Stage-Restricted Non-Metabolic Function for Aconitase Enzymes in Erythropoietin Programming of Erythroid Growth and Differentiation in Vivo

Abstract Erythropoietin (EPO) is the main cytokine responsible for red blood cell production in the human marrow. Signal transduction through the EPO receptor consists of multiple limbs that promote the proliferation, differentiation, and survival of erythroid progenitors. Regulatory loops that modulate EPO signaling are not fully understood, but could prove critical in providing new therapeutic approaches to EPO-refractory anemias. One such poorly understood loop is the regulation of EPO-driven erythropoiesis by iron. Previously presented work has established that iron deprivation acts in a lineage specific manner to block erythroid development and inhibit the activity of aconitase, a Krebs cycle enzyme that interconverts citrate and isocitrate. In the current studies, the causal relationship between aconitase inhibition and erythroid blockade was characterized in normal donor-derived mobilized human primary hematopoietic CD34+ cells and in adult wild type C57BL/6 mice. We hypothesized that critical thresholds of aconitase activity are required for specific facets of EPO signaling during discrete phases of erythroid development. CD34+ cells in erythroid-promoting culture conditions (EPO and iron) were strongly growth inhibited in a dose-dependent manner when treated with fluoroacetate, a reversible aconitase inhibitor. Interestingly, there was no increase in overall cell death in the growth impaired cultures, providing the first evidence that growth and survival signals in primary erythroid precursors can be dissociated. Aconitase blockade also inhibited EPO-dependent erythroid maturation, with decreased glycophorin A upregulation, diminished CD34 downregulation, and a sharp decrease in globin chain protein levels after 5 days of culture. Biochemical analysis of known EPO targets showed no alterations in the phosphorylation status of STAT5, Akt and PKCalpha, effectively ruling out a role for the corresponding pathways in the impaired growth and differentiation. Moreover, intracellular ATP levels were unaffected by aconitase inhibition, and alterations in AMPK activation, an intracellular sensor of the ATP:AMP ratio, could not be detected. These results argue against energy starvation as the cause of the observed developmental defects. Cell cycle analysis using propidium iodide showed no evidence for phase-specific arrest, excluding standard checkpoint mechanisms. Delayed addition or washout of fluoroacetate at various time points during cultures identified the existence of aconitase-dependent and subsequent aconitase-independent phases of human erythroid development. To extend these studies to in vivo erythropoiesis, C57BL/6 mice underwent fluoroacetate (n=10) or saline (n=10) treatment with continuous infusion pumps at a drug dose of 4 mg/kg/day. In vivo blockade of aconitase over a two-week period resulted in an anemia characterized by a significant decrease in mean hemoglobin (11.6 vs. 14.7 g/dL, P<0.001), hematocrit (37.3 vs. 44%, P<0.001) and red cell number (7.49 vs. 9.32 × 1012 cells/liter, P<0.001). No thrombocytopenia or neutropenia was noted. Reticulocytes were diminished (6.6 vs. 7.9%, P<0.001), and mean serum EPO levels were 3 fold higher in the treated animals (533.2 vs. 172.3 pg/ml, P<0.001). Flow cytometric analysis of the marrow erythroid compartment consistently showed accumulation of cells at a Ter119-bright C71-intermediate stage (n=3 for each group). In summary, our data establish a new function for aconitase in EPO-driven erythropoiesis that is distinct from its metabolic role in cellular energy homeostasis. Sustained aconitase activity is required for the proliferation and maturation of erythroid precursors, but does not impact survival or specific cell cycle phases. Furthermore, this requirement for aconitase activity is stage specific and restricted to an early phase of EPO-dependent erythroid development. Taken together, these results suggest the existence of a new regulatory loop in which levels of aconitase activity modulate EPO-mediated growth and maturation. This novel checkpoint could provide new targets in the treatment of many disorders of red cell production.

