Leukemia Cells Remodel Adipocyte Niches and Their Progenitor Functions to Generate Leukemia Favoring Niche

Abstract Increasing evidence suggests that the cancer cells take shelter in different osteoblastic and adipocytic niches, where they hide from chemotherapy and continue to survive. As yet, how leukemia cells alter the bone marrow (BM) adipocytic niches to facilitate their expansion and assist them in evading chemotherapy is unclear. We have previously shown that the acute myeloid leukemia (AML) cells directly or through their exosomes, reprogram BM osteoblastic niche which facilitates their expansion and suppress normal hematopoiesis(Kumar B et al, Leukemia 2018,32(3):575-587). In this study , we provide further evidences that AML and Acute lymphoid leukemia (ALL) transformed the BM adipocytic niche to facilitate their expansion and suppress normal hematopoiesis. Using MLL-AF9 (AML) knock-in mouse, MLL-AF9 or BCR-ABL(p190, ALL) HSC transduction transplantation leukemia mice models, we performed flow cytometry analysis to show that the leukemia cells stimulate expansion of BM derived CD45-Ter119-CD31-CD166-Sca1+CD140a+(PaS)MSCs population compared to normal mice (p=0.04, p=0.001 and p=0.002 respectively).Further, the BM osteoblasts specific Osteocalcin mRNA expression in sorted stroma cells and flow cytometry based osteoblasts population (CD45-Ter119-CD31- CD166+Sca1-) numbers were also significantly reduced in AML (p=0.04) and ALL (p=0.02) mice models suggesting bone loss with the leukemia development. Similar to osteoblasts loss, mature adipocytes (Perilipin, PPARg mRNA) were also significantly reduced in the ALL/AML mice compared to control. The triglyceride content and white adipose tissue(WAT) mass was diminished in leukemic mice , suggesting leukemia may have utilized adipocyte for survival. Adipocyte loss in the leukemia mice was accompanied by long term hematopoietic stem cells(LT-HSC ) and erythroid megakaryocyte progenitor (MEP) populations reduction in the leukemic mice (p=0.01 and p=0.02 respectively). To dissect the mechanism of adipocytes reduction is either due to adipocyte loss or adipocytes maturation defect in leukemic mice, we analyzed different stromal progenitors in normal and leukemic mice and identified that the leukemia cells stimulate the growth of BM derived adipocytic committed progenitors (CD45-Ter119-CD31-CD166-Sca1+CD140a+CD29+CD24-) and blocked the chondrocyte/osteoblastic/adipocytic multipotent progenitors (CD45-Ter119-CD31- CD166-Sca1+CD140a+CD29+CD24+) population (p=0.02,p=0.01 respectively).Despite the increase in number of MSCs and adipocytic progenitors, the in-vitro adipocytic differentiation potential of sorted adipocyte committed progenitors was severely compromised in ALL and AML compared to control. The WAT western blot analysis showed significantly increased expression of ATGL and pHSL(Ser-563) expression involved in triglyceride lipolysis in the leukemic mice .The ALL leukemic adipocytic stroma had increased expression of IL-1β and IL-6 cytokine levels compared to normal stroma and provided more survival advantage to leukemia cells in in-vitro co-culture experiments in nutrient deprived conditions and during chemo-radiotherapy treatment. Further, the ATGL and HSL pharmacological inhibitors rescued leukemia induced lipolysis, reduced leukemia proliferation and increased chemotherapy induced apoptosis in leukemia cells. Overall, this data strongly suggests the notion of progressive decline in functional LT-HSCs & normal hematopoiesis, adipocytes and osteoblasts numbers with leukemia progression due to activation of lipolytic enzymes resulting in increased availability of fatty acids for leukemia expansion and is a common feature in both lymphoid and myeloid leukemias. Disclosures No relevant conflicts of interest to declare.

