Early Clonal Hematopoiesis Detected in Gata2 heterozygosity By Color-Barcoding of Hematopoietic Stem Cells in Zebrafish

Abstract Germline GATA2 haploinsufficiency underlies an inherited syndrome of predisposition to myeloid malignancies. The mechanism by which GATA2 mutations lead to leukemia in children and young adults remains to be determined. We generated gata2b heterozygous mutant zebrafish using CRISPR/Cas9 technology to study this process. We used a color-barcoding system in zebrafish called Zebrabow that labels each hematopoietic stem cell (HSC) born during embryogenesis with a different color. Color-barcoding was induced by a blood-specific promoter-driven Cre recombinase during development at 24 hours post fertilization when there are on average 20 HSC clones. We then examined adult hematopoiesis in wildtype and gata2b+/- zebrafish. Surprisingly, at steady state the marrow of gata2b+/- fish showed color dominance with a single color clone contributing to over 30% of granulocytes, while the myeloid output in wildtype clutchmates was polyclonal. This suggested a baseline oligoclonal state in gata2b+/- hematopoiesis. Gata2b+/- fish also had a concomitant mild myelocytopenia with 20% reduction of myeloid cells in the marrow as early as 2 months post fertilization (mpf), reminescent of monocytopenia often present in GATA2 patients. Myelocytopenia with concurrent expansion of progenitor population was more profound in gata2b-/- fish with up to 4-fold increase of progenitor to myeloid cell ratio at 3mpf. Gata2b-/- fish are adult viable, possibly due to partial compensation by a duplicated gata2a gene. Germline GATA2-associated MDS and AML often harbor additional acquired mutations in epigenetic regulators. To study the effect of somatic mutations in GATA2 heterozygosity, we induced mosaic mutagenesis in wild-type or gata2b+/-embryos by injecting 1-cell embryos with Cas9 mRNA and guide RNAs targeting zebrafish orthologs of ASXL1 and STAG2, both of which have been associated with GATA2 mutations in MDS or AML. The marrow analysis of injected gata2b+/- fish showed an increase of hematopoietic progenitor cells and worse myelocytopenia at 7 mpf compared to control injected wildtype fish (progenitor/myeloid ratio of 1.7, p < 0.05). Single color expansion was observed in over 60% of injected gata2b+/-fish as measured by Shannon index of diversity, and correlated with stag2a and stag2b frame-shift mutations with variant allele fraction of >20%. This mutations result in the predicted loss of at least 2 of the 4 total stag2 alleles. In summary, gata2b deficiency alone results in aberrant hematopoiesis with reduced HSC diversity and myelocytopenia. Mutations in stag2 lead to early enhanced clonal dominance in gata2b+/- but not wildtype fish. Our studies suggest a model in which germline GATA2 mutations lead to a state of reduced HSC diversity during development. Such a state predisposes to clonal events due to acquired mutations in epigenetic regulators resulting in clonal hematopoiesis and myeloid leukemias, establishing a link between abnormal developmental hematopoiesis and leukemia risk later in life. Disclosures No relevant conflicts of interest to declare.

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Crispra: A Promising Tool for β-Thalassemia Treatment

Blood ◽

10.1182/blood-2020-141196 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-8

Author(s):

Xiaoqing Jia ◽

Qi Yao ◽

Hui Li ◽

Jieping Chen

Keyword(s):

