Radiotherapy and immunotherapy converge on elimination of tumor-promoting erythroid progenitor cells through adaptive immunity

Tumor-induced CD45−Ter119+CD71+ erythroid progenitor cells, termed “Ter cells,” promote tumor progression by secreting artemin (ARTN), a neurotrophic peptide that activates REarranged during Transfection (RET) signaling. We demonstrate that both local tumor ionizing radiation (IR) and anti–programmed death ligand 1 (PD-L1) treatment decreased tumor-induced Ter cell abundance in the mouse spleen and ARTN secretion outside the irradiation field in an interferon- and CD8+ T cell–dependent manner. Recombinant erythropoietin promoted resistance to radiotherapy or anti–PD-L1 therapies by restoring Ter cell numbers and serum ARTN concentration. Blockade of ARTN or potential ARTN signaling partners, or depletion of Ter cells augmented the antitumor effects of both IR and anti–PD-L1 therapies in mice. Analysis of samples from patients who received radioimmunotherapy demonstrated that IR-mediated reduction of Ter cells, ARTN, and GFRα3, an ARTN signaling partner, were each associated with tumor regression. Patients with melanoma who received immunotherapy exhibited favorable outcomes associated with decreased expression of GFRα3. These findings demonstrate an out-of-field, or “abscopal,” effect mediated by adaptive immunity, which is induced during local tumor irradiation. This effect, in turn, governs the therapeutic effects of radiation and immunotherapy. Therefore, our results identify multiple targets to potentially improve outcomes after radiotherapy and immunotherapy.

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Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3147 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3147-3153 ◽

Cited By ~ 88

Author(s):

G A Blobel ◽

C A Sieff ◽

S H Orkin

Keyword(s):

Bone Marrow ◽

Estrogen Receptor ◽

Transcription Factor ◽

Progenitor Cells ◽

Erythroid Cell ◽

High Dose ◽

Dependent Manner ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

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A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽

10.1182/blood.v75.7.1446.1446 ◽

1990 ◽

Vol 75 (7) ◽

pp. 1446-1454 ◽

Cited By ~ 11

Author(s):

N Katayama ◽

M Nishikawa ◽

F Komada ◽

N Minami ◽

S Shirakawa

Keyword(s):

Progenitor Cells ◽

Colony Formation ◽

Hematopoietic Progenitor Cells ◽

Dependent Manner ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Gm Csf ◽

Erythroid Progenitor Cells ◽

Dependent Inhibition ◽

Dose Dependent

Abstract A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

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Neuromedin U Peptide Activates STAT5 and S6 in a JAK-2 Dependent Manner and Promotes Erythroid Cell Growth in Primary Erythroid Progenitor Cells

Blood ◽

10.1182/blood.v120.21.1241.1241 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1241-1241

Author(s):

Rebecca Lenzo ◽

Martha Dua-Awereh ◽

Martin Carroll ◽

Susan E. Shetzline

Keyword(s):

Cell Growth ◽

Progenitor Cells ◽

Surrogate Marker ◽

Janus Kinase ◽

Erythroid Cell ◽

Dependent Manner ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Erythroid Progenitor Cells ◽

Pi3k Activation

Abstract Abstract 1241 Erythropoiesis is a multi-step process during which hematopoietic stem cells terminally differentiate into red blood cells (RBCs). Erythropoietin (EPO) is the only known cytokine regulator of terminal erythroid differentiation. Previously, we reported that the neuropeptide, neuromedin U (NmU), which interacts with NmU receptor type 1 (NMUR1), functions as a novel extracellular cofactor with EPO to promote the expansion of early erythroblasts, which are CD34−, CD71+, glycophorin A (GlyA)dim(Gambone et al, Blood. 2011). Here, we describe studies to understand the mechanism whereby NmU augments EPO effects on erythroid cell growth. EPO triggers Janus kinase (Jak)-2 dependent activation of signal transducer and activator of transcription (STAT) 5 and phosphatidylinositol 3-kinase (PI3K) to promote the proliferation and/or survival of erythroid progenitor cells. We hypothesized that NmU peptide would cooperate with EPO to promote the proliferation of early erythroblasts through STAT5 and/or PI3K activation. To address this hypothesis, we cultured primary human CD34+ cells in 2-stage liquid culture with IL-3, IL-6, and stem cell factor (SCF) from day 0 to day 6. On day 6, 2U/mL of EPO was added, and the cells were cultured for an additional 5 days to expand erythroid progenitors. On day 11, cells were briefly serum starved and then stimulated with EPO and/or NmU in the absence or presence of a Jak-1/2 inhibitor. Activation of STAT5 and S6, a surrogate marker for PI3K activation, were assessed by phospho-flow in ERY3 (CD34−, CD71+, GlyA+) and ERY4 (CD34−, CD71dim, GlyA+) cells. As expected, EPO alone activated STAT5 and S6 in ERY3 cells only, and the presence of a Jak-1/2 inhibitor diminished STAT5 activation. Interestingly, STAT5 and S6 were activated by NmU peptide alone in ERY3 and ERY4. Surprisingly, in the presence of a Jak-1/2 inhibitor, NmU peptide, which binds to NMUR1 a G-protein coupled receptor, did not activate STAT5 or S6 in ERY3 or 4 cells, suggesting that NmU functions through a JAK kinase in erythroid cells. No additive or synergistic activation of STAT5 and S6 is observed in the presence of both EPO and NmU peptide when EPO was used at a dose of 2 U/mL. The mechanism whereby NmU activates a JAK dependent signaling pathway is under investigation. Preliminary evidence suggests that EPO induces the physical association of NMUR1 with EPO receptor (EPOR). Taken together, we propose that NmU is a neuropeptide expressed in bone marrow cells that cooperates to regulate erythroid expansion during early erythropoiesis through the activation of cytokine receptor like signaling pathways and perhaps through direct interaction with EPOR. NmU may be useful in the clinical management of anemia in patients unresponsive to EPO or other erythroid-stimulating agents. Disclosures: No relevant conflicts of interest to declare.

