MicroRNA Maestros of Hematopoiesis

Abstract Abstract SCI-32 MicroRNAs (miRs) inhibit stability and/or translation of mRNAs, usually by binding to specific sites in the 32′UTRs of their target mRNAs. Due to imperfect (i.e. partially complementary) miR:mRNA base-pairing, miRs can block translation of many mRNAs and serve as powerful master switches to regulate cell functions. Therefore, we profiled miR expression in human CD34+ hematopoietic stem-progenitor cells (HSPCs) and combined human HSPC miR expression, mRNA expression, and miR-mRNA target predictions to hypothesize that certain HSPC-expressed miRs (HE-miRs) target several mRNAs critical to hematopoiesis. On this informatic basis, we formulated a model of hematopoietic differentation in which many genes specifying hematopoietic differentiation are expressed by early HSPCs, but held in check by miRs [1]. In addition, we noted that the miR-23a cluster (i.e. adjacent, co-transcribed miR-23a, miR-27a, and miR-24-2) is not expressed or is expressed at levels >2-fold lower in 50% of acute myeloid leukemias and 80% of acute lymphoid leukemias tested compared to normal human HSPCs. ‘Re-expressing’ 1 or more of these miR-23a cluster members in leukemia cells promotes their apoptosis and reduces their proliferation, thus suggesting that these miRs have a tumor suppressive role. We have identified YWHAQ (14-3-3q) and several other 14-3-3 isoforms, which are anti-apoptotic and have established roles as oncogenes, as miR-23a cluster target molecules. Artificial manipulation of these HE-miRs and their target genes may lead to novel strategies for leukemia treatment and/or for expansion of normal HSPCs. Since the CD34+ HSPCs that we studied initially include rare stem cells and various stages of progenitors, we have expanded our miR profiling to more highly purified subsets of mouse HSPCs. Several previously described (e.g. miR-155 [1], miR-451 [2], miR-146 [3]) and novel HE-miRs are expressed differentially in lineages/stages of HSPCs, and their selective expression has been confirmed in human HSPC subsets. We are using cellular gain- and loss-of-function approaches with hematopoietic functional assays to determine whether these HE-miRs control human hematopoiesis. Understanding the effects of HE-miRs in hematopoiesis may elucidate hematopoietic and general stem cell biologic mechanisms. 1. Georgantas RW, 3rd, Hildreth R, Morisot S, Alder J, Liu CG, Heimfeld S, Calin GA, Croce CM, Civin CI. CD34+ hematopoietic stem-progenitor cell microRNA expression and function. A circuit diagram of differentiation control. Proc Natl Acad Sci USA. 2007;104:2750–2755. 2. Dore LC, Amigo JD, Dos Santos CO, Zhang Z, Gai X, Tobias JW, Yu D, Klein AM, Dorman C, Wu W, Hardison RC, Paw BH, Weiss MJ. A GATA-1-regulated microRNA locus essential for erythropoiesis. Proc Natl Acad Sci USA. 2008;105:3333–3338. 3. Starczynowski DT, Kuchenbauer F, Argiropoulos B, Sung S, Morin R, Muranyi A, Hirst M, Hogge D, Marra M, Wells RA, Buckstein R, Lam W, Humphries RK, Karsan A. Identification of miR-145 and miR-146a as mediators of the 5q- syndrome phenotype. Nat Med. 2010;16:49–58. Disclosures: No relevant conflicts of interest to declare.

