Chd7 Is a Cell Autonomous Regulator of Chromatin In Hematopoietic Stem Cells.

Abstract Abstract 1596 Hematopoietic stem cells (HSC) are specified during embryogenesis, and the induction process involves not only transcription factors but also epigenetic factors that modulate chromatin to regulate the hematopoietic transcriptional programs. Here, we performed a reverse genetic screen to identify all the chromatin factors that are required for HSC induction in zebrafish. The zebrafish homologs of 350 human chromatin factors were identified by reciprocal BLAST and knocked down by injecting morpholinos designed against each homolog into the single cell embryo. Morphants were then analyzed for changes in blood formation by in situ hybridization for β-globin e3 expression in primitive erythrocytes at 16 somite stage and for c-myb and runx1 in definitive stem cells at 36 hours post fertilization. From the screen, we have identified known regulators of hematopoiesis such as bmi1, in which knock down results in loss of stem cell formation. We have also identified one novel HSC regulator chd7. Chd7 is a member of the chromodomain helicase DNA-binding domain family that functions at gene enhancer elements and in ribosomal RNA synthesis. Zebrafish embryos injected with chd7 morpholino had higher levels of β-globin e3 and c-myb/runx1 expression. Additional markers such as scl, gata1, fli1, and lmo2 were also upregulated, although vascular markers flk1 and ephrinB2 were downregulated. Early mesodermal markers eve1 and ntl expression appeared normal, suggesting that the effects of chd7 knock down occurs when the mesodermal precursor cell population becomes an HSC. Transplants of chd7 deficient Tg(c-myb:GFP) blastomeres into Tg(lmo2:DsRed) blastulas resulted in more chimeric embryos compared to controls, demonstrating that the phenotype is cell autonomous. In humans, haploinsufficiency for CHD7 is the main cause of CHARGE syndrome, and it has been recognized more recently that these patients are immunodeficient, though the etiology remains unknown. Our studies indicate a new role for chd7 in hematopoiesis in which it functions to repress HSC formation during embryogenesis. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Hematopoietic Stem Cells Develop in the Absence of Endothelial Cadherin 5 Expression

Blood ◽

10.1182/blood.v126.23.1165.1165 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1165-1165

Author(s):

Heidi Anderson ◽

Taylor Patch ◽

Pavan Reddy ◽

Elliott Hagedorn ◽

Owen J. Tamplin ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Hematopoietic Stem Cells ◽

Board Of Directors ◽

Research Funding ◽

Hemogenic Endothelium ◽

Employment Equity ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership

Abstract Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from Cadherin 5 (Cdh5, VE-cadherin)+ endothelial precursors, and isolated populations of Cdh5+ cells from mouse embryos and embryonic stem (ES) cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Since Cdh5-/- mice embryos die before the first HSCs emerge, it is unknown if Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlbbw306), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Utilizing time-lapse imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5-/- GFP+/+ cells inchimeric mice, we demonstrated that Cdh5-/- GFP+/+ HSCs emerging from E10.5 and E11.5 AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multi-lineage adult blood. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs. Disclosures Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Zon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder. Orkin:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Pfizer: Research Funding; Sangamo Biosciences: Consultancy.

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Msi2 Maintains Normal Hematopoietic Stem Cell Self-Renewal

Blood ◽

10.1182/blood.v118.21.222.222 ◽

2011 ◽

Vol 118 (21) ◽

pp. 222-222 ◽

Cited By ~ 1

Author(s):

