Novel Roles for the Homeobox Gene, HoxA1, and Its Truncated Form, HoxA1-T, in the Regulation of Hematopoietic Stem Cell Self-Renewal.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1706-1706 ◽  
Author(s):  
Stewart A. Fabb ◽  
Gemma Haines ◽  
Seb Dworkin ◽  
Paul J. Simmons ◽  
Lorraine J. Gudas ◽  
...  
Keyword(s):  
Hox Genes ◽  
Marrow Cell ◽  
Homeobox Gene ◽  
Donor Cell ◽  
Transplant Recipients ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Liquid Suspension ◽  
Donor Cells ◽  
Alternatively Spliced

Abstract There is increasing evidence that homeobox (Hox) genes play critical roles in the regulation of hematopoiesis. We found that both forms of the earliest HoxA gene, HoxA1 and its alternatively spliced transcript, HoxA1-T, which lacks the homeobox domain, are expressed in immature populations of hematopoietic stem cells (HSCs) and progenitor cells. In more mature bone marrow cell (BM) populations the levels of HoxA1-T increases relative to HoxA1 with both transcripts absent in mature peripheral blood (PB) cells. Roles for either of these Hox transcripts in hematopoiesis have not yet been described. We overexpressed either HoxA1 or HoxA1-T in BM using a modified GFP-containing MSCV vector (MXIE). Overexpression of either HoxA1 transcript did not affect expression levels of other Hox genes relative to control (empty vector). Overexpression of HoxA1-T significantly reduced the numbers of colony-forming cells (CFCs) produced by 500 GFP+ BM compared to control GFP+ BM (mean±SEM control 73±6.4; HoxA1 57±6.8; HoxA1-T 26.7±5.4; n=6; P<0.01 control vs. HoxA1-T). Interestingly, colonies generated by HoxA1 GFP+ BM contained ~3-fold more cells (mean±SEM: control 22,000±900 cells; HoxA1 61,700±12,000 cells; HoxA1-T 20,600±6,360 cells; n=6; P<0.005 HoxA1 vs. control and HoxA1-T). In each of 2 experiments 10,000 HoxA1 overexpressing BM grew in liquid suspension culture for up to 15 weeks, increasing by ~239-fold weekly. In contrast, control BM proliferated for only 3 weeks, with weekly ~53-fold increases, whereas HoxA1-T BM expired after 2 weeks of culture, with weekly cell increases of only ~7-fold (control v HoxA1 P<0.01; HoxA1 v HoxA1-T P<0.001). There was no difference in the number of day 12 colony-forming unit-spleen (CFU-S) formed from 2500 control or HoxA1 GFP+ BM (11.2±0.47 and 12.6±1.26 respectively). In contrast, 2500 HoxA1-T GFP+ BM produced significantly fewer CFU-S (8.9±0.61) compared to both control and HoxA1 BM (n=4, P<0.02). To assess HSC potential, lethally irradiated CD45.2+ recipients (n=6/group) were injected with 5x106 congenic CD45.1+ BM immediately post-transduction without selection (all groups had similar transduction efficiencies). All recipients had >75% donor cells (CD45.1+, GFP+/−) in their PB at 3 months post-transplant. The average %GFP+ cells in recipients were similar for control (24.5±7.0%) and HoxA1 cells (20.8±2.5%). Strikingly, HSCs overexpressing HoxA1-T had markedly reduced repopulating ability (3.5±0.6% GFP+, P<0.05 HoxA1-T vs. control or HoxA1). Poisson statistics were used to quantitate HSC frequency in secondary transplant recipients. Mice were injected with 5x103, 5x104, 2x105 or 1x106 BM from control or HoxA1 primary recipients together with 2x105 congenic CD45.2+ BM (n=10/group). The frequency of HSCs was markedly higher (~32-fold) in the HoxA1 cells (~1 HSC per 1.8x105 BM) compared to control (~1 HSC per 5.8x106 BM; P<0.001). Although secondary recipients injected with 1x106 HoxA1-T BM showed donor (CD45.1+) contribution (15.3±1.8%), none of the 10 recipients had GFP+ donor cell reconstitution. These data therefore suggest that HoxA1 enhances HSC self-renewal whereas HoxA1-T rapidly promotes HSC differentiation. These novel findings highlight important roles for the two HoxA1 transcripts in the regulation of HSCs, the mechanisms of which are currently being assessed.

