Tumor Suppressor Role of HIF-1alpha in Leukemia-Initiating Cells in Flt3-ITD-Induced Myeloproliferative Neoplasm

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3578-3578
Author(s):  
Talia Velasco ◽  
Jörg Cammenga
Keyword(s):  
Stem Cells ◽  
Tumor Suppressor ◽  
Hematopoietic Stem Cells ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Molecular Response ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hypoxia-induced signalling is a major regulator in normal and malignant hematopoiesis. The transcription factor HIF-1alpha plays a crucial role in the quiescence and self-renewal of hematopoietic stem cells as well as leukemia-initiating cells (LICs) of acute myeloid leukemia and chronic myeloid leukemia. Better understanding of the requirement of the molecular response to hypoxia in LICs could lead to new therapies targeting this pathway. We have therefore investigated the effect of HIF-1alpha loss on the phenotype and biology of FLT3-ITD induced myeloproliferative neoplasm (MPN). Using a combined transgenic mouse model (Mx1-Cre; Hif-1alphafl/fl; Flt3ITD/+) we showed that deletion of HIF-1alpha leads to a more severe MPN phenotype reflected by higher numbers of white blood cells and myeloid cells in peripheral blood, as well as a more severe splenomegaly. Loss of long-term hematopoietic stem cells (LT-HSCs: Lin- Sca1+ cKit+ CD48- CD150+) and increased number of lineage-restricted progenitors (Lin- Sca1+ cKit+ CD48+ CD150-) were the most pronounced effects on a cellular level upon the loss of HIF-1alpha. The proliferative effect of the HIF-1alpha loss was cell intrinsic and not at the expense of the ability of the LICs to self-renew because the disease was transplantable into secondary recipients recapitulating the same phenotype. Mice transplanted with FLT3-ITD induced MPN lacking HIF-1alpha succumbed to their disease (average survival of 35 weeks after transplant), while animals transplanted with MPN with wild-type HIF-1alpha suffered from MPN but did not die in the observation period of 60 weeks. These findings are in contrary to what has been previously described for the role of HIF-1alpha in leukemia initiating cells and lead us to propose that HIF-1alpha could act as a tumor suppressor gene, inhibiting proliferation in myeloid malignancies. Our results provide evidence that targeting HIF-1alpha can lead to disease progression of MPN while not affecting self-renewal of LICs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Suv39h1 Represses the Progression of MLL-Rearranged Myeloid Leukemia Via Hoxb13

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3878-3878
Author(s):  
Yajing Chu ◽  
Yangpeng Chen ◽  
Huidong Guo ◽  
Mengke Li ◽  
Jun Shi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tumor Suppressor ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Leukemic Cells ◽  
Moderate Increase ◽  
Hematopoietic Stem ◽  
Clinical Databases