Michael G Kharas ◽

Christopher Lengner ◽

Fatima Al-Shahrour ◽

Benjamin L. Ebert ◽

George Q. Daley

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Board Of Directors ◽

Bone Marrow Cells ◽

Advisory Committees ◽

Self Renewal ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Marrow Cells

Abstract Abstract 222 Genes that regulate normal hematopoietic stem cells are commonly dysregulated in hematopoietic malignancies. Recently we published that the Msi2 RNA binding protein is an important modulator in both normal hematopoietic stem cells and leukemia (Kharas et al, Nat. Medicine 2010). The closely related Msi1 protein has been shown to regulate mRNA translation through binding to the 3'UTR. Based on the high homology in the RNA recognition motifs, Msi2 has been considered to have similar functions. Moreover, increased MSI2 expression in chronic myelogenous leukemia blast crisis and acute myeloid leukemia predicts a worse clinical prognosis. Previous studies have mainly utilized shRNAs to functionally assess the role of Msi2 in the hematopoietic compartment. However, it remains unclear how Msi2 affects hematopoietic stem cells (HSC) and what are its critical mRNA targets. To develop a model focusing on the HSC compartment and to avoid potential compensatory mechanisms during development, we created Msi2 conditional knockout mice and crossed them with Mx1-Cre mice. We induced excision with poly(I):poly(C), (pIpC), and tested the peripheral blood, bone marrow cells and splenocytes by Southern blotting and QPCR analysis to verify Msi2 deletion. Loss of Msi2 mRNA was confirmed in the Lineagelo, Sca1+ and c-Kit+ (LSK) population. Msi2 deleted bone marrow contained reduced myeloid colony forming capacity and replating efficiency. Mice conditionally deleted for Msi2 had normal white blood cell counts but smaller spleens. In addition, we observed normal percentages of the mature hematopoietic populations, including the myeloid and lymphoid compartments. Nevertheless, absolute numbers of long-term HSCs in the bone marrow were reduced by 3-fold. Bone marrow cells non-competitively transplanted into primary and secondary recipient mice showed a dramatic reduction in HSC chimerism. This defect was also observed when bone marrow was transplanted first to allow engraftment followed by Msi2 deletion. Furthermore, we were able to recapitulate this defect in vitro using the cobblestone-forming activity assay. These results indicate that Msi2 is both an important regulator of normal HSC maintenance and required for efficient engraftment. Most interestingly, Msi2 HSCs failed to maintain a normal quiescent HSC population. We performed microarrays to identify the pathways altered in the LSK population. The Msi2 deficient LSKs showed a reduced self-renewal and increased differentiation gene signature. Gene expression analysis indicates a defective self-renewal program in Msi2-deficient HSCs that is identical to the program gained in leukemic stem cells. These data suggest that MSI2 is a critical modulator of HSCs and may help explain its requirement in the most aggressive myeloid leukemias. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

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Neonatal Recipients Offer Permissive Hematopoietic Microenvironment for Engraftment of Embryonic Murine Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2344.2344 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2344-2344

Author(s):

Natasha Arora ◽

Shannon McKinney-Freeman ◽

Garrett C Heffner ◽

Il-Ho Jang ◽

Pamela L. Wenzel ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Board Of Directors ◽

Early Embryo ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Hematopoietic Microenvironment ◽

Equity Ownership

Abstract Abstract 2344 The first hematopoietic stem cells (HSC) that give rise to robust, long-term, multi-lineage reconstitution in irradiated adult recipients arise in the murine embryo at embryonic day 11.5 (E11.5). However, long-term multi-lineage engraftment in neonatal recipients has been observed from E9.0 yolk sac, suggesting that the neonatal hematopoietic microenvironment is more permissive for engraftment of embryonic HSCs. To resolve the apparent discrepancy between the numbers of candidate HSCs detected by direct visualization in the early embryo, relative to the numbers that can be measured by limiting dilution, we sought to characterize engraftment of neonatal recipients versus adult recipients with hematopoietic populations dissected from the aorta-gonad-mesonephros (AGM) region of the early embryo, the first putative site of intraembryonic origin of definitive HSCs. We dissected whole AGM from E11.5 embryos and injected cell dilutions from 2 embryo equivalents (ee) to 0.25 ee into the facial vein of day 1–2 neonatal recipients that had received sublethal conditioning with 350 rad irradiation. In neonatal recipients we detected robust, long-term, multi-lineage hematopoietic engraftment from as little as 0.25 ee. From less than 1 ee of whole AGM, the engraftment chimerism ranged from 5–20%. With 2 ee, chimerism was as high as 70%. Most animals showed balanced donor derived myeloid and lymphoid contribution by 10 weeks post-transplant. Interestingly, some animals had predominantly myeloid reconstitution for as long as 18 weeks, suggesting the presence of a novel long-term, myeloid-restricted, embryonic HSC. We also explored the neonatal engraftment potential of VE-cadherin+ CD45+ and VE-cadherin+ CD45− populations. As expected from the literature, only the VE-cadherin+ CD45+ population engrafted the neonatal recipients. Our data indicate that the neonate harbors a more permissive hematopoietic microenvironment that enables more robust engraftment of early embryonic hematopoietic populations, thereby allowing us to identify potentially novel classes of embryonic hematopoietic progenitors. We are currently exploring the neonatal engraftment potential of E9.5 and E10.5 embryonic populations, additional FACS-purified populations, and hematopoietic populations derived from pluripotent stem cells in vitro. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

