. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A04.

Spatiotemporal Regulation of Hematopoietic Stem Cell Niches By Dual Cholinergic Signaling

Hematopoietic stem cells (HSCs) are known to be heterogeneous, but it is unclear whether, or how, different bone marrow (BM) microenvironments can imprint distinct HSC states. In the BM, quiescent HSCs have been found enriched in endosteal niches, whereas activated HSCs traffic in and out the BM through sinusoids localized further from bone. However, how these separate niches are integrally regulated to maintain BM homeostasis remains largely unknown. We have shown previously that sympathetic adrenergic nerve terminals innervating nestin+ mesenchymal stem cells (MSCs) regulate HSC egress from BM to circulation (Méndez-Ferrer S et al, Nature 2008 and 2010). This regulatory network is required to control HSC proliferation and migration, since its damage caused by mutated HSCs can lead to the manifestation of diseases such as myeloproliferative neoplasms and its protection can block the progression of these disorders (Arranz L et al, Nature 2014). Here we aimed to study the possible cooperation of both branches of the autonomic nervous system, the parasympathetic and the sympathetic nervous systems, which have antagonistic actions in other systems, in HSC regulation in endosteal and sinusoidal niches. We describe for the first time different cholinergic neural signals derived from both autonomic branches that regulate spatially and temporally distinct BM HSC niches. We used mice deficient in cholinergic nerve fibers (secreting acethylcholine, the main postsynaptic neurotransmitter of the parasympathetic nervous system), due to the lack of the neurturin receptor Gfrα2, a member of the glial-cell-derived family of neurotrophic factors (Rossi J et al. Neuron 1999). We found that Gfrα2-/- mice exhibited a circadian-specific defect in HSC traffic. Parasympathetic deficiency caused exacerbated sympathetic tone, manifested by ~2-fold increased BM sympathetic adrenergic fibers and nocturnal urine norepinephrine. Circulating HSCs, measured by long-term competitive repopulating assays, were 3-fold higher in Gfrα2-/- mice only during the resting period, and this was rescued by deletion of the β3-adrenergic-receptor. Therefore, systemic parasympathetic cholinergic signals antagonize BM sympathetic adrenergic activity during the resting phase, contributing to β3-adrenergic-receptor-orchestrated circadian HSC traffic through sinusoidal niches. On the other hand, sympathetic cholinergic nerve fibers, described here for the first time in the BM and running along Haversian canals of bone, regulate HSC maintenance and quiescence in endosteal niches. In neonatal mice, we found that some endosteal sympathetic nerve fibers, sensitive to chemical sympathectomy by 6-hydroxydopamine, switch from catecholaminergic to cholinergic fate and help direct developmental HSC migration to BM. This migration, dependent in perinatal life on Cxcl12 produced by BM nestin+ MSCs (Isern et al, eLife 2014), was impaired in Gfrα2-/- mice, which exhibited one week after birth a reversible ~40% reduction in BM HSCs, associated with the lack of BM sympathetic cholinergic fibers. In adult mice, these sympathetic cholinergic fibers activate nicotinic receptors and induce Cxcl12 expression in bone-associated nestin+ MSCs, an effect reproduced in vitro with the MS-5 stromal cell line. Cxcl12 expression was 2.5-fold higher in Nes-GFP+ cells associated with the bone, compared with those that were not, and was 4-fold lower in Gfrα2-/- Nes-GFP+ cells localized only in the endosteal BM. Concomitantly, Gfrα2-/- mice exhibited reduced allocation of quiescent HSPCs in the endosteal BM, compared with control mice, in which the HSPC fraction in the G0 cell cycle phase was 2-fold higher in the endosteal than in non-endosteal BM. Moreover, HSCs from Gfrα2-/- mice showed increased proliferation, even 6 months after transplantation into secondary wild-type recipients. As a result, Gfrα2-/- mice exhibit accelerated hematopoietic recovery after myeloablation, a phenotype mimicked by mice deficient in the α7-nicotinic receptor. Increased HSC proliferation was associated with loss of self-renewal, since bone-associated HSCs from Gfrα2-/- mice exhibited 50% reduced reconstituting capacity and myeloid potential 24 weeks after competitive transplantation into wild-type recipients. Thus, both branches of the autonomic nervous system regulate HSC maintenance and function in temporally and spatially separate niches. DisclosuresNo relevant conflicts of interest to declare.

A novel view of the adult bone marrow stem cell hierarchy and stem cell trafficking.

