Bleeding Diathesis in Mice Lacking JAK2 in Platelets

The tyrosine kinase JAK2 is a critical component of intracellular JAK/STAT cytokine signaling cascades that is prevalent in hematopoietic cells such as hematopoietic stem and progenitor cells (HSPCs), megakaryocytes (MKs), and platelets. Individuals expressing somatic activating JAK2 mutations such as JAK2V617F commonly develop myeloproliferative neoplasms (MPNs) associated with serious complications, including venous and arterial thrombosis, a leading cause of mortality. Here, we investigated the role of JAK2 in hemostasis and thrombosis using Jak2fl/flPf4-Cre (Jak2Plt-/-) mice specifically lacking JAK2 within the platelet lineage. Jak2Plt-/- mice developed severe thrombocytosis with a 5-fold increase in circulating platelet number, MK hyperplasia, and splenomegaly. Jak2Plt-/-platelets were of normal size and the expression of major membrane surface glycoproteins was indistinguishable from controls, except for the integrin β3, which was reduced by 20%. Despite the thrombocytosis, Jak2Plt-/- mice had a severe bleeding diathesis, as evidenced by: 1) prolonged tail bleeding time; 2) failure to occlude in a ferric chloride-induced carotid artery injury model; and 3) failure to form stable thrombi in a laser-induced cremaster muscle injury model. Jak2Plt-/- platelets spread poorly on immobilized collagen or on immobilized fibrinogen following GPVI stimulation with the collagen-related peptide (CRP). Jak2Plt-/- platelets had defective α-granule secretion and integrin αIIbβ3 activation following stimulation with CRP, but not thrombin, and showed aggregation defects with low-doses of CRP. Together, the data support a GPVI-specific impairment in platelets lacking JAK2, a notion that was supported by impaired intracellular signaling following GPVI stimulation, as assessed by protein tyrosine phosphorylation. Jak2Plt-/- platelets adhered poorly to type I collagen under arterial shear rates in whole blood. However, JAK2 deletion in platelets did not alter plasma von Willebrand factor (VWF) levels or botrocetin-mediated binding of plasma VWF to GPIbα. Together, the results underline a critical role for JAK2 in platelet GPVI signaling and hemostatic function, which likely contributes to venous and arterial thrombosis observed in patients with MPNs with the activating JAK2V617F mutation. Disclosures No relevant conflicts of interest to declare.

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Bleeding diathesis in mice lacking JAK2 in platelets

Blood Advances ◽

10.1182/bloodadvances.2020003032 ◽

2021 ◽

Vol 5 (15) ◽

pp. 2969-2981

Author(s):

Nathan Eaton ◽

Saravanan Subramaniam ◽

Marie L. Schulte ◽

Caleb Drew ◽

David Jakab ◽

...

Keyword(s):

Intracellular Signaling ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Cytokine Signaling ◽

Hematopoietic Stem ◽

Shear Rates ◽

Granule Secretion ◽

Static Conditions ◽

Activation Motif

Abstract The tyrosine kinase JAK2 is a critical component of intracellular JAK/STAT cytokine signaling cascades that is prevalent in hematopoietic cells, such as hematopoietic stem cells and megakaryocytes (MKs). Individuals expressing the somatic JAK2 V617F mutation commonly develop myeloproliferative neoplasms (MPNs) associated with venous and arterial thrombosis, a leading cause of mortality. The role of JAK2 in hemostasis remains unclear. We investigated the role of JAK2 in platelet hemostatic function using Jak2fl/fl Pf4-Cre (Jak2Plt−/−) mice lacking JAK2 in platelets and MKs. Jak2Plt−/− mice developed MK hyperplasia and splenomegaly associated with severe thrombocytosis and bleeding. This notion was supported by failure to occlude in a ferric chloride carotid artery injury model and by a cremaster muscle laser-induced injury assay, in which Jak2Plt−/− platelets failed to form stable thrombi. Jak2Plt−/− platelets formed thrombi poorly after adhesion to type 1 collagen under arterial shear rates. Jak2Plt−/− platelets spread poorly on collagen under static conditions or on fibrinogen in response to the collagen receptor GPVI-specific agonist, collagen-related peptide (CRP). After activation with collagen, CRP, or the CLEC-2 agonist rhodocytin, Jak2Plt−/− platelets displayed decreased α-granule secretion and integrin αIIbβ3 activation or aggregation, but showed normal responses to thrombin. Jak2Plt−/− platelets had impaired intracellular signaling when activated via GPVI, as assessed by tyrosine phosphorylation. Together, the results show that JAK2 deletion impairs platelet immunoreceptor tyrosine-based activation motif signaling and hemostatic function in mice and suggest that aberrant JAK2 signaling in patients with MPNs affects GPVI signaling, leading to hemostatic platelet function.