Abstract Acute myeloid leukemia (AML) is the most frequent and heterogeneous malignancy in adult leukemic patients. Genome-wide analyses revealed that genes involved in epigenetic modifications are among the most often re-occurring mutations in AML, suggesting a crucial role of epigenetic regulation in leukemogenesis and leukemia relapse. As a mammalian lysine methyltransferase, SUV39H1 catalyzes di- and tri-methylation of histone 3 lysine 9, and is the predominant H3K9 methyltransferase expressed in hematopoietic stem cells (HSCs). Previous studies have shown that in MLL-rearranged leukemic cells, the normal localization of Suv39h1 and Sirt1 was interrupted due to the DNA binding of Dot1L to DNA. However, the biological role of SUV39H1 in MLL-rearranged leukemia remains unexplored. In this study, we investigated the role and the underlying mechanism of Suv39h1 during leukemia progression. By analyzing the clinical databases, we found a significantly reduced expression of SUV39H1 in AML cells in comparison with normal bone marrow (BM) cells. More importantly, we found that low expression of SUV39H1 predicts poorer survival in AML patients. In MLL-fusion induced AML mouse models (MLL-AF9/MA9 and MLL-NRIP3/MN3), Suv39h1 also exhibited lower expression in leukemia stem cells (LSCs, defined as c-Kit+ or Lin-Sca1-IL-7R-c-Kit+CD34+CD16/32+ L-GMP cells) when compared with normal HSPCs. These data suggest a potential role of SUV39H1 in leukemic progression and/or maintenance. To explore if Suv39h1 functions as a tumor suppressor in MLL-fusion driven leukemogenesis, we overexpressed Suv39h1 in MA9 BM AML cells. Western blotting analysis confirmed the overexpression of Suv39h1 with a moderate increase in global H3K9me3 levels in Suv39h1-overexpressed (SUV-OE) MA9 AML cells. Interestingly, Suv39h1 overexpression prolonged the survival of recipient AML mice in both secondary and tertiary transplantation groups. Both the frequency and the absolute number of phenotypic LSCs in BM and SP were significantly reduced in SUV-OE groups as manifested by flow cytometry. Furthermore, limiting dilution assays revealed a significant six-fold decrease of functional LSCs in SUV-OE AML cells (1/314 LSCs in SUV-OE AML cells vs 1/56 in controls). Cell cycle analysis of control and SUV-OE LSCs from BM revealed a significantly decreased proportion of SUV-OE cells in the S/G2/M phase concordant by an increased proportion of G0/G1 phases when compared with control cells. In contrast, a similar apoptotic ratio of L-GMPs in BM was observed between control and SUV-OE groups. Taken together, these data demonstrated that overexpressing Suv39h1 in AML cells reduces the frequency of functional LSCs by suppression its proliferation. To explore the underlying mechanisms, gene expression profiles were assessed by RNA-Seq of SUV-OE and control mouse AML c-Kit+ cells. A total of 69 genes were differentially expressed with fold change ≥ 4. Among these genes, Hoxb13 was of particular interesting since it was reported to be recurrently mutated in several types of cancers including leukemia. ChIP-qPCR revealed a two-fold increase of H3K9m3 distribution at the promoter of Hoxb13 in SUV-OE groups, indicating Hoxb13 may be a direct downstream target of Suv39h1. Restoring the expression of Hoxb13 in SUV-OE AML cells diminished the effect of SUV-OE-mediated prolonged survival of SUV-OE AML mice. Interestingly, overexpression of Hoxb13 alone in MA9 cells had no significant effect on the survival of MA9 AML mice, indicating that Hoxb13 is a downstream effector of Suv39h1, rather than MA9, and Suv39h1 itself is a downstream mediator of MA9. To summarize, we here for the first time, demonstrate that Suv39h1 is significantly down-regulated in AMLs and could function as a tumor suppressor in MLL-rearranged leukemia by epigenetically inhibiting the Hoxb13 expression. The molecular mechanism mediated by Suv39h1-Hoxb13 axis in tumor suppression could potentially provide us novel therapeutic strategies for MLL-rearranged leukemia. YJ.C, YP.C and HD.G contributed equally to this work. Corresponding authors: WP.Y and MJ.X. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

The Role of Mel18 Overlaps with BMI1 In the Self-Renewal of Human Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2619-2619
Author(s):  
Yasmin Reyal ◽  
Dominique Bonnet
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Polycomb Group ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Starting Point

Abstract Abstract 2619 Complex mechanisms regulate the ability of hematopoietic stem cells (HSCs) to self-renew, some of which may be exploited by leukemic stem cells. BMI1, a member of the polycomb group (PcG) proteins is known to be a positive regulator of this process, largely by repressing the p16/INK4a locus. However the role of other PcG proteins is unclear. We initially screened HSCs and progenitor populations from umbilical cord blood (CB), for the expression patterns of a number of PcG genes. Levels of expression were heterogeneous, indicating that there may be different roles for different PcG in HSCs versus progenitors. As a starting point we have focused on Mel18 (PCGF2) as it has been suggested in murine hematopoiesis that it acts to counteract BMI1. Lineage negative CB cells were transduced with lentiviral vectors expressing shRNA against Mel18, BMI1 and a control sequence. Specific knockdown by these constructs was confirmed at the RNA level to be at least 80% for both genes and was verified at the protein level by Western blot. Our data indicates that knockdown of Mel18 impairs the proliferation of primitive cord blood cells in both stromal-dependent and -independent culture, in a similar manner to BMI1. Furthermore Mel18 deficiency impedes both primary and secondary colony formation of all myeloid lineages in methylcellulose. These findings have been confirmed in vivo with significant reduction in engraftment of CB lineage negative cells in NOD/SCID mice at twelve weeks. We are investigating whether over expression of Mel18 can rescue BMI1 deficient cells to establish if these homologous genes have redundant functions. The data so far suggests that BMI1 is not unique in its role in HSC self - renewal, and Mel18 may share overlapping functions. This highlights a possible difference between human and murine hematopoiesis. Moreover it is likely that other members of the PcG family are also important in human HSCs. It will be of interest to investigate whether like BMI1, they are also implicated in the maintenance of the leukemic stem cell. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Deletion of PI3-Kinase Promotes Myelodysplasia Through Dysregulation of Autophagy in Hematopoietic Stem Cells

