scholarly journals Dose-Dependent Role of the Cohesin Complex in Normal and Malignant Hematopoiesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 435-435
Author(s):  
Aaron D. Viny ◽  
Christopher J. Ott ◽  
Barbara Spitzer ◽  
Martin A Rivas ◽  
Cem Meydan ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Board Of Directors ◽  
Cohesin Complex ◽  
Wild Type ◽  
Advisory Committees ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Regulatory Architecture ◽  
Dose Dependent

Abstract Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation, however cohesin-mutant leukemias do not show genomic instability suggesting an alternate role in malignant transformation. We hypothesized reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic stem/progenitor cells. We therefore investigated the impact of both complete loss and haploinsufficiency of Smc3, an obligate member of the cohesin complex, in normal hematopoiesis and in myeloid transformation by developing a conditional Smc3 knockout allele. Somatic loss of Smc3 in hematopoietic cells induced lethal bone marrow aplasia (median survival 11 days; p<0.001), with premature sister chromatid separation and abnormal nucleolar organization. Competitive transplant assays showed that Smc3 loss completely abrogated stem cell self-renewal in vivo. These data are consistent with an absolute requirement for the cohesin complex in hematopoietic stem/progenitor cells. By contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, with increased serial replating, expanded hematopoietic stem/progenitor cells, and a self-renewal/engraftment advantage in competitive transplantation assays in vivo (Figure a). Smc3 haploinsufficiency altered coordinated transcriptional output, including reduced expression of master regulatory transcription factors governing lineage commitment. Consistent with these data, Smc3 loss resulted in expanded Cd150+ Cd48+ ST-HSC (p=0.008), reduction in Cd150+ Cd48- LT-HSC (p=0.001), and altered chromatin architecture with dysregulated expression of genes with specific chromatin architecture footprints. Smc3 haploinsufficiency cooperated with Flt3ITD to induce acute leukemia in vivo (Figure b), with dysregulated expression of hematopoietic master regulators and altered nucleolar topology similar to that observed in germline cohesinopathy syndromes and in AML patients with cohesin mutations (Figure c). To further explore the mechanism by which Smc3 loss cooperates with Flt3ITD to induce leukemia, we investigated chromatin cis-regulatory architecture with transposase hypersensitivity assays (ATAC-seq). We hypothesized that increased accessibility at cis-regulatory elements and the alterations in gene expression seen in cells with combined Smc3 haploinsufficiency and Flt3ITD may be in a large part driven by potentiated Stat signaling at chromatin. We analyzed 146 transcription factor recognition motifs within the THS differentially observed in Smc3Δ/+Flt3ITD and wild-type cells. Chromatin accessibility gained in Smc3Δ/+Flt3ITD cells are enriched in Stat family transcription factor binding sites, including Stat5. We also observed enrichment of the Stat5 gene expression signature in the Smc3Δ/+Flt3ITD cells compared to Smc3Δ/+, Flt3ITD and wild-type cells, suggesting the divergent mutations cooperate to potentiate oncogenic Stat5 signaling in HSPCs. Our results demonstrate a key dose-dependent role for the cohesin complex in hematopoiesis, and show that reduced cohesin functions to alter enhancer-mediated transcription and contribute to aberrant self-renewal and myeloid transformation. Figure 1. Figure 1. Disclosures Levine: Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

The Role of Chromatin Factors in Hematopoietic Development

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-48-SCI-48
Author(s):  
Leonard I. Zon
Keyword(s):  
Progenitor Cells ◽  
Board Of Directors ◽  
Large Scale ◽  
Erythroid Progenitors ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Conditional Allele ◽  
Stem And Progenitor Cells ◽  
Equity Ownership

