TET2: Mechanism and Functional Consequences of Hydroxymethylation

Abstract Abstract SCI-32 TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Somatic TET2 mutations are frequently observed in myeloid neoplasms in humans. Bone marrow samples from patients with mutant TET2, as well as some patients with wild type TET2, display low levels of 5hmC in genomic DNA compared to healthy controls. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool to tailor therapies and assess responses to anticancer drugs. We have developed novel and specific approaches to profile the genomic localization of 5hmC and will describe their application to profiling 5hmC in mouse hematopoietic progenitor cells that express or lack Tet2. Disclosures: No relevant conflicts of interest to declare.

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Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽

10.1182/blood.v120.21.1234.1234 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1234-1234

Author(s):

Robert S Welner ◽

Giovanni Amabile ◽

Deepak Bararia ◽

Philipp B. Staber ◽

Akos G. Czibere ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cells ◽

Quiescent State ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

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Use of Human Pluripotent Stem Cells to Model the Hematopoietic Defect and Repair of Human Telomerase Deficiency

Blood ◽

10.1182/blood.v126.23.1212.1212 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1212-1212

Author(s):

George Scaria ◽

Trevor Argall ◽

Shyam S. Jose ◽

Laura Bendzick ◽

Dan S. Kaufman

Keyword(s):

Bone Marrow ◽

Telomere Length ◽

Pluripotent Stem Cells ◽

Bone Marrow Failure ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Differentiation ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Diploid Genome ◽

Mutant Cells

Abstract Telomeres are repetitive DNA protein structures that cap the ends of chromosomes, protect chromosome ends from degradation and fusion, and are essential for maintenance of genomic integrity. Telomere length has been shown to gradually shorten over time as cells divide. When telomeres become critically short, the cells enter a state of senescence. As such, telomere shortening has been implicated in accelerated aging. Defects in telomerase function have been associated with the development of bone marrow failure. Patients with inherited mutations in telomerase components have significantly shortened telomere lengths and reductions in telomere length have been associated with a worse prognosis in myelodysplastic syndrome (MDS) and Aplastic anemia (AA). We have isolated fibroblasts from patients with a novel mutation in the telomerase RNA component (TERC), a 6 nucleotide in frame duplication at position +334, which results in bone marrow failure. We have derived several lines of human induced pluripotent stem cells (iPSCs) from these patients. We have demonstrated that these telomerase-deficient iPSCs appropriately express markers of pluripotency: Oct4, Sox2, SSEA4, and Nanog. Using quantitative real time PCR, we were able to measure the average telomere length as a T/S ratio of kilobases of telomere length per genome. We have determined that reprogramming results in significant increase in telomere length of control fibroblasts. However despite the typical induction of endogenous TERT expression during reprograming in our telomerase-deficient iPSCs, these TERC-mutant iPSCs did not demonstrate significant telomere elongation. We found telomerase-deficient fibroblasts have telomere lengths of 130-150kb/diploid genome compared to normal human fibroblasts with telomeres of ~250kb/diploid genome. After reprogramming, the iPSCs generated from wild type fibroblasts can have markedly increased telomere lengths to as high as 2000kb/diploid genome. The telomere lengths in TERC-mutant iPSCs from two different patients are less than 300kb/diploid genome (replicates=3). Expression of telomerase components TERT and TERC, DKC was compared to the mRNA level by qRT-PCR in the TERC-mutant iPSCs. We found significant variation in mRNA expression levels of telomerase components the different lines of telomerase deficient iPSCs, and even variation among different clones of the same telomerase deficient line. We have also found that mutations in TERC results in defective hematopoietic differentiation from these iPSCs in in vitro assays. In the TERC-mutant iPSCs, the proportion of CD34+CD45+ hematopoietic cells was reduced compared to wild type controls. Wild type iPSCs produce 30% CD34+ cells compared to 15-20% in TERC-mutant iPSCs (n=3). Additionally, wild type iPSC controls produce 9-10% CD34+CD45+ hematopoietic progenitor cells compared to 1-2% of TERC mutant cells. Interestingly, approximately equal proportions (8-9%) of wild type and TERC-mutant cells differentiate into CD34+CD31+ endothelial cells, suggesting this pathway is less affected by the TERC mutation. The TERC-mutant iPSCs demonstrate reduced development of hematopoietic progenitor cells in standard hematopoietic colony forming cell assays: 100 CFCs per 50,000 differentiated wild type iPSC-derived cells compared to 65 CFCs per 50,000 differentiated TERC mutant iPSC-derived cells. In order to rescue the hematopoietic defect in telomerase deficient TERC-mutant iPSCs we have used the Sleeping Beauty transposon system to over express these telomerase components TERC and TERT in the TERC mutant iPSCs. We are currently characterizing the effect of overexpression of these telomerase components on hematopoietic differentiation to determine if this provides a strategy to enable use of gene-corrected iPSCs to provide a future therapy for patients with bone marrow failure due to defined telomerase deficiencies. Disclosures No relevant conflicts of interest to declare.

