A Novel MBT-Containing Protein, Hemp, Plays Essential Roles In Skeletal Formation and Hematopoietic Stem Cell Function.

Abstract Abstract 1597 Hemp (hematopoietic expressed mammalian polycomb, also denoted as mbt-containing 1) gene was originally identified in the hematopoietic stem cell (HSC)-enriched fraction of the mouse fetal liver (FL). It encodes a protein containing a putative Cys2-Cys2 zinc-finger region, followed by four tandem malignant brain tumor (MBT) repeats, which is frequently observed in polycomb gene (PcG) proteins. The structural characteristics strongly suggest that Hemp functions as an epigenetic regulator, but its biological role remains unknown. To address this issue, we generated hemp-deficient (hemp–/–) mice. Hemp–/– mice died soon after birth. Although no abnormalities were detected in internal organs, skeletal analysis exhibited a variety of malformations. Severe deformities were observed in the thoracic cavity, strongly suggesting that hemp–/– mice died of respiratory failure. Interestingly, they showed malformations of cervical and thoracic vertebrae, which were different from typical homeotic transformations observed in PcG-deficient mice. These results suggest that Hemp governs downstream target genes in distinct manners from conventional PcG proteins. The hematopoietic analysis of hemp in the FL showed that hemp is preferentially expressed in CD150+LSK and CD150–LSK HSC fractions in the hematopoietic hierarchy. Hemp–/– FL contained a significantly reduced number of hematopoietic cells and produced fewer number of hematopoietic colonies as compared to hemp+/+ FL. The decreases correlated with reduced number of CD150+LSK HSCs in hemp–/– FL, which generated much fewer hematopoietic colonies in the HPP-CFC assay. In addition, the competitive repopulation assay exhibited that the hematopoietic reconstitution ability of hemp–/– FL CD150+LSK HSCs was significantly impaired. Moreover, microarray analysis revealed that expression levels of several genes, such as Prdm16, Sox4, and Erdr1 were altered in hemp–/– FL HSCs. Since hemp–/– mice died at neonate, the role of Hemp in adult hematopoiesis remains to be elucidated. To address this issue, we generated hemp conditional knockout (cKO) mice. Acquired deletion of Hemp in the hematopoietic tissues was successfully achieved by crossing hempflox/flox mice with MxCre mice and stimulating the compound mice with pIpC. Analysis of the hematopoietic tissues revealed that the cell numbers of Mac+Gr1– and Mac+Gr1+ fractions in the hemp cKO bone marrow (BM) were significantly increased and decreased, respectively, as compared to those of the wild-type BM. However, no apparent differences have so far been observed between hemp cKO and wild-type littermates in functional analyses, such as colony forming activity and competitive repopulation ability of the BM cells. Here, we report that a novel MBT-containing protein, Hemp, plays essential roles in skeletal formation and HSC function during embryogenesis and also contributes to myeloid differentiation in adult hematopoiesis. Since Hemp likely functions as an epigenetic regulator, further studies will be required to clarify whether and what methylated lysine residues Hemp interacts with through the MBT repeats, what kind of genes are direct targets of Hemp, and how Hemp exerts its biological activity. Disclosures: No relevant conflicts of interest to declare.

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TWIST1 Preserves Hematopoietic Stem Cell Function Via Repression of Cacna1b Expression

Blood ◽

10.1182/blood-2020-139005 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-12

Author(s):

Nan Wang ◽

Jing Yin ◽

Na You ◽

Dan Guo ◽

Yangyang Zhao ◽

...

