Hematopoietic Cell Populations Research Articles

Acetyl-CoA Carboxylase 1 Is Essential for Hematopoietic Stem Cell Homeostasis

Though fatty acid oxidation has been shown to be essential for hematopoietic stem cell maintenance, the importance of fatty acid uptake from the bone marrow microenvironment vs. de novo lipogenesis has not been elucidated. The process of de novo lipogenesis begins with the generation of malonyl-CoA from acetyl-CoA and is catalyzed by the enzyme acetyl-CoA carboxylase 1 (ACC1), encoded by Acaca. In leukemia, both ACC1 activation and degradation of ACC1 have been shown to enhance disease progression, suggesting an important role in leukemogenesis. The function of ACC1 in normal hematopoiesis remains unknown. To characterize the role of ACC1 in normal hematopoiesis, we bred mice that harbored an Acaca allele in which exons 22 through 26 were flanked by loxp sites. We crossed these animals to a mouse line harboring the Mx1-Cre transgene, generating Acaca fl/fl; Mx1-Cre + (ACC1 KO) and Acaca fl/fl; Mx1-Cre -(ACC1 WT) mice. Animals were then treated with intraperitoneal poly(I:C) injection to drive Mx1-Cre expression and Acaca excision. By 4-6 weeks after poly(I:C) injection, ACC1 KO animals developed expansion of hematopoietic stem cells and progenitors, including Lineage - Sca-1 +cKit hi (LSK) and the LSK CD48 -CD150 + long-term hematopoietic stem cell (LT-HSC) populations. In addition, these animals developed splenomegaly with extramedullary LSK expansion and enhanced myelopoiesis. ACC1 KO LSKs showed increased cell cycle activity and LT-HSCs demonstrated a reduced quiescent fraction compared to ACC1 WT controls. These data suggest that ACC1 deficiency results in increased cell cycle activity among primitive hematopoietic progenitors and biases cell fate to the myeloid lineage. To further test the impact of ACC1 deletion on LT-HSC function, we established competitive chimerism maintenance assays in which equal numbers of CD45.2 +Acaca fl/fl; Mx1-Cre +or CD45.2 +Acaca fl/fl; Mx1-Cre - bone marrow cells were mixed with CD45.1 + competitor bone marrow and transplanted into lethally irradiated recipient mice. Transplants were then given 8 weeks to establish stable engraftment. After documenting comparable engraftment, 5-poly(I:C) injections were administered every other day. Over the course of 16 weeks of tracking, the ACC1 KO graft showed a significant reduction in trilineage hematopoietic output as indicated by a reduction in B cell, T cell, and myeloid populations in the peripheral blood. After 16 weeks, animals were sacrificed and the LT-HSC compartment was analyzed, showing a significant reduction in ACC1 KO LT-HSCs. Together, these data suggest that the loss of ACC1 significantly impairs the ability of LT-HSCs to maintain long term hematopoiesis in competitive transplants. Collectively, these data are the first report of a critical role for ACC1 in hematopoiesis and LT-HSC function. Mechanistically, in the absence of ACC1, FACS sorted LSK demonstrated increased ATP content compared to WT controls, suggesting an altered metabolic state in these progenitor cells. Further studies are ongoing to determine if these findings are due to an essential function for ACC1 within the context of de novo lipogenesis, or if ACC1 participates in an as of yet uncharacterized tumor suppressive role. DisclosuresNo relevant conflicts of interest to declare.

Clonal Expansion of Tet2 +/- hematopoiesis Is Driven By Inflamm-Ageing Associated IL-1 Increase in Mice

Clonal Hematopoiesis of Indeterminate Potential (CHIP) is defined as the presence of an expanded somatic blood cell clone carrying a mutation in genes that are drivers of hematologic malignancy including DNMT3A, TET2, ASXL1, TP53, JAK2, and SF3B1, at a variant allele frequency (VAF) of at least 2% in the absence of other hematological abnormalities. CHIP has a prevalence of about 10% in the 70-80 year old population, further increases with ageing and associates with an increased risk of hematological malignancies, cardiovascular disease and all-cause mortality (Genovese et al. NEJM 2014; Jaiswal, S. et al. NEJM 2014). Recent studies indicate that higher pre-malignant clonal size and mutational burden increase the chances of malignant transformation in individuals carrying CHIP (Desai, P. Nat. Med. et al., 2018; Abelson, S. et al. Nature, 2018). While age is the best predictor of CHIP development and correlates directly with pre-malignant clonal size, the specific cellular-extrinsic factors promoting CHIP clonal expansion in the context of physiological aging are still unclear. We hypothesized that ageing associated low-grade inflammation (termed “inflamm-ageing”) is a driver of CHIP clonal expansion.We used standard bone marrow (BM) chimera models and developed a novel, irradiation independent, hematopoietic specific and tamoxifen inducible genetic mosaicism mouse model of Tet2 +/- driven CHIP (HSC-Scl-Cre-ERT; Tet2+/flox; R26 +/floxstop-EGFP triple transgenic mice) to determine the contribution of inflamm-aging factors to Tet2 +/- hematopoieticclonal expansion. Using these complementary models, we observe that peripheral Tet2 +/- clonal expansion rates increase with age (evident in erythroid, myeloid, lymphoid B and T lineages), which is paralleled by a significant expansion of Tet2 +/- hematopoietic stem and progenitor cell (HSPCs) populations in aged mice (12-14 months old). Importantly, Tet2 +/- clonal expansion associates with increased levels of inflammatory cytokine IL-1 in aged mice, which derives partially from Tet2 +/- mutant mature hematopoietic cells. To test the contribution of IL-1 to Tet2 +/- clonal expansion, we administered IL-1 (0.5ug/day for 14 days) to young CHIP carrying mice (2-4 months of age) and observed an IL-1R1-dependent expansion of Tet2 +/- hematopoietic mature lineages and HSPCs. Dissection of the cellular mechanisms operating downstream of IL-1/IL-1R1 revealed that Tet2 +/- clonal expansion results from increased multilineage differentiation and associates with increased HSPC cell-cycle progression (while not depending on IL-1-mediated effects on HSPC viability). Moreover, Tet2 +/- HSPCs show a higher in vitro and in vivo repopulation capacity in response to prolonged IL-1 exposure compared to their WT counterparts. Finally, to directly test the contribution of IL-1 to drive Tet2 +/- clonal expansion in the context of physiological aging, we set up genetic (BM chimeras using donor BM from Tet2 +/-; Il-1r1-/- compound mutants) or pharmacological inhibited IL-1 signaling (Anakinra, hIL-1ra) during mouse ageing. Strikingly, both approaches prevented ageing-dependent Tet2 +/- clonal expansion, thus confirming IL-1 as key “inflamm-ageing” driver of Tet2 +/- clonal expansion. Overall, our data provide proof-of-concept that IL-1 production derived from aged BM cells is a relevant and targetable driver of Tet2 +/- clonal expansion in aged mice. DisclosuresManz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

