MSI2 Is Required for Maintaining the Activated Myelodysplastic Syndrome Stem Cell

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 714-714
Author(s):  
Tzu-Chieh Ho ◽  
James Taggart ◽  
Elianna Amin ◽  
Haiming Xu ◽  
Trevor Barlowe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Rna Binding ◽  
Fold Change ◽  
Conditional Knockout ◽  
Gene Set Enrichment Analysis ◽  
Poor Survival ◽  
Hematopoietic Stem ◽  
Log2 Fold Change ◽  
Progenitor Compartment

Abstract + The first four authors contributed equally to this project. Myelodysplastic syndromes (MDS) are a group of blood cell disorders, characterized by ineffective hematopoiesis and severe cytopenias, which often transform to acute leukemia. MDS is also considered to be a clonal stem cell disease driven by alterations that are both genetic and epigenetic. However, it remains unclear how stem cell function is dysregulated and what factors drive these alterations in MDS HSCs. MSI2 is an important RNA-binding protein in normal HSC maintenance and can promote aggressive myeloid leukemia. Our preliminary data indicate that MSI2 expression is increased in high-risk MDS compared to low-risk MDS and correlates with poor survival. In order to model the role of MSI2 in MDS, we utilized the NUP98-HOXD13 transgenic (NHD13) model, which recapitulates many salient features of MDS including, leukopenia, severe anemia, erythroid dysplasia and leukemic transformation. Despite the lethal MDS or AML disease found in primary NHD13 animals, bone marrow cells transplanted into congenic mice generate a non-lethal MDS that rarely transform. Depletion of Msi2 utilizing a conditional knockout (NHD13-Msi2f/f -MX1-Cre) reversed the MDS phenotype and after one month the diseased HSPCs were eliminated. Conversely, we found that tetracycline inducible MSI2 overexpression in the context of the NHD13 transgene (NHD13/MSI2 mice) resulted in a worse MDS disease and a fully penetrant and lethal transformation to an AML, which was further accelerated during serial transplantation. AML arising in NHD13/MSI2 mice remained dependent on sustained MSI2 overexpression as mice removed from doxycycline demonstrated improved survival. Most interestingly, MSI2 overexpression expanded and maintained a more activated (G1) MDS hematopoietic stem and progenitor compartment (HSPC) in NHD13 cells. Gene expression profiling of the LSKs (Lineagelo, c-Kit+, Sca1+) before disease progression identified 891 significant genes, of which 137 genes were up-regulated (log2 fold change > 0) and 754 genes were down-regulated (log2 fold change <= 0). Furthermore, Gene Set Enrichment Analysis (GSEA) demonstrated a more progenitor like gene expression signature, enrichment in an NRAS activated signature, and a reduced quiescent phenotype. Unsupervised hierarchical clustering of the NHD13/MSI2 LSK gene signature in MDS patients resulted in four distinct clusters. Clusters segregated MSI2 high expressing MDS patients and this "MSI2 high cluster" predicted poor survival. In summary, our findings suggest that MSI2 plays a critical functional role in the maintenance of the hematopoietic stem and progenitor compartment in MDS and highlights it as a novel therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals JMML Fetal Identity Results Either from Retention of a Physiologic Signature or Aberrant Activation of Master Oncofetal Regulators

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-5
Author(s):  
Marion Strullu ◽  
Aurélie Caye-Eude ◽  
Loïc Maillard ◽  
Chloé Arfeuille ◽  
Elodie Lainey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Board Of Directors ◽  
Hematopoietic Stem Cell ◽  
Hierarchical Clustering ◽  
Research Funding ◽  
Gene Set Enrichment Analysis ◽  
Hematopoietic Progenitors ◽  
Advisory Committees ◽  
Hematopoietic Stem