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Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽

10.1182/blood-2019-126283 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3704-3704

Author(s):

Aldona A Karaczyn ◽

Edward Jachimowicz ◽

Jaspreet S Kohli ◽

Pradeep Sathyanarayana

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Quiescent State ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

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Identification of the Human Unipotent Megakaryocyte Progenitor, and Its Pathophysiological Roles in Human Thrombopoietic Disorders

Blood ◽

10.1182/blood.v126.23.2362.2362 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2362-2362

Author(s):

Kohta Miyawaki ◽

Hiromi Iwasaki ◽

Koichi Akashi

Keyword(s):

Culture Condition ◽

Conflicts Of Interest ◽

Morphological Characteristics ◽

Gene Set Enrichment Analysis ◽

Jak2 Mutation ◽

Erythroid Progenitor ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Lineage Specific Genes

Abstract In human hematopoiesis, the megakaryocyte (Meg) lineage is known to diverge from bipotent megakaryocytic/erythroid progenitor (MEP), which resides downstream of common myeloid progenitor (CMP). However, the definition of unipotent Meg progenitor (MegP) is still controversial. To shed light on the heterogeneity of hematopoietic stem/progenitor cells (HSPCs), we analyzed the expression level of 96 lineage-specific genes in a single cell. While most of MEPs exclusively expressed erythroid- but not Meg-specific genes, a fraction of CMP cells (6.25%) showed distinct and exclusive expression pattern of platelet or Meg-specific genes. Among them were the cell surface molecules including CD41, the contents of alpha-granules of platelets and Meg-lineage specific transcription factors. Based on these data, we hypothesized that MegP might exist within the CD41+ CMP fraction. In fact, a fraction (7.9 ± 3.2 %) of CMPs expressed significant level of CD41 in FACS analysis, but none of the other CD34+ cells including hematopoietic stem cells (HSCs), MEPs and granulocyte-macrophage progenitors (GMPs) expressed CD41. Purified CD41+CMPs exhibited myeloblast-like immature morphological characteristics. Interestingly, cells with 4N-nuclei were exclusively found in CD41+CMPs (1%), indicating the initiation of endomitosis and polyploidization process has already taken place at this early stage of hematopoiesis. A conventional in vitro culture condition revealed that CD41+CMPs completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials, whereas CD41-CMPs generated all types of myeloid colonies. To analyze Meg-lineage potential, we cultured them at a serum-free culture condition that is highly-optimized for Meg differentiation. The majority of single CD41+CMPs gave rise to pure megakaryocyte colonies. In addition, the novel immunodeficient mouse strain, which was recently developed by us and could support human Meg-lineage hematopoiesis much more than any strains available today, enabled us to evaluate in vivo differentiation potential of lineage-restricted progenitors. As a result, CD41+CMPs showed unipotent and robust Meg-lineage differentiation potential compared to MEPs. In order to elucidate gene expression profiling of CD41+CMPs more precisely, we performed cDNA microarray experiment. GSEA (Gene Set Enrichment Analysis) revealed that CD41+CMPs up-regulated Meg lineage-specific genes related to megakaryocyte development, platelet production, platelet activation and aggregation, and down-regulated erythroid and GM lineage-affiliated genes, clearly reflecting their lineage potentials. We also evaluated lineal relationship of these populations in vitro. While MEP did not give rise to CD41+CMPs, CD41-CMPs successfully generated MegPs, suggesting that CD41+CMPs could be the progeny of CD41-CMPs. Therefore, we defined the CD41+CMP as a human MegP. The Meg colony-producing potential of MegPs was almost thirty times as strong as that of MEPs on per-cell-basis, indicating that this population is the major source of Megs rather than MEP. Finally, to evaluate the pathophysiological significance of MegPs in human diseases, we analyzed population frequencies of each stem/progenitor population in essential thrombocythemia (ET) patients' bone marrow. The MegP pool dramatically expanded in ET BM as compared to that in normal BM (24.2 ± 8.6 % vs. 7.9 ± 3.2 % of CMP). We also evaluated JAK2 mutation (V617F) burden in each stem/progenitor population by utilizing the digital PCR method with allele specific probes and the amplicon sequencing-based detection. As a result, JAK2 mutation is highly concentrated to MegP population in accordance with expansion of MegP, suggesting that this population should play an important role in pathogenesis of ET. In summary, we have prospectively identified the unipotent MegP population, which was endowed with robust Meg potential and emerged directly from CMPs bypassing MEPs. The newly-identified MegPs robustly contribute to physiological and pathological human megakaryopoiesis including ET. Disclosures No relevant conflicts of interest to declare.

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Trajectory of chemical cocktail-induced neutrophil reprogramming

Journal of Hematology & Oncology ◽

10.1186/s13045-020-01008-8 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Yi Zhou ◽

Chuijin Wei ◽

Shumin Xiong ◽

Liaoliao Dong ◽

Zhu Chen ◽

...