The Disparity Observed Between Murine Short-term and Long-term Hematopoietic Stem Cells in Their Ability to Migrate and Engraft When Introduced IV is Due to Distinct Adhesion Molecule Expression

CD34 has traditionally been used as a marker to enrich human bone marrow stem cells for clinical bone marrow transplants for over five decades. However, several studies support that the most primitive hematopoietic stem cells (HSCs) responsible for long-term hematopoiesis-long-term HSCs (LT-HSCs) -are found within the CD34 negative (CD34 neg) fraction of bone marrow cells. LT-HSCs are challenging to study due to their scarcity and lack of specific markers to identify them. Once these stem cells enter the circulation, they must find their way, through multistep process controlled by a variety of cell surface adhesion molecules, into the bone marrow. In contrast to the CD34 pos stem cells, studies have suggested that LT-HSCs have trouble migrating and engrafting when introduced intravenously. In order to better understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by various murine hematopoietic stem cell populations to migrate. To this end, we assayed the homing effectors that are either necessary in the first step, i.e., the cell surface adhesion molecule Sialyl Lewis X (sLe x) and its interaction with selectins-mainly E-selectin-on the bone marrow endothelium, or the second step, i.e. the chemokine receptor CXCR4 and its binding to the bone marrow stroma resident chemokine SDF-1α, of cell migration.Specifically focusing on murine populations of short term HSCs (ST-HSCs; Flk2 negCD34 pos) and LT-HSCs (Flk2 negCD34 neg), flow cytometric and gene expression analysis clearly illustrated distinctive expression patterns of homing effectors with the highest contrast observed in sLe x where Flk2 negCD34 neg HSCs exhibited low expression (< 10%) and Flk2 negCD34 pos HSCs exhibited much higher expression (> 60%) which correlated to higher binding to E-selectin. Of the common E-selectin ligands identified, both Flk2 negCD34 pos and Flk2 negCD34 neg HSCs expressed CD44 and PSGL-1 to equivalent levels while CD43 was expressed at significantly lower levels on Flk2 negCD34 neg HSCs. Moreover, our data indicates that the variation observed in sLe x expression/E-selectin binding correlates with differences in the expression of key glycosyltransferase genes involved in the creation of the sLe x epitope on these E-selectin ligands. mRNA expression analysis of glycosyltransferases revealed that, in general, sialyltransferase (ST3Gals) genes were expressed at higher levels in Flk2 negCD34 neg HSCs while only one fructosyltransferase (FT4) gene was expressed at higher levels in this HSC population. Thus, these data suggest that α2,3 sialylation is intact in Flk2 negCD34 neg HSCs but α1,3 fucosylation is lacking. The expression levels of homing molecules involved in the stages of cell migration after E-selectin binding were also assessed. No differences in the expression of the integrins CD49d, CD49e and CD29 were observed between Flk2 negCD34 neg and Flk2 negCD34 pos HSCs. Interestingly, higher levels of the chemokine receptor CXCR4 were measured on the surface of Flk2 negCD34 pos HSCs compared to Flk2 negCD34 neg HSCs. Likewise, this was reflected in a much lower ability of the Flk2 negCD34 neg HSCs to migrate towards the CXCR4 ligand, SDF-1, in a transwell assay.Based on these findings that show that both E-selectin binding and CXCR4 mediated migration are compromised in Flk2 negCD34 neg HSCs, we sought to enhance this LT-HSC populations ability to migrate using well-known treatments including recombinant human fucosyltransferase 6 (rhFVI) to create the glycans necessary for selectin binding, as well as the CD26 inhibitor (Diprotin A; Dip A) to enhance the binding of chemokines to their receptors.Interestingly, although both CD26 inhibition and cell surface fucosylation remarkably enhanced the expression of homing molecules in both HSC populations in vitro, only pretreatment of Flk2 negCD34 neg HSCs with Dip A significantly enhanced engraftment in vivo after transplantation into C57BL/6N mice.This study represents the first direct analysis of E-selectin ligand expression on murine LT-HSC populations and strongly support that by temporarily enhancing the expression of migration-associated molecules on the surface of these cells, prolonged and efficient HSC engraftment can be achieved. We hope our finding will potentially shed light on methods to optimally utilize these very valuable long-term HSCs in clinical bone marrow and cord blood transplants worldwide. DisclosuresNo relevant conflicts of interest to declare.

Mouse Breast Carcinoma Monocytic/Macrophagic Myeloid-Derived Suppressor Cell Infiltration as a Consequence of Endothelial Dysfunction in Shb-Deficient Endothelial Cells Increases Tumor Lung Metastasis.

Metastasis reflects both the inherent properties of tumor cells and the response of the stroma to the presence of the tumor. Vascular barrier properties, either due to endothelial cell (EC) or pericyte function, play an important role in metastasis in addition to the contribution of the immune system. The Shb gene encodes the Src homology-2 domain protein B that operates downstream of tyrosine kinases in both vascular and immune cells. We have investigated E0771.lmb breast carcinoma metastasis in mice with conditional deletion of the Shb gene using the Cdh5-CreERt2 transgene, resulting in inactivation of the Shb-gene in EC and some hematopoietic cell populations. Lung metastasis from orthotopic tumors, tumor vascular and immune cell characteristics, and immune cell gene expression profiles were determined. We found no increase in vascular leakage that could explain the observed increase in metastasis upon the loss of Shb expression. Instead, Shb deficiency in EC promoted the recruitment of monocytic/macrophagic myeloid-derived suppressor cells (mMDSC), an immune cell type that confers a suppressive immune response, thus enhancing lung metastasis. An MDSC-promoting cytokine/chemokine profile was simultaneously observed in tumors grown in mice with EC-specific Shb deficiency, providing an explanation for the expanded mMDSC population. The results demonstrate an intricate interplay between tumor EC and immune cells that pivots between pro-tumoral and anti-tumoral properties, depending on relevant genetic and/or environmental factors operating in the microenvironment.

Targeting miR-126 disrupts maintenance of myelodysplastic syndrome stem and progenitor cells.

