Hematopoietic Stem Cells Are Dependent On Homologous Recombination Only During Active Cell Cycle in Nuclease-Mutant Exonuclease1mut mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1225-1225
Author(s):  
Amar Desai ◽  
Stanton L. Gerson ◽  
Yulan Qing
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Stem Cell ◽  
Homologous Recombination ◽  
Conflicts Of Interest ◽  
Whole Body ◽  
Hematopoietic Stem ◽  
Increased Sensitivity ◽  
Quiescent Hscs

Abstract Abstract 1225 Hematopoietic stem cell (HSC) maintenance and self-renewal is crucial for long term tissue repopulation and immune function. HSC populations require functional DNA repair pathways in order to maintain their reconstitution capabilities but the pathways involved and the mechanisms of regulation are still being elucidated. It has been proposed that quiescent HSCs rely on the error prone non homologous end joining pathway for DNA double strand break (DSB) repair while HSCs in cycle use both NHEJ and the high fidelity homologous recombination (HR), but functional in vivo studies have not yet been completed. Exonuclease 1 participates in homologous recombination. We used Exo1mut fibroblasts to demonstrate that loss of Exo1 function results in a defective HR response, increased sensitivity to DSB inducing agents, and aberrant DNA damage signaling. However, Exo1mut mice did not appear to require HR to maintain quiescent HSCs at steady state or to respond to DNA damage. Exo1mutmice were able to sustain long term serial repopulation, displayed no defect in competitive repopulation or quiescence maintenance, and did not display increased sensitivity to whole body ionizing radiation (IR). In contrast, when Exo1mut HSCs were pushed into cell cycle with 5-Fluorouracil, the hematopoietic population and HSCs became hypersensitive to IR stress relative to WT B6 mice, as shown by decreased bone marrow cellularity, colony forming unit defects, loss of the HSC population, and finally animal death. Thus, loss of Exo1, and in turn fully functional HR, in quiescent HSC is not critical to stem cell function, survival, or recovery after DNA damage, whereas HR mediated repair of DNA damage is essential for HSC maintenance after cell cycle entry. In HSCs, DNA damage repair response, and sensitivity is dependent on cell cycle. Disclosures: No relevant conflicts of interest to declare.

TIMP-1 Deficiency Subverts Cell Cycle Dynamics in Long-Term HSCs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2514-2514
Author(s):  
Lara Rossi ◽  
Margaret Goodell
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Biological Behavior ◽  
Cell Cycle Distribution ◽  
Matrix Remodeling ◽  
Hematopoietic Stem ◽  
Stem Cell Quiescence

Abstract Abstract 2514 Poster Board II-491 In addition to the consolidated role in extracellular matrix remodeling, the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) has been suggested to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. We found that TIMP-1−/− mice have decreased HSC numbers and, consistent with this finding, TIMP-1−/− HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1−/− LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G1 phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Indeed, HSCs exhibit a higher proliferation rate, leading to an increased formation of CFU-C in vitro and spleen colonies (CFU-S) after transplant. Of note, TIMP-1−/− HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Our findings establish TIMP-1 role in HSC function, suggesting a novel mechanism presiding over stem cell quiescence and potentially involved in the development of hematological diseases. Disclosures: No relevant conflicts of interest to declare.

Slug Is Required for a Functional Hematopoietic Stem Cell (HSC) Niche

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2579-2579
Author(s):  
Yan Sun ◽  
Zack Zhengyu Wang ◽  
Wen-Shu Wu
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Adipocyte Differentiation ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Whole Body ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
B Lymphoid