Keyword(s):

Cell Types ◽

Lymphoid Cells ◽

Great Promise ◽

Target Cells ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

First Time

AbstractHematopoietic reprogramming holds great promise for generating functional target cells and provides new angle for understanding hematopoiesis. We reported before for the first time that diverse differentiated hematopoietic cell lineages could be reprogrammed back into hematopoietic stem/progenitor cell-like cells by chemical cocktail. However, the exact cell types of induced cells and reprogramming trajectory remain elusive. Here, based on genetic tracing method CellTagging and single-cell RNA sequencing, it is found that neutrophils could be reprogrammed into multipotent progenitors, which acquire multi-differentiation potential both in vitro and in vivo, including into lymphoid cells. Construction of trajectory map of the reprogramming procession shows that mature neutrophils follow their canonical developmental route reversely into immature ones, premature ones, granulocyte/monocyte progenitors, common myeloid progenitors, and then the terminal cells, which is stage by stage or skips intermediate stages. Collectively, this study provides a precise dissection of hematopoietic reprogramming procession and sheds light on chemical cocktail-induction of hematopoietic stem cells.

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Treatment of Post- HSCT Immunodeficiency By Infusion of Ex Vivo- Generated T Cell Precursors from Adult and Cord Blood Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v126.23.1887.1887 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1887-1887

Author(s):

Laura Simons ◽

Corinne De La Chappedelaine ◽

Christian Reimann ◽

Elodie Elkaim ◽

Sandrine Susini ◽

...

Keyword(s):

Cell Differentiation ◽

T Cell ◽

Cord Blood ◽

Conflicts Of Interest ◽

T Cell Differentiation ◽

Cell Compartment ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

T Cell Progenitors

Abstract Non-HLA identical hematopoietic stem cell transplantation (HSCT) provides a corrective therapy for most life-threatening primary immunodeficiencies (PID) and some malignant hemopathies. Despite advances made, severe complications following the treatment such as the prolonged persistence of T cell immunodeficiency still limit the use of this partially incompatible HSCT. After HSCT, the reconstitution of a functional T cell compartment relies on the availability of T cell precursors to rapidly seed the thymus and differentiate into mature T cells. We have previously demonstrated that an in vitro culture system based on the use of a modified Delta-like-4 (DLL4) Notch ligand and T cell cytokines allows for the effective generation of human T cell precursors from cord blood within 7 days. Moreover, once injected into NOD/SCID/gcko mice, T cell precursors generated in this system were able to colonize the thymus and generate a diversified and functional T-cell compartment. Here, we aimed at testing the capacity of adult HSPCs in this reconstitution system. We found that, like their CB- derived counterparts, T cell precursors generated from adult HPSCs phenotypically resembled thymic CD34+CD7+ cells with high in vitro T-cell differentiation potential. Interestingly, the peak of T cell progenitors for adult HSPCs occurred around day 3, compared to day 7 in CB. At this timepoint, T cell precursors derived from adult HSPC already expressed all critical genes for T cell lineage development, as well as the major chemokine receptors implicated in thymus homing. The introduction of retronectin further improved differentiation and proliferation of T cell progenitors from both HPSC sources in our in vitro system. Comparative molecular analysis of adult- and CB- derived progenitors suggested, that differential requirements for Notch receptor/ligand interactions may explain the differences in kinetics observed during the culture of the two types of HSPC. It remains to be further evaluated, whether targeted modifications of the Notch signaling pathway can improve the outcome of this in vitro T cell differentiation system for adult HPSCs. Overall our results suggest that adult HSPCs, like their CB- derived counterparts, provide an effective source of in vitro cultured T cell progenitors harboring all the necessary requirements for the in vivo -reconstitution of a functional T cell compartment. This is particularly important in the context of future clinical applications in HSCT where adult HSPCs are more available and more frequently used than CB HSPCs. Based on our results, we propose that upon injection into a patient, DLL4- cultured T cell precursors from both HSPC sources could significantly accelerate the reconstitution of the adaptive immune system after a partially HLA-incompatible HSCT. Currently, we are translating these results into a phase I clinical trial including adult and pediatric patients transplanted for malignant hemopathies or PIDs requiring an allogeneic HSCT from a HLA-partially mismatched donors. Disclosures No relevant conflicts of interest to declare.

