Retention Of Hematopoietic Stem Cells Research Articles

IGF1R signaling in perinatal mesenchymal stem cells determines definitive hematopoiesis in bone marrow

During the transition from embryonic to adult life, the sites of hematopoiesis undergo dynamic shifts across various tissues. In adults, while bone marrow becomes the primary site for definitive hematopoiesis, the establishment of the bone marrow niche for accommodating hematopoietic stem cells (HSCs) remains incompletely understood. Here, we reveal that perinatal bone marrow mesenchymal stem cells (BMSCs) exhibit highly activated insulin-like growth factor 1 receptor (IGF1R) signaling compared to adult BMSCs. Deletion of Igf1r in perinatal BMSCs hinders the transition of HSCs from the fetal liver to the bone marrow in perinatal mice and disrupts hematopoiesis in adult individuals. Conversely, the deletion of Igf1r in adult BMSCs, adipocytes, osteoblasts, or endothelial cells does not affect HSCs in the bone marrow. Mechanistically, IGF1R signaling activates the transcription factor nuclear factor of activated T cells c1 (NFATc1) in perinatal BMSCs, which upregulates CXCL12 and other niche factors for HSC retention. Overall, IGF1R signaling in perinatal BMSCs regulates the development of the bone marrow niche for hematopoiesis.

Mobilization dynamics of bone marrow hematopoietic stem cells during hematopoietic regeneration

Under stress hematopoiesis, previous studies have suggested the migration of hematopoietic stem cells (HSCs) from bone marrow (BM) to extramedullary sites such as the spleen. However, there is little direct evidence of HSC migration from the BM to the spleen. Here, we induced myeloablation via 5-fluorouracil (5-FU) and showed direct evidence of HSC migration from BM to spleen during hematopoietic regeneration via a photoconvertible fluorophore. Moreover, during steady state, HSCs preferentially migrated to BM rather than spleen, but during hematopoietic regeneration, HSCs preferred spleen as a migration site equivalently or greater. Furthermore, in the early phase, HSCs egressed from BM through the attenuated HSC retention. However, HSCs in the late phase gained significantly enhanced cell-autonomous motility, which was independent of chemotaxis. Collectively, HSC mobilization from BM, before the migration to the spleen, was dynamically changed from passive to active events during hematopoietic regeneration.

From Marrow to Bone and Fat: Exploring the Multifaceted Roles of Leptin Receptor Positive Bone Marrow Mesenchymal Stromal Cells.

The bone marrow (BM) stromal cell microenvironment contains non-hematopoietic stromal cells called mesenchymal stromal cells (MSCs). MSCs are plastic adherent, form CFU-Fs, and give rise to osteogenic, adipogenic, chondrogenic progenitors, and most importantly provide HSC niche factor chemokine C-X-C motif ligand 12 (CXCL12) and stem cell factor (SCF). Different authors have defined different markers for mouse MSC identification like PDGFR+Sca-1+ subsets, Nestin+, or LepR+ cells. Of these, the LepR+ cells are the major source of SCF and CXCL12 in the BM microenvironment and play a major role in HSC maintenance and hematopoiesis. LepR+ cells give rise to most of the bones and BM adipocytes, further regulating the microenvironment. In adult BM, LepR+ cells are quiescent but after fracture or irradiation, they proliferate and differentiate into mesenchymal lineage osteogenic, adipogenic and/or chondrogenic cells. They also play a crucial role in the steady-state hematopoiesis process, as well as hematopoietic regeneration and the homing of hematopoietic stem cells (HSCs) after myeloablative injury and/or HSC transplantation. They line the sinusoidal cavities, maintain the trabeculae formation, and provide the space for HSC homing and retention. However, the LepR+ cell subset is heterogeneous; some subsets have higher adipogenic potential, while others express osteollineage-biased genes. Different transcription factors like Early B cell factor 3 (EBF3) or RunX2 help maintain this balance between the self-renewing and committed states, whether osteogenic or adipogenic. The study of LepR+ MSCs holds immense promise for advancing our understanding of HSC biology, tissue regeneration, metabolic disorders, and immune responses. In this review, we will discuss the origin of the BM resident LepR+ cells, different subtypes, and the role of LepR+ cells in maintaining hematopoiesis, osteogenesis, and BM adipogenesis following their multifaceted impact.

Local and Systemic Overexpression of COMP-Ang1 Induces Ang1/Tie2-Related Thrombocytopenia and SDF-1/CXCR4-Dependent Anemia.

While supplemental angiopoietin-1 (Ang1) improves hematopoiesis, excessive Ang1 induces bone marrow (BM) impairment, hematopoietic stem cell (HSC) senescence, and erythropoietic defect. Here, we examined how excessive Ang1 disturbs hematopoiesis and explored whether hematopoietic defects were related to its level using K14-Cre;c-Ang1 and Col2.3-Cre;c-Ang1 transgenic mice that systemically and locally overexpress cartilage oligomeric matrix protein-Ang1, respectively. We also investigated the impacts of Tie2 inhibitor and AMD3100 on hematopoietic development. Transgenic mice exhibited excessive angiogenic phenotypes, but K14-Cre;c-Ang1 mice showed more severe defects in growth and life span with higher presence of Ang1 compared with Col2.3-Cre;c-Ang1 mice. Dissimilar to K14-Cre;c-Ang1 mice, Col2.3-Cre;c-Ang1 mice did not show impaired BM retention or senescence of HSCs, erythropoietic defect, or disruption of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis. However, these mice exhibited a defect in platelet production depending on the expression of Tie2 and globin transcription factor 1 (GATA-1), but not GATA-2, in megakaryocyte progenitor (MP) cells. Treatment with Tie2 inhibitor recovered GATA-1 expression in MP cells and platelet production without changes in circulating RBC in transgenic mice. Consecutive AMD3100 administration not only induced irrecoverable senescence of HSCs but also suppressed formation of RBC, but not platelets, via correlated decreases in number of erythroblasts and their GATA-1 expression in B6 mice. Our results indicate that genetic overexpression of Ang1 impairs hematopoietic development depending on its level, in which megakaryopoiesis is preferentially impaired via activation of Ang1/Tie2 signaling, whereas erythropoietic defect is orchestrated by HSC senescence, inflammation, and disruption of the SDF-1/CXCR4 axis.

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.