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Bone Morphogenetic Proteins Regulate the Developmental Program of Human Hematopoietic Stem Cells

Journal of Experimental Medicine ◽

10.1084/jem.189.7.1139 ◽

1999 ◽

Vol 189 (7) ◽

pp. 1139-1148 ◽

Cited By ~ 270

Author(s):

Mickie Bhatia ◽

Dominique Bonnet ◽

Dongmei Wu ◽

Barbara Murdoch ◽

Jeff Wrana ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Bone Morphogenetic Proteins ◽

Transforming Growth Factor ◽

Critical Role ◽

Type I ◽

Hematopoietic Tissue ◽

Hematopoietic Stem ◽

Proliferation And Differentiation

The identification of molecules that regulate human hematopoietic stem cells has focused mainly on cytokines, of which very few are known to act directly on stem cells. Recent studies in lower organisms and the mouse have suggested that bone morphogenetic proteins (BMPs) may play a critical role in the specification of hematopoietic tissue from the mesodermal germ layer. Here we report that BMPs regulate the proliferation and differentiation of highly purified primitive human hematopoietic cells from adult and neonatal sources. Populations of rare CD34+CD38−Lin− stem cells were isolated from human hematopoietic tissue and were found to express the BMP type I receptors activin-like kinase (ALK)-3 and ALK-6, and their downstream transducers SMAD-1, -4, and -5. Treatment of isolated stem cell populations with soluble BMP-2, -4, and -7 induced dose-dependent changes in proliferation, clonogenicity, cell surface phenotype, and multilineage repopulation capacity after transplantation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Similar to transforming growth factor β, treatment of purified cells with BMP-2 or -7 at high concentrations inhibited proliferation yet maintained the primitive CD34+CD38− phenotype and repopulation capacity. In contrast, low concentrations of BMP-4 induced proliferation and differentiation of CD34+ CD38−Lin− cells, whereas at higher concentrations BMP-4 extended the length of time that repopulation capacity could be maintained in ex vivo culture, indicating a direct effect on stem cell survival. The discovery that BMPs are capable of regulating repopulating cells provides a new pathway for controlling human stem cell development and a powerful model system for studying the biological mechanism of BMP action using primary human cells.

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PRMT5 Methyltransferase Activity Is Required to Sustain Adult Hematopoiesis

Blood ◽

10.1182/blood.v124.21.4335.4335 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4335-4335

Author(s):

Fan LIU ◽

Guoyan Cheng ◽

Fabiana Perna ◽

Xu Haiming ◽

Pierre-Jacques Hamard ◽

...

Keyword(s):