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Comparative Studies of B Cell Precursor ALL Pathogenesis and Translational Biology Using a Conditional E2a-PBX1 Transgenic Mouse Model

Blood ◽

10.1182/blood.v124.21.283.283 ◽

2014 ◽

Vol 124 (21) ◽

pp. 283-283

Author(s):

Jesus Duque-Afonso ◽

Jue Feng ◽

Florian Scherer ◽

Zhong Wang ◽

Michael L. Cleary

Keyword(s):

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Disease Free Survival ◽

Leukemia Cells ◽

Mouse Strains ◽

Driver Gene ◽

Hematopoietic Stem

Abstract Pediatric acute lymphoblastic leukemia (ALL) represents a collection of orphan diseases that are defined by their genomic abnormalities. Their genetic diversity and low prevalence serve as major barriers to investigations of their molecular pathogenesis and translational biology. To address this, we have engineered novel mouse strains that conditionally activate and express E2a-PBX1, the fusion oncogene derived from chromosomal translocation t(1;19), present in 5-7% of pediatric ALL. Somatic activation of the oncogene is accomplished by Cre-recombinase expressed under the control of specific B-lineage promoters CD19 or Mb1, or in hematopoietic stem cells using the Mx1 interferon-inducible promoter. The three mouse strains show similar pre-leukemic and leukemic phenotypes. At the time of disease, mice exhibit leukocytosis, anemia and thrombocytopenia as well as lymphadenopathy and hepatosplenomegaly and infiltration of several organs including kidney, lung and central nervous system. Leukemia cell phenotypes (CD117+, CD19+, CD43+, CD45+, CD25+, sIgM-, Bp-1+, CD24+, CD127+, CD79a+ and TdT+) correspond to the phenotypic fraction B-C’ (or pro-B/large pre-B II stage, Basel nomenclature) that is very similar to human E2a-PBX1+ pre-B-ALL. Hence, we detected productive VDJ rearrangements and cytoplasmic heavy chain in 12.5 % of cases, a characteristic of human E2a-PBX1 leukemias. Leukemia incidence varies from 5-50% depending on the Cre driver gene and the median latency is about 10 months in E2a.PBX1/Mb1.Cre and Mx1.Cre lines, suggesting the need for secondary mutations. Whole exome sequencing detected secondary genetic aberrations, which were validated in a larger cohort of leukemias. Spontaneous deletions of Pax5, which are present in ~45% of pediatric ALLs with E2a-PBX1 gene fusions, were found in about 30 % of mouse E2a-PBX1 leukemias. Conditional deletion of Pax5 and E2a-PBX1 expression expanded progenitor B cell subpopulations in healthy 3-months old preleukemic mice. Consequently, Pax5 haplo-insufficiency in mice cooperates with E2a-PBX1 increasing the penetrance and shortening the latency of leukemia, providing the first evidence for cooperative oncogenic effects of Pax5 haplo-insufficiency. Tumor suppressor genes as Trp53 and Cdkn2a/b were inactivated by secondary mutations and deletions, respectively. Additionally, secondary recurrent activating mutations were detected in key signaling pathways such as Ras/Mapk and Jak/Stat on which the leukemia cells are strongly dependent. Furthermore, leukemia cells displayed higher basal levels of phosphorylated pSTAT5 and pAKT, pERK1/2, and were hyper-sensitive to stimulation with IL-7 and thymic stromal lymphopoietin (TSLP) as seen by induction of pSTAT5 and supported growth in colony-forming assays. The JAK1/2 inhibitor ruxolitinib blocked the induction of pSTAT5 by IL-7 and TSLP, inhibited colony formation in vitro, and increased disease-free survival after in vivo treatment. Human E2a-PBX1 primary cells and cell lines showed hypersensitivity to IL-7/pSTAT5 activation compared to other ALL karyotypes and pre-treatment with ruxolitinib blocked induction of pSTAT5 by IL-7. In summary, we have developed conditional transgenic E2a-PBX1 mouse models that consistently develop leukemias that resemble human pre-B-ALL carrying the t(1;19) and identified key cooperating oncogenic pathways. This model provides experimental validation of the multistep pathogenesis for a subset of ALL previously inferred from genomic analyses and provides a platform for comparative mechanistic and preclinical studies. Disclosures No relevant conflicts of interest to declare.

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All-Trans Retinoic Acid Synergizes With FLT3 Tyrosine Kinase Inhibition To Eliminate FLT3/ITD-Expressing Leukemia Cells

Blood ◽

10.1182/blood.v122.21.3960.3960 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3960-3960

Author(s):

Hayley S Ma ◽

Sarah M Greenblatt ◽

Eric Jung ◽

Amy S. Duffield ◽

Li Li ◽

...