EPCR Signaling Controls the Activity of Hematopoietic Stem Cells Independent of Coagulation Regulation

BackgroundAll mature blood cells are derived from multipotent hematopoietic stem cells (HSCs) which are activated to meet the demand of the host during inflammation and injury. The endothelial cell protein C receptor (EPCR) is a marker for primitivity and quiescence of HSCs but the relative contributions of EPCR signaling versus anticoagulant functions in HSC maintenance are incompletely defined.AimsWe aimed to dissect functions of EPCR by studying anticoagulant and signaling function in HSC of EPCR C/S mice carrying a single intracellular point mutation abolishing normal trafficking of EPCR through endo-lysosomal compartments. We assessed the contributions of EPCR signaling to stem cell maintenance by analyzing HSC mobilization and leukemia progression.MethodsWe studied the frequency and cell cycle activity of bone marrow (BM) hematopoietic stem and progenitor cells (HSPC) by multicolor flow cytometry. Furthermore, we analyzed changes in hematopoiesis in steady state, after granulocyte colony stimulating factor (G-CSF)-induced mobilization, in the context of aging and in the context of leukemia, using the MLL-AF9-induced acute myeloid leukemia (AML) model.ResultsHSCs, lungs and isolated lung-derived smooth muscle cells of EPCR C/S mice showed protein expression levels and anticoagulant function indistinguishable from wildtype (WT). We found an increase of circulating HSCs in the peripheral blood of EPCR C/S mice compared to control under steady state conditions. Isolated HSC displayed diminished polarization of CDC42 and VLA-4 (α 4β 1 integrin) affinity to VCAM-1 in EPCR C/S versus strain-matched EPCR wt mice, indicating that EPCR signaling directly controls HSC retention via integrin affinity to the BM niche. In addition, we noticed a higher cell cycle activity in myeloid-restricted progenitors of EPCR C/S mice compared to control. G-CSF treatment led to increased mobilization of both BM neutrophils and HSCs into the peripheral blood of EPCR C/S mice compared to EPCR wt mice. A myeloid bias was also seen in serially transplanted aged mice, resulting in increased frequencies of myeloid-biased progenitors in the BM of EPCR C/S mice compared to control mice, accompanied by an increase of circulating neutrophils in the blood. Consistent with higher cell cycle activity of myeloid progenitors and an overall increase of myeloid-biased output in EPCR C/S mice, induction of AML by retroviral transduction of EPCR C/S BM cells with MLL-AF9-expressing retrovirus resulted in an increase of cell cycle activity of Lin - MLL-AF9 + leukemic BM blasts and a higher leukemic load in the peripheral blood of mice transplanted with MLL-AF9 + EPCR C/S BM compared to control. As a result, MLL-AF9 + EPCR C/S leukemia showed a more aggressive disease with shortened survival times compared to control. In contrast, chemotherapy of MLL-AF9 + EPCR C/S leukemia reduced leukemic load in the peripheral blood and decelerated disease progression. These data demonstrate that increased leukemia cell cycle activity conferred chemosensitivity.ConclusionWith a site-specific EPCR mutant knock-in mouse, we here demonstrate that EPCR signaling and anticoagulant function can be separated. We provide direct evidence that EPCR signaling plays a crucial role in maintaining HSC retention via VLA-4 affinity to VCAM-1, controls cell cycle activity and myeloid output in normal, stress-induced, and malignant hematopoiesis with implications for therapeutic approaches in acute myeloid leukemia. DisclosuresRuf: MeruVasimmune: Other: Ownership Interest; ARCA bioscience: Consultancy, Patents & Royalties; ICONIC Therapeutics: Consultancy.

G-CSF Signaling Counteracts TGF-β—PAI-1-Dependent Retention of Hematopoietic Stem Cells in the Niche

Hematopoietic stem cells (HSCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but are usually stayed in the niche. Recently, we have reported that niche cell-producing TGF-β induces intracellular PAI-1 expression in HSCs, which inhibits the proteolytic activity of proprotein convertase Furin-dependent maturation of MT1-MMP, a protease involved in the mobilization of HSPCs, thereby keeping HSCs retaining in the BM niche. Pharmacological inhibition or genetic disruption of the TGF-β−PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSCs from the TGF-β-expressing niche cells, demonstrated a crucial role for TGF-β−PAI-1 signal in the retention of HSCs in the BM. On the other hand, HSCs frequently egress from the BM to circulation and replenish blood supply in order to maintain the homeostasis of blood system. At this moment, however, how environmental stimuli, such as a blood G-CSF concentration rise, counteract the TGF-β-PAI-1 signaling and releases the motility restriction of HSCs remains unknown.To identify the molecular mechanism that govern the dynamic motion of HSCs in the niche, we chose to investigate the cellular activities of HSCs in response to G-CSF. Here, we provide evidence that G-CSF inhibits Smad-dependent signaling by regulating cAMP-PKA-dependent signaling pathway in HSCs. G-CSF-induced activation of this pathway inhibits Smad-dependent expression of PAI-1 through an increase in phosphorylation levels of CREB protein. Mechanistically, increased pCREB competitively inhibited the Smad from binding to the p300/CBP, which ultimately resulting in a transcriptional suppression of Smad-p300/CBP transcription regulatory complex-dependent PAI-1 gene expression. In line with this, administration of G-CSF to mice strongly downregulated intracellular PAI-1 expression in HSCs, which resulted in enhanced Furin-dependent MT1-MMP maturation. The enhanced expression of MT1-MMP results in MT1-MMP-mediated CD44 cleavage as well as CXCR4 and VLA-4 activation, which in turn stimulates detachment of HSPCs from the niche and active trafficking into the blood stream. Expectedly, pharmacological inhibition of intracellular PAI-1 activity synergized with G-CSF-induced HSC mobilization.Our study reveals a novel role for G-CSF signaling in controlling TGF-β−PAI-1-dependent retention of HSCs to recruit them into the circulation, which potentially improve clinical HSC mobilization and transplantation protocols. DisclosuresAndo:CHUGAI PHARMACEUTICAL: Other: Donation to institute; Meiji Seika Pharma: Other: Donation to institute; NOVARTIS: Other: Donation to institute; TOYAMA CHEMICAL: Other: Donation to institute; MOCHIDA PHARMACEUTICAL: Other: Donation to institute; Sumitomo Dainippon Pharma: Other: Donation to institute; Eisai: Other: Donation to institute; Bristol-Myers Squibb: Other: Donation to institute; MSD: Other: Donation to institute; Kyowa Hakko Kirin: Other: Donation to institute; ALEXION: Other: Donation to institute; Takeda: Other: Donation to institute; Japan Blood Products Organization: Other: Donation to institute; Nippon Shinyaku: Other: Donation to institute; NIHON PHARMACEUTICAL: Other: Donation to institute; TAIHO: Other: Donation to institute; Asahi KASEI: Other: Donation to institute.

