scholarly journals NIPA As a Novel Regulator of Aging and Stress Response of the Primitive HSC Pool

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1155-1155
Author(s):  
Stefanie Kreutmair ◽  
Rouzanna Istvanffy ◽  
Cathrin Klingeberg ◽  
Christine Dierks ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stress Response ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Age Related

Abstract Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Although HSCs numerically expand with age, their functional activity declines over time and the protection mechanism from DNA damage accumulation remains to be elucidated. NIPA (Nuclear Interaction Partner of ALK) is highly expressed in hematopoietic stem and progenitor cells, especially in the most primitive long-term repopulating HSCs (CD34-Flt3-Lin-Sca1+cKit+). Loss of NIPA leads to a significant exhaustion of primitive hematopoietic cells, where Lin-Sca1+cKit+ (LSK) cells were reduced to 40% of wildtype (wt) littermates (p<0.001). All LSK-subgroups, LT-HSCs (p<0.001), ST-HSCs (CD34+Flt3-LSK; p<0.01) and MPPs (CD34+Flt3+LSK; p<0.05) of NIPA deficient animals are affected and failed to age-related increase, whereas the lineage differentiation of Nipako/ko progenitor cells showed no gross differences. Myeloid depression by 5-FU treatment led to severely reduced HSC self renewal in Nipako/ko mice independent of age (p<0.001). Moreover, weekly 5-FU activation showed reduced survival of Nipako/ko vs. wt animals (11 vs. 14.5 days). To further examine the role of NIPA in HSC maintenance and exhaustion, we performed in vivo repopulationexperiments, where Nipa deletion causes bone marrow failure in case of competition, as Nipako/ko cells contributed to less than 10% of transplanted BM cells 6 month after transplantation (TX). According to this, colony formation assays and limiting dilution transplantation showed a dramatic reduction of competitive repopulation units (p<0.0001) in Nipako/ko animals. Serial LSK transplantation assays revealed loss of Nipa-deficient LSKs shortly after TX, whereas long-term repopulation capacity seemed to be maintained, suggesting a role of NIPA in critical stress response. To further investigate the stress response in Nipa-deficient HSCs, we irradiated LSKs with 3 Gy and stained for DNA-Damage foci by pH2ax. Remarkably, loss of NIPA led to significant higher numbers of pH2ax foci in irradiated HSCs (46% > 6 foci vs. 17% > 6 foci in wt cells) and highly increased the rates of apoptotic cells especially in the primitive CD34-LSK population. Taken together our results highlight the importance of the DNA damage response at HSC level for lifelong hematopoiesis and establish NIPA as a novel regulator of aging and stress response of the primitive HSC pool. Disclosures No relevant conflicts of interest to declare.

