Cell competition between wild-type and JAK2V617F mutant cells in a murine model of a myeloproliferative neoplasm

List Type ◽

Wild Type ◽

Hematopoietic Stem ◽

Increased Risk ◽

Mutant Cells

AbstractThe myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by overproduction of mature blood cells and increased risk of transformation to frank leukemia. The acquired kinase mutation JAK2V617F plays a central role in a majority of these disorders. The hematopoietic stem cell (HSC) compartment in MPN is heterogeneous with the presence of both JAK2 wild-type and JAK2V617F mutant cells in most patients with MPN. Utilizing in vitro co-culture assays and in vivo competitive transplantation assays, we found that the presence of wild-type cells altered the behavior of co-existing JAK2V617F mutant cells, and a mutant microenvironment (niche) could overcome the competition between wild-type and mutant cells, leading to mutant clonal expansion and overt MPN. We also demonstrated that competition between wild-type and JAK2V617F mutant cells triggered a significant immune response, and there was a dynamic PD-L1 deregulation in the mutant stem/progenitor cells caused by their interactions with the neighboring wild-type cells and the microenvironment. Therefore, while accumulation of oncogenic mutations is unavoidable during aging, our data suggest that, if we could therapeutically enhance normal cells’ ability to compete, we might be better able to control neoplastic cell expansion and prevent the development of a full-blown malignancy.Key PointsThe presence of wild-type cells alters the behavior of co-existing JAK2V617F mutant cellsA mutant microenvironment overcomes the competition between wild-type and JAK2V617F mutant cells, leading to the development of a MPN

Determining the role of inflammation in the selection of JAK2 mutant cells in myeloproliferative neoplasms

10.1101/079020 ◽

2016 ◽

Author(s):

Jie Zhang ◽

Angela Fleischman ◽

Dominik Wodarz ◽

Natalia L. Komarova

Keyword(s):

Hematologic Malignancy ◽

Clonal Evolution ◽

Wild Type ◽

Hematopoietic Stem ◽

Evolutionary Advantage ◽

Functional Forms ◽

Mutant Cells

AbstractMyeloproliferative neoplasm (MPN) is a hematologic malignancy characterized by the clonal outgrowth of hematopoietic cells with a somatically acquired mutation most commonly in JAK2 (JAK2V 617F). This mutation endows upon myeloid progenitors cytokine independent growth and consequently leads to excessive production of myeloid lineage cells. It has been previously suggested that inflammation may play a role in the clonal evolution of JAK2V 617F mutants. In particular, it is possible that one or more cellular kinetic parameters of hematopoietic stem cells (HSCs) are affected by inflammation, such as division or death rates of cells, and the probability of HSC differentiation. This suggests a mechanism that can steer the outcome of the cellular competition in favor of the mutants, initiating the disease. In this paper we create a number of mathematical evolutionary models, from very abstract to more concrete, that describe cellular competition in the context of inflammation. It is possible to build a model axiomatically, where only very general assumptions are imposed on the modeling components and no arbitrary (and generally unknown) functional forms are used, and still generate a set of testable predictions. In particular, we show that, if HSC death is negligible, the evolutionary advantage of mutant cells can only be conferred by an increase in differentiation probability of HSCs in the presence of inflammation, and if death plays a significant role in the dynamics, an additional mechanism may be an increase of HSC’s division-to-death ratio in the presence of inflammation. Further, we show that in the presence of inflammation, the wild type cell population is predicted to shrink under inflammation (even in the absence of mutants). Finally, it turns out that if only the differentiation probability is affected by the inflammation, then the resulting steady state population of wild type cells will contain a relatively smaller percentage of HSCs under inflammation. If the division-to-death rate is also affected, then the percentage of HSCs under inflammation can either decrease or increase, depending on other parameters.

Effects of the JAK2 mutation on the hematopoietic stem and progenitor compartment in human myeloproliferative neoplasms

Blood ◽

10.1182/blood-2010-12-327593 ◽

2011 ◽

Vol 118 (1) ◽

pp. 177-181 ◽

Cited By ~ 43

Author(s):

Shubha Anand ◽

Frances Stedham ◽

Philip Beer ◽

Emma Gudgin ◽

Christina A. Ortmann ◽

...