An Unbalanced Monocyte Macrophage Polarization in the Bone Marrow Microenvironment of Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

BackgroundPoor graft function (PGF) remains a life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the underlying mechanisms have not yet been elucidated. Considerable evidence from murine studies has demonstrated that effective hematopoiesis depends on the specific bone marrow (BM) microenvironment, where hematopoietic stem cells (HSCs) reside. In this regard, we previously reported that PGF patients had impaired BM endosteal cells and endothelial progenitor cells, as well as dysregulated T cell responses in the BM microenvironment, which may be involved in the occurrence of PGF (BBMT 2013; BMT2016; Oncotarget 2016; BBMT 2016; Blood 2016; J Transl Med2017). Macrophage is one of the important components of BM immune microenvironment. Meanwhile, Murine studies suggest that macrophages regulate the retention of HSCs by regulating osteoblastic cell activity and nestin-expressing cells. Increasing evidence shows that BM resident macrophages are indispensable for HSCs function and BM erythroid output. However, little is known about the quantity and function of BM macrophages and whether the unbalanced BM macrophages directly interact with HSCs in PGF patients post allo-HSCT.AimsTo compare the number and function of BM macrophages between patients with PGF and GGF after allo-HSCT. Moreover, to investigate whether the unbalanced BM macrophages directly interact with HSCs in PGF patients.MethodsThis prospective nested case-control study enrolled 30 patients with PGF, 60 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 30 healthy donors (HD). Standard monocyte subsets, defined by cluster of differentiation CD14 and CD16, were quantified by flow cytometry. In addition, based on the phenotype of polarized macrophages in vitro, classically activated inflammatory macrophage (M1:CD68+CCR2+) and alternatively activated anti-inflammatory macrophage (M2:CX3CR1+CD163+/CD206+) from BM samples were analyzed by flow cytometry. BM CD14+ monocytes were isolated from BM mononuclear cell (BMMNCs) and were cultured with 100 ng/ml recombinant human M-CSF for 10 days to obtain macrophages. The functions of BM derived macrophages were evaluated by migration assay and phagocytosis assay. CD34+ cells from healthy donors were co-cultured with BM derived macrophages from PGF patients, and the levels of reactive oxygen species (ROS) and apoptosis in CD34+ cells were analyzed by flow cytometry after 5 days co-culture.ResultsAlterations in standard monocyte subsets (classical, intermediate and non-classical) were found when comparing subjects among PGF, GGF and normal controls. In particular, elevated intermediate and non-classical monocyte subsets were observed in PGF patients when compared with those in GGF patients. Moreover, PGF patients displayed an imbalanced M1/M2 ratio compared with the GGF and normal controls, attributable to a reduction in M2 and an increase in M1. Dysfunctional BM derived macrophages, which were characterized by impaired proliferation, migration, phagocytosis and higher level of apoptosis, were revealed in patients with PGF. BM CD34+ cells from healthy donors were co-cultured respectively with BM derived macrophages from patients with PGF and normal controls. Apoptosis and intracellular levels of ROS were markedly increased in CD34+ bone marrow cells which were co-cultured with PGF patients' BM derived macrophages compared with normal controls.Summary/ConclusionIn summary, the current study demonstrated the abnormal number and impaired function of BM macrophages and unbalanced BM macrophages directly interact with HSCs in PGF patients. We speculate that reduction of anti-inflammatory M2 monocytes while increase of pro-inflammatory M1 monocytes in PGF patient BM have a negative impact on HSCs in BM microenvironment. Thus, our results indicate that unbalanced BM M1/M2 and dysfunctional BM macrophages may play an important role in the pathogenesis of PGF following allo-HSCT. Therefore, it would be of value to investigate the appropriate approach to balance the M1/M2 and enhance function of BM macrophages, which may be a promising therapeutic approach for PGF patients. DisclosuresNo relevant conflicts of interest to declare.

ACE Inhibition Modulates Myeloid Hematopoiesis after Acute Myocardial Infarction and Reduces Cardiac and Vascular Inflammation in Ischemic Heart Failure.

Aims: Angiotensin-converting-enzyme inhibitors (ACE inhibitors) are a cornerstone of drug therapy after myocardial infarction (MI) and improve left ventricular function and survival. We aimed to elucidate the impact of early treatment with the ACE inhibitor ramipril on the hematopoietic response after MI, as well as on the chronic systemic and vascular inflammation. Methods and Results: In a mouse model of MI, induced by permanent ligation of the left anterior descending artery, immediate initiation of treatment with ramipril (10 mg/k/d via drinking water) reduced cardiac inflammation and the number of circulating inflammatory monocytes, whereas left ventricular function was not altered significantly, respectively. This effect was accompanied by enhanced retention of hematopoietic stem cells, Lin−Sca1−c-Kit+CD34+CD16/32+ granulocyte–macrophage progenitors (GMP) and Lin−Sca1−c-Kit+CD150−CD48− multipotent progenitors (MPP) in the bone marrow, with an upregulation of the niche factors Angiopoetin 1 and Kitl at 7 d post MI. Long-term ACE inhibition for 28 d limited vascular inflammation, particularly the infiltration of Ly6Chigh monocytes/macrophages, and reduced superoxide formation, resulting in improved endothelial function in mice with ischemic heart failure. Conclusion: ACE inhibition modulates the myeloid inflammatory response after MI due to the retention of myeloid precursor cells in their bone marrow reservoir. This results in a reduction in cardiac and vascular inflammation with improvement in survival after MI.