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Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

Blood ◽

10.1182/blood.v124.21.1591.1591 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1591-1591

Author(s):

Juliana M. Xavier ◽

Lauremilia Ricon ◽

Karla Priscila Vieira ◽

Longhini Ana Leda ◽

Carolina Bigarella ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Progenitor Cells ◽

Bone Marrow Niche ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Short Term ◽

Hematopoietic Stem

Abstract The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown. In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/-mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays. Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant. The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/-recipient (meanÂ± SD) (2.07 Â± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 Â± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 Â±1.39; Arhgap21+/-: 2.17 Â± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 Â± 3.85; Arhgap21+/-: 14.07 Â±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/-bone marrow and spleen were retained in the blood stream. In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 Â±0.03; Arhgap21+/-0.12Â±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 Â±1.1; Arhgap21+/-94.3Â±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal. The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation. In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. Figure 1 Reduced homing and short term reconstitution in Arhgap21 +/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means Â±SD of 2 independent experiments. Figure 1. Reduced homing and short term reconstitution in Arhgap21+/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means Â±SD of 2 independent experiments. Disclosures No relevant conflicts of interest to declare.

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Tumor necrosis factor (TNF) is a physiologic regulator of hematopoietic progenitor cells: increase of early hematopoietic progenitor cells in TNF receptor p55-deficient mice in vivo and potent inhibition of progenitor cell proliferation by TNF alpha in vitro

Blood ◽

10.1182/blood.v86.8.2930.bloodjournal8682930 ◽

1995 ◽

Vol 86 (8) ◽

pp. 2930-2937 ◽

Cited By ~ 3

Author(s):

Y Zhang ◽

A Harada ◽

H Bluethmann ◽

JB Wang ◽

S Nakao ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Hematopoietic Progenitor Cells ◽

Tnf Alpha ◽

Tnf Receptor ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Deficient Mice ◽

Necrosis Factor

Murine bone marrow cells with lineage phenotypes (Lin)-Sca-1+c-kit+ and Lin-Sca-1-c-kit+ cells represent primitive hematopoietic stem cells (HSCs) and committed hematopoietic progenitor cells, respectively. The number of Lin-Sca-1+c-kit+ HSCs in bone marrow was significantly increased in tumor necrosis factor (TNF) receptor p55-deficient (TNF-R55–1-) mice compared with the TNF-R55+/+ wild-type mice without a marked change in bone marrow cellularity. In both the methylcellulose culture and a single-cell proliferation assay, mouse TNF alpha (mTNF alpha) inhibited in vitro the proliferation of wild-type mouse-derived Lin-Sca-1+c-kit+ cells in response to a combination of multiple growth factors. The same is true for that of Lin-Sca-1+c-kit+ cells stimulated with granulocyte colony-stimulating factor (G-CSF) plus stem cell factor (SCF). Moreover, mTNF alpha significantly arrested the entry into S-phase from G0/G1 phase of Lin-Sca-1+c-kit+ cells stimulated with multiple growth factors and Lin-Sca-1-c-kit+ cells stimulated with G-CSF plus SCF. In contrast, mTNF alpha failed to affect the growth and cell cycle progression of Lin-Sca-1+c-kit+ cells and Lin-Sca-1-c-kit+ cells that were obtained from TNF-R55-deficient mice. These data suggest that TNF may be an important physiologic regulator of hematopoiesis and that TNF-R55 may be essentially involved in TNF-mediated inhibition of the growth of both primitive stem and more committed progenitor cells.

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Tumor necrosis factor (TNF) is a physiologic regulator of hematopoietic progenitor cells: increase of early hematopoietic progenitor cells in TNF receptor p55-deficient mice in vivo and potent inhibition of progenitor cell proliferation by TNF alpha in vitro

Blood ◽

10.1182/blood.v86.8.2930.2930 ◽

1995 ◽

Vol 86 (8) ◽

pp. 2930-2937 ◽

Cited By ~ 97

Author(s):

Y Zhang ◽

A Harada ◽

H Bluethmann ◽

JB Wang ◽

S Nakao ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Hematopoietic Progenitor Cells ◽

Tnf Alpha ◽

Tnf Receptor ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Deficient Mice ◽

