Development of Hras-Induced Zebrafish Leukemia Models

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2459-2459
Author(s):  
Elisa Alghisi ◽  
Martina Konantz ◽  
Marina Mione ◽  
Claudia Lengerke
Keyword(s):  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Experimental Models ◽  
Pcr Analysis ◽  
Human Leukemia ◽  
Hematopoietic Tissue ◽  
Hematopoietic Stem ◽  
Primitive Hematopoiesis ◽  
Transgenic Lines ◽  
Early Lethality

Abstract Background: Over the last years, the zebrafish has emerged as a versatile novel experimental model for hematopoietic studies. The major genetic pathways have proven conserved between fish and mammalian hematopoiesis. Several oncogenes involved in human leukemia have been successfully overexpressed in a transient fashion in zebrafish embryos. However, despite first encouraging results these experimental models often failed to fully recapitulate human myeloid malignancy, perhaps due to early lethality caused by off-target expression or lack of secondary events necessary for full malignant transformation. Material and Methods: Here we took advantage of the Gal4/UAS binary system and of existing transgenic lines to overexpress the human oncogenic HRAS gene in zebrafish hematopoietic cells under control of specific promoters (fli.1, pu.1, runx.1). HRAS-transgenic lines were generated and fish followed by microscopy from early development until, if possible, sexual maturity. Hematopoietic cell development was studied at embryonic (fli.1, pu.1), larval (pu.1, runx1) and adult stages (runx1) by in situ hybridization and real-time PCR analysis of hematopoietic gene expression, flow cytometry, immunohistochemistry and/or blood smear morphological assessment. Results: All HRAS transgenic lines showed hematopoietic abnormalities. However, different phenotypes were observed depending on the promoter driving the oncogene expression. HRAS induction via the early hematopoietic promoter fli.1 affected primitive hematopoiesis inducing a myelo-erythroid proliferation characterized by the expansion of the caudal hematopoietic tissue, enhanced expression of myelo-erythroid genes and delayed erythrocyte maturation (Alghisi et al. 2013). Surprisingly, no obvious effects were noted on the emerging hematopoietic stem cells (HSCs) in the aorta-gonado-mesonephros (AGM) region and studies at later stages were hampered by early lethality of the fish due to vascular defects and cardiac edema. The lethality at early stages was also observed using the myeloid promoter pu.1 that induced the expansion of primitive myeloid cells along with severe developmental defects. In contrast, HRAS expression driven by runx1, a known HSC marker, did not affect primitive hematopoiesis and allowed studies at later developmental stages. Supporting the results obtained with the fli.1 promoter, no alteration was noted in the AGM of runx1-HRAS induced fish. However, at 1 month post-fertilization, flow cytometry analyses revealed a prominent cellular expansion of the hematopoietic stem/progenitor cells in the kidney marrow, the zebrafish definitive hematopoietic compartment. Kidney marrow cytospin preparation and flow cytometry analysis confirmed high numbers of undifferentiated cells indicating that HRAS -overexpression in this model induced stem/progenitor cell proliferation. Interestingly, first analyses suggested that the numbers of differentiated cells might be reduced in these fish, implying that HRAS-induced stem/progenitors have impaired differentiation capacity. Outlook and conclusion: We are currently further investigating the effects of runx1-driven HRAS on the hematopoietic compartment and generate tools to explore potential cooperation of HRAS with other oncogenes during leukemogenesis. Successfully established zebrafish leukemia models shall be eventually used for identification of therapeutically active compounds in small molecule screens. Disclosures No relevant conflicts of interest to declare.

A New Xenograft Model for Studying Multiple Myeloma and Drug Response

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4073-4073
Author(s):  
Varda Deutsch ◽  
Yona Farnoushi ◽  
Michal Cipok ◽  
Sigi Kay ◽  
Elizabeth Naparstek ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Xenograft Model ◽  
In Vivo Model ◽  
Pcr Analysis ◽  
Human Leukemia ◽  
In Vivo Models