Abstract Abstract 2579 Slug belongs to the highly conserved Slug/Snail family of zinc-finger transcription factors found in diverse species ranging from C. elegans to humans. Our previous study has already demonstrated that Slug serves intrinsic role in the regulation of HSC fates (Blood, 2010, 115(9):1709-1717). There is bone defect in Slug-/- mice, while Slug is expressed in osteoblast cells which are niche cells for HSCs, suggesting that Slug might be also extrinsically required for HSC regulation. To address this issue, the following experiments and assays were performed. Firstly, the cell proliferation of wild-type (WT) hematopoietic stem/progenitor cells (HS/PCs) (loaded with carboxyfluorescein succinimidyl ester (CFSE)) placed on bone marrow (BM) stromal layers (prepared from either Slug-/- or WT mice) was analyzed. Co-culture proliferation assay illustrated a clear increasing in the ability of Slug-/-BM stroma to support normal proliferation of WT HS/PCs, suggesting a significant and qualitative change in Slug-/- BM stromal cells so that they were able to support normal proliferation by Slug-competent HS/PCs. Secondly, BM microenvironment altered by Slug deficiency was defined by RT-PCR. Slug-/- BM had altered cell cycle profiles associated with increased stromal Notch1, N-cadherin, Vcam and Angiopoietin-1 expression. These findings indicate that Slug may participate in signaling in BM niche cells and thus is capable of influencing their function. Thirdly, the transplantation analysis was performed to address whether Slug impact was stem cell stroma dependent. WT hematopoietic cells in Slug knockout environment (8 weeks after transplantation) displayed a dramatic increase in LSK and LSK-Flk-2 ratio and in total number in BM, but not in spleen, indicating a stroma-determined effect by Slug on HSCs. It also showed a mobilization phenotype similar to that originally observed in Slug+/+ environment. Moreover, the annexin V apoptosis assay and cell-cycle analysis by the Edu incorporation assay suggested that Slug deficiency microenvironment promoted HSC expansion largely by increasing their proliferation, but not cell survival. Fourthly, each cell lineage in BM and spleen after WT BM transplanted into Slug+/+ and Slug-/- recipients was fully analyzed. The total number and ratio of T cells (CD3e, CD4+CD8-, CD4-CD8+ and CD4+CD8+) was markedly decreased in BM, but not in spleen, while the other lineages (granulocytes CD11b+Gr-1+, macrophages CD11b+, immature B lymphoid IgM-B220+, mature B lymphoid IgM+B220+, mature Erythroid CD71-Ter119+, and immature erythroid CD71+Ter119+) had an equivalent number and ratio in BM and spleen. Overall, Slug-/- BM microenvironment results in T cell loss. Finally, the recent study shows BM adipocytes as negative regulators of haematopoietic microenvironment, while the in vivo and in vitro evidences indicate that Slug is a key regulator of the adipocyte differentiation. Thus, in this study, we try to address whether Slug as a key factor adjusts adipocytes in BM niche. The fat of the whole body and the femurs and tibias were measured by DXA (Dual-energy X-ray Absorptiometry). Our data showed Slug-/- mice at 8 wk of age had lowest total fat, as well as ROI (region of interest) fat compared to Slug+/+ mice. In vitro adipocyte differentiation assay indicated there was less adipocyte formation from Slug-/- MSCs from BM. Furthermore, a dramatic loss of adipocytes in the femur in Slug-/- recipient was observed in 8 weeks after transplanting WT hematopoietic cells into Slug+/+ and Slug-/- recipients. Combining the above-mentioned data from WT hemtopoisis in Slug-/- microenvironment showed a dramatic increase in LSK and LSK-Flk-2 ratio and total numbers, lack of BM adipocytes after irradiation in fatless mice (Slug-/- recipient), suggesting a role of Slug-/- BM microenvironment in enhancing haematopoietic progenitor expansion via inhibition of BM adipocytes and post-transplant recovery. Taken together, our previously and present findings demonstrate that Slug serves intrinsic and extrinsic roles in the regulation of HSC fates. Disclosures: No relevant conflicts of interest to declare.