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Effects of NO-Donors on Thrombus Formation and Microcirculation in Cerebral Vessels of the Rat

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650532 ◽

1996 ◽

Vol 76 (01) ◽

pp. 111-117 ◽

Cited By ~ 10

Author(s):

Yasuto Sasaki ◽

Junji Seki ◽

John C Giddings ◽

Junichiro Yamamoto

Keyword(s):

Blood Flow ◽

Thrombus Formation ◽

Dependent Manner ◽

Red Cell ◽

Cell Velocity ◽

Red Cell Velocity ◽

The Mean ◽

Wall Shear ◽

Dose Dependent

SummarySodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 Μg/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 Μg/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction

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A Novel Transcriptional Repression Domain Mediates p21WAF1/CIP1 Induction of p300 Transactivation

Molecular and Cellular Biology ◽

10.1128/mcb.20.8.2676-2686.2000 ◽

2000 ◽

Vol 20 (8) ◽

pp. 2676-2686 ◽

Cited By ~ 88

Author(s):

Andrew W. Snowden ◽

Lisa A. Anderson ◽

Gill A. Webster ◽

Neil D. Perkins

Keyword(s):

Cell Cycle ◽

Transcriptional Activation ◽

Transcriptional Repression ◽

Kinase Inhibitor ◽

Core Promoter ◽

Dependent Manner ◽

Creb Binding Protein ◽

Cycle Dependent ◽

Repression Domain

ABSTRACT The transcriptional coactivators p300 and CREB binding protein (CBP) are important regulators of the cell cycle, differentiation, and tumorigenesis. Both p300 and CBP are targeted by viral oncoproteins, are mutated in certain forms of cancer, are phosphorylated in a cell cycle-dependent manner, interact with transcription factors such as p53 and E2F, and can be found complexed with cyclinE-Cdk2 in vivo. Moreover, p300-deficient cells show defects in proliferation. Here we demonstrate that transcriptional activation by both p300 and CBP is stimulated by coexpression of the cyclin-dependent kinase inhibitor p21WAF/CIP1. Significantly this stimulation is independent of both the inherent histone acetyltransferase (HAT) activity of p300 and CBP and of the previously reported carboxyl-terminal binding site for cyclinE-Cdk2. Rather, we describe a previously uncharacterized transcriptional repression domain (CRD1) within p300. p300 transactivation is stimulated through derepression of CRD1 by p21. Significantly p21 regulation of CRD1 is dependent on the nature of the core promoter. We suggest that CRD1 provides a novel mechanism through which p300 and CBP can switch activities between the promoters of genes that stimulate growth and those that enhance cell cycle arrest.

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Reconstruction of Sickle Cell Disease with Circulating Sickling Red Blood Cells in Novel Humanized Cytokines and Liver Mistrg Mice

Blood ◽

10.1182/blood-2020-141603 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 29-30

Author(s):