Dual Role Of MERIT40 In Hematopoietic Stem and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 797-797
Author(s):  
Krasimira Rozenova ◽  
Jing Jiang ◽  
Chao Wu ◽  
Junmin Wu ◽  
Bernadette Aressy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Adaptor Protein ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is maintained by cell intrinsic and extrinsic mechanisms, including tight regulation of signaling pathways such as Tpo-Mpl and SCF-ckit. Posttranslational modifications, such as phosphorylation and ubiquitination, regulate these pathways. While the role of protein phosphorylation is well established, the importance of ubiquitination in HSC self-renewal has not been well addressed. It is known that of the seven different lysines on ubiquitin, Lys48 polyubiquitination is a marker for protein degradation, and Lys63 polyubiquitination is associated with regulation of kinase activity, protein trafficking, and localization. In this study, we provide evidence that the adaptor protein MERIT40 has multiple roles in hematopoietic stem/progenitor cells (HSPCs). MERIT40 is a scaffolding protein shared by two distinct complexes with Lys63 deubiquitinase (DUB) activities: the nuclear RAP80 complex with a known role in DNA damage repair in breast/ovarian cancer cells, whereas the functions of the cytoplasmic BRISC remains less characterized. MERIT40 is important for integrity of both complexes, and its deficiency leads to their destabilization and a >90% reduction in deubiquitinase activity. By using MERIT40 knockout (M40-/-) mice, we found that lack of MERIT40 leads to a two-fold increase in phenotypic and functional HSCs determined by FACS and limiting dilution bone marrow transplantation (BMT), respectively. More importantly, M40-/- HSCs have increased regenerative capability demonstrated by increased chimerism in the peripheral blood after BMT of purified HSCs. The higher self-renewal potential of these HSCs provides a survival advantage to M40-/- mice and HSCs after repetitive administration of the cytotoxic agent 5-flurouracil (5FU). MERIT40 deficiency also preserves HSC stemness in culture as judged by an increase in peripheral blood chimerism in recipient mice transplanted with M40-/- Lin-Sca1+Kit+ (LSK) cells cultured in cytokines for nine days compared to recipient mice receiving cultured wildtype (WT) LSK cells. In contrast to the increased HSC homeostasis and superior stem cell activity due to MERIT40 deficiency, M40-/- mice are hypersensitive to DNA damaging agents caused by inter-cross linking (ICL), such as Mitomycin C (MMC) and acetaldehydes that are generated as side products of intracellular metabolism. MMC injection caused increased mortality in M40-/- mice compared to WT controls attributable to DNA damage-induced bone marrow failure. MMC-treated M40-/- mice showed marked reduction in LSK progenitor numbers accompanied by increased DNA damage, in comparison to WT mice. Consistent with the in vivo studies, M40-/- progenitor cells are hypersensitive to MMC and acetaldehyde treatment in a cell-autonomous manner in colony forming assays. ICL repair is known to require Fanconi Anemia (FA) proteins, an ICL repair network of which mutations in at least 15 different genes in humans cause bone marrow failure and cancer predisposition. Thus, M40-/- mice represent a novel mouse model to study ICL repair in HSPCs with potential relevance to bone marrow failure syndromes. Taken together, our data establishes a complex role of MERIT40 in HSPCs, warranting future investigation to decipher functional events downstream of two distinct deubiquitinating complexes associated with MERIT40 that may regulate distinct aspects of HSPC function. Furthermore, our findings reveal novel regulatory pathways involving a previously unappreciated role of K63-DUB in stem cell biology, DNA repair regulation and possibly bone marrow failure. DUBs are specialized proteases and have emerged as potential “druggable” targets for a variety of diseases. Hence, our work may provide insights into novel therapies for the treatment of bone marrow failure and associated malignancies that occur in dysregulated HSCs. Disclosures: No relevant conflicts of interest to declare.

G-CSF Treatment Induces Toll-Like Receptor Signaling and Regulates Hematopoietic Stem Cell Function

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1181-1181 ◽  
Author(s):  
Laura G. Schuettpelz ◽  
Joshua N. Borgerding ◽  
Priya Gopalan ◽  
Matt Christopher ◽  
Molly Romine ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Intestinal Flora ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Tlr Signaling ◽  
Hematopoietic Stem

Abstract Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are unclear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect. RNA profiling and flow cytometry studies of HSCs from G-CSF treated mice show that multiple toll- like receptors (TLRs) are upregulated in HSCs upon G-CSF treatment, and gene set enrichment analysis shows enhancement of TLR signaling in G-CSF-treated HSCs. G-CSF-induced expansion of phenotypic HSCs is reduced in mice lacking the TLR signaling adaptors MyD88 or Trif, and the induction of quiescence is abrogated in mice lacking these adaptors. Furthermore, loss of TLR4 mitigates the G-CSF-mediated HSC repopulating defect. Interestingly, baseline HSC function is also dependent on TLR signaling. We show that HSC long-term repopulating activity is enhanced in Tlr4-/- and MyD88-/- mice, but not Trif-/- mice. One potential source of TLR ligands affecting HSC function in the bone marrow is the gut microbiota. Indeed, we show that in mice treated with antibiotics to suppress intestinal flora, G-CSF induced HSC quiescence and hematopoietic progenitor mobilization are attenuated. Moreover, in germ free mice, HSC long-term repopulating activity is enhanced. Collectively these data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling. Our finding of enhanced TLR signaling upon G-CSF treatment, and the mitigation of G-CSF’s effects in mice deficient for TLR signaling or commensal organisms, suggest that TLR antagonists and/or agonists may ultimately be used clinically to enhance engraftment following bone marrow transplantation or applied toward the treatment of patients with bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