This review presents a novel view and working hypothesis about the hierarchy within the adult bone marrow stem cell compartment and the still-intriguing question of whether adult bone marrow contains primitive stem cells from early embryonic development, such as cells derived from the epiblast, migrating primordial germ cells or yolk sac-derived hemangioblasts. It also presents a novel view of the mechanisms that govern stem cell mobilization and homing, with special emphasis on the role of the complement cascade as a trigger for egress of hematopoietic stem cells from bone marrow into blood as well as the emerging role of novel homing factors and priming mechanisms that support stromal-derived factor 1-mediated homing of hematopoietic stem/progenitor cells after transplantation.

Macrophages and Bone Marrow Stem Cell Niches: The Roles of PGE2 and Coagulation

Blood-forming hematopoietic stem cells (HSC) functionally express the chemokine receptor CXCR4, which is required to control their directional motility, cell cycle, adhesion, and bone marrow (BM) repopulation. HSC are mostly BM-retained in a quiescent, non-motile mode via CXCR4-induced adhesion interactions with BM niche cells, which functionally express surface CXCL12. Surface CXCL12-mediated adhesion interactions with BM stromal cells protect quiescent CXCR4+ stem cells from DNA damaging agents while preserving their developmental potential. On the other hand, CXCL12 secretion by BM stromal cells and its release to the blood increase CXCR4 expression and signaling in hematopoietic stem and progenitor cells (HSPC), inducing their egress and clinical mobilization. Finally, CXCR4+ HSC also follow CXCL12 gradients to the BM when they home back from the blood. Intriguingly, dynamic CXCR4/CXCL12 signaling cascades control not only HSC retention in the BM, but also their homing to the BM and their release and mobilization to the blood. In addition to their central role in host defense, myeloid cells participate in organ homeostasis, including HSC localization. Monocyte-derived bone resorbing osteoclasts cleave and release endosteal membrane-bound CXCL12, stem cell factor, and osteopontin, factors needed for HSC adhesion and BM retention, leading to CXCL12/CXCR4-mediated HSPC mobilization. Recently, we identified a rare population of BM αSMA+ macrophages that highly express, which protects HSC from inflammatory insults via COX-2-mediated PGE2 secretion and reactive oxygen species inhibition. In vitro PGE2 upregulates CXCR4 on enriched human cord blood CD34+ and murine HSC via cAMP activation, leading to enhanced CXCL12-induced migration, homing and BM repopulation. However, preliminary results reveal that in vivo PGE2 treatment reduced CXCR4 expression on BM HSC by increasing surface CXCL12 expression by stromal cells. Surface CXCL12 upregulation is due to PGE2-mediated secretion and autocrine signaling of lactate, which is followed by cAMP inhibition in BM stromal cells. Indeed, antagonizing COX-2 or activating cAMP induced HSPC mobilization via BM CXCL12 secretion and increased CXCR4 expression and signaling in HSPC. Coagulation cascades also navigate HSC localization. We revealed that anticoagulant microenvironments in the BM mediate HSC adhesion and retention via inhibition of nitric oxide (NO) production and HSC migration. Physiologic stress induced extensive production of the pro-coagulant factor thrombin, which activates NO generation, CXCL12 secretion and enhanced CXCR4+ stem cell motility and mobilization. We found multinucleated pre-osteoclast cell clusters in femoral metaphysis expressing Tissue Factor (TF) a potent initiator of coagulation leading to thrombin generation. Osteoclast maturation and stress signals also activate TF induced pro-coagulation cascades, mediating HSPC egress and recruitment to the circulation. Daily light and darkness cues regulate many physiological processes, including osteoclast/osteoblast bone remodeling, BM CXCL12 production and secretion and CXCR4+ HSC egress. In addition to the previously identified morning peak of HSC egress to the blood, we identified two peaks of BM HSC proliferation: a morning peak in conjunction with stem cell egress and an evening peak of HSC expansion without egress. Higher BM levels of the HSC-protecting aSMA+ monocyte/macrophages documented in the evening and their associate “low-CXCR4/high CXCL12” BM microenvironment provide the mechanism for these fluctuating patterns. Finally, we found higher PGD2 levels and reduced PGE2 levels in monocyte/macrophages in the morning, whereas at night we documented the opposite patterns. PGD2 induced CXCL12 secretion and HSC egress, while PGE2 induced their retention. Our studies attribute central roles for BM myeloid cells in HSC regulation. Disclosures No relevant conflicts of interest to declare.