Abstract Abstract 4073 While new treatment options are available, multiple myeloma (MM) still remains an incurable malignancy of plasma cells with a grim prognosis. Practical in vivo models to study human MM may enable a better understanding of the biology of the disease, and better optimization of therapeutic strategies. The best current xenograft model, the immune-deficient NOD/SCID mice, recapitulates MM in vivo, however, the price is very costly and maintenance complex, with >1 month required to establish engraftment. Our goal was to develop a user friendly rapid alternative xenograft system for the preclinical assessment of MM growth and therapy. We recently described this new in-vivo system for studying human leukemia in the pre-immune turkey embryo 1,2. These embryos are inexpensive, require no maintenance, and are easily manipulated experimentally. Described here are the first attempts at application of this novel system to study MM and test therapies. Cell lines ARH-77 and CAG line and fresh patient cells (5 × 106/embryo) were injected IV into turkey egg chorioallantoic membrane veins on embryonic day E11. Engraftment of human cells in hematopoietic organs, bone marrow (BM) and liver was detected 7 days later (E18) by RTPCR, immunohistochemistry and flow cytometry and by circulating free light chain (6-25 mg/L) in the peripheral blood of 100% of the injected cell lines and 50% of patients myelomas. Treatment with Velcade (Bortezomib) or Revlimid IV on E13 (48 hours after MM cell injection), at drug levels that were precalibrated to be non-toxic to the developing embryonic BM, dramatically reduced engraftment, demonstrating the utility of this new model for testing drug activity in vivo. ARH-77 cells, detected by flow cytometry of the embryonic BM cells with anti-human CD19, CD38 and CD138, were inhibited from 8.5% engraftment to 0.72% after a single Velcade treatment, with an 18 fold decrease compared to untreated embryos in the ratio of human to avian cells in BM tissue. determined by Q-RT-PCR analysis of human alpha satellite and avian GAPDH DNA normalized per cell. Very similar results were obtained with Revlimid. The results presented suggest that with further work the turkey embryo model may provide an affordable, rapid and practical xenograft system in vivo for studying the biology of MM, for affordably testing MM therapies, as well for developing a new method for individualized patient screening for response or resistance to particular therapeutic agents. 1. Taizi M, Deutsch VR, Leitner A, Ohana A, Goldstein RS. A novel and rapid in vivo system for testing therapeutics on human leukemias. Exp Hematol. 2006;34:1698-1708. 2. Grinberg I, Reis A, Ohana A, et al. Engraftment of human blood malignancies to the turkey embryo: a robust new in vivo model. Leuk Res. 2009;33:1417-1426. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inflammasome-Mediated Regulation of Hematopoiesis in the Vertebrate Embryo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 330-330 ◽  
Author(s):  
Jenna M Frame ◽  
Timothy Long ◽  
Caroline Schuster-Kubaczka ◽  
Virginie Esain ◽  
Sung Eun Lim ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Glucose Metabolism ◽  
Conflicts Of Interest ◽  
Metabolic Activation ◽  
Ros Generation ◽  
Inflammasome Activation ◽  
Hemogenic Endothelium ◽  
Hematopoietic Tissue ◽  
Glucose Stimulation ◽  
Hematopoietic Stem