Characterization of Hematopoietic Stem Cell Function and Sensitivity in the Homologous Recombination Deficient Exonuclease1mut Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1293-1293
Author(s):  
Amar Desai ◽  
Yulan Qing ◽  
Stanton L. Gerson
Keyword(s):  
Dna Repair ◽  
Stem Cell ◽  
Homologous Recombination ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Strand Break ◽  
Hematopoietic Stem ◽  
Quiescent Hscs

Abstract Abstract 1293 Hematopoietic stem cell (HSC) maintenance is essential for sustained longevity and tissue function. The HSC population has lifelong self-renewing capabilities and gives rise to subsets of multipotent progenitor cells, and in turn a progeny of terminally differentiated mature cells consisting of all subtypes of the myeloid and lymphoid lineages. Long term reconstituting HSCs are necessary to replace these differentiated cells after losses caused by normal degradation or damage accumulation, with failure to replenish these stores being linked to a variety of human pathogeneses as well as aging phenotypes. HSC populations require functional DNA repair pathways in order to maintain their reconstitution capabilities but little is known about the pathways involved or the mechanism of regulation. While the majority of HSCs are quiescent at steady state, endogenous or exogenous stress can force these cells into proliferation, and previous evidence has suggested that the HSC reliance on DNA repair changes with this mobilization. Quiescent HSCs are believed to depend on non-homologous end joining (NHEJ) for repair but prior literature has shown that once forced into cycle, the DNA repair dependency shifts and is shared between homologous recombination (HR) and NHEJ. We use Exo1 deficiency as a model for homologous recombination loss in mice and demonstrate in vivo that HR is dispensable in quiescent HSCs. This is in contrast to loss of the complementary double strand break repair pathway NHEJ which has been shown to result in severe defects in HSC function. However when we force mobilize HSCs into cycle in vivo using the anti metabolite 5-fluorouracil we are able to demonstrate that the HR defects become detrimental to the animal as shown by increased cellular IR sensitivity and subsequent animal death. Additionally we use competitive repopulation studies to show that indeed the Exo1mut HSC population is more radiation sensitive after forced mobilization. This work begins to elucidate the consequences of the loss of homologous recombination in hematopoietic stem cells as well as the interplay between cell cycle status and DNA repair dependency. Disclosures: No relevant conflicts of interest to declare.

T(15;17)-PML/RAR-Induced Leukemogenesis: Long-Term Repopulating Hematopoietic Stem Cells as the Initial Target and More Mature Progenitors as the Potential Targets for Final Leukemic Transformation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3980-3980 ◽  
Author(s):  
Claudia Oancea ◽  
Brigitte Rüster ◽  
Jessica Roos ◽  
Afsar Ali Mian ◽  
Tatjana Micheilis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Conflicts Of Interest ◽  
Cell Model ◽  
Leukemic Cell ◽  
Hematopoietic Stem ◽  
Proliferation Potential

Abstract Abstract 3980 Poster Board III-916 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed PML/RAR in Sca1+/lin- cells and enriched this population for LT- (lin-/Sca1+/c-Kit+/Flk2-) and ST-HSC (lin-/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia without signs of differentiation in the group transplanted with the lin-/Sca1+/c-Kit+/Flk2- population but not from that transplanted with lin-/Sca1+/c-Kit+/Flk2+ cells. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine PML/RAR leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

Cycling Marrow Stem Cells Are Lost with Purification.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2308-2308
Author(s):  
Laura R Goldberg ◽  
Mark S Dooner ◽  
Mandy Pereira ◽  
Michael DelTatto ◽  
Elaine Papa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tritiated Thymidine ◽  
Hematopoietic Stem ◽  
Cell Purification ◽  
Marrow Stem Cell ◽  
Marrow Cells