Cell Biology ◽

Conflicts Of Interest ◽

Critical Role ◽

Conditional Knockout ◽

Major Type ◽

Cytokine Signaling ◽

Methyltransferase Activity ◽

Hematopoietic Stem ◽

Arginine Residues

Abstract Epigenetic regulators have been shown to play critical roles in normal hematopoiesis, and their activity is frequently altered in hematopoietic cancers. Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMTs, catalyzing the symmetric di-methylation of arginine residues in histones (H2A, H3 and H4) and non-histone proteins. PRMT5 is over-expressed in several cancers, including acute leukemia and non-Hodgkin’s lymphoma. To define the role of PRMT5 in normal adult hematopoiesis, we generated PRMT5 conditional knockout mice using Mx1-cre. The induced deletion of both alleles of PRMT5 leads to severe pancytopenia and bone marrow aplasia with subsequent lethality in two weeks. First, loss of PRMT5 triggers the impaired proliferation and rapid disappearance of progenitor cells. At the same time, PRMT5 deficient HSCs show increased cell cycling and a transient HSC accumulation, which is rapidly followed by stem cell exhaustion. Mechanistically, we show that deletion of PRMT5 severely impairs cytokine signaling. It also up-regulates p53 protein level and the expression of p53 target genes. These effects likely account for the critical role of PRMT5 in HSPCs. We have conducted many additional experiments to show that these effects of PRMT5 deletion on hematopoiesis are cell autonomous; and also that the methyltransferase activity of PRMT5 is required to sustain normal hematopoiesis. Thus, we identify PRMT5 as a critical regulator of normal hematopoietic stem and progenitor cell biology. Disclosures No relevant conflicts of interest to declare.

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Syndecan-1 Is Expressed on a Subset of Hematopoietic Stem Cells and Affects Their Function and Susceptibility to Malignant Transformation.

Blood ◽

10.1182/blood.v106.11.1376.1376 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1376-1376

Author(s):

Erik A. Ranheim ◽

Sean P. McDermott ◽

Caroline M. Alexander

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Knockout Mice ◽

Critical Role ◽

Cellular Migration ◽

Cytokine Signaling ◽

Ovarian Carcinomas ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract Syndecan-1 (CD138) is a heparan-sulfate proteoglycan (HSPG) known to be involved in cytokine signaling and cellular migration in the extracellular matrix. Knockout mice lacking CD138 expression exhibit a profound resistance to Wnt-1 induced mammary carcinoma that may be secondary to changes in mammary progenitor cell number or function. To determine whether this represented a more global phenomenon, we assessed the effects of CD138 expression on tumorigenesis induced by IP administration of the chemical carcinogen, DMBA, to post-natal mice. CD138−/− mice were highly resistant to DMBA-induced lung, liver, and ovarian carcinomas with an 80% reduction in tumor incidence compared to CD138+/+ mice. Absence of CD138 also protected from DMBA-induced hematopoietic tumors, with 4/58 (6.9%) KO versus 11/56 (19.6%) WT animals developing hematopoietic proliferations. Moreover, upon histologic and phenotypic analysis, the tumors arising in WT animals all proved to be CD4+CD8+ T-lymphoblastic lymphomas, which was seen in only a single KO animal. The other CD138−/− mice instead developed a myeloproliferative disorder with extensive splenic extramedullary hematopoiesis. We hypothesized that differences in tumor susceptibility between WT and KO mice may be secondary to abnormalities in tissue specific stem cell numbers and/or function. Therefore, we examined the expression and function of CD138 on mouse hematopoietic stem cells (HSC) and found that CD138 is expressed on approximately one third of mouse long-term HSC (LT-HSC), as defined by the Kit+Thy1.1loLin−Sca+ (KTLS) phenotype. Knockout mice lacking CD138 expression have mildly reduced numbers of HSC in the bone marrow with Lin−Sca+Kit+ cells making up 0.073±0.017% of marrow cells in CD138−/− mice versus 0.098±0.019% in WT mice (p = 0.038, n=6). While apparently adequate for hematopoiesis throughout the first 18 months of life, these HSC are functionally defective in competitive reconstitution assays in wild-type recipients compared to syngeneic, CD138+/+ LT-HSC. CD45.2+Kit+Flk2-Lin−Sca+ LT-HSC from CD138−/− or control CD138+/+ C57/Bl6 mice were flow sorted and 200 purified LT-HSC were injected into lethally irradiated CD45 congenic recipients along with 3 x 105 syngeneic whole bone marrow cells. Peripheral blood was monitored for reconstitution of T, B, and myeloid cells by analysis of CD45 allele expression; and as shown in the figure, LT-HSC from CD138−/− (KO) donor mice gave significantly less total leukocyte (p = 0.03 at 12 wks.) and myeloid (CD11b+ cells) reconstitution versus WT donor LT-HSC. These data suggest that CD138 plays a critical role in HSC function and provide a potential mechanism for the tumor resistant phenotype of CD138−/− mice. We are currently assessing function (in vivo and in vitro) of CD138 positive and negative WT KTLS-defined LT-HSC to determine whether CD138 may further subdivide this heterogeneous population. Figure Figure