Abstract The initiation of blood-specific programs is orchestrated by key transcription factors. To generate a complete compendium of chromatin factors that establish the epigenetic code during developmental hematopoiesis, a large-scale reverse genetic screen was conducted targeting orthologs of 425 human chromatin factors in zebrafish. A set of chromatin regulators was identified that function at distinct steps of primitive and definitive blood formation, including factors not previously implicated in blood development. We identified 15 factors that regulate development of primitive erythroid progenitors and 29 factors that regulate development of definitive stem and progenitor cells. These chromatin factors are associated with SWI/SNF and ISWI chromatin remodeling, SET1/MLL methyltransferase, CBP/P300/HBO1/NuA4 acetyltransferase, Sin3A/NuRD deacetylase, and Polycomb repressive complexes. Knockdown of a class of chromatin factors led to an expansion of hematopoietic stem cells (HSCs). In collaboration with Nancy Speck’s laboratory, we have investigated the activity of one of these chromatin factors, CHD7, that led to an expansion of hematopoietic stem and progenitor cells in the aorta. Using a T-cell line, a CBFβ protein pulldown, and a mass spectrometric sequencing approach led to the finding that CBFβ immunoprecipitated RUNX1 and CHD7. By studying a conditional allele of murine Chd7, inactivation does not have an effect on peripheral blood counts, but Chd7-deleted HSCs purified based on phenotypic markers contain an increased number of functional HSCs. Our studies suggest that CHD7 suppresses hematopoiesis, and provides a novel control mechanism for the regulation of HSCs. Our work provides a comprehensive view of how specific chromatin factors and their associated complexes play a major role in the establishment of hematopoietic cells in vivo. Disclosures: Zon: Scholar Rock: Consultancy, Equity Ownership, Founder, 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; FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Oncogenic Wilms Tumor 1 (WT1) Mutation Augments Hematopoietic Progenitor Cell Clonogenicity and Promotes Expansion Of The Long-Term Hematopoietic Stem Cell (LT-HSC) Compartment: Implications For WT1-Mediated Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1269-1269
Author(s):  
Colleen E. Annesley ◽  
Rachel E. Rau ◽  
Daniel Magoon ◽  
David Loeb ◽  
Patrick Brown
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Wilms Tumor ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Exon 9

Abstract Background The WT1 gene encodes for a zinc finger-containing transcription factor involved in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with expression maintained in the kidney and in CD34+ hematopoietic progenitor cells. Inactivating mutations of this tumor suppressor gene are well-described in sporadic Wilms tumor and as germline mutations in Wilms tumor predisposition syndromes. WT1 mutations have been reported in approximately 10% of both adult and pediatric patients with cytogenetically-normal acute myeloid leukemia (CN-AML), and have been associated with treatment failure and a poor prognosis. These reported mutations consist of insertions, deletions or point mutations. Many are frameshift mutations in exon 7, can occur as biallelic double mutations, and result in truncated proteins which may alter DNA-binding ability. Missense mutations in exon 9 have also been identified, and reports suggest that these may act in a dominant-negative manner, resulting in a loss of function. Despite these observations, the functional contribution of WT1 mutations to leukemogenesis is still largely undetermined. Methods/Results We obtained a novel knock-in WT1 mutant mouse model, which is heterozygous for the missense mutation R394W in exon 9, and homologous to exon 9 mutations seen in human AML. We hypothesized that WT1 mutations may have an aberrant effect on hematopoiesis, and specifically, could alter progenitor cell differentiation or proliferation. To investigate this, we collected lineage-negative bone marrow (lin- BM) cells from two-month old WT1 mutant (WT1mut) and wild-type (wt) mice. We performed methylcellulose colony-forming assays, serially replating cells every 10-12 days. Strikingly, WT1mut progenitor cells showed higher in vitro colony-forming capacity and an increased ability to serially replate, suggesting aberrantly enhanced self-renewal capability. Furthermore, WT1mut colonies from secondary and tertiary passages were larger and more cohesive than wild-type colonies, demonstrating increased proliferation and morphology consistent with blast colony-forming units (CFU-blast). Flow cytometric analysis of these WT1mut cells at tertiary replating revealed an immature, largely c-Kit+ population. Next, in order to study the effects of WT1mut on HSCs in vivo, we performed serial competitive transplantation of HSC-enriched, lineage-depleted BM into lethally irradiated mice. At 14 weeks post-transplant, the donor bone marrow cells were harvested and analyzed by flow cytometry. We observed a significant expansion of the LT-HSC compartment in the WT1mut mice compared to wild-type mice. These data provide new insight into the biology and functional role of WT1 mutations in the aberrant regulation of hematopoietic stem and progenitor cell expansion. Conclusion Oncogenic WT1 mutations confer enhanced proliferation and renewal of myeloid progenitor cells in vitro and expansion of LT-HSCs in vivo. Our findings suggest that WT1 mutations enhance stem cell self-renewal, potentially priming these cells for leukemic transformation upon acquisition of cooperative events. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dose-dependent role of the cohesin complex in normal and malignant hematopoiesis