Abstract Abstract 2308 Hematopoietic stem cell biologists have amassed a tremendous depth of knowledge about the biology of the marrow stem cell over the past few decades, facilitating invaluable basic scientific and translational advances in the field. Most of the studies to date have focused on highly purified populations of marrow cells, with emphasis placed on the need to isolate increasingly restricted subsets of marrow cells within the larger population of resident bone marrow cells in order to get an accurate picture of the true stem cell phenotype. Such studies have led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to homogeneity. However, work from our laboratory, focusing on the stem cell potential in un-separated whole bone marrow (WBM), supports an alternate view of marrow stem cell biology in which a large population of marrow stem cells are actively cycling, continually changing phenotype with cell cycle transit, and therefore, cannot be purified to homogeneity. Our studies separating WBM into cell cycle-specific fractions using Hoechst 33342/Pyronin Y or exposing WBM to tritiated thymidine suicide followed by competitive engraftment into lethally irradiated mice revealed that over 50% of the long-term multi-lineage engraftment potential in un-separated marrow was due to cells in S/G2/M. This is in stark contrast to studies showing that highly purified stem cell populations such as LT-HSC (Lineage–c-kit+sca-1+flk2−) engraft predominantly when in G0. Additionally, by performing standard isolation of a highly purified population of stem cells, SLAM cells (Lineage–c-kit+sca-1+flk2−CD150+CD41−CD48−), and testing the engraftment potential of different cellular fractions created and routinely discarded during this purification process, we found that 90% of the potential engraftment capacity in WBM was lost during conventional SLAM cell purification. Incubation of the Lineage-positive and Lineage-negative fractions with tritiated thymidine, a DNA analogue which selectively kills cells traversing S-phase, led to dramatic reductions in long-term multi-lineage engraftment potential found within both cellular fractions (over 95% and 85% reduction, respectively). This indicates that the discarded population of stem cells during antibody-based stem cell purification is composed largely of cycling cells. In sum, these data strongly support that 1) whole bone marrow contains actively cycling stem cells capable of long-term multi-lineage engraftment, 2) these actively cycling marrow stem cells are lost during the standard stem cell purification strategies, and 3) the protean phenotype of actively cycling cells as they transit through cell cycle will render cycling marrow stem cells difficult to purify to homogeneity. Given the loss of a large pool of actively cycling HSC during standard stem cell isolation techniques, these data underscore the need to re-evaluate the total hematopoietic stem cell pool on a population level in addition to a clonal level in order to provide a more comprehensive study of HSC biology. Disclosures: No relevant conflicts of interest to declare.

Metabolic Regulation of Hematopoietic Stem Cells During Stress

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-42-SCI-42
Author(s):  
Toshio Suda
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Metabolic Regulation ◽  
Conflicts Of Interest ◽  
Hypoxia Inducible Factor ◽  
Ros Generation ◽  
Hematopoietic Stem

Abstract Abstract SCI-42 Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1a (HIF-1a) protein, and generate ATP by anaerobic metabolism. In HIF-1a deficiency, HSCs became metabolically aerobic, lost cell cycle quiescence, and finally became exhausted. An increased dose of HIF-1a protein in VHL-mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the bone marrow (BM), which were not transplantable. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species (ROS), but leaves HSCs susceptible to changes in redox status (1). We have performed the metabolomic analysis in HSCs. Upregulation of pyruvate dehydrogenase kinases enhanced the glycolytic pathway, cell cycle quiescence, and stem cell capacity. Thus, HSCs directly utilize the hypoxic microenvironment to maintain their slow cell cycle by HIF-1a-dependent metabolism. Downregulation of mitochondrial metabolism might be reasonable, since it reduces ROS generation. On the other hand, at the time of BM transplantation, HSCs activate oxidative phosphorylation to acquire more ATP for proliferation. Autophagy also energizes HSCs by providing amino acids during transplantation. ATG (autophagy-related) 7 is essential for transplantation and metabolic homeostasis. The relationship between mitochondrial heat shock protein, mortalin, and metabolism in HSCs will also be discussed. Disclosures: No relevant conflicts of interest to declare.