Role of thrombomodulin expression on hematopoietic stem cells

BackgroundActivation of protease‐activated receptor 1 (PAR1) by either thrombin or activated protein C (aPC) differentially regulate the quiescence and bone marrow (BM) retention of hematopoietic stem cells (HSC). Murine HSC co‐express THBD, PAR1, and endothelial protein C receptor (EPCR), suggesting that HSC sustain quiescence in a quasi‐cell autonomous manner due to the binding of thrombin present in the microenvironment to THBD, activation of EPCR‐bound protein C by the thrombin‐THBD‐complex, and subsequent activation of PAR1 by the aPC‐EPCR complex. ObjectiveTo determine the role of THBD expression on HSC for sustaining stem cell quiescence and BM retention under homeostatic conditions. MethodsHematopoietic stem cell function was analyzed in mice with constitutive or temporally controlled complete THBD‐deficiency by flow cytometry, functional assays, and single cell RNA profiling. ResultsTHBD was expressed in mouse, but not human, HSC, progenitors, and immature B cells. Expression in vascular endothelium was conserved in humans' BM. Mice with constitutive THBD deficiency had a normal peripheral blood profile, altered BM morphology, reduced numbers of progenitors and immature B cells, pronounced extramedullary hematopoiesis, increased HSC frequency, and marginally altered transcriptionally defined HSC stemness. Transplantation experiments indicated near normal engraftment and repopulating ability of THBD‐deficient HSC. Transgenic aPC supplementation normalized BM histopathology and HSC abundance, and partially restored transcriptional stemness, but had no effect on B cell progenitors and extramedullary hematopoiesis. Temporally controlled THBD gene ablation in adult mice did not cause the above abnormalities. ConclusionTHBD expression on HSPC has minor effects on homeostatic hematopoiesis in mice, and is not conserved in humans.

CXCR4 Inhibition with BL-8040 in Combination with Nelarabine in Patients with Relapsed or Refractory T-Cell Acute Lymphoblastic Leukemia / Lymphoblastic Lymphoma

Background: The chemokine receptor, CXCR4, mediates the trafficking and retention of hematopoietic stem cells to the marrow through its interaction with the chemokine, CXCL12. CXCR4 is the most highly expressed chemokine receptor on T-ALL cells, and preclinical data demonstrate that CXCR4 is critical for the growth and survival of T-cell acute lymphoblastic lymphoma (T-ALL). Genetic loss or pharmacologic blockade of CXCR4 signaling suppresses T-ALL cell growth and leukemia initiating activity in murine models. BL-8040 is a novel synthetic peptide antagonist of the chemokine receptor, CXCR4. Compared to other CXCR4 inhibitors, BL-8040 is a more potent mobilizer of normal HSCs with sustained occupancy and inhibition of CXCR4. We previously reported that BL-8040 potently suppresses T-ALL cell growth in xenotransplantation models of T-ALL. To test the efficacy of BL-8040 in T-ALL by conducting a multicenter, open label pilot study of BL-8040 in combination with nelarabine in patients with relapsed or refractory disease (ClinicalTrials.gov: NCT0276338). Methods: Adults with relapsed or refractory T-ALL/LBL, age ≥18 years and ECOG PS ≤2, were eligible for the study. A peripheral blood lymphoblast count ≤50,000/mm3 was required prior to starting treatment. During cycle 1, subjects received daily subcutaneous administration of BL-8040 1.5 mg/kg from Day 1 to Day 6 with nelarabine 1,500 mg/m2 intravenously over 2 hours on Days 2, 4 and 6. In subsequent 21-day cycles, BL-8040 1.5 mg/kg was administered daily on days 1-5 with nelarabine 1,500 mg/m2 given on days 1, 3 and 5. Up to 4 cycles of treatment was allowed. Results: Nine patients with median age of 29 years (range 20-75) have been enrolled in the study out of which including fout patients were in untreated 1st relapse, one in 2nd relapse, two with relapsed and refractory disease, and two primary refractory patient. Two patients had failed prior allogeneic hematopoietic cell transplant. The rate of complete remission is 56% (5/9) with all responses occurring after 1 cycle of treatment. Adverse events occurred in 2 subjects requiring dose modification. Consistent with the known mechanism of BL-8040, one subject developed G3 leukocytosis after BL-8040 administration with the peripheral WBC increasing from 5.3 to 273 h/mm3 with evidence of spontaneous tumor lysis requiring interruption of BL-8040 and leukapheresis. A second patient developed injection site pain requiring omission of the final dose of BL-8040. Peripheral blood analyzed at baseline and 24 hours after the first dose of BL-8040 (prior to nelarabine) showed the following: 1) sustained blockade of CXCR4 on leukemic blasts; 2) mobilization of leukemic blasts into the blood; and 3) no induction of leukemia blast apoptosis as measured by activated caspase 3 expression. Conclusions: BL-8040 can be combined with nelarabine in subjects with relapsed or refractory T-ALL with encouraging CR rates. Evidence of on target effects on CXCR4 has been observed in this study with sustained CXCR4 receptor occupancy at 24 hours after administration along with tumor mobilization of ALL blasts into the peripheral circulation. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Kadia:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bioline RX: Research Funding; BMS: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding. Stock:Astellas: Membership on an entity's Board of Directors or advisory committees; Daiichi: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; UpToDate: Honoraria; Agios: Membership on an entity's Board of Directors or advisory committees; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Brammer:Verastem, Inc: Research Funding; Viracta Therapeutics, Inc.: Research Funding; Bioniz Therapeutics, Inc.: Research Funding. Bohana-Kashtan:BiolineRx: Employment, Equity Ownership. Vainstein:BiolineRx: Employment, Equity Ownership. Sorani:BiolineRx: Employment, Equity Ownership. Chen:BiolineRx: Employment. DiPersio:Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; NeoImmune Tech: Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Incyte: Consultancy, Research Funding; Karyopharm Therapeutics: Consultancy. OffLabel Disclosure: BL-8040 for ALL