BackgroundMyelodysplastic syndrome (MDS) arises from a rare population of aberrant hematopoietic stem and progenitor cells (HSPCs). These cells are relatively quiescent and therefore treatment resistant. Understanding mechanisms underlying their maintenance is critical for effective MDS treatment.MethodsWe evaluated microRNA‐126 (miR‐126) levels in MDS patients’ sample and in a NUP98‐HOXD13 (NHD13) murine MDS model along with their normal controls and defined its role in MDS HSPCs’ maintenance by inhibiting miR‐126 expression in vitro and in vivo. Identification of miR‐126 effectors was conducted using biotinylated miR‐126 pulldown coupled with transcriptome analysis. We also tested the therapeutic activity of our anti‐miR‐126 oligodeoxynucleotide (miRisten) in human MDS xenografts and murine MDS models.ResultsmiR‐126 levels were higher in bone marrow mononuclear cells from MDS patients and NHD13 mice relative to their respective normal controls (P < 0.001). Genetic deletion of miR‐126 in NHD13 mice decreased quiescence and self‐renewal capacity of MDS HSPCs, and alleviated MDS symptoms of NHD13 mice. Ex vivo exposure to miRisten increased cell cycling, reduced colony‐forming capacity, and enhanced apoptosis in human MDS HSPCs, but spared normal human HSPCs. In vivo miRisten administration partially reversed pancytopenia in NHD13 mice and blocked the leukemic transformation (combination group vs DAC group, P < 0.0001). Mechanistically, we identified the non‐coding RNA PTTG3P as a novel miR‐126 target. Lower PTTG3P levels were associated with a shorter overall survival in MDS patients.ConclusionsMiR‐126 plays crucial roles in MDS HSPC maintenance. Therapeutic targeting of miR‐126 is a potentially novel approach in MDS.

Deficiencies in the DNA Binding Protein ARID3a Alter Chromatin Structures Important for Early Human Erythropoiesis.

ARID3a is a DNA-binding protein important for normal hematopoiesis in mice and for in vitro lymphocyte development in human cultures. ARID3a knockout mice die in utero with defects in both early hematopoietic stem cell populations and erythropoiesis. Recent transcriptome analyses in human erythropoietic systems revealed increases in ARID3a transcripts implicating potential roles for ARID3a in human erythrocyte development. However, ARID3a transcript levels do not faithfully reflect protein levels in many cells, and the functions and requirements for ARID3a protein in those systems have not been explored. We used the erythroleukemic cell line K562 as a model to elucidate functions of ARID3a protein in early human erythropoiesis. ARID3a knockdown of hemin-stimulated K562 cells resulted in lack of fetal globin production and modifications in gene expression. Temporal RNA sequencing data link ARID3a expression with the important erythroid regulators Gata1, Gata2, and Klf1 Ablation of ARID3a using CRISPR-Cas9 further demonstrated it is required to maintain chromatin structures associated with erythropoietic differentiation potential. These data demonstrate that the ARID3a protein is required for early erythropoietic events and provide evidence for the requirement of ARID3a functions for proper maintenance of appropriate chromatin structures.

IL-1 mediates microbiome-induced inflammaging of hematopoietic stem cells in mice

AbstractAging is associated with impaired hematopoietic and immune function caused in part by decreased fitness in the hematopoietic stem cell (HSC) population and an increased myeloid differentiation bias. The reasons for this aging-associated HSC impairment are incompletely understood. Here we demonstrate that older specific pathogen free (SPF) wild-type (WT) mice in contrast to young SPF mice produce more interleukin-1a and interleukin-1b (IL-1a/b) in steady-state bone marrow (BM), with most of the IL-1a/b being derived from myeloid BM cells. Furthermore, blood from steady-state older SPF WT mice contains higher levels of microbe-associated molecular patterns, specifically TLR4 and TLR8 ligands. In addition, BM myeloid cells from older mice produce more IL-1b in vitro, and older mice show higher and more durable IL-1a/b responses upon stimulation with lipopolysaccharide in vivo. To test whether HSC aging is driven by IL-1a/b, we evaluated HSCs from IL-1 receptor 1 (IL-1R1) knockout (KO) mice. Indeed, older HSCs from IL-1R1KO mice show significantly mitigated aging-associated inflammatory signatures. Moreover, HSCs from older IL-1R1KO and from germ-free mice maintain unbiased lymphomyeloid hematopoietic differentiation upon transplantation, thus resembling this functionality of young HSCs. Importantly, in vivo antibiotic suppression of microbiota or pharmacologic blockade of IL-1 signaling in older WT mice was similarly sufficient to reverse myeloid-biased output of their HSC populations. Collectively, our data define the microbiome/IL-1/IL-1R1 axis as a key, self-sustaining and also therapeutically partially reversible driver of HSC inflammaging.

Single-cell analysis at the protein level delineates intracellular signaling dynamic during hematopoiesis

BackgroundHematopoietic stem and progenitor cell (HSPC) subsets in mice have previously been studied using cell surface markers, and more recently single-cell technologies. The recent revolution of single-cell analysis is substantially transforming our understanding of hematopoiesis, confirming the substantial heterogeneity of cells composing the hematopoietic system. While dynamic molecular changes at the DNA/RNA level underlying hematopoiesis have been extensively explored, a broad understanding of single-cell heterogeneity in hematopoietic signaling programs and landscapes, studied at protein level and reflecting post-transcriptional processing, is still lacking. Here, we accurately quantified the intracellular levels of 9 phosphorylated and 2 functional proteins at the single-cell level to systemically capture the activation dynamics of 8 signaling pathways, including EGFR, Jak/Stat, NF-κB, MAPK/ERK1/2, MAPK/p38, PI3K/Akt, Wnt, and mTOR pathways, during mouse hematopoiesis using mass cytometry.ResultsWith fine-grained analyses of 3.2 million of single hematopoietic stem and progenitor cells (HSPCs), and lineage cells in conjunction with multiparameter cellular phenotyping, we mapped trajectories of signaling programs during HSC differentiation and identified specific signaling biosignatures of cycling HSPC and multiple differentiation routes from stem cells to progenitor and lineage cells. We also investigated the recovery pattern of hematopoietic cell populations, as well as signaling regulation in these populations, during hematopoietic reconstruction. Overall, we found substantial heterogeneity of pathway activation within HSPC subsets, characterized by diverse patterns of signaling.ConclusionsThese comprehensive single-cell data provide a powerful insight into the intracellular signaling-regulated hematopoiesis and lay a solid foundation to dissect the nature of HSC fate decision. Future integration of transcriptomics and proteomics data, as well as functional validation, will be required to verify the heterogeneity in HSPC subsets during HSC differentiation and to identify robust markers to phenotype those HSPC subsets.