2020 ◽  
Author(s):  
Kristina Ames ◽  
Imit Kaur ◽  
Yang Shi ◽  
Meng Tong ◽  
Taneisha Sinclair ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Myeloid Leukemia ◽  
Chromosomal Abnormalities ◽  
Akt Pathway ◽  
Hematopoietic Growth Factors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Phosphoinositide 3 Kinase

AbstractHematopoietic stem cells (HSCs) maintain the blood system through a delicate equilibrium between self-renewal and differentiation. Most hematopoietic growth factors and cytokines signal through phosphoinositide 3-kinase (PI3K) via three Class IA catalytic PI3K isoforms (P110α, β, and δ), encoded by Pik3ca, Pik3cb, and Pik3cd, respectively. The PI3K/AKT pathway is commonly activated in acute myeloid leukemia (AML), and PI3K is a common therapeutic target in cancer. However, it is not known whether PI3K is required for HSC differentiation or self-renewal. We previously demonstrated that individual PI3K isoforms are dispensable in HSCs1,2. To determine the redundant roles of PI3K isoforms in HSCs, we generated a triple knockout (TKO) mouse model with deletion of all three Class IA PI3K isoforms in the hematopoietic system. Surprisingly, we observed significant expansion of TKO HSCs after transplantation, with decreased differentiation capacity and impaired multilineage repopulation. Additionally, the bone marrow of TKO mice exhibited myelodysplastic features with chromosomal abnormalities. Interestingly, we found that macroautophagy (thereafter autophagy) is impaired in TKO HSCs, and that pharmacologic induction of autophagy improves their differentiation. Therefore, we have uncovered important roles for PI3K in autophagy regulation in HSCs to maintain the balance between self-renewal and differentiation.

MicroRNA-29a Is An Oncogene in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 683-683
Author(s):  
Christopher Y. Park ◽  
Yoon-Chi Han ◽  
Govind Bhagat ◽  
Jian-Bing Fan ◽  
Irving L Weissman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Mirna Expression ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract microRNAs (miRNAs) are short, non-protein encoding RNAs that bind to the 3′UTR’s of target mRNAs and negatively regulate gene expression by facilitating mRNA degradation or translational inhibition. Aberrant miRNA expression is well-documented in both solid and hematopoietic malignancies, and a number of recent miRNA profiling studies have identified miRNAs associated with specific human acute myeloid leukemia (AML) cytogenetic groups as well as miRNAs that may prognosticate clinical outcomes in AML patients. Unfortunately, these studies do not directly address the functional role of miRNAs in AML. In fact, there is no direct functional evidence that miRNAs are required for AML development or maintenance. Herein, we report on our recent efforts to elucidate the role of miRNAs in AML stem cells. miRNA expression profiling of AML stem cells and their normal counterparts, hematopoietic stem cells (HSC) and committed progenitors, reveals that miR-29a is highly expressed in human hematopoietic stem cells (HSC) and human AML relative to normal committed progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors is sufficient to induce a myeloproliferative disorder (MPD) that progresses to AML. During the MPD phase of the disease, miR-29a alters the composition of committed myeloid progenitors, significantly expedites cell cycle progression, and promotes proliferation of hematopoietic progenitors at the level of the multipotent progenitor (MPP). These changes are manifested pathologically by marked granulocytic and megakaryocytic hyperplasia with hepatosplenomegaly. Mice with miR-29a-induced MPD uniformly progress to an AML that contains a leukemia stem cell (LSC) population that can serially transplant disease with as few as 20 purified LSC. Gene expression analysis reveals multiple tumor suppressors and cell cycle regulators downregulated in miR-29a expressing cells compared to wild type. We have demonstrated that one of these genes, Hbp1, is a bona fide miR-29a target, but knockdown of Hbp1 in vivo does not recapitulate the miR-29a phenotype. These data indicate that additional genes are required for miR-29a’s leukemogenic activity. In summary, our data demonstrate that miR-29a regulates early events in normal hematopoiesis and promotes myeloid differentiation and expansion. Moreover, they establish that misexpression of a single miRNA is sufficient to drive leukemogenesis, suggesting that therapeutic targeting of miRNAs may be an effective means of treating myeloid leukemias.