Objectives: Juvenile myelomonocytic leukemia (JMML) is a rare but aggressive myeloproliferative/myelodysplastic neoplasm affecting infants and young children. The narrow age-window of onset suggests that a prenatal environment is needed for JMML oncogenesis. In search of a transcriptional reminiscence of embryo-fetal characteristics that would confirm this hypothesis, we investigated how the gene expression profile of JMML hematopoietic progenitors compared to their healthy counterpart isolated at different stages of ontogeny. Methods: Hematopoietic stem cell and progenitor cell (HSPC) fractions of JMML (n=16), bone marrow (BM) of healthy children (n=7), fetal liver (FL; n=3) and fetal BM (FBM; n=2) were phenotyped and sorted using signatures validated in the fetal and adult BM (Notta et al, Science 2011). RNAseq was performed using the TruSeq® Stranded Total RNASample preparation kit. Unsupervised hierarchical clustering analysis was done with the Bioconductor edgeR package. Differentially expressed genes were identified with the Bioconductor limma package. Results: To eliminate the impact of variations in the HSPC distribution, the JMML transcriptome was assessed on FACS-sorted common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) and megakaryocyte-erythroid progenitor (MEP) cell fractions from 16 JMML and compared to healthy counterparts at different stages of ontogeny (FL, FBM, age-matched children BM). Unsupervised hierarchical clustering separated the samples into 4 groups (C1-4), primarily according to ontogeny, with 14/15 embryo-fetal fractions in C1 and all healthy post-natal progenitors in C2 (CMP, MEP) or C3 (GMP). Most JMML fractions clustered either with the prenatal fractions (C1; 17/47 fractions from 8/16 patients) or in a separate group containing no healthy sample (C4; 23/47 samples from 10/16 patients). Two groups were defined accordingly: one with JMML resembling embryo-fetal samples ('Fetal-JMML'; n=6/16), and a JMML-specific group ('Onco-JMML'; 8/16). Patients with Onco-JMML tended to be older, with a more severe presentation and elevated fetal hemoglobin levels. All PTPN11-mutated JMML were in this group whereas 5/6 Fetal-JMML had NRAS or KRAS mutations. Analysis of differential gene expression between Fetal and Onco-JMML highlighted 344 up-regulated genes versus 19 up-regulated genes in Onco-JMML. Surprisingly, LIN28B and WT1, both known to activate fetal pathways, were the most up-regulated genes in Onco-JMML. These key transcription factors were deregulated as early as the hematopoietic stem cell (HSC) compartment. Gene Set Enrichment Analysis (GSEA) confirmed enrichment in LIN28B and WT1-related signatures and showed enrichment in an AML signature in Onco-JMML. On the other hand, Fetal-JMML showed striking overexpression of monocytic /dendritic cell markers and inflammasome and innate immunity components. GSEA confirmed the strong monocyte identity of Fetal-JMML progenitors compared to onco-JMML or healthy postnatal progenitors. Part of the monocytic markers 'aberrantly' expressed in JMML progenitors was expressed in healthy fetal progenitors. Analysis of the HSC and multipotent progenitor (MPP) fractions showed that up regulation of monocytic markers was limited to the JMML progeny compartments. As we were able to confirm the transcriptional and functional identity of the sorted progenitors, these data suggest an early monocytic priming in these JMML progenitors, reminiscent of the monocyte-biased myelopoiesis characterizing physiologic fetal hematopoiesis. Conclusion: Our findings give a striking example of how ontogeny-related features are involved in childhood oncogenesis. They highlight a strong but complex link beween JMML and development, with a fetal identity resulting either from retention of a physiologic fetal monocytic signature or from aberrant (re)activation of master oncofetal regulators. Intriguingly, although LIN28B is thought to reprogram hematopoietic progenitors into a fetal-like state, its activation does not lead to an overall fetal profile in JMML, suggesting a regulatory mechanism distinct from that of physiological development. These two ontogeny-based signatures are likely to uncover the biology underlying previous classifiers based on AML-like profile or DNA methylation and suggest that a subset of JMML patient may benefit from immunomodulating therapies. Disclosures Dalle: Bellicum: Consultancy, Honoraria; bluebird bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Consultancy, Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; AbbVie Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Orchard: Consultancy, Honoraria; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees. Baruchel:Jazz Pharmaceuticals: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria; Astra Zeneca: Consultancy; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Shire: Research Funding; Bellicum: Consultancy.