Abstract Hematopoietic stem and progenitor cells (HSPCs) arise in the embryonic dorsal aorta (DA) through a Runx1-dependent process of endothelial-to-hematopoietic transition (EHT). Subsequently, HSPCs colonize secondary niches to proliferate, differentiate and maintain lifelong hematopoiesis. We previously reported that elevated glucose metabolism and sterile inflammatory signaling each stimulate zebrafish HSPC production from hemogenic endothelium. Consistent with the hypothesis that transient metabolic activation induces an inflammatory response to influence HSPC formation, we found that glucose stimulation from 12-36 hours post fertilization (hpf) increased expression of pro-inflammatory cytokines and receptors by quantitative PCR. In particular, morpholino-mediated knockdown of il1b blunted the inductive effects of glucose metabolism on runx1 expression in the DA and numbers of CD41+ HSPCs in the caudal hematopoietic tissue (CHT). In contrast, overexpression of mature il1b increased HSPC numbers as assessed by flow cytometry and runx1/cmyb in situ hybridization. As IL-1β is cleaved and activated by the inflammasome, which is a well-known sensor of metabolic stimuli, we treated embryos with validated inflammasome activators, such as nigericin, and found that they also increased runx1/cmyb expression and Flk1+cMyb+ HSPCs. Inflammasome stimulation required NF-κB activity, and upregulated canonical IL-1β targets, including IL-6 and IL-8, which have been previously shown by our laboratory to play significant roles in embryonic HSPC formation. Importantly, loss of the inflammasome adapter pycard or effector caspa reduced HSPC numbers, and prevented expansion of the HSPC pool in response to glucose stimulation. In further support of a role for sterile inflammatory activity in regulating HSPC number downstream of metabolic activation, we found that inflammasome-mediated IL-1β action bypassed the suppressive effect of antioxidant treatment on runx1/cmyb expression in the DA. Conversely, inflammasome inhibition partially blocked effects of metabolism-associated HIF1α activation on runx1/cmyb expression, indicating that the inflammasome acts downstream of reactive-oxygen-species (ROS) generation and HIF1α activation to promote HSPC production and/or expansion from hemogenic endothelium. Inflammasome components are highly expressed in myeloid cells; targeted knockdown and cell ablation studies indicate that macrophages are necessary to mediate the effects of inflammasome stimulation on HSPC numbers in the CHT at 72hpf. Interestingly, prolonged inflammasome stimulation also expands myeloid and lymphoid progenitors, as cmyb, rag1 and mpo expression were each elevated at 120hpf; increased numbers of Rag2+ and Mpo+ cells were confirmed by flow cytometry. To determine whether the effects of inflammasome activation were conserved in higher vertebrates, we induced inflammasome activation during differentiation of human induced pluripotent stem cells (iPSC) into hematopoietic progenitors. In this system, nigericin treatment of iPSC-derived hemogenic endothelial cells yielded an increased frequency of functional colony-forming units by day 7 of EHT culture. Taken together, inflammasome-mediated IL-1β action appears to serve as an integrator of metabolic activity, downstream of ROS/ HIF1α, to promote HSPC formation and development of myeloid and lymphoid lineages in vivo and in vitro. These studies identify the inflammasome as a promising target to promote human HSPC production from iPSCs for therapeutic purposes. Disclosures No relevant conflicts of interest to declare.

scholarly journals CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1054-1054 ◽  
Author(s):  
Hongxing Liu
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Lymph Nodes ◽  
Conflicts Of Interest ◽  
Sh2 Domain ◽  
Janus Kinase ◽  
Gingival Hyperplasia ◽  
Hematopoietic Stem ◽  
T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

Expression and Function of the CXCR7 Chemokine Receptor in Leukemias

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2423-2423
Author(s):  
Sergej Konoplev ◽  
Hongbo Lu ◽  
Michael A Fiegl ◽  
Zhihong Zeng ◽  
Wenjing Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
And Function

Abstract Background: Bone marrow produced stromal-derived factor-1a (SDF-1a) is a key chemokine involved in chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. While the majority of well-defined functions of SDF-1a are mediated via its receptor CXCR4, recent studies have characterized CXCR7 as an alternative receptor capable of binding SDF-1a. Although the functions of CXCR7 are still incompletely understood, the receptor was reported to promote migration and adhesion in certain cell types and function as a pro-survival factor in breast cancer cells. CXCR7 expression and function in human leukemia cells has not been characterized. In this study, we examined CXCR7 expression in leukemia cell lines and primary samples from patients with acute lymphoblastic leukemia (ALL) and utilized a small molecule inhibitor of CXCR7 to probe CXCR7’s function. Materials and methods: CXCR4 and CXCR7 expression was determined by flow cytometry, real-time PCR (RT-PCR) and immunocytochemistry (ICC) in leukemic cell lines including AML (OCI-AML2, OCI-AML3, HL60, U937 NB4, Molm13), ALL (REH, Raji, RS4; 11, Nalm6, Molt4) and CML (KBM5, K562) cells. In primary ALL patient samples, CD34+CD19+ gating was applied to detect CXCR7 expression on pre-B leukemic cells by flow cytometry. The migration of leukemic cells towards SDF-1a was studied using a transwell system. CXCR4 inhibitor AMD3100 was purchased from Sigma, and CXCR7 inhibitor CCX-733 was provided by ChemoCentryx Inc., Mountain View, CA. Results: CXCR4 was found to be ubiquitously expressed on the cell surface of all leukemic cell lines tested. CXCR7 mRNA and protein expression was detectable only in Burkitt lymphoma Raji cells, as analyzed by flow cytometry (clone 11G8, R&D systems), RT-PCR and ICC. Curiously, CXCR7 expression was significantly induced in MOLM13 cells under hypoxic (6% O2) conditions (p=0.01). Low levels of surface CXCR7 were found in 8 of the 9 primary ALL samples by flow cytometry. To determine the respective roles of CXCR4 and CXCR7 in migration of leukemic cells, we utilized CXCR4 inhibitor AMD3100 and CXCR7 inhibitor CCR733 in Raji (CXCR7 positive) and RS4;11 (CXCR7 negative) cells. AMD3100 at 25μM significantly inhibited SDF-1a induced migration (from 38.5% to 12%); CCR733 at 10μM also inhibited SDF-1a induced migration (from 38.5% to 24%) and the combination of AMD3100 and CCR733 resulted in 81% inhibition of migration (from 38.5% to 7.2%). AMD3100 blocked SDF-1a induced migration of CXCR4+CXCR7− RS4;11 cells (from 36.5% to 15.8%), while CCR733 had no effect (36.5% and 39.2%). In conclusion, these studies demonstrate functional expression of the SDF-1 receptor CXCR-7 on Raji and primary ALL cells and suggest that CXCR7 plays an active role in the migration of leukemic cells. CXCR-7 may serve as an alternative receptor to CXCR4. Studies addressing the role of CXCR7 in adhesion, SDF-1a-mediated signaling and survival of leukemic cells are in progress.