Identification of a Developmentally-Restricted Hematopoietic Stem Cell That Gives Rise to Innate-like Lymphocytes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4302-4302
Author(s):  
Anna E Beaudin ◽  
Scott W. Boyer ◽  
Gloria Hernandez ◽  
Camilla E Forsberg
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Lineage Tracing ◽  
Lymphoid Cells ◽  
Gfp Expression ◽  
Hematopoietic Stem

Abstract The generation of innate-like immune cells distinguishes fetal hematopoiesis from adult hematopoiesis, but the cellular mechanisms underlying differential cell production during development remain to be established. Specifically, whether differential lymphoid output arises as a consequence of discrete hematopoietic stem cell (HSC) populations present during development or whether the fetal/neonatal microenvironment is required for their production remains to be established. We recently established a Flk2/Flt3 lineage tracing mouse model wherein Flk2-driven expression of Cre recombinase results in the irreversible switching of a ubiquitous dual-color reporter from Tomato to GFP expression. Because the switch from Tom to GFP expression in this model involves an irreversible genetic excision of the Tomato gene, a GFP+ cell can never give rise to Tom+ progeny. Using this model, we have definitively demonstrated that all functional, adult HSC remain Tomato+ and therefore that all developmental precursors of adult HSC lack a history of Flk2 expression. In contrast, adoptive transfer experiments of Tom+ and GFP+ fetal liver Lin-cKit+Sca1+ (KLS) fractions demonstrated that both Tom+ and GFP+ fetal HSC support serial, long-term multilineage reconstitution (LTR) in irradiated adult recipients. We have therefore identified a novel, developmentally restricted HSC that supports long-term multilineage reconstitution upon transplantation into an adult recipient but does not normally persist into adulthood. Developmentally-restricted GFP+ HSC display greater lymphoid potential, and regenerated both innate-like B-1 lymphocytes and Vg3-expressing T lymphocytes to a greater extent than coexisting Tom+ FL and adult HSC. Interestingly, whereas developmental regulation of fetal-specific B-cell subsets appears to be regulated cell-instrinsically, as fetal HSC generated more innate-like B-cells than adult HSC even within an adult environment, T-cell development may be regulated both cell intrinsically and extrinsically, as both the cell-of-origin and the fetal microenvironment regulated the generation of innate-like T-cells. Our results provide direct evidence for a developmentally restricted HSC that gives rise to a layered immune system and describes a novel mechanism underlying the source of developmental hematopoietic waves. As early lymphoid cells play essential roles in establishing self-recognition and tolerance, these findings are critical for understanding the development of autoimmune diseases, allergies, and tolerance induction upon organ transplantation. Furthermore, by uncoupling self-renewal capacity in situ with that observed upon transplantation, our data suggests that transplantation- and/or irradiation-induced cues may allow for the engraftment of developmental HSC populations that do not normally persist in situ. As LTR upon transplantation has served as the prevailing definition of adult HSC origin during development, our data challenge the current conceptual framework of adult HSC origin. Disclosures No relevant conflicts of interest to declare.

scholarly journals GATA-3 regulates hematopoietic stem cell maintenance and cell-cycle entry

Blood ◽  
2012 ◽  
Vol 119 (10) ◽  
pp. 2242-2251 ◽  
Author(s):  
Chia-Jui Ku ◽  
Tomonori Hosoya ◽  
Ivan Maillard ◽  
James Douglas Engel
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Normal Number ◽  
Cell Cycle Entry ◽  
Null Mutant Mice ◽  
Th2 Differentiation

Abstract Maintaining hematopoietic stem cell (HSC) quiescence is a critical property for the life-long generation of blood cells. Approximately 75% of cells in a highly enriched long-term repopulating HSC (LT-HSC) pool (Lin−Sca1+c-KithiCD150+CD48−) are quiescent, with only a small percentage of the LT-HSCs in cycle. Transcription factor GATA-3 is known to be vital for the development of T cells at multiple stages in the thymus and for Th2 differentiation in the peripheral organs. Although it is well documented that GATA-3 is expressed in HSCs, a role for GATA-3 in any prethymic progenitor cell has not been established. In the present study, we show that Gata3-null mutant mice generate fewer LT-HSCs and that fewer Gata3-null LT-HSCs are in cycle. Furthermore, Gata3 mutant hematopoietic progenitor cells fail to be recruited into an increased cycling state after 5-fluorouracil–induced myelosuppression. Therefore, GATA-3 is required for the maintenance of a normal number of LT-HSCs and for their entry into the cell cycle.