Msi2 Maintains the MLL Leukemia Stem Cell Regulatory Program

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 836-836
Author(s):  
Sun Mi Park ◽  
Mithat Gönen ◽  
Ly P. Vu ◽  
Gerard Minuesa ◽  
Patrick Tivnan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Myeloid Leukemia ◽  
Rna Binding ◽  
Expression Profiles ◽  
Conditional Knockout ◽  
Transcriptional Analysis ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Renewal Program

Abstract Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. LSCs are characterized by their gain of a self-renewal program that is normally associated with hematopoietic stem cells (HSCs). Previously we have shown that the RNA binding protein, Msi2 contributes to both HSC and myeloid leukemia function. Elevated MSI2 expression predicts a poor prognosis in a variety of leukemias and shRNA-mediated depletion in human AML cell lines reduces proliferation, increases differentiation and induces apoptosis. Despite these in vitroand correlative studies, MSI2’s molecular mechanism is not known and its role in LSC function has not been assessed. To elucidate MSI2’s role in LSC function, we utilized the MLL-AF9 leukemia mouse model. Initially we found MSI2 was elevated in the LSC enriched compartment (c-KitHigh cells) compared to non-LSCs (c-KitLow cells) based on flow cytometric intracellular staining. Therefore, to establish a model to study Msi2 and its contribution to myeloid LSCs, we have utilized the Msi2 conditional knockout mice that we previously crossed (Msi2f/f) into an Mx1-Cre background to generate the Msi2Δ/Δallele (injection of polyinositol-polycytosine; pIpC). In order to test if Msi2 is critical for MLL-AF9 mediated initiation, we transduced control Msi2f/f and Msi2Δ/ΔLin- Sca1+ c-Kit+cells (LSKs) with MLL-AF9 expressing retroviruses co-expressing GFP. Msi2 deleted LSKs or granulocyte-monocyte progenitors (GMPs) transduced with MLL-AF9 demonstrated delayed leukemogenesis with dramatically reduced diseased burden. Msi2 deficient leukemias were found to have a 4-fold reduced phenotypic LSC population and were more differentiated based on cellular morphology. Msi2 deficient leukemias failed to transplant into secondary recipients demonstrating that Msi2 is required for maintaining LSCs. Deletion of Msi2after leukemia engraftment led to a delay in leukemogenesis indicating that Msi2 is also important for leukemia maintenance. Gene expression profiling of the Msi2 ablated LSCs resulted in a loss of the HSC/LSC program and an increase in differentiation gene sets. The gene signature from the Msi2 deleted murine LSCs (121 genes) was overlapped and subjected to unsupervised clustering with the gene expression profiles from 336 AML patients (ECOG1900 dataset). This analysis resulted in distinct clusters that had differential MSI2 expression and the MSI2“high” cluster predicted a worse clinical outcome when compared to the other clusters. Overlapping of the differential transcriptional analysis of the Msi2 deleted murine LSCs with our global MSI2 direct mRNA targets (HITS-CLIP) led us to identify that MSI2 binds to transcripts that are associated with the downstream MLL self-renewal program, including Myc and Ikzf2. Ikzf2 is a member of the Ikaros transcription factor family and is known to regulate lymphocyte development by controlling regulatory T-cell function and chromatin remodeling. Ikzf2 shRNA mediated depletion resulted in reduced colony formation, decreased proliferation and increased apoptosis. The MLL associated targets were also reduced, which included Bcl-2 and Hoxa9. In contrast to its tumor suppressor role in hypodiploid B-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell maintenance. Thus, we provide evidence that MSI2 maintains the oncogenic LSC epigenetic program and the rationale for clinically targeting MSI2 in myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of novel regulators of hematopoietic stem cell development through refinement of stem cell localization and expression profiling