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High Resolution Imaging Reveals Hematopoietic Stem Cells in the Perivascular Niche Are Anchored to Mesenchymal Stromal Cells That Orient Their Divisions

Blood ◽

10.1182/blood.v124.21.770.770 ◽

2014 ◽

Vol 124 (21) ◽

pp. 770-770

Author(s):

Owen J. Tamplin ◽

Ellen M. Durand ◽

Logan A. Carr ◽

Sarah J. Childs ◽

Elliott H. Hagedorn ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Stem Cell ◽

High Resolution ◽

Board Of Directors ◽

Stromal Cells ◽

Stromal Cell ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership

Abstract Hematopoietic stem cells (HSC) reside in a highly structured microenvironment called the niche. There is two-way communication between a stem cell and its niche that determines important cell fate decisions. HSC must remain quiescent to persist throughout life but also divide and contribute progenitors that will replenish the blood supply. Although there have been a number of elegant studies that have imaged the mammalian bone marrow, we still lack a high-resolution real-time view of endogenous HSC behaviors and interactions within the niche. To overcome these challenges, we developed a transgenic zebrafish line that expresses GFP or mCherry in HSC. We generated this line using the previously described mouse Runx1 +23 kb intronic enhancer. We confirmed the purity of these stem cells by adult-to-adult limiting dilution transplantation with as few as one cell. Based on long-term multi-lineage engraftment, we estimated a stem cell purity of approximately 1/35, which is similar to the KSL (Kit+Sca1+Lin-) population in mouse. Using a novel embryo-to-embryo transplantation assay that is unique to zebrafish, we estimated an even higher stem cell purity of 1/2. These experiments have defined the most pure HSC population in the zebrafish. Using this novel transgenic reporter we have tracked HSC as they migrate in the live zebrafish embryo. This allowed us to image HSC as they interact with other cell types in their microenvironment, including endothelial cells and mesenchymal stromal cells. We have shown that a small group of endothelial cells remodel around a single HSC soon after it lodges in the niche. Recently, we have also found that a single stromal cell can anchor an HSC as it divides. In most cases, we observed that an HSC divides perpendicular to the stromal cell, with one daughter cell remaining attached to the stromal cell and the other migrating away. To gain a much higher resolution view of these cellular events than is possible with confocal microscopy we looked for an alternative approach. A combined method is called “Correlative Light and Electron Microscopy” (CLEM), and involves identification of cells by confocal microscopy, followed by processing of the same sample for EM scanning. We have applied this method by: 1) tracking endogenous HSC in the live embryo; 2) fixing the same embryo for serial block-face scanning EM; 3) reconstructing 3D models from high resolution serial EM sections. We used easily visible blood vessels as anatomical markers that allowed us to pinpoint a single cell in a relatively large block of scanned tissue. As expected, the identified HSC was round, had a distinctive large nucleus, scant cytoplasm, and ruffled membrane. The HSC was surrounded by a small group of 5-6 endothelial cells, as predicted from our confocal live imaging. However at this very high resolution (10 nm/pixel), we could see that only part of the HSC surface was contacted and wrapped by an endothelial cell. Other regions of the HSC surface were contacted by small endothelial cell protrusions. Much of the HSC surface was surrounded by a narrow extracellular space with endothelial and stromal cells lying opposite. Strikingly, we were able to identify the firm anchored attachment between a single stromal cell and HSC that we showed previously oriented the plane of division. By combining confocal live imaging of a novel zebrafish HSC reporter, and serial block-face scanning EM, we have created the first high-resolution 3D model of an endogenous stem cell in its niche. Disclosures Tamplin: Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Ontology of Megakaryopoesis.