Oncogenic Nras Increases Hematopoietic Stem Cell Proliferation and Self-Renewal Through a Bimodal Effect

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 119-119
Author(s):  
Qing Li ◽  
Natacha Bohin ◽  
Tiffany Wen ◽  
Kevin M. Shannon ◽  
Sean J. Morrison
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Mek Inhibitor ◽  
Ras Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Activating Mutations ◽  
Cycle Regulation

Abstract Abstract 119 Accumulating evidence suggests that most leukemias are initiated by rare leukemic stem cells (LSC) that are transformed from the normal hematopoietic stem cells and progenitors (HSC/P) by genetic lesions that lead to activation of oncogenes and inactivation of tumor suppressor genes. However, the signaling mechanisms by which these genes transform HSC/P into LSC are poorly understood. Activating mutations of NRAS and KRAS are highly prevalent in acute myeloid leukemia (AML), some myeloproliferative neoplasm (MPN) and myelodysplastic syndromes (MDS). In addition other leukemia associated genetic lesions, such as the BCR-ABL fusion, PTPN11 mutations, FLT3 internal tandem duplications, and NF1 inactivation all deregulate Ras signaling. We previously developed a mouse strain that conditionally expresses an oncogenic NrasG12D allele from the endogenous locus. This consistently resulted in an indolent MPD with delayed onset and prolonged survival in Mx1-cre, NrasG12D/+ mice (referred to as NrasG12D). Oncogenic NrasG12D, however, cooperated with the MOL4070LTR retrovirus to induce AMLs that share molecular and morphologic features with human M4/M5 AML. Here we report that NrasG12D directly affects HSC/P functions. While normal HSCs must remain quiescent to maintain the long term self-renewal capacity and mutations that drive HSC into cycle often lead to HSC depletion, NrasG12D increased HSC proliferation but at the same time increased the self-renewal and competitiveness of HSCs. Serial transplantations revealed that NrasG12D HSCs were able to give higher level of reconstitution than wild-type (WT) HSCs and gave rise to long term multi-lineage reconstitution in lethally irradiated mice after up to four rounds of transplantation while WT HSCs failed to reconstitute beyond two rounds. These effects were not associated with the development of leukemia suggesting oncogenic Nras dys-regulates HSC at a pre-leukemic stage and therefore plays an important role in leukemia initiation. Using histone-2B-GFP (H2B-GFP) label-retaining assays, we further detected a “bimodal” effect of NrasG12D on HSCs: NrasG12D induced a subpopulation of rapid “cycling” HSCs that lost GFP labeling and reconstitution activity faster than WT HSC but another HSC subpopulation that remained more “quiescent” than WT HSCs and retained higher reconstitution when transplanted to irradiated mice. The canonical Ras effector, ERK, was not activated in NrasG12D HSC/Ps and inhibition of ERK with a MEK inhibitor, PD325901, did not have any effect on the Nras induced increase of HSC proliferation. Stat5, on the other hand, was significantly activated in NrasG12D HSC/Ps and heterozygous knockout of Stat5ab abolished the increased proliferation in NrasG12D HSCs, suggesting that Stat5 signaling mediates at least part of the Nras induced increase in HSC proliferation. Nras is thus the first signaling pathway that simultaneously increases HSC proliferation, self-renewal and competitiveness without inducing frank leukemogenesis. This is likely through a “bimodal” effect of Nras signaling on HSC cell cycle regulation. Our studies also identified Stat5 as a novel therapeutic target to inhibit early events in Ras mediated leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

GPI-80 Defines Self-Renewal Ability In Hematopoietic Stem Cells During Human Development