Blood ◽  
2009 ◽  
Vol 114 (21) ◽  
pp. 4645-4653 ◽  
Author(s):  
Maria I. Mascarenhas ◽  
Aimée Parker ◽  
Elaine Dzierzak ◽  
Katrin Ottersbach
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Expression Profiling ◽  
Gene Expression Analysis ◽  
Kinase Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Hematopoietic Stem ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Cell Localization ◽  
Before And After

Abstract The first adult-repopulating hematopoietic stem cells (HSCs) are detected starting at day 10.5 of gestation in the aorta-gonads-mesonephros (AGM) region of the mouse embryo. Despite the importance of the AGM in initiating HSC production, very little is currently known about the regulators that control HSC emergence in this region. We have therefore further defined the location of HSCs in the AGM and incorporated this information into a spatial and temporal comparative gene expression analysis of the AGM. The comparisons included gene expression profiling (1) in the newly identified HSC-containing region compared with the region devoid of HSCs, (2) before and after HSC emergence in the AGM microenvironment, and (3) on populations enriched for HSCs and their putative precursors. Two genes found to be up-regulated at the time and place where HSCs are first detected, the cyclin-dependent kinase inhibitor p57Kip2/Cdkn1c and the insulin-like growth factor 2, were chosen for further analysis. We demonstrate here that they play a novel role in AGM hematopoiesis. Interestingly, many genes involved in the development of the tissues surrounding the dorsal aorta are also up-regulated during HSC emergence, suggesting that the regulation of HSC generation occurs in coordination with the development of other organs.

scholarly journals Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1204-1204
Author(s):  
Xi Jin ◽  
Tingting Qin ◽  
Nathanael G Bailey ◽  
Meiling Zhao ◽  
Kevin B Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Double Mutant ◽  
Mutant Mice ◽  
Cytokine Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Mutant ◽  
Tet2 Mutation

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Post-transcriptional regulation in hematopoiesis: RNA binding proteins take control

2019 ◽  
Vol 97 (1) ◽  
pp. 10-20 ◽  
Author(s):  
Laura P.M.H. de Rooij ◽  
Derek C.H. Chan ◽  
Ava Keyvani Chahi ◽  
Kristin J. Hope
Keyword(s):  
Transcriptional Regulation ◽  
Stem Cell ◽  
Cell Fate ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Control Of Gene Expression ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Post Transcriptional Regulation

Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP–RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.

scholarly journals Pericardial Effusion after Hematopoietic Stem Cell Transplant Is Associated with Poor Survival

2020 ◽  
Vol 26 (3) ◽  
pp. S140-S141
Author(s):  
Rebecca Wiersma ◽  
Vasant Chinnabhandar ◽  
Qing Cao ◽  
Kari Erickson ◽  
Troy C. Lund ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pericardial Effusion ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem Cell Transplant ◽  
Stem Cell Transplant ◽  
Cell Transplant ◽  
Poor Survival ◽  
Hematopoietic Stem

scholarly journals PDGFB-expressing mesenchymal stem cells improve human hematopoietic stem cell engraftment in immunodeficient mice

2019 ◽  
Vol 55 (6) ◽  
pp. 1029-1040 ◽  
Author(s):  
Xiuxiu Yin ◽  
Linping Hu ◽  
Yawen Zhang ◽  
Caiying Zhu ◽  
Hui Cheng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Human Mesenchymal Stem Cells ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Bm Niche