2015 ◽  
Vol 212 (11) ◽  
pp. 1819-1832 ◽  
Author(s):  
Aaron D. Viny ◽  
Christopher J. Ott ◽  
Barbara Spitzer ◽  
Martin Rivas ◽  
Cem Meydan ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Cohesin Complex ◽  
Hematopoietic Stem ◽  
Regulatory Architecture ◽  
Absolute Requirement ◽  
Transcriptional Output ◽  
Dose Dependent

Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation; however, cohesin mutant leukemias do not show genomic instability. We hypothesized that reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic progenitors. We investigated the consequences of Smc3 deletion in normal and malignant hematopoiesis. Biallelic Smc3 loss induced bone marrow aplasia with premature sister chromatid separation and revealed an absolute requirement for cohesin in hematopoietic stem cell (HSC) function. In contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, including competitive transplantation. Smc3 haploinsufficiency reduced coordinated transcriptional output, including reduced expression of transcription factors and other genes associated with lineage commitment. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce acute leukemia in vivo, with potentiated Stat5 signaling and altered nucleolar topology. These data establish a dose dependency for cohesin in regulating chromatin structure and HSC function.

scholarly journals A Genetic Disorder Reveals a Hematopoietic Stem Cell Regulatory Network Co-Opted in Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 861-861
Author(s):  
Richard Voit ◽  
Liming Tao ◽  
Fulong Yu ◽  
Blake Cohen ◽  
Liam Cato ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Gene Network ◽  
Genetic Disorder ◽  
Regulatory Elements ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Chromatin Reorganization

Abstract The molecular regulation of human hematopoietic stem cell (HSC) self-renewal and maintenance is of substantial interest, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. To better discern in vivo HSC function in humans, we have studied a rare genetic disorder due to MECOM haploinsufficiency that is characterized by neonatal aplastic anemia with an early-onset absence of HSCs in vivo. To establish a faithful model of MECOM haploinsufficiency, we performed CRISPR/Cas9 editing of MECOM in human hematopoietic stem and progenitor cells (HSPCs) and achieved predominantly heterozygous editing in phenotypic long-term (LT)-HSCs, as substantiated through both bulk and single cell assessments. Following MECOM editing, HSPCs showed a significant reduction in the number of phenotypic LT-HSCs as well as multipotential progenitor colonies in vitro, and had impaired engraftment following xenotransplantation into immunodeficient and Kit mutant mice. Next, we sought to use this model of MECOM haploinsufficiency to define the regulatory networks driven by MECOM that are critical for HSC maintenance. We therefore performed single-cell RNA sequencing on several thousand phenotypic LT-HSCs following MECOM editing and identified a list of 724 subtly but significantly differentially expressed genes compared to controls, including 322 genes that are downregulated after MECOM perturbation. Given the profound phenotypic effects associated with loss of MECOM and dysregulation of these gene sets, we sought to identify other cooperating factors that control the MECOM dependent gene network which underlies HSC self-renewal. To do so, we used integrative genomic approaches involving accessible chromatin and gene expression correlations, as well as long range chromatin interaction data across human hematopoiesis, to comprehensively define associations between genes and putative regulatory elements. Inspection of the nominated cis-regulatory elements controlling genes impacted by MECOM perturbation revealed significant enrichment for motifs and chromatin occupancy by several key cooperating transcription factors, including RUNX1, FLI1, and GATA2. In addition to the identification of cooperating transcription factors, we also discovered a strong binding motif enrichment for, and chromatin occupancy by, CTCF. CTCF is crucial to enable differentiation of LT-HSCs, and we found that MECOM regulated genes frequently had associated cis-regulatory elements that were occupied by CTCF. Moreover, these occupied cis-elements became more highly enriched for binding during hematopoietic differentiation. Chromatin conformation analysis revealed that the MECOM regulated genes with cis-elements bound by CTCF underwent chromatin reorganization and became more highly looped as LT-HSCs underwent differentiation, suggesting opposing functions of MECOM and CTCF in the regulation of the MECOM gene network. In light of these findings, we performed tandem perturbation of MECOM and CTCF and demonstrated rescue of LT-HSC loss with the dual perturbation, thereby illuminating a key role for MECOM in constraining CTCF-dependent chromatin reorganization that occurs as HSCs undergo differentiation. Finally, based on the observation that elevated MECOM expression is associated with high-risk myeloid malignancies, we investigated the role of the MECOM-regulated HSC gene network in acute myeloid leukemias (AML). Across three independent AML datasets, we found that the MECOM regulated gene network had an independent and strong prognostic prediction ability that enabled risk stratification beyond currently used approaches, including a variety of molecular criteria and the previously described LSC17 signature. To validate these correlative observations, we performed CRISPR/Cas9 editing of MECOM in the MUTZ-3 AML cell line that is characterized by MECOM overexpression. We found that MECOM editing results in a loss of CD34 + leukemia progenitors and that the same transcriptional network that we identified in LT-HSCs is similarly altered upon MECOM perturbation in these AML cells. Collectively, we use the study of a rare experiment of nature due to MECOM haploinsufficiency resulting in neonatal aplastic anemia to illuminate a gene regulatory network necessary for HSC self-renewal and maintenance that is co-opted in high-risk forms of AML. Disclosures Regev: Genentech: Current Employment; Celsius Therapeutics: Current equity holder in publicly-traded company, Other: Co-founder; Immunitas: Current equity holder in publicly-traded company; ThermoFisher Scientific: Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Neogene Therapeutics: Membership on an entity's Board of Directors or advisory committees; Asimov: Membership on an entity's Board of Directors or advisory committees. Sankaran: Forma: Consultancy; Ensoma: Consultancy; Novartis: Consultancy; Cellarity: Consultancy; Branch Biosciences: Consultancy.