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4839-4839
Author(s):  
Sacha L. Prashad ◽  
Vincenzo Calvanese ◽  
Catherine Yao ◽  
Joshua Kaiser ◽  
Rajkumar Sasidharan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Human Development ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Surface Protein ◽  
Surface Proteins ◽  
Proliferative Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Advances in pluripotent stem cell and reprogramming technologies have provided hope of generating transplantable hematopoietic stem cells (HSC) in culture. However, better understanding of the identity and regulatory mechanisms that define the self-renewing HSC during human development is required. We discovered that the glycophosphatidylinositol-anchored surface protein GPI-80 (Vanin-2), previously implicated in neutrophil diapedesis, distinguishes a functionally distinct subpopulation of human fetal hematopoietic stem and progenitor cells (HSPC) that possess self-renewal ability. CD34+CD90+CD38-GPI80+ HSPCs were the only population that could maintain proliferative potential and undifferentiated state in co-culture on supportive stroma, and displayed engraftment potential in sublethally irradiated NSG mice. GPI-80 expression also enabled tracking of human HSC during development as they migrate across fetal hematopoietic niches, including early fetal liver and bone marrow. Microarray analysis comparing CD34+CD90+CD38-GPI80+ HSPC to their immediate progeny (CD34+CD90+CD38-GPI80-) identified novel candidate self-renewal regulators. Knockdown of GPI80, or the top enriched transcripts encoding surface proteins (ITGAM) or transcription factors (HIF3a) documented the necessity of all three molecules in sustaining human fetal HSC self-renewal. These findings provide new insights to the poorly understood regulation of human HSC development and suggest that human fetal HSCs utilize common mechanisms with leukocytes to enable cell-cell interactions critical for HSC self-renewal. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Smad4 is critical for self-renewal of hematopoietic stem cells

2007 ◽  
Vol 204 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Göran Karlsson ◽  
Ulrika Blank ◽  
Jennifer L. Moody ◽  
Mats Ehinger ◽  
Sofie Singbrant ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
In Vitro Proliferation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Early Embryonic Lethality

Members of the transforming growth factor β (TGF-β) superfamily of growth factors have been shown to regulate the in vitro proliferation and maintenance of hematopoietic stem cells (HSCs). Working at a common level of convergence for all TGF-β superfamily signals, Smad4 is key in orchestrating these effects. The role of Smad4 in HSC function has remained elusive because of the early embryonic lethality of the conventional knockout. We clarify its role by using an inducible model of Smad4 deletion coupled with transplantation experiments. Remarkably, systemic induction of Smad4 deletion through activation of MxCre was incompatible with survival 4 wk after induction because of anemia and histopathological changes in the colonic mucosa. Isolation of Smad4 deletion to the hematopoietic system via several transplantation approaches demonstrated a role for Smad4 in the maintenance of HSC self-renewal and reconstituting capacity, leaving homing potential, viability, and differentiation intact. Furthermore, the observed down-regulation of notch1 and c-myc in Smad4−/− primitive cells places Smad4 within a network of genes involved in the regulation HSC renewal.

scholarly journals Self-Renewal Pathways in Acute Myeloid Leukemia Stem Cells

2020 ◽  
Author(s):  
Jonason Yang ◽  
Nunki Hassan ◽  
Sheng Xiang Franklin Chen ◽  
Jayvee Datuin ◽  
Jenny Y. Wang
Keyword(s):  
Stem Cells ◽  
Monoclonal Antibody ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Myeloid Leukemia ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Clinical Potential ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a difficult-to-treat blood cancer. A major challenge in treating patients with AML is relapse, which is caused by the persistence of leukemia stem cells (LSCs). Self-renewal is a defining property of LSCs and its deregulation is crucial for re-initiating a new leukemia after chemotherapy. Emerging therapeutic agents inhibiting aberrant self-renewal pathways, such as anti-RSPO3 monoclonal antibody discovered in our recent study, present significant clinical potential that may extend beyond the scope of leukemogenesis. In this chapter, we provide an overview of normal and malignant hematopoietic stem cells, discuss current treatments and limitations, and review key self-renewal pathways and potential therapeutic opportunities in AML.

scholarly journals Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3704-3704
Author(s):  
Aldona A Karaczyn ◽  
Edward Jachimowicz ◽  
Jaspreet S Kohli ◽  
Pradeep Sathyanarayana
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Quiescent State ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

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