scholarly journals Dnmt3a Is Essential for Hematopoietic Stem Cell Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 386-386 ◽  
Author(s):  
Grant A. Challen ◽  
Deqiang Sun ◽  
Mira Jeong ◽  
Min Luo ◽  
Jaroslav Jelinek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Dna Methyltransferase ◽  
Donor Cell ◽  
The Self ◽  
Blood Cell Count ◽  
Serial Transfer ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Differentiation Capacity

Abstract Abstract 386 Aberrant genomic DNA methylation patterns are widely reported in human cancers but the prognostic value and pathological consequences of these marks remain uncertain. CpG methylation is catalyzed by a family of DNA methyltransferase enzymes comprised of three members – Dnmt1, Dnmt3a and Dnmt3b. Mutations in the de novo DNA methyltransferase enzyme DNMT3A have now been reported in over 20% of adult acute myeloid leukemia (AML) and 10–15% of myelodysplastic syndrome (MDS) patients. However, analysis of promoter methylation and gene expression in these patients has thus far failed to yield any mechanistic insight into the pathology of DNMT3A mutation-driven leukemia. In this study, we have used a conditional knockout mouse model to study the role of Dnmt3a in normal hematopoiesis. Hematopoietic stem cells (HSCs) from Mx1-Cre:Dnmt3afl/fl mice were serially transplanted into lethally irradiated recipient mice to study the effect of loss of Dnmt3a on HSC self-renewal and differentiation. We show that loss of Dnmt3a progressively impedes HSC differentiation over four-rounds of serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Examination of the bone marrow post-transplant revealed that control HSCs showed a gradual decline in their ability to regenerate the HSC pool at each successive round of transplantation, while in contrast Dnmt3a-KO HSCs show a remarkably robust capacity for amplification, generating 40,000 – 100,000 HSCs per mouse. Quantification of peripheral blood differentiation on a per HSC basis demonstrated in the absence of Dnmt3a, a cell division is more likely to result in a self-renewal rather than differentiation fate (Figure 1). Using semi-global reduced representation bisulfite sequencing (RRBS), we show that Dnmt3a-KO HSCs manifest both increased and decreased methylation at distinct loci, including dramatic CpG island hypermethylation. Global transcriptional analysis by microarray revealed that Dnmt3a-KO HSCs show upregulation of HSC multipotency genes coupled with simultaneous downregulation of early differentiation factors (e.g. Flt3, PU.1, Mef2c), likely inhibiting the initial stages of HSC differentiation. Upregulation of key HSC regulators including Runx1, Gata3 and Nr4a2 was associated with gene-body hypomethylation and activated chromatin marks (H3K4me3) in Dnmt3a-KO HSCs. Finally, we show that Dnmt3a-KO HSCs are unable to methylate and transcriptionally repress these key HSC multipotency genes in response to chemotherapeutic ablation of the hematopoietic system, leading to inefficient differentiation and manifesting hypomethylation and incomplete repression of HSC-specific genes in their limited differentiated progeny. In conclusion, we show that Dnmt3a plays a specific role in permitting HSC differentiation, as in its absence, phenotypically normal but impotent stem cells accumulate and differentiation capacity is progressively lost. This differentiation-deficit phenotype is reminiscent of Dnmt3a/Dnmt3b-null embryonic stem (ES) cells while markedly distinct from that of Dnmt1-KO HSCs which show premature HSC exhaustion and lymphoid-deficient differentiation, demonstrating distinct roles for the different DNA methyltransferase enzymes in HSCs. In light of the recently-identified DNMT3A mutations in AML and MDS patients, these studies are the first biological models linking mutation of Dnmt3a with inhibition of HSC differentiation which may be one of the first pathogenic steps occuring in such patients.Figure 1Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal.Figure 1. Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal. Disclosures: Issa: Novartis: Honoraria; GSK: Consultancy; SYNDAX: Consultancy; Merck: Research Funding; Eisai: Research Funding; Celgene: Research Funding; Celgene: Honoraria; J&J: Honoraria.

scholarly journals Purified murine hematopoietic stem cells function longer on nonirradiated W41/Wv than on +/+ irradiated stroma