Keyword(s):

Stem Cell ◽

Retinoic Acid ◽

Tyrosine Kinase ◽

Cell Lines ◽

Conflicts Of Interest ◽

Apoptotic Cell ◽

Leukemia Cells ◽

Cure Rate ◽

Hematopoietic Stem

Abstract FLT3 is one of the most frequently mutated genes in AML with approximately 1/3 of patients affected, and the internal tandem duplication (ITD) mutations portending a poor prognosis. To improve the cure rate for FLT3 mutant AML a number of FLT3 tyrosine kinase inhibitors (TKIs) have been developed to inhibit FLT3 signaling. While several recent FLT3 TKIs are proving increasingly successful at achieving high levels of FLT3 inhibition, there remain several problems in treating FLT3 mutant AML. One is that these drugs used as monotherapy achieve limited clinical responses and do not cure patients. While there is hope that combination with chemotherapy will achieve an improved cure rate in these patients without the necessity for hematopoietic stem cell transplantion (HSCT), the ultimate goal of studies are to achieve cures without chemotherapy, eliminating the short and long-term side effects that its use engenders. Thus, finding additional molecular targets that might synergize with FLT3 inhibition will move the field towards the goal of eliminating chemotherapy. One pathway known to play important roles in leukemia stem cell (LSC) survival and differentiation is the retinoic acid (RA) pathway. We therefore explored the combination of molecularly targeting the RA pathway together with FLT3 TKIs to determine the effect on FLT3 mutant leukemia cells. FLT3/ITD+ AML cell lines (Molm14 and MV411), along with FLT3/WT cell lines (THP-1, SEMK2 and NB4), were treated with FLT3 TKI alone (AC220 and sorafenib), ATRA alone, FLT3 TKI plus ATRA, or vehicle controls. Proliferative, apoptotic, cell-cycle and differentiation effects on the cells were assessed by MTT, annexin V binding, propidium iodide (PI) staining, CD11b staining and cell count assays. Highly synergistic effects were observed for the combination of ATRA with FLT3 TKIs against FLT3/ITD+ cells, with combination index (CI) values of 0.1-0.6. Colony forming unit (CFU) assays further demonstrated decreased clonogenicity of Molm14 and MV411 cells upon treatment with ATRA and sorafenib. A series of experiments were performed using a genetic model of spontaneous leukemia that we developed in which FLT3/ITD knock-in mice are bred with NUP98- HOXD13 (NHD13) transgenic mice, resulting in AML that is highly penetrant, lethal and transplantable. Cohorts of leukemic mice were generated by transplanting lineage negative (Lin-) BM cells from leukemic FLT3/ITD-NHD13 mice, and treated with either vehicle, sorafenib, ATRA or a combination of both drugs. We determined that treatment with sorafenib plus ATRA greatly decreases the level of engraftment at 2 and 8 weeks, and increases median survival, with some mice even cured of their disease. We also directly assessed the effect of the treatments (FLT3 TKI alone, ATRA alone, FLT3 TKI plus ATRA, or vehicle alone) on the proliferation of LSCs by using the different treatments on Lin- BM cells harvested from sick FLT3/ITD NHD13 mice ex vivo and found that ATRA further increased the anti-proliferative effect of FLT3 TKIs, with additive/synergistic CI values of 0.6-1. Lin- BM cells were also assessed in vitro in CFU assays of differentiation and clonogenicity, and combinatorial effects were observed. The results of these experiments show good synergy for this drug combination in vitro and improved survival/reduced LSC frequency in vivo. We believe these preclinical data are encouraging for the development of a clinical trial of ATRA plus FLT3 TKI in relapsed/refractory FLT3 mutant AML patients. Disclosures: No relevant conflicts of interest to declare.

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Prospective Isolation Of Human Erythroid Lineage-Committed Progenitors

Blood ◽

10.1182/blood.v122.21.3418.3418 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3418-3418

Author(s):

Yasuo Mori ◽

Jun Seita ◽

John V. Pluvinage ◽

James Y. Chen ◽

Irving L. Weissman

Keyword(s):

Bone Marrow ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Marrow Cell ◽

Significant Loss ◽

Refractory Anemia ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Murine Bone Marrow