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Ing4-Deficiency Enhances Hematopoietic Stem Cell Quiescence and Confers Resistance to Inflammatory Stress

Blood ◽

10.1182/blood-2021-154353 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1095-1095

Author(s):

Zanshé Thompson ◽

Georgina A Anderson ◽

Seth Gabriel ◽

Melanie Rodriguez ◽

Vera Binder ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Target Genes ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Critical Role ◽

Loss Of Function ◽

Wild Type ◽

Hematopoietic Stem

Abstract In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, loss of Ing4 has been shown to promote stem cell-like characteristics in malignant cells and it is a frequent target of inactivation in various types of cancer. Mutations in Ing4 cause deregulation of both NF-kB and c-Myc target gene expression. We have also identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- mice have aberrant hematopoiesis and elevated cytokine expression in bone marrow cells. Using RNA-sequencing, we found that Ing4-deficient HSPCs express high levels of c-Myc target genes and genes associated with oxidative phosphorylation and ribosomal biogenesis. Yet, Ing4 deficiency induces G 0 arrest in HSPCs and they have low levels of reactive oxygen species. This places Ing4-deficient HSPCs in a poised state, where they are quiescent, but express elevated levels of genes associated with differentiation. Under stress hematopoiesis following low-dose irradiation, Ing4-deficient long-term hematopoietic stem cells (LT-HSCs) do not expand, but short-term hematopoietic stem cells (ST-HSCs) function comparably to wild-type. Similarly, under transplantation stress, LT-HSCs fail to contribute to multilineage chimerism, while ST-HSCs contribute at levels equal to wild-type cells. These results are striking, particularly when compared to other models of enhanced NF-kB activity, where HSPCs cannot contribute to multilineage chimerism in transplantation. We sought to target the misregulated pathways in Ing4-deficient HSCs to rescue to effects of Ing4 deficiency. To this end, we chose to target the c-Myc pathway for several reasons: c-Myc target genes are over-represented in our RNA-seq data, c-Myc lies upstream of several of the misregulated pathways observed in Ing4-/- HSCs, and Ing4 has previously been reported to negatively regulate c-Myc activity directly. When treated with the c-Myc inhibitor, 10058-F4, both LT-HSCs and ST-HSCs are pushed into cycling, but this treatment also resulted in fewer cells overall. These results suggest that dampening of the c-Myc pathway can partially rescue Ing4 loss of function. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. Disclosures No relevant conflicts of interest to declare.

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The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10533-10542.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10533-10542 ◽

Cited By ~ 59

Author(s):

Marc-Werner Dobenecker ◽

Christian Schmedt ◽

Masato Okada ◽

Alexander Tarakhovsky

Keyword(s):

T Cell ◽

Adaptor Protein ◽

Regulatory Function ◽

Loss Of Function ◽

Thymic Development ◽

Cell Functions ◽

Carboxy Terminal ◽

And Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

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Enhanced Megakaryocyte and Erythroid Development From Normal Human CD34+ Cells: Consequence of Enforced Expression of SCL

Blood ◽

10.1182/blood.v91.10.3756 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3756-3765 ◽

Cited By ~ 54

Author(s):

Ngaire J. Elwood ◽

Helen Zogos ◽

Daniel S. Pereira ◽

John E. Dick ◽

C. Glenn Begley

Keyword(s):

Bhlh Transcription Factor ◽

Cell Compartment ◽

Cell Cycle Time ◽

Hematopoietic Stem ◽

Helix Loop Helix ◽

Proliferative Effect ◽

Human Cd34 ◽

Cd34 Cells ◽

Normal Human ◽

Erythroid Development

Abstract The product of the SCL gene is a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of hematopoietic stem cells in both the embryo and the adult. However, once the stem cell compartment is established, the function of SCL in subsequent differentiation and commitment events within normal hematopoietic cells remains undefined. The aim of the current study was to investigate this role using purified normal human hematopoietic CD34+ cells. An SCL retrovirus was used to transduce CD34+ cells isolated from human bone marrow, peripheral blood, and umbilical cord blood. Enforced expression of SCL increased by a median of twofold the number of erythroid colonies, with an increase in both colony size and the rate of hemoglobinization. Unexpectedly, enforced expression in CD34+ cells also significantly increased the number of megakaryocyte colonies, but with no impact on the size of colonies. There was no consistent effect on the number nor size of granulocyte-macrophage (GM) colonies. The proliferative effect of enforced SCL expression on erythroid cells was attributed to a shortened cell cycle time; the self-renewal capacity of erythroid or GM progenitors was unchanged, as was survival of cells within colonies. These results demonstrate a role for SCL in determining erythroid and megakaryocyte differentiation from normal human hematopoietic CD34+ cells.