scholarly journals CDK6 Antagonizes TP53-Induced Responses during Tumorigenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-15-SCI-15
Author(s):  
Veronika Sexl ◽  
Karoline Kollmann ◽  
Florian Bellutti
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Conflicts Of Interest ◽  
Induced Responses ◽  
Hematopoietic Stem ◽  
Hematopoietic Malignancies ◽  
Independent Functions ◽  
Oncogenic Stress

Inhibitors directed against cyclin dependent kinases (CDKs) have raised much interest as anti-cancer therapeutics over the last years. In particular, inhibitors directed against CDK4/6 have been declared as a major breakthrough in cancer therapy by the FDA. CDK4 and CDK6 bind to D-type cyclins and subsequently phosphorylate the RB protein to allow cells to progress through the G1 phase of the cell cycle. The effectiveness of CDK4/6 inhibitors was primarily assigned to their ability to block cell cycle progression. In hematopoietic malignancies high levels of CDK6, but not CDK4, are frequently found. Over the last years we have assigned a novel and unexpected role for CDK6 as global transcriptional regulator. ChIP-Seq experiments identified more than 20.000 specific CDK6 binding sites in leukemic cells with the majority located in the promoter regions. CDK6 binding to chromatin does not require kinase activity whereas transcriptional control is regulated in a kinase- dependent as well as kinase-independent manner. Overlaying ChIP-Seq and RNA-Seq experiments showed that CDK6 contributes to the induction or repression of genes. Target genes of CDK6 which are important for leukemia progression include PIM1, c-MYC, AURKA, AURKB, AKT and VEGF-A. Murine leukemia models verified the importance of CDK6 for myeloid and lymphoid tumor formation downstream of a variety of oncogenes including FLT3-ITD, NPM/ALK, MLL/AF9, BCR/ABL or JAK2V617F. CDK6 contributes to disease development by regulating proliferation, cell survival, angiogenesis and cytokine production. In hematopoietic stem cells and leukemic stem cells kinase-independent functions dominate and CDK6 controls a network of transcription factors regulating stem cell quiescence and activation. The importance of kinase-dependent transcriptional effects is pronounced under conditions of stress and transformation. Upon oncogenic stress, CDK6 induces a set of genes that counteract pro-apoptotic TP53 responses including MDM4, PRMT5, PPM1D and BCL2. This response is induced by a CDK6 - dependent phosphorylation of the transcription factors SP1 and NFYA as verified by phospho-chromatome analysis. Murine Cdk6-deficient cells only survive oncogenic stress by mutating Tp53. The link between CDK6 and TP53 is conserved in human hematopoietic malignancies. Kollmann K, Heller G, Schneckenleithner C, et al. A kinase-independent function of CDK6 links the cell cycle to tumor angiogenesis. Cancer Cell. 2013;24(2):167-181.Scheicher R, Hoelbl-Kovacic A, Bellutti F, et al. CDK6 as a key regulator of hematopoietic and leukemic stem cell activation. Blood. 2015;125(1):90-101.Uras IZ, Walter GJ, Scheicher R, et al. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6. Blood. 2016;127(23):2890-2902.Bellutti F, Tigan AS, Nebenfuehr S, et al. CDK6 antagonizes P53-induced responses during tumorigenesis. Cancer Discov. 2018;8(7):884-897.Uras IZ, Maurer B, Nivarthi H, et al. CDK6 coordinates JAK2V617F mutant MPN via NF-kB and apoptotic networks. Blood. 2019;133(15):1677-1690. Disclosures No relevant conflicts of interest to declare.

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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