Abstract Anemia is one of the findings most frequently seen in patients with myelodysplastic syndrome (MDS); however, in our hands, significant loss of megakaryocyte/erythrocyte progenitors (MEP) is not observed among these patients (PNAS, 2013, 110(8):3011-6). Unipotent erythroid-committed progenitors (EPs), estimated to exist downstream of bipotent MEPs, may be involved in the pathogenesis of refractory anemia among MDS patients. Previously, we identified mouse (m) EPs (mEPs) in bone marrow. The expression of endoglin (CD105) was a key marker to isolate mEPs: mEPs were the Lineage(lin)-Sca-1-c-kit+CD16/32-CD150+CD105+CD41- population in murine bone marrow (Cell Stem Cell, 2007, 1(4):428-42). Here we show that the human (h) counterpart (hEPs) is prospectively isolatable in human bone marrow. We analyzed the expression of hCD105 in addition to hCD71 (known to be an early erythroid marker) in human stem and progenitor populations, and found a fraction (32.9±13.6%, n = 5) of common myeloid progenitors (CMPs; lin-CD34+CD38+CD45RA-IL-3Ra+) and a major part (85.1±5.5%) of MEPs (lin-CD34+CD38+CD45RA-IL-3Ra-) expressed hCD71. A portion of CD71+ MEPs co-expressed CD105 (CD105+MEPs; 32.3±6.5% of MEPs). Neither hCD105 nor hCD71 was detectable in hematopoietic stem cells (HSCs; lin-CD34+CD38-CD45RA-), common lymphoid progenitors (CLPs; lin-CD34+CD38+CD10+), or granulocyte/monocyte progenitors (GMPs; lin-CD34+CD38+CD45RA+IL-3Ra+) by FACS. In vitro, CD71+ cells within the CMP fraction showed differentiation potential skewed toward MegE lineage, representing the transitional stage to MEPs. CD71+MEPs mostly generated BFU-E, although they still retained some MegK potential, while output of CD105+MEPs was completely restricted to erythroid lineage, and colonies from this fraction contained a small number of mature (enucleated) erythrocytes (CFU-E type). Thus we termed CD71+MEPs and CD105+MEPs as human erythroid-biased MEPs (E-MEPs) and hEPs, respectively. In short-term liquid culture, hE-MEPs gave rise to hEPs in the presence of SCF, TPO and EPO, whereas hEPs could not generate hE-MEPs, suggesting a hierarchical progression from E-MEPs to EPs. These newly classified populations might be a very useful tool for understanding the molecular mechanisms of human erythroid development, and should be analyzed in patients with erythroid-related disorders (e.g., MDS, polycythemia vera, or aplastic anemia). Disclosures: No relevant conflicts of interest to declare.

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Megakaryocytes Support Viability Proliferation and Protection of Primary Pre-B ALL Cells from Chemotherapy

Blood ◽

10.1182/blood.v126.23.3763.3763 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3763-3763

Author(s):