scholarly journals Germline Runx1 Mutations Induce Inflammation in Hematopoietic Stem and Progenitor Cells and Predispose to Hematologic Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2201-2201
Author(s):  
Mohd Hafiz Ahmad ◽  
Mahesh Hegde ◽  
Waihay J. Wong ◽  
Andrew Dunbar ◽  
Anneliese Carrascoso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Hematologic Malignancies ◽  
Advisory Committees ◽  
Hematopoietic Stem

Abstract Patients with Familial Platelet disorder (FPD) have a germline RUNX1 mutation and are at high risk to developing hematologic malignancies (HM), primarily myelodysplastic syndrome and acute myeloid leukemia (lifetime risk~40%). To understand how germline RUNX1 mutations predispose to HM in vivo, we developed a Runx1 R188Q/+ mouse strain , mimicking the FPD-associated R201Q missense mutation. Analysis of the bone marrow cells in Runx1 R188Q/+ mice revealed a significant increase in the total number of bone marrow cells. Immunophenotypic analysis using Sca-1 and Cd86 markers revealed a significant increase in Sca-1 expression in hematopoietic stem and multi-potential progenitor cells, indicating a systemic inflammation in the bone marrow. In addition, the frequency of common-myeloid, granulocytic-monocytic and granulocytic progenitor cells were found significantly increased in the Runx1 R188Q/+ bone marrow. Accordingly, their colony-forming unit capacity was increased when compared to wildtype controls (wt/Runx1 R188Q/+ CFU average = 45/85), indicating a myeloid bias. The number and size of platelets were not altered in Runx1 R188Q/+ mice. However, platelet function was significantly reduced. The activation of the Cd41/Cd61 fibrinogen receptor complex in membrane after thrombin treatment was reduced in Runx1 R188Q/+ platelets. Similarly, the translocation of P-selectin by alpha granules and the secretion of serotonin by the dense granules were also reduced. Hematopoietic progenitor cells isolated from Runx1 R188Q/+ mice revealed a significant reduction in DNA-damage repair response in vitro. Quantitative analysis of nuclei with 53bp1-positive foci in response to ionizing radiation showed a marked increase in 53bp1-positive foci in Runx1 R188Q/+ nuclei, suggesting that Runx1 R188Q/+ cells have a defective repair of double strand DNA breaks. Furthermore, expression of DNA-damage repair pathway-associated Pmaip1 (Noxa) was significantly reduced in irradiated Runx1 R188Q/+ hematopoietic progenitor cells. To understand underlying mechanism responsible for the observed myeloid bias in Runx1 R188Q/+ cells, transcription profiling analysis was performed in myeloid progenitors from wildtype and Runx1 R188Q/+ mice, utilizing RNA-sequencing. A total of 39 genes were significantly deregulated (&gt; 1.5 FC; FDR&lt;0.05), including 8 up- and 31 down-regulated genes. The expression of three repressed genes with important function in hematopoietic differentiation and malignancy (Cdh1, Gja1, and Fcer1a) were validated by qRT-PCR. To study the FPD-associated pre-leukemic process in vivo, wildtype and Runx1 R188Q/+ mice were monitored for 20 months. Although Runx1 R188Q/+ mice remained healthy for 18 months, somatic mutations in their leukocytes were evident at 12 months. Targeted sequencing of 578 cancer genes (mIMPACT panel) in leukocyte DNA of two Runx1 R188Q/+ mice identified somatic mutations in Kdm6a, Setd1b, Amer1, and Esco1 (variant allele frequencies between 0.5% and 2.8%). These mutations were confirmed at stable frequency for eight following months. Since loss of the second Runx1 allele is a frequent somatic event in progression to FPD/HM, we evaluated the predisposition to HM in Mx1Cre-Runx1 R188Q/fl mice over time. Unlike Runx1 R188Q/+ mice, Runx1 R188Q/Δ mice succumbed to myeloid leukemia with a median latency of 37.5 weeks and full penetrance. In addition, the expression of oncogenic Nras-G12D, in Runx1 R188Q/Δ mice reduced the median latency to 14.7 weeks. These studies demonstrate that FPD-associated Runx1 germline mutations induce inflammation in hematopoietic stem cells, induce myeloid expansion with defective DNA-damage response and predispose to HM over time. These studies suggest that anti-inflammatory therapies in pre-symptomatic FPD patients may reduce clonal expansion and predisposition to HM. Disclosures Ebert: Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Skyhawk Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Deerfield: Research Funding; GRAIL: Consultancy. Levine: Isoplexis: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Lilly: Honoraria; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Incyte: Consultancy; Astellas: Consultancy; Amgen: Honoraria.