Yuanbin Song ◽

Rana Gbyli ◽

Liang Shan ◽

Wei Liu ◽

Yimeng Gao ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Red Cell ◽

Cell Disease ◽

Ineffective Erythropoiesis ◽

Cd34 Cells

In vivo models of human erythropoiesis with generation of circulating mature human red blood cells (huRBC) have remained elusive, limiting studies of primary human red cell disorders. In our prior study, we have generated the first combined cytokine-liver humanized immunodeficient mouse model (huHepMISTRG-Fah) with fully mature, circulating huRBC when engrafted with human CD34+ hematopoietic stem and progenitor cells (HSPCs)1. Here we present for the first time a humanized mouse model of human sickle cell disease (SCD) which replicates the hallmark pathophysiologic finding of vaso-occlusion in mice engrafted with primary patient-derived SCD HSPCs. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Murine models of SCD exclusively express human globins in mouse red blood cells in the background of murine globin knockouts2 which exclusively contain murine erythropoiesis and red cells and thus fail to capture the heterogeneity encountered in patients. To determine whether enhanced erythropoiesis and most importantly circulating huRBC in engrafted huHepMISTRG-Fah mice would be sufficient to replicate the pathophysiology of SCD, we engrafted it with adult SCD BM CD34+ cells as well as age-matched control BM CD34+ cells. Overall huCD45+ and erythroid engraftment in BM (Fig. a, b) and PB (Fig. c, d) were similar between control or SCD. Using multispectral imaging flow cytometry, we observed sickling huRBCs (7-11 sickling huRBCs/ 100 huRBCs) in the PB of SCD (Fig. e) but not in control CD34+ (Fig. f) engrafted mice. To determine whether circulating huRBC would result in vaso-occlusion and associated findings in SCD engrafted huHepMISTRG-Fah mice, we evaluated histological sections of lung, liver, spleen, and kidney from control and SCD CD34+ engrafted mice. SCD CD34+ engrafted mice lungs showed an increase in alveolar macrophages (arrowheads) associated with alveolar hemorrhage and thrombosis (arrows) but not observed control engrafted mice (Fig. g). Spleens of SCD engrafted mice showed erythroid precursor expansion, sickled erythrocytes in the sinusoids (arrowheads), and vascular occlusion and thrombosis (arrows) (Fig. h). Liver architecture was disrupted in SCD engrafted mice with RBCs in sinusoids and microvascular thromboses (Fig. i). Congestion of capillary loops and peritubular capillaries and glomeruli engorged with sickled RBCs was evident in kidneys (Fig. j) of SCD but not control CD34+ engrafted mice. SCD is characterized by ineffective erythropoiesis due to structural abnormalities in erythroid precursors3. As a functional structural unit, erythroblastic islands (EBIs) represent a specialized niche for erythropoiesis, where a central macrophage is surrounded by developing erythroblasts of varying differentiation states4. In our study, both SCD (Fig. k) and control (Fig. l) CD34+ engrafted mice exhibited EBIs with huCD169+ huCD14+ central macrophages surrounded by varying stages of huCD235a+ erythroid progenitors, including enucleated huRBCs (arrows). This implies that huHepMISTRG-Fah mice have the capability to generate human EBIs in vivo and thus represent a valuable tool to not only study the effects of mature RBC but also to elucidate mechanisms of ineffective erythropoiesis in SCD and other red cell disorders. In conclusion, we successfully engrafted adult SCD patient BM derived CD34+ cells in huHepMISTRG-Fah mice and detected circulating, sickling huRBCs in the mouse PB. We observed pathological changes in the lung, spleen, liver and kidney, which are comparable to what is seen in the established SCD mouse models and in patients. In addition, huHepMISTRG-Fah mice offer the opportunity to study the role of the central macrophage in human erythropoiesis in health and disease in an immunologically advantageous context. This novel mouse model could therefore serve to open novel avenues for therapeutic advances in SCD. Reference 1. Song Y, Shan L, Gybli R, et. al. In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice. Blood. 2019;134:338. 2. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science. 1997;278(5339):873-876. 3. Blouin MJ, De Paepe ME, Trudel M. Altered hematopoiesis in murine sickle cell disease. Blood. 1999;94(4):1451-1459. 4. Manwani D, Bieker JJ. The erythroblastic island. Curr Top Dev Biol. 2008;82:23-53. Disclosures Xu: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Flavell:Zai labs: Consultancy; GSK: Consultancy.

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A novel Amh-Treck transgenic mouse line allows toxin-dependent loss of supporting cells in gonads

Reproduction ◽

10.1530/rep-14-0171 ◽

2014 ◽

Vol 148 (6) ◽

pp. H1-H9 ◽

Cited By ~ 8

Author(s):

Mai Shinomura ◽

Kasane Kishi ◽

Ayako Tomita ◽

Miyuri Kawasumi ◽

Hiromi Kanezashi ◽

...

Keyword(s):

Germ Cells ◽

Granulosa Cells ◽

Sertoli Cells ◽

Seminiferous Tubules ◽

Specific Cell ◽

Partial Recovery ◽

Dependent Manner ◽

Mouse Line ◽

Cell Degeneration

Cell ablation technology is useful for studying specific cell lineages in a developing organ in vivo. Herein, we established a novel anti-Müllerian hormone (AMH)-toxin receptor-mediated cell knockout (Treck) mouse line, in which the diphtheria toxin (DT) receptor was specifically activated in Sertoli and granulosa cells in postnatal testes and ovaries respectively. In the postnatal testes of Amh-Treck transgenic (Tg) male mice, DT injection induced a specific loss of the Sertoli cells in a dose-dependent manner, as well as the specific degeneration of granulosa cells in the primary and secondary follicles caused by DT injection in Tg females. In the testes with depletion of Sertoli cell, germ cells appeared to survive for only several days after DT treatment and rapidly underwent cell degeneration, which led to the accumulation of a large amount of cell debris within the seminiferous tubules by day 10 after DT treatment. Transplantation of exogenous healthy Sertoli cells following DT treatment rescued the germ cell loss in the transplantation sites of the seminiferous epithelia, leading to a partial recovery of the spermatogenesis. These results provide not only in vivo evidence of the crucial role of Sertoli cells in the maintenance of germ cells, but also show that the Amh-Treck Tg line is a useful in vivo model of the function of the supporting cell lineage in developing mammalian gonads.