Stephanie Nicole Shishido ◽

EunJi Gang ◽

Ruth Xu ◽

Hye Na Kim ◽

Yann Duchartre ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Count ◽

Stromal Cells ◽

Leukemia Cells ◽

P Value ◽

Hematopoietic Stem

Abstract BACKGROUND: The bone marrow is known to shelter leukemia cells from chemotherapy and contributes to the survival of chemotherapy resistant residual cells, termed minimal residual disease (MRD). We have studied in situ the location of MRD+ ALL cells using a xenograft model of primary ALL cells and have found a novel co-localization of megakaryocytes (MK) with ALL cells. Mature hematopoietic cells have been implicated in modifying the local normal hematopoietic stem cell environment including MK. We hypothesize that MK are associated with the survival of MRD+ ALL cells. For this purpose, we tested the role of MK cells in maintenance and chemoprotection of ALL cells. METHODS: Patient-derived (primary) pre-B ALL cells were engrafted into non-irradiated NOD/SCID IL2Rγ-/- (NSG) mice. Leukemia-bearing mice received 4 weeks of chemotherapy treatment (Vincristine, Dexamethasone, L-Asparaginase; VDL). MRD status of mice was confirmed by detection of human CD45 + CD19+ leukemia cells in the bone marrow by flow cytometry. In situ location of the MRD+ ALL cells was determined by histological analysis and quantitation was performed by Fiji Image J. For in vitro studies, primary pre-B ALL cells were co-cultured for up to 2 days with murine calvaria-derived stromal OP9 cells or MK isolated from C57/BL6 BM primed for with murine thrombopoietin (mTPO) and sorted by flow cytometry for CD41+ MK. Annexin V/7-AAD staining was used for viability determination by flow cytometry. Boyden chamber system with either OP9 cells or MK cells seeded on the bottom and ALL cells on the top of the system was used for migration assays. RESULTS: In situ analysis of MRD+ ALL recipient mice showed that cells MRD+ ALL cells (huCD45+) are located in close proximity to MKs, with 11.57±2.94% of MRD+ ALL cells lying directly adjacent to MKs (0-5μm distance to MK). To further assess the role of MKs in ALL survival in vitro, we compared if MK cells can sustain proliferation and viability of primary leukemia cells like the OP stromal co-culture model that we have established previously. MKs were isolated from BM of C57BL/6 mice by FACS sorting for CD41+ cells from BM primed with mTPO. The sorted population showed a 90% purity of CD41+ cells. MKs were able to maintain ALL cell proliferation 1.90e6 cell count ± 0.48e6 cell count on day 2) and provide chemoprotection from VDL treatment (77.24 ± 2.03% on day 2), which was similar to the effect of OP9 cells on sustained proliferation and viability. Interestingly ALL cells cultured with MKs had a slight reduction in G2/M-phase (8.46±0.31%) 3 days after culture set up without treatment compared to cultures with OP9 stromal cells (13.59±0.14%; P-value < 0.0005). In a migration assay, MKs stimulated migration of ALL cells (5.42e5 ±0.72 migrated cells) significantly more than OP9 stromal cells (2.92e5 ±0.72 migrated cells) over a 24 hour period (P-value = 0.0132). Using the SDF1α inhibitor AMD3100 (100µM), migration of ALL cells was only partially inhibited (2.92e5 ±0.72), suggesting additional MK produced factors influence mobilization of human leukemia cells besides SDF1α. Using the recombinant forms of stromal cell-derived factor 1 (SDF1α) and von Willebrand factor (VWF), ALL cell migration was successfully stimulated over 24 hours (6.25e4 ±1.25 and 3.33e4 ±0.72 migrated cells, respectively), and this effect was inhibited using AMD3100, small molecule inhibitor of CXCR4, and anti-VWF antibody respectively. CONCLUSION: Here we show for the first time that co-culture of MK and primary pre-B ALL cells supports their proliferation, viability and protection from chemotherapy similar to murine OP9 stromal cells. Our data warrants further investigation of the underlying mechanism. Disclosures No relevant conflicts of interest to declare.

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Essential Role of Endogenous MOZ in Aberrant HoxA9 and Meis1 Expressions Induced By MOZ Fusion Gene

Blood ◽

10.1182/blood.v124.21.2191.2191 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2191-2191

Author(s):

Takuo Katsumoto ◽

Kazutsune Yamagata ◽

Yoko Ogawara ◽

Takuro Nakamura ◽

Issay Kitabayashi

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Molecular Mechanisms ◽

Zinc Finger Protein ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Leukemia Cells ◽

Hematopoietic Stem

Abstract Monocytic leukemia Zinc finger protein (MOZ), a histone acetyltransferase, is involved in chromosome translocations associated with FAB M4/M5 types of acute myeloid leukemia (AML). In normal hematopoiesis, MOZ is essential for self-renewal of hematopoietic stem cells (HSCs) and for expression of HoxA9/Meis1 in hematopoietic stem/progenitor cells (HSPCs). Previously we found that endogenous MOZ is critical for MOZ-TIF2-induced AML. Although MOZ-/- cells expressing the MOZ-fusion serially generated colonies in vitro, they did not induce AML after transplantation into recipient mice. In these cells, up-regulation of Meis1 was impaired, while HoxA9 expression was induced. However, roles of endogenous MOZ in MOZ fusion induced leukemia remained unclear. To elucidate molecular mechanisms, we performed experiments described below. First, to reveal mechanisms in defect of Meis1 expression in MOZ-/- MOZ-fusion leukemia cells, we performed chromatin immune-precipitation assays on Meis1 locus. Coincident with gene expression, active histone marks (H3K9ac, H3K27ac etc.) were disrupted. In contrast, repressive histone modifications (H3K9me2, H3K27me3) were elevated. Next we analyzed requirement of HoxA9 and Meis1 in MOZ fusion induced AML development. When mice were transplanted with MOZ-/- HSPCs simultaneously introduced with MOZ-fusion and Meis1 genes, AML development were induced. On the other hand, when Meis1 was conditionally deleted in MOZ-fusion leukemia cells, AML development was significantly delayed. Mice transplanted with MOZ-/- HSPCs, which were introduced with both HoxA9 and Meis1 genes elicited AML development. Furthermore, we analyzed gene expression profiles of MOZ-/- MOZ fusion leukemia cells. In these cells, expressions of monocyte/macrophage lineage characteristic genes (C/EBPa, Irf8, CD68 etc.) and MLL fusion target genes (Meis1, Mef2c) were decreased. In contract, other hematopoietic lineage characteristic genes (GATA1-3, FOG-1, CD41, Aiolos, Helios, Eag, Epx etc.) were increased. In addition, expression of CDK inhibitor INK4A was also up-regulated. Finally, we tested requirement of endogenous MOZ in various cellular conditions. Previous report showed that AML development was induced by introduction of MOZ-TIF2 not only in hematopoietic stem cells but also in more differentiated Common myeloid progenitors (CMPs) and Granulocyte/Monocyte progenitors (GMPs) (Huntly et al, Cancer Cell 2004). So we introduced MOZ fusion genes in HSCs and CMPs collected from E14.5 MOZ-/- fetal liver. MOZ-/- HSCs, not CMPs, expressing MOZ-TIF2 continuously formed colonies in vitro. In the CMPs expressing MOZ-TIF2, expression of both Meis1 and HoxA9, were abolished. These results suggest that high levels of HoxA9 and Meis1 expressions were respectively required for MOZ-TIF2-induced AML development, and that endogenous MOZ is critical for MOZ-TIF2-induced AML development. Disclosures No relevant conflicts of interest to declare.