Macrophage Transfer to HSCs Assigns Residence in Bone Marrow

Hematopoietic stem cells (HSCs), residing at the apex of the hematopoietic hierarchy, are a rare and heterogeneous cell population. Although HSC subsets with various repopulation capacities and lineage bias have been identified, there is no available information about whether each HSC has an equal chance of being mobilized or whether there are specific pools of mobilizable or non-mobilizable HSCs in bone marrow (BM). Here, we identify a BM resident HSC subset based on the expression of the macrophage marker F4/80. Flow cytometry analysis revealed that F4/80 was expressed on 75% of HSCs (Lin- Sca1+cKit+CD150+CD34-; hereafter referred to as HSCM) in the BM. In competitive transplantation assays, HSCM exhibited significantly lower engraftment potential, but no lineage bias, compared to F4/80- HSCs (HSCM: 19.7±4.8%; F4/80- HSC: 37.1±4.5%, P<0.05). We next analyzed the ability of HSCM and F4/80- HSCs to be mobilized after G-CSF treatment and found that F4/80- HSCs, but not HSCM, were exclusively mobilized (F4/80- HSC: 5042±912.5; HSCM: 6.6±6.6 per mL blood, P<0.001). Similar selective mobilization was observed after administration of AMD3100 or macrophage depletion using clodronate liposomes. To confirm this observation in a genetic model, we crossed transgenic mice expressing Cre recombinase knocked into the Cd169 locus, a marker of BM macrophages (MΦ), with ROSA26-loxP-stop-loxP-tdTomato (CD169/Tomato). We found CD169/Tomato labelled a large fraction of HSCM (31.7±8.4%), in contrast to F4/80- HSCs which were largely unlabelled (2.1±1.2%). Remarkably, CD169/Tomato+ HSCs were not mobilized into the circulation after G-CSF treatment (CD169/Tomato- HSC: 4573±1416; CD169/Tomato+ HSC: 90.6±90.6 per mL blood, P<0.01). Although HSCs are reported to express Csf1r, the expression of macrophage markers on HSCs (F4/80 and CD169) was unexpected. To ascertain the expression origin, we transplanted CD169/Tomato+ and CD169/Tomato- sorted HSCs into lethally irradiated CD45.1 congenic mice. If HSCs expressed CD169, we would expect that donor HSC tomato expression would be retained. However, to our surprise, we found that the proportion of Tomato+ HSCs was higher in the CD169/Tomato- group, suggesting that F4/80 and CD169 expression may be acquired, rather than expressed within HSCs. Indeed, co-culture of TdTomato+ BM MΦ with GFP+ HSCs consistently revealed the acquisition of tdTomato (GFP+ TdTomato+: 9.9±1.9%). Transwell experiments revealed that cell contact was important as GFP+ cells did not show any TdTomato expression in the absence of direct cell contact (GFP+TdTomato+: 0%). To investigate the mechanism of HSC retention, we analyzed CXCR4 expression on HSCM and F4/80- HSC subsets. These analyses revealed that CXCR4 was expressed at higher expression levels on HSCM compared to F4/80- HSCs (HSCM: 79.4±9.1% CXCR4+;F4/80- HSC: 45.6±8.5% CXCR4+, P<0.001). In accordance with their increased CXCR4 expression, HSCM exhibited a 4.7-fold increase in pERK1/2 after CXCL12 stimulation, while CXCL12 only triggered a modest increase (1.5-fold) in pERK1/2 in F4/80- HSCs, suggesting that HSCM have a higher signaling response to CXCL12, and thus exhibit enhanced BM retention. To further confirm that the cell transfer occurred in vivo, we used xenografted mice in which human HSCs were transplanted into NOG mice. We found a significant presence of murine F4/80 on human CD34+ HSCs in xenografted huNOG mice, confirming the F4/80 transfer from host MΦ into human HSCs in vivo. To investigate further the mechanism of cell transfer, we carried out co-culture experiments of GFP+ lineage-depleted BM cells with TdTomato+ MΦ in the presence of inhibitors known to prevent cell transfer through membrane and cytoskeleton remodeling. Of all conditions tested, only the presence of the GAP junction inhibitor carbenoxolone (CBX) could inhibit the transfer of TdTomato from MΦ to GFP+ cells (control: 21.4±3.0%; CBX: 12.6±2.1%, P<0.001). Furthermore, treatment of wild-type mice with CBX led to a marked suppression of F4/80 transfer in vivo, similar to that observed in vitro, suggesting that GAP junctions are responsible for the direct transfer of cellular content from BM MΦ to HSCs. Our results thus identify a novel mechanism by which macrophages can assign HSC residence in BM. Manipulation of macrophage-mediated transfer may enhance the mobilizable HSC pool and provide a new method to improve HSC yields after mobilization. Disclosures Frenette: Cygnal Therapeutics: Equity Ownership; Ironwood Pharmaceuticals: Research Funding; Albert Einstein College of Medicine, Inc: Patents & Royalties; Pfizer: Consultancy.

Abstract 2181: A novel CXCR4 antagonist strongly mobilizes hematopoietic stem cells in vivo

Abstract Introduction: The transplantation of hematopoietic stem cells (HSCs) has been the standard of care for the treatment of chemotherapy sensitive relapsed acute leukemia. HSCs mainly reside in the bone marrow with only few HSCs circulating in the peripheral blood (PB), due to homing effect mediated by the interaction of chemokine receptor CXCR4 expressed on HSCs with chemotactic ligand SDF-1α rich in the bone marrow. It is clinically desirable to enhance the number of HSCs in the PB for collection during the transplantation procedure. Since SDF-1α/CXCR4 axis and interaction mediates the homing and retention of HSCs in the bone marrow, our laboratory has recently discovered and developed a novel small molecule, HFX51116, that is capable of binding CXCR4 and blocking its interaction with SDF-1α, raising the possibility that this compound can be used for releasing HSCs from the bone marrow to the PB useful for the transplantation of HSCs. Method and Results: In vitro, there was almost no toxicity of HFX51116 at high concentrations in bone marrow stromal cells that were originally extracted from rats. In addition, HFX51116 was stable in rat serum. In vivo, we tested the short-term and long-term mobilization efficacy of HFX51116. For the short-term efficacy study, we determined the dynamic change of efficacy at different times and doses of HFX51116 in HSC mobilization assays. Furthermore, different strains of mice, such as C57/BL6, C3H/HEJ and DBA/2, were sensitive to HFX51116. HFX51116, when used in combination with G-CSF, had higher efficacy than G-CSF alone. For the long-term repopulation study, we employed the CD45.1/CD45.2 competitive assay to demonstrate that the HSCs mobilized by G-CSF and HFX51116 had the repopulation ability when compared with the bone marrow cells. Conclusion: Our studies have demonstrated high HSC mobilization efficacy of HFX51116 in vivo and the promise of this new lead compound for further preclinical and clinical studies. Citation Format: Xiao Fang, Qian Meng, Xiong Fang, Yujia Mao, Yan Xu, Jing An, Ziwei Huang. A novel CXCR4 antagonist strongly mobilizes hematopoietic stem cells in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2181.