Keyword(s):

Stem Cell ◽

Steady State ◽

Calcium Channel ◽

Cell Fate ◽

Hematopoietic Stem Cell ◽

Cell Function ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Voltage Gated ◽

Voltage Gated Calcium Channel

The mitochondria of hematopoietic stem cell (HSC) play crucial roles in regulating cell fate and in preserving HSC functionality and survival. However, the mechanism underlying its regulation remain poorly understood. Here, we identify transcription factor TWIST1 as a novel regulator of HSC maintenance through modulating mitochondrial function. We demonstrate that Twist1 deletion results in a significantly decreased long-term HSC (LT-HSC) frequency, markedly reduced dormancy and self-renewal capacities and skewed myeloid differentiation in steady-state hematopoiesis. Twist1-deficient LT-HSC are more compromised in tolerance of irradiation and 5 fluorouracil-induced stresses, and exhibit typical phenotypes of senescence and higher levels of DNA damage and apoptosis. Mechanistically, Twist1 deficiency upregulates the expression of voltage-gated calcium channel Cacna1b in HSC, leading to noticeable increases in mitochondrial calcium levels, biogenesis, metabolic activity and reactive oxygen species production. Suppression of voltage-gated calcium channel by a calcium channel blocker largely rescues the phenotypic and functional defects in Twist1-deleted HSCs under both steady-state and stress conditions. Collectively, our data, for the first time, characterize TWIST1 as a critical regulator of HSC function acting through CACNA1B/Ca2+/mitochondria axis, and highlight the importance of Ca2+ in HSC maintenance. These observations provide new insights into the mechanisms for the control of HSC fate. Disclosures No relevant conflicts of interest to declare.

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Activity of a Heptad of Transcription Factors Is Associated with Stem Cell Programs and Clinical Outcome in Acute Myeloid Leukaemia

Blood ◽

10.1182/blood.v120.21.3525.3525 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3525-3525

Author(s):

Eva Diffner ◽

Dominik Beck ◽

Emma Gudgin ◽

Julie Thoms ◽

Kathy Knezevic ◽

...

Keyword(s):

Risk Factor ◽

Stem Cell ◽

Transcription Factors ◽

Clinical Outcome ◽

Hematopoietic Stem Cell ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Wild Type ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Abstract 3525 Leukaemic transformation is driven by aberrant transcriptional programs often in combination with abnormal proliferative signalling. These programs operate in normal hematopoiesis where they are involved in hematopoietic stem cell (HSC) proliferation and maintenance. ERG is a component of normal and leukemic stem cell signatures and high ERG expression has been proposed as a risk factor for poor prognosis in acute myeloid leukemia (AML). However, mechanisms that underlie ERG expression in AML and how its expression relates to leukemic stemness are unknown. We report that ERG expression in AML is associated with activity of the ERG+85 stem cell enhancer (SCE) and a heptad of transcription factors that combinatorially regulate genes in normal HSCs. Gene expression signatures derived from ERG+85 stem cell enhancer (Fig A) and heptad activity (Fig B) predict clinical outcome in a cytogenetically normal cohort of AML (CN-AML) patients when ERG expression alone fails. The heptad signature is an independent risk factor for poor overall and event-free survival (Fig C). There were no long-term survivors amongst patients with a heptad signature, FLT3 mutations and wild-type NPM1 (Fig D) pointing to a hitherto unappreciated link between aberrant signaling and transcriptional mediators of hematopoietic stem cell identity. In two independent cohorts, the heptad signature was as closely associated with wild-type NPM1 AML as the HOX signature was with mutant NPM1 AML (Fig E–F) suggestive of a collective role for these transcription factors in mediating the leukemic signature in the former. Taken together, these results show that key transcriptional regulators cooperate in establishing stem cell signatures in leukemic cells and that the underlying spectrum of somatic mutations contributes to the development of these signatures and modulate their influence on clinical outcome. Disclosures: No relevant conflicts of interest to declare.

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Conditional Deletion of STAT5 in Non-Myeloablated Recipient Mice Promotes Donor Hematopoietic Stem Cell Engraftment.