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SHP-2 Knockdown Results in Profound Inhibition of Human CD34+ Hematopoietic Stem/Progenitor Cell Survival, Proliferation and Differentiation in Response to Growth Factor Stimulation

Blood ◽

10.1182/blood.v112.11.3859.3859 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3859-3859

Author(s):

Li ang Li ◽

Tinisha McDonald ◽

Hardik Modi ◽

Arjun Sehgal ◽

Ravi Bhatia

Keyword(s):

Growth Factor ◽

Cell Survival ◽

Progenitor Cell ◽

The Other ◽

Hematopoietic Stem ◽

Cell Numbers ◽

Human Cd34 ◽

Cd34 Cells ◽

Normal Human ◽

Cells Cultured

Abstract SHP-2 (ptpn11), a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase, is expressed at high levels in hematopoietic cells and regulates downstream signaling from growth factor (GF) receptors. SHP-2 has been shown to play an important role in murine hematopoiesis. Moreover, several SHP-2 activating mutations have been identified in myeloid malignancies and there is interest in the development of SHP-2 inhibitors for cancer treatment. On the other hand previous report suggested that SHP-2 inhibition was associated with enhanced GF responsiveness in human hematopoietic cell lines. However the role of SHP-2 signaling in normal human hematopoietic stem and progenitor cell growth has not been studied. Here we investigated the function of SHP-2 in normal human hematopoiesis by inhibiting SHP-2 expression in cord blood (CB) CD34+ cells with stable SHP-2 shRNA expression. We transduced CB CD34+ cells with lentivirus vectors coexpressing SHP-2 specific shRNAs (Si-1 or Si-2) or a control shRNA (Ctrl) and RFP and selected RFP expressing CD34+ cells by flow cytometry sorting. We observed >80% inhibition of SHP-2 expression by Western blotting in Si-1 or Si-2 shRNA transduced cells compared with Ctrl shRNA transduced cells. We observed that culture with increasing concentrations of GF was associated with markedly reduced GF-induced stimulation of proliferation of SHP-2-knockdown CD34+ cells compared to controls. In addition we observed significantly increased apoptosis of SHP-2-knockdown CD34+ cells cultured under low and high GF conditions compared to controls, but little increase in apoptosis in GF-deprived cells, indicating markedly reduced response of SHP-2-knockdown cells to GF-mediated promotion of cell survival. SHP-2-knockdown CD34+ cells also demonstrated significantly reduced expansion in cell numbers following culture in high GF conditions compared with controls (115.3, 25.5 and 10.4 fold expansion for Ctrl, Si-1 and Si-2 at day 7). Analysis of the nature of cells generated in GF culture showed significantly reduced generation of both myeloid (CD33+, CD11b+ and CD14+) and erythroid cells from SHP-2-knockdown CD34+ cells compared with controls, with relatively greater inhibition of myeloid compared with erythroid differentiation. On the other hand CD34+ cell numbers were retained at levels similar to controls after culture. We also observed significantly reduced cell expansion and differentiation and higher apoptotic rates of SHP-2-knockdown cells cultured under either myeloid promoting (IL-3+SCF+G-CSF+GM-CSF) or erythroid promoting (SCF+EPO) GF conditions. SHP-2-knockdown cells demonstrated reduced activation of MAPK and STAT5 but not Akt on Western blotting that was associated with reduced MCL-1 expression, consistent with their reduced GF mediated proliferation and survival. Expression of the transcription factors SCL1, GATA-1, NF-E2 and FOG-1 was increased in SHP-2 knockdown CD34+ cells compared to controls, consistent with the relatively higher retention of CD34+ and erythroid cells compared with myeloid cells after culture. In conclusion, we show that SHP-2 knockdown in human CD34+ cells results in markedly decreased responsiveness to GF stimulation with significantly increased apoptosis, markedly diminished proliferation and reduced generation of differentiated cells during GF culture. A relative retention of the CD34+ cell population was seen despite increased apoptosis, which may be the result of reduced cell turnover and altered transcription factor expression in SHP-2-knockdown cells, and is in contrast to reduced stem cell self-renewal observed following SHP-2 knockdown in murine models. These results indicate a critical role for SHP-2 in GF mediated signaling responses in human hematopoietic stem/progenitor cells. These studies also caution that therapeutic SHP-2 inhibition could be associated with significant hematopoietic toxicity.