Aberrant Bone Marrow Perivascular Niches are Involved in Multiple Myeloma Progression: Role of Notch–Delta Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2800-2800
Author(s):  
Sara Lamorte ◽  
Marta Costa ◽  
Giovanni Camussi ◽  
Sergio Dias
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Pcr Analysis ◽  
Adherent Cells ◽  
Feeder Layer ◽  
Hematopoietic Stem ◽  
Lower Chamber

Abstract Abstract 2800 Poster Board II-776 Bone marrow (BM) angiogenesis is implicated in Multiple Myeloma (MM) progression. In this study, we tested the hypothesis that MM progression occurs when aberrant BM perivascular niches are established. We isolated BM endothelial cells derived from MM patients (MM-BMECs) from BM aspirates using anti-CD31Ab coupled to magnetic beads. FACS analysis showed that of all the cell lines isolated were endothelial: more than 95% expressed Ulex Europaeus Agglutinin-1 and Factor VIII and were negative for monocyte-macrophage (CD14) and plasma cell markers (CD38). To test the hypothesis that in MM patients BM perivascular niches are aberrant we analyzed how MM-BMECs modulate hematopoietic stem cells (HSCs) properties using a BM microvascular endothelial cell line isolated from a healthy donor (BMECs) as control. We co-cultured cord blood cells CD34+ HSCs in the presence of MM-BMECs or BMECs feeder layer and we analyzed the ability of MM-BMECs compared with BMECs to modulate HSCs adhesion, chemotaxis and apoptosis. The results show that MM-BMECs promote CD34+ HSCs adhesion, recruitment and protect them from apoptosis. In detail, we showed that after 24h of co-culture there was a significant increase in the number of adherent HSCs on MM-BMECs than on BMECs: 43±9% versus 25±6%. Moreover, when HSCs were cultured for 48 hours in 1% of serum in the presence of MM-BMECs they were less sensitive to apoptosis (9±11% of Annexin V+ cells) than HSCs cultured in the presence of BMECs (14±1% of Annexin V+ cells) or without a feeder layer, as control (17±3% of Annexin V+ cells). For the migration assay a transwell chamber system, in which the upper and the lower chambers were separated by 5-μm pore-size filter, was used. BMECs, MM-BMECs or nothing was plated in the lower chamber, while HSCs were seeded into the upper chamber. Both chambers were loaded with unsupplemented EBM-2 plus 2% of serum. Cell migration was studied over a 6-8 hours period and evaluated as number of cells migrated into the lower chamber. The results showed a significantly greater migration of HSCs in the presence of MM-BMECs than BMECs: 12±2% versus 5±1% of migrated cells. Taken together, these data showed that MM-BMECs promoted HSCs migration, adhesion and survival. Next we evaluated how MM-BMECs modulate the hemopoiesis recovery after irradiation in a NOD-SCID mouse model. When injected into sub-lethally irradiated (3 Grey) NOD-SCID mice MM-BMECs were detected in the BM integrated within the murine BM vessels and promoted hematopoietic recovery. In detail, MM-BMECs provided signals favoring the commitment towards lymphoid lineage. In fact, 7 days after injection, the BM of mice injected with MM-BMECs showed an increase in the percentage of lymphoblast (2.7%), compared with mice injected with BMECs or PBS, as control (respectively, 1.5% and 1.4%); followed, 14 days after injection, by a significant increase in the percentage of peripheral blood lymphocytes in mice injected with MM-BMECs (75±6%) versus mice injected with BMECS and PBS (respectively 60±0.5% and 47±7%). Since MM is a plasma cells disorder and the Notch-Delta pathway has been shown to play a central role in regulating HSCs properties, including the decisions of HSCs to undergo T- or B-cell differentiation, we investigated the involvement of this pathway in MM-BMECs and HSCs interaction. As determined by FACS and RT-PCR analysis, MM-BMECs, compared to BMECs, over expressed Delta-like Notch ligand 4 (DII4). Thus, we investigated the role of DII4 in the MM-BMECs/BMECs-HSCs adhesion. The first results showed that the expression of DII4 by MM-BMECs is necessary to promote HSCs adhesion. In fact, using a blocking antibody against DII4 (AbαDII4) at 50ug/ml there was an impairment in HSCs adhesion to MM-BMECs (43±9% versus 24±2% of adherent cells without and with AbαDII4 treatment), but not on BMECs (25±6% versus 26±1.4% of adherent cells without and with AbαDII4 treatment). Ongoing experiments are focusing on the role of DII4 in the modulation of HSCs proliferation, protection against apoptosis and in vitro-in vivo B commitment by MM-BMECs. Taken together, all these data suggest that BMECs in MM may function as “aberrant perivascular niches”, modulating HSCs properties. This aberrant phenotype could be due to an alteration of the Notch-Delta pathway in BMECs that favors malignant clonal growth by protecting it from apoptosis, favoring migration, adhesion and providing self-renewing and/or proliferative cues. Disclosures: No relevant conflicts of interest to declare.