Keyword(s):

Jak2 V617f ◽

Myeloid Differentiation ◽

Jak2 Mutation ◽

Hematopoietic Stem ◽

Progenitor Compartment ◽

Proliferative Advantage

Abstract The JAK2 V617F mutation is present in the majority of patients with a myeloproliferative neoplasm (MPN) and is sufficient to recapitulate an MPN in murine models. However, the consequences of JAK2 mutations for myeloid differentiation are poorly understood. After systematic analyses of a large cohort of JAK2-mutated MPN patients, we demonstrate in vivo that JAK2 mutations do not alter hematopoietic stem and progenitor cell com-partment size or in vitro behavior but generate expansion of later myeloid differentiation compartments, where homozygous expression of the mutation confers an added proliferative advantage at the single-cell level. In addition, we demonstrate that these findings may be partially explained by the expression pattern of JAK2, which markedly increases on myeloid differentiation. Our findings have potential clinical relevance, as they predict that JAK2 inhibitors may control myeloproliferation, but may have limited efficacy in eradicating the leukemic stem cells that sustain the human MPN.

EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

10.1101/387043 ◽

2018 ◽

Author(s):

Stephanie Grainger ◽

Nicole Nguyen ◽

Jenna Richter ◽

Jordan Setayesh ◽

Brianna Lonquich ◽

...

Keyword(s):

Stem Cell ◽

Wnt Signaling ◽

Hematopoietic Stem Cell ◽

Cell Biology ◽

Embryonic Stem ◽

Cell Development ◽

List Type ◽

Hematopoietic Stem

SummaryThe mechanisms of Wnt-Frizzled (Fzd) signaling selectivity and their biological implications remain unclear. We demonstrate for the first time that the epidermal growth factor receptor (EGFR) is required as a co-factor for Wnt signaling. Using genetic studies in zebrafish, paired within vitrocell biology and biochemistry, we have determined that Fzd9b signals specifically with Wnt9ain vivoandin vitroto elicit β-catenin dependent Wnt signals that regulate hematopoietic stem and progenitor cell (HSPC) development in the dorsal aorta. This requirement is conserved in the derivation of HSPCs from human embryonic stem cells. Wnt9a-Fzd9b specificity requires two intracellular domains in Fzd9b, which interact with EGFR as a required co-factor to promote signal transduction. EGFR phosphorylates one tyrosine residue on Fzd9b, a requirement for the Wnt signal. These findings indicate that Wnt signaling interactions can be exquisitely specific and inform protocols for derivation of HSPCsin vitro.HighlightsAnin vitrosignaling screen identifies Fzd9b as a Wnt9a-specific receptor.Fzd9b and Wnt9a regulate hematopoietic stem cell development as a cognate pair.WNT9A and FZD9 are required for HSPC derivation from human pluripotent cellsin vitro.EGFR confers specificity to Wnt9a-Fzd9b signaling in zebrafish and human cells.

Synergic Crosstalk between Inflammation, Oxidative Stress, and Genomic Alterations in BCR–ABL-Negative Myeloproliferative Neoplasm

Antioxidants ◽

10.3390/antiox9111037 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1037

Author(s):

Alessandro Allegra ◽

Giovanni Pioggia ◽

Alessandro Tonacci ◽

Marco Casciaro ◽

Caterina Musolino ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cell ◽

Chronic Inflammation ◽

Tumor Stage ◽

Driver Mutations ◽

Hematopoietic Stem ◽

Chronic Myeloproliferative Neoplasms ◽

Inflammatory State

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have recently been revealed to be related to chronic inflammation, oxidative stress, and the accumulation of reactive oxygen species. It has been proposed that MPNs represent a human inflammation model for tumor advancement, in which long-lasting inflammation serves as the driving element from early tumor stage (over polycythemia vera) to the later myelofibrotic cancer stage. It has been theorized that the starting event for acquired stem cell alteration may occur after a chronic inflammation stimulus with consequent myelopoietic drive, producing a genetic stem cell insult. When this occurs, the clone itself constantly produces inflammatory components in the bone marrow; these elements further cause clonal expansion. In BCR–ABL1-negative MPNs, the driver mutations include JAK 2, MPL, and CALR. Transcriptomic studies of hematopoietic stem cells from subjects with driver mutations have demonstrated the upregulation of inflammation-related genes capable of provoking the development of an inflammatory state. The possibility of acting on the inflammatory state as a therapeutic approach in MPNs appears promising, in which an intervention operating on the pathways that control the synthesis of cytokines and oxidative stress could be effective in reducing the possibility of leukemic progression and onset of complications.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽

10.1182/blood.v95.9.2813.009k20_2813_2820 ◽

2000 ◽

Vol 95 (9) ◽

pp. 2813-2820 ◽

Cited By ~ 215

Author(s):

Lisa Gallacher ◽

Barbara Murdoch ◽

Dongmei M. Wu ◽

Francis N. Karanu ◽

Mike Keeney ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Surface Markers ◽

Cell Surface Markers ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect

Eukaryotic Cell ◽

10.1128/ec.1.6.906-914.2002 ◽

2002 ◽

Vol 1 (6) ◽

pp. 906-914 ◽

Cited By ~ 18

Author(s):

Thomas Schreiner ◽

Martina R. Mohrs ◽

Rosemarie Blau-Wasser ◽

Alfred von Krempelhuber ◽

Michael Steinert ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Gene Replacement ◽

Actin Binding ◽

Wild Type ◽

Hyperosmotic Shock ◽

Latex Beads ◽

Knockout Mutants ◽

Mutant Cells

ABSTRACT Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F–positive essential thrombocythemia

Blood ◽

10.1182/blood-2009-12-259747 ◽

2010 ◽

Vol 116 (9) ◽

pp. 1528-1538 ◽

Cited By ~ 136

Author(s):

Juan Li ◽

Dominik Spensberger ◽

Jong Sook Ahn ◽

Shubha Anand ◽

Philip A. Beer ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Essential Thrombocythemia ◽

Cell Function ◽

Jak2 V617f ◽

Transgenic Models ◽

Disease Phenotype ◽

Hematopoietic Stem

The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F–positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2V617F mice develop reduced numbers of lineage−Sca-1+c-Kit+ cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2V617F mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.

Growth Suppressors IFI-204 and IFI-205 Inhibit Primitive Hematopoietic Cell Proliferation In Vitro and in Vivo.

Blood ◽

10.1182/blood.v104.11.1691.1691 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1691-1691

Author(s):

Kimberly Klarmann ◽

Daniel Gough ◽

Benyam Asefa ◽

Chris Clarke ◽

Katie Renn ◽

...

Keyword(s):

Stem Cell ◽

Cell Growth ◽

Cell Line ◽

Constitutive Expression ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Inhibit Cell Growth ◽

Stem And Progenitor Cells

Abstract Members of the interferon inducible-200 (IFI-200) family of proteins inhibit cell growth and may be important mediators of differentiation. We examined IFI-204 and IFI-205 mRNA expression in purified populations of hematopoietic stem and progenitor cells at different stages of maturation using quantitative RT-PCR and found that their expression markedly increased during myeloid maturation. To evaluate the effect of IFI-205 and IFI-204 on hematopoietic stem cell (HSC) growth, we transduced these genes into mouse bone marrow cells (BMC) using retroviral vectors. The presence IFI-204 or IFI-205 resulted in a decrease in cell growth in response to hematopoietic growth factors. Further analysis revealed the infected cells were 98% c-Kit+ Sca-1+, indicative of the stem cell surface phenotype, suggesting they may be blocked in a primitive stage of maturation. When transplanted, BMC transduced with IFI-204 or IFI-205 failed to engraft lymphoid, myeloid, or erythroid lineages in both short and long term reconstitution assays, suggesting that constitutive expression of IFI-204 and IFI-205 inhibited HSC development both in vitro and in vivo. However, based on the quantitative RT-PCR results, which show that IFI-205 increased during myeloid differentiation, we know its endogenous, regulated expression must permit the cells to mature. Therefore, to study of the effects of these genes on differentiation we transduced the mulitpotential EML (erythroid, myeloid, lymphoid) cell line with IFI-204 and IFI-205 to circumvent severe growth inhibition caused by expression of IFI-204 and Ifi-205 in normal cells. Single cell analysis of EMLs transduced with IFI-205 demonstrated that expression of IFI-205 in this cell line did not significantly inhibit cell growth. We have isolated EML clones from the transduced cells and verified IFI-205 expression. In addition, we generated transgenic mice that express IFI-205 under control of the Vav and MRP8 promoters, and we identified transgenic lines that express IFI-205 at higher levels compared to wild type controls. Analysis of hematopoiesis in these animals is currently in progress. Altogether, our data demonstrate 3 findings: 1) IFI-204 and IFI-205 expression increases during myeloid development based on quantitative RT-PCR analysis, 2) constitutive expression of IFI-204 and -205 results in potent inhibition of growth and maturation of normal hematopoietic stem and progenitor cells in vivo and in vitro and 3) these genes did not significantly inhibit the proliferation of the EML cell line, which provides us with a means to study the mechanism by which these molecules regulate myeloid maturation. Finally, the considerable inhibitory effects of these family members on normal hematopoietic cell growth suggest their potential as therapeutic modalities for treatment of leukemia.