Blood ◽

10.1182/blood.v110.11.178.178 ◽

2007 ◽

Vol 110 (11) ◽

pp. 178-178 ◽

Cited By ~ 2

Author(s):

Zhengqi Wang ◽

Geqiang Li ◽

Eleonora Haviernikova ◽

William Tse ◽

Kevin D. Bunting

Keyword(s):

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell ◽

De Novo ◽

Genetic Interaction ◽

Conditional Knockout ◽

Wild Type ◽

Myeloablative Conditioning ◽

Hematopoietic Stem ◽

Cell Engraftment

Abstract Currently there is a major need in stem cell transplantation to develop innovative conditioning regimens that allow a wider range of patients to recieve therapy. Signal transducer and activator of transcription-5 (STAT5) plays major roles in hematopoietic stem and progenitor cell engraftment. Our previous studies have reported that one injection of 1 x 107 wild-type bone marrow (BM) cells into unconditioned newborn STAT5-deficient mice resulted in high levels of donor engraftment. Here we describe studies to develop novel non-myeloablative conditioning approaches based on suppression of STAT5 in vivo followed by donor cell injection (1 x 107 cells, newborns; 5 x 106 cells, adults). We have used two genetic models for STAT5 deficiency in order to test feasibility; true null STAT5 knockout mice containing deleted STAT5a and STAT5b loci or a conditional knockout mouse with STAT5ab loci flanked by loxP sites. These allowed us to examine engraftment of donor cells into STAT5 deficient hosts. We found that STAT5ab+/null hosts could be engrafted to very high levels (9/13 mice engrafted at 10–33%) when injected with BM cells as newborn pups but not surprisingly engraftment was much lower in adult 4–6 week old STAT5ab+/null mice (5/6 mice engrafted at 1–3%). In both cases, the levels of engraftment were significantly higher than in wild-type control mice. Next, we utilized conditional Mx1-Cre/STAT5abflox/null mice to determine the effects of de novo loss of STAT5 in adult mice. Adult 4–6 week old mice were treated with 16 mg/kg pI:pC on days 1, 3, 5 to bring down STAT5 levels further. pI:pC treatment did not present toxicity and resulted in complete deletion in circulating Gr-1+ and B220+ cells. Notably, the CD4+ T-lineage remained ∼50% undeleted indicating a strong selection for the undeleted T cell progenitors. In sorted primitive c-Kit+LinnegSca-1+ cells, deletion was >95%. STAT5 deletion and reduced circulating B cells and myeloid cells were sustained up to 16 weeks after the initial round of treatment without a re-emergence of the undeleted cells in non-T cell lineages. When mice treated with this regimen were injected with donor BM on day 7 we found that treatment lead to high level sustained multilineage engraftment of primary and secondary hosts at times up to 16 weeks following injection (4/4 mice engrafted with donor Gr-1+, B220+, Ter119+, and CD4+ cells between 12–78%). These data demonstrate that STAT5 can be conditionally knocked out in transplant recipients and this leads to sustained donor reconstitution in the absence of any myeloablative conditioning. Since the extremely high levels of deletion of STAT5 might be difficult to achieve therapeutically, we have also begun exploring whether there is a benefit to the combined blockage of Gab2 signaling and/or PI3-kinase in STAT5ab+/null hosts based on our observations of STAT5/Gab2 genetic interaction during hematopoiesis (Li et al., ASH 2007 abstract). In this very stringent model of a more clinically applicable situation, we have found that STAT5ab+/nullGab2−/− mice (2/8 mice engrafted at >4%) are more highly and frequently engrafted than STAT5ab+/nullGab2+/+ mice (0/8). Overall, these studies provide strong genetic proof-of-principle for modulation of STAT5 as an adjuvant for transplantation conditioning. New methods for non-myeloablative hematopoietic stem cell engraftment through combinatorial signaling inhibition may ultimately be applicable in combination with cell and gene therapies for hematologic disorders.