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1489-1496 ◽  
Author(s):  
F Vecchini ◽  
KD Patrene ◽  
SS Boggs
Keyword(s):  
Stem Cells ◽  
Wheat Germ ◽  
Donor Cell ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Donor Cells ◽  
Nonadherent Cell ◽  
Specific Increase

Abstract Mouse bone marrow (BM) was separated into low-density, lineage- negative, wheat germ agglutinin-positive (WGA+), Rhodamine-123 bright (Rhbright) or dim (Rhdim) cells to obtain populations that were highly enriched for committed progenitors (Rhbright cells) or for more primitive stem cells (Rhdim). When 2,500 Rhbright or Rhdim cells were seeded onto 6-week-old irradiated (20 Gy) long-term BM cultures (LTBMC), the nonadherent cell production from Rhbright cells was transient and ended after 5 weeks. Production from Rhdim cells did not begin until week 3, peaked at week 5, and ended at week 8, when the irradiated stroma seemed to fail. Termination of cell production from Rhdim cells did not occur in nonirradiated LTBMC from W41/Wv mice. During peak nonadherent cell production, 25% to 30% of the cells in the nonirradiated LTBMC from W41/Wv mice had donor cell markers. Two approaches were tested to try to enhance the proportion or number of donor cells. Addition of Origen-HGF at the time of seeding Rhdim cells caused a nonspecific increase in both host and donor cell production, but a specific increase in production of donor cells was obtained by seeding the cultures at 2 weeks rather than 6 weeks. Limiting dilution of Rhdim cells gave the same frequency of wells producing cells on both irradiated +/+ and nonirradiated W41/Wv or W/Wv cultures.

Murine hematopoietic stem cell distribution and proliferation in ablated and nonablated bone marrow transplantation

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3521-3526 ◽  
Author(s):  
Jiang F. Zhong ◽  
Yuxia Zhan ◽  
W. French Anderson ◽  
Yi Zhao
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Donor Cell ◽  
Cytotoxic Agents ◽  
Hematopoietic Stem ◽  
Donor Bone Marrow ◽  
Donor Cells ◽  
Murine Hematopoietic Stem Cell

The engraftment of donor bone marrow (BM) cells in nonablated mice is inefficient. Niche availability has been thought to be the reason, and cytoablation with irradiation or cytotoxic agents is routinely used with the belief that this frees the preoccupied niches in recipients. In this study, donor cell redistribution and proliferation in ablated and nonablated mice were compared by implanting donor cells directly into the femur cavity of sedated mice. The redistribution of Lin− donor cells into BM was similar between ablated and nonablated mice. Poor engraftment in nonablated mice was shown to be the result of inefficient donor cell proliferation rather than because of a lack of space. Competitive repopulation assays demonstrated that the donor hematopoietic stem cells (HSCs) were present in nonirradiated recipients for at least 6 months after transplantation, but that they did not expand as did their counterparts in lethally irradiated mice. This study suggests that efficient bone marrow transplantation in nonablated recipients may be possible as a result of better understanding of HSC proliferative regulation and appropriate in vitro manipulation.

Donor Cell Leukemia: A Clinicopathological Study of 9 Cases and a Comprehensive Review of Literature.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3466-3466
Author(s):  
Charles Blake Hutchinson ◽  
Jennifer H. Crow ◽  
Qin Huang ◽  
Chuanyi M. Lu ◽  
Siby Sebastain ◽  
...  
Keyword(s):  
Donor Cell ◽  
The Other ◽  
Fish Analysis ◽  
Cell Leukemia ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Cell Source ◽  
Benign Group ◽  
Donor Cell Leukemia