Disease-Relevant Expression Of TRAF6 In Mouse HSC Results In An MDS-Like Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 101-101
Author(s):  
Jing Fang ◽  
Xiaona Liu ◽  
Brenden Barker ◽  
Lyndsey Bolanos ◽  
Yue Wei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Bone Marrow Failure ◽  
Gene Signature ◽  
Sublethal Dose ◽  
Hematopoietic Tissue ◽  
Hematopoietic Stem ◽  
Disease Evolution ◽  
Cd34 Cells

Abstract Overexpression of immune-related genes is widely reported in Myelodysplastic Syndrome (MDS), and chronic immune stimulation increases the risk for developing MDS. We find that TNF receptor associated factor 6 (TRAF6), an innate immune protein, is overexpressed approximately 2-fold in CD34+ cells from 40% of MDS patients, and may explain immune pathway activation in the MDS-initiating hematopoietic stem/progenitor cell (HSPC). In support of these observations and our hypothesis that TRAF6 is important in the pathophysiology of MDS, a gene expression analysis revealed that TRAF6 controls an MDS gene signature in human cells. We, and others, have previously shown that retroviral overexpression of TRAF6 in mouse HSPC results in MDS and Acute Myeloid Leukemia (AML). However, interpretations of these findings are hampered by supra-physiological levels of TRAF6 (>10-fold overexpression) and the stress associated with HSPC transduction/transplantation. To investigate the consequences of TRAF6 overexpression to MDS, we generated a transgenic mouse model overexpressing TRAF6 from a hematopoietic-specific Vav promoter. Expression of TRAF6 in HSPC was approximately 2-fold higher as compared to endogenous TRAF6 and in line with MDS patient CD34+ cells. By 15 months of age, half of Vav-TRAF6 mice succumbed to a hematologic disease resembling MDS associated with bone marrow failure (BMF). In contrast to the retroviral overexpression approach, Vav-TRAF6 mice did not develop AML. Examination of sick mice revealed stage-specific disease evolution. Initially, all Vav-TRAF6 mice exhibit an inversion of myeloid/lymphoid proportions. For Vav-TRAF6 mice that develop a fatal disease, they present with a hypocellular marrow, dysplasic myeloid cells, and neutropenia. A subset of mice also display anemia with nucleated red blood cells, poikilocytosis, and extramedullular erythropoiesis. In support of a BMF phenotype, HSPC from Vav-TRAF6 mice form fewer colonies in methylcellulose. To investigate the consequences of an acute exposure to pathogen, early-stage Vav-TRAF6 mice were treated with a single sublethal dose of lipopolysaccharide (LPS). Unlike wild-type (WT) mice, Vav-TRAF6 mice developed a rapid and reversible anemia, suggesting environmental factors can influence the severity of the disease. To gain insight into the mechanism contributing to BMF, gene expression profiling was performed in WT and Vav-TRAF6 HSPC. One of the enriched pathways consisted of AKT activation and FOXO downregulation. Consistent with the microarray analysis, AKT is constitutively phosphorylated at Thr308 in hematopoietic tissue from Vav-TRAF6 mice. SOD2, a transcriptional target of FoxO3a that is suppressed by activated AKT, is decreased in Vav-TRAF6 HSPC. Given that AKT/FOXO regulate reactive oxygen species (ROS) in cells, we investigated ROS levels in HPSC from Vav-TRAF6 and WT mice. Intracellular ROS is significantly elevated in BM cells from Vav-TRAF6 mice, and restored to normal levels when AKT was inhibited. In conclusion, we propose the potential role of TRAF6 in the development of MDS-associated BMF, partly due to constitutive activation of AKT and subsequent ROS elevation in HSPC cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Clinical Utility of Flow Cytometry Immunophenotyping in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5191-5191
Author(s):  
Gustavo Henrique de Medeiros Oliveira ◽  
Lenilton Silva DA Silva Júnior ◽  
Antonia Eduarda Martins Oliveira Elói Silva ◽  
João Pedro Andrade Lima ◽  
Victor lima Soares ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Clinical Laboratory ◽  
Conflicts Of Interest ◽  
Demographic Data ◽  
Hematopoietic Tissue ◽  
Diagnostic Challenge ◽  
Acute Leukemias ◽  
Acute Myeloid