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14-3-3 Regulates the Lnk/JAK2 Pathway In Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v116.21.86.86 ◽

2010 ◽

Vol 116 (21) ◽

pp. 86-86

Author(s):

Joanna Balcerek ◽

Jing Jiang ◽

Alexey Bersenev ◽

Yiwen Song ◽

Chao Wu ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Large Fraction ◽

Nodal Point ◽

Adaptor Protein ◽

Serine Phosphorylation ◽

Cytokine Signaling ◽

Spectrometric Analysis ◽

Loss Of Function ◽

Hematopoietic Stem

Abstract Abstract 86 Hematopoietic stem and progenitor cell (HSPC) homeostasis is regulated by intricate signaling networks. The tyrosine kinase JAK2 plays an essential role in cytokine signaling during hematopoiesis and its dysregulation can lead to hematologic malignancies. Recently activating mutations in JAK2 were found in a large fraction of patients with myeloproliferative neoplasms (MPNs). We previously demonstrated that lymphocyte adaptor protein (Lnk) binds JAK2 and attenuates its activity, thereby limiting HSPC expansion (Bersenev et al., JCI, 2008;118:2832-2844). We further showed that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPNs in mice (Bersenev et al., JCI, 2010;120:2058-2069). Lnk directly inhibits oncogenic JAK2 as well as acting through JAK2- independent pathways to constrain MPN development. Consistently, aged Lnk–/– mice spontaneously developed a CML-like MPN (Bersenev et al., JCI, 2010;120:2058-2069). More importantly, loss-of-function mutations in Lnk are found in human MPN patients (Oh, et. al., Blood, 2010, in press). This work suggests that Lnk plays a pivotal role in regulating both normal and malignant HSPC expansion. However, it remains to be determined how Lnk attenuates JAK2 activity since Lnk itself does not possess any enzymatic activity. Therefore, we began exploring Lnk regulatory mechanisms by identifying novel Lnk partners through protein purification and mass spectrometric analysis. This resulted in the identification of a number of novel Lnk binding proteins, which include the 14-3-3 proteins as the most robust interactors. 14-3-3s are a group of scaffold proteins that regulate many disease-relevant gene products and play important functions in many aspects of cellular processes. We found that Lnk is phosphorylated at two serine residues, which serve as the critical binding sites for 14-3-3. 14-3-3 abrogates the Lnk-JAK2 interaction thereby alleviating Lnk inhibitory function in both JAK2 signaling and cell growth. Furthermore, 14-3-3 binding is necessary and sufficient to maintain Lnk in an inactive state. We also investigated the signals that regulate Lnk phosphorylation, revealing Lnk as a signaling nodal point that integrates multiple signaling pathways in controlling HSPC homeostasis. The physiological significance of the Lnk-14-3-3 interaction in HSPC development will be discussed. Thus, our data implicate previously unappreciated serine phosphorylation events in Lnk-dependent hematopoietic function and regulation of JAK2. In addition, identification of novel signaling molecules that influence hematopoiesis might facilitate stem cell therapies and provide novel therapeutic targets for the treatment of MPNs. Disclosures: No relevant conflicts of interest to declare.