Differential hMLH1 Gene Expression after Temozolomide Selection Linked to Microsatellite Instability in a Subset of Hematopoietic Stem/Progenitor Cells in Old Versus Young and Cancer Versus Normal Patient Samples.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 509-509
Author(s):  
Jonathan Kenyon ◽  
Emily Thomas ◽  
Karen Lingas ◽  
Stanton L. Gerson
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Microsatellite Instability ◽  
Progenitor Cells ◽  
Mismatch Repair ◽  
Dna Methyltransferase ◽  
Apoptotic Cell ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Age Related

Abstract The etiology of hematologic pathologies such as leukemia, myelodysplasia, anemia, bone marrow failure, altered immune function, and how they are associated with aging, remains unclear. Our hypothesis is that these diseases are caused or aggravated by a subset of hematopoietic stem/progenitor cells (HSC) lacking effective mismatch repair (MMR) and therefore exhibiting a hypermutator phenotype. Microsatellite instability (MSI) is a marker of MMR deficiency. We used cord blood, bone marrow, and bone core samples to isolate and then clonally expand HSC for MSI analysis. Five microsatellite loci previously used in the diagnosis of the MMR defective disease HNPCC (BAT 25, BAT 26, D2S123, D5S346, and D17S250) were analyzed for insertions and deletions. We have analyzed 38 patient samples between the ages of 0 and 86 years, including 8 cancer patients. These data show an age-dependent increase in the frequency of high grade microsatellite instability (MSI-H), i.e. those CFU with microsatellite instability at >20% of loci tested. Additionally, samples obtained from individuals older than 50 years were 6 times more likely to have a > 10% frequency of MSI-H CFU than samples obtained from younger individuals, suggesting an inflection point for the onset of hematopoietic diseases. In all instances this instability is seen only within a subset of human HSC clones. To further characterize the origin of this deficiency, a method to select for MMR deficient hematopoietic cells was developed that first selected for survival of MMR deficient HSC, and then allowed for the examination of expression status of key MMR pathway genes hMLH1 and hMSH2 and their protein products. First, CD34+ HSC were isolated from various aged patient samples. To avoid possible effects of other repair pathways, the cells were treated with O6-Benzylguanine (BG) to remove O6-methylguanine DNA-methyltransferase (MGMT) activity and prevent removal of O6-methylguanine lesions. Next, temozolomide (TMZ) at concentrations of 50–125 μM was used to induce O6-methylguanine (O6-mG) lesions that persist in the presence of BG. These O6-mG lesions mispair with cytosine and are recognized as DNA mismatches by the mismatch repair (MMR) pathway inducing apoptotic cell death. TMZ selected cells that fail to recognize the mispair due to a lack of MMR survive this selection. In these TMZ resistant clones, RT-PCR amplification of hMLH1 transcripts from total RNA isolated reveal a defect in hMLH1 but not hMSH2 expression. In the one AML sample obtained thus far HSC treated with 200 uM TMZ we have observed 0 to 30% of hMLH1 expression within TMZ resistant CFU was observed when compared to untreated controls. Together this data links MSI to MMR defects of a subpopulation of hematopoietic precursors in older individuals. This is the first examination of MMR gene expression in clones of HSC that has shown specific MMR functional deficiencies. Our study suggests that a MMR pathway deficiency in a subset of stem cells could contribute to age related hematopoietic disease processes including stem cell failure and malignant transformation.