Conflicts Of Interest ◽

Gene Activation ◽

Fold Increase ◽

293T Cell ◽

Genomic Locus ◽

Hematopoietic Stem ◽

Nb4 Cells ◽

Guide Rnas ◽

Promising Tool

Induction of hemoglobin γ expression is a reliable strategy to treat β-thalassemia. Gene editing using CRISPR/Cas9 technology has been widely used. However, application in vivo is limited due to the uncertainty on genomic cleavages of Cas9. In contrast, CRISPR/Cas9-based gene activation (CRISPRa) can only locate genomic locus but not interrupt sequence. Here, we use SAM system of CRISPRa to locate and activate HBG1 and HBG2, exploring the great potential of CRISPRa for β-thalassemia treatment. WWe designed 8 single-guide RNAs (sgRNAs) online and cloned into vector SAM V2, which fused dCas9 and VP64. To test the over-expression efficiency, vector containing sgRNA and MPH (fused HSF1, p65 and MS2) were transfected into 293T cell. After 72h transfection, 293T cells were collected. Q-PCR data showed that two sgRNAs were excellent on activating HBG expression with over 1000-fold increase. WTo test the activating function in hematological cell and the persistence of hemoglobin γexpression, two screened sgRNA were transfected into NB4 cells using lentivirus system. We harvested NB4 cells at different time-point (3 day, 1 week and 2 weeks), and implemented q-PCR assay. HBG expression were increased 50-hold and 1000-hold, respectively. However, the expression were reducing over time and the intrinsic mechanism is unknown. WThis study set out to increase HBG without interrupt genome using CRISPRa system. This study has found two sgRNA to activate the expression of HBG in 293T cell and NB4 cell. Further research is required to vertify the efficiency of sgRNA in hematopoietic stem cells and prolong the expression time. Figure Disclosures No relevant conflicts of interest to declare.

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Osteocytes Embedded Inside Bone Are Essential for G-CSF-Induced Hematopoietic Stem/Progenitor Mobilization

Blood ◽

10.1182/blood.v118.21.721.721 ◽

2011 ◽

Vol 118 (21) ◽

pp. 721-721 ◽

Cited By ~ 2

Author(s):

Noboru Asada ◽

Yoshio Katayama ◽

Mari Sato ◽

Kentaro Minagawa ◽

Kanako Wakahashi ◽

...

Keyword(s):

Bone Cells ◽

Conflicts Of Interest ◽

Morphological Changes ◽

Fibroblast Growth Factor 23 ◽

Bone Matrix ◽

Fold Increase ◽

Nerve Fibers ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Endosteal Niche

Abstract Abstract 721 Hematopoietic stem/progenitor cells (HSPCs) are released from the bone marrow (BM) to the circulation by granulocyte-colony stimulating factor (G-CSF) via sympathetic nervous system (SNS)-mediated osteoblast suppression (Katayama et al. Cell 2006). We further elucidated that vitamin D receptor is essential for this neuronal control of endosteal niche (Kawamori et al. Blood 2010). Osteoblasts are known to adopt three fates: die by apoptosis, become bone-lining cells, or become embedded in osteoid and then in mineralized bone matrix to terminally differentiate into osteocytes, which constitute more than 95% of bone cells. Osteocytes have been shown to control the functional balance between osteoblast and osteoclast via mechanotransduction. In order to address the role of bone-embedded osteocytes in HSPCs niche function, we first quantified mRNA expression of bone-related genes in the femur of wild-type (WT) mice to examine if osteocytic function changes during G-CSF treatment (125μg/kg/dose, 8 divided doses, every 12 hours). Whereas markers relating to osteoblast function, osteocalcin and osteopontin, started to decrease late at 6 doses of G-CSF administration when mild mobilization of HSPCs had occurred, osteocyte-specific genes, including neuropeptide y, SOST, MEPE, E11/gp38 and Phex, were rapidly suppressed at 1 dose when no mobilization was observed. These data suggest that osteocytes respond to G-CSF with altered gene expression much earlier than osteoblasts. Further, the number and thickness of osteocyte projections extending toward the endosteal surface were markedly reduced, as assessed by fluorescently labeled phalloidin, at 8 doses of G-CSF treatment when full mobilization was achieved; these morphological changes were observed specifically in newly-embedded osteoid osteocytes, but not in mature osteocytes embedded deep inside mineralized bone. These findings suggest that osteoid osteocytes may sense the signal triggered by G-CSF. We confirmed the presence of β2-adrenergic receptor in osteoid osteocytes and tyrosine hydroxylase-positive nerve fibers in the vicinity by immunofluorecence staining, suggesting that osteoid osteocytes may be regulated by SNS. To directly address osteocyte involvement in G-CSF-induced mobilization, we utilized a transgenic (TG) mice in which inducible and specific ablation of osteocytes is achieved through targeted expression of diphtheria toxin (DT) receptor under DMP-1 promoter. A single injection of DT in TG mice generates “osteocyte-less (OL)” mice. We found that mobilization by G-CSF was drastically impaired in OL mice for progenitors (CFU-Cs, mean±SEM, WT vs Tg: 1673±271 vs 242±94/ml blood, n=6-13, p<0.01; lineage-Sca-1+c-kit+ (LSK) cells, WT vs Tg: 6878±1209/ml vs 1763±502/ml, n=6-13, p<0.01) and stem cells (repopulating units at 4 months, WT vs Tg: 2.5±0.7 vs 0.5±0.2, n=6-7, p<0.05), while the OL BM showed normal HSPC number. The levels of CXCL12 mRNA and protein in BM and bone were markedly decreased during G-CSF treatment even in OL mice despite the mobilization defect, and a CXCR4 antagonist AMD3100 induced mobilization normally in the absence of osteocytes. Thus, osteocytes embedded within the bone are indispensable for G-CSF-induced mobilization through a CXCL12-independent mechanism. Although most of bone-related genes exhibited drastic decreases following G-CSF treatment, we found that fibroblast growth factor 23 (fgf23) mRNA displayed a 4-fold increase at 6 doses of G-CSF. FGF23 is mainly produced by osteocytes and Klotho is an obligate coreceptor for FGF23 to bind and activate FGF receptors. Since we confirmed that klotho hypomorphic (kl/kl) mice showed remarkably disrupted osteocyte network, we injected G-CSF into these mice. As we expected, G-CSF induced virtually no mobilization in kl/kl mice while the number of HSPCs in the BM remained comparable to control mice. Collectively, our results demonstrate a novel function of bone-embedded osteocytes as a critical regulator of HSPC trafficking perhaps by controlling the endosteal niche and establish the important physiologic function of skeletal tissue for hematopoietic microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Correlation of Morphological Dysplasia and Immunophenotypic Features in Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v128.22.5548.5548 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5548-5548