Loss of Notch Receptor-Ligand Engagement Leads to Increased Hematopoietic Stem and Progenitor Cell Egress and Mobilization

BACKGROUNDNotch has long been recognized as an important molecule that regulates stem cell self-renewal and differentiation. Despite that Notch is required for the embryonic hematopoietic stem cell (HSC) generation, and that Notch is expressed in the adult HSC, the role of Notch pathway in adult HSC remains unclear. Recently it has been shown that Notch activation and the ability of interacting with Notch ligand Jagged1 is a signature of human primitive HSC and supports HSC regenerative potential. Here we study the physiological significance of Notch in the adult HSC population focusing on how Notch-ligand engagement regulates HSC quiescence and niche retention. METHODSTo better understand the role of Notch in adult HSC homeostasis, we examined HSC frequency, quiescence maintenance, niche occupancy, and HSC mobilization in mice with either conditional lack of RBP-JK, which mediates the canonical Notch signaling activity, or in mice with conditional lack of Pofut1 that catalyzes O-fucosylation of Notch EGF-like repeats and the generation of O-fucose glycans important for the binding of Notch ligands. We also tested Notch ligand neutralizing antibodies and Notch1 and Notch2 inhibitory antibodies to examine the effect of blocking Notch receptor-ligand engagement or the block of Notch signaling activation on HSC homeostasis. RESULTSWe report here that Pofut1-deficient hematopoietic stem and progenitor cells (HSPCs) display enhanced cell cycling and proliferation cell autonomously. These changes are accompanied by G-CSF-independent increased HSPC egress from the marrow to the periphery and other hematopoietic organs, and their enhanced sensitivity to mobilizing stimuli of G-CSF plus the CXCR4 antagonist, AMD3100. This phenotype is caused by reduced adhesion of Pofut1-deficient HSPC to Notch ligand-expressing stromal cells and the osteoblastic lineage cells. Adhesion to these cells by wild type but not Pofut1-deficient HSPCs can be blocked by recombinant Notch ligand Dll1 or Dll4. In addition, adhesion to these cells inhibits wild type but not Pofut1-deficient HSPC cycling, independent of RBP-Jk-mediated Notch signaling. Further, Pofut1-deficient HSPCs exhibit normal expression of key adhesion molecules and normal SDF-1-mediated chemotaxis, but show scattered and distal occupancy in the endosteal or osteoblastic niche. In support of roles for Notch-ligand engagement in facilitating HSPC niche retention, we show that mice receiving 4 doses of neutralizing antibodies to the Notch ligand Dll4 or Jagged1, but not Dll1, display ~2-3-fold increased HSC and progenitor egress when compared to mice receiving isotype control antibody, and further display 60% increased HSC mobilization when compared to mice receiving control antibody and treated similarly with G-CSF and AMD3100. Dll4 blockade also increases the sinusoidal endothelial cell population and HSPC cell cycling. In comparison, only a mild HSPC proliferation and egress is found in RBP-JK-deficient mice, or in mice receiving Notch2 inhibitory antibody. However, Notch2 blockade but not Notch1 blockade induces unique features of HSC and myeloid progenitor mobilization responding to G-CSF plus AMD3100. CONCLUSIONSBased on these findings, we conclude that HSPC quiescence and retention in the marrow niche is facilitated by the interaction between Notch-expressing HSPCs and Jagged1- or Dll4-expressing niche cells, and is likely also contributed by Notch signaling activation. In addition, Notch receptor-ligand engagement in this process is strengthened by O-fucose modification of Notch receptors. Finally, the observations from our studies may provide a therapeutic indication. Inadequate mobilization in HSPC transplantation remains a clinical problem. Our findings that targeting Notch receptor-ligand interaction and/or inhibiting Notch2 activation increase HSPC emigration suggests a novel approach for enhancing mobilization of stem and progenitor cells for those patients who respond poorly to current mobilizing regimes. DisclosuresShim:Genentech: Employment, Equity Ownership. Yan:Genentech: Employment, Equity Ownership. Lowe:Genentech: Employment, Equity Ownership. Siebel:Genentech: Employment, Equity Ownership.