Involvement of Transcriptional Mediator Subunit TRAP220/MED1 in Action of Niche Cells to Support Hematopoietic Stem/Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3581-3581
Author(s):  
Kana Inoue ◽  
Akiko Sumitomo ◽  
Natsumi Hasegawa ◽  
Ayuko Kasai ◽  
Kenji Yonezawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Progenitor Cells ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Live Cells ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract The mammalian TRAP/Mediator complex is a master transcriptional regulatory complex that integrates signals of diverse activators and recruits RNA polymerase II and other general factors to activate transcription. The TRAP220/MED1 subunit was originally identified as a ligand-dependent coactivator specific for nuclear receptors. We have previously shown through biochemical and mouse genetic studies that MED1 is essential for embryogenesis, cell growth/differentiation and homeostasis, and that it stimulates nuclear receptor-mediated myelomonopoiesis. MED1 also integrates other activators such as GATA-1 and C/EBPβ and appears to mediate erythropoiesis as well. The niche cells in the bone marrow plays a pivotal role in the maintenance of hematopoietic stem/progenitor cells (HSPCs). In this study, we employed mouse embryonic fibroblasts (MEFs) as a model to analyze the role of MED1 in the niche, since MEFs have a mesenchymal feature with the osteoblastic precursor lineage and are known to support HSPCs. To establish an experimental system, we crossed Med1 and p53 double knockouts to obtain Med1+/+/p53−/− and Med1−/−/p53−/− E10.0 embryos from a single female and prepared stable MEF lines. Then the Med1−/−/p53−/− MEFs were stably transfected with a MED1 expression vector (Rev-Med1−/− MEFs) or a control empty vector. When normal mouse bone marrow cells were cocultured with these MEFs treated with mitomycin C for a short period of 2 weeks, cell counts, live cells (MTT assay) and a DNA synthesis (BrdU incorporation) of marrow cells were measured. The number of live cells as well as DNA synthesis on Med1−/− MEFs was significantly decreased during this period, but those on Rev-Med1−/− MEFs recovered to the control levels. Thus the growth stress on MEFs appears to be attenuated on Med1−/− MEFs. When apoptosis of the marrow cells was measured, both the FITC-dUTP incorporation by TdT and annexin V/PI double positive cells were lower for Med1−/− MEFs, indicating that apoptosis was also attenuated. We next assessed the role of MED1 in MEFs to support long-term bone marrow culture. After bone marrow cells were cultured on mitomycin C-treated MEFs for 8 weeks in Myelocult M5300 (StemCell Technologies) or IMDM supplemented with BIT9500 (StemCell Technologies) and LDL, progenitor cells (adherent and nonadherent) were collected and cultured in complete methylcellulose (Methocult M3434; StemCell Technologies), and colonies were counted. The number of both myeloid and erythroid colonies were significantly attenuated (0 to 40% depending on experimental conditions) for cells on Med1−/− MEFs, but colonies for cells cultured on Rev-Med1−/− MEFs recovered to the control level. In order to exclude the possibility that lot differences among MEFs or p53 depletion might have affected the results, we next prepared primary Med1+/+ and Med1−/− MEFs by crossing Med1+/− mice and conducted the long-term culture experiments using these MEFs. The attenuated number of colonies for cells cocultured with Med1−/− MEFs (circa 10% of the control) was reproduced repeatedly, indicating that the observed role of MED1 in MEFs to support HSPCs is intrinsic. Since MED1 converges signals from a series of activators on specific promoters and activates transcription, one or some products of the downstream target genes in MEFs may be responsible for the observed activity to maintain HSPCs. In search for candidate MED1 target gene products among a series of known molecules that possess an activity on HSPCs, only the expression of osteopontin was found to be attenuated in Med1−/− MEFs and reverted in Rev-Med1−/− MEFs. Other factors including Angiopoietin-1 and Jagged-1 were comparable. This fact contrasts with the previous observation of osteopontin knockouts where the null niche cells that restricted the size of HSPC number overexpressed these factors. We next assessed the role of MED1 on the osteopontin promoter. We focused on vitamin D receptor (VDR) and Runx2 among the activators and tested MEFs by luciferase reporter assays. The basal level of transcription without any activators in Med1−/− MEFs was about half of the control. Moreover, both the activation by Runx2 and the liganddependent VDR function were significantly attenuated in Med1−/− MEFs. These results indicate that transcriptional coactivator MED1 in niche cells plays an important role in HSPCs support, and that osteopontin may be one of the downstream candidate target genes for MED1.