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽

10.1182/blood.v104.11.1293.1293 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1293-1293

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

The Rho Family GTPase Cdc42 Regulates Multiple Hematopoietic Stem/Progenitor Cell Functions and Erythropoiesis.

Blood ◽

10.1182/blood.v104.11.32.32 ◽

2004 ◽

Vol 104 (11) ◽

pp. 32-32

Author(s):

Lei Wang ◽

Linda Yang ◽

Marie–Dominique Filippi ◽

David A. Williams ◽

Yi Zheng

Keyword(s):

Bone Marrow ◽

Fetal Liver ◽

Brdu Incorporation ◽

Low Density ◽

Actin Assembly ◽

Wild Type ◽

Hematopoietic Stem ◽

Rho Family

Abstract The Rho family GTPase Cdc42 has emerged as a key signal transducer in cell regulation. To investigate its physiologic function in hematopoiesis, we have generated mice carrying a gene targeted null allele of cdc42gap, a major negative regulatory gene of Cdc42 and mice with conditional targeted cdc42 allele (cdc42flox/flox). Deletion of the respective gene products in mice was confirmed by PCR genotyping and Western blotting. Low-density fetal liver or bone marrow cells from Cdc42GAP−/− mice displayed ~3 fold elevated Cdc42 activity and normal RhoA, Rac1 or Rac2 activity, indicating that cdc42gap deletion has a specific effect on Cdc42 activity. The Cdc42GAP-deficient hematopoietic stem/progenitor cells (HSC/Ps, Lin−c-Kit+) generated from Cdc42GAP−/− E14.5 fetal liver and the Cdc42−/− HSC/Ps derived by in vitro expression of Cre via a retrovirus vector from Cdc42flox/flox low density bone marrow showed a growth defect in liquid culture that was associated with increased apoptosis but normal cell cycle progression. Cdc42GAP-deficient HSC/Ps displayed impaired cortical F-actin assembly with extended actin protrusions upon exposure to SDF–1 in vitro and a punctuated actin structure after SCF stimulation while Cdc42−/− but not wild type HSC/Ps responded to SDF-1 in inducing membrane protrusions. Both Cdc42−/− and Cdc42GAP−/− HSC/Ps were markedly decreased in adhesion to fibronectin. Moreover, both Cdc42−/− and Cdc42GAP−/− HSC/Ps showed impaired migration in response to SDF-1. These results demonstrate that Cdc42 regulation is essential for multiple HSC/P functions. To understand the in vivo hematopoietic function of Cdc42, we have characterized the Cdc42GAP−/− mice further. The embryos and newborns of homozygous showed a ~30% reduction in hematopoietic organ (i.e. liver, bone marrow, thymus and spleen) cellularity, consistent with the reduced sizes of the animals. This was attributed to the increased spontaneous apoptosis associated with elevated Cdc42/JNK/Bid activities but not to a proliferative defect as revealed by in vivo TUNEL and BrdU incorporation assays. ~80% of Cdc42GAP−/− mice died one week after birth, and the surviving pups attained adulthood but were anemic. Whereas Cdc42GAP−/− mice contained small reduction in the frequency of HSC markers and normal CFU-G, CFU-M, and CFU-GM activities, the frequency of BFU-E and CFU-E were significantly reduced. These results suggest an important role of Cdc42 in erythropoiesis in vivo. Taken together, we propose that Cdc42 is essential for multiple HSC/P functions including survival, actin cytoskeleton regulation, adhesion and migration, and that deregulation of its activity can have a significant impact on erythropoiesis. Cdc42 regulates HSC/P functions and erythropoiesis Genotype/phenotype Apoptosis increase Adhesion decrease Migration decrease F-actin assembly HSC frequency decrease BFU-E, CFU-E decrease The numbers were indicated as fold difference compared with wild type. ND:not determined yet. Cdc42GAP−/− 2.43, p<0.005 0.97, p<0.01 1.01, p<0.01 protrusion (SDF-1); punctruated (SCF) 0.34, p<0.05 0.92, p<0.01; 0.38, p<0 Cdc42−/− 3.68, p<0.005 0.98, p<0.001 3.85, p<0.005 protrusion (SDF-1) ND ND