Abstract Abstract 3466 Donor cell leukemia (DCL) in the setting of bone marrow/hematopoietic stem cell transplant (HCT) has not been well characterized. We analyzed 9 cases of DCL and performed a literature review (table). The indications for transplant and subtypes of DCL are shown (table). The 6 myelodysplastic syndrome (MDS) cases included 1 case of refractory cytopenia with multilineage dysplasia (RCMD), 2 cases of refractory anemia and 3 cases which were unclassifiable. Conventional cytogenetic analysis was performed on all 9 cases of DCL (table). All 9 cases had engraftment studies performed either by short tandem repeat analysis (3) or FISH analysis for donor gonosomal complement (6) when DCL was diagnosed. Seven cases had either engraftment studies or cytogenetic analysis performed periodically after HCT to test the donor cell engraftment and engraftment was confirmed in all. FISH analysis for monosomy 7, del(7q) and del(5q) was retrospectively performed on preserved donor cells in 4 cases after DCL was diagnosed. A low level of abnormalities was observed in preserved donor cells for the cases with del(7q) (2.9%) and del(5q) (8.2%). The 2 cases of AML received chemotherapy. Of the MDS cases, 2 received donor cell infusion, 1 received 6 cycles of revlimid, and 3, along with the case of CLL, received either supportive therapy or were simply observed. Six cases have clinical follow up ≥ 5 months and of these, 1 died of disease (AML) while the other 5 are alive, including 4 MDS and the 1 CLL. The disproportionate detection of DCL in sex mismatched HCT suggests a probable under-detection in the sex-matched population. In our analysis, the interval between HCT and diagnosis of DCL (table) falls within the range of currently reported cases. When stratified by type of DCL, the T-LGL group demonstrates presentation significantly earlier than other groups (Fig. A), indicating pathogenesis of T-LGL may involve a distinct pathway. When stratified by types of primary disease, the interval of the neoplastic group is shorter than that of benign group (Fig. B), implying that pre-HCT treatment may play a role in the pathogenesis of DCL. When stratified by stem cell sources, UCB group shows shorter latency than the other sources (Fig. C), suggesting a higher risk of DCL in this cell source. The low level cytogenetic abnormalities of preserved donor cells in our series and the longer latency of the benign group suggest that donor cells with an intrinsic defect may be predisposed to evolve into DCL. Total cases (%) Reported cases (%) Current cases (%) Number of cases 83 74 9 Age (years)     Median/range 37.0/3~70 36.0/4~62 53.0/3~70 Gender     Male 43 (52.4) 38 (52.0) 5 (55.6)     Female 39 (47.6) 35 (48.0) 4 (44.4) Primary disease     Neoplasms 76 (91.6) 67 (90.5) 9 (100)     Non-neoplasms 7 (8.4) 7 (9.5) 0 (0.0) Donor     Related 59 (72.0) 54 (74.0) 5 (55.6)     Unrelated 23 (28.0) 19 (26.0) 4 (44.4)     Sex-matched 28 (34.6) 27 (37.5) 1 (11.1)     Sex-mismatched 53 (65.4) 45 (62.5) 8 (88.9) Donor cell source     BM 48 (63.2) 44 (65.7) 4 (44.4)     BHSC 16 (21.0) 13 (19.4) 3 (33.3)     UCB 12 (15.8) 10 (14.9) 2 (22.2) 2nd neoplasm (DCL)     AML 31 (37.4) 29 (39.2) 2 (22.2)     MDS/MPN* 27 (32.5) 21 (28.4) 6 (66.7)     ALL 20 (24.1) 20 (27.0) 0 (0.0)     T-LGL 4 (4.8) 4 (5.4) 0 (0.0)     CLL 1 (1.2) 0 (0.0) 1 (11.1) Interval (months)     Median/range 24.0/1~312 24.0/2~312 26.0/1~193 Cytogenetics     Normal 21 (28.0) 20 (30.3) 1 (11.1)     Abnormal 54 (72.0) 46 (69.7) 8 (88.9)     -7 or del(7q)** 15 (27.8) 10 (21.7) 5 (62.5)     +8** 2 (3.7) 2 (4.4) 0 (0.0)     Del(20)** 4 (7.4) 2 (4.4) 2 (25.0)     Del(5q)** 2 (3.7) 1 (2.2) 1 (12.5)     11q23** 3 (5.6) 3 (6.5) 0 (0.0) Other abnormalities** 28 (51.9) 28 (60.9) 0 (0.0) Follow up (months)     Median/range 8.5/1~108 9.0/1~108 6.0/1~68     Died of disease 28 (46.7) 27 (52.9) 1 (11.1) DCL, donor cell leukemia; BM, bone marrow; BHSC, blood hematopoietic stem cells; UCB, umbilical cord blood; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; ALL, acute lymphoblastic leukemia (including B-cell and T-cell ALL); T-LGL, T-cell large granular lymphocyte leukemia; CLL, chronic lymphocytic leukemia. All the numbers represent the cases with data available. * One case of myeloproliferative neoplasm is included in this category. ** The percentage is calculated using number of total cytogenetic abnormalities in each column as denominator. Disclosures: No relevant conflicts of interest to declare.