INTRUDUCTION: Acute myeloid leukemia (AML) is a heterogeneous group of clonal disease of hematopoietic tissue, characterized by proliferation of abnormal myeloid progenitor cells, suppressing the normal hematopoietic activity and constituting a great diagnostic challenge. With the advent of immunophenotyping by flow cytometry, the diagnosis of these neoplasms became more faithful, facilitating the treatment and follow-up of the patients. METHODS: In this study a flow cytometric immunophenotyping study was performed in bone marrow aspirate and / or peripheral blood samples from 38 patients with AML, using a panel of monoclonal antibodies specific for acute leukemias, also investigating clinical, laboratory and demographic data of these patients. RESULTS AND DISCUSSION: Of the 38 subjects studied, 23 were male and 15 females. In relation to the age, we found a higher number of cases in adult patients. According to clinical data, splenomegaly and hepatomegaly were present in most cases. The immunophenotyping demonstrated a characteristic profile of AML with expression of CD13 and CD33 in all cases and CD34 and CD117 in most cases. The CD14 was reactive in monocytic leukemia (M4 and M5-AML). Were observed negativity for lymphoid antigens CD19, CD10 and CD3 and positivity to CD7, CD56 and CD2, present in 10, 5 and one cases respectively. Regarding cytomorphology, there was a direct correlation with FAB classification, with the prevalence of type myeloid-monocytic (AML-M4). CONCLUSION: These data demonstrate the importance of immunophenotyping in the differential diagnosis of AML and monitoring of these neoplasms. Disclosures No relevant conflicts of interest to declare.

A New In Vivo Platform for Studying the Growth of Hematopoietic Cancer Initiating Blood Cells and Response to Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4142-4142
Author(s):  
Michal Cipok ◽  
Yona Farnoushi ◽  
Sigi Kay ◽  
Igor Grinberg ◽  
Arbel Reis ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Pcr Analysis ◽  
Human Leukemia ◽  
Human Stem Cell ◽  
Cell Leukemia ◽  
Myeloma Cells ◽  
Fold Reduction ◽  
Stem Cell Leukemia