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Embryologic Origin of Functional Granulopoiesis

Blood ◽

10.1182/blood.v124.21.228.228 ◽

2014 ◽

Vol 124 (21) ◽

pp. 228-228

Author(s):

Kathleen E McGrath ◽

James Palis ◽

Katherine H. Fegan ◽

Seana C Catherman

Keyword(s):

Flow Cytometry ◽

Mast Cells ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Early Embryo ◽

Yolk Sac ◽

Morphological Evidence ◽

Mammalian Embryo ◽

Hematopoietic Stem

Abstract The first progenitors with granulocyte potential arise in the murine yolk sac beginning at embryonic day 8.5 (E8.5), 48 hours before hematopoietic stem cells (HSCs) are formed. These granulocyte progenitors are part of a wave of definitive erythro-myeloid progenitors (EMPs) that display a unique immunophenotype. By E10.5, we observe EMPs in the bloodstream and enriched in the liver, the site of fetal hematopoiesis, consistent with previous reports of GM-CFC presence in the early embryo. HSCs begin to colonize the fetal liver between E11.5 and E12.5, where they subsequently expand and differentiate. Thus, the presence of maturing blood cells at this time-point likely represent the output of EMP that colonize the fetal liver before HSCs. In vitro culture of purified EMPs results in the complete myeloid repertoire found in the adult, including neutrophils, basophils, eosinophils and mast cells. To see which of these potentials is actually realized in the embryo, we examined myelopoiesis in the liver at E11.5- E12.5. There was no morphological evidence of eosinophils, basophils or mast cells in the early fetal liver, and there were no lineage specific transcripts for these cells types (EPX, FCepsilonR) detected by qPCR before E15.5. However, rare cells with neutrophil morphology were found in the fetal liver and in the bloodstream at E12.5. We utilized flow cytometry to enumerate granulocytes (GF1+, Mac1+) in both liver and the bloodstream during early embryogenesis. In order to rule out contamination from maternal cells, we analyzed embryos generated from GFP+ male mice mated with wild-type females. Per embryo equivalent, we consistently found a small number of granulocytes already present in both fetal liver and circulation at E11.5. The number of granulocytes increases to over one hundred at E12.5 and thousands by E13.5. We used imaging flow cytometry to examine the maturational state of the granulocytes in the fetal liver. Consistent with their recent differentiation, fetal liver granulocytes were predominately at the most immature stages as compared to bone marrow samples. Interestingly, at E11.5 and E12.5, a large proportion the circulating granulocytes were maternal derived (GFP-). The presence of these maternal granulocytes could not be accounted for by contamination of maternal blood given the levels of maternal RBCs in the samples. These data indicate that both maternal- and embryonic EMP-derived neutrophils co-circulate in the early embryo. Furthermore, when fetal blood was stimulated with bacteria-like BioParticles, fetal derived (GFP+) and maternal granulocytes (GFP-) each responded with oxidative bursts. Taken together, these data indicate that the early mammalian embryo utilizes both yolk sac-derived transient definitive progenitors and maternal granulocytes to provide host defense before HSC-derived hematopoiesis is established. Disclosures No relevant conflicts of interest to declare.

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