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Whole Exome Sequencing of a Patient with Atypical Congenital Pure Red Cell Aplasia Has Enabled the Identification of a Novel Key Actor of Erythropoiesis That May be Involved in CDAI Physiopathology

Blood ◽

10.1182/blood-2020-136078 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 3-4

Author(s):

Elia Colin ◽

Genevieve Courtois ◽

Lydie Da Costa ◽

Carine Lefevre ◽

Michael Dussiot ◽

...

Keyword(s):

Cell Cycle ◽

Homologous Recombination ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Research Funding ◽

Pure Red Cell Aplasia ◽

Red Cell ◽

Whole Exome

Background: The development of next generation sequencing techniques has brought important insights into the molecular mechanisms of erythropoiesis and how these processes can be perturbed in human diseases. This strategy may be valuable in some hereditary erythroid disorders where a subset of patients does not carry any mutations in the supposed causal gene and for which transgenic mouse models do not recapitulate the phenotype, suggesting that additional genetic events may be involved in pathogenesis. Here, we report the case of an adult patient presenting with atypical pure red cell aplasia associated with facial dysmorphy and chronic leg ulcers. Whole exome sequencing revealed a heterozygous missense mutation (R725W) in the CDAN1 gene, which has been previously reported in congenital dyserythropoietic anemia type I (CDAI). However, this mutation was also detected in her healthy brother, suggesting that this event alone was not sufficient to explain her phenotype. According to this hypothesis, we found an additional germline heterozygous nonsense mutation (Q732X) in the MMS22L gene, which was not shared by her unaffected relatives. MMS22L is a protein involved in homologous recombination-dependent repair of stalled or collapsed replication forks. Additionally, MMS22L is able to bind newly synthesized soluble histones H3 and H4 and exhibits a histone chaperone activity. MMS22L loading onto ssDNA during homologous recombination is promoted by the histone chaperone ASF1. Interestingly, CDAN1 acts as a negative regulator of ASF1 by mediating its sequestration in the cytoplasm, which results in the blocking of histone delivery. Aims: As MMS22L has never been reported in erythropoiesis before, we aimed to investigate the role of MMS22L in human erythropoiesis. Based on the data summarized above, the purpose of this study was also to determine the effect of combined inactivation of MMS22L and CDAN1 on in vivo erythropoiesis, while exploring the functional cooperation between both proteins. Results: To decipher the role of MMS22L in human erythropoiesis, we assessed the consequences of complete MMS22L inactivation in human cord blood CD34+ progenitors as well as in CD36+ immature erythroblasts using shRNA lentiviruses. This resulted in a severe decrease of cell proliferation and differentiation due to G1 cell cycle arrest, with a slight increase of apoptosis. Interestingly, this phenotype was not observed when MMS22L was inactivated in the granulo-monocytic lineage, in which differentiation was maintained, suggesting that erythroid cells, that are highly proliferative, are more sensitive to MMS22L inactivation. To better understand the effect of combined CDAN1 and MMS22L haploinsufficiency observed in the proband, we used zebrafish as an in vivo model. Mms22l and cdan1 expression were simultaneously or separately downregulated by about 50% using antisens morpholino oligomers. 48 hours later, zebrafish embryos were stained with o-dianisidine to detect hemoglobin-containing cells. We found that combined knock-down of mms22l and cdan1 resulted in severe anemia, while knock-down of mms22l or cdan1 alone did not lead to any erythroid disorder. This experiment provides a proof-of-concept, indicating that the phenotype of the proband is indeed caused by the combination of both MMS22L and CDAN1 mutations. Finally, in order to decipher the cooperation between MMS22L and CDAN1 we used the human erythroid UT-7 cell line. We found that CDAN1 inactivation resulted in a severe decrease in MMS22L expression within the nucleus, suggesting that CDAN1 may regulate MMS22L expression or localization. We therefore wanted to confirm these results by assessing MMS22L expression in B-EBV cell lines established from two CDAI patients with CDAN1 compound heterozygous mutations. We found a great decrease in MMS22L expression within the nucleus of the CDAI patients' cells when compared to three control B-EBV cell lines. Based on these results, we suggest that impairment of MMS22L trafficking to the nucleus could be involved in CDA1 physiopathology. Conclusion: Through comprehensive genetic analysis of a single case with atypical congenital anemia, we demonstrated for the first time that MMS22L, a cell cycle regulator, is essential for the process of erythropoiesis. The crosstalk between MMS22L and CDAN1 is currently under investigation and could bring important new insights into the physiopathology of CDAI. Disclosures Hermine: Novartis: Research Funding; Alexion: Research Funding; AB Science: Consultancy, Current equity holder in publicly-traded company, Honoraria, Patents & Royalties, Research Funding; Celgene BMS: Consultancy, Research Funding; Roche: Consultancy.