Author(s):

Elvira D Rodrigues Pereira Velloso ◽

Rosana M Cosentino ◽

Liliana Mitie Suganuma ◽

Rodrigo de Souza Barroso ◽

Nydia Strachman Bacal ◽

...

Keyword(s):

Clinical Data ◽

Myelodysplastic Syndromes ◽

Conflicts Of Interest ◽

Intraclass Correlation ◽

Myeloid Cell ◽

High Sensitivity ◽

World Health ◽

Hematopoietic Stem ◽

Morphological Criteria ◽

Increased Risk

Abstract The Myelodysplastic Syndromes (MDS) are a group of clonal hematopoietic stem cell diseases characterized by cytopenias, dysplasia in one or more myeloid cell lines, ineffective hematopoiesis and increased risk for development of acute myeloid leukemia (AML).The pathological hallmark of myelodysplastic syndromes (MDS) is marrow dysplasia, which represents the basis of the World Health Organization (WHO) classification of these disorders. However, morphological diagnosis of MDS may have difficulties, with poor inter-observer agreement.Recently, Della Porta and col.(on behalf of Rete Ematologica Lombarda (REL) Network), described a new morphological score system to define dysplasia, that showed high sensitivity and specificity (>90%) andbetter inter-operator agreement (Leukemia 2015;29:66-75). In addition, new toolsfor diagnostic and prognostic purposes were developed.Flow cytometry (FCM) was addedin standard European Leukemia Net guidelinesasrecommended tool for diagnosis and prognosis in MDS, and a simple MDS-FCM scoring system based on four parameters (mainly CD34 and granulocytic cells) by Ogata and col. showed a good specificity (Haematologica 2009;94:1066-1074). The aim of this study is to correlate morphological and immunophenotypic features in suspicion of MDS. The study comprised two steps: the first used for morphological validation, included 33 bone marrow (BM) smears from patients with suspected MDS with or without therapy, reviewed by four experts hematologists blinded to clinical data. Dysplasia was evaluated using WHO 2008 criteria and Della Porta's score: 100 erythroblasts, 100 granulocytic cells and 30 megakaryocytes for dysplasia and 500 nucleated cells for blast cells percentage. The second step included 69 BM samples from patients with suspected MDS (27 with final diagnosis of MDS) and was used to correlate cytology and immunophenotypic findings. The morphological aspects were reviewed by one of the expert hematologists, using the same previous criteria and blinded to clinical data. Inter-operator reproducibility of morphological analyses was assessed by Intraclass Correlation Coefficient (ICC). Acceptable reproducibility was defined as ICC > 0.4. The correlation of minimal morphological criteria with immunophenotypic criteria of Ogata and col. was assessed by Discordance rate and Spearman correlation. The results are shown in table. In conclusion our study shows good inter observer reproducitibility, in both used criteria. The use of new criteria of Della Porta to the erythroid series was slightly better, suggesting that can be useful in diagnosing myelodisplasias. Immunophenotypic data had a good agreement with morphologic data in confirmed cases of MDS. Table 1 Table 1. Disclosures No relevant conflicts of interest to declare.

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The Impact of Aging of Hematopoietic Stem and Progenitor Cells (HSPCs) in Non-Human Primates As Interrogated By Genetic Barcode Clonal Tracking

Blood ◽

10.1182/blood.v126.23.1151.1151 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1151-1151

Author(s):

Kyung-Rok Yu ◽

Chuanfeng Wu ◽

Diego Espinoza ◽

Idalia Yabe ◽

Sandhya R. Panch ◽

...

Keyword(s):

Conflicts Of Interest ◽

Clustering Algorithms ◽

Autologous Transplantation ◽

Shannon Index ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Poor Prognostic Factor ◽

Age Related ◽

Close Phylogenetic Relationship ◽

The Impact

Abstract Aging of the hematopoietic system is associated with a number of observations, including diminished regenerative potential, skewed lineage differentiation, increased incidence of anemia, and higher rates of neoplastic transformation. Despite advanced age being a strong poor prognostic factor, an increasing number of older patients are receiving hematopoietic stem and progenitor cell (HSPC) transplantation. Most previous investigations of the effects of aging on hematopoiesis have been obtained in murine models. The rhesus macaque is a powerful model to study human hematopoiesis and aging, based on a close phylogenetic relationship to humans, and similar telomere lengths, lifespans and aging phenotypes. To quantitatively elucidate the age-related changes that compromise hematopoietic function at a clonal level, we applied a genetic barcoding approach to quantitatively track the clonal behavior of HSPCs in young versus old macaques following autologous transplantation. We delivered high diversity barcodes via lentiviral transduction of CD34+ HSPC (detailed in our prior study: Wu et al Cell Stem Cell, 2014), allowing quantitative tracking of the output of thousands of individual HSPC clones labeled by unique barcodes, over time and in various lineages post-transplantation. We successfully transplanted barcoded HSPCs into 2 macaques aged 19 and 25 years, constituting "old" macaques based on an average lifespan in captivity of 20-30 years, and compared results to clonal patterns observed in 5 "young" macaques aged 3-5 years. Both old macaques engrafted promptly, and CD3+ T cells, CD3-CD20+ B cells, CD33+ Granulocytes (Gr), CD3-CD20-CD14+ Monocytes (Mo), and CD3-CD20-CD14-CD16+/or CD56+ NK cells were purified from the peripheral blood monthly following transplantation. In terms of overall polyclonality and diversity (Shannon index), analyzed through 4 months to date, there were no marked differences between the old and young recipients, with thousands of individual clones contributing to hematopoiesis in both sets of animals during the initial post-transplant time period studied. However, there were marked differences in the patterns of clonal lineage relationships between young and old animals, as assessed via pairwise Pearson correlations of all contributing clones as well as clustering algorithms allowing interrogation of patterns of clonal behavior. In both young and old, there was no correlation (i.e. no shared clones and thus no shared progenitors) between lineages at 1m, and clones contributing at 1m did not contribute to any lineage at 3m or later, indicating the existence of short-term, lineage-restricted progenitors in both age groups. By 3m in young animals, B and Gr/Mo became correlated, and by 3-6m, B/T/Mo/Gr multilineage clones appeared and constituted the majority of hematopoietic output. However, in old animals clones contributing to Gr/Mo versus B or T lineages remained almost completely distinct or markedly biased, up to 4m studied to date, without evidence for multi-lineage clones (Fig 1). In both young and old animals, the NK lineage remained clonally distinct, as previously reported for young animals. In summary, we transplanted 2 aged macaques with barcoded CD34+ HSPCs, and discovered a pattern of clonal reconstitution distinct from that in young animals, with persistent unilineage or highly-biased myeloid and B lymphoid progenitors in the aged animals. Longer follow-up will be required to determine if this biased pattern persists, and will be presented. This approach should improve our understanding of disorders of hematopoiesis in the elderly, and help improve transplantation and other therapies in this vulnerable patient population. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Calreticulin mutations Are Present in Polyclonal As Well As Clonal ET

Blood ◽

10.1182/blood.v124.21.4590.4590 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4590-4590

Author(s):

Xylina Gregg ◽

Sabina Swierczek ◽

Soo Jin Kim ◽

Josef T. Prchal

Keyword(s):