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Novel Mutations: Dissecting Pathogenetic Contribution

Blood ◽

10.1182/blood.v116.21.sci-34.sci-34 ◽

2010 ◽

Vol 116 (21) ◽

pp. SCI-34-SCI-34

Author(s):

William Vainchenker ◽

Francois Delhommeau ◽

Isabelle Plo ◽

Jean-Luc Villeval ◽

Olivier Bernard

Keyword(s):

Stem Cell ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Cytokine Signaling ◽

Regulation Of Transcription ◽

Loss Of Function ◽

Gene Repair ◽

Hematopoietic Stem ◽

Myeloid Malignancies ◽

Tet2 Mutation

Abstract Abstract SCI-34 BCR-ABL negative myeloproliferative neoplasms (MPN) such as Polycythemia Vera, Essential Thrombocytemia and Primary Myelofibrosis are clonal stem cell disorders associated with an increase production of mature blood cells usually affecting a single cell lineage. All three disorders may progress to leukemia. Cytokine hypersensitivity was likely common to all three disorders and recent. experimental evidences including the discovery of JAK2V617F, JAK2 exon 12, MPLW515 and LNK mutations, validated this hypothesis. Indeed these mutations, gain-of-function for JAK2 and MPL and loss-of-function for LNK, lead to a deregulated cytokine signaling. Furthermore JAK2 and MPL mutations were shown to target a hematopoietic stem cell. In different murine models expression of JAK2 and MPL mutations recapitulated most stages of MPN, except progression to leukemia, although JAK2V617F was capable to modify gene repair mechanisms and may therefore contribute to a form of genomic instability. Recently, genomic analyses of MPN allowed the identification of mutations affecting the TET2, ASXL1 and EZH2 genes, that may be associated together and with the others mutations. TET2, ASXL1 and EZH2 proteins are involved in the epigenetic regulation of transcription. Interestingly, ASXL1 and TET2 are mutated in a large spectrum of myeloid malignancies including, acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and MDS/MPN, suggesting that all three classes of myeloid malignancies may arise from a common genomic hit. The first discovered gene of this class was the TET2 gene, which belongs to a family of three genes that include also TET1 and TET3. In vitro experiments demonstrated that the TET1 protein is able to hydroxylate the 5-methylcytosine, resulting in the generation of 5-hydroxymethylcytosine, a previously unknown modified base in mammalian DNA with unknown function. TET2 and TET3 are very likely to have the same propriety. Analysis of 800 patients with classical MPN revealed that TET2 variants are present in an average 14% of MPN with some differences among these disorders (20% in PMF, 13% in PV, 11% in ET and 20–30% in post-MPN AML. The frequency of TET2 mutation is similar in AML and much higher in CMML (50%). All variants observed are compatible with loss-of-function of the protein and biallelic mutations are found in 1/4 of the cases. Precise analysis of TET2 mutation occurrence during MPN progression has shown that TET2 mutations may precede or follow JAK2V617F mutations. There is increasing evidence that JAK2V617F is only giving a proliferative advantage during differentiation, but not in stem cells, thus allowing a clonal dominance in late stages of differentiation. Whereas acquisition of a TET2 mutation in a MPN might be associated with its progression, initial TET2 mutations might be responsible for the clonal dominance at early stages of hematopoiesis. Further studies on the functions of these mutants in hematopoiesis may permit to decipher their precise role in the pathogenesis of MPN and other hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

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Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms

PLoS ONE ◽

10.1371/journal.pone.0247858 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0247858

Author(s):

Ron Baik ◽

Stacia K. Wyman ◽

Shaheen Kabir ◽

Jacob E. Corn

Keyword(s):

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Cytokine Signaling ◽

Competitive Growth ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Myeloproliferative neoplasms (MPNs) cause the over-production of blood cells such as erythrocytes (polycythemia vera) or platelets (essential thrombocytosis). JAK2 V617F is the most prevalent somatic mutation in many MPNs, but previous modeling of this mutation in mice relied on transgenic overexpression and resulted in diverse phenotypes that were in some cases attributed to expression level. CRISPR-Cas9 engineering offers new possibilities to model and potentially cure genetically encoded disorders via precise modification of the endogenous locus in primary cells. Here we develop “scarless” Cas9-based reagents to create and reverse the JAK2 V617F mutation in an immortalized human erythroid progenitor cell line (HUDEP-2), CD34+ adult human hematopoietic stem and progenitor cells (HSPCs), and immunophenotypic long-term hematopoietic stem cells (LT-HSCs). We find no overt in vitro increase in proliferation associated with an endogenous JAK2 V617F allele, but co-culture with wild type cells unmasks a competitive growth advantage provided by the mutation. Acquisition of the V617F allele also promotes terminal differentiation of erythroid progenitors, even in the absence of hematopoietic cytokine signaling. Taken together, these data are consistent with the gradually progressive manifestation of MPNs and reveals that endogenously acquired JAK2 V617F mutations may yield more subtle phenotypes as compared to transgenic overexpression models.