AbstractThe bone marrow (BM) niche regulates multiple hematopoietic stem cell (HSC) processes. Clinical treatment for hematological malignancies by HSC transplantation often requires preconditioning via total body irradiation, which severely and irreversibly impairs the BM niche and HSC regeneration. Novel strategies are needed to enhance HSC regeneration in irradiated BM. We compared the effects of EGF, FGF2, and PDGFB on HSC regeneration using human mesenchymal stem cells (MSCs) that were transduced with these factors via lentiviral vectors. Among the above niche factors tested, MSCs transduced with PDGFB (PDGFB-MSCs) most significantly improved human HSC engraftment in immunodeficient mice. PDGFB-MSC-treated BM enhanced transplanted human HSC self-renewal in secondary transplantations more efficiently than GFP-transduced MSCs (GFP-MSCs). Gene set enrichment analysis showed increased antiapoptotic signaling in PDGFB-MSCs compared with GFP-MSCs. PDGFB-MSCs exhibited enhanced survival and expansion after transplantation, resulting in an enlarged humanized niche cell pool that provide a better humanized microenvironment to facilitate superior engraftment and proliferation of human hematopoietic cells. Our studies demonstrate the efficacy of PDGFB-MSCs in supporting human HSC engraftment.

scholarly journals HIF-1α deletion partially rescues defects of hematopoietic stem cell quiescence caused by Cited2 deficiency

Blood ◽  
2012 ◽  
Vol 119 (12) ◽  
pp. 2789-2798 ◽  
Author(s):  
Jinwei Du ◽  
Yu Chen ◽  
Qiang Li ◽  
Xiangzi Han ◽  
Cindy Cheng ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Knockout Mice ◽  
Gene Dosage ◽  
Fetal Liver ◽  
Conditional Knockout ◽  
Negative Regulator ◽  
Transcriptional Level ◽  
Double Knockout ◽  
Hematopoietic Stem

Abstract Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence- and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1α gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1α double-knockout mice. Additional deletion of HIF-1α in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1–dependent and HIF-1–independent pathways.

Leukemic CD34+CD38- Cells Display Conserved Differential Expression of Many ATP Binding-Cassette Transporter Genes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1380-1380
Author(s):  
Marc H.G.P. Raaijmakers ◽  
Elke P.L.M. de Grouw ◽  
Louis T.F. van de Locht ◽  
Bert A. van der Reijden ◽  
Theo J.M. de Witte ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Abc Transporters ◽  
Cell Biology ◽  
Cell Fraction ◽  
Atp Binding ◽  
Stem Cell Biology ◽  
Hematopoietic Stem ◽  
Atp Binding Cassette

Abstract In most cases of acute myeloid leukemia (AML) CD34+CD38− cells are considered to be stem cells, responsible for the maintenance and relapse of AML. ATP binding cassette transporters function in the extrusion of xenobiotics and chemotherapeutical compounds, and may be involved in therapy resistance. Elucidation of mechanisms conferring drug resistance to CD34+CD38− cells is essential to provide novel targets for stem cell eradication in AML. We studied gene expression of all 45 transmembrane ABC transporters (the complete ABCA, B, C, D and G family) in human hematopoietic CD34+CD38− cells and more committed CD34+CD38+ progenitor cells, from healthy donors and patients with non-hematological diseases (N=11) and AML patients (N=11). Gene expression was assessed using a novel real-time RT-PCR approach with micro fluidic cards. In normal CD34+CD38− cells 36 ABC transporters were expressed, 22 of these displayed significant higher expression in the CD34+CD38− cell fraction compared to the CD34+CD38+ cell fraction. In addition to the known stem cell transporters (ABCB1, ABCC1 and ABCG2) these differential expressed genes included many members not previously associated with stem cell biology. In AML the ABC transporter expression profile was largely conserved, including expression of all 13 known drug transporters. These data suggest an important role for many ABC transporters in hematopoietic stem cell biology. In addition, the preferential expression of a high number of drug transport related transporters predicts that broad spectrum inhibition of ABC transporters is likely to be required for CD34+38− stem cell eradication in AML. This approach will, apart from affecting the leukemic stem cells, equally affect the normal stem cells.

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