Purified murine hematopoietic stem cells function longer on nonirradiated W41/Wv than on +/+ irradiated stroma

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1489-1496
Author(s):  
F Vecchini ◽  
KD Patrene ◽  
SS Boggs
Keyword(s):  
Stem Cells ◽  
Wheat Germ ◽  
Donor Cell ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Donor Cells ◽  
Nonadherent Cell ◽  
Specific Increase

Mouse bone marrow (BM) was separated into low-density, lineage- negative, wheat germ agglutinin-positive (WGA+), Rhodamine-123 bright (Rhbright) or dim (Rhdim) cells to obtain populations that were highly enriched for committed progenitors (Rhbright cells) or for more primitive stem cells (Rhdim). When 2,500 Rhbright or Rhdim cells were seeded onto 6-week-old irradiated (20 Gy) long-term BM cultures (LTBMC), the nonadherent cell production from Rhbright cells was transient and ended after 5 weeks. Production from Rhdim cells did not begin until week 3, peaked at week 5, and ended at week 8, when the irradiated stroma seemed to fail. Termination of cell production from Rhdim cells did not occur in nonirradiated LTBMC from W41/Wv mice. During peak nonadherent cell production, 25% to 30% of the cells in the nonirradiated LTBMC from W41/Wv mice had donor cell markers. Two approaches were tested to try to enhance the proportion or number of donor cells. Addition of Origen-HGF at the time of seeding Rhdim cells caused a nonspecific increase in both host and donor cell production, but a specific increase in production of donor cells was obtained by seeding the cultures at 2 weeks rather than 6 weeks. Limiting dilution of Rhdim cells gave the same frequency of wells producing cells on both irradiated +/+ and nonirradiated W41/Wv or W/Wv cultures.

scholarly journals Growth Factor Receptor-Bound Protein 10 (Grb10) Regulates Hematopoietic Stem Cell (HSC) Self-Renewal Via Control of mTOR Signaling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1160-1160
Author(s):  
Xiao Yan ◽  
Heather A Himburg ◽  
Phuong L Doan ◽  
Mamle Quarmyne ◽  
Evelyn Tran ◽  
...  
Keyword(s):  
Stem Cell ◽  
Growth Factor ◽  
Hematopoietic Stem Cell ◽  
Growth Factor Receptor ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Duke University