Abstract 4792: Elucidating stem cell-specific metabolic pathways in normal and malignant hematopoiesis to target human acute myeloid leukemia stem cells

Abstract Metabolic alterations are a cancer hallmark but the metabolic requirements of hematopoietic stem cells (HSC) in general, and leukemic stem cells (LSCs) specifically are poorly understood, particularly beyond energy metabolism. By analyzing a comprehensive transcriptional roadmap of human hematopoiesis, and comparing this to a LSC signature developed from 84 primary human acute myeloid leukemia (AML) samples, we uncovered two unexpected findings: (1) several metabolic pathways, specifically in bioactive lipids, distinguish HSC from progenitors and are essential for their function; and LSC are more similar to HSC overall, but also possess specific metabolic pathways that are more comparable to those of normal progenitors. We compiled a comprehensive list of 67 genes involved in fatty acid metabolism based on literature and database information and found that 36 of these were differentially expressed between HSC and progenitors, with 24 of them higher in HSC. In particular, sphingolipid enzymes are differentially regulated throughout the hematopoietic hierarchy. Interesting, sphingosine-1-phosphate (S1P) plays a role in HSC egress and ceramide vs S1P levels serve as a rheostat to regulate growth and cell survival. We defined these 24 genes as a lipid stem signature, and find that it is enriched in the LSC gene expression profiles from our 84 primary AML cohort by GSEA analysis indicating that fatty acid metabolism is differentially regulated in LSCs and non-LSCs, as it is for HSCs vs. normal progenitors. Our signature was found to be prognostic for patient survival in a Dutch cohort of 181 cytogenetically normal AMLs. To determine if lipid metabolism plays a functional role in AML biology, we screened a fatty acid compound library using a novel AML cell line (8227) that retains hierarchical organization and assessed cell viability, phenotypic LSC content, and differentiation. We identified myriocin, which targets serine palmitoyltransferase (1st step of sphingolipid synthesis) in this screen. Myriocin decreased 8227 viability and altered differentiation in vitro and reduced leukemia burden in vivo following transplantation into NOD/SCID mice transgenic for human cytokines. Treatment of mice bearing primary AML xenografts, including those from therapy resistant and relapsed patients, with myriocin or its derivative FTY720, a S1P mimetic, resulted in reduction of leukemic engraftment. Moreover, serial transplantation of AML samples by limiting dilution showed decreased LSC frequency of FTY720- and myriocin-treated cells in secondary mice compared to vehicle-treated cells. Importantly, myriocin or FTY720 treatment do not disrupt engraftment in mice bearing normal hematopoietic grafts. Thus, sphingolipid biology in LSC is different from that of HSC, and we present a novel AML therapeutic strategy targeting bioactive sphingolipids as a means to eradicate LSC while sparing HSC. Citation Format: Stephanie Z. Xie, Elisa Laurenti, John E. Dick. Elucidating stem cell-specific metabolic pathways in normal and malignant hematopoiesis to target human acute myeloid leukemia stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4792. doi:10.1158/1538-7445.AM2014-4792

Polymorphism of CD44 Influences the Efficacy of CD34+ Cells Mobilization in Patients with Hematological Malignancies

In the last decade, peripheral blood was the main source of hematopoietic stem cells (HSC) for autologous and allogeneic transplantation. The exact mechanisms of HSC mobilization are still not clear and the efficacy of the procedure is hardly predictable. Ligand-receptor interactions of adhesion molecules, such as SDF1/CXCR4, VLA4/VCAM-1, or CD44/osteopontin, play an important role in homing of HSC in the hematopoietic niche. There is some evidence that disruption of the ligand-receptor complex leads to the egress of HSCs to the peripheral blood. The aim of the present study was the evaluation of constitutive polymorphism of genes encoding cytokines and receptors present in the HSC niche and their impact on the efficacy of mobilization of HSCs in patients with hematological malignancies. We enrolled 110 patients (60 females and 50 males) in the study. The median age of the patients was 55 (range, 22 to 69) years. The group consisted of patients with multiple myeloma (n = 74), non-Hodgkin lymphoma (n = 19), Hodgkin lymphoma (n = 15), or acute myeloid leukemia (n = 2). The mobilization procedures comprised chemotherapy and subsequent granulocyte-colony stimulating factor (G-CSF) at a dose of 10 μg/kg daily. The poor mobilizers group was defined according to Italian National Bone Marrow Transplant Registry criteria: patients with peak CD34+ in the peripheral blood < 20/μL or total yield < 2 × 106 CD34+ cells/kg body weight in maximum 3 aphereses. Genotyping was performed using standard PCR-based assays. The group of patients (N = 108) who achieved minimal threshold for collections (CD34+ at least 10/μL) proceeded to apheresis. The median total yield of CD34+ in this group was 5.6 × 106 cells/kg body weight, whereas the median number of cells collected during the first apheresis was 3.3 × 106 cells/kg body weight. Median number of days of G-CSF treatment before first apheresis was 10. Fifteen patients fulfilled the criteria for poor mobilizer. The group of poor mobilizers had higher frequency of TT genotype in rs13347 (CD44) gene (CC+ CT versus TT P = .047). Patients homozygous for T allele had a lower total yield of CD34+ cells/kg body weight than the group with allele C (median, 3.7 × 106/kg versus 5.8 × 106/kg; P = .019) and a lower number of CD34+ cells gathered during first apheresis (.95 × 106/kg versus 3.3 × 106/kg, P = .04). Multivariate logistic regression analysis revealed that the CD44 TT genotype was the only factor associated with 5-fold higher risk of poor mobilization (P = .037). Polymorphic variants of CXCR4 and VCAM-1 did not significantly influence the efficacy of HSCs mobilization in our group of patients. In conclusion, our results indicate that among investigated single nucleotide polymorphisms (SNPs), only CD44 rs13347 has an impact on the efficacy of HSCs mobilization in patients with hematologic malignancies. CD44 SNPs analysis may be helpful for predicting the poor mobilizers population who may benefit from newer modalities using adhesion molecules inhibitors.