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AMD3100-Mobilized CD34+ Cells Are Phenotypically Different and Better Targets for Retroviral Transduction Than G-CSF-Mobilized CD34+ Cells in Rhesus Macaques.

Blood ◽

10.1182/blood.v104.11.2686.2686 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2686-2686

Author(s):

Andre Larochelle ◽

Allen Krouse ◽

Donald Orlic ◽

Robert E. Donahue ◽

Cynthia E. Dunbar ◽

...

Keyword(s):

Cell Cycle ◽

Steady State ◽

Genetic Manipulation ◽

Rhesus Macaques ◽

Post Transplantation ◽

Alternative Source ◽

Cd34 Cells

Abstract AMD3100 (AMD) has recently been shown to rapidly mobilize primitive hematopoietic cells in mice and humans, but little is known about the properties of cells mobilized with this agent. We initiated a study to determine retroviral (RV) in vivo gene marking efficiency in AMD-mobilized CD34+ cells in rhesus macaques. CD34+ cells collected 3 hours after administration of AMD to 2 animals were transduced using RV vectors containing the NeoR gene. Animals were irradiated and cells reinfused immediately after transduction. By molecular analysis, the levels of PB MNC and granulocyte NeoR gene marking at steady-state (up to 12 months post-transplantation) was 1–2% in animal RC909 and 30–40% in RQ2851. In two additional rhesus macaques, CD34+ cells were collected from steady-state BM and from the PB after mobilization with AMD or G-CSF (G). The two PB populations from each animal were transduced with one of two distinguishable NeoR vectors and simultaneously reinfused into irradiated animals. In animal RQ3590, 2% in vivo gene marking at steady-state (up to 4 months post-transplantation) was derived from AMD-mobilized cells compared to 0.05% from the G-mobilized fraction. Animal RQ3636 showed 10% in vivo marking from the AMD-mobilized fraction and no detectable marking from the G-mobilized cells. We also compared phenotypic and functional characteristics of CD34+ cells from BM, AMD-PB and G-PB. An average of 31% of the AMD-mobilized cells were in the Go phase of the cell cycle, compared to 79% of G-mobilized cells (p=0.02), and 45% for the BM fraction (p=0.24). In contrast, 64% AMD-mobilized cells were in G1 compared to 17% of G-mobilized cells (p=0.03) and 44% for the BM fraction (p=0.15). Flow cytometry showed CXCR4 expression on 59% AMD-mobilized cells, in comparison to 11% G-mobilized cells (p=0.02) and 22% BM cells (p=0.07). Similar results were obtained when comparing VLA-4 expression. The increased expression of CXCR4 on AMD-mobilized CD34+ cells correlated with their increased ability to migrate towards SDF-1α in vitro (45%) compared to G-mobilized cells (8%, p=0.01) and BM cells (17%, p=0.08). Our data indicate efficient long-term in vivo gene marking in the rhesus macaque model, validating the ability of AMD to induce mobilization of true long-term repopulating HSCs. AMD-mobilized PB HSCs represent an alternative source of HSCs amenable to genetic manipulation with integrating RV vectors, with potential applications in gene therapy approaches for patients with sickle cell anemia; documented complications have precluded mobilization using G or G/SCF in these patients. Also, cell cycle status and surface phenotype of AMD-mobilized CD34+ cells are more comparable to steady-state BM cells than G-mobilized PB HSCs. AMD-mobilized CD34+ cells are more actively cycling than G-mobilized CD34+ cells, correlating with the increased efficiency of replication-dependent retrovirus-mediated gene transduction. The increased expression of the adhesion receptors CXCR4 and VLA-4 on primitive AMD-mobilized cells compared to G-mobilized cells suggests fundamental differences in the mechanisms of AMD-mediated and cytokine-mediated stem cell mobilization.