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LDL and HDL Counteract Hematopoietic Stem/Progenitor Cells in Regulation of Inflammation and Atherosclerosis

Blood ◽

10.1182/blood.v118.21.1288.1288 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1288-1288

Author(s):

Yingmei Feng

Keyword(s):

Progenitor Cells ◽

High Fat Diet ◽

Conflicts Of Interest ◽

Density Lipoprotein ◽

Brdu Incorporation ◽

High Fat ◽

Hematopoietic Stem ◽

Differential Effects ◽

And Function ◽

Opposing Effects

Abstract Abstract 1288 Low-density lipoprotein (LDL) is an independent risk factor in cardiovascular disease. Hypercholesterolemia-associated monocytosis and transformation of monocytes into macrophages are the main features underlying the proatherogenic property of LDL. Different from LDL, high-density lipoprotein (HDL) and its major apolipoprotein, apoA-I, counteract atherosclerosis by reversing cholesterol transport, inhibiting inflammatory cell adhesion to plaques, maintaining endothelial integrity, inhibiting oxidation, and as has recently been shown, suppressing hematopoietic stem cell (HSC) proliferation. As inflammatory cells in atherosclerotic plaques are exclusively derived from hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM), we hypothesized that differential effects of LDL and HDL on HSPC proliferation and differentiation may play a role in atherosclerotic plaque development. We explored the effect of HDL and LDL on HSPC number and function using LDL receptor knockout (LDLr ko) mice, fed with high fat diet, and C57BL/6 mice infused with purified apoA-I and or reconstituted (r)HDL. Compared to mice on normal diet, the number of lineage−/cKit+Sca-1+ (KLS) cells was 5-fold higher in LDLr ko mice on high-fat diet. By contrast, infusion of either 8 mg/kg apoA-I or 8, 12 and 16 mg/kg rHDL decreased the KLS cell frequency in BM. Using BrdU incorporation, we demonstrated that LDL induced but HDL inhibited KLS cell proliferation. When lineage negative cells, exposed to LDL or rHDL were used in competitive repopulation studies, HDL-treated cells supported greater chimerism than untreated controls, whereas LDL-treated cells competed less well. In addition, for HDL-treated Lin− cells, skewing of reconstitution to the B-lymphoid lineage at the expense of the granulocyte/monocyte lineage was seen. In vitro, LDL promoted KLS cell differentiation towards the monocytic lineage, which was abrogated by addition of HDL. In conclusion, LDL and HDL have opposing effects on HSPC number and function. These differential effects may contribute to the opposing effects of HDL and LDL on atherosclerosis development. Disclosures: No relevant conflicts of interest to declare.

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MiR-150 Suppresses MLL-AF9 Associated Leukemia Through Simultaneously Targeting Multiple Oncogenes,

Blood ◽

10.1182/blood.v118.21.3461.3461 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3461-3461

Author(s):

Beiyan Zhou

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Target Genes ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Gene Profiling ◽