Blood ◽

10.1182/blood.v114.22.sci-8.sci-8 ◽

2009 ◽

Vol 114 (22) ◽

pp. SCI-8-SCI-8

Author(s):

Amy E. Geddis

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Hematopoietic Stem Cells ◽

Board Of Directors ◽

Cell Surface Markers ◽

Developmental Relationships ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Common Precursor ◽

Review Current

Abstract Abstract SCI-8 Compared to red cells, whose passage from embryonic to adult stages is marked by the expression of distinct forms of hemoglobin, the development of megakaryocytes during embryogenesis is less well understood. However, certain shared characteristics between megakaryocytes, endothelial cells, hematopoietic stem cells and erythrocytes infer developmental relationships between these lineages. Recent data support the model that hematopoietic stem cells derive from the hemangioblast, and that megakaryocytes and erythrocytes develop from a common precursor both in primitive and adult hematopoiesis. Evidence of these common origins can be found in the genetic programs that are activated during hematopoiesis, in that many of the cell surface markers and transcriptions factors that are characteristic of megakaryocytes can also be found in endothelial cells, stem cells and erythrocytes. In this session I will review current views on developmental thrombopoiesis, key megakaryocytic transcription factors and the experimental and clinical phenotypes associated with their disruption, and current controversies in lineage choice during megakaryocyte differentiation. Disclosures Geddis: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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IKZF1 Mutation Mediate Resistance to IMiDs in Human Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v126.23.3003.3003 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3003-3003

Author(s):