Necrosis Factor

Abstract Murine bone marrow cells with lineage phenotypes (Lin)-Sca-1+c-kit+ and Lin-Sca-1-c-kit+ cells represent primitive hematopoietic stem cells (HSCs) and committed hematopoietic progenitor cells, respectively. The number of Lin-Sca-1+c-kit+ HSCs in bone marrow was significantly increased in tumor necrosis factor (TNF) receptor p55-deficient (TNF-R55–1-) mice compared with the TNF-R55+/+ wild-type mice without a marked change in bone marrow cellularity. In both the methylcellulose culture and a single-cell proliferation assay, mouse TNF alpha (mTNF alpha) inhibited in vitro the proliferation of wild-type mouse-derived Lin-Sca-1+c-kit+ cells in response to a combination of multiple growth factors. The same is true for that of Lin-Sca-1+c-kit+ cells stimulated with granulocyte colony-stimulating factor (G-CSF) plus stem cell factor (SCF). Moreover, mTNF alpha significantly arrested the entry into S-phase from G0/G1 phase of Lin-Sca-1+c-kit+ cells stimulated with multiple growth factors and Lin-Sca-1-c-kit+ cells stimulated with G-CSF plus SCF. In contrast, mTNF alpha failed to affect the growth and cell cycle progression of Lin-Sca-1+c-kit+ cells and Lin-Sca-1-c-kit+ cells that were obtained from TNF-R55-deficient mice. These data suggest that TNF may be an important physiologic regulator of hematopoiesis and that TNF-R55 may be essentially involved in TNF-mediated inhibition of the growth of both primitive stem and more committed progenitor cells.

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Lack of the Transcription Factor NFAT (Nuclear Factor of Activated T cells) c2 in Hematopoietic Progenitor Cells Results in Profound Hematological Abnormalities in Mice

Blood ◽

10.1182/blood.v118.21.1296.1296 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1296-1296

Author(s):

Laleh S. Arabanian ◽

Michael Haase ◽

Ivonne Habermann ◽

Malte von Bonin ◽

Claudia Waskow ◽

...

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Extramedullary Hematopoiesis ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Hematological Abnormalities

Abstract Abstract 1296 Understanding the transcriptional mechanisms that control hematopoiesis and the interaction between hematopoietic stem cells and the bone marrow microenvironment in vivo is of considerable interest. We have previously shown that aged mice lacking the transcription factor NFATc2 develop bone marrow hypoplasia, anemia, and extramedullary hematopoiesis in spleen and liver. The proliferation and differentiation of NFATc2-deficient hematopoietic progenitor cells (HPC) ex vivo, however, was found to be intact. It remained therefore unclear whether the disturbed hematopoiesis in NFATc2-deficient mice was caused by the hematopoietic or the stroma component of the bone marrow hematopoietic niche. In the current study we dissected the relative contribution of hematopoietic and stroma cells to the phenotype of the NFATc2-deficent mice by transplanting immunomagnetically purified NFATc2-deficient (ko) HPCs to lethally irradiated wildtype (wt) mice, and vice versa. After a posttransplantation period of 6–8 months, peripheral blood, bone marrow as well as spleen and liver of the transplanted animals were analyzed and compared to wt and ko mice transplanted with control cells. Transplantation of NFATc2-deficient HPCs into wt recipients (ko → wt) induced similar hematological abnormalities as those occurring in non-transplanted ko mice or in ko mice transplanted with ko cells (ko → ko). Compared to wt mice transplanted with wt cells (wt → wt), ko → wt mice showed evidence of anemia, thrombocytopenia and a significantly reduced number of hematopoietic cells in their bone marrow. Likewise, ko → wt mice developped clear signs of extramedullary hematopoiesis in spleen and liver, which was not the case in wt → wt control animals. Our data demonstrate for the first time, that NFAT transcription factors directly regulate the intrinsic function of hematopoietic progenitor cells in vivo. The transcriptional targets for NFAT in these cells are yet unknown and are the focus of further investigations. Disclosures: No relevant conflicts of interest to declare.

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Abstract 545: Redox-Sensitive Regulation of Bone Marrow Hematopoietic Progenitor Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a545 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Shi Pan ◽

Nadan Wang ◽

Shey-Shing Sheu

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Progenitor Cells ◽

Blood Cells ◽

Bone Marrow Failure ◽

Hematopoietic Progenitor Cells ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Significant Difference

Bone marrow failure is a group of hematopoietic stem cell disorders that affect one or more lineages of blood cells. Hematopoietic progenitor cells are highly sensitive to reactive oxygen species (ROS)-induced oxidative stress. ROS impair the self-renewal of hematopoietic progenitor cells in the bone marrow and cause bone marrow failure. Glutaredoxin (Grx) is an antioxidant enzyme important for many cellular processes such as resistance against oxidative stress, DNA synthesis, sulfur assimilation, apoptosis, and cellular differentiation. However, the role of Grx in hematopoietic progenitor cells remains unknown. We study the role of Grx in hematopoietic progenitor cells using Grx KO mice as the model system. Bone marrow cells were isolated from 9-10 weeks old wild type and Grx KO mice and the ratio of KSL cells, total blood count and p38 activation was studied. Our results showed no significant difference on the ratio of KSL cells between wild type and Grx KO mice in basal condition. There is also no significant difference in the numbers of red blood cells, lymphocytes, white blood cells and platelet in basal condition between wild type mice and Grx KO. Interestingly, upon hydrogen peroxide treatment, there is higher level of p38 activation in Grx KO mice compared to wild type mice. Our results suggest that Grx is involved in redox-sensitive regulation of hematopoietic progenitor cells. Grx may play a regulatory role in the prevention of bone marrow failure.

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