Fusion Genes ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Mll Gene

Abstract Abstract 3461 The mixed lineage leukemia (MLL) gene codes for an evolutionarily conserved histone methyltransferase that is crucial for early hematopoiesis. As a result of a chromosomal translocation involving locus 11q23 results in formation of chimeras composed of the 5' part of the MLL gene fused with more than 60 partner genes lead to disruption of normal function of MLL as a histone methytransferase and acquisition of transcriptional properties conferred by the partner genes. MLL fusion genes (MLL-FG) are often the causal mutations for aggressive acute myeloid and lymphoid leukemias (AML and ALL) that correlated with poor prognosis. In order to treat or even eliminate MLL-associated leukemias, extensive studies on the regulatory mechanism underlying MLL associated transformation and progression have been carried out. Leukemic stem cells (LSC) can derive from either hematopoietic stem or progenitor cells with the recruitment of MLL-fusion genes (MLL-FG) and wild type MLL protein. We report that miR-150, a key hematopoietic regulatory microRNA (miRNA) and one of the most downregulated miRNAs in MLL-associated leukemias, acts as a tumor suppressor to block the leukemogenic potency of leukemic stem cells. When expression of miR-150 was restored, a significantly suppressed leukemic stem cell potency of MLL-AF9 cells was observed both in vivo and in vitro. Gene profiling analysis demonstrated that elevated miR-150 altered various aspects of gene expression patterns in MLL-AF9 cells, including stem cell signatures, cancer pathways, and cell survival. By screening more than 30 predicted target genes, we identified multiple leukemia-associated oncogenes as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the tumor suppressive effects observed after ectopically expression of miR-150 in MLL-AF9 cells. Disclosures: No relevant conflicts of interest to declare.

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Role of Notch Signaling in Hematopoietic Stem Cell Function

Blood ◽

10.1182/blood.v118.21.sci-15.sci-15 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-15-SCI-15

Author(s):

Lluis Espinosa ◽

Anna Bigas

Keyword(s):

Stem Cell ◽

Cell Function ◽

Conflicts Of Interest ◽

Notch Pathway ◽

Cell Types ◽

Loss Of Function ◽

Hematopoietic Stem ◽

On Chip ◽

Stem Cell Populations

Abstract Abstract SCI-15 The Notch pathway controls the generation of different cell types in most tissues including blood, and dysregulation of this pathway is strongly associated with oncogenic processes. In many systems, Notch is also required for the maintenance of the stem cell populations. However, in the adult hematopoietic system this link between Notch and stemness has not been established. Instead, work of several groups, including ours, has clearly demonstrated that Notch has a prominent role in the generation of hematopoietic stem cells (HSC) during embryonic development. Although the first wave of blood cells appears in the mouse embryo around day 7.5 of development and is independent of Notch function, embryonic HSC are formed around day 10 of development from endothelial-like progenitors that reside in the embryonic aorta surrounded by the gonad and mesonephros, also called AGM region. By analyzing different Notch pathway mutant mouse embryos, we have demonstrated the involvement of the Jagged1-Notch1-GATA2 axis in this event. However, the formal demonstration that Notch regulates the GATA2 gene during HSC generation is still lacking. We have now found that GATA2 is a direct Notch target in vivo during embryonic HSC generation. However, whereas Notch positively activates GATA2 transcription in the HSC precursors, it simultaneously activates hes1 transcription, which acts a repressor of the same GATA2 gene. This finding directly implicates hes1 in the regulation of HSC development although further studies using loss-of-function mutant embryos are still needed. Altogether, our results indicate that both Notch and hes1 are required to finely regulate the levels, distribution, and likely the timing of GATA2 expression through an incoherent feed-forward loop. In parallel, we have identified other downstream targets of Notch in the AGM region by ChIP-on-chip and expression microarray analysis that we are currently characterizing. Disclosures: No relevant conflicts of interest to declare.

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MiR-29a Maintains Hematopoietic Stem Cell Self-Renewal and Is Required For Myeloid Leukemogenesis

Blood ◽

10.1182/blood.v122.21.1190.1190 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1190-1190

Author(s):

Wenhuo Hu ◽

James Dooley ◽

Stephen S. Chung ◽

Safak Yalcin ◽

Yu Sup Shin ◽

...