Shirong Li ◽

Jing Fu ◽

Jing Wu ◽

Markus Y Mapara ◽

Suzanne Lentzsch

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Board Of Directors ◽

Cell Expansion ◽

Lineage Commitment ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Introduction: Previously we have shown that the immune modulatory drugs (IMiDs) downregulate GATA1 and PU.1 resulting in maturational arrest of granulocytes with accumulation of immature myeloid precursors and subsequent neutropenia. Our studies further revealed that similar to MM cells cereblon (CRBN) is critical for the mediation of the effects of IMiDS in hematopoietic stem cells (HSCs) and associated with decrease of IKZF1-dependent transcription factors such as GATA1 and PU.1, which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Here we investigated the mechanism how IMIDs induce degradation of IKZF1 and confirmed our studies in vivo by using the humanized NOD/SCID/Gamma-c KO (NSG) mouse model. Methods and Results After we had shown that knockdown of CRBN in HCS mediates resistance to IMIDs (2014 ASH abstract 418) we assessed the impact of IKZF1 inhibition using two different approaches. First, we knocked down IKZF1 expression in CD34+ cells by shRNA lentivirus transduction. As expected, IKZF1 knockdown in CD34+ cells mimicked the effects of IMiDs resulting in increased CD34+ cell proliferation, CD33+ cell expansion (flow cytometry) and shift of lineage commitment from BFU-E to CFU-G (colony assay). Knockdown of IKZF1 was associated with decreased GATA1 and PU.1 expression at both mRNA and protein levels. Next, we generated a mutant IKZF1 by substituting Glutamine Q146 to Histidine, which abrogates IKZF1 ubiquitination induced by CRBN. CD34+ cells were transduced with lentiviral constructs to overexpress IKZF1-WT or IKZF1-Q146H. POM failed to induce IKZF1 degradation in IKZF1-Q146H-OE CD34+ cells, indicating CRBN binding to IKZF1 and subsequent ubiquitination is critical in this process. Functional assays further confirmed that IKZF1-Q146H CD34+ cells were resistant to POM induced CD33+ cell expansion and shift in lineage commitment from BFU-E to CFU-G. Since conventional mouse models are not applicable to test IMIDs in vivo due to the fact that IMIDs do not bind to mouse CRBN (Kronke, Fink et al. 2015), we established a humanized mouse model resembling human hematopoiesis. In this model, NOD/SCID/Gamma-c KO (NSG) mice received human fetal thymus grafts and 105 CD34+ fetal liver cells to generate human hematopoiesis including functional T-cells. After establishing human hematopoiesis mice were injected with POM (0.3 mg/kg) i.v every 2 days for 3 weeks. Analysis of bone marrow revealed that POM treatment significantly induced granulocyte/macrophage progenitor cells (CD34+ CD38+ CD45RA+ cells) at the expense of common lymphoid progenitors (CD34+ CD10+ cells). The shift into myelopoiesis is consistent with our in vitro finding that IMiDs affect lineage commitment. Conclusion: In summary, our results demonstrate that IMiDs affect CD34+ cell fate via CRBN and IKZF1 mediated mechanism. These results will be helpful to elucidate the mechanism of IMiDs on lineage commitment and maturation in HSCs. Also establishment of the humanized xenograft mice model may provide an advanced platform for the analysis of human hematopoiesis and human immune responses to IMiDs as well development of secondary hematologic malignancies in vivo. Disclosures Lentzsch: Axiom: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

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A Network Of Epigenetic Regulators Guide Developmental Hematopoiesis In Vivo

Blood ◽

10.1182/blood.v122.21.1174.1174 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1174-1174

Author(s):

Katie L Kathrein ◽

Hsuan-Ting Huang ◽

Abby Barton ◽

Zachary Gitlin ◽

Yue-Hua Huang ◽

...

Keyword(s):

Stem Cells ◽

Board Of Directors ◽

Large Scale ◽

Chromatin Domain ◽

Advisory Committees ◽

Self Renewal ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Marker Expression