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A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽

10.1182/blood.v75.7.1446.bloodjournal7571446 ◽

1990 ◽

Vol 75 (7) ◽

pp. 1446-1454

Author(s):

N Katayama ◽

M Nishikawa ◽

F Komada ◽

N Minami ◽

S Shirakawa

Keyword(s):

Progenitor Cells ◽

Colony Formation ◽

Hematopoietic Progenitor Cells ◽

Dependent Manner ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Gm Csf ◽

Erythroid Progenitor Cells ◽

Dependent Inhibition ◽

Dose Dependent

A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

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Mortalin-2 (mthsp70/PBP74/Grp75), a Novel Mediator of Erythropoietin Signal.

Blood ◽

10.1182/blood.v106.11.3141.3141 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3141-3141

Author(s):

Rie Ohtsuka ◽

Yasunobu Abe ◽

Tomomi Fujii ◽

Eriko Nagasawa ◽

Hirotoshi Shimada ◽

...

Keyword(s):

Progenitor Cells ◽

Intracellular Signaling ◽

Antigen Processing ◽

Control Culture ◽

Janus Kinase ◽

Dependent Manner ◽

Transient Transfection Assay ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Peptide Mass

Abstract Erythropoietin (EPO) stimulates erythroid growth by enhancing the proliferation, maturation and survival of late stage erythroid progenitor cells. Intracellular signaling molecules such as Janus kinase 2 (JAK2) and phosphoinositide-3 kinase (PI-3K)/Akt are considered mediators of the EPO signal, however the entire process of EPO stimulation remains undetermined. To further clarify the intracellular mechanism by which EPO affects the growth of erythroid progenitor cells, we analyzed protein obtained from purified human erythroid colony forming cells (ECFCs) cultured with, or without EPO, by 2-dimensional gel electrophoresis. Five protein spots were apparently related to EPO stimuli, and these proteins were then examined by peptide mass fingerprinting. One of the 5 was identified as mortalin-2, which is a member of the heat shock protein 70 family of chaperones. Mortalin-2 is thought to perform multiple functions relevant to stress response, intracellular trafficking, antigen processing, control of cell proliferation and differentiation. When the expression of mortalin-2 was confirmed by Western blotting, the amount of mortalin-2 was found to be greater in ECFCs cultured with EPO. The amount of mortalin-2 mRNA was then analyzed by quantitative real time PCR. In the presence of EPO, the amount of mortalin-2 mRNA gradually increased during erythroid maturation, peaked on day 6, and then decreased in the terminal stage of maturation. When day 6 ECFCs were incubated with different concentrations of EPO, the amount of mortalin-2 mRNA increased in a dose-dependent manner. Next, we designed a small interfering RNA (siRNA) to RNA encoding mortalin-2, and used this in a transient transfection assay. When the siRNA was transfected into day 6 ECFCs, the expression of mortalin-2 mRNA was suppressed to 40–60% of that seen in the control culture. In the presence of different concentrations of EPO (0–1.0 u/ml), the effect of the siRNA on ECFC growth was determined by MTT assay. The siRNA significantly suppressed the growth of ECFCs in the presence of relatively low concentrations of EPO (0.5 u/ml). These data suggest that mortalin-2 mediates the EPO signal to stimulate the growth of erythroid progenitor cells.

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Metformin induces FOXO3-dependent fetal hemoglobin production in human primary erythroid cells

Blood ◽

10.1182/blood-2017-11-814335 ◽

2018 ◽

Vol 132 (3) ◽

pp. 321-333 ◽

Cited By ~ 16

Author(s):

Yankai Zhang ◽

Alireza Paikari ◽

Pavel Sumazin ◽

Carly C. Ginter Summarell ◽

Jacy R. Crosby ◽

...