Further Evidence That HO-1 Regulates SDF-1 Expression in the Bone Marrow Microenvironment and That HO-1-Deficient Mice Show a Defect in the SDF-1–CXCR4 Retention Axis of Hematopoietic Stem/Progenitor Cells in Bone Marrow and Thus Are Easy Mobilizers - Studies in HO-1 Mutant Mice, Irradiation Chimeras, and the Effect of in Vivo Pharmacological Inhibition of HO-1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 344-344
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Crucial Role ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Deficient Mice

Abstract Abstract 344 Background: Stromal derived factor-1 (SDF-1), which binds to the CXCR4 receptor expressed on the surface of hematopoietic stem/progenitor cells (HSPCs), plays an important role in the retention of HSPCs in BM niches. Heme oxygenase (HO-1) is a stress-responsive enzyme that catalyzes the degradation of heme and plays an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury, atherosclerosis, and cancer. Interestingly, it has also been reported that HO-1 regulates the expression of SDF-1 in myocardium (J Mol Cell Cardiol. 2008;45:44–55). Aim of study: Since SDF-1 plays a crucial role in retention and survival of HSPCs in BM, we become interested in whether HO-1 is expressed by BM stromal cells and whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach: To address this issue, we employed several complementary strategies to investigate HO-1–/–, HO-1+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) the chemotactic responsiveness of HSPCs to an SDF-1 gradient as well as to other chemoattractants, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and extracellular nucleotiodes (ATP, UTP), iv) the adhesiveness of clonogenic progenitors to immobilized SDF-1 and stroma, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100, assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. We also exposed mice to the small HO-1 molecular inhibitor tin protoporphyrin IX (SnPP) and studied the effect of this treatment on G-CSF- or AMD3100-induced mobilization of HSPCs. Finally, to prove an environmental HSPC retention defect in HO-1-deficient mice, we created radiation chimeras, wild type mice transplanted with HO-1-deficient BM cells, and, vice versa, HO-1-deficient mice reconstituted with wild type BM cells. Results: Our data indicate that under normal, steady-state conditions, HO-1–/– and HO+/– mice have normal PB cell counts and numbers of circulating CFU-GM, while a lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. However, while BMMNCs from HO-1–/– have normal expression of the SDF-1-binding receptor, CXCR4, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1–/– animals respond more robustly to an SDF-1 gradient, and HO-1–/– animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into PB in response to G-CSF and AMD3100. Both G-CSF and AMD3100 mobilization were also significantly enhanced in normal wild type mice after in vivo administration of HO-1 inhibitor. Finally, mobilization studies in irradiation chimeras confirmed the crucial role of the microenvironmental SDF-1-based retention mechanism of HSPCs in BM niches. Conclusions: Our data demonstrate for the first time that HO-1 plays an important and underappreciated role in modulating the SDF-1 level in the BM microenvironment and thus plays a role in retention of HSPCs in BM niches. Furthermore, our recent data showing a mobilization effect by a small non-toxic molecular inhibitor of HO-1 (SnPP), suggest that blockage of HO-1 could be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate the role of HO-1 in homing of HSPCs after transplantation to BM stem cell niches. Disclosures: No relevant conflicts of interest to declare.

scholarly journals ROS-mediated iron overload injures the hematopoiesis of bone marrow by damaging hematopoietic stem/progenitor cells in mice

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Xiao Chai ◽  
Deguan Li ◽  
Xiaoli Cao ◽  
Yuchen Zhang ◽  
Juan Mu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Iron Overload ◽  
Progenitor Cells ◽  
Bone Marrow Failure ◽  
Hereditary Hemochromatosis ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Organ Dysfunctions

Abstract Iron overload, caused by hereditary hemochromatosis or repeated blood transfusions in some diseases, such as beta thalassemia, bone marrow failure and myelodysplastic syndrome, can significantly induce injured bone marrow (BM) function as well as parenchyma organ dysfunctions. However, the effect of iron overload and its mechanism remain elusive. In this study, we investigated the effects of iron overload on the hematopoietic stem and progenitor cells (HSPCs) from a mouse model. Our results showed that iron overload markedly decreased the ratio and clonogenic function of murine HSPCs by the elevation of reactive oxygen species (ROS). This finding is supported by the results of NAC or DFX treatment, which reduced ROS level by inhibiting NOX4 and p38MAPK and improved the long-term and multi-lineage engrafment of iron overload HSCs after transplantation. Therefore, all of these data demonstrate that iron overload injures the hematopoiesis of BM by enhancing ROS through NOX4 and p38MAPK. This will be helpful for the treatment of iron overload in patients with hematopoietic dysfunction.