Abstract Long-term hematopoietic stem cells (HSCs) are capable of self-renewal and differentiation into all mature hematopoietic lineages. This process is regulated by transcription factors interact with co-factors to orchestrate chromatin structure and facilitate gene expression. To generate a compendium of factors that establish the epigenetic code in HSCs, we have undertaken the first large-scale in vivo reverse genetic screen targeting chromatin factors. We have designed and injected antisense morpholinos to knockdown expression of 488 zebrafish orthologs of conserved human chromatin factors. The resultant morphants were analyzed by whole embryo in situ hybridization at 36 hours post fertilization for expression of two HSC marker genes, c-myb and runx1, which are expressed in the developing blood stem cells. Morphants were categorized into five groups based on HSC marker expression, ranging from no change to mild, intermediate, or strong reduction in expression or an increase in expression. 29 morpholinos caused a complete or near complete knockdown of HSC marker expression, while 4 were found to increase HSC marker expression. As ubiquitous knockdown of chromatin factors could interfere with vascular development and the establishment of proper arterial identity, a crucial upstream event for HSC formation, we subsequently analyzed morphants with the most robust HSC phenotypes using two vascular markers: kdr for overall vasculogenesis and ephrinb2a for arterial formation. We found that of the 29 morpholinos that caused reduced marker expression, only 9 showed reduced overall vascular or arterial marker staining, suggesting that the majority of morphants with HSC phenotypes are specific to HSC formation. For the 4 morphants with increased HSC marker expression, vasculature appeared normal. These factors likely function as potent negative regulators of HSC development. Several genes known to be essential for HSC self-renewal and maintenance were identified in the screen. For example, knockdown of Mll or Dot1, which are also present in leukemia fusion proteins, fail to specify HSCs, as indicated by a nearly complete reduction in expression of the HSC markers in embryos tested. Of the remaining hits, many represent factors with no previous function ascribed in hematopoiesis. By incorporating protein interaction data, we have defined a handful of complexes necessary for HSC specification, including the SWI/SNF, ISWI, SET1/MLL, CBP/P300/HBO1/NuA4, HDAC/NuRD, and Polycomb complexes. As chromatin factors associated with the same complex likely share target binding sites, we analyzed 34 published ChIP-seq datasets in K562 erythroleukemia cells of chromatin factors tested in the screen, including hits from our screen: SIN3A, CHD4, HDAC1, TAF1, and JARID1C associated with the HDAC/NuRD complex and RNF2, SUZ12, CBX2, and CBX8 from the Polycomb complexes. We ranked triplet combinations of these factors together with all other groups of three factors based on the percent overlap of target genes. The HDAC/NuRD and PRC1/2 complex combinations predicted from our screen fell within the top 20% of all possible combinations of 3 factors, suggesting that our screen has identified chromatin factors that function in distinct complexes to regulate hematopoietic development. Our work has been compiled into a web-based database that will be made publicly available upon publication. Within this database, users can search by gene names and aliases, chromatin domain names and human or zebrafish genes. All experimental data, including experimental design, materials, protocols, images, and all further analyses of the 33 most robust morphants is included. Our large-scale genetic analysis of chromatin factors involved in HSC development provides a comprehensive view of the programs involved in epigenetic regulation of the blood program, offering new avenues to pursue in the study of histone modifications in HSCs and for therapeutic alternatives for patients with blood disorders and leukemia. Disclosures: Zon: FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

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Valine as a Key Metabolic Regulator of Hematopoietic Stem Cell Maintenance

Blood ◽

10.1182/blood.v130.suppl_1.sci-20.sci-20 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. SCI-20-SCI-20

Author(s):

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Amino Acid ◽

Board Of Directors ◽

Bone Marrow Niche ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Conditioning Approach

Abstract Hematopoietic stem cells (HSCs) are maintained by a specialized bone marrow microenvironment (niche) and are largely quiescent during the steady-state conditions1. However, upon stimulation or transplantation, HSCs can expand and differentiate into any mature hematopoietic cell type. This ability of donor HSCs to reform a recipient's hematopoietic system is key to the success of HSC transplantation (HSCT). For donor HSCs to engraft, the recipient bone marrow niche must first be emptied via myeloablative irradiation or chemotherapy. However, myeloablative conditioning can cause severe complications and even mortality. As an alternative, we have recently developed a metabolic conditioning approach for HSCT. By screening the amino acid requirements of HSCs, we identified the essential amino acid valine as indispensable for the expansion and maintenance of HSCs2. Both mouse and human HSCs failed to expand when cultured in valine-restricted conditions. In mice fed a valine-restricted diet, HSC frequency fell dramatically within one week. Dietary valine restriction thereby emptied the mouse bone marrow niche and just a two-week diet afforded long-term donor-HSC engraftment without chemoirradiative myeloablation. We conclude that valine plays a critical role in HSC maintenance and suggest dietary valine restriction as a conditioning regimen that may reduce iatrogenic complications in HSCT. These findings, and recent efforts to optimize this metabolic conditioning approach through mechanistic understanding of the HSC valine dependency, will be presented. Sudo K, Ema H, Morita Y, Nakauchi H. Age-associated characteristics of murine hematopoietic stem cells. J Exp Med. 2000;192:1273-1280.Taya Y, Ota Y, Wilkinson AC, et al. Depleting dietary valine permits nonmyeloablative mouse hematopoietic stem cell transplantation. Science. 354:1152-1155. Disclosures Nakauchi: ReproCELL Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Megakaryon Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; iBUKI Corp: Equity Ownership; iCELL Inc: Equity Ownership; Advanced Immunothearpy Inc: Equity Ownership.