Polymorphism Of CD44 Influences Efficacy Of CD34+Cells Mobilization In Patients With Hematological Malignancies

▪ BackgroundIn the last decade peripheral blood was the main source of hematopoietic stem cells (HSC) for autologous (auto-SCT) and allogeneic (allo-SCT) transplantation. The exact mechanisms of HSC mobilization are still not clear and efficacy of the procedure is hardly predictable. Numerous clinical factors including age, number of previous intensive regimens, radiotherapy and type of disease can influence efficacy of CD34+ cell mobilization for auto-SCT in patients with hematologic malignancies. Recently it has been stated that ligand- receptor interactions of adhesion molecules such as SDF1/CXCR4, VLA4/VCAM-1 or CD44/osteopontin play important role in homing of HSC in hematopoietic niche. There is evidence that disruption of the ligand-receptor complex leads to egress of HSC to peripheral blood. Influence of single nucleotide polymorphisms in CD44, VCAM-1 and CXCR4 on efficacy of HSC mobilization was evaluated in healthy donors, but not in patients with hematological disorders.The aim of the present study was evaluation of constitutive polymorphism of genes encoding cytokines and receptors present in HSC niche and their impact on efficacy of mobilization of HSC in patients with hematological malignancies. Patients and Methods110 (60 females and 50 males) were enrolled to the study. The median age of the patients was 55 (range 22-69) years. All of the patients evaluated were eligible for autologous HSC mobilization and transplantation. The group consisted of patients with multiple myeloma (74), non-Hodgkin lymphoma (19), Hodgkin lymphoma (15) or acute myeloid leukemia (2). The mobilization procedures comprised chemotherapy and then G-CSF at a dose of 10µg/kg daily. ‘Poor mobilizers' group was defined according to GITMO criteria: patients with peak CD34+ in peripheral blood < 20/μL or total yield <2x 106 CD34+/kg in maximum 3 aphereses. Genotyping was performed using standard PCR-based assays. Three subgroups were established for the genotype in each polymorphism (homozygous more frequent, heterozygous and homozygous less frequent). ResultsThe group of patients (N=108) who achieved minimal threshold for collections (CD34+ at least 10/µl) proceeded to aphereses. Median total yield of CD34+ in this group of patients was 5,6 x 106//kg, while median number of cells collected during first apheresis was 3,3x 106//kg. Median number of days of G-CSF treatment before first apheresis was 10. Fifteen patients fulfilled the criteria for ‘poor mobilizer'.The group of ‘poor mobilizers' had higher frequency of TT allele in rs13347 (CD44) gene (CC+ CT vs TT p=0,047), the difference was even more pronounced in patients with multiple myeloma (N=72, p=0,027). TT homozygous genotype resulted in reduced CD44 mRNA expression at the time of apheresis in comparison with carries of C allele (median=40 in TT group, median=77 in CT and median=85 in CC group, p<0,001). Patients with TT allele had lower total yield CD34+/kg than the group with allele C (Median=3,7x 106//kg vs. 5,8x 106//kg, p=0.019) and lower number of CD34+cells gathered during first apheresis (0,95x 106//kg vs. 3,3x 106//kg, p=0.04). Multivariate logistic regression analysis including age, sex, diagnosis (multliple myeloma vs. others), number of previous treatment lines and CD44 polymorhism (TT vs CT+CC) revealed TT allele was the only factor associated with 5 fold higher risk of poor mobilization (p=0,037).Polymorphic variants of CXCR4 and VCAM-1 did not influence significantly efficacy of HSC mobilization in our group of patients.In conclusion, our results indicate that among investigated SNPs, only CD44 rs13347 has impact on efficacy HSC mobilization in patients with hematologic malignancies. CD44 SNPs analysis may be helpful for predicting of ‘poor mobilizers' population that may benefit from newer modalities using adhesion molecules inhibitors. Disclosures:No relevant conflicts of interest to declare.