The Zebrafish Provides Mechanistic Insights into the Role of Poly(A)-Specific Ribonuclease (PARN) in Hematopoietic Stem Cell (HSC) Homeostasis and Bone Marrow Failure

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 668-668
Author(s):  
Adam P Deveau ◽  
Andrew J Coombs ◽  
Santhosh Dhanraj ◽  
Gretchen Wagner ◽  
Yigal Dror ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Phosphohistone H3 ◽  
Biallelic Mutations ◽  
Insight Into

Abstract Development of tissues during embryogenesis and their homeostasis after formation are highly regulated by expression of coding and non-coding RNAs. Deadenylation is a core mechanism that regulates RNA function and fate by controlling turnover, abundance and maturation of RNA. Factors that promote or inhibit deadenylation control hematopoietic stem cell (HSC) homeostasis, and inhibition of deadenylation limits differentiation of the HSCs. Importantly, RNA biogenesis has emerged as a mechanism underlying several inherited bone marrow failure syndromes (IBMFSs), such as Diamond Blackfan anemia, dyskeratosis congenita (DC) and Shwachman-Diamond syndrome. Poly(A)-specific ribonuclease (PARN) is a major deadenylation factor and demonstrates high specificity for single-stranded poly (A) tails of various RNA species. We recently identified biallelic mutations in PARN as a cause of hematopoietic failure and profound hypomyelination, similar to the severe form of DC, Hoyeraal-Hreidersson syndrome. We developed a zebrafish model to characterize the hematopoietic phenotype of a patient identified to have severe inherited bone marrow failure resulting from a combined deletion of PARN on one allele and missense mutation in the other. Zebrafish posses a single parn ortholog. Zebrafish parn protein shares homology and high sequence identity (~64%) to its human counterpart. Embryos were injected with either translation start-site or splice-site-blocking morpholino at the one-cell stage. Both morpholino injections resulted in anemic embryos at 48 hours post fertilization (hpf), as evidenced by reduced o-dianisidine staining and gata1 expression by whole-mount in situ hybrization and GFP+ red cell numbers by fluorescence-activated cell sorting (FACS). Morphant embryos also demonstrated reduced expression of myeloid cell markers including l-plastin, myeloperoxidase, and macrophage expressed gene 1 and were leukopenic as evidenced by reduced number of GFP+ myeloid cells. FACS analysis revealed that fluorescently labeled HSCs were increased in parn morphants. Early hematopoietic markers, lmo2 and fli1, expressed in hemogenic and vascular tissue respectively, were also overexpressed in parn morphants. Furthermore, there was reduced global cell proliferation in morphant embryos as determined by phosphohistone H3 antibody staining. These findings suggest that the absence of parn results in a developmental arrest at the HSC stage with an inability to differentiate into leukocyte or erythroid lineages. Similarly, human cell culture data from PARN-deficient HSC/progenitor cells demonstrated markedly reduced colony forming capacity. By modeling parn deficiency in the zebrafish, we validate for the first time an IBMFS that results from biallelic mutations in a major deadenylating protein. Moreover, our zebrafish studies provide insight into the role of parn in maintaining HSC homeostasis/differentiation as the origin of the pancytopenia observed in this patient. Permanent knockouts in the zebrafish using CRISPR/Cas9 technology are underway, which will enable tracking the hematopoietic phenotype into adulthood. These studies have set the stage for critical translational research in a rare form of bone marrow failure as well as new insight into HSC regulation. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Depletion of TGF-β Co-Receptor CD109 Induces Erythroid Differentiation of TF-1 Cells: A Model of Preferential Commitment of PIGA-Mutated Hematopoietic Stem Cells in Immune-Mediated Bone Marrow Failure

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3874-3874
Author(s):  
Tanabe Mikoto ◽  
Noriharu Nakagawa ◽  
Kohei Hosokawa ◽  
Luis Espinoza ◽  
Kana Maruyama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Bone Marrow Failure ◽  
Erythroid Differentiation ◽  
Healthy Individuals ◽  
Hematopoietic Stem ◽  
Immune Mediated