Abstract Elucidation of the mechanisms governing HSC regeneration has been impeded by difficulty in isolating HSCs early following genotoxic injury, such as total body irradiation (TBI). Using multiparametric flow cytometric cell sorting of BM ckit+sca-1+lin- cells coupled with gene expression analysis, we identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed by BM KSL cells at the earliest detectable point of regeneration (day +10) following TBI (3.3-fold, p<0.0001). Grb10 is a member of the imprinted gene family which is predominately expressed in the stem cells of a variety of tissues, including embryonic stem cells, bone marrow, skin and muscle. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) detected in 7-day culture (p=0.03 and p=0.002, respectively). Furthermore, mice competitively transplanted with Grb10-deficient HSCs displayed 10-fold lower donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed a greater than 10-fold deficit in long-term repopulating capacity in Grb10-deficient KSL cells compared to Grb10-expressing KSL cells (p=0.006 for %CD45.1+ donor cells). In order to determine if Grb10 was necessary for HSC maintenance and normal hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8 week old Grb10m/+ (1 mutant allele, 1 wild type allele) had 10-fold decreased Grb10 expression in BM lin-cells. BM CFCs and SLAM+ KSL cells were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006 and p=0.04, respectively). Competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (p=0.003 for %CD45.1+ donor cells). Mice transplanted with BM cells from homozygous Grb10-/- mice showed a similar decrease in donor-derived hematopoietic repopulation compared to mice transplanted with BM cells from Grb10+/+ mice (p=0.02 at 20 weeks post-transplantation). These results confirmed that Grb10 regulates HSC self-renewal capacity in vivo. To determine whether Grb10 regulates HSC regeneration after myelotoxic injury, we irradiated Grb10m/+ mice with 550cGy TBI, and monitored hematopoietic recovery over time in comparison to Grb10+/+ controls. Interestingly, Grb10m/+ mice displayed accelerated hematopoietic regeneration early following TBI. At day+10 after 550cGy, Grb10m/+ mice contained significantly increased numbers of BM SLAM+ KSL cells (p=0.04) and CFCs (p=0.009), compared to Grb10+/+ littermates. Similarly, mice transplanted with BM cells from irradiated, Grb10m/+ mice displayed 5-fold increased donor hematopoietic repopulation at 20 weeks post-transplantation compared to mice transplanted with BM cells from irradiated, Grb10+/+ mice (p=0.006). These data suggest that Grb10 deficiency accelerates hematopoietic recovery in the early period following myelosuppressive radiation injury. Mechanistically, Grb10-deficiency caused an increase in the percentage of BM KSL cells in G1 and G2/S/M phase of cell cycle compared to Grb10+/+ KSL cells (p=0.003). We also observed significantly increased levels of mTOR activation in Grb10m/+ BM KSL cells compared to Grb10+/+ BM KSL cells (p=0.001 for pS6, p=0.001 for pS6k and p=0.02 for p4EBP1). Furthermore, mTOR inhibition via siRNA-mTOR targeting rescued the defect in BM hematopoietic progenitor content (colony forming cells) in Grb10-deficient BM cells (p<0.0001). Taken together, our results suggest that Grb10 is necessary for HSC maintenance in steady state, while, paradoxically, Grb10 inhibition accelerates HSC regeneration early following injury. Furthermore, our data suggest that Grb10 mediates these effects via regulation of mTOR signaling. Selective modulation of Grb10 signaling has the potential to augment HSC self-renewal in steady state and to accelerate HSC regeneration following myelotoxic injury. Disclosures Himburg: Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.

scholarly journals Activation of Hottip lncrna Perturbs HSC Function Leading to AML like Disease in Mice

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3877-3877
Author(s):  
Huacheng Luo ◽  
Ganqian Zhu ◽  
Jie Zha ◽  
Bowen Yan ◽  
Ying Guo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hox Genes ◽  
Chromatin Domain ◽  
Active Chromatin ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Chromatin Signature ◽  
Genes Expression ◽  
Hoxa Genes

Abstract Several HOX loci associated long noncoding RNAs (lncRNAs) have been shown to regulate transcription of HOX genes through influencing epigenetic landscape. Especially, the posterior HOXA domain associated lncRNA HOTTIP acts as an epigenetic regulator that recruits WDR5/MLL complex to coordinate active chromatin modifications and HOXA genes expression in the development of animal digits. Despite HOX genes, especially HOXA genes, are highly expressed in many acute myeloid leukemia (AML) patients, it remains largely unknown whether and how HOTTIP lncRNA regulates hematopoietic stem cell (HSC) function and contributes to leukemogenesis. We showed previously that disruption of the CTCF boundary located between HOXA7 and HOXA9 genes (CBS7/9) resulted in reduced lncRNA HOTTIP and HOXA genes expression in MLL rearranged AML suggesting that HOTTIP may play a role in ectopic expression of the posterior HOXA gene. We employed a pooled CRISPR-Cas9 KO library to specifically screen lncRNAs in four HOX gene loci and identify HOTTIP as acritical regulator in controlling oncogenic HOX chromatin signature and associated gene expression patterns in AML by collaborating with posterior HOXA chromatin boundary. HOTTIP is upregulated in AML patients with MLL-rearrangement or NPM1 mutation. AML patients with high HOTTIP expression exhibits a significantly shortened survival compared to low HOTTIP expressing patients. To test whether HOTTIP acts to coordinate posterior chromatin domain and HOXA genes activation in AML, we manipulated HOTTIP lncRNA expression levels in the MLL-AF9 rearranged MOLM13 by loss-of-function KO and gain-of function rescue, as well as carried out genome wide chromatin and transcriptomic analysis to intterrogate the role of HOTTIP in control of AML specific posterior HOXA chromatin domain. We found that knock-out of HOTTIP lncRNA led to a loss of active chromatin structure and invasion of repressive H3K27me3 mark over the posterior HOXA domain. HOTTIP KO attenuated progression of AML in the transplanted AML mouse model resembling the effect of CBS7/9 boundary disruption, while transcriptional activation of HOTTIP lncRNA in the CBS7/9 boundary-disrupted AML cells restored HOXA locus chromatin signature and gene expression as well as reversed the CBS7/9-mediated anti-leukemic effects. To further determine the role of HOTTIP lncRNA in regulating HSC function and leukemogenesis, we generated transgenic mice that expresses Hottip lncRNA under the control of the hematopoietic specific Vav1 enhancer and promoter. The Hottip transgenic (Tg) mice exhibited increased WBC and neutrophil counts and developed splenomegaly indicating that enforced expression of Hottip lncRNA resulted in perturbation of hematopoiesis. Furthermore, overexpression of Hottip lncRNA in mice bone marrow hematopoietic compartment strongly perturbed hematopoietic stem and progenitor cell (HSC/HPC) function by altering self-renewal and differentiation property of HSC/HPCs through affecting homeotic gene associated oncogenic transcription program. Approximately 20% of Hottip lncRNA transgenic mice developed abnormal hematopoietic phenotypes resembling AML-like disease. RNA-seq and ATAC-seq analysis indicated that overexpression of Hottip enhanced promoter chromatin accessibility and stimulates transcription of genes and pathways involved in HSC function and leukemogenesis, including WNT signaling, hematopoietic cell lineage, cell cycle, Hoxa9, Hoxa13, and Meis1, Runx1, and Twist1 genes. Thus, Hottip lncRNA overexpression acts as an oncogenic event to promote HSC self-renewal and HPC proliferation by reprograming leukemic associated chromatin signature and transcription programs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bone Marrow Transplantation in Acid Sphingomyelinase-Deficient Mice: Engraftment and Cell Migration Into the Brain as a Function of Radiation, Age, and Phenotype