T Cells ◽

X Chromosome ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis ◽

Calr Mutations ◽

Calr Mutation

Abstract First and second authors contributed equally During female embryogenesis, most of the genes in either the maternal or paternal X-chromosome are randomly inactivated in each cell, a process that remains remarkably constant in their progeny. X-chromosome inactivation has been used to define clonality in myeloproliferative neoplasms (MPNs) such polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET). One such method to determine clonality uses a quantitative, transcriptional clonality assay based on conservative exonic polymorphisms in five X-chromosome genes (MPP1, FHL1, IDS, BTK, and G6PD). Females who are heterozygous for any of these polymorphisms are considered “informative” and can be studied for clonality by interrogating their platelets’ and granulocytes’ RNA allelic usage ratio. JAK2 mutations occur in >95% of PV and 50-60% of ET and PMF; cMPL mutations are found in another 5-10% of ET and MF. Somatic calreticulin (CALR) mutations have been identified in a majority of patients with ET and MF who lack JAK2 and cMPL mutations. CALR mutations are reported to be associated with a more favorable prognosis and are believed to be acquired early in the disease course. More than 30 CALR mutations have been described, but type 1 (52-bp deletion; c.1092_1143del) and type 2 (5-bp insertion; c.1154_1155insTTGTC) mutations are the most frequent. We analyzed 61 females informative for a transcriptional clonality assay and 44 males with unexplained thrombocytosis or marrow fibrosis and no detectable JAK2 or cMPL mutations for CALR mutations in their granulocytes. With the exception of an absence of a clonal marker, these patients met WHO criteria for ET or PMF. A CALR mutation (20 type 1 and 17 type 2) was present in 37 of these 105 patients (22 females and 15 males). One of the CALR mutated females had a paternal grandmother with JAK2V617F –positive PV, confirming a previous report that, in familial clustering of MPNs, affected individuals may carry different disease-defining somatic mutations. In those CALR positive patients who had available T cells, no detectable CALR mutations were found in their T cells. In one of these subjects, CD34+ cells were available and had a similar mutation level as in the granulocytes. Of the 22 females with a CALR mutation, 19 had clonal hematopoiesis, but 3 had polyclonal hematopoiesis; all 3 had previously unexplained thrombocytosis. None of these patients had any prior treatment for thrombocytosis. Clonal hematopoiesis was present in 26 of the 39 females without a CALR mutation. All female patients with myelofibrosis had clonal hematopoiesis, regardless of CALR mutation status. In contrast to the polyclonal hematopoiesis seen in some CALR positive ET patients, 166 informative PV and JAK2V617F-positive ET or PMF females all had clonal hematopoiesis. We report that CALR mutations are associated with polyclonal hematopoiesis in some ET patients. This finding differs from JAK2V617F-positive ET and PMF and PV females, where clonal hematopoiesis was always seen. This indicates that CALR mutated clones have a weaker suppressive effect on residual normal hematopoietic stem cells than JAK2 mutated clones and may contribute to the possibly more benign course of CALR mutated ET. The CALR mutation was not detected in T cells, which also differs from JAK2V617F mutated MPNs, where a small level of the JAK2 mutation is often detected in T cells. Similar to other reports, we found a lower prevalence of the CALR mutation in JAK2 or cMPL non-mutated ET and PMF than initially described. Disclosures No relevant conflicts of interest to declare.

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Growth Factor Independence 1 b (Gfi1b) as a New Regulator of Hematopoietic Stem Cell Fate

Blood ◽

10.1182/blood.v116.21.837.837 ◽

2010 ◽

Vol 116 (21) ◽

pp. 837-837

Author(s):

Cyrus Khandanpour ◽

Lothar Vassen ◽

Marie-Claude Gaudreau ◽

Christian Kosan ◽

Tarik Moroy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Fate ◽

Conflicts Of Interest ◽

Fold Increase ◽

Surface Proteins ◽

Expression Array ◽

Hematopoietic Stem

Abstract Abstract 837 Donor matched transplantation of bone marrow or hematopoietic stem cells (HSCs) are widely used to treat hematological malignancies, but are associated with high mortality. Methods for expansion of HSC numbers and their mobilization into the bloodstream of a donor could significantly improve therapy. We show here that the zinc finger transcriptional repressor Gfi1b is highly expressed in hematopoietic stem cells (defined as CD 150+, CD 48-, Lin-, Sca1+ and c-kit+) cells and is down-regulated more than 10 fold upon differentiation into multipotential progenitors (defined as CD 150+ or CD150-, CD 48+, Lin-, Sca1+ and c-kit+). Constitutive germline deletion of Gfi1b is lethal at midgestation due to impaired development of erythrocytes and megakaryocytes. We have therefore developed a conditional knock-out of Gfi1b to study its role specifically in the adult hematopoietic system. Deletion of Gfi1b leads to a 30-fold increase of HSC numbers in bone marrow and around a100 fold increase in spleen and peripheral blood. This was due to a higher rate of HSCs undergoing cell cycling. Concomitantly, the number of quiescent HSCs was reduced 5–6 times. We then performed an gene expression array of wt and Gfi1b deficient HSCs and observed that loss of Gfi1b leads to an altered RNA expression of integrins and adhesion molecules, for instance CXCR4, VCAM-1 and Tenascin C, which usually retain HSCs in a dormant state in the endosteal niche. These changes were also confirmed on protein level. Finally, we could observe a higher levels of Reactive Oxygen Species (ROS) in the Gfi1b deficient HSCs compared to wt HSCs. We verified whether elevated level of ROS are causative for the expansion of HSCs and noticed that application of N-Acetyl-Cystein, which counteracts the effects of ROS, limits significantly the expansion of HSCs, underscoring the important role of ROS in the expansion of Gfi1b deficient HSCs. Despite markedly increased proliferation, Gfi1b-/- HSCs can reconstitute lymphoid and myeloid lineages to the same extent as wt HSCs when transplanted in competition with wt HSCs. Furthermore, Gfi1b deficient HSCs also feature an expansion after transplantation and expand 5–10 fold more than wt HSC when transplanted initially in equal numbers with wt HSCs. It is possible that lower expression of CXCR4, VCAM-1 and other surface proteins leads to release and egression of Gfi1b deficient HSCs from the hypoxic endosteal stem cell niche and exposes the HSCs to more oxygen which in turn increases ROS levels. Elevated ROS could promote entry of Gfi1b-/- HSCs into cell cycle. In conclusion Gfi1b regulates HSC dormancy, pool size and potentially also the egress and mobilization of HSCs and might offer a new therapeutic approach to improve human HSC transplantation. Disclosures: No relevant conflicts of interest to declare.

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Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽

10.1182/blood.v118.21.1919.1919 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1919-1919

Author(s):

Iman Hatem Fares ◽

Jalila Chagraoui ◽

Jana Krosl ◽

Denis-Claude Roy ◽

Sandra Cohen ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Fold Increase ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

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Regulatory T Cells of G-CSF Mobilized Stem Cell Donors Qualify for Clinical Applications,

Blood ◽

10.1182/blood.v118.21.4055.4055 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4055-4055

Author(s):

Sya N. Ukena ◽

Sarvari Velaga ◽

Goudeva Lilia ◽

Philipp Ivanyi ◽

Arnold Ganser ◽

...

Keyword(s):

Clinical Trials ◽

T Cells ◽

Stem Cell ◽

Regulatory T Cells ◽

Conflicts Of Interest ◽

Interleukin 2 ◽

Fold Increase ◽

Hematopoietic Stem ◽

Cell Numbers

Abstract Abstract 4055 Recent clinical studies demonstrate the high potency of human regulatory T cells (Tregs) to control graft-versus-host disease following hematopoietic stem cell transplantation (SCT). Isolation of Tregs after recombinant G-CSF induced mobilization of stem cells would simplify the design of clinical trials in allogeneic SCT. However, there is growing evidence that G-CSF also exerts profound immune modulatory effects in the adaptive immune system. Therefore, we analyzed Tregs isolated by FACS based cell sorting from stem cell donors before (n=8) and after (n=13) G-CSF administration regarding their phenotype, stability, functional and expansion properties. Absolute CD4+ T cell (3.2 fold increase of mean after G-CSF; p<0.05) and CD4+CD25hi Treg cell numbers (4.1 fold increase of mean after G-CSF; p<0.01) were significantly increased after G-CSF administration. Analysis of the Foxp3 TSDR demethylation level displays a stable Foxp3 phenotype of G-CSF encountered Tregs (mean value 97.1% vs. 95.0 % after G-CSF administration). Moreover, the CD4+CD25hi Tregs of G-CSF treated SC donors suppress the proliferation of effector T cells with no significant differences to Tregs isolated from healthy donors before G-CSF treatment (mean values 42.1% vs. 49.9% after G-CSF administration at a Treg/ T effector cell ratio of 1:1). In vitro expansion of Tregs isolated after G-CSF application with anti-CD3 and anti-CD28 dynabeads in the presence of interleukin-2 led to comparable cell numbers as for the stimulation of control Treg cells. However, differences could be detected for the thymic derived marker molecule CD31 and those associated with activation (LAP, CD69, CD62L) and migration (CxCR3) as detected by FACS analysis. Our results show no significant differences regarding suppressive function and stability of Tregs isolated from stem cell donors before and after G-CSF administration. These data support the application of G-CSF mobilized Tregs for clinical trials which in turn opens up new possibilities for the adoptive transfer of Tregs in HSCT. Disclosures: No relevant conflicts of interest to declare.

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Circulatory Glycan-Modifying Enzymes As Systemic Regulators In Blood Cell Development

Blood ◽

10.1182/blood.v122.21.3686.3686 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3686-3686

Author(s):

Joseph Lau

Keyword(s):

Cell Surface ◽

Blood Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Genetically Modified ◽

Fold Increase ◽

Sialic Acids ◽

Cell Surfaces ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem

Abstract Regulating blood cell production is crucial to meeting physiologic demands and to maintaining hematopoietic equilibrium. Elucidating the systemic factors and how they contribute to the interaction of hematopoietic progenitors with their bone marrow microenvironments is crucial to the mechanistic understanding of hematopoietic regulation and development of novel therapeutic strategies. Glycosyltransferases are enzymes that normally reside within the intracellular secretory apparatus to assemble glycans on nascent proteins and lipids in transit, but they are also present in abundance in the extracellular spaces, especially in systemic circulation. One such blood-borne enzyme is the ST6Gal-1 sialyltransferase that mediates the attachment of sialic acids in a6-linkages to glycoproteins. By virtue of their presence on cell surfaces, sialyl-glycans, which are conserved structures in mammals, occupy the critical interphase between cells and their external environment. We hypothesize that remodeling of these cell surface glycans can drastically modulate the communication of systemic cues among marrow hematopoietic cells. A mouse model genetically modified for low circulating ST6Gal-1 has increased numbers of hematopoietic progenitors, increased ability to produce inflammatory cells upon acute challenge, and faster recovery from cyclophosphamide-induced myelosuppression. In ex vivo cultures, addition of ST6Gal-1 strikingly delays expansion and differentiation of murine LSK (Linneg sca-1pos cKitpos) and human CD34pos cells. We developed a subcutaneously implantable B16-F10 model genetically modified to overexpress circulatory ST6Gal-1 (B16st6gal1). Mice bearing B16st6gal1 had >10-fold elevation in circulating ST6Gal-1 levels, accompanied by >50% reduction in marrow granulocyte and B cell populations. B16st6gal1 bearing mice also had 2-fold increase in the number of Linneg cells, suggesting a blockade in development of progenitors into end-effector cells. Murine LSK cell surfaces are decorated with a6-sialic acids but they do not endogenously express ST6Gal-1. Bone chimeras show that the a6-sialic acids on LSK surfaces require the distally synthesized ST6Gal-1 enzyme in circulation. Taken together, our data indicates the glycan-modifying enzyme, the ST6Gal-1 in circulation, is a potent systemic regulator of hematopoiesis by remodeling of cell surface glycan architecture of marrow hematopoietic stem and progenitors. (Funded by NIH Program of Excellence in Glycosciences award P01HL107146 and NIH R01AI56082) Disclosures: No relevant conflicts of interest to declare.

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Ezh2 and Runx1 Mutations Targeted to Early Lymphoid Progenitors Collaborate to Promote Early Thymic Progenitor Leukemia

Blood ◽

10.1182/blood.v126.23.846.846 ◽

2015 ◽

Vol 126 (23) ◽

pp. 846-846

Author(s):

Christopher Booth ◽

Nikolas Barkas ◽

Wen Hao Neo ◽

Elizabeth Soilleux ◽

Hanane Boukarabila ◽

...

Keyword(s):

Gene Expression ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

High Sensitivity ◽

Response To Therapy ◽

Ras Signaling ◽

Specific Cell ◽

Cell Of Origin ◽

Hematopoietic Stem ◽

Lymphoid Progenitors

Abstract Understanding the specific cell populations responsible for propagation of leukemia is an important step for development of effective targeted therapies. Recently, the lymphoid-primed multipotent progenitor (LMPP) has been proposed to be a key propagating population in acute myeloid leukemia (AML; PMID 21251617). We have also shown that LMPPs share many functional and gene expression properties with early thymic progenitors (ETPs; PMID 22344248). This finding is of particular interest as ETP leukemias have recently been described: a distinct and poor prognostic disease entity with a transcriptional profile reminiscent of murine ETPs, showing co-expression of hematopoietic stem cell (HSC) and myeloid markers (PMID 19147408). Together, this raises the question whether ETPs can act as a leukemia-initiating/propagating cell population; however, relevant disease models to test this hypothesis are currently lacking. Analysis of the genetic landscape of ETP leukemias has revealed frequent coexistence of inactivating mutations of EZH2 and RUNX1 (PMID 22237106). We therefore generated mice with deletions of Ezh2 and Runx1 specifically targeted to early lymphoid progenitors using Rag1Cre (Ezh2fl/flRunx1fl/flRag1Cre+; DKO mice). As anticipated, HSCs lacked significant recombination in DKO mice whereas close to 100% of purified ETPs (Lin- CD4- CD8- CD44+ CD25- Kit+ Flt3+) showed deletion of Ezh2 and Runx1. Strikingly, despite a 16-fold reduction in thymus cellularity caused by a block in thymocyte maturation at the DN2-DN3 transition, absolute numbers of ETPs within the thymus of DKO mice were markedly expanded (12-fold; p<0.0001). In contrast, Ezh2 or Runx1 deletion alone had no impact on numbers of ETPs. RNA-sequencing of the expanded ETPs in DKO mice revealed upregulation of HSC- and myeloid-associated transcriptional programs, reminiscent of ETP leukaemia e.g. Pbx1 (log2FC=3.0; p<0.0001) and Csf3r (log2FC=1.9; p=0.0038). Single-cell gene expression analysis confirmed co-expression of HSC and myeloid programs with lymphoid genes within individual DKO ETPs. Further, some key regulators of T-cell maturation which are aberrantly expressed in ETP leukemia were also disrupted in DKO ETPs e.g. Tcf7 (log2FC=-9.5; p<0.0001). Gene expression associated with aberrant Ras signalling was also present. However, despite a continued expansion of the ETP population with age, we did not observe leukemia in DKO mice with over 1 year of follow-up. Since ETP leukemias frequently feature activating mutations in genes regulating RAS signaling, we hypothesised that the expanded "pre-leukemic" ETPs in DKO mice would be primed for leukemic transformation by signalling pathway mutation. We therefore crossed DKO mice with a Flt3ITD/+ knock-in mouse line, as internal tandem duplications (ITD) of FLT3 are frequent in ETP leukemias. Ezh2fl/flRunx1fl/flRag1Cre+Flt3ITD/+ (DKOITD) mice showed dramatically reduced survival (median 9.3 weeks) resulting from an aggressive, fully penetrant acute leukemia showing a predominantly myeloid phenotype (e.g. Mac1) but with co-expression of some lymphoid antigens (e.g. intracellular CD3). Crucially, this leukaemia could be propagated in wild-type recipients upon transplantation of the expanded ETPs. DKOITD ETPs were transcriptionally very similar to DKO ETPs, retaining expression of lymphoid alongside HSC- and myeloid-associated genes. Finally, in a lympho-myeloid cell line model (EML cells) we demonstrated that Ezh2 inactivation-induced loss of H3K27me3 is associated with a corresponding increase in H3K27Ac, a transcriptional activating signal that recruits bromodomain proteins. As such, we reasoned that our ETP leukemia model might be sensitive to bromodomain inhibitors such as JQ1. Indeed, we observed high sensitivity of expanded DKOITD ETPs to JQ1, raising the possibility of a new therapeutic approach for ETP leukemias. This novel mouse model of ETP-propagated leukemia, driven by clinically relevant mutations, provides intriguing evidence that leukemias with a predominant myeloid phenotype, but co-expressing lymphoid genes, may initiate within a bona fide early lymphoid progenitor population. Since the functional characteristics of the cell of origin of a leukaemia may direct its progression and response to therapy, these findings could have important implications for future stratification and treatment of both AML and ETP leukemias. Disclosures No relevant conflicts of interest to declare.

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