Abstract [Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may play an important role in the regulation of the HSPC commitment, given the fact that a lack of GPI-APs due to PIGA mutations allows HSPCs to preferentially undergo commitment into mature blood cells under immune pressure against HSPCs in patients with acquired aplastic anemia. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs has not been clarified. TF-1 is one of a few myeloid leukemia cell lines that express CD109, the proliferation of which is dependent on GM-CSF. Since TF-1 undergoes erythroid differentiation in response to δ-5-aminolevulinic acid (δ-ALA), and its differentiation is reportedly inhibited by TGF-β, a lack of GPI-APs due to PIGA mutation and/or the knockout (KO) of CD109 may affect the differentiation of TF-1 cells. [Objectives/Methods] To gain insights into the role of GPI-APs on HSPCs, we established a PIGA-mutated TF-1 cell line by culturing TF-1 in the presence of α-toxin for several months, and a CD109 KO TF-1 cell line using a CRISPR-Cas 9 system. The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) on TF-1 cells using flow cytometry (FCM) and iron staining. We also determined the CD109 expression by HSPCs from healthy individuals and C57BL/6 mice using FCM and a quantitative PCR. [Results] Both GPI-AP-deficient TF-1 cells that had a PIGA mutation (7 nucleotide deletion at position 291-297 [TTGTCAC] in exon 2) and CD109 KO TF-1 cells showed slower proliferation than wild-type (WT) TF-1 cells. Similarly to TF-1 cells treated with δ-ALA, both mutant cells expressed GPA, exhibited erythroid morphology, and were positive for iron granules, suggesting that GPI-APs inhibited the erythroid differentiation of WT TF-1 cells that were cultured in RPMI1640 containing 10% fetal bovine serum (FBS), and that the GPI-AP that plays a key role in the inhibition of erythroid differentiation is CD109. Since low levels (1-2 ng/ml) of TGF-β in the serum-containing culture medium were suspected to inhibit the erythroid differentiation of WT TF-1 through its binding to CD109, WT TF-1 cells were cultured in a serum-free medium Expi293 Expression Medium for 10 days. While control TF-1 cells cultured in the serum-containing RPMI1640 were negative for the expression of GPA, 77.0-84.5% of the cultured TF-1 cells expressed GPA and exhibited erythroid morphology. CD109 was expressed by 12.1-18.3% of CD34+CD38- cells, 4.5-7.4% of common myeloid progenitor cells (CMPs), 20.8-42.4% of megakaryocyte-erythrocyte progenitor cells (MEPs), and 14.2-22.0% of granulocyte macrophage progenitor cells (GMPs) in the bone marrow of healthy individuals, while murine CD48-CD150+CD34- LSK cells were negative for either CD109 protein or mRNA. [Conclusions] CD109 protects TF-1 cells from differentiating into erythroid cells in serum-containing culture. In contrast to keratinocytes, the CD109 on TF-1 cells, and possibly on HSPCs, may enhance TGF-β signaling, and the lack of the GPI-AP might make PIGA-mutated HSPCs insensitive to TGF-β, leading to the preferential commitment of mutant HSPCs to mature blood cells in immune-mediated bone marrow failure. Disclosures Nakao: Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

scholarly journals Mutant ASXL1 Promotes Expansion of the Phenotypic Hematopoietic Stem Cell Compartment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 821-821
Author(s):  
Takeshi Fujino ◽  
Susumu Goyama ◽  
Yuki Sugiura ◽  
Daichi Inoue ◽  
Satoshi Yamasaki ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Bone Marrow Cells ◽  
Mtor Pathway ◽  
Mitochondrial Activity ◽  
Hematopoietic Stem ◽  
Rapamycin Treatment ◽  
Age Related ◽  
Age Related Changes