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 444-452 ◽  
Author(s):  
Silvia R.P. Miranda ◽  
Shai Erlich ◽  
Jan W.M. Visser ◽  
Shimon Gatt ◽  
Arie Dagan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Donor Cell ◽  
Cell Number ◽  
Acid Sphingomyelinase ◽  
Radiation Doses ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
The Brain

Types A and B Niemann-Pick disease (NPD) result from the deficient activity of the lysosomal hydrolase, acid sphingomyelinase (ASM). A long-term goal of our research is to evaluate the effects of bone marrow transplantation (BMT) and hematopoietic stem cell gene therapy (HSCGT) on the NPD phenotype. As an initial step toward this goal, we have undertaken a study aimed at optimizing hematopoietic cell engraftment in acid sphingomyelinase “knock-out” (ASMKO) mice. Several parameters were analyzed, including the effects of radiation and donor cell number on survival and engraftment of newborn and adult animals, the number of donor cells detected in the brain posttransplantation, and the levels of ASM activity achieved in the brain. A total of 202 ASMKO and normal animals were transplanted and studied, and the overall conclusions were: (1) newborn ASMKO animals were more susceptible to radiation-induced mortality than normal animals, (2) at low radiation doses, increasing the donor cell number improved engraftment, while this was less evident at the higher radiation doses, (3) engraftment was easier to achieve in normal as compared with ASMKO animals, (4) among newborn transplants, the number of donor cells detected in the brain was directly correlated with engraftment in the blood, (5) more donor cells were detected in the brains of newborn ASMKO animals as opposed to newborn normal animals, and (6) no donor cells were found in the brains of animals transplanted as adults, including those that were highly engrafted in the blood. These results provide important information regarding the design of future BMT and HSCGT studies in ASMKO mice and other mouse models and demonstrate the potential of altering the NPD phenotype by these therapeutic strategies.

scholarly journals The Mechanism By Which Mutant Nucleophosmin (NPM1) Creates Leukemic Self-Renewal Is Readily Reversed

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 444-444 ◽  
Author(s):  
Xiaorong Gu ◽  
Reda Z. Mahfouz ◽  
Quteba Ebrahem ◽  
Francis Enane ◽  
Tomas Radivoyevitch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hox Genes ◽  
Cell Model ◽  
Expression Patterns ◽  
Monocytic Differentiation ◽  
Self Renewal ◽  
Produce Cell ◽  
Hematopoietic Stem ◽  
Renewal Program