Keyword(s):

Stem Cell ◽

Colony Formation ◽

Target Genes ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Null Mouse ◽

Cell Cycle Regulators ◽

Self Renewal ◽

Hematopoietic Stem

Abstract microRNAs (miRNAs) are important regulators of both embryonic and adult tissue stem cell self-renewal. We previously showed that ectopic expression of miR-29a, a miRNA highly expressed in HSCs as well as in human acute myeloid leukemia (AML) stem cells, in immature mouse hematopoietic cells is sufficient to induce a myeloproliferative disorder that progresses to AML. During the early phase of this disease, miR-29a induces aberrant self-renewal of committed myeloid progenitors, strongly suggesting a role for miR-29a in regulating HSC self-renewal. In order to determine the role of miR-29a in HSC function, we have evaluated our recently described miR-29a/b1 null mouse. Homozygous deletion of miR-29a/b1 resulted in reduced bone marrow cellularity and reduced colony forming capacity of hematopoietic stem and progenitor cells (HSPCs). The phenotype was mediated specifically by miR-29a since miR-29b expression was not significantly altered in HSCs and reconstitution of miR-29a/b1 null HSPCs with miR-29a, but not miR-29b, rescued in vitro colony formation defects. Self-renewal defects were observed in miR-29a deficient HSCs in both competitive and non-competitive transplantation assays, and these deficits were associated with increased HSC cell cycling and apoptosis. Gene expression studies of miR-29a deficient HSCs demonstrated widespread gene dysregulation including a number of up-regulated miR-29a target genes including DNA methylation enzymes (Dnmt3a, -3b) and cell cycle regulators (e.g. Cdk6, Tcl1, Hbp1, Pten). Knockdown of one of these targets, Dnmt3a, in miR-29a deficient HSCs resulted in partial restoration of colony formation, providing functional validation that Dnmt3a mediates part of miR-29a null HSPCs functional defects. miR-29a loss also abrogated leukemogenesis in the MLL-AF9 retroviral AML model. Together, our results demonstrate that miR-29a positively regulates HSC self-renewal and is required for myeloid leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Viral Infection Sentinel Rig-I Maintains Hematopoietic Stem Cell Function Through Regulating DNA-Damage Response

Blood ◽

10.1182/blood.v122.21.1166.1166 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1166-1166

Author(s):

Wu Zhang ◽

Meng-Lei Ding ◽

Xian-Yang Li ◽

He-Zhou Guo ◽

Hong-Xin Zhang ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cell Function ◽

Conflicts Of Interest ◽

Dna Lesions ◽

Type I ◽

Hematopoietic Stem ◽

Damage Response ◽

Underlying Mechanisms ◽

And Function

Abstract Throughout life hematopoietic stem cells (HSCs) have to cope with various kinds of insults from inflammation to DNA damage constantly to maintain the integrity of stemness. It is possible that certain core factors are commonly implicated in the maintenance of HSC pool and function under discrete physiological and pathological conditions. However, the underlying mechanisms remain largely unexplored. Previous works have demonstrated that retinoic acid inducible gene I (Rig-I) plays an essential role in recognizing viral RNA and activating type I IFN transcription, but whether Rig-I is involved in the core program governing HSCs’ behaviors is unclear. Here, we report that in the steady status Rig-I deficiency significantly increased HSC number by dysregulating the cell-cycling status of HSCs in mice. However, HSCs in Rig-I-/- mice were actually more sensitive to genotoxic treatments such as irradiation as compared to wild type HSCs, causing more Rig-I-/- mice to die of hematopoietic exhaustion. In accordance, HSC transplantation assays showed a significant impact of Rig-I loss on the hematopoietic regeneration capacity. Mechanistically, we found that Rig-I represented a pivotal component of the molecular pathways that mediate DNA-damage response and the repair of DNA lesions. Taken together, these data indicate a crucial role of innate immunity-regulatory factor Rig-I in the maintenance of HSCs. Disclosures: No relevant conflicts of interest to declare.

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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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