Somatic mutations of the ASXL1 gene are recurrently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations causes CH are not understood. Here, using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we investigated the effect of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). 　To examine the influence of ASXL1-MT on hematopoiesis, we bred the ASXL1-MT-KI mice with Vav-Cre transgenic mice. Young ASXL1-MT-KI mice (6-12 weeks) did not show significant changes in hematological parameters and differentiation status of peripheral blood. We observed the decreased frequency of hematopoietic stem and progenitor cells (HSPCs), including long-term HSCs (LT-HSCs). Competitive transplantation assays showed the reduced repopulation ability in ASXL1-MT-KI HSPCs. Thus, ASXL1-MT decreased the number and impaired the function of HSPCs in young mice. 　Next, we examined age-related changes in hematopoiesis caused by ASXL1-MT. Aged ASXL1-MT-KI mice displayed a myeloid-biased differentiation and hypocellular bone marrow, indicating the dysfunction of hematopoiesis. Interestingly, ASXL1-MT markedly increased the frequency of phenotypic LT-HSCs (pLT-HSCs) in aged mice (20-24 months). Competitive transplantation assays showed the impaired repopulation potential of pLT-HSCs from aged ASXL1-MT-KI mice. These data demonstrate that the increased pLT-HSCs in aged ASXL1-MT-KI mice are not functional HSCs with long-term repopulation potential. 　To elucidate how ASXL1-MT drives HSPC dysfunction, we conducted RNA-Seq analysis using HSPCs from young mice. This analysis revealed upregulation of mitochondrial genes in ASXL1-MT-KI HSPCs. In addition, MitoTracker staining, extracellular flux analyses and metabolome analyses demonstrated the enhanced mitochondrial metabolism in ASXL1-MT-KI HSPCs. We also found that the aberrantly elevated mitochondrial activity induced ROS overproduction and increased DNA damage, resulting in HSPC dysfunction. 　As a mechanism underlying the enhanced mitochondrial activity of ASXK1-MT-KI HSPCs, we revealed that ASXL1-MT activated the Akt/mTOR pathway in HSPCs. Treatment with an Akt inhibitor perifosine or an mTOR inhibitor rapamycin normalized the mitochondrial membrane potential and ROS levels in ASXL1-MT-KI HSPCs. Moreover, rapamycin treatment improved engraftment of ASXL1-MT-KI bone marrow cells after transplantation. These data indicate that the activated Akt/mTOR signaling leads to the enhanced mitochondrial activity, elevated ROS levels, and HSPC dysfunction in ASXL1-MT-KI mice. 　To assess the impact of the enhanced Akt/mTOR signaling on age-related changes in ASXL1-MT-KI mice, we administered rapamycin to aged ASXL1-MT-KI mice. Intriguingly, rapamycin treatment decreased the frequency of pLT-HSCs, and normalized the bone marrow cellularity in aged ASXL1-MT-KI mice. Cell cycle analysis revealed that pLT-HSCs in G0 phase were decreased in aged ASXL1-MT-KI mice, which was normalized by rapamycin treatment. These data demonstrate that the activated Akt/mTOR pathway provokes the aberrant expansion of pLT-HSCs in aged ASXL1-MT-KI mice. 　We next attempted to clarify the underlying mechanism of Akt activation in ASXL1-MT-KI mice. Immunoprecipitation experiments revealed that ASXL1-MT/BAP1 complex deubiquitinated AKT in 293T cells. To determine the role of endogenous Bap1 on Akt signaling, we assessed the effect of Bap1 deletion in murine bone marrow cells transformed by combined expression of SETBP1-D868N and ASXL1-MT (cSAM cells). A time course experiments showed Akt phosphorylation induced by IL-3 stimulation was attenuated and shortened in Bap1-depleted cSAM cells. These data suggest that ASXL1-MT/BAP1 complex deubiquitinate and stabilize phosphorylated Akt. 　In summary, we demonstrated that ASXL1-MT cooperated with BAP1 to promote AKT deubiquitination and activation. The activated Akt/mTOR pathway led to enhanced mitochondrial metabolism, elevated ROS levels and increased DNA damage. These molecular bases underlie the age-associated expansion of the pLT-HSC compartment. Our results underscore the possibility that CH can originate from a pLT-HSC with a limited repopulation potential. A pharmacological inhibition of the Akt/mTOR pathway could be a promising therapeutic intervention to individuals with CH harboring ASXL1 mutations. Disclosures No relevant conflicts of interest to declare.

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