Keyword(s):

Progenitor Cells ◽

Fetal Hemoglobin ◽

Dependent Manner ◽

Metformin Treatment ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Functional Studies ◽

Erythroid Progenitor Cells ◽

Key Points ◽

Hemoglobin Production

Key Points Functional studies in human primary erythroid progenitor cells support a role for FOXO3 in γ-globin regulation. Metformin treatment of human primary erythroid progenitor cells increases fetal hemoglobin in a partially FOXO3-dependent manner.

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Atiprimod Inhibits the Growth of JAK2 Tyrosine Kinase Over-Expressing Cells “In Vitro” and “Ex Vivo”.

Blood ◽

10.1182/blood.v108.11.4924.4924 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4924-4924

Author(s):

Roberto Nussenzveig ◽

Taghi Manshouri ◽

Hagop Kantarjian ◽

Francis Giles ◽

Jorge Cortes ◽

...

Keyword(s):

Tyrosine Kinase ◽

Progenitor Cells ◽

Cell Lines ◽

Annexin V ◽

Dependent Manner ◽

Wild Type ◽

Erythroid Progenitor ◽

Myeloid Metaplasia ◽

Erythroid Progenitor Cells ◽

Therapeutic Benefits

Abstract Background: A novel somatic single point non-synonymous mutation (1849 G to T) in the tyrosine kinase JAK2, rendering the enzyme constitutively active, has been reported in classical myeloproliferative disorders (MPD), specifically, in greater than 90% of patients with polycythemia vera (PV), and 50% of patients with essential thrombocythemia (ET) and myelofibrosis with myeloid metaplasia (MMM). Consequently, JAK2 tyrosine kinase has become a significant target for the development of specific inhibitors of its activity with possible therapeutic benefits for patients with classical MPD. We report here the results of a study using atiprimod, an anti-inflammatory and anti-cancer compound belonging to the azaspirane class of cationic amphiphilic drugs in JAK2 over-expressing cell lines and patient samples. In contrast to TKI inhibitors, this drug is causing downregulation of JAK2 protein by facilitating its degradation, possibly through the ubiquitin-proteosome pathway (Faderl, S. et al, Leukemia Research 2006 in press). Methods and Results: We have examined the growth inhibitory effect of atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R) and, either wild-type JAK2 (JAK2WT) or mutant JAK2 (JAK2V617F). Atiprimod growth inhibition, measured using 72 hours MTS assay, was significant against both mutant JAK2V617F cells (IC50 0.42μM) and wild-type JAK2WT cells (IC50 0.65μM). Atiprimod was found to induce apoptosis in cell lines as evidenced by an increase in the mitochondrial membrane potential and augmented annexin V positivity by flow cytometry. Mononuclear cells, isolated from peripheral blood of PV patients (JAK2V617F positive), were cultured in cytokine supplemented serum free media for expansion of erythroid progenitors. Expansion spanned a three week, three step sequential treatment of the erythroid progenitor cells with specific combinations of cytokines. Finally, treatment of expanded erythroid progenitor cells with atiprimod for 24 and 48 hours resulted in increased annexin V positive cells, in both, a dose and time dependent manner. Conclusion: Our preliminary pre-clinical results suggest that atiprimod might be a beneficial medication for MPD patients carrying the JAK2V617F mutation. Atiprimod is currently being tested in a phase I clinical study.

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Human thymic epithelial cells maintain long-term survival of clonogenic myeloid and erythroid progenitor cells in vitro

British Journal of Haematology ◽

10.1046/j.1365-2141.2000.02337.x ◽

2000 ◽

Vol 111 (1) ◽

pp. 363-370 ◽

Cited By ~ 1

Author(s):

Katsuto Takenaka ◽

Mine Harada ◽

Tomoaki Fujisaki ◽

Koji Nagafuji ◽

Shinichi Mizuno ◽

...

Keyword(s):

Epithelial Cells ◽

Progenitor Cells ◽

Thymic Epithelial Cells ◽

Erythroid Progenitor ◽

Term Survival ◽

Long Term Survival ◽

Erythroid Progenitor Cells

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Fetal erythropoiesis in steel mutant mice. III. Defect in differentiation from BFU-E to CFU-E during early development

Blood ◽

10.1182/blood.v51.3.539.539 ◽

1978 ◽

Vol 51 (3) ◽

pp. 539-547 ◽

Cited By ~ 18

Author(s):

DH Chui ◽

SK Liao ◽

K Walker

Keyword(s):

Progenitor Cells ◽

Early Development ◽

The Other ◽

Mutant Mice ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Other Hand ◽

Colony Forming Units ◽

Fetal Erythropoiesis

Abstract Erythroid progenitor cells in +/+ and Sl/Sld fetal livers manifested as burst-forming units-erythroid (BFU-E) and colony-forming units- erythroid (CFU-E) were assayed in vitro during early development. The proportion of BFU-E was higher as mutant than in normal fetal livers. On the other hand, the proportion of CFU-E was less in the mutant than in the normal. These results suggest that the defect in Sl/Sld fetal hepatic erythropoiesis is expressed at the steps of differentiation that effect the transition from BFU-E to CFU-E.

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