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Cell-to-Cell Contact Is Critical for the Survival of Hematopoietic Progenitor Cells on Osteoblasts.

Blood ◽

10.1182/blood.v104.11.1285.1285 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1285-1285

Author(s):

Jianhua Wang ◽

Russell Taichman ◽

Younghun Jung ◽

Aaron Havens ◽

Yanxi Sun ◽

...

Keyword(s):

Progenitor Cells ◽

Intracellular Signaling ◽

Hematopoietic Cells ◽

Differentially Expressed ◽

Hematopoietic Progenitor Cells ◽

Cell Contact ◽

Type I ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem

Abstract Osteoblasts constitute part of the stromal cell support system in marrow for hematopoiesis, however little is known as to how they interact with hematopoietic stem cells (HSCs). In vitro studies have demonstrated that the survival of HSCs in co-culture with osteoblasts requires intimate cell-to-cell contact. This suggests that the osteoblast-derived factor(s) that supports stem cell activities are either produced in very small quantities, are rapidly turned over, may be membrane-anchored and/or requires the engagement of cell-cell adhesion molecules yet to be determined. In the present report we found that survival of hematopoietic progenitor cells on osteoblasts is dependent upon the engagement of VLA-4 (α4β1) and VLA-5 (α5ß1) receptors using function blocking antibodies. Surprisingly, cell-to-cell contact is not absolutely required to support progenitor activity, but does not require receptor-ligand engagement of the VLA-4 and LFA-1 complexes, which can in part be replaced through the use of recombinant ligands (fibronectin, ICAM-1, VCAM-1). Moreover conditioned once these receptors were engaged, medium derived from HSCs grown on osteoblasts ligands supported significantly greater hematopoietic progenitors in vitro than did osteoblast-conditioned or HSC-conditioned medium alone. As an initial attempt to identify the activity we examined which genes are activated following the establishment of osteoblast-CD34+ cell co-cultures nine separate co-cultures were establsihed and the RNA was pooled and analyized on Affymetrix HG-U133A chips at 24 hours. Initially our analysis revealed that there were 259 genes that are up regulated at 24 hours, and 14 genes that are down regulated. Inspection revealed that 30 of these signals were repeated at least once suggesting that 206 genuine gene candidates were differentially expressed resulting from the co-culture. A significant proportion of the differentially expressed cDNAs represent intracellular signaling ligands 16.5% (n=34) and cell surface receptors 13.5% (n=28). Molecules associated with assembly of the extra cellular matrix or its degradation comprised 7.2% (n=15) of the differentially up regulated molecules. Molecules associated with intracellular signaling, novel sequences and intermediate metabolism comprised the majority of the remaining activities. Amoung the candidates of extra cellular signaling molecules, we noted that IL-6, LIF, MIP-1alpha and SDF-1 were identified in the microarray analysis. This observation was most gratifying as we had previously reported that IL-6, LIF and MIP-1α activities are critical components of an HSC-osteoblast microenvironment. Other notable cytokine messages for BMP-2, CCL7, FGF2b, GRO1α, GRO3, IGF1, IL1ß, IL-8, IL-11, LIF, PDGF-D and the receptors for CCL7 (CCR7). Elevations in mRNA for fibronectin, lysine hydroxylase-like proteins, laminin and Type I collagen suggest that the presence of hematopoietic cells also induces osteoblastic activities. While the identity of those molecules present in the co-cultured medium remain to be identified, the data suggests that hematopoietic cells cooperate with osteoblasts to assemble the various marrow microenvironments by directing the synthesis of osteoblast-derived cytokines to improve HSC survival.