Genesis - a Phase III Randomized Double-Blind, Placebo-Controlled, Multi-Center Trial Evaluating the Safety and Efficacy of BL-8040 and G-CSF in Mobilization of Hematopoietic Stem Cells for Autologous Transplantation in Multiple Myeloma – Lead-in Period Results

Background CXCR4 mediates retention of hematopoietic stem cells (HSCs) in the bone marrow (BM) niche. BL-8040, a novel, high affinity CXCR4 antagonist is a potent mobilizer of HSCs to the peripheral blood with numerous potential clinical applications, including mobilization of CD34+ cells for autologous HSC transplantation (auto-HSCT) in Multiple Myeloma (MM). This study aims to evaluate the efficacy of single dose BL-8040 plus G-CSF in mobilization of ≥6.0 × 106 CD34+ cells/kg in up to 2 apheresis sessions for auto-HSCT in MM. Methods A Phase III study composed of an open-label, single-arm lead-in Part1 followed by a randomized, double-blinded, placebo-controlled Part2. Eligible MM patients age 18-78 will receive G-CSF (10 µg/kg; SC) daily for up to 8 days and one dose of BL-8040 (1.25 mg/kg; SC) or placebo on day 4 followed by up to 2 apheresis sessions; and if needed a second dose of BL-8040 or placebo on day 6 followed by up to 2 apheresis sessions. Part1 included up to 3 cohorts (∼10 patients/cohort), with Data Monitoring Committee (DMC) review after each cohort. Part2 will include 177 patients randomized 2:1. Results Part1 enrolled 11 patients, median age 61 (57-70). 9/11 patients (82%) reached the primary endpoint of ≥6 × 106 CD34+ cells/kg with one dose of BL-8040 and up to 2 apheresis sessions. 7/11 patients (64%) collected ≥6 × 106 CD34+ cells/kg in one apheresis session. Administration of BL-8040 resulted in a 7.86-fold average increase in circulating peripheral CD34+ cells (range 1.62-15.75, median 7.5, n=9). Additional CD34+ immunophenotyping/subset analyses are currently underway. BL-8040 plus G-CSF was found to be safe and well tolerated. Following these promising results, DMC recommended early continuation to Part2 of the Phase III trial. Conclusions The GENESIS lead-in results demonstrate BL-8040 is a potent mobilizer of HSCs, with potential to improve mobilization rates while minimizing mobilization-related healthcare costs.

Microfluidic Shear Force Assay to Determine Cell Adhesion Forces.

Cell adhesion is implicated in many physiological settings such as the retention of hematopoietic stem cells (HSCs) in their bone marrow niches or their migration into the bloodstream. During HSC mobilization these adhesion sites are cleaved and have to be newly formed during HSC homing and engraftment. To determine the adhesive properties of HSCs on different extracellular matrix (ECM) molecules, we present a microfluidic shear force assay, where a laminar flow is used to detach a semi-adherent cell population, the HSC model cell line KG-1a, from an ECM protein-coated substrate. This technique combines the high throughput of population-based assays with the ability to observe cell detachment in real time. Additionally, it is suitable for weakly adherent cells, as the setup allows cell incubation on various substrates and application of shear stress ranging several orders of magnitude in one setup without additional washing or transfer steps. As a measure for the adhesion strength of the studied cell population on the substrate, the critical shear force τ50 is determined which is required to remove 50% of the initially adherent cell fraction.

Characterization and Validation of Antibodies for Immunohistochemical Staining of the Chemokine CXCL12.

Chemokines and their receptors have been implicated in cancer biology. The CXCL12/CXCR4 axis is essential for the homing and retention of hematopoietic stem cells in bone marrow niches, and has a significant role in neonatal development. It is also implicated in multiple facets of cancer biology including metastasis, angiogenesis/neo-vasculogenesis, and immune cell trafficking at the tumor microenvironment (TME). Immunohistochemistry (IHC) is an ideal method for investigating involvement of CXCL12 in the TME. Three antibodies were evaluated here for their suitability to stain CXCL12. Both D8G6H and K15C gave apparent specific staining in both lymphoid and tumor tissue, but with converse staining patterns. D8G6H stained cells in the parafollicular zone whereas K15C showed staining of lymphoid cells in the interfollicular zone of tonsil tissue. Using a cell line with high CXCL12 expression, TOV21G, as a positive control, it was found that D8G6H gave strong staining of TOV21G cells whereas no staining was observed with K15C indicating that D8G6H specifically stains CXCL12. Significant staining of CXCL12 in the ovarian TME using tissue microarray was observed using D8G6H. These data demonstrate the importance of antibody characterization for IHC applications, and provide further evidence for the involvement of CXCL12 in ovarian cancer biology.

Interferon-alfa2 Treatment of Patients with Polycythemia Vera and Related Neoplasms Impacts Deregulation of Oxidative Stress Genes and Antioxidative Defence Mechanisms. Potential Implications of IFN-Alfa Induced Changes in TP53, NRF2 and CXCR4 for Genomic Instability and CD34+ Mobilisation