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

Blood ◽

10.1182/blood.v118.21.1293.1293 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1293-1293

Author(s):

Amar Desai ◽

Yulan Qing ◽

Stanton L. Gerson

Keyword(s):

Dna Repair ◽

Stem Cell ◽

Homologous Recombination ◽

Hematopoietic Stem Cell ◽

Cell Function ◽

Conflicts Of Interest ◽

Strand Break ◽

Hematopoietic Stem ◽

Quiescent Hscs

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

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Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cell Function and Promotes G-CSF Mediated HSC Mobilization

Blood ◽

10.1182/blood.v124.21.4334.4334 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4334-4334

Author(s):

Angela Herman ◽

Molly Romine ◽

Darlene Monlish ◽

Laura G. Schuettpelz

Keyword(s):

Bone Marrow ◽

Immune Response ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Immune Cell ◽

Conflicts Of Interest ◽

Wild Type ◽

Hematopoietic Stem ◽

Tlr2 Signaling

Abstract Toll like receptors (TLRs) are a family of pattern recognition receptors that play a central role in pathogen recognition and shaping the innate immune response. While most of the studies of the role of TLRs have focused on mature immune cell populations, recent reports suggest that TLR signaling may regulate the immune response from the level of the hematopoietic stem cell (HSC). In this study, we sought to further elucidate the effects of systemic TLR ligand exposure on HSCs and determine the cell-intrinsic versus extrinsic effects of such exposure. We specifically focused on TLR2 signaling, as although TLR2 is expressed on HSCs, it’s role in their regulation is not clear. Furthermore, enhanced TLR2 signaling is associated with myelodysplastic syndrome (Wei et al, Leukemia 2013), suggesting that aberrant signaling through this receptor may have clinically significant effects on HSC function. To elucidate the role of TLR2 signaling in regulating HSCs, we used mice with genetic loss of TLR2, as well as a synthetic agonist of TLR2 (PAM3CSK4) to determine the effects of TLR2 signaling loss or gain, respectively, on HSC cycling, mobilization and function. While TLR2 expression is not required for normal HSC function, treatment of wild-type mice with PAM3CSK4 leads to expansion of HSCs in the bone marrow and spleen, increased HSC cycling, and loss of HSC function in competitive bone marrow transplantation experiments. As TLR2 is expressed on a variety of stromal and hematopoietic cell types, we used bone marrow chimeras (Tlr2-/- + Tlr2+/+ marrow transplanted into Tlr2+/+ recipients) to determine if the effects of PAM3CSK4 treatment are cell intrinsic or extrinsic. The data suggests that HSC cycling and expansion in the marrow and spleen upon PAM3CSK4 treatment are extrinsic (occurring in both transplanted HSC populations), and are associated with increased serum levels of G-CSF. Indeed, inhibition of G-CSF using either a neutralizing antibody or mice lacking the G-CSF receptor (Csf3r-/-) leads to even further enhanced HSC bone marrow expansion upon G-CSF treatment but significantly reduced numbers of spleen HSCs compared to similarly treated wild-type mice. This suggests mobilization in response to TLR2 signaling is an indirect, G-CSF-mediated process. Ongoing studies are aimed at determining the contribution of G-CSF to the PAM3CSK4- induced loss of HSC function, and determining the source (stromal vs hematopoietic) of G-CSF production upon PAM3CSK4 exposure. Collectively, this data suggest that TLR2 signaling affects HSCs in a largely extrinsic fashion, with G-CSF playing a major role in regulating the effects of TLR2 ligand exposure on HSCs. Disclosures No relevant conflicts of interest to declare.

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Hemp, an mbt domain-containing protein, plays essential roles in hematopoietic stem cell function and skeletal formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1003403108 ◽

2011 ◽

Vol 108 (6) ◽

pp. 2468-2473 ◽

Cited By ~ 12

Author(s):

H. Honda ◽

K. Takubo ◽

H. Oda ◽

K. Kosaki ◽

T. Tazaki ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Function ◽

Hematopoietic Stem ◽

Skeletal Formation

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RhoA GTPase Is Dispensable for Hematopoietic Stem Cell Maintenance but Essential for Multipotent Progenitor and Lower Hierarchical Hematopoietic Differentiation.