Abstract Acute myeloid leukemia (AML) is self-renewal by immature myeloid precursors that fail to differentiate. An influential 'leukemia stem cell' model thus proposes that leukemogenic proteins augment or introduce a stem cell-like self-renewal program into cells, e.g., by upregulating signaling or transcription factors (TF) emblematic of stem cells (e.g., HOX). We investigated how the most recurrently mutated protein in AML, mutant nucleophosmin (mNPM1), causes leukemic cell expansion. The results challenge this model, but most importantly, open the door to rational targeted therapy for mNPM1 AML. One way of examining for stem cell programs in AML cells is to look at expression patterns of master TF that regulate expression of hundreds of genes and dictate cell fates. Of these select TF, the master TF that create hematopoietic stem cells (HLF etc.) are minimally or not expressed. Instead, there are very high levels of the master TF that drive monocyte and granulocyte lineage fates, PU.1 (SPI1) and CEBPA. Clearly, however, the lineage-programs intended by PU.1/CEBPA are inefficiently executed if at all - mNPM1 AML patient bone marrows had 85-97% cells with a granulocyte-monocyte progenitor phenotype, accumulated at the expense of downstream mature cells (Quek et al, JEM 2016). This aggregation at a lineage-committed, intermediate, naturally proliferative level of the hematopoietic hierarchy suggests an alternative model - instead of introducing a poorly-defined stem cell self-renewal program, mutant proteins disable differentiation programs which usually quench MYC-driven proliferation intrinsic to lineage-progenitors. To better understand how mNPM1 interacts with cellular machinery, we used mass-spectrometry to comprehensively document the protein interactions of endogenous NPM1 in AML cell nuclear and cytoplasmic fractions, the first analysis of this kind. Notably, the NPM1 protein interactome was enriched for PU.1. Critically, wild-type (wt) NPM1/PU.1 was in the nucleus of wtNPM1 AML cells, but mNPM1/PU.1 was in the cytoplasm of mNPM1 AML cells. This was evident clearly also by Western blot of cell fractions and by IF microscopy of primary AML cells and cell lines. Is cytoplasmic dis-location of PU.1 sufficient to explain persistent hematopoietic precursor proliferation? We used murine Pu.1 knock-out hematopoietic precursors transduced to express Pu.1 fused with the estrogen receptor (Pu.1-ER) to answer this question - Pu.1 relocation from the cytoplasm to the nucleus by tamoxifen triggered monocytic differentiation that terminated proliferation. Moreover, Pu.1-ER cells, like mNPM1 AML cells, highly express Hox genes, rapidly suppressed upon Pu.1 relocation to the nucleus. Thus, Pu.1 dominantly controls Hox and proliferation, as befitting of a master TF, and persistent HOX expression, like persistent progenitor proliferation, can be caused by Pu.1 loss-of-function. Protein macromolecules like NPM1 require transport factors to exit (exportins) the nucleus. A specific exportin, XPO1, was the major exportin found in the NPM1 interactome. XPO1 interactions with transported cargo can be inhibited by the small molecule drug KPT330. KPT330 10-20 nM rapidly re-located mNPM1 and PU.1 to the nucleus, downregulated MYC, upregulated p27/CDKN1B, upregulated monocyte but not granulocyte differentiation markers, induced morphologic changes of monocyte differentiation, and terminated proliferation of mNPM1 AML cells. The same low nanomolar treatment did not induce differentiation of wtNPM1 AML cells (THP1). Moreover, these KPT330 levels are not toxic to normal hematopoiesis (also shown by others). Thus, rather than gain-of-function of elusive stem cell-like self-renewal, the most frequently mutated protein in AML creates self-renewal by disabling a differentiation program that quenches intrinsic MYC-driven proliferation of lineage-progenitors. These observations are a mechanistic rationale to select refractory/relapsed mNPM1 AML patients for treatment with low well-tolerated doses of KPT330, with a defined molecular pharmacodynamic objective of returning PU.1 to the nucleus, to produce cell cycle exits by differentiation rather than p53-mediated apoptosis (to address chemotherapy resistance), to spare precious normal HSC (good therapeutic index), and directly reverse the basis for leukemic self-renewal (proliferation without differentiation). Figure. Figure. Disclosures Landesman: Karyopharm Therapeutics Inc: Employment, Other: stockholder. Saunthararajah:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine.

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