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Promotion of Erythropoietic Differentiation in Hematopoietic Stem Cells by SOCS3 Knock-Down

PLoS ONE ◽

10.1371/journal.pone.0135259 ◽

2015 ◽

Vol 10 (8) ◽

pp. e0135259 ◽

Cited By ~ 4

Author(s):

Yu-xiao Liu ◽

Xing Dong ◽

Feng Gong ◽

Ning Su ◽

Su-bo Li ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem ◽

Knock Down

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Epigenetic Activation of the pH Regulator MCT4 in Acute Myeloid Leukemia Exploits a Fundamental Metabolic Process of Enhancing Cell Growth through Proton Shifting

Blood ◽

10.1182/blood-2019-123604 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3765-3765

Author(s):

Cheuk-Him Man ◽

David T. Scadden ◽

Francois Mercier ◽

Nian Liu ◽

Wentao Dong ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Growth ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Monocarboxylate Transporter ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Acute Myeloid

Acute myeloid leukemia (AML) cells exhibit metabolic alterations that may provide therapeutic targets not necessarily evident in the cancer cell genome. Among the metabolic features we noted in AML compared with normal hematopoietic stem and progenitors (HSPC) was a strikingly consistent alkaline intracellular pH (pHi). Among candidate proton regulators, monocarboxylate transporter 4 (MCT4) mRNA and protein were differentially increased in multiple human and mouse AML cell lines and primary AML cells. MCT4 is a plasma membrane H+and lactate co-transporter whose activity necessarily shifts protons extracellularly as intracellular lactate is extruded. MCT4 activity is increased when overexpressed or with increased intracellular lactate generated by glycolysis in the setting of nutrient abundance. With increased MCT4 activity, extracellular lactate and protons will increase causing extracellular acidification while alkalinizing the intracellular compartment. MCT4-knockout (MCT4-KO) of mouse and human AMLdid not induce compensatory MCT1 expression, reduced pHi, suppressed proliferation and improved animal survival. Growth reduction was experimentally defined to be due to intracellular acidification rather than lactate accumulation by independent modulation of those parameters. MCT4-KOmetabolic profiling demonstrated decreased ATP/ADP and increased NADP+/NADPH suggesting suppression of glycolysis and the pentose phosphate pathway (PPP) that was confirmed by stable isotopic carbon flux analyses. Notably,the enzymatic activity of purified gatekeeper enzymes, hexokinase 1 (HK1), pyruvate kinase M2 isoform (PKM2) and glucose-6-phosphate dehydrogenase (G6PDH) was sensitive to pH with increased activity at the leukemic pHi (pH 7.6) compared to normal pHi (pH 7.3). Evaluating MCT4 transcriptional regulation, we defined that activating histonemarks, H3K27ac and H3K4me3, were enriched at the MCT4 promoter region as were transcriptional regulators MLL1 and Brd4 by ChIP in AML compared with normal cells. Pharmacologic inhibition of Brd4 suppressed Brd4 and H3K27ac enrichment and MCT4 expression in AML and reduced leukemic cell growth. To determine whether MCT4 based pHi changes were sufficient to increase cell proliferation, we overexpressed MCT4 in normal HSPC and demonstrated in vivo increases in growth in conjunction with pHi alkalization. Some other cell types also were increased in their growth kinetics by MCT4 overexpression and pHi increase. Therefore, proton shifting may be a means by which cells respond to nutrient abundance, co-transporting lactate and protons out of the cell, increasing the activity of enzymes that enhance PPP and glycolysis for biomass generation. Epigenetic changes in AML appear to exploit that process by increasing MCT4 expression to enforce proton exclusion thereby gaining a growth advantage without dependence on signaling pathways. Inhibiting MCT4 and intracellular alkalization may diminish the ability of AML to outcompete normal hematopoiesis. Figure Disclosures Scadden: Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership.

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