Blood ◽

10.1182/blood.v116.21.2618.2618 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2618-2618

Author(s):

Xuan Zhou ◽

Jaime Melendez ◽

Yuxin Feng ◽

Richard Lang ◽

Yi Zheng

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Conflicts Of Interest ◽

Annexin V ◽

Small Gtpases ◽

Dominant Negative ◽

Conditional Knockout ◽

Dominant Negative Mutant ◽

Hematopoietic Stem ◽

Cell Counts

Abstract Abstract 2618 Rho family small GTPases are critical regulators of multiple functions of blood cells, including adhesion, migration, proliferation, survival and gene expression. Elucidation of their roles in hematopoiesis could be fundamental for understanding the mechanisms of various blood diseases and improving therapeutic outcomes for hematopoietic abnormalities. The Rho GTPases Rac1, Rac2 and Cdc42 have been found essential in maintaining hematopoietic stem cell (HSC) niche residency and regulating myelopoiesis and lymphopoiesis in previous mouse gene targeting studies. Using a dominant-negative mutant overexpression approach, an earlier study showed that suppression of RhoA activity enhanced HSC proliferation and engraftment potential; however, the bone fide role of RhoA in blood development remains unknown given limitations of such a mutant overexpression approach in specificity, dosage effect, and physiological relevance. Here, we stringently define the function of RhoA in HSC maintenance and hematopoiesis using an interferon inducible RhoA conditional knockout mouse model (Mx-cre+; RhoAloxp/loxp). Systematic deletion of RhoA caused lethality of the mice 7 days post polyIC induction due to hematopoietic failure that was accompanied by drastically decreased bone marrow (BM) cellularity (to ∼1/3 of wild type controls), a loss of splenocytes, and a significant reduction of cell counts of most cell lineages in peripheral blood, suggesting RhoA is required for multiple blood cell lineage differentiation and production. Syngenic transplant experiments yielded similar results, demonstrating that these effects are intrinsic to the hematopoietic compartment. The observed cytopenia resulting from RhoA loss was associated with the exhaustion of BM phenotypic HSPC (Lin−Sca1+c-kit+, LSK) and the hierarchical progenitor cells (Lin−c-kit+, LK) in number and frequency, a complete loss of colony forming activities, and a total engraftment failure. In addition, BrdU chase labeling and Annexin V/7-AAD staining revealed that RhoA deletion caused a transient increase of proliferation (1.7 fold increase in S phase) and reduction of survival (16.8 fold reduction in Annexin V− 7-AAD−) of remaining LSK in the BM. These results indicate that RhoA plays an indispensible, cell autonomous role in HSPC maintenance and hematopoiesis. In a competitive transplantation model where Mx-cre+;RhoAloxp/loxp or Mx-cre−;RhoAloxp/loxp CD45.2+ BM cells and WT CD45.1+ competitor cells were transplanted at 1:1 ratio into syngenic CD45.1+ recipients prior to polyIC induction, RhoA deletion also caused a complete extinction of donor derived (CD45.2+) Mac1+Gr1+ myeloid cells, B220+ B cells, as well as CD3+ T cells, in the peripheral. Interestingly, distinct from these more differentiated lineages, BM CD45.2+ LSK population was only marginally affected (88.9 % of pre-polyIC induction level) while the CD45.2+ LK cells and later hierarchical lineages were rapidly eliminated after RhoA deletion in this competitive transplant model. This was associated with increased apoptosis in CD45.2+ RhoA−/− LK, but not LSK, cells, suggesting a specific requirement of RhoA in the myeloid progenitor cell survival. Further, the CD45.2+ RhoA−/− LSK and LSKCD150+ cell populations, not differentiated donor-derived progenitors, from the primary competitive transplant BM, were able to be maintained in secondary transplant recipients 5 months post-transplantation, indicating that RhoA serves as a key regulator at an early progenitor differentiation step. Interestingly, RhoA deletion did not affect lin− cell p-MLC and p-cofilin contents and in vitro expansion in response to SCF stimulation, suggesting that RhoA is not required for actomyosin signaling nor SCF induced proliferation. Taken together, our results implicate RhoA as a unique and essential regulator of multipotent progenitor differentiation and survival that controls multi-lineage hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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