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Excessive R-Loops Trigger an Inflammatory Cascade Leading to Aberrant Hematopoietic Stem and Progenitor Cell Expansion

Blood ◽

10.1182/blood-2019-124888 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 772-772 ◽

Cited By ~ 1

Author(s):

Joshua Weinreb ◽

Varun Gupta ◽

Teresa V. Bowman

Keyword(s):

Nucleic Acid ◽

Target Genes ◽

Conflicts Of Interest ◽

Critical Role ◽

Transgenic Zebrafish ◽

Type I ◽

Inflammatory Signaling ◽

Hematopoietic Stem ◽

Inflammatory Cascade ◽

Flow Cytometric

Mutations in DEAD-box Helicase 41 (DDX41) are observed in patients with myelodysplastic syndromes (MDS) with inferior overall survival, implicating this factor in disease pathogenesis. DDX41 is an understudied factor with links to inflammation and RNA processing, but whose function in hematopoiesis is unknown. Using a novel zebrafish model of Ddx41-deficiency, we unveiled a critical role for Ddx41 in regulating hematopoietic stem and progenitor cells (HSPCs). Mutants for ddx41 have more HSPCs than their siblings as measured by in situ hybridization for the HSPC marker runx1 as well as quantification of cd41:gfp+ and runx1:mcherry+ HSPCs by flow cytometry. To uncover potential mechanisms driving HSPC expansion in ddx41 mutants, we performed RNA-seq analysis of purified HSPCs from ddx41 mutants and sibling controls. Expression of inflammatory target genes was significantly elevated in ddx41 mutants including those regulated by the transcription factor NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells). As inflammatory signaling and NFκB signaling in particular is elevated in MDS, we next explored Ddx41 regulation of NFκB signaling. Using an in vivonfkb:gfp reporter, we demonstrated that NFκB signaling was indeed elevated in ddx41 mutants. DDX41 is implicated in the STING (STimulator of INterferon Genes) and TBK1 (TANK binding kinase 1) pathway that mediates type I interferon production in response to foreign or damaged endogenous DNAs. By lowering pathway activity via knockdown of sting or pharmacological inhibition of TBK1, we determined that the STING/TBK1 pathway triggered excessive NFκB activation in ddx41 mutants. Moreover, the elevated inflammatory signaling was detrimental to normal HSPC homeostasis as lowering STING/TBK1 pathway activation significantly reduced HSPC expansion in ddx41 mutants, as measured by runx1in situ hybridization and cd41:gfp flow cytometric quantification. R-loops, nucleic acid structures consisting of RNA:DNA hybrids and displaced ssDNAs, can activate the STING/TBK1 pathway via the nucleic acid sensor cGAS (cyclic GMP-AMP synthase). Recent proteomic analysis showed that DDX41 can bind R-loops. Via immunofluorescence quantification of RNA:DNA hybrid levels in ddx41 mutants versus sibling controls, we revealed that Ddx41 acts as a suppressor of R-loop accumulation. Using a RNASEH1-GFP transgenic zebrafish line that permits inducible depletion of R-loops, we revealed that excess R-loops in ddx41 mutants promote HSPC expansion as measured by runx1in situ hybridization and runx1:mcherry flow cytometric quantification. Moreover, using RT-qPCR analysis of NFκB target genes, we determined that R-loop depletion dampened NFκB activation in ddx41 mutants, suggesting the R-loop-mediated effects on HSPC numbers was via inflammatory signaling. We deciphered cGAS activity was critical for this effect as both nfkb:gfp reporter activity and HSPC levels in ddx41 mutants were diminished following treatment with a pharmacological inhibitor of cGAS. Our data demonstrate that DDX41 insufficiency triggers an R-loop-mediated inflammatory cascade leading to aberrant HSPC expansion and is the first study to delineate a functional consequence for R-loops in HSPC biology. Furthermore, recent studies showed R-loop accumulation in other common forms of MDS suggesting our novel in vivo findings are more broadly applicable to the MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

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Single Cell Mass Cytometry Reveals Hyperactivated Signaling Networks in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v124.21.1884.1884 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1884-1884

Author(s):

Daniel A.C. Fisher ◽

Olga Malkova ◽

Mary C. Fulbright ◽

Gregory K. Behbehani ◽

Garry P. Nolan ◽

...