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Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.

Blood ◽

10.1182/blood.v108.11.83.83 ◽

2006 ◽

Vol 108 (11) ◽

pp. 83-83

Author(s):

Alex J. Tipping ◽

Cristina Pina ◽

Anders Castor ◽

Ann Atzberger ◽

Dengli Hong ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Zinc Finger ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Colony Number

Abstract Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.

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The PI3K Specific Inhibitor BKM120 Results Effective and Synergizes With Ruxolitinib In Preclinical Models Of Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v122.21.1599.1599 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1599-1599 ◽

Cited By ~ 1

Author(s):

Niccolò Bartalucci ◽

Costanza Bogani ◽

Serena Martinelli ◽

Carmela Mannarelli ◽

Jean-Luc Villeval ◽

...

Keyword(s):

Cell Cycle ◽

Colony Formation ◽

Myeloproliferative Neoplasms ◽

Janus Kinase ◽

Preclinical Models ◽

Mtor Inhibition ◽

Jak2 Inhibitor ◽

Cd34 Cells

Abstract Background and Aims A gain-of-function mutation in Janus kinase 2 (JAK2V617F) is at the basis of the majority of chronic myeloproliferative neoplasms (MPN). The dual JAK1/JAK2 inhibitor ruxolitinib (ruxo) determined rapid and sustained responses in splenomegaly and symptomatic improvement in patients with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling. Enhanced activation of other downstream pathways including the PI3K/mTOR pathway has been documented as well. We previously reported (Bogani et al, PlosOne 2013;8:54828) that targeting mTOR by the allosteric inhibitor RAD001 resulted in inhibition of JAK2VF mutated cells and produced clinical benefits in a phase I/II trial (Guglielmelli et al, Blood 2011;118:2069). In this study we evaluated the effects of BKM120, a specific PI3K inhibitor, alone and in combination with ruxolitinib, in in-vitro and in-vivo MPN models. Methods To evaluate cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status we used mouse BaF3 and BaF3-EPOR cells expressing wild type (WT) or VF mutated JAK2, the human VF-mutated HEL and SET2 cell lines, and primary MPN CD34+ cells from patients with MF or polycythemia vera (PV). Effect of drug combination was analyzed according to Chou and Talalay calculating the combination index (CI); a CI <1 indicates synergistic activity. For in vivo studies we used two mouse models: (1) SCID mice receiving iv BaF3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on day 6 to different treatment groups based on baseline luminescence. (2) C57Bl6/J JAK2 VF Knock-in mice were generated by insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies with VF heterozygous expression (Hasan et al, Blood 2013;Epub). Mice were treated for 15 days, then blood, spleen and bone marrow cells were analyzed. Results We found that BKM120 preferential inhibited BAF3 VF and BaF3-EpoR VF cells (IC50: 364±200nM and 1100±207nM, respectively) compared to their respective WT counterpart (5300±800nM and 3122±1000nM: p<.05). HEL and SET2 cells resulted also sensitive to BKM120 (2000±500nM and 1000±300nM). Interestingly we found that BKM120 significantly increased G2/M phase and decreased S phase of cell cycle (p<.01) and induced apoptosis (IC50, SET2=10µM, BaF3-EPOR VF=1.8 µM). Western blot analysis showed marked reduction of phospho-mTOR and its target phospho-4EBP1 as well as downregulation of phospho-STAT5 at 6 and 24h of treatment. BKM120 impaired colony formation from MF and PV CD34+ cells at doses 2 to 8-fold lower than healthy controls (p<.01). BKM120 strongly inhibited EEC colony growth from PV pts (IC50, 9±4nM). Co-treatment of BKM120+ruxo resulted in synergistic inhibition of proliferation of SET2 (median CI=0.45) and BaF3-EPOR VF (median CI=0.8) cells. Triple combinations including BKM120/ruxo plus either RAD001 (Torc1 inhibitor) or PP242 (Torc1/2 inhibitor) resulted highly synergistic (median CI=0.27 and 0.52) to indicate the importance of complete mTOR inhibition. BKM120 at 45mpk and 60mpk increased mean lifespan of BaF3 VF luc mouse model from 21d in control mice to 27.2d and 28d in BMK120 treated mice. In KI mice, co-treatment with 60mpk BKM120 + 60mpk ruxo resulted in improvement of splenomegaly (median spleen weight: 1.4, 0.82, 0.8 and 0.6 g respectively for controls, 60mpk BKM120, 60mpk ruxo and 60mpk BKM120+60mpk ruxo) and reduction of leukocytosis and reticulocyte count. The level of phosho-STAT5 and -4EBP1 in the spleen was significantly reduced in mice receiving BKM120+ruxo as compared to single drug treatment. We finally analyzed the effects of BKM120+/-ruxo on the in-vitro clonogenic growth of BM cells from VF and WT KI mice mixed in a 1:1 ratio. The proportion of VF-positive colonies resulted reduced in a dose dependent manner by 19%, 33% and 44% (p<.03) compared to controls with 50nM, 100nM and 300nM BKM120 respectively. A 25% and 39% of VF-positive colonies reduction was achieved with 50nM and 100nM ruxolitinib. The combined treatment with 100nM BKM120 + 50nM ruxo resulted in a 50% decrease of the number of mutated colonies (p<.02). Conclusions Inhibition of PI3K by BKM120 alone and combined with JAK2 inhibitor ruxolitinib resulted in enhanced activity in preclinical models of MPN, providing a rationale for the ongoing combination clinical trial. Disclosures: Vannucchi: Novartis: Membership on an entity’s Board of Directors or advisory committees.