A Primer on the Biology of MDS

Introduction Myelodysplastic syndromes (MDS) arise from a small population of disease-initiating hematopoietic stem cells (HSCs) that persist and expand through conventional therapies and are major contributors to disease progression and relapse.1 In the last decade, genomic technologies coupled with mouse genetic studies have greatly improved our understanding of the genetic elements driving MDS initiation and progression and the ways in which such elements functionally contribute to specific aspects of the disease’s pathobiology.2–6 These studies have revealed that MDS are typically driven by multistep processes that affect a recurrent set of genes, which leads to the clonal expansion of mutant HSCs over their normal counterparts. Disease onset can be accelerated by exposure to cytotoxic chemotherapy, the presence of germ-line predisposition mutations, or any type of stress (e.g., inflammation) that confers a fitness advantage to the mutant clone. Progression from MDS to secondary acute myeloid leukemia (sAML) is mostly associated with the expansion of HSC clones carrying preexisting or newly acquired recurrent mutations affecting hematopoietic transcription factor genes that abrogate normal differentiation or those carrying activating mutations in signaling pathways that control cellular proliferation.7

Heat-inactivated Escherichia coli promotes hematopoietic regeneration after irradiation with IL-1β

Background aimsHematopoietic stem and progenitor cells (HSPCs) are known to produce short-lived mature blood cells via proliferation and differentiation in a process that depends partially on regulatory cytokines from the bone marrow (BM) microenvironment. Delayed BM recovery after tremendous damage to the hematopoietic system can lead to neutropenia, anemia, thrombopenia and even death. However, efficiently promote BM recovery is still a big problem to be solved. Here, the authors aim to use heat-inactivated Escherichia coli (HIEC) to enhance BM recovery and further to find out the potential mechanism. MethodsX-rad was used to establish HIEC/IL-1β-induced radioprotection model. Single-cell RNA sequencing, RT-PCR, and western blotting were performed to detect the expression of IL-1R1 on HSPCs. Flow cytometry and automated hematology analyzer were used to analyze the percentage and absolute number of different populations of hematopoietic cells. The effects of IL-1β on HSPCs were studied using in vivo and in vitro experiments. ResultsHIEC/IL-1β pre-treatment can significantly increase the survival rate of lethally irradiated mice, and these mice showed better hematopoietic regeneration compared with control group. IL-1R was expressed on HSPCs, and IL-1β could directly function on HSPCs to promote the proliferation and differentiation of HSPCs, and inhibit the apoptosis of HSPCs. ConclusionsHIEC pre-treatment can rescue lethally irradiated mice by promoting hematopoietic recovery via IL-1β/IL-1R1 signaling, which can promote the proliferation of HSPCs by enhancing the cell cycle and attenuating the apoptosis of HSPCs.

A comprehensive RNA editome reveals that edited Azin1 partners with DDX1 to enable hematopoietic stem cell differentiation

AbstractAdenosine-to-inosine RNA editing and the catalyzing enzyme adenosine deaminase are both essential for hematopoietic development and differentiation. However, the RNA editome during hematopoiesis and the underlying mechanisms are poorly defined. Here, we sorted 12 murine adult hematopoietic cell populations at different stages and identified 30 796 editing sites through RNA sequencing. The dynamic landscape of the RNA editome comprises stage- and group-specific and stable editing patterns, but undergoes significant changes during lineage commitment. Notably, we found that antizyme inhibitor 1 (Azin1) was highly edited in hematopoietic stem and progenitor cells (HSPCs). Azin1 editing results in an amino acid change to induce Azin1 protein (AZI) translocation to the nucleus, enhanced AZI binding affinity for DEAD box polypeptide 1 to alter the chromatin distribution of the latter, and altered expression of multiple hematopoietic regulators that ultimately promote HSPC differentiation. Our findings have delineated an essential role for Azin1 RNA editing in hematopoietic cells, and our data set is a valuable resource for studying RNA editing on a more general basis.

Identification of a Hematopoietic Cell Population Emerging From Mouse Bone Marrow With Proliferative Potential In Vitro and Immunomodulatory Capacity.

There is continuing interest in therapeutic applications of bone marrow-derived mesenchymal stromal cells (MSC). Unlike human counterparts, mouse MSC are difficult to propagate in vitro due to their contamination with adherent hematopoietic cells that overgrow the cultures. Here we investigated the properties of these contaminating cells, referred to as bone marrow-derived proliferating hematopoietic cells (BM-PHC). The results showed that both BM-PHC and MSC had strong immunomodulatory properties on T cells in vitro, with PGE2 and NO involved in this mechanism. However, BM-PHC were stronger immunomodulators than MSC, with CCL-6 identified as putative molecule responsible for superior effects. In vivo studies showed that, in contrast to BM-PHC, MSC endorsed a more rapid xenograft tumor rejection, thus indicating a particular context in which only MSC therapy would produce positive outcomes. In conclusion, bone marrow contains two cell populations with immunomodulatory properties, which are valuable sources for therapeutic studies in specific disease-relevant contexts.

The Orphan Nuclear Receptor Nr4a1 Specifies Long-Term, Myeloid-Biased HSCs and Responds To PGE2

The Nr4a family of transcription factors regulates the fate of cells from multiple tissues. The absence of Nr4a1 and Nr4a3 in the hematopoietic system results in acute myeloid leukemia, suggesting that this protein family is critically involved in the control of blood cell proliferation and development. In order to better understand the role played by Nr4a1 in particular, we have taken advantage of our Nr4a1(Nur77)GFP reporter mouse and investigated Nr4a1 expression during hematopoiesis. We find that Nr4a1 is expressed within the hematopoietic stem cell (HSC) population and most highly expressed among quiescent, long term (LT)-HSC. The association of Nr4a1 expression with LT-HSCs was directly confirmed by engraftment experiments. Sorted Lin-Sca+c-Kit+ (LSK) Nr4a1GFPhigh cell populations from CD45.2+ donors reconstituted the blood of CD45.1+ irradiated recipients for at least 20 weeks after transplant; an average of 20.3% of blood cells were CD45.2+. In contrast, Nr4a1GFPlow populations of LSK cells only transiently reconstituted the blood; well below 1% of blood cells were CD45.2+. Furthermore, blood cells arising from Nr4a1GFPhigh LSKs were dominated by myeloid cells: the lymphoid (L) to myeloid (M) ratio (ρ) was 13.7. The few Nr4a1GFPlow cells that remained after 20 weeks were almost exclusively lymphoid (L/M ratio = 99.3). Examination of the bone marrow from engrafted mice at 20 weeks showed that HSC populations derived from Nr4a1GFPhigh cells included a higher percentage of CD150+CD48- LT-HSCs than cells arising from whole bone marrow competitors. Given the association of Nr4a1 expression with long-term engraftment potential, and given that Nr4a1 has been shown to be highly responsive to environmental stimuli in a variety of tissues, we tested whether HSCs express Nr4a1 in response to signals generated by the bone marrow stroma. We evaluated the influence of a variety of factors, including GCSF, SCF, IL-6, TNFa, and PGE2. We found that PGE2 and, to a lesser extent, TNFa specifically upregulate Nr4a1 in HSCs. In summary, we show that Nr4a1 is expressed by a population of long-term, myeloid-biased HSCs, which closely resemble the potent long-term, myeloid biased HSC subset recently identified by Oguro et al. We further show that Nr4a1 expression is responsive to PGE2, which is known to enhance HSC engraftment. We hypothesize that Nr4a1 expression by HSCs reflects and integrates bone marrow environmental inductive cues critical for long-term stem cell homeostasis, including quiescence. Disclosures:No relevant conflicts of interest to declare.

Abstract 2799: Multiple myeloma derived mitochondrial DAMPs induce a pro-inflammatory signature in the bone marrow microenvironment to promote pro-tumoral expansion

Abstract Multiple myeloma (MM) is an incurable malignancy of antibody (Ig) secreting differentiated B cells (plasma cells) characterized by the accumulation and localization of tumor cells in the bone marrow microenvironment (BMM). Mitochondrial DNA (mtDNA) is a damage associated molecular pattern (DAMP), as the mitochondrial genome contains islands of unmethylated CpG nucleotide motifs that have been shown to activate and promote memory B cell proliferation and antibody secretion. Recent studies have indicated that mtDNA is elevated in the circulation of trauma and cancer patients. Here we investigate the functional purpose of elevated mtDNA within the BM microenvironment of MM.We hypothesize that multiple myeloma cells secrete mitochondrial DAMPs into the bone marrow microenvironment promoting a state of chronic inflammation that drives the progression and expansion of multiple myeloma. NSG immunocompromised mice engrafted with human MM1S myeloma cell line showed elevated levels of MM derived mtDNA in the serum, detected by real-time PCR. Next we engrafted C57BL/6 mice with murine 5TGM1 myeloma cell line to establish a syngeneic mouse model. Flow cytometry analysis of the haematopoietic stem and progenitor cell (HSPC) populations showed that 5TGM1 induced an inflammatory expansion of the stem cell niche To determine the role of mtDNA in HSPC expansion we treated C57BL/6 mice with multiple doses of CpG oligodeoxynucleotides to mimic mtDNA export by MM. Results showed similar expansion of haematopoietic stem and progenitor cell populations. To understand the effects of mtDAMPs on the inflammatory cells of the BMM, we show that bone marrow derived macrophages treated with mtDNA and CpG had increased expression of pro-inflammatory cytokines including IL-6. In vivo, isolated F4/80+ bone marrow macrophages from 5TGM1 and CpG treated mice also showed increased expression of pro-inflammatory cytokines. Finally, to understand the role of mtDAMPs in regulating HSPC expansion we used blocking antibodies to TLR9 (toll-like receptor 9 for mtDNA) and FPR1 (receptor for formylated mitochondrial proteins) in 5TGM1 engrafted animals. Blocking these receptors resulted in reduced myeloma tumor burden compared to control animals. Here we establish that MM releases mtDNA into the microenvironment and highlight the involvement of mtDAMPs in creating a pro-inflammatory BMM that aids in MM disease progression. This data suggests the potential for the targeting of TLR9 or FPR1 signaling pathways as a novel therapeutic approach for MM. Citation Format: Aisha Jibril, Prakrit Kumar, Charlotte Hellmich, Jamie A. Moore, Jayna J. Mistry, Victoria Willimott, Kristian M. Bowles, Stuart A. Rushworth. Multiple myeloma derived mitochondrial DAMPs induce a pro-inflammatory signature in the bone marrow microenvironment to promote pro-tumoral expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2799.

Hematopoietic Stem Cell Potential within the Primary Sorted Lineage Positive Population: What Are We Losing with Hematopoietic Stem Cell Purification?

Lineage positive cells within murine whole bone marrow (WBM) are classically considered to be devoid of stem cell capacity. As such, lineage depletion prior to further selection for stem cell marker positive, lineage negative cells dominates virtually all hematopoietic stem cell (HSC) purification protocols. Here, we present data showing a significant population of long-term multi-lineage hematopoietic stem cells within the primary sorted lineage positive (Lin+) murine marrow population. Using limiting dilution competitive bone marrow transplantation assays, we found significant long-term multi-lineage engraftment capacity within the total primary sorted Lin+ population (cells positive for erythroid (TER119), myeloid (GR1, CD11b) or lymphoid (B220, CD4, CD8) markers). When 0.25 x 106 Lin+ cells were competed against 1 x 106 competitor WBM cells, average percent donor chimerism in recipient mice at 6 months post-transplant was 7.2% ± 2.9%. Donor chimerism was multi-lineage. There was still 1.9% ± 0.5% donor chimerism when only 62,500 primary sorted Lin+ cells were competed against 1 x 106 competitor WBM cells at 3 months post transplant, and further limiting dilution assays are ongoing to quantitate the stem cell compartment within the primary sorted Lin+ population. These levels of engraftment were similar to that found within un-separated donor WBM at the same dilutions, and multi-lineage engraftment capacity within the primary sorted Lin+ population persisted in secondary transplant. When further sub-fractionated into erythroid, myeloid or lymphoid sub-populations, every primary sorted individual sub-population contained long-term multi-lineage engraftment capacity at 6 months post transplant. Primary sorts were between 96-97.5% pure. When primary Lin+ sub-sets (B220+ cells, GR1+ cells, or CD11b+ cells) were double sorted, there was loss of engraftment potential within the double sorted persistently positive Lin+ subsets, but surprisingly high engraftment within those cells negative for only the single Lin+ marker on double sort (31%-59% donor chimerism at 6mo post-transplant; 1 x 105 donor cells + 3 x 105 competitor WBM cells). Immunophenotype analysis showed that these populations were not enriched in c-Kit+/Sca-1+/CD150+ cells. The high engraftment capacity of cells within the primary sorted Lin+ population, further isolated solely based on their being negative for a single Lin+ marker on the double sort prompted us to directly compare their engraftment capacity to that of purified Lineage negative (Lin-) cells. Interestingly, although isolated solely based on the presence or absence of GR1 on double sort, we found that the average percent donor chimerism at 6 months post-transplant was equal between purified Lin- cells (58.3% ± 17%) and the population of GR1 positive cells that became negative for GR1 on double sort (58.3% ± 26%; 1 x 105 donor cells + 3 x 105 competitor WBM cells). Using tritiated thymidine suicide, we found that the stem cell activity within these populations isolated from the primary sorted Lin+ fraction was due to actively cycling cells. These data further indicate that the engraftment capacity within the primary sorted Lin+ population is not simply due to conventionally purified quiescent HSCs contaminating our primary sort. In sum, there is a cycling population of stem cells within the primary sorted Lin+ fraction that appears to be distinct from quiescent highly purified HSCs. As primary sorted Lin+ cells, routinely discarded in most HSC studies, comprise up to 98% of WBM, engraftment capacity within this population represents a potentially large and understudied stem cell population within WBM worthy of further characterization. Work is currently underway to further quantitate and define this population, and delineate how cell cycle-related changes influence the phenotype and function of this cycling murine HSC population. DisclosuresNo relevant conflicts of interest to declare.

Correlative Lightsheet and Electron Microscopy Applied to a Zebrafish Model of Fetal Bone Marrow: Resolving the Ultrastructure of a Single Hematopoietic Stem Cell in the Niche

Hematopoietic stem and progenitor cells (HSPC) emerge from the hemogenic endothelium of the dorsal aorta. Next, HSPC migrate to the fetal liver where they expand exponentially before migrating again to the fetal bone marrow. This early stage of bone marrow colonization is poorly understood. Current imaging technology does not allow direct visualization of the fetal bone marrow niche. Zebrafish provide a model to observe these events because they develop externally, are transparent, and ontogeny of the hematopoietic system is highly conserved. The adult marrow niche in zebrafish is in the kidney and is colonized 4 to 5 days post fertilization. To track HSPC during this dynamic process we used our previously characterized HSPC-specific transgenic reporter lines (Runx:GFP and Runx:mCherry). We demonstrated by transplantation these cells mark a highly pure population of long-term engrafting hematopoietic stem cells. To image the kidney marrow live we applied lightsheet microscopy. This technology uses three objective lenses: two that illuminate the sample in the XY plane, and one that takes images in the Z plane. This allows hundreds of optical sections to be captured in seconds. The temporal and spatial resolution of this technique produces a highly dynamic view of the entire developing marrow niche. By imaging Runx+ HSPC together with flk+ endothelial cells in a double transgenic embryo, we could track interactions between HSPC and the perivascular niche. Previously, we showed that HSPC arrival in the caudal hematopoietic tissue, the zebrafish equivalent of the fetal liver, triggers remodeling of endothelial cells into a surrounding pocket. In this study, we found a similar cellular structure forms in the kidney marrow niche. We wanted to resolve the ultrastructure of the HSPC and surrounding cells in the context of the larger developing kidney organ. To do this we applied Correlative Light and Electron Microscopy (CLEM) to match fluorescent cells tracked by lightsheet in the same embryo fixed and stained for EM. Micro-CT was used to orient and trim the embedded sample to isolate the kidney niche. Serial block-face scanning EM was used to capture high resolution serial sections (volume dimensions: x, y, z (um) = 236, 114, 105; pixel size: x, y, z (nm) = 9.5, 9.5, 70). Blood vessels observed in light and EM 3D data sets provided the anatomical markers needed to match the position of cells found using both techniques. As we previously found in the caudal hematopoietic tissue, a single round HSPC with a large nucleus, little cytoplasm, and a ruffled membrane, was lodged just under the vessel wall. This single HSPC was in a distinct anatomical location compared to larger clusters of HSPC in an abluminal space. This observation is suggestive of a marrow with distinct anatomical niche sites that may support different functions, such as proliferation versus quiescence. This unique system for analysis of the ultrastructure of an endogenous HSPC in an unperturbed niche will allow further functional and mechanistic studies at the subcellular level. Our goal is to gain novel insights into intercellular interactions between HSPC and the surrounding supportive cells in the perivascular niche. DisclosuresNo relevant conflicts of interest to declare.

A Precise Tool to Edit Human Hematopoietic Stem Cells at High Efficiency and Decipher and Optimize the Different Double Strand Break Repair Outcomes

The CRISPR/Cas9 discovery has revolutionized precise editing of the genome. However, Cas9-induced site-specific double-strand breaks (DSB) are predominantly repaired by the ‘error-prone’ non-homologous end-joining (NHEJ)/alternate NHEJ (aNHEJ) mechanisms, with relatively low precise correction using homology-directed repair (HDR). The HDR-corrected cells can be selected with drugs/surface reporter in cell lines, induced pluripotent cells, mouse embryonic cells. However, selection of gene edited/corrected hematopoietic stem cells (HSC) is not feasible in vitro. HSC remain the most promising targets because of their unique self-renewing, expansion and differentiation potential. Assessment and optimization of editing/correction of the rare human HSC comprising 1-2% of the CD34+ hematopoietic stem and progenitor cell (HSPC) population is particularly challenging, as their selection before/after often results in insufficient cell numbers for detailed molecular analysis; and selection post-editing for clinical translation can reduce recovery/engraftment potential from increased processing. Currently, the readout of the edited HSC is by molecular analysis for NHEJ- and aNHEJ-induced insertions/deletions and HDR detection of total CD34+ cells and assessment of long term repopulating potential (LTRP) in in vivo models. Herein, we report a unique platform that can precisely quantify NHEJ, aNHEJ, HDR and random integration of the HDR donor template in the human HSC using a flow cytometry. We targeted the CD45 surface reporter (PTPRC), present on all leukocytes and HSPC, to induce a DSB in exon 2 of the PTPRC gene using CRISPR/Cas9, and an in-frame promoter-less GFP containing homology template and a downstream tNGFR expression cassette (GFP/tNGFR HDR donor). HSC and HSPC that repaired Cas9-induced DSB via NHEJ on a single allele in diploid cells lost CD45 expression by flow cytometry. Furthermore, the gRNA/Cas9 induced DSB site was designed to have a 3bp micro-homology so that DSB repaired via aNHEJ resulted in cells express CD45 in frame, but a 6bp deletion detectable by TIDE assay. Provision of the GFP/tNGFR HDR donor repair template resulted in GFP and CD45 expressing, but not tNGFR expressing cells, but if the GFP/tNGFR HDR donor integrated randomly, cells were tNGFR+. Using this system, we optimized editing of hematopoietic progenitor cell lines (K562, Kasumi cells) and EBV transformed B-lymphoblasts to >95% efficiency, and molecular analysis of total and CD45+, and CD45- and CD45+GFP+ cell populations confirmed the precision of the flow cytometry based quantification of NHEJ, aNHEJ. In fact, molecular analysis of the CD45+ cells led to the discovery that K562 cells had a high rate of aNHEJ, while this editing via aNHEJ was absent in LCL- and Kasumi- B lymphoid cells. Inhibition of the aNHEJ pathway with small molecule inhibitors/siRNA resulted in loss of the 6bp aNHEJ-mediated deletion in K562 cells. We also found that random integration in LCL, Kasumi and CD34+ cells was a rare event, but occurred with high frequency in K562 cells. Due to the rapid detection of editing events by flow cytometry, we were able to optimize different editing and cell parameters in both cord blood and mobilized peripheral blood derived CD34+ HSPC and CD34+38-90+RA- HSC populations to achieve high viability and 80-87% editing efficiency (CD45-) in HSPC and HSC, and the FACS-based results were similar to molecular analysis. The edited CD34+ HSPC, when injected into NSG mice resulted in robust human cell engraftment, with 45±10% total engraftment, and 39±16% edited cell engraftment (n=12 mice). Using this platform, we improved HDR frequency to 30% and reduced NHEJ events to 60% in hematopoietic cell lines and similar optimization of HDR in HSPC/HSC is underway. In summary, we have developed a novel platform that allows rapid quantification of gene editing by flow cytometry of human HSC at a single cell and a single allele level, in a single step; with concurrent molecular analysis that identifies different DNA repair pathways utilized by different cell types. This platform allows rapid identification of edited events in the rare HSC population, rather than bulk CD34+ HSPC. Furthermore, ability to sort the edited HSC allows precise determination of the genotoxicity of gene editing, and optimization of DSB repair pathway needed, and optimize editing parameters that result in engraftable edited HSC with LTRP. DisclosuresNo relevant conflicts of interest to declare.

Niche Regulation of Residual Hematopoietic Stem Cell Protein Synthesis By Extracellular Vesicle (EV) Trafficking in the Acute Myeloid Leukemia (AML) Microenvironment

Work by several groups has shown that the presence of Acute Myeloid Leukemia (AML) deregulates critical functions of the bone marrow microenvironment, including hematopoiesis, in part through indirect stroma-secreted factors. We recently reported that Extracellular Vesicles (EV) released from AML blasts directly suppress the clonogenicity of hematopoietic progenitors via the action of miRNA-150 and miR-155 on cMyb, a transcription factor highly expressed in differentiating progenitors. The fate of long-term hematopoietic stem cells (LT- HSC) in the AML niche has not been widely studied. Using a xenograft model of human AML (Molm-14, U937 and HL-60 cell lines), we first ascertained the in vivo uptake of AML-EVs and found different internalization efficiencies among hematopoietic stem and progenitor cell (HSPC) populations, including LT-HSC (CD150+CD48−KSL) by imaging and flow cytometry. Live-cell microscopy studies of LT-HSCs revealed AML-EV binding and uptake within minutes following in vitro exposure. To determine the functional implications of EV trafficking, we next evaluated the relative frequency of the hematopoietic populations in low chimerism xenografts and found a consistent increase in the LT-HSC population, but not multi-potent progenitors, suggestingthe proportional accumulation of LT-HSC population caused by mobilization and lack of expansion and differentiation of other hematopoietic cells. To gain insight into how AML-EVs may regulate cell fate and function following internalization, we performed a proteomic profiling of HSPC cells exposed to EVs from two cell lines representing different AML subtypes. Bioinformatics analysis found significant enrichment for the ribosomal biogenesis pathways and negative cell cycle regulators in the EV-exposed cells. Because LT-HSC have been shown to be particularly susceptible to impairment of ribosome biogenesis, we wished to determine if AML-EVs modulate the LT-HSC fate by suppressing protein synthesis. We therefore injected AML-EVs into the femurs of both immuno-competent and immuno-compromised mice and evaluated the cellular protein synthesis by quantifying O-Propargyl-Puromycin (OPP) incorporation. Results show that AML-EVs significantly suppress the protein synthesis in the LT-HSC, but not in the HSPC pool population. The mTOR signaling pathway is known to be greatly involved in ribosome biogenesis via phosphorylation of the ribosomal protein S6 (S6RP) which in turn regulates several repressors of rRNA and ribosomal proteins. Consistent with the notion of translational suppression, we found that AML-EVs reduced the phosphorylation of S6RP in LT-HSC. In correlation with the observation that ribosomal stress induction via suppression of ribosomal proteins we found upregulation of P53 and its transcriptional target p21, as well as a gain in quiescence (G0) in the LT-HSC population. To address the functional fate of deregulated protein synthesis and quiescence, we transplanted KSL cells purified from male mice engrafted with AML cells into irradiated female recipient mice and measured the donor chimerism via qPCR for Sex-determining Region Y (SRY). Early repopulation capacities between cohorts of mice transplanted with KSL from AML xenograft versus those from control non-engrafted mice are similar (with long-term evaluation ongoing) in the host non-leukemic microenvironment. This suggesting that hematopoietic impairment by AML-EVs is reversible upon transplantation to a different host environment. Collectively, our results provide the first evidence that AML-EV impair LT-HSC via suppressing of ribosomal biogenesis. Understanding the regulation of LT-HSC by AML-EV will help reduce the morbidity and mortality from hematopoietic suppression in patients with leukemia. DisclosuresNo relevant conflicts of interest to declare.

MTOR-C/EBPβ-c-Myc Axis Regulates the Behavior of Myeloid-Biased Hematopoietic Stem Cells Under Stress Conditions

In stress or regenerative conditions, hematopoietic stem cell (HSC) population drastically alters its behavior; they rapidly enter into cell cycle, differentiate to multipotent progenitors (MPPs) at the expense of self-renewal activity, and are reprogrammed toward predominantly myeloid-biased hematopoiesis. In these processes, essential roles of myeloid transcription factors in regenerating HSCs are anticipated. C/EBPβ regulates both proliferation and differentiation of granulocytic precursors in stress conditions. However, its functions in HSCs during stresses are largely unknown.In the past several years, we have been investigating the roles of C/EBPβ in HSCs under stress conditions, especially focusing on the expression pattern of C/EBPβ isoforms and their respective functions. Cebpb is a unique single exon gene, from which two long protein isoforms (LAP* and LAP) and one truncated isoform lacking N-terminal activating domain (LIP) are translated by different usage of initiating codons. To monitor the expression pattern of these isoforms in scarce cells, we have devised an intracellular double staining method for flow cytometric analysis using two distinct anti-C/EBPβ antibodies recognizing N- and C-terminus, respectively (57th ASH annual meeting, #3580).Here, we refined this method to reveal the characteristic expression pattern of C/EBPβ isoforms in HSCs in vivo in response to stresses. In regenerative conditions after either 5-FU treatment or transplantation, we detected remarkable LIP-dominant upregulation in LT-HSCs at early phases, which was followed by increase of LAP*/LAP at later phases. This stress-induced translational upregulation and alteration of C/EBPβ were significantly suppressed by short-term administration of rapamycin, a selective inhibitor of mTOR, suggesting its dependence on mTORC1 pathway at least in part.We further found that the stress-induced LIP-dominant upregulation of C/EBPβ was remarkable in LT-HSCs with higher surface CD150 expression (CD150high LT-HSCs), which possess myeloid-biased potential. Cell cycle analyses at early phases after 5-FU treatment, when LIP was predominantly expressed, revealed that CD150high HSCs were significantly less quiescent than CD150low HSCs but this difference was abolished in Cebpb-/- HSCs, suggesting that C/EBPβ specifically activated the cell cycle of CD150high “myeloid-biased” HSCs.We next examined the functions of C/EBPβ isoforms in HSCs in vivo . We transplanted WT BM cells retrovirally transduced with each C/EBPβ isoform into sublethally irradiated WT mice, and then analyzed the transduced cells in the recipients. LAP*- or LAP-transduced cells disappeared as early as 3 weeks after transplantation, probably due to their rapid myeloid differentiation. LIP-transduced HSCs were significantly less quiescent, exhibited more accelerated differentiation to MPPs, especially lymphoid-primed MPP4s, and produced more lymphoid-biased outputs. These results indicate that LIP induces cell cycle activation of HSCs and expansion of MPP pool, while antagonizing the other long isoforms that induce rapid myeloid commitment and premature exhaustion of HSCs.As a candidate for the downstream target of C/EBPβ, especially LIP, in HSCs, we focused on Myc (encoding c-Myc). LIP-transduced EML cells, a mouse HSC line, expressed 1.5-fold higher level of Myc mRNA than those transduced with control, and LIP-transduced HSCs in vivo expressed 2-fold higher than control. We next compared Myc mRNA expression between WT and Cebpb-/- HSCs at early phases of regeneration post 5-FU treatment and transplantation, when LIP was predominantly upregulated. Then, we found that significantly lower level of Myc transcripts was expressed in Cebpb-/- HSCs. When we performed a ChIP-qPCR analysis using C/EBPβ-transduced EML cells, remarkable enrichment of C/EBPβ was observed at the distal enhancer of Myc gene. These data suggest that both forced overexpression and physiological elevation of LIP positively regulate Myc transcription in HSCs likely through direct binding to Myc enhancer.In summary, our findings illustrate that stress-induced upregulation and alteration of C/EBPβ protein, mediated by mTORC1 and modulating Myc transcription, is significantly involved in stress-mode behavior of HSCs especially with myeloid bias. DisclosuresNo relevant conflicts of interest to declare.

Podoplanin Expression in the Bleeding Complications of Acute Promyelocytic Leukemias

Background: Acute promyelocytic leukemia (APL), characterized by the t(15;17) translocation, represents a favorable-risk subgroup of AML patients when treated with ATRA-based regimens. The leading cause of early death and the major unmet medical need in this disease are the bleeding complications, which are mainly attributed to aberrant expression of Tissue Factor (TF) and Annexin A2 (ANXA2) on leukemic promyelocytes, leading to disseminated intravascular coagulation and to hyperfibrinolysis, respectively. APL patients are also at increased risk of venous and arterial thromboses. The mechanisms underlying APL-associated hemostatic complications are not fully elucidated.Aims and Methods: We analyzed the transcriptome of 30 APL specimens comprised in the Leucegene 430 AML cohort, as well as normal hematopoietic cell populations (n=63) aiming to better understand the hemostasis-related transcriptomic landscape of this subgroup.Results: We identified podoplanin (PDPN) as the most significantly and differentially overexpressed gene in APL (median 2.6 vs 0 RPKM for APL and AML, respectively, q-value = 7.3 x 10-29) and determined that PDPN gene expression correlates with protein surface expression as assessed by flow cytometry on primary APL cells (r=0.89). TF (6.6 vs 1.6, p=1.8x10-9) and ANXA2 (142.8 vs 69.1, p=1.9x10-5) are also more expressed in APL, but less specifically than PDPN. Podoplanin is a glycoprotein that physiologically binds to its receptor, CLEC2, on platelets to induce platelet aggregation causing the separation of blood and lymphatic vessels during embryogenesis (Uhrin et al Blood 2010). As anticipated, we found that PDPN is not expressed in all studied sorted cell subpopulations from normal blood or bone marrow specimens, including in promyelocytes (median of 0 RPKM for all), indicating that platelets are most likely not exposed to PDPN in the adult vasculature and that this protein is ectopically expressed on APL promyelocytes.Next, we used lentiviral gene transfer to engineer OCI-AML5 cells, which do not express PDPN, to ectopically express this protein (AML5PDPN). When mixed with platelet-rich plasma (PRP), we found that AML5PDPN cells have much greater platelet-binding and activating capacity than AML5CTRL cells. Using light transmission aggregometry, only AML5PDPN cells, not AML5CTRL cells, could induce platelet aggregation at tested concentrations. These results indicate that PDPN expression on leukemia cells is sufficient to induce platelet binding, activation and aggregation. Using patient-derived cells, we similarly found that PDPN-expressing primary APL cells (n=3 samples), but not PDPN-negative APL (n=2) or AML (n=5) cells, have the capacity to bind and activate platelets and to induce platelet aggregation. Our results suggest that PDPN could contribute to APL-related thromboses, as was recently reported in brain tumors (Riedl et al Blood 2017).We next tested the hypothesis that PDPN expression on human myeloid blasts leads to platelet consumption, thrombocytopenia and bleeding in vivo . NSG mice were transplanted with either AML5CTRL or AML5PDPN cells and were monitored until they showed clinical signs of leukemia. Platelet counts rapidly and significantly dropped in the AML5PDPN-cohort at day 25 and beyond when compared to control animals (median 343 vs 1005 x 109/L, p < 0.0001). Mice platelet counts were inversely correlated with circulating PDPN load (r=-0.88) and animals with PDPN+ leukemias had markedly prolonged bleeding time (median > 15:00 vs 7:45, p = 0.0003).Finally, we hypothesized that ATRA reduces bleeding complications in patients, at least partially, by decreasing PDPN expression. We found that PDPN surface protein on primary APL promyelocytes is markedly decreased within the first 24 hours of ATRA treatment in vitro, and levels reached less than 10% on day 4 when compared to untreated cells (DMSO). Arsenic trioxide or dexamethasone treatment led to a modest or no reduction in PDPN expression, respectively.Conclusion: PDPN expression is specific to APL and, by inducing platelet binding, activation and aggregation, it contributes to platelet consumption and to bleeding complications. Our findings may also expose a new promising angle for the treatment of the early hemostatic complications found in APL patients. DisclosuresNo relevant conflicts of interest to declare.