scholarly journals A Network Of Epigenetic Regulators Guide Developmental Hematopoiesis In Vivo

Blood ◽  
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.

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

Blood ◽  
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.

Slug Plays an Essential Role in the Radioprotection of Hematopoietic Progenitors In Vivo by Antagonizing p53-Mediated Apoptotic Pathways.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 31-31
Author(s):  
Wen-Shu Wu ◽  
Dong Xu ◽  
Stefan Heinrichs ◽  
A. Thomas Look
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Γ Irradiation ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Myeloid Progenitors

Abstract An antiapoptotic role for Slug/Snail in mammals was suggested by studies in C. elegans, where CES-1/Scratch, a member of the Slug/Snail superfamily, was found to control the apoptotic death of NSM sister neurons by acting as a transcriptional repressor of EGL-1, a BH3-only proapoptotic protein. Identification of Slug as the target gene of the E2A-HLF oncoprotein in human pro-B leukemia cells led us to demonstrate its antiapoptotic function in IL-3-dependent murine pro-B cells. In contrast to its aberrant expression in pro-B leukemia cells, endogenous Slug is normally expressed in both LT-HSC and ST-HSC, as well as committed progenitors of the myeloid series, but not in pro-B and pro-T cells, implying its function in myelopoiesis. Using Slug−/− mice produced in our laboratory, we showed that these knockouts are much more radiosensitive than Slug+/− and wild-type mice, and that apoptotic cells increase significantly in the hematopoietic progenitor cells of Slug−/− mice as compared to wild-type mice following γ-irradiation, indicating a radioprotective function in vivo. We showed here that although the development of myeloid progenitors is not impaired under steady-state conditions, their repopulation is incomplete γ-irradiated in in Slug−/− mice. We demonstrate further the radiation-induced death of Slug−/− mice is exclusively a result of bone marrow failure with no apparent contribution from systemic injures to other tissues. By two-way bone marrow transplantation, we provide firm evidence that Slug protects mice from γ-irradiation-induced death in a cell-autonomous manner. Interestingly, regenerative capacity of hematopoietic stem cells (HSC) was retained in irradiated Slug−/− mice, which could be rescued by wild-type bone marrow cells after irradiation, indicating that Slug exerts its radioprotective function in myeloid progenitors rather than HSCs. Furthermore, we establish that Slug radioprotects mice by antagonizing downstream of the p53-mediated apoptotic signaling through inhibition of the p53-resposive proapoptotic gene Puma, leading in turn to inhibition of the mitochondria-dependent apoptotic pathway activated by γ-irradiation in myeloid progenitors. More interestingly, we observed that Slug is inducible by γ-irradiation in a p53-dependent manner. Together, our findings implicate a novel Slug-mediated feedback mechanism by which p53 control programmed cell death in myeloid progenitor cells in vivo in response to γ-irradiation.