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ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v124.21.4316.4316 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4316-4316

Author(s):

Hendrik Folkerts ◽

Maria Catalina Gomez Puerto ◽

Albertus T.J. Wierenga ◽

Koen Schepers ◽

Jan Jacob Schuringa ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Target Genes ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

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Sequence specificity of docetaxel (Doc) and the proteasome inhibitor PS-341 combinations in androgen-independent (AI) prostate cancer (CaP)

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.13150 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 13150-13150

Author(s):

W. S. Holland ◽

P. N. Lara ◽

T. Kimura ◽

T. Kenosi ◽

D. R. Gandara ◽

...

Keyword(s):

Cell Cycle ◽

Patient Outcomes ◽

Tumor Volume ◽

Specific Cell ◽

Sequence Specificity ◽

Cycle Arrest ◽

Best Response ◽

Altered Cell

13150 Background: AI CaP is an invariably fatal disease. While treatment with Doc, a microtubule-stabilizing taxane, improves survival, patient outcomes remain suboptimal. PS-341 inhibits degradation of cell cycle and tumor suppressor proteins resulting in cycle arrest and apoptosis. We hypothesized that the combination of Doc with PS-341 would abrogate the abnormal survival response seen in AI CaP and lead to improved tumor cell kill, but that results would be dependent on administration schedule due to interactive cell cycle kinetics. Methods: The PC3 cell line model of AI CaP was evaluated in vitro and in vivo to determine response to Doc or PS-341 alone, and in combination in sequences of PS-341→Doc, Doc→PS-341, and simultaneous (PS-341 + Doc). Cell cycle and protein analyses were performed by flow cytometry and Western blotting, respectively. For nu/nu mouse xenografts, 5 × 106 cells were injected subcutaneously into each flank. The agents were administered either together or 24hr apart, with all regimens given weekly [IP doses: Doc: 10 mg/kg; PS-341: 0.5 mg/kg]. Results: in vitro: Each combination showed an increased apoptotic sub-G1 population versus untreated cells, in addition to altered cell cycling in a sequence-specific manner. Of the combinations, PS-341 + Doc showed the largest sub-G1 while Doc→PS-341 had the lowest sub-G1 but the largest S-phase content; in vivo: PS-341 + Doc showed a cytotoxic effect (reduction in tumor volume) while the combinations of Doc→PS-341 and PS-341→Doc both showed growth inhibition (stabilization of tumor growth) as best response. Conclusions: Combinations of PS-341 and Doc have sequence specific cell cycle effects leading to increases in apoptosis (PS-341 + Doc) or cell cycle arrest (Doc→PS-341). Clinical validation of these findings is warranted. (ACS: CRTG-0019701-CCE) No significant financial relationships to disclose.

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