Abstract Abstract 4142 There are few in-vivo models for studying human leukemia and its therapy. These include the high cost immune-deficient NOD/SCID mice and large mammalian fetuses, which both require weeks to assess treatment response. We described a rapid and low-cost alternative in-vivo system for human leukemia in the preimmune chick embryo (Taizi et al, Exp Hem 34;1698-708,2006). We recently demonstrated that the turkey embryo provides a more robust model for the preclinical assessment of human leukemia infiltration (Grinberg et al, Leukemia Res 33;1417-26 2009). Here we describe the application of this powerful and inexpensive model for rapid preclinical assessment of anti-cancer therapies and basic research of blood malignancies. BM engraftment was robust occurring in 95% of the embryos using cells lines and 40% using fresh samples. Leukemia cells homed to the BM where they were already detected at 20 hours after injection and reached highest levels on days E19-24, using FACS and RT-PCR. Serial engraftment in secondary recipients of all three human stem cell leukemia lines and one fresh sample was detected in embryos injected with BM harvested 8-10 days after the first inoculation, validating the engraftment of cancer initiating cells. Human stem cell leukemia lines K562 and LAMA, (both BCR/Abl+) and CHRF (c-Kit+), and myeloma cell lines ARH-77 and CAG and fresh patient samples were injected IV into turkeys, on embryonic day E 11, using 5×106 cells from lines or 107 fresh patient cells. Engraftment of human leukemia and myeloma cells (cell lines and fresh samples) was detected 8-14 days later (E19-24), in the BM and in several hematopoietic organs at a frequency of 0.5->20%, by real-time PCR, immunohistochemistry and flow cytometry. CAG and ARH-77 myeloma cells engraftment was also detected by the presence of human monoclonal free light chain (6-10 mg/L) in blood collected from vessels of the chorioallantoic membrane, one week after cell injection. The growth of leukemias treated with doxorubicin or the tyrosine-kinase inhibitor Imatinib and myeloma with Velcade, at levels that were not toxic to the developing embryonic BM, was dramatically inhibited in vivo when the drug was injected together with cells on E11 or 48-72 hours after injection of the cells and homing to the BM. Using flow cytometry analysis the frequency of CHRF cells (detected with anti-human CD33) was reduced from 8% to 0.01% and K562 and LAMA (detected with anti-human CD71) from 1%-3% engraftment to <0.17% following treatment with Imatinib. Q-PCR analysis supported these results showing an average 8 fold reduction of CHRF and a 2-5 fold reduction of K562 and LAMA cells in the Imatinib treated turkey embryos. The ARH-77 cells (detected with anti-human CD38 and CD138) were inhibited from 8.5% to 0.72% after Velcade treatment, with a 16.5 fold reduction determined by Q-PCR analysis compare to untreated embryos. These results prove the efficacy and demonstrate the utility of the turkey embryo as a new complementary in-vivo model for studying cancer initiating cells and the growth of human blood malignancies and their response to treatment. With further improvements, it may provide an affordable, rapid in vivo system for studying the growth blood malignancies and help reduce time and cost of drug development. Disclosures: No relevant conflicts of interest to declare.

Targeting HRASV12G Expression to the Zebrafish Early Hemogenic Progenitors Induces a Myeloproliferative Disorder by Repressing the Notch Pathway

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4676-4676
Author(s):  
Elisa Alghisi ◽  
Michele Malagola ◽  
Cristina Santoriello ◽  
Martin Distel ◽  
Christiaan Henkel ◽  
...  
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Juvenile Fish ◽  
Notch Pathway ◽  
Myeloproliferative Disorders ◽  
Experimental Models ◽  
Hematopoietic Tissue ◽  
Erythroid Progenitors ◽  
Oncogene Expression ◽  
Notch Pathways

Abstract Abstract 4676 Introduction Myeloproliferative diseases (MPDs) are a group of haematological disorders characterized by the hyper proliferation of different blood cells in peripheral blood and other hematopoietic organs. The clinical heterogenity of these neoplasms reflects the different gene pathways involved, most of which are only partially known. Given the genetic homology and the physiological similarity to mammals, zebrafish has emerged as an ideal model to study human normal and malignant haematopoiesis. In the last decade several oncogenes involved in the development of hematopoietic neoplasms have been used to model leukemia in zebrafish with the aim to discovery new molecular pathways involved in malignant transformation. Despite the first encouraging results these experimental models failed to fully recapitulate human myeloproliferative disorders. Methods We took advantage of the Gal4/UAS binary system to induce the expression of human oncogenic HRASV12G in the zebrafish hematopoietic compartment. We used a specific transgenic line that drives oncogene expression in zebrafish early hematopoietic progenitors under control of the FLI.1 (Friend Leukemia virus Integration 1) promoter. Results We observed the development of a myelo-erythroid proliferative disease in few days in zebrafish transgenic larva. The pathological phenotype is characterized by the expansion of the hematopoietic tissue, an increased expression of myelo-erythroid specific genes (PU.1, gata1, mpx, c-mpl) associated with a slight increase of staminality markers (lmo2, scl, c-myb, runx.1), and a higher number of l-plastin expressing cells. Moreover blood smear of pathological larva displayed leukemic blasts and the arrest of erythrocyte differentiation whereas kidney marrow of juvenile fish displayed abnormal myelopoiesis characterized by the increase of erythro-myeloid progenitors. We found that the pathological phenotype is associated with a down regulation of the Notch pathway as shown by the decreased gene expression of notch pathways target genes (notch1, notch3, her6). Furthermore we discovered a novel set of genes involved in neoplastic transformation induced by HRASV12 expression through RNA-Seq analysis of pathological larva. Conclusions The expansion of the zebrafish hematopoietic compartment characterized by the hyper-proliferation of the myelo-erythroid progenitors that we found in this model reproduces some of the pathological features of human myeloproliferative disorders. This study showed that forcing oncogene expression in the hemogenic endothelial cells induces the transdifferentiation of the early hemogenic pluripotent stem cells into abnormal myeloerythoid progenitors by repressing the Notch pathways. Transcriptome analysis identified a number of potential effectors of this transformation. Disclosures: No relevant conflicts of interest to declare.