Differential Expression of Mll-AF9 Up-Regulated Genes Correlates with Enhanced Self-Renewal of Hematopoietic Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 733-733 ◽  
Author(s):  
Ashish R. Kumar ◽  
Wendy A. Hudson ◽  
Weili Chen ◽  
Rodney A. Staggs ◽  
Anne-Francoise Lamblin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Cell Types ◽  
Third Generation ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
The Third

Abstract In order to understand the pathophysiology of leukemia, we need to study the effects of leukemic oncogenes on the rare hematopoietic stem and progenitor cells. We investigated the self-renewal capabilities of the various hematopoietic cell types derived from Mll-AF9 knock-in mice. We used the murine knock-in model since it offers the advantage of a single copy of the Mll-fusion gene under the control of the endogenous promoter present in every hematopoietic stem/progenitor cell. In methylcellulose cultures, we compared myeloid colony formation of Mll-AF9 cells to wild type progenitor populations over three generations of plating. In the first generation of plating, the Mll-AF9 common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) formed more colonies than the hematopoietic stem cells (HSCs) and common lymphoid progenitors (CLPs). However, at the third generation of plating, colony numbers formed by Mll-AF9 HSCs and CLPs were significantly greater than those formed by CMPs and GMPs. By the third generation only occasional colonies were found in the wild type groups. These results demonstrate that while Mll-AF9 led to an increase in self-renewal of all 4 cell types studied, these effects were more pronounced in HSCs and CLPs. To identify the downstream genes that mediate the growth deregulatory effects of Mll-AF9, we compared gene expression profiles of Mll-AF9 derived cells to their wild type counterparts. To assess gene expression levels, we extracted RNA from wild type and Mll-AF9 HSCs, CLPs, CMPs and GMPs. We then amplified and labeled the RNA for analysis by Affymetrix murine 430 2.0 genome arrays. In an unsupervised analysis, the various Mll-AF9 cells clustered with their corresponding wild type counterparts, indicating that the expression of most genes was not significantly altered by Mll-AF9. To identify the genes that are differentially expressed in the Mll-AF9 derived cells, we performed a two-way ANOVA (with the genotype and cell type as the two variables) allowing for a false discovery rate of 10%. In this analysis, we found that 76 genes were up-regulated in all Mll-AF9 progenitor cells compared to their wild-type counterparts. This list included known targets of Mll-fusion proteins Hoxa5, Hoxa7, Hoxa9 and Hoxa10. Also included were Evi1 and Mef2c, two genes that have been implicated in promoting enhanced self-renewal of murine hematopoietic cells. Importantly, in wild type mice, these 6 genes were expressed at higher levels in HSCs and CLPs compared to CMPs and GMPs (average 3–25 fold). While we observed an average 2–10 fold increase in expression of these genes in all Mll-AF9 cell types compared to their respective wild type controls, the expression level was 3–8 fold higher in Mll-AF9 HSCs and CLPs compared to CMPs and GMPs. Thus, the expression of genes known to be intrinsically related to self-renewal is further enhanced as a result of the Mll-AF9 fusion gene. In conclusion, while activation of the Mll-AF9 genetic program and the resulting enhanced self-renewal occurs in all 4 cell types studied, these effects are greatest in HSCs and CLPs. Thus, HSCs and CLPs are likely to be more efficient than CMPs and GMPs in producing cellular expansion and targets for cooperating mutations resulting in leukemia.

In Vivo Genome Editing of Liver Albumin for Therapeutic Gene Expression: Rescue of Hemophilic Mice Via Integration of Factor 9

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 751-751 ◽  
Author(s):  
Xavier M Anguela ◽  
Rajiv Sharma ◽  
Yannick Doyon ◽  
Sunnie Y Wong ◽  
David E Paschon ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Transgene Expression ◽  
Factor Ix ◽  
Safe Harbor ◽  
Wild Type ◽  
Advisory Committees ◽  
Aav Vector ◽  
Children's Hospital ◽  
Children’S Hospital