Introduction: The Philadelphia-negative myeloproliferative neoplasms are associated with chronic inflammation and accumulation of reactive oxygen species (ROS). Whole blood transcriptional profiling studies have most recently identified deregulation of several oxidative and anti-oxidative stress genes. Amongst the genes significantly downregulated is TP53 and the NFE2L2 or Nrf2 gene, the latter having a key role in the regulation of the oxidative stress response and in modulating both migration and retention of hematopoietic stem cells (HSCs). During MPN-disease progression, the HSC pool is steadily expanding with the egress of CD34+cell from stem cell niches into the circulation. In addition to Nrf2 several other genes are involved in this process, including CXCR4, which is also significantly downregulated in MPNs. Interferon-alpha2 (IFN) is recognized as a highly efficacious and promising agent in the treatment of MPNs. Taken into account that chronic inflammation with ROS accumulation is likely involved in the pathogenesis of MPNs, ultimately implying the induction of an altered redox balance of pivotal significance for stem cell mobilization in MPNs, we herein report the first study on the impact of IFN upon oxidative stress and anti-oxidative defence genes.Methods: The HG-U133 Plus 2.0 microarray was used to profile expression of 38.500 genes in whole blood from ET (n=19), PV (n=41), PMF (n=9) and controls (n=21) (data set 1) and in ET (n=8), PV (n=21), and PMF (n=4) before and during treatment with IFN (data set 2). All patients received treatment with IFN, in the large majority in a dosage ranging from 45-90 ug x 1 sc/week. Total RNA was purified from whole blood amplified to biotin-labeled RNA, and hybridized to microarrays. The R statistical software was applied to perform data preprocessing and statistical analysis of microarray data.Results: Single gene analysis of 154 genes found to be included in previous studies focusing on deregulation of oxidative stress genes in various diseases were chosen for further analysis. In response to treatment with IFN, 19 genes were upregulated in ET including CXCR4 and TP53, and 11 genes were downregulated including FOXO3, PRDX2, and PRDX6. In patients with PV, ATOX1, CXCR4, SEPP1, and TP53 were among the 29 upregulated genes, and FOXO3 and PRDX2 were among the 14 downregulated genes. In response to treatment with IFN, 2 genes were upregulated in PMF which were CYBB and MSRA, and 9 genes were downregulated including PRDX2, and FOXO3 (all P<0.05). In a recent microarray study (data set 1), we have shown significant downregulation of genes associated with oxidative stress including ATM, CYBA, NRF2, PTGS1, SIRT2, TTN, and significant upregulation of AKR1B1, CCND1, DEFB122, GPX8 in ET, PV, and PMF vs controls. These genes were not significantly deregulated during IFN-treatment in any of the disease entities. Similarly, the significant downregulation of the NFR2 and the CXCR4 genes before IFN-treatment was no longer present after IFN treatment.Discussion and Conclusions: The present study has shown IFN to have a major impact upon deregulated oxidative stress genes and antioxidative defence genes, implying a gene signature that mostly corresponds to decreased oxidative stress and enhancement of antioxidative defence genes. In regard to TP53 - being significantly downregulated before IFN treatment - this gene was significantly upregulated in ET and PV and no longer deregulated in PMF during IFN treatment. Downregulation of TP53 implies genomic instability due to an increased burden of oxidative stress upon the genome, which accordingly is reduced by IFN treatment. In addition to being the master regulator of the antioxidant response, Nrf2 also has a major role for normal stem cell function. Thus, our findings of a change in the Nrf2 gene expression from being significantly downregulated before IFN exposure to not being deregulated during treatment with IFN may imply an improvement in the antioxidant defence mechanisms against increased oxidative stress and accumulation of ROS. In conclusion, our findings of a major impact of IFN upon several oxidative stress genes and antioxidative defence genes may imply an IFN mediated dampening of genotoxic damage to hematopoietic cells and stromal cells as well, thereby ultimately diminishing the risk of additional mutations that might drive the malignant clone towards myelofibrotic and leukemic transformation. [Display omitted] DisclosuresHasselbalch:Novartis: Research Funding.

Hippo Kinase Inactivation Contributes to Del(20q) Malignancies and Cooperates with JAK2-V617F to Promote Myelofibrosis in Mice

Recurring chromosome abnormalities are frequent events in cancer and are especially prevalent in hematologic neoplasms. Somatic heterozygous deletions on chromosome 20q are detected in a variety of hematopoietic malignancies including myelodysplastic syndrome (MDS), classical myeloproliferative neoplasm (MPN), MDS/MPN overlap disorders such as chronic myelomonocytic leukemia (CMML), and acute leukemias. Del(20q) is especially prevalent in MPN patients (~10-15%), where it is the most commonly detected cytogenetic abnormality associated with primary myelofibrosis (PMF) and post-polycythemia vera myelofibrosis (MF). This suggests that heterozygous loss of genes in the del(20q) common deleted region (CDR) may contribute to adverse MPN progression. Despite these observations, relatively few genes located within the CDR have been unambiguously implicated, highlighting a significant need for further investigation. To identify genes that may play an important role in the biology of del(20q)-associated malignancies we utilized a published gene expression dataset of bone-marrow derived CD34+ cells from MDS patients and healthy controls (Gerstung et al, 2015). Comparison of the patients harboring del(20q) to healthy controls revealed STK4 (encoding Hippo kinase MST1) to be the most significantly downregulated gene (mean: 3.5-fold) among those located within the chromosome 20q CDR. We therefore set out to assess the role of Hippo kinase inactivation in hematologic malignancy using conditional gene inactivation in mice.We found that complete inactivation of both Hippo kinases (Stk4 and Stk3) within the hematopoietic system using Vav1-Cre (Stk4-/-Stk3-/-) resulted in a lethal bone marrow failure (median survival: 7 weeks) associated with myelodysplastic features and frequent extramedullary hematopoiesis in the spleen. A single copy of Stk4 rescued the lethality due to bone marrow failure, however sub-haploinsufficient mice displayed thrombocytopenia with a trend towards mild anemia; phenotypes that closely resemble those observed in MDS patients with isolated del(20q). Both a reduced number of mature megakaryocytes and the presence of dysplastic megakaryocytes were apparent in bone marrow sections. Inducible Hippo kinase inactivation in adult mice using the Mx1-Cre system similarly recapitulated several phenotypic features of both MDS and MPN. In competitive bone marrow transplant assays we found that Stk4-/-Stk3-/- hematopoietic stem cells (HSC) completely lacked engraftment potential and failed to reconstitute normal hematopoiesis, revealing a potential role for Hippo kinase function in HSC homing and retention in the bone marrow. Heterozygous HSCs maintained relatively normal steady-state hematopoiesis in peripheral blood and bone marrow for up to 48 weeks in primary and secondary transplantations, although upon aging these mice were prone to development of thrombocytopenia with increased mean platelet volume.Given the high frequency of del(20q) in MPN, especially PMF, we asked whether heterozygous Hippo kinase inactivation may cooperate with the common driver mutation JAK2-V617F to accelerate disease progression. Using an HSC-enriched retroviral transduction/transplantation model in C57BL/6 recipient mice, we monitored MPN progression for 36 weeks in heterozygous Stk4+/-Stk3+/-, or control (Vav1-Cre-), cells with or without expression of JAK2-V617F. While both JAK2-V617F groups initially displayed a similar degree of polycythemia relative to controls, we found heterozygous Hippo kinase inactivation to promote accelerated disease progression towards lethal bone marrow fibrosis during the course of observation. Recipients in this group showed significantly reduced overall survival, which was associated with higher grade fibrosis in bone marrow, elevated peripheral granulocyte counts, enhanced splenomegaly, and increased frequencies of hematopoietic stem and progenitor populations in the spleen. Together, these findings implicate aberrant Hippo kinase loss-of-function in the pathogenesis of del(20q)-associated hematologic malignancies, and shed new light on the molecular events that contribute to adverse MPN progression. DisclosuresBejar:Genoptix: Consultancy; Modus Outcomes: Consultancy; Celgene: Consultancy, Honoraria; Takeda: Research Funding; Astex/Otsuka: Consultancy, Honoraria; AbbVie/Genentech: Consultancy, Honoraria; Foundation Medicine: Consultancy. Guan:Vivace: Equity Ownership.

Acute Myeloid Leukemia Induced Remodelling of the Human Bone Marrow Niche Predicts Clinical Outcome

The hematopoietic stem cell (HSC) niche consists of different cellular and non-cellular constituents which regulate HSC maintenance and retention in the bone marrow. It has been shown in a number of murine models of myeloid neoplasia how leukemia infiltration alters the HSC niche to reinforce malignancy. Acute myeloid leukemia (AML) is characterized in human by a high relapse rate indicating that leukemia initiating cells are protected by its niche. However, despite our knowledge in murine models little is known about the bone marrow architecture in human and the impact of the leukemic niche on clinical outcome.In this study, we combined immunohistochemical stainings (IHC) with protein and global gene expression analyses together with clinical data to dissect the human bone marrow architecture in AML and assess its clinical impact. Human bone marrow was collected from AML patients at first diagnosis and matching non-leukemic donors. To evaluate the bone marrow architecture CD271+ mesenchymal stem and progenitor cells (MSPCs) were automatically quantified on bone marrow sections. In fact, AML patients showed 1.5-fold increase in bone marrow MSPCs compared to non-leukemic donors (Median (IQR), AML: 5.5% (2.8-9.5), n=36; control: 3.7% (2.1-5.7), n=58; p < 0.01). MSPCs proved to produce reticular fibers, an extracellular matrix protein frequently associated with different malignancies. In AML bone marrow these fibers were also found to be more abundantly expressed (Median (IQR), AML: 3.4% (1.8-4.5), n=37; control: 1.6% (1.1-3.3), n=19; p < 0.05).Next, to globally assess the gene expression profile of MSPCs in AML bone marrow we performed microarray analyses (ClariomTM S Human Assay) of freshly isolated uncultured lineage- CD146+ CD271+ MSPCs. Strikingly, HSC-regulating genes in particular CXCL12, ANGPT1 and VCAM1 showed lower expression in AML MSPCs which correlated with the degree of hematopoietic failure in AML patients. Along with the increased number of MSPCs, geneset enrichment analysis (GSEA) revealed higher proliferation of MSPCs in AML. In murine models loss of quiescence of MSPCs was previously found to be due to bone marrow sympathetic neuropathy. We therefore measured catecholamines and neurotrophic factor in the bone marrow extracellular fluid of AML patients and non-leukemic donors at first diagnosis. In fact, noradrenalin and brain-derived neurotrophic factor (BDNF) showed a 2-fold (p=0.26) resp. 4-fold (p<0.0001) lower expression in AML bone marrow. Importantly, BDNF is proved to be essential for sympathetic neuron proliferation and differentiation.In order to get an overview of alterations of canonical pathways in bone marrow MSPCs upon AML infiltration, we applied QIAGEN's Ingenuity® Pathway Analysis software. Several of the major differently regulated pathways proved to involve differentiation and mineralization of MSPCs. We therefore assessed bone metabolism in AML patients at first diagnosis and quantified serum osteocalcin levels. Notably, AML patients showed 30% lower osteocalcin levels than non-leukemic donors (Median (IQR), AML, 12.15ng/ml (7.53-16.28) n=58; control, 17.2ng/ml (12.5-23.45) n=31; p < 0.05). To evaluate if the deficiency in osteoblast mineralization is specifically due to AML infiltration we performed in vitro co-culture assays. Both MSPCs and an osteoblast-like cell line (SaOS2) showed significant impaired mineralization in presence of certain AML cell lines as well as primary human AML cells, while healthy mononuclear cells did not affect mineralization. Strikingly, this AML-induced defect in osteoblast mineralization proved to be of clinical significance. Patients with low osteocalcin levels (<11ng/ml) showed inferior overall survival with 1-year survival rate of 38.7% while patients with high osteocalcin levels reached 66.8% (n=58; median duration of follow-up 9.7 months).In summary, we globally characterized the bone marrow architecture in AML patients in comparison to non-leukemic donors and assessed its clinical significance. This increasing understanding of the human AML bone marrow microenvironment might open the window for new niche-targeted therapies to eradicate leukemic stem cells and eventually decrease the high relapse rate in AML. DisclosuresDuehrsen:Amgen: Research Funding; Roche: Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Janssen: Honoraria.

Mesenchymal stromal cells induce a permissive state in the bone marrow that enhances G-CSF-induced hematopoietic stem cell mobilization in mice

Mesenchymal stromal cells (MSCs) support hematopoietic stem cells (HSCs) in vivo and enhance HSC engraftment and hematopoietic recovery upon cotransplantation with HSCs. These data have led to the hypothesis that MSCs may affect the HSC niche, leading to changes in HSC retention and trafficking. We studied the effect of MSC administration on the HSC compartment in the bone marrow (BM) in mice. After injection of MSCs, HSC numbers in the BM were decreased coinciding with an increased cell cycle activity compared with phosphate-buffered saline (PBS)-injected controls. Furthermore, the frequency of macrophages was significantly reduced and niche factors including Cxcl12, Scf, and Vcam were downregulated in endosteal cells. These BM changes are reminiscent of events associated with granulocyte colony-stimulating factor (G-CSF)-induced hematopoietic stem and progenitor cell (HSPC) mobilization. Interestingly, coadministration of MSCs and G-CSF resulted in a twofold increase in peripheral blood HSPC release compared with injection of G-CSF alone, whereas injection of MSCs alone did not induce HSPC mobilization. After intravenous administration, MSCs were only observed in the lungs, suggesting that they exert their effect on the HSC niche through a soluble mediator. Therefore, we tested the hypothesis that MSC-derived extracellular vesicles (EVs) are responsible for the observed changes in the HSC niche. Indeed, administration of EVs resulted in downregulation of Cxcl12, Scf, and Vcam and enhanced G-CSF-induced HSPC mobilization at similar levels as MSCs and G-CSF. Together, these data indicate that MSCs induce a permissive state in the BM, enhancing HSPC mobilization through the release of EVs.