Keyword(s):

Single Cell ◽

Signaling Pathways ◽

Intracellular Signaling ◽

Colony Formation ◽

Myeloproliferative Neoplasms ◽

Cell Mass ◽

Innovative Technology ◽

Signaling Networks ◽

Mass Cytometry ◽

Hematopoietic Stem

Abstract Myeloproliferative neoplasms (MPNs) including myelofibrosis (MF) are characterized by chronic hyperactivation of a signaling axis downstream of the JAK2 kinase. Pharmacologic inhibitors of JAK2 ameliorate constitutional symptoms and splenomegaly in MF patients. However, these agents do not appear to be capable of eradicating the malignant clone, nor have they have been shown to prevent transformation to secondary acute myeloid leukemia (sAML). These findings suggest that aberrant activation of additional signaling pathways, either downstream of JAK2, or via alternative mechanisms, may contribute to MPN pathogenesis. To develop more effective therapeutic strategies, a fuller understanding of these altered signaling pathways in MPNs is needed. Mass cytometry is an innovative technology that enables the characterization of dysregulated signaling networks at the single cell level. We utilized this approach to examine intracellular signaling phenotypes of seven MF patients, five sAML patients, and five normal controls across two independent experiments. Patient CD34+ hematopoietic stem and progenitor cells (HSPCs) frequently exhibited basal (unstimulated) signaling abnormalities suggestive of chronic hyperactivation of the JAK-STAT, MAP kinase/PI3 kinase, and NFκB signaling pathways. HSPCs from individual patients also exhibited hypersensitive responses to stimulation by the cytokines thrombopoietin (TPO), G-CSF, and/or TNFα. Elevated phosphorylation of the signaling molecules AKT, ERK, CREB, and S6 suggests an extensive network of hyperactivated signaling in MF and sAML HSPCs. Evidence of NFκB signaling hyperactivation was identified as indicated by (1) elevated phosphorylation of the NFκB subunit p65/RELA and supranormal abundance of IκBα in unstimulated cells; and (2) hypersensitive responses to TNFα, in the form of TNFα stimulated p65/RELA phosphorylation and IκBα degradation. Pronounced NFκB signaling hyperactivation was observed in a subset of MF and sAML patients from these experiments. Elevated NFκB signaling was predominantly insensitive to ex vivo exposure to the JAK inhibitor ruxolitinib, but was partly sensitive to the IκB kinase inhibitor IKKiVII. The relevance of NFκB signaling to myeloproliferation was tested by colony-forming unit (CFU) assays with MF patient HSPCs. IKKiVII inhibited myeloid colony formation from MF CD34+ cells with a potency similar to that observed for ruxolitinib. Inhibition of colony formation by IKKiVII was enhanced in combination with ruxolitinib. Similarly, growth of the JAK2 mutant HEL cell line was inhibited by IKKiVII with a potency similar to that observed for ruxolitinib, and the combination of IKKiVII and ruxolitinib gave substantially greater inhibition than either inhibitor alone. This suggests that NFκB signaling may be an important component of myeloproliferation, particularly in the context of hyperactive JAK2. These findings suggest that co-targeting of JAK2 and NFκB could be beneficial therapeutically. Ongoing experiments are focused on further characterizing the extent of dysregulated signaling in MPNs, as well as the prevalence of hyperactive NFκB signaling in MF and sAML. These experiments will identify components of myeloproliferative signaling which are abnormally active in MF and sAML, and may represent targets for improved therapeutic intervention. Disclosures Oh: Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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