Abstract Abstract 751 Gene correction using zinc finger nuclease (ZFN) technology can be applied to target virtually any locus in the human genome. Beyond correcting mutated genes causative of disease, ZFNs can also be utilized to target transgene insertion into genomic “safe harbors.” Ideally, specific gene targeting to such “safe harbor” sites would (i) ensure therapeutically relevant levels of transgene expression and (ii) tolerate transgene addition without deleterious effect on the host organism. For liver-derived protein replacement, albumin represents an attractive target locus. Firstly, albumin is very highly expressed exclusively in the liver, thus targeting of a relatively small percentage of alleles should yield therapeutically relevant levels of liver-specific transgene expression. Second, the reduction or complete absence of albumin in animals and even humans (analbuminemia) produces surprisingly few symptoms. Here, we sought to investigate whether ZFN-mediated targeted insertion of a promoter-less copy of the human F9 cDNA at the mouse albumin locus could result in human Factor IX production and successfully correct the hemophilic phenotype in mice. To address this question, we constructed an AAV vector encoding a pair of ZFNs targeting intron 1 of the mouse albumin locus (AAV8-mAlb-ZFN) and a donor AAV vector (AAV8-Donor) harboring a partial cDNA cassette containing exons 2–8 of the wild-type human F9 gene flanked by sequences lacking significant homology to the mouse genome. Co-delivery of 1e11 vg of AAV8-mAlb-ZFN along with 5e11vg of AAV8-Donor resulted in stable (>12wk) circulating F.IX levels of 1600–3200 ng/mL (32–64% of normal). As a control, mice injected with the AAV8-Donor along with an AAV vector encoding a ZFN pair targeting an unrelated locus exhibited background F.IX levels (∼50 ng/mL). A dose-response study was performed by administering a fixed dose of donor (5e11 vg/mouse) with decreasing doses of AAV8-mAlb-ZFN (1e11, 1e10 and 1e9 vg/mouse). Human F.IX levels increased as a function of ZFN dose in the range tested (3260±480, 225±43 and 31±4 ng/mL at the high, medium and low dose, respectively). Importantly, these results showed that donor homology to the target site is not required to achieve robust levels of gene addition to the albumin locus in adult mice, thus permitting the design of donor vectors harboring corrective copies of transgenes up to the maximum AAV packaging capacity of ∼4.7 Kb. Albumin and factor IX are both synthesized as pre-propeptides and turned into propeptides after the signal peptide is removed. Expression of human F9 exons 2–8 spliced with mouse albumin exon 1 is expected to yield a chimeric propeptide. The first 2 N-terminal amino acids would originate from proalbumin, followed by a Val to Leu mutation at position −17 of the hF.IX propeptide and 16 aa encoded by human F9. To evaluate whether this chimeric human F.IX derived from gene addition to the albumin locus would be processed correctly and normalize the prolonged clotting times in hemophilia B (HB) mice, we injected 1e11 vg of AAV8-mAlb-ZFN and 5e11vg of AAV8-Donor into HB animals. Two weeks post-treatment, hF.IX antigen levels were in the range of 20% of normal and activated partial thromboplastin time, a measurement of clot formation, was corrected to wild-type levels (42 seconds), from an average of 70 seconds pre-treatment. Thus expression of a therapeutic protein (F.IX) from the albumin locus is shown to correct the HB disease phenotype in vivo. In summary, these data provide the first demonstration of ZFN-mediated in vivo genome editing of a safe harbor locus for therapeutic protein production. While we provide here a proof of principle establishing phenotypic correction of hemophilia B, appropriately designed donors could expand this strategy. Most importantly the magnitude of albumin expression (>15 g / day) should enable production of a diverse range of transgenes at therapeutically consequential levels. Disclosures: Anguela: The Children's Hospital of Philadelphia: Patents & Royalties. Sharma:The Children's Hospital of Philadelphia: Patents & Royalties. Doyon:Sangamo BioSciences, Inc.: Employment. Wong:Sangamo BioSciences, Inc.: Employment. Paschon:Sangamo BioSciences, Inc.: Employment. Gregory:Sangamo BioSciences, Inc.: Employment. Holmes:Sangamo BioSciences, Inc.: Employment. Rebar:Sangamo BioSciences, Inc.: Employment. High:Shire Pharmaceuticals: Consultancy; Sangamo Biosciences, Inc: Collaborator, Collaborator Other; Novo Nordisk: Visiting Professor, Visiting Professor Other; Genzyme, Inc: Membership on an entity's Board of Directors or advisory committees; The Children's Hospital of Philadelphia: Patents & Royalties; Bluebird Bio, Inc: Membership on an entity's Board of Directors or advisory committees.

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