The novel CXCR4 antagonist POL5551 mobilizes hematopoietic stem and progenitor cells with greater efficiency than Plerixafor.

Mobilized blood has supplanted bone marrow (BM) as the primary source of hematopoietic stem cells for autologous and allogeneic stem cell transplantation. Pharmacologically enforced egress of hematopoietic stem cells from BM, or mobilization, has been achieved by directly or indirectly targeting the CXCL12/CXCR4 axis. Shortcomings of the standard mobilizing agent, granulocyte colony-stimulating factor (G-CSF), administered alone or in combination with the only approved CXCR4 antagonist, Plerixafor, continue to fuel the quest for new mobilizing agents. Using Protein Epitope Mimetics technology, a novel peptidic CXCR4 antagonist, POL5551, was developed. In vitro data presented herein indicate high affinity to and specificity for CXCR4. POL5551 exhibited rapid mobilization kinetics and unprecedented efficiency in C57BL/6 mice, exceeding that of Plerixafor and at higher doses also of G-CSF. POL5551-mobilized stem cells demonstrated adequate transplantation properties. In contrast to G-CSF, POL5551 did not induce major morphological changes in the BM of mice. Moreover, we provide evidence of direct POL5551 binding to hematopoietic stem and progenitor cells (HSPCs) in vivo, strengthening the hypothesis that CXCR4 antagonists mediate mobilization by direct targeting of HSPCs. In summary, POL5551 is a potent mobilizing agent for HSPCs in mice with promising therapeutic potential if these data can be corroborated in humans.

Role of sphingosine 1-phosphate in trafficking and mobilization of hematopoietic stem cells

The 'mobilization' of hematopoietic stem cells (HSCs) out of the bone marrow and into the peripheral blood is used clinically to obtain HSCs for transplantation. Although generally successful, mobilization protocols remain imperfect and the mechanisms involved are not fully understood. This review discusses the latest findings in respect to the mechanisms involved in the egress of HSCs from the bone marrow into the circulation and the potential for these recent developments to improve mobilization procedures. It has recently become apparent that the bioactive lipid sphingosine 1-phosphate (S1P) plays an active role in attracting HSCs into the peripheral blood. S1P is the first factor identified that provides a chemoattractant gradient promoting the movement of HSCs into the peripheral blood. Drugs that mimic S1P are available with others in development, raising the possibility of increasing the strength of the egress signal and thereby improving the efficacy of mobilization procedures. S1P is the first egress factor described for HSCs, but the details of the underlying biology are only just emerging. Although manipulating the S1P axis to enhance mobilization protocols is an exciting possibility, much needs to be learned before improvements in mobilization strategies can be realized.

Sphingosine-1-phosphate facilitates trafficking of hematopoietic stem cells and their mobilization by CXCR4 antagonists in mice

AbstractCXCL12 and VCAM1 retain hematopoietic stem cells (HSCs) in the BM, but the factors mediating HSC egress from the BM to the blood are not known. The sphingosine-1-phosphate receptor 1 (S1P1) is expressed on HSCs, and S1P facilitates the egress of committed hematopoietic progenitors from the BM into the blood. In the present study, we show that both the S1P gradient between the BM and the blood and the expression of S1P1 are essential for optimal HSC mobilization by CXCR4 antagonists, including AMD3100, and for the trafficking of HSCs during steady-state hematopoiesis. We also demonstrate that the S1P1 agonist SEW2871 increases AMD3100-induced HSC and progenitor cell mobilization. These results suggest that the combination of a CXCR4 antagonist and a S1P1 agonist may prove to be sufficient for mobilizing HSCs in normal donors for transplantation purposes, potentially providing a single mobilization procedure and eliminating the need to expose normal donors to G-CSF with its associated side effects.

Diabetic Stem-Cell “Mobilopathy”

Obtaining hematopoietic stem cells (HSCs) for transplantation depends on effective egress of HSCs from bone marrow into the peripheral circulation on induction. This process is impaired in a mouse model of diabetes and in patients undergoing autologous transplantation.