Erythropoietin Treatment Enhanced Cell Surface CXCR4 Expression On Neonatal CD34-Positive Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4724-4724
Author(s):  
Norioki Ohno ◽  
Seiichi Hayakawa ◽  
Shuhei Karakawa ◽  
Masao Kobayashi
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cell Surface ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cxcr4 Expression ◽  
Stem Cell Marker ◽  
Cell Marker ◽  
Low Oxygen ◽  
Hematopoietic Stem

Abstract Abstract 4724 Erythropoietin (EPO), which is used to treat anemic immature neonates, is thought to be effective for treating neonatal hypoxic encephalopathy by stimulating nervous system cells or vascular stem cells. On stem cells, CXCR4 is an important chemokine receptor for cell migration and proliferation. Previously, we reported that increased expression of CXCR4 on cord blood (CB)-derived CD34-positive cells, caused by low oxygen tension, enhanced cell homing activity and engraftment (Ohno et al., ASH. 2010). In human endothelial stem cells, CXCR4 expression is controlled by hypoxia-inducible factor-1α (HIF-1α), and EPO expression is enhanced by hypoxia via HIF-1α. In this study, we examined the effect of EPO treatment on CXCR4 expression on premature neonatal endothelial stem cells and cultured CB-derived CD34-positive cells. First, we examined the cell surface markers CD34, CD133, and CXCR4 in preterm infants diagnosed with anemia and treated with EPO. The 10 enrolled neonates were first administered EPO when they were over 32 weeks old and were not given oxygen therapy. Blood samples were collected twice before and once 24 h after EPO administration. Mononuclear cells were isolated from peripheral blood and stained for the hematopoietic stem cell marker CD34, vascular endothelial progenitor cell marker CD133, and surface CXCR4. The cells were analyzed by flow cytometry. No change in the proportions of CD34- and CD133-positive cells were found after the treatment (CD34 positive: 0.59%→0.57%; CD133 positive: 0.52%→0.49%). The proportion of cell surface CXCR4 on the CD133-positive cells did not change after EPO administration, whereas the proportion of cell surface CXCR4 on the CD34-positive cells increased significantly after EPO was administered (average: 7.6%→11.0%, p < 0.01). In particular, slightly CD34-positive cells showed enhanced cell surface CXCR4. Next, we examined CXCR4 enhancement in EPO-treated CB-derived CD34-positive enriched samples. Samples were divided into three aliquots and cultured. The first aliquot was incubated for 24 h in RPMI-1640 medium alone as a control; the second, for 24 h in RPMI-1640 medium with EPO (10 U/ml); and the third, for 24 h in RPMI-1640 medium with EPO (10 U/ml) containing the specific HIF-1 antagonist rapamycin. Flow cytometry revealed significantly increased surface CXCR4 expression on CD34-positive cells after incubation with EPO compared with the control expression (average: 51.7% vs. 30.3%, p< 0.05). This enhancement was inhibited completely by the addition of rapamycin. Intracellular HIF-1 was enhanced significantly in the EPO-treated cells compared with the control expression on flow cytometry. The enhanced CXCR4 expression on CD34-positive cells reflects an amplification of the chemotactic and homing abilities of stem cells. The CXCR4 enhancement caused by EPO may increase tissue migration of CD34-positive cells, although this is probably a result of an active HIF-1 pathway. Disclosures: No relevant conflicts of interest to declare.

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