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We show that pro-inflammatory oncostatin M (OSM) is an important regulator of hematopoietic stem cell (HSC) niches in the bone marrow (BM). Treatment of healthy humans and mice with granulocyte colony-stimulating factor (G-CSF) dramatically increases OSM release in blood and BM. Using mice null for the OSM receptor (OSMR) gene, we demonstrate that OSM provides a negative feed-back acting as a brake on HSPC mobilization in response to clinically relevant mobilizing molecules G-CSF and CXCR4 antagonist. Likewise, injection of a recombinant OSM molecular trap made of OSMR complex extracellular domains enhances HSC mobilization in poor mobilizing C57BL/6 and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Mechanistically, OSM attenuates HSC chemotactic response to CXCL12 and increases HSC homing to the BM signaling indirectly via BM endothelial and mesenchymal cells which are the only cells expressing OSMR in the BM. OSM up-regulates E-selectin expression on BM endothelial cells indirectly increasing HSC proliferation. RNA sequencing of HSCs from Osmr-/- and wild-type mice suggest that HSCs have altered cytoskeleton reorganization, energy usage and cycling in the absence of OSM signaling in niches. Therefore OSM is an important regulator of HSC niche function restraining HSC mobilization and anti-OSM therapy combined with current mobilizing regimens may improve HSPC mobilization for transplantation.

Anemia of inflammation (AI) is the second most prevalent anemia after iron deficiency anemia and results in persistent low blood erythrocytes and hemoglobin, fatigue, weakness, and early death. Anemia of inflammation is common in people with chronic inflammation, chronic infections, or sepsis. Although several studies have reported the effect of inflammation on stress erythropoiesis and iron homeostasis, the mechanisms by which inflammation suppresses erythropoiesis in the bone marrow (BM), where differentiation and maturation of erythroid cells from hematopoietic stem cells (HSCs) occurs, have not been extensively studied. Here we show that in a mouse model of acute sepsis, bacterial lipopolysaccharides (LPS) suppress medullary erythroblastic islands (EBIs) and erythropoiesis in a TLR-4- and MyD88-dependent manner with concomitant mobilization of HSCs. LPS suppressive effect on erythropoiesis is indirect as erythroid progenitors and erythroblasts do not express TLR-4 whereas EBI macrophages do. Using cytokine receptor gene knock-out mice LPS-induced mobilization of HSCs is G-CSF-dependent whereas LPS-induced suppression of medullary erythropoiesis does not require G- CSF-, IL- 1-, or TNF-mediated signaling. Therefore suppression of medullary erythropoiesis and mobilization of HSCs in response to LPS are mechanistically distinct. Our findings also suggest that EBI macrophages in the BM may sense innate immune stimuli in response to acute inflammation or infections to rapidly convert to a pro-inflammatory function at the expense of their erythropoietic function.

The interactions of leukemia cells with the bone marrow (BM) microenvironment is critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E-(endothelial)-selectin is a key niche component that directly mediates acute myeloid leukemia (AML) chemo-resistance, revealing E-selectin as a promising therapeutic target. To understand how E-selectin promotes AML survival, we investigated the potential receptors on AML cells involved in E-selectin-mediated chemo-resistance. Using CRISPR-Cas9 gene editing to selectively suppress canonical E-selectin receptors CD44 or P-selectin glycoprotein ligand-1 (PSGL-1/CD162) from human AML cell line KG1a, we show that CD162, but not CD44, is necessary for E-selectin-mediated chemo-resistance in vitro. Using preclinical models of murine AML, we then demonstrate that absence of CD162 on AML cell surface leads to a significant delay in the onset of leukemia and a significant increase in sensitivity to chemotherapy in vivo associated with a more rapid in vivo proliferation compared to wild-type AML and a lower BM retention. Together, these data reveal for the first time that CD162 is a key AML cell surface receptor involved in AML progression, BM retention and chemo-resistance. These findings highlight specific blockade of AML cell surface CD162 as a potential novel niche-based strategy to improve the efficacy of AML therapy.

Hematopoietic stem cells (HSCs) with superior reconstitution potential are reported to be enriched in the endosteal compared to central bone marrow (BM) region. To investigate whether specific factors at the endosteum may contribute to HSC potency, we screened for candidate HSC niche factors enriched in the endosteal compared to central BM regions. Together with key known HSC supporting factors Kitl and Cxcl12, we report that prostacyclin/prostaglandin I2 (PGI2 ) synthase (Ptgis) was one of the most highly enriched mRNAs (>10-fold) in endosteal compared to central BM. As PGI2 signals through receptors distinct from prostaglandin E2 (PGE2 ), we investigated functional roles for PGI2 at the endosteal niche using therapeutic PGI2 analogs, iloprost and cicaprost. We found PGI2 analogs strongly reduced HSC differentiation in vitro. Ex vivo iloprost pulse treatment also significantly boosted long-term competitive repopulation (LT-CR) potential of HSCs upon transplantation. This was associated with increased tyrosine-phosphorylation of transducer and activator of transcription-3 (STAT3) signaling in HSCs but not altered cell cycling. In vivo, iloprost administration protected BM HSC potential from radiation or granulocyte colony-stimulating factor (G-CSF)-induced exhaustion, and restored HSC homing potential with increased Kitl and Cxcl12 transcription in the BM. In conclusion, we propose that PGI2 is a novel HSC regulator enriched in the endosteum that promotes HSC regenerative potential following stress. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: We discovered prostacyclin as a novel HSC regulator enriched in endosteal bone marrow niches. Ex vivo and in vivo treatment with clinical prostacyclin analogs enhanced HSC reconstitution potential and protect HSC from stress-mediated exhaustion. These findings open new therapeutic avenues to improve HSC engraftment after autologous transplantations by short ex vivo pulse treatment with prostacyclin analogs; and limit loss of HSC and acquired bone marrow failure by protecting HSC in patients undergoing radiation or chemotherapy treatment for non-hematologic malignancies. Prostacyclin analogs are safe, used clinically for vascular diseases, and thus have potential for swift repurposing for the therapies mentioned above.

The unique interactions of leukemia cells with the bone marrow (BM) microenvironment (niche) are critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E-selectin is a key niche component mediating acute myeloid leukemia (AML) chemoresistance, highlighting E-selectin as a promising therapeutic target. In this study, we found canonical E-selectin receptors CD44 and CD162 to be crucial for E-selectin adhesion, as mouse AML cells lacking both receptors failed to bind to E-selectin. We then developed an in vitro model to assess the chemo-sensitivity of mouse AML blasts adhering to various vascular adhesion molecules; this showed that E-selectin uniquely boosts AML cell survival to chemotherapy, but only when CD44/CD162 are present. Likewise when transplanted into recipient mice, CD44/CD162-/- AML cells were significantly more sensitive to chemotherapy compared to wildtype AML. Together these results suggest that CD44 and/or CD162 are key E-selectin receptors involved in AML chemoresistance. To validate these findings in human, we used CRISPR-Cas9 gene editing to selectively suppress CD44 or CD162 from the human AML cell line KG1a. Using our in vitro chemo-sensitivity assay, we showed that E-selectin could not promote AML survival in the absence of either CD44 or CD162, confirming our findings in mice. However interestingly, KG1a cells could still bind to E-selectin in the absence of CD44 or CD162, suggesting the involvement of several E-selectin receptors that can play different roles – either binding or signalling. To conclude, we described a novel form of niche-mediated chemoresistance that can be modelled in vitro, and identified CD44 and CD162 as key AML cell surface receptors involved. These findings highlight blockade of E-selectin or its receptors as a novel strategy to improve the treatment of AML.

Neurological heterotopic ossification (NHO) is the abnormal formation of bone in soft tissues as a consequence of spinal cord or traumatic brain injury. NHO causes pain, ankyloses, vascular and nerve compression and delays rehabilitation in this high-morbidity patient group. The pathological mechanisms leading to NHO remain unknown and consequently there are no therapeutic options to prevent or reduce NHO. Genetically modified mouse models of rare genetic forms of heterotopic ossification (HO) exist, but their relevance to NHO is questionable. Consequently, we developed the first model of spinal cord injury (SCI)-induced NHO in genetically unmodified mice. Formation of NHO, measured by micro-computed tomography, required the combination of both SCI and localized muscular inflammation. Our NHO model faithfully reproduced many clinical features of NHO in SCI patients and both human and mouse NHO tissues contained macrophages. Muscle-derived mesenchymal progenitors underwent osteoblast differentiation in vitro in response to serum from NHO mice without additional exogenous osteogenic stimuli. Substance P was identified as a candidate NHO systemic neuropeptide, as it was significantly elevated in the serum of NHO patients. However, antagonism of substance P receptor in our NHO model only modestly reduced the volume of NHO. In contrast, ablation of phagocytic macrophages with clodronate-loaded liposomes reduced the size of NHO by 90%, supporting the conclusion that NHO is highly dependent on inflammation and phagocytic macrophages in soft tissues. Overall, we have developed the first clinically relevant model of NHO and demonstrated that a combined insult of neurological injury and soft tissue inflammation drives NHO pathophysiology.

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

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

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

Potent functional HSCs are enriched at the endosteal bone marrow (BM), which comprises ∼10% of total BM. To identify novel niche factors that regulate HSCs, we performed a gene expression microarray seeking genes overexpressed in the endosteal relative to central BM. Prostacyclin/prostaglandin I2 (PGI2) synthase (Ptgis) was one of the highest enriched genes in the endosteal versus central BM (27-fold). PTGIS is the sole enzyme for biosynthesis of PGI2. PGI2 has no reported roles in HSC regulation in the BM and was chosen for further investigation. We found PGI2 analogue iloprost treatment of sorted BM lineage- Kit+ Sca1- (LKS+) cells in vitro for 7 days potently reduced proliferation and differentiation compared to vehicle control. BM cells pulse treated ex vivo with iloprost for 1 hour also resulted in 14-fold increased multilineage reconstitution potential compared to vehicle controls in competitive transplant assay. Iloprost administration in vivo partially rescued BM HSC reconstitution potential in mice sub-lethally (6.5 Gy) irradiated or administered pro-inflammatory granulocyte colony stimulating factor (G-CSF) for 3 days. These data suggest that PGI2 protects HSC from stress. Mechanistically, we found iloprost pulse treatment in vitro was associated with increased pSTAT3 and cell cycle progression in HSCs. Additionally, we found ∼4-fold greater proportion of untreated LKS+ HSPC homing to the BM of recipients co-administered iloprost + G-CSF compared to vehicle + G-CSF at 4 hours post-transplant. We found that G-CSF administration also decreased Scf and Cxcl12 mRNA expression in BM, which were partially rescued with iloprost co-administration. These data suggest that PGI2 protects HSC during stress by acting directly on HSCs and indirectly to preserve BM niche functions. In summary, we identified PGI2 as a novel HSC niche factor abundant in the endosteal BM.

The central macrophage (MΦ) in erythroblastic islands (EI) is critical to erythropoiesis by providing iron and growth factors, and mediating enucleation of the maturing erythroblasts. As MΦ are key effectors of inflammation, we investigated the effect of lipopolysaccharides (LPS) on erythropoiesis in vivo. LPS (2.5mg/kg/d for 2 days) in C57BL/6 mice caused the whitening of the bone marrow (BM) with dramatically decreased erythroblast and reticulocyte numbers. Imaging flow cytometry was used to visualize structural changes and quantify numbers of EI. LPS treatment in vivo caused a marked loss of EI in the BM. This suppressive effect of LPS on BM erythropoiesis was TLR4-dependent as it was absent in TLR4 KO mice. By qRT-PCR and flow cytometry, TLR4 is not expressed by BM erythroblasts but by myeloid cells including EI MΦ. Together with the fact that addition of 100ng/mL LPS into BFU-E assays does not inhibit BFU-E colony growth, these suggest that the suppressive effect of LPS on erythroblasts is indirectly mediated. It is known that LPS mobilizes HSC in a G-CSF-dependent manner. As MΦ express the G-CSF receptor (GCSFR), we explored LPS effects in GCSFR KO mice. Surprisingly, although HSC did not mobilize in response to LPS in GCSFR KO mice, BM erythropoiesis was still suppressed with reduced numbers of erythroblasts. Unexpectedly however, the BM from LPS-treated GCSFR KO mice was still red with high numbers of erythrocytes. The explanation of this paradox is that LPS treatment causes BM vascular leakage in GCSFR KO mice with blood plasma volume in the BM 2.9-fold higher compared to LPS-treated WT mice (measured by i.v. injection of Evans Blue). In conclusion our data show that LPS suppresses medullar erythropoiesis indirectly in a G-CSF-independent manner but GCSFR-mediated signaling is necessary to maintain the integrity of the BM vasculature in response to LPS.

Haematopoietic stem cell (HSC) transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs) or osteoblasts. Sorted Lineage(-) Sca-1(+) c-kit(+) (LSK) haematopoietic stem/progenitor cells (HPC) demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.

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

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

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

Despite the fact that many hypoxia-inducible genes are important in hematopoiesis, the spatial distribution of oxygen in the bone marrow (BM) has not previously been explored in vivo. Using the hypoxia bioprobe pimonidazole, we showed by confocal laser scanning microscopy that the endosteum at the bone-BM interface is hypoxic, with constitutive expression of hypoxia-inducible transcription factor-1α (HIF-1α) protein in steady-state mice. Interestingly, at the peak of hematopoietic stem and progenitor cell (HSPC) mobilization induced by either granulocyte colony-stimulating factor or cyclophosphamide, hypoxic areas expand through the central BM. Furthermore, we found that HSPC mobilization leads to increased levels of HIF-1α protein and increased expression of vascular endothelial growth factor A (VEGF-A) mRNA throughout the BM, with an accumulation of VEGF-A protein in BM endothelial sinuses. VEGF-A is a cytokine known to induce stem cell mobilization, vasodilatation, and vascular permeability in vivo. We therefore propose that the expansion in myeloid progenitors that occurs during mobilization depletes the BM hematopoietic microenvironment of O2, leading to local hypoxia, stabilization of HIF-1α transcription factor in BM cells, increased transcription of VEGF-A, and accumulation of VEGF-A protein on BM sinuses that increases vascular permeability.Disclosure of potential conflicts of interest is found at the end of this article.

p21 WAF1/CIP1 , a cyclin-dependent kinase inhibitor and a critical regulator of cell cycle, is controlled transcriptionally by p53-dependent and -independent mechanisms and posttranslationally by the proteasome. We have identified WISp39, a tetratricopeptide repeat (TPR) protein that binds p21. WISp39 stabilizes newly synthesized p21 protein by preventing its proteasomal degradation. WISp39, p21, and hsp90 form a trimeric complex in vivo. The interaction of WISp39 with Hsp90 is abolished by point mutations within the C-terminal TPR domain of WISp39. Although this WISp39 TPR mutant binds p21 in vivo, it fails to stabilize p21. Our results suggest that WISp39 recruits Hsp90 to regulate p21 protein stability. WISp39 downregulation by siRNA prevents the accumulation of p21 and cell cycle arrest after ionizing radiation. The results demonstrate the importance of posttranslational stabilization of p21 protein by WISp39 in regulating cellular p21 activity.

p53-mediated increase in cyclin-dependent kinase inhibitor p21WAF1 protein is thought to be the major mediator of cell cycle arrest after DNA damage. Previously p21 protein levels have been reported to increase or to decrease after UV irradiation. We show that p21 protein is degraded after irradiation of a variety of cell types with low but not high doses of UV. Cell cycle arrest occurs despite p21 degradation via Tyr15 inhibitory phosphorylation of cdk2 and differs from the classical p21-dependent checkpoint elicited by ionizing radiation. In contrast to the basal turnover of p21, degradation of p21 switches to ubiquitin/Skp2-dependent proteasome pathway following UV irradiation. ATR activation after UV irradiation is essential for signaling p21 degradation. Finally, UV-induced p21 degradation is essential for optimal DNA repair. These results provide novel insight into regulation of p21 protein and its role in the cellular response to DNA damage.

Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches.

Hypoxia-inducible factor (HIF)-1α accumulation promotes hematopoietic stem cells' quiescence and is necessary to maintain their self-renewal. However, the role of HIF-2α in hematopoietic cells is less clear. We investigated the role of HIF-2α in leukemia and lymphoma cells. HIF-2α expression was high in subsets of human and mouse leukemia and lymphoma cells, whereas it was low in normal bone marrow leukocytes. To investigate the role of HIF-2α, we transduced human HIF-2α cDNA in mouse syngeneic models of myeloid preleukemia and a transgenic model of B lymphoma. Ectopic expression of HIF-2α accelerated leukemia cell proliferation in vitro. Mice transplanted with cells transduced with HIF-2α died significantly faster of leukemia or B lymphoma than control mice transplanted with empty vector-transduced cells. Conversely, HIF-2α knockdown in human myeloid leukemia HL60 cells decreased proliferation in vitro and significantly prolonged animal survival following transplantation. In human acute myeloid leukemia (AML), HIF-2α mRNA was significantly elevated in several subsets such as the t(15;17), inv(16), complex karyotype and favorable cytogenetic groups. However, patients with high HIF-2α expression had a trend to higher disease-free survival in univariate analysis. The different effects of HIF-2α overexpression in mouse models of leukemia and human AML illustrates the complexity of this mutliclonal disease. Refereed/Peer-reviewed

Many patients with hematological neoplasms fail to mobilize sufficient numbers of hematopoietic stem cells (HSCs) in response to granulocyte colony-stimulating factor (G-CSF) precluding subsequent autologous HSC transplantation. Plerixafor, a specific antagonist of the chemokine receptor CXCR4, can rescue some but not all patients who failed to mobilize with G-CSF alone. These refractory poor mobilizers cannot currently benefit from autologous transplantation. To discover alternative targetable pathways to enhance HSC mobilization, we studied the role of hypoxia-inducible factor-1α (HIF-1α) and the effect of HIF-1α pharmacological stabilization on HSC mobilization in mice. We demonstrate in mice with HSC-specific conditional deletion of the Hif1a gene that the oxygen-labile transcription factor HIF-1α is essential for HSC mobilization in response to G-CSF and Plerixafor. Conversely, pharmacological stabilization of HIF-1α with the 4-prolyl hydroxylase inhibitor FG-4497 synergizes with G-CSF and Plerixafor increasing mobilization of reconstituting HSCs 20-fold compared with G-CSF plus Plerixafor, currently the most potent mobilizing combination used in the clinic.

Similarly to other tissues, the bone marrow contains subsets of resident tissue macrophages, which are essential to maintain bone formation, functional hematopoietic stem cell (HSC) niches, and erythropoiesis. Pharmacologic doses of granulocyte colony-stimulating factor (G-CSF) mobilize HSC in part by interfering with the HSC niche-supportive function of BM resident macrophages. Because bone marrow macrophages are key to both maintenance of HSC within their niche and erythropoiesis, we investigated the effect of mobilizing doses of G-CSF on erythropoiesis in mice. We now report that G-CSF blocks medullar erythropoiesis by depleting the erythroid island macrophages we identified as co-expressing F4/80, vascular cell adhesion molecule-1, CD169, Ly-6G, and the ER-HR3 erythroid island macrophage antigen. Both broad macrophage depletion, achieved by injecting clodronate-loaded liposomes, and selective depletion of CD169 + macrophages, also concomitantly depleted F4/80 + VCAM-1 + CD169 + ER-HR3 + Ly-6G + erythroid island macrophages and blocked erythropoiesis. This more precise phenotypic definition of erythroid island macrophages will enable studies on their biology and function in normal settings and on diseases associated with anemia. Finally, this study further illustrates that macrophages are a potent relay of innate immunity and inflammation on bone, hematopoietic, and erythropoietic maintenance. Agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.

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

Harvest of granulocyte colony-stimulating factor (G-CSF)-mobilized hematopoietic stem cells (HSCs) begins at day 5 of G-CSF administration, when most donors have achieved maximal mobilization. This is based on surrogate markers for HSC mobilization, such as CD34(+) cells and colony-forming activity in blood. However, CD34(+) cells or colony-forming units in culture (CFU-C) are heterogeneous cell populations with hugely divergent long-term repopulation potential on transplantation. HSC behavior is influenced by the vascular bed in the vicinity of which they reside. We hypothesized that G-CSF may mobilize sequentially cells proximal and more distal to bone marrow venous sinuses where HSCs enter the blood. We addressed this question with functional serial transplantation assays using blood and bone marrow after specific time points of G-CSF treatment in mice. We found that in mice, blood collected after only 48 hours of G-CSF administration was as enriched in serially reconstituting HSCs as blood collected at 5 days of G-CSF treatment. Similarly, mobilized Lin(-)CD34(+) cells were relatively enriched in more primitive Lin(-)CD34(+)CD38(-) cells at day 2 of G-CSF treatment compared with later points in half of human donors tested (n = 6). This suggests that in both humans and mice, hematopoietic progenitor and stem cells do not mobilize uniformly according to their maturation stage, with most potent HSCs mobilizing as early as day 2 of G-CSF.

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

Osteoblasts are necessary to B lymphopoiesis and mobilizing doses of G-CSF or cyclophosphamide inhibit osteoblasts, whereas AMD3100/Plerixafor does not. However, the effect of these mobilizing agents on B lymphopoiesis has not been reported. Mice (wild-type, knocked-out for TNF-α and TRAIL, or over-expressing Bcl-2) were mobilized with G-CSF, cyclophosphamide, or AMD3100. Bone marrow, blood, spleen and lymph node content in B cells was measured. G-CSF stopped medullar B lymphopoiesis with concomitant loss of B-cell colony-forming units, pre-pro-B, pro-B, pre-B and mature B cells and increased B-cell apoptosis by an indirect mechanism. Overexpression of the anti-apoptotic protein Bcl2 in transgenic mice rescued B-cell colony forming units and pre-pro-B cells in the marrow, and prevented loss of all B cells in marrow, blood and spleen. Blockade of endogenous soluble TNF-α with Etanercept, or combined deletion of the TNF-α and TRAIL genes did not prevent B lymphopoiesis arrest in response to G-CSF. Unlike G-CSF, treatments with cyclophosphamide or AMD3100 did not suppress B lymphopoiesis but caused instead robust B-cell mobilization. G-CSF, cyclophosphamide and AMD3100 have distinct effects on B lymphopoiesis and B-cell mobilization with: 1) G-CSF inhibiting medullar B lymphopoiesis without mobilizing B cells in a mechanism distinct from the TNF-α-mediated loss of B lymphopoiesis observed during inflammation or viral infections; 2) CYP mobilizing B cells but blocking their maturation; and 3) AMD3100 mobilizing B cells without affecting B lymphopoiesis. These results suggest that blood mobilized with these three agents may have distinct immune properties.

The microenvironment, or niche, surrounding a stem cell largely governs its cellular fate. Two anatomical niches for hematopoietic stem cells (HSCs) have been reported in the bone marrow, but a distinct function for each of these niches remains unclear. Here we report a new role for the adhesion molecule E-selectin expressed exclusively by bone marrow endothelial cells in the vascular HSC niche. HSC quiescence was enhanced and self-renewal potential was increased in E-selectin knockout (Sele(-/-)) mice or after administration of an E-selectin antagonist, demonstrating that E-selectin promotes HSC proliferation and is a crucial component of the vascular niche. These effects are not mediated by canonical E-selectin ligands. Deletion or blockade of E-selectin enhances HSC survival threefold to sixfold after treatment of mice with chemotherapeutic agents or irradiation and accelerates blood neutrophil recovery. As bone marrow suppression is a severe side effect of high-dose chemotherapy, transient blockade of E-selectin is potentially a promising treatment for the protection of HSCs during chemotherapy or irradiation.

An important factor contributing to hematopoietic stem cell (HSC) mobilization is the ability of mobilizing cytokines and chemotherapy to disturb the cellular components of HSC niches, particularly osteoblasts and their progenitors, and to inhibit the production of HSC supportive cytokines and chemokines. Although the mechanisms by which niche cells are inhibited by mobilizing treatments is still incompletely understood, it has recently emerged that bone marrow macrophages play a critical role in maintaining osteoblasts, bone formation, and the expression of CXCL12, KIT ligand, and angiopoietin-1 necessary to HSC maintenance. In this chapter, we describe how to mobilize HSC into the blood in mice by depleting macrophages with clodronate-loaded liposomes and compare this mode of mobilization to mobilization induced by granulocyte colony-stimulating factor and cyclophosphamide. Detailed methods to analyze mobilization of phenotypic and functional reconstituting HSC are described with examples.

The hematopoietic system is highly proliferative in the bone marrow (BM) due to the short half-life of granulocytes and platelets in the blood. Analysis of cell cycling and cell proliferation in vivo in specific populations of the mouse BM has highlighted some key properties of adult hematopoietic stem cells (HSCs). For instance, despite their enormous proliferation and repopulation potential, most true HSC are deeply quiescent in G(0) phase of the cell cycle and divide very infrequently, while less potent lineage-restricted progenitors divide rapidly to replace the daily consumption of blood leukocytes, erythrocytes, and platelets. In response to stress, e.g., following ablative chemotherapy or irradiation, HSC must enter the cell cycle to rapidly repopulate the BM with progenitors. Due to their extreme rarity in the BM, at least five color flow cytometry for cell surface antigens has to be combined with staining for DNA content and nuclear markers of proliferation to analyze cell cycle and proliferation of HSC in vivo. In this chapter, we describe two methods to stain mouse HSC to (1) distinguish all phases of the cell cycle (G(0), G(1), S, and G(2)/M) and (2) analyze the divisional history of HSC in vivo by incorporation of the thymidine analog 5-bromo-2-deoxyuridine.

Identification of the precise location, where hematopoietic stem cells (HSCs) reside in the bone marrow, has made a great leap forward with the advance of live time-lapse video 2-photon fluorescent microscopy. These studies have shown that HSCs preferentially resides in the endosteal region of the BM, at an average of two cell diameters from osteoblasts covering endosteal bone surfaces. However, this equipment is very sophisticated and only a very few laboratories can perform these studies. To investigate functional attributes of these niches, we have developed a flow cytometry technique in which mice are perfused with the cell-permeable fluorescent dye Hoechst33342 in vivo before bone marrow cells are collected and antibody stained. This method enables to position phenotypic HSC, multipotent and myeloid progenitors, as well as BM nonhematopoietic stromal cells relative to blood flow in vivo. This technique enables prospective isolation of HSCs based on the in vivo perfusion of the niches in which they reside.

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

BACKGROUND: Tissue inhibitor of metalloproteinases-3 (TIMP-3) inhibits matrix metalloproteinases and membrane-bound sheddases. TIMP-3 is associated with the extracellular matrix and is expressed in highly remodeling tissues. TIMP-3 function in the hematopoietic system is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We now report that TIMP-3 is highly expressed in the endosteal region of the bone marrow (BM), particularly by osteoblasts, endothelial and multipotent mesenchymal stromal cells which are all important cellular components of hematopoietic stem cell (HSC) niches, whereas its expression is very low in mature leukocytes and hematopoietic stem and progenitor cells. A possible role of TIMP-3 as an important niche component was further suggested by its down-regulation during granulocyte colony-stimulating factor-induced mobilization. To further investigate TIMP-3 function, mouse HSC were retrovirally transduced with human TIMP-3 and transplanted into lethally irradiated recipients. TIMP-3 overexpression resulted in decreased frequency of B and T lymphocytes and increased frequency of myeloid cells in blood and BM, increased Lineage-negative Sca-1(+)KIT(+) cell proliferation in vivo and in vitro and increased colony-forming cell trafficking to blood and spleen. Finally, over-expression of human TIMP-3 caused a late onset fatal osteosclerosis. CONCLUSIONS/SIGNIFICANCE: Our results suggest that TIMP-3 regulates HSC proliferation, differentiation and trafficking in vivo, as well as bone and bone turn-over, and that TIMP-3 is expressed by stromal cells forming HSC niches within the BM. Thus, TIMP-3 may be an important HSC niche component regulating both hematopoiesis and bone remodeling.

Several regulatory proteins control cell cycle progression. These include Emi1, an anaphase-promoting complex (APC) inhibitor whose destruction controls progression through mitosis to G1, and p21WAF1, a cyclin-dependent kinase (CDK) inhibitor activated by DNA damage. We have analyzed the role of p21WAF1in G2-M phase checkpoint control and in prevention of polyploidy after DNA damage. After DNA damage, p21+/+cells stably arrest in G2, whereas p21−/−cells ultimately progress into mitosis. We report that p21 down-regulates Emi1 in cells arrested in G2 by DNA damage. This down-regulation contributes to APC activation and results in the degradation of key mitotic proteins including cyclins A2 and B1 in p21+/+cells. Inactivation of APC in irradiated p21+/+cells can overcome the G2 arrest. siRNA-mediated Emi1 down-regulation prevents irradiated p21−/−cells from entering mitosis, whereas concomitant down-regulation of APC activity counteracts this effect. Our results demonstrate that Emi1 down-regulation and APC activation leads to stable p21-dependent G2 arrest after DNA damage. This is the first demonstration that Emi1 regulation plays a role in the G2 DNA damage checkpoint. Further, our work identifies a new p21-dependent mechanism to maintain G2 arrest after DNA damage.

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

Rb (retinoblastoma protein) inhibits E2F-1-induced cell death. We now show that the ability of Rb to inhibit E2F-1-induced cell death is dependent on a functional LXCXE-binding site in Rb, thereby suggesting that proteins that bind the LXCXE-binding site in Rb may regulate the anti-apoptotic activity of Rb. HDAC1, an LXCXE protein that plays a critical role in Rb-mediated transcription repression, abrogates the effect of Rb on E2F-1-induced cell death. In contrast, RF-Cp145, another LXCXE protein, cooperates with Rb to inhibit E2F-1-induced cell death. Both proteins exert their effect in an LXCXE-dependent manner. Rb regulates E2F-induced cell death by acting upstream of p73. Rb represses the p73 promoter. Our results further suggest a model in which Rb-E2F-1 complexes mediate the anti-apoptotic activity of Rb through active repression